WO2025235697A1 - Cotton transgenic event gh_bcs246002 and methods for detection and uses thereof - Google Patents

Cotton transgenic event gh_bcs246002 and methods for detection and uses thereof

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Publication number
WO2025235697A1
WO2025235697A1 PCT/US2025/028295 US2025028295W WO2025235697A1 WO 2025235697 A1 WO2025235697 A1 WO 2025235697A1 US 2025028295 W US2025028295 W US 2025028295W WO 2025235697 A1 WO2025235697 A1 WO 2025235697A1
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WIPO (PCT)
Prior art keywords
cotton
seq
bcs246002
dna
event
Prior art date
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Pending
Application number
PCT/US2025/028295
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French (fr)
Inventor
Aqeel Ahmad
Jeffrey Ahrens
Sarah L. BROWN
Cheryl CLONINGER
Arlene R. HOWE
Jiyan Ma
Linda RYMARQUIS
Jason STELZER
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Monsanto Technology LLC
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Monsanto Technology LLC
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Application filed by Monsanto Technology LLC filed Critical Monsanto Technology LLC
Publication of WO2025235697A1 publication Critical patent/WO2025235697A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8286—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for insect resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/13—Plant traits
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the present invention relates to transgenic cotton plants, plant parts, seeds, cells and agricultural products containing cotton event GH_BCS246002 or a modified cotton event GH_BCS246002, recombinant DNA molecules present in and/or isolated from cotton event GH_BCS246002 or a modified cotton event GH_BCS246002, as well as methods of using the same and detecting the presence of DNA for a cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 in a sample.
  • the present invention also relates to transgenic cotton plants, plant parts, seed and cells containing cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 DNA that exhibit resistance to Lepidopteran insect infestations.
  • the present invention also relates to methods for modifying the cotton event GH_BCS246002 using genome editing techniques to produce a modified cotton event GH_BCS246002.
  • Cotton is an important crop in many areas of the world. The methods of biotechnology have been applied to cotton for improvement of the agronomic traits and quality of the product.
  • One such agronomic trait is insect resistance, which is accomplished through the expression of heterologous insect toxins, also known as transgenes, inserted into the genome of the cotton plant.
  • transgenic events in cotton that have been described in the art that provide various types of insect resistance, particularly to Lepidopteran species, and these include, MON531, MON 15985, DAS-24236 (281 -24-236), DAS-21023 (3006-210-23), COT102, GHB 119, and T304-40.
  • the Lcpidoptcran resistance trait conferred by these transgenic events have been in use commercially in a variety of geographies across the globe for an extended period of time, and resistance to many of the expressed toxins in these events by targeted insect pests has developed in many of the geographic regions where these transgenic traits have been deployed.
  • novel transgenic events in cotton that confer novel traits for controlling Lepidopteran target pest species, and preferably the novel transgenic events confer resistance to the target insect pests, including those races that have evolved resistance to the existing commercially deployed traits.
  • Such transgenic traits will preferably use modes of action that are different than the modes of action previously deployed in existing commercial embodiments. Described herein is an example of a novel transgenic event in cotton that confers resistance to Lcpidoptcran infestations, including resistance to Lcpidoptcrans that have evolved resistance to commercial embodiments that have been previously deployed.
  • a novel transgenic cotton event GH_BCS246002 or a modified cotton event GH_BCS246002, that provides insecticidal control over Lcpidoptcran pests of cotton is provided.
  • transgenic plants, plant cells, seed, plant parts, and commodity products comprising cotton event GH_BCS246002 DNA or a modified cotton event GH_BCS246002 DNA.
  • Novel DNA molecules and constructs that are specific and unique to the event GH_BCS246002 or a modified cotton event GH_BCS246002 event and comprise the inserted transgenic DNA segment and the novel DNA segments that are adjacent to the inserted transgenic DNA segment.
  • Adjacent DNA segments are described herein as the junction sequences which correspond to the DNA sequences spanning the flanking chromosomal or genomic DNA and a portion of the adjacent transgenic DNA insert at or near a chromosomal breakpoint where the transgenic inserted DNA has been introduced into the genome of a cotton plant.
  • the orientation of the inserted DNA (SEQ ID NO: 9) is specified herein in reference to the adjacent flanking chromosomal or genomic DNA. Because the transgenic DNA insert is a linear arrangement of elements with a 5’ end and 3’ end relative to the orientation of the insert and expression cassettes and the surrounding genome as depicted, for example, in FIGs. 1 and 2.
  • the 5’ junction is represented herein by the sequence as set forth in SEQ ID NO: 1
  • the 3’ junction is represented herein by the sequence as set forth in SEQ ID NO: 2
  • longer sequences described herein arc also examples of both junction sequences, although SEQ ID NO: 1 or SEQ ID NO: 2, as applicable, are included within the respective longer sequences.
  • 5’ junction sequences containing SEQ ID NO: 1 include but are not limited to SEQ ID NO: 3
  • SEQ ID NO: 5 and SEQ ID NO: 7, and 3’ junction sequences containing SEQ ID NO: 2 include but are not limited to SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8.
  • polynucleotides and molecules are provided that are specific and unique to, and capable of use for identifying and detecting the presence of cotton event GH_BCS246002 DNA or a modified cotton event GH_BCS246002 DNA in a biological sample containing or derived from a cotton plant, plant part, tissue, cell or other plant material, such progeny plants and commodity products, comprising cotton event GH_BCS246002 DNA or a modified cotton event GH_BCS246002 DNA.
  • methods related to selecting a plant, plant part, plant cells and/or seed comprising the cotton event GH_BCS246002 DNA or a modified cotton event GH_BCS246002 DNA, and detection of the presence (or absence) of cotton event GH_BCS246002 DNA or a modified cotton event GH_BCS246002 DNA in a sample are provided, such methods provided for the purpose of confirming that the event GH_BCS246002 DNA is, or is not, present in a particular sample subjected to the method or to methods reliant upon polynucleotide sequences that are the subject of this disclosure.
  • detection of a polynucleotide sequence in a sample from cotton tissue, cells, seed, plants, or plant parts that contain cotton genomic DNA and one or both of SEQ ID NO: 1 or SEQ ID NO: 2, is determinative of the presence of the cotton event GH_BCS246002 DNA in said sample.
  • the present disclosure provides recombinant DNA molecules comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, and a complete complements thereof.
  • RNA sequence corresponding to any of the foregoing polynucleotide sequences could also be used for similar purposes and would include a uridine residue in place of any thymidine residue depicted in the applicable DNA provided herein and complements thereof, and references to DNA herein may also be applied to a corresponding RNA sequence and complements thereof.
  • the recombinant DNA molecules provided herein may be derived from cotton event GH_BCS246002 in a sample of seed containing the event and containing the unique and specific DNA segments as provided herein corresponding to cotton event GH_BCS246002 seed which has been deposited as ATCC Accession No. PTA-127733.
  • Another aspect of the present disclosure provides a DNA molecule comprising a polynucleotide segment of sufficient length to function as a nucleic acid probe that hybridizes specifically under stringent hybridization conditions with cotton event GH_BCS246002 DNA in a sample, wherein detecting hybridization of the probe to the event DNA in the sample under the stringent hybridization conditions is diagnostic for, or characteristic of, confirming the presence of cotton event GH_BCS246002 DNA in that sample.
  • an absence of detection of hybridization of the probe to the cotton DNA could be used to demonstrate the absence of the event in the sample.
  • the sample may comprise whole or process cotton seeds, cotton fiber, cotton oil and derivatives of cotton oil, cotton protein, cotton meal, animal feed comprising cotton, paper comprising cotton, cotton biomass, candle wicks, cotton string, cotton rope, cotton balls, cotton batting, cotton fuel products, and cotton cellulose products, wherein such cotton and cotton products contain detectable amounts of cotton event GH_BCS246002 DNA or detectable amounts of the novel toxin proteins produced by such transgenic cotton plants, cells and the like, to contain the cotton event GH_BCS246002.
  • Yet another aspect of the invention provides a pair of DNA molecules comprising a first DNA molecule and a second DNA molecule different from the first DNA molecule, wherein the pair of DNA molecules can be used together as DNA primers in an amplification reaction containing the appropriate and necessary reagents with a sample that may or may not contain cotton event GH_BCS246002 template DNA to produce an amplicon diagnostic for, or characteristic of, the presence of said cotton event GH_BCS246002 DNA if present in said sample.
  • the amplicon produced will contain at least the nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, and the complete complements thereof.
  • Another aspect of the present disclosure provides a method of detecting or determining the presence or absence of a DNA segment diagnostic for confirming the presence or absence of cotton event GH_BCS246002 DNA in a sample.
  • methods are provided that may be conducted by contacting the sample with a probe DNA molecule that hybridizes specifically to DNA uniquely associated with cotton event GH_BCS246002, then subjecting the sample and the probe DNA molecule to stringent hybridization conditions to allow the probe to bind to the appropriate complement segment of cotton event GH_BCS246002 specific DNA. Detecting hybridization of the probe DNA molecule to the DNA in the sample is conclusive, diagnostic, or determinative that the DNA in the sample contains the cotton event GH_BCS246002 DNA.
  • methods may comprise the steps of contacting a biological sample with a pair of DNA molecules that function as nucleic acid primers specific for amplification of a DNA segment specific for the cotton event GH_BCS246002 DNA, performing an amplification reaction sufficient to produce the DNA amplicon, and detecting the presence or absence of the DNA amplicon in the reaction.
  • Detection of the DNA amplicon is diagnostic for, or characteristic of, the presence of a detectable amount of the cotton event GH_BCS246002 DNA in the sample, and the amplicon should contain all or a portion of the nucleotide sequence targeted for amplification that lies between the primer hybridization positions, which may include a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, and the complete complements thereof.
  • the lack of detection of the DNA amplicon would be diagnostic for, or characteristic of, the absence of at least a detectable amount of the cotton event GH_BCS246002 DNA in the sample.
  • Another embodiment of the invention is a method of detecting the presence of protein diagnostic for or characteristic of cotton event GH_BCS246002 in a sample, said method comprising an immunoassay in which one or more of the antibodies specific for the respective toxin proteins encoded by one or more of the cassettes of cotton event GH_BCS246002 to detect the presence or absence of one or more of the toxins produced by the cotton event, the method comprising: (a) contacting said sample with a first, second, and/or third monoclonal antibody, wherein the first monoclonal antibody binds specifically to one or more epitopes of Cry IB.3 toxin protein, the second monoclonal antibody binds specifically to one or more epitopes of CrylDa_7 toxin protein, and the third monoclonal antibody binds specifically to one or more epitopes of Vip3Cbl .1 toxin protein; (2) incubating the immunoassay for a sufficient amount of time to allow for binding of
  • the assay can be selected from the group consisting of an Enzyme-linked Immunosorbent Assay (ELISA), a Radioimmunoassay, and a Lateral flow immunochromatographic assay.
  • ELISA Enzyme-linked Immunosorbent Assay
  • Radioimmunoassay a Radioimmunoassay
  • Lateral flow immunochromatographic assay The detection step can be colorimetric, radiologic, or fluorescent relative to a control samples, such as a negative control and/or a positive control for the detecting the presence of the respective toxin protein.
  • Another embodiment of the invention is a cotton plant, cotton plant part, cotton cell, or part thereof comprising a recombinant polynucleotide molecule comprising the nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, and the complete complement thereof.
  • SEQ ID NOs arc each the specific and selective DNA that defines the cotton event GH_BCS246002.
  • This cotton plant, cotton plant part, cotton cell, or part thereof is insecticidal when provided in the diet of a Lepidopteran insect pest.
  • Lepidopteran insect target pests intended to be controlled include Cotton bollworm (Helicoverpa zed), Tobacco budworm (Heliothis virescens), and/or Fall armyworm (Spodopiera frugiperdd), and any combination of any two or all three of the foregoing pest species.
  • the cotton plant can be further defined as progeny of any generation of a cotton plant comprising the cotton event GH_BCS246002, provided that the progeny also contains the specific and selective DNA that defines the cotton event GH_BCS246002.
  • Yet another aspect of the present disclosure provides methods for protecting a cotton plant from insect infestation, wherein said method comprises providing in the diet of a Lepidopteran insect pest an insecticidally effective amount of cotton cells or tissue of a cotton plant comprising cotton event GH_BCS246002.
  • Contemplated Lepidopteran insect pests include Cotton bollworm (Helicoverpa zea), Tobacco budworm (Heliothis virescens), and Fall armyworm (Spodoptera frugiperdd).
  • Another aspect of the present disclosure provides methods of producing an insect resistant cotton plant comprising: a) manually crossing by conventional breeding, two different cotton plants to produce progeny, wherein at least one of the two different cotton plants contains the cotton event GH_BCS246002 DNA or DNA from a modified cotton event GH_BCS246002; b) confirming in the seed arising from the breeding activity, and in the progeny plants and tissue grown from such seed, the presence of a DNA segment diagnostic for, or characteristic of, cotton event GH_BCS246002 or a modified cotton event GH_BCS246002; and c) selecting the progeny comprising the cotton event GH_BCS246002 DNA or DNA from a modified cotton event GH_BCS246002.
  • Such seed and progeny are Lepidopteran resistant cotton plants.
  • a further aspect of the present disclosure provides cotton seeds, nonliving plant materials, or microorganisms comprising a detectable amount of the polynucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, and complete complements thereof.
  • Yet another aspect of the present disclosure is a commodity cotton product comprising a detectable amount of a recombinant DNA molecule unique to cotton event GH_BCS246002, wherein the recombinant DNA molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, and complete complements thereof.
  • Commodity cotton products may include, but are not limited to, whole or process cotton seeds, cotton fiber, cotton oil and derivatives of cotton oil, cotton protein, cotton meal, animal feed comprising cotton, paper comprising cotton, cotton biomass, candle wicks, cotton string, cotton rope, cotton balls, cotton batting, cotton fuel products, and cotton cellulose products.
  • Another aspect of the present disclosure is a cotton plant, cotton plant part, cotton seed, cotton tissue or cell comprising a DNA molecule functional as a template in a DNA amplification assay to produce an amplicon diagnostic for, or characteristic of, the presence of cotton event GH_BCS246002 DNA.
  • Yet another aspect of the present disclosure are methods of determining the zygosity of a cotton plant, plant part or seed comprising DNA specific to the cotton event GH_BCS246002 or a modified cotton event GH_BCS246002.
  • the zygosity can be determined in a series of consecutive steps.
  • a sample comprising cotton DNA can be contacted with a first primer pair that is capable of producing an amplicon in a DNA amplification assay that is diagnostic for, or characteristic of, DNA that is specific for and uniquely present in cotton event GH_BCS246002.
  • the sample comprising the cotton DNA can be contacted with a second primer pair that is designed to produce an amplicon of an internal standard known to be single-copy and homozygous in the cotton plant.
  • Such methods may additionally include contacting the DNA sample with a probe set which contains at least a first probe that specifically hybridizes to a cotton event GH_BCS246002 DNA, and a second probe that specifically hybridizes to an internal genomic DNA standard known to be single-copy and homozygous in the cotton plant.
  • the DNA amplification reaction in these methods may be performed using real-time PCR to determine the cycle thresholds (Ct values) of the amplicon corresponding the allele of cotton event GH_BCS246002 and the single-copy, homozygous internal standard.
  • the difference (delta-Ct) between the Ct value of the single-copy, homozygous internal standard amplicon, and the Ct value of the allele for cotton event GH_BCS246002 amplicon may be calculated.
  • zygosity is determined wherein a delta-Ct of about zero (0) indicates homozygosity of the inserted T-DNA of cotton event GH_BCS246002, and a delta-Ct of about one (1) indicates heterozygosity of the inserted T-DNA of cotton event GH_BCS246002.
  • the primer pairs are selected from the group consisting of SEQ ID NO: 15 combined with SEQ ID NO: 16, and SEQ ID NO: 18 combined with SEQ ID NO: 19; and wherein the probes are SEQ ID NO: 17 and SEQ ID NO: 20.
  • the delta-Ct of about one (1) indicating heterozygosity of the inserted T-DNA of cotton event GH_BCS246002 is in the range of 0.75 to 1.25.
  • a delta-Ct of about zero (0) may be about 0, 0.05, 0.1, 0.15, 0.2, or 0.25, in other embodiments, a delta-Ct of about one (1) may be about 0.75, 0.8, 0.85, 0.9, 0.95, 1.0, 1.05, 1.1, 1.15, 1.2, or 1.25.
  • a delta-Ct of about one (1) may be in the range of 0.75 to 1.25, 0.8 to 1.25, 0.85 to 1.25, 0.9 to 1.25, 0.95 to 1.25, 1.0 to 1.25, 1.05 to 1.25, 1.1 to 1.25, 1.15 to 1.25, 1.2 to 1.25, 0.75 to 1.2, 0.8 to 1.2, 0.85 to 1.2, 0.9 to 1.2, 0.95 to 1.2, 1.0 to 1.2, 1.05 to 1.2, 1.1 to 1.2, 1.15 to 1.2, 0.75 to 1.15, 0.8 to 1.15, 0.85 to 1.15, 0.9 to 1.15, 0.95 to 1.15, 1.0 to 1.15, 1.05 to 1.15, 1.1 to 1.15, 0.75 to 1.1, 0.8 to 1.1, 0.85 to 1.1, 0.9 to 1.1, 0.95 to 1.1, 1.0 to 1.1, 1.05 to 1.1, 0.75 to 1.05, 0.8 to 1.1, 0.85 to 1.1, 0.9 to 1.1, 0.95 to 1.1, 1.0 to
  • a further aspect of the present disclosure provides methods of determining the zygosity of a cotton plant, plant part, seed, pollen, ovum, tissue or cell, in which such biological material from cotton suspected of containing cotton event GH_BCS246002 DNA may be subjected to the following method: (a) contacting a sample containing cotton DNA obtained from the biological material with at least two different sets of primers, in which (i) a first primer pair consisting of a first primer and a second primer (different from the first primer) that, when used in an amplification reaction with the cotton DNA containing sample, arc capable of producing a first amplicon diagnostic for, or characteristic of, the presence of cotton event GH_BCS246002 DNA in the sample, and (ii) a second primer pair consisting of the first primer and a third primer (different from the first primer and from the second primer) that, when used in a DNA amplification reaction with the cotton DNA, are capable of producing a second amplicon diagnostic for, or characteristic of, native
  • step (b) may comprise contacting a sample comprising cotton DNA from a cotton biological material with a probe set which contains at least a first probe that specifically hybridizes to DNA specific for cotton event GH_BCS246002 and at least a second probe that specifically hybridizes to cotton genomic DNA spanning the segment of chromosomal DNA that was disrupted by insertion of the heterologous transgenic DNA insert (SEQ ID NO: 9) of cotton event GH_BCS246002 and which does not hybridize to DNA from cotton event GH_BCS246002 DNA, and then hybridizing the probe set with a sample under stringent hybridization conditions, wherein detecting the hybridization of only the first probe under the hybridization conditions is diagnostic for, or characteristic of, a homozygous allele of cotton event GH_BCS246002 DNA in the sample, and wherein detecting hybridization of both the first probe and the second probe under the hybridization conditions is diagnostic for, or characteristic of, a heterozygous allele of cotton event GH_BCS246002
  • the set of primer pairs may comprise SEQ ID NO: 15 combined with SEQ ID NO: 16, and SEQ ID NO: 15 combined with SEQ ID NO: 21.
  • the probe set may comprise SEQ ID NO: 17 and SEQ ID NO: 22.
  • Another aspect of the present disclosure provides a recombinant DNA molecule comprising: (a) a first nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8; and (b) a second nucleotide sequence that (i) comprises at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1,000, at least 1,500, or at least 2,000 consecutive nucleotides of SEQ ID NO: 11 or 43, or (i) a
  • the recombinant DNA molecule can further comprise: (c) a third nucleotide sequence that (i) comprises at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1,000, at least 1,500, or at least 2,000 consecutive nucleotides of SEQ ID NO: 12 or 44, or (ii) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%
  • the recombinant DNA molecule can have a deletion of one or more consecutive nucleotides between the second nucleotide sequence and the nucleotide sequence relative to SEQ ID NO: 10, 12, or 44.
  • the second recombinant DNA molecule is selected from the group consisting of SEQ ID NOs: 45-144.
  • the recombinant DNA molecule can have a deletion of one or more consecutive nucleotides between the first nucleotide sequence and the second nucleotide sequence relative to SEQ ID NO: 10, 11, or 43.
  • Another aspect of the present disclosure provides a recombinant DNA molecule comprising: (a) a first nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9; and (b) a second nucleotide sequence that (i) comprises at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least
  • the recombinant DNA molecule can have a deletion of one or more consecutive nucleotides between the first nucleotide sequence and the second nucleotide sequence relative to SEQ ID NO: 10, 12, or 44.
  • the recombinant DNA molecule further comprises nucleotides 1-1,000 or 16,130-17,129 of SEQ ID NO: 10.
  • the recombinant DNA molecule is comprised in a cotton plant, cotton plant part, cotton plant cell, cotton plant seed, cotton progeny plant, or commodity product made from cotton and cotton plant parts.
  • the recombinant further comprises an amplicon diagnostic for the presence of DNA comprising a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9.
  • the recombinant DNA molecule is derived from a cotton plant, cotton plant part, cotton seed, processed cotton seed, cotton plant cell or tissue, cotton fiber, cotton oil and derivatives of cotton oil, cotton protein, cotton meal, animal feed comprising cotton, paper comprising cotton, cotton biomass, candle wicks, cotton string, cotton rope, cotton balls, cotton batting, cotton fuel products, and cotton cellulose products.
  • Another aspect of the present disclosure is a recombinant DNA molecule comprising a polynucleotide segment of sufficient length to function as a DNA probe that hybridizes specifically under stringent hybridization conditions with a polynucleotide having a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 43, and SEQ ID NO: 44.
  • a further embodiment of the invention are a pair of DNA molecules that can function as DNA primers when used together in an amplification reaction comprising a first DNA molecule and a second DNA molecule, wherein the first DNA molecule and the second DNA molecule are different, (a) wherein the first DNA molecule is: (i) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% identical or 100% identical to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50
  • the first DNA molecule and the second DNA molecule can be used together in an amplification reaction with a sample from a plant, plant part, plant seed, plant cell, food or animal feed, or commodity or fuel product made from a plant or plant part to produce an amplicon diagnostic for the presence of a modified cotton event GH_BCS246002 DNA in said sample.
  • the resulting amplicon comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10.
  • a further aspect of the present disclosure provides methods of detecting or determining the presence or absence of a DNA segment diagnostic for a modified cotton event GH_BCS246002 DNA in a sample, which may be unique to the modified cotton event GH_BCS246002 DNA, said method comprising: (a) contacting said sample with a DNA probe that binds or hybridizes specifically to the modified cotton event DNA; (b) subjecting said sample and said DNA probe to stringent hybridization conditions; and (c) detecting hybridization of said DNA probe to said DNA segment in said sample, wherein said detection is diagnostic for the presence of said modified cotton event GH_BCS246002 DNA in said sample.
  • Another aspect of the present disclosure provides methods for detecting or determining the presence or absence of a DNA segment diagnostic for a modified cotton event GH_BCS246002 DNA in a sample, said method comprising: (a) contacting said sample with the pair of DNA molecules that can function as DNA primers when used together in an amplification reaction comprising a first DNA molecule and a second DNA molecule, wherein the first DNA molecule and the second DNA molecule are different (b) performing an amplification reaction sufficient to produce a DNA amplicon; and (c) detecting the presence of said DNA amplicon in said reaction, wherein the presence of said DNA amplicon is diagnostic for the presence of said modified cotton event GH_BCS246002 DNA in said sample.
  • a further aspect of the present disclosure provides methods of detecting the presence of a DNA segment diagnostic for a modified cotton event GH_BCS246002 DNA in a sample, said method comprising performing a sequencing reaction with a sample, wherein the production in the sequencing reaction of a target nucleotide sequence comprises at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 consecutive nucleotides of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:
  • SEQ ID NO: 5 is diagnostic for the modified cotton event GH_BCS246002 DNA in a sample.
  • modified cotton plant, cotton plant part, cotton seed, cotton tissue or cotton cell comprising a modified cotton event GH_BCS246002.
  • the modified cotton plant, cotton plant part, cotton seed, cotton tissue or cotton cell comprises (a) a recombinant DNA molecule or DNA segment comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO:
  • the modified cotton plant, cotton plant part, cotton seed, cotton tissue or cotton cell may be further defined as a progeny plant of any generation of a cotton plant comprising a modified cotton event GH_BCS246002, or a cotton plant part, cotton seed, or cotton cell derived therefrom.
  • the modified cotton plant, cotton plant part, cotton seed, cotton tissue or cotton cell may exhibit resistance to a Lepidopteran insect pest species.
  • Another aspect of the present disclosure provides a DNA kit comprising: (a) the DNA probes as described herein; and (b) the pair(s) of DNA primers as described herein.
  • a further aspect of the present disclosure provides methods of producing a progeny cotton plant comprising a modified cotton event GH_BCS246002 comprising: (a) sexually crossing a first modified cotton plant that comprises a modified cotton event GH_BCS246002 with itself or a second cotton plant; (b) collecting one or more seeds produced from said cross; (c) growing said seed to produce one or more progeny plants; and (d) selecting at least a first progeny plant or seed comprising a modified cotton event GH_BCS246002.
  • the method can be used to make a hybrid modified cotton plant or seed comprising a modified cotton event GH_BCS246002.
  • the method may further comprise: (e) collecting seed from said at least first progeny plant comprising a modified cotton event GH_BCS246002.
  • Another aspect of the present disclosure provides a nonliving, non-viable or non- regenerable cotton plant material comprising a detectable amount of the recombinant DNA molecule of a modified cotton event GH_BCS246002 as described herein.
  • a recombinant DNA molecule of a modified cotton event GH_BCS246002 is provided as described herein, which may be comprised within a host cell, such as a cotton cell or a microorganism (e.g., a bacterial cell). Inn present embodiments, the microorganism can be a plant cell.
  • a commodity product is provided comprising a recombinant DNA molecule of a modified cotton event GH_BCS246002 as described herein.
  • the commodity product is produced from a modified cotton plant or a modified cotton plant part, cotton seed, or cotton tissue or cell comprising a modified cotton event GH_BCS246002.
  • the commodity product may be further selected from the group consisting of whole or processed cotton seed, animal feed comprising cotton, cotton oil, cotton meal, cotton flour, cotton flakes, cotton bran, cotton biomass, and fuel products produced using cotton and cotton plant parts.
  • Another aspect of the present disclosure provides methods of producing a commodity product, said method comprising: (a) obtaining a modified cotton plant, cotton plant part, or cotton seed comprising a modified cotton event GH_BCS246002; and (b) producing a commodity product from the transgenic cotton plant, cotton plant part, or cotton seed.
  • the present disclosure provides a cotton plant, cotton plant pail, or cotton seed comprising a DNA molecule or segment functional as a template in a DNA amplification method to produce an amplicon diagnostic for the presence of a modified cotton event GH BCS246002 DNA.
  • a further aspect of the present disclosure provides methods of determining the zygosity of a cotton plant, cotton plant part, or cotton seed comprising a modified cotton event GH_BCS246002 comprising: (a) contacting a sample comprising DNA from the cotton plant, cotton plant pail, or cotton seed with a primer pair capable of producing a first amplicon diagnostic for the modified cotton event GH_BCS246002 and a second amplicon diagnostic for native cotton genomic DNA not comprising the modified cotton event GH_BCS246002; (b) performing a nucleic acid amplification reaction with the sample and the set of primer pairs; and (c) detecting the first amplicon and the second amplicon, wherein the presence of only the first amplicon is diagnostic of a cotton plant, cotton plant part, or cotton seed homozygous for the modified cotton event GH_BCS246002, the presence of only the second amplicon is diagnostic of a cotton plant, cotton plant part, or cotton seed homozygous for native cotton genomic DNA not comprising
  • Another aspect of the present disclosure provides methods for determining the zygosity of a cotton plant, cotton plant part, or cotton seed comprising a modified cotton event GH_BCS246002 comprising: (a) contacting a sample comprising DNA from the cotton plant, cotton plant part, or cotton seed with a probe set which contains at least a first probe that specifically hybridizes to the modified cotton event GH_BCS246002 and at least a second probe that specifically hybridizes to cotton genomic DNA that was disrupted by insertion of the heterologous DNA of cotton event GH_BCS246002 and is disrupted by the modified cotton event GH BCS246002 DNA, wherein the second probe does not hybridize to the modified cotton event GH_BCS246002 DNA; and (b) hybridizing the probe set with the sample under stringent hybridization conditions, wherein detecting hybridization of only the first probe under the hybridization conditions is diagnostic for a cotton plant, cotton plant part, or cotton seed homozygous for the modified cotton event GH_BCS24
  • each transgenic cotton plant comprises a modified cotton event GH_BCS246002.
  • Each of the transgenic cotton plants of the population may exhibit resistance to one or more Lepidopteran insect pest species.
  • a further aspect of the present disclosure provides a method of modifying a cotton plant, the method comprising: (a) introducing a site-specific nuclease or a recombinant DNA construct comprising an expression cassette encoding a sit-specific nuclease into at least one cell of an explant of a cotton plant comprising cotton event GH_BCS246002, or a plant part thereof, to produce a modified cotton event GH_BCS246002 via a genome editing technique; and (b) developing or regenerating a modified cotton plant from the explant, wherein the modified cotton plant comprises the modified cotton event GH_BCS246002.
  • the site-specific nuclease is a zinc- finger nuclease (ZFN), a meganuclease, an RNA-guided endonuclease, a TALE-endonuclease (TALEN), a recombinase, or a transposase.
  • ZFN zinc- finger nuclease
  • TALEN TALE-endonuclease
  • recombinase a recombinase
  • transposase a transposase.
  • the site-specific nuclease is an RNA-guided endonuclease or a CRISPR/Cas nuclease.
  • the introducing step (a) comprises introducing the recombinant DNA construct into the at least one cell of the explant, and wherein the recombinant DNA construct further comprises an expression cassette encoding a first guide RNA (gRNA).
  • the recombinant DNA construct further comprises an expression cassette encoding a second guide RNA (gRNA).
  • the introducing step (a) further comprises introducing a first guide RNA (gRNA) or a second recombinant DNA construct comprising an expression cassette encoding a first guide RNA (gRNA) into the at least one cell of the explant.
  • the introducing step (a) comprises introducing at least two guide RNAs (gRNAs) comprising the first gRNA and a second gRNA into the at least one cell of the explant.
  • the second recombinant DNA construct further comprises an expression cassette encoding a second guide RNA (gRNA).
  • the introducing step (a) comprises introducing a first gRNA and second recombinant DNA construct comprising an expression cassette encoding a second guide RNA (gRNA) into the at least one cell of the explant.
  • the site-specific nuclease has a first target site in the genome of the cotton plant at or near cotton event GH_BCS246002.
  • the site-specific nuclease has a second target site in the genome of the cotton plant at or near cotton event GH_BCS246002.
  • the introducing step (a) comprises introducing a second site-specific nuclease or a recombinant DNA construct comprising an expression cassette encoding a second site specific nuclease into at least one cell of the explant, and wherein the second site-specific nuclease has a second target site in the genome of the cotton plant at or near cotton event GH_BCS246002.
  • the first gRNA has a first target site in a flanking DNA sequence, 5 ' flank, 3 ' flank, junction sequence, or insertion sequence of cotton event GH_BCS246002, or a complement thereof.
  • the first gRNA has a first target site comprising a target sequence that is: (i) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 10, at least 11, at least 12, at least 13, at least 14,
  • the first gRNA has a second target site comprising a target sequence that is: (i) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 consecutive nucleotides of SEQ ID NO: 11 or 43, or a complement thereof; or (ii) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least
  • the first gRNA has a first target site comprising a target sequence that is: (1 ) at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 consecutive nucleotides of SEQ ID NO: 11 or 43, or a complement thereof; or (2) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 9
  • the modified cotton event GH_BCS246002 comprises a deletion or excision of intervening genomic DNA between the first target site and the second target site, relative to the cotton event GH_BCS246002.
  • the method further comprises: (c) selecting the modified cotton plant comprising the modified cotton event GH_BCS246002; and (d) sexually crossing the modified cotton plant with itself or a second cotton plant to produce one or more modified progeny cotton plants.
  • Another aspect of the present disclosure provides methods of introducing a target site into a cotton plant, the method comprising: (a) introducing a cognate target site into the cotton event GH_BCS246002 locus of at least one cell of a cotton plant or cotton plant part comprising the cotton event GH_BCS246002 or an explant thereof via a targeted genome editing technique, wherein the cognate target site is identical or similar to an originator target site for a site-specific nuclease present in the cotton event GH_BCS246002 locus; and (b) developing or regenerating a modified cotton plant comprising a modified cotton event GH_BCS246002 comprising the cognate target site.
  • the method further comprises: (c) introducing a site-specific nuclease or a recombinant DNA construct comprising an expression cassette encoding a site-specific nuclease into at least one cell of an explant of a cotton plant comprising the modified cotton event GH_BCS246002 or a plant part thereof, to produce a further modified cotton event GH_BCS246002 via a targeted genome editing technique, wherein the target site of the sitespecific nuclease includes the cognate target site and the originator target site; and (d) developing or regenerating a second modified cotton plant comprising the further modified cotton event GH_BCS246002.
  • a further aspect of the present disclosure provides methods of introducing a target site into a cotton plant, the method comprising: (a) introducing a cognate target site into the cotton event GH_BCS246002 locus of at least one cell of a cotton plant or cotton plant part comprising the cotton event GH_BCS246002 or an explant thereof via a targeted genome editing technique to produce a modified cotton event GH_BCS246002 comprising the cognate target site, wherein the cognate target site is identical or similar to an originator target site for a site- specific nuclease present in the cotton event GH_BCS246002 locus; (b) introducing a site-specific nuclease or a recombinant DNA construct comprising an expression cassette encoding a site specific nuclease into at least one cell of an explant of a modified cotton plant comprising the modified cotton event GH_BCS246002, or a plant part thereof, to produce a further modified cotton event GH_BCS24600
  • the further modified cotton event GH_BCS246002 of the second modified cotton plant comprises a deletion or excision of intervening genomic DNA between the originator target site and the cognate target site, relative to the cotton event GH_BCS246002 or modified cotton event GH_BCS246002.
  • the method further comprises: selecting the second modified cotton plant or a progeny plant of the second modified cotton plant comprising the further modified cotton event GH_BCS246002, and sexually crossing the second modified cotton plant or the progeny plant with itself or another cotton plant to produce one or more modified progeny cotton plants comprising the further modified cotton event GH_BCS246002.
  • Another aspect of the present disclosure provides methods of producing a progeny cotton plant comprising a modified cotton event GH_BCS246002 comprising: (a) sexually crossing a first modified cotton plant that comprises a modified cotton event GH_BCS246002 with itself or a second cotton plant; (b) collecting one or more seeds produced from said cross; (c) growing said seed to produce one or more progeny plants; and (d) selecting at least a first progeny plant or seed comprising a modified cotton event GH_BCS246002.
  • Another aspect of the present disclosure provides methods for insertion of a supplemental expression cassette into a sequence within cotton event GH_BCS246002 or a modified cotton event GH_BCS246002, wherein said supplemental expression cassette comprising at least a heterologous plant expressible promoter operably linked to an open reading frame that encodes a RNA or protein conferring a trait upon a cotton plant or plant part when expressed in the plant or plant part, which is further operably linked to a plant functional transcription termination sequence.
  • Such a trait may be selected from the group consisting of an insect control trait, an herbicide tolerance trait, a nematode resistance trait, a trait that improves yield of cotton fiber, a stay-green trait, a drought tolerance trait, and a trait that confers a unique phenotype to the cotton fiber of the cotton plant.
  • the method for insertion of the transgenic DNA may rely upon a lox site for Cre-recombinase integration, or site directed integration of the supplemental expression cassette using a targeted genome editing technique.
  • Another aspect of the present disclosure provides methods for editing of an inserted DNA and/or junction sequence of cotton event GH_BCS246002 or a modified cotton event GH BCS246002.
  • the protoxin domain of the Cry IB or the Cry ID expression cassettes may be deleted from the coding sequence or open reading frame of the respective expression cassette, or a translation termination codon, whether amber, opal or ocher, is inserted or engineered into the coding sequence or open reading frame of the respective toxin coding sequence, to eliminate the expression of the protoxin coding domain, resulting in a modified cotton event GH_BCS246002.
  • Yet another aspect of the present disclosure are methods of determining the zygosity of a cotton plant, plant part or seed comprising DNA specific to the cotton event GH_BCS246002 or a modified cotton event GH_BCS246002.
  • the zygosity can be determined in a series of consecutive steps.
  • a sample comprising cotton DNA can be contacted with a first primer pair that is capable of producing an amplicon in a DNA amplification assay that is diagnostic for, or characteristic of, the presence of a coding sequence or expression cassette encoding the CrylB.3, CrylDa_7, or Vip3Cbl.l protein within the allele corresponding to cotton event GH_BCS246002.
  • the sample comprising the cotton DNA can be contacted with a second primer pair that is designed to produce an amplicon of an internal standard known to be single-copy and homozygous in the cotton plant.
  • Such methods may additionally include contacting the DNA sample with a probe set which contains at least a first probe that specifically hybridizes to the coding sequence or expression cassette encoding the Cry IB.3, CrylDa_7, or Vip3Cbl .l protein within cotton event GH_BCS246002 DNA, and a second probe that specifically hybridizes to an internal genomic DNA standard known to be single-copy and homozygous in the cotton plant.
  • the DNA amplification reaction in these methods may be performed using real-time PCR to determine the cycle thresholds (Ct values) of the amplicon corresponding the allele of cotton event GH BCS246002 and the single-copy, homozygous internal standard. After the amplification, the difference (delta-Ct) between the Ct value of the single-copy, homozygous internal standard amplicon, and the Ct value of the allele for cotton event GH_BCS246002 amplicon may be calculated.
  • zygosity is determined wherein a delta-Ct of about zero (0) indicates homozygosity of the inserted T-DNA of cotton event GH_BCS246002, and a delta-Ct of about one (1) indicates heterozygosity of the inserted T-DNA of cotton event GH_BCS246002.
  • the DNA amplification assay is diagnostic for, or characteristic of, the presence of a coding sequence or expression cassette encoding the Vip3Cbl.l protein within the allele corresponding to cotton event GH_BCS246002.
  • the primer pairs are selected from the group consisting of SEQ ID NO: 245 combined with SEQ ID NO: 246, and SEQ ID NO: 18 combined with SEQ ID NO: 19; and wherein the probes are SEQ ID NO: 247 and SEQ ID NO: 20.
  • the delta-Ct of about one (1) indicating heterozygosity of the inserted T-DNA of cotton event GH_BCS246002 is in the range of 0.75 to 1.25.
  • a delta-Ct of about zero (0) may be about 0, 0.05, 0.1, 0.15, 0.2, or 0.25, in other embodiments, a delta-Ct of about one (1) may be about 0.75, 0.8, 0.85, 0.9, 0.95, 1.0, 1.05, 1.1, 1.15, 1.2, or 1.25.
  • a delta-Ct of about one (1) may be in the range of 0.75 to 1.25, 0.8 to 1.25, 0.85 to 1.25, 0.9 to 1.25, 0.95 to 1.25, 1.0 to 1.25, 1.05 to 1.25, 1.1 to 1.25, 1.15 to 1.25, 1.2 to 1.25, 0.75 to 1.2, 0.8 to 1.2, 0.85 to 1.2, 0.9 to 1.2, 0.95 to 1.2, 1.0 to 1.2, 1.05 to 1.2, 1.1 to 1.2, 1.15 to 1.2, 0.75 to 1.15, 0.8 to 1.15, 0.85 to 1.15, 0.9 to 1.15, 0.95 to 1.15, 1.0 to 1.15, 1.05 to 1.15, 1.1 to 1.15, 0.75 to 1.1, 0.8 to 1.1, 0.85 to 1.1, 0.9 to 1.1, 0.95 to 1.1, 1.0 to 1.1, 1.05 to 1.1, 0.75 to 1.05, 0.8 to 1.1, 0.85 to 1.1, 0.9 to 1.1, 0.95 to 1.1, 1.0 to
  • FIG. 1 is a graphical depiction of the orientation and alignment of the DNA elements, segments and sequences that are present within the nucleotide sequence of SEQ ID NO: 10, represented in the drawing as [10], which is the sequence of the inserted transgenic DNA and adjacent 5' and 3' genomic sequences of the cotton event GH_BCS246002.
  • SEQ ID NO: 1 is represented by [1] and is a fifty (50) nucleotide junction sequence or segment spanning twenty- five (25) nucleotides of the 5’ end of the inserted DNA and twenty-five (25) nucleotides of the flanking cotton chromosomal or genomic DNA sequence immediately adjacent to the 5’ end of the inserted DNA.
  • SEQ ID NO: 2 is represented by [2] and is a fifty (50) nucleotide junction sequence or segment spanning twenty-five (25) nucleotides of the 3’ end of the inserted DNA and twenty five (25) nucleotides of the flanking cotton chromosomal or genomic DNA sequence immediately adjacent to the 3’ end of the inserted DNA.
  • SEQ ID NO: 1 is embedded within SEQ ID NO: 3 represented by [3], which is a one hundred (100) nucleotide junction sequence or segment spanning fifty (50) nucleotides of the 5’ end of the inserted DNA and fifty (50) nucleotides of the flanking cotton chromosomal or genomic DNA sequence immediately adjacent to the 5’ end of the inserted DNA.
  • SEQ ID NO: 2 is embedded within SEQ ID NO: 4 represented by [4], which is a one hundred (100) nucleotide junction sequence or segment spanning fifty (50) nucleotides of the 3’ end of the inserted DNA and fifty (50) nucleotides of the flanking cotton chromosomal or genomic DNA segment immediately adjacent to the 3’ end of the inserted DNA.
  • SEQ ID NO: 3 is embedded within SEQ ID NO: 5 represented by [5], which is a two hundred (200) nucleotide junction sequence or segment spanning one hundred (100) nucleotides of the 5’ end of the inserted DNA and one hundred (100) nucleotides of the flanking cotton chromosomal or genomic DNA sequence immediately adjacent to the 5’ end of the inserted DNA.
  • [5] is a two hundred (200) nucleotide junction sequence or segment spanning one hundred (100) nucleotides of the 5’ end of the inserted DNA and one hundred (100) nucleotides of the flanking cotton chromosomal or genomic DNA sequence immediately adjacent to the 5’ end of the inserted DNA.
  • SEQ ID NO: 4 is embedded within SEQ ID NO: 6 represented by [6], which is a two hundred (200) nucleotide junction sequence or segment spanning one hundred (100) nucleotides of the 3’ end of the inserted DNA and one hundred (100) nucleotides of the flanking cotton chromosomal or genomic DNA sequence immediately adjacent to the 3’ end of the inserted DNA.
  • SEQ ID NO:7 represented as [7] is one thousand (1,000) consecutive nucleotides of the flanking cotton chromosomal or genomic DNA sequence immediately adjacent to the 5' end of the inserted DNA and two hundred (200) consecutive nucleotides of the 5’ end of the inserted DNA.
  • SEQ ID NO: 8 represented as [8] is one thousand (1,000) consecutive nucleotides of the flanking cotton chromosomal or genomic DNA sequence immediately adjacent to the 3' end of the inserted DNA and two hundred (200) consecutive nucleotides of the 3' end of the inserted DNA.
  • SEQ ID NO: 9 represented as [9] is the full length nucleotide sequence or segment of the inserted transgenic DNA.
  • the arrows, and the labels below each arrow, represent the orientation of the direction of transcription and translation, as applicable, from the applicable expression elements that are positioned within each of the cassettes within the inserted DNA.
  • RB and LB represent the positions of the right and left borders of the Agrobacterium T-DNA transformation vector
  • letter P represents the positions of the promoter elements in the respective constructs or cassettes
  • letter L represents the positions of leader sequences (5' untranslated regions, 5' UTR) in the respective constructs or cassettes
  • letter I represents the position of the intron sequence in the respective constructs or cassettes
  • the letter T represents the positions of the transcription termination sequences (3' untranslated regions, 3' UTR) in the respective constructs or cassettes.
  • Each series of elements from a P to the immediately following T in the graphic depiction of [9] and [10] represents a single expression cassette or construct.
  • SEQ ID NO: 11 represented as [11] is the flanking cotton chromosomal or genomic DNA sequence immediately adjacent to the 5' end of the inserted DNA.
  • SEQ ID NO: 12 represented as [12] is the flanking cotton chromosomal or genomic DNA sequence immediately adjacent to the 3' end of the inserted DNA.
  • SEQ ID NO: 14 represented as [14] is a LoxP Cre-recombinase recognition sequence which remained in the cotton event GH_BCS246002 after Cre/Lox autoexcision was performed to remove the antibiotic resistance selection marker and the Cre-recombinase expression cassette at the Ri generation (see FIG. 2).
  • SEQ ID NOs: 15, 16 and 21 are primer sequences represented as [15], [16] and [21], respectively, and SEQ ID NO: 17 is a probe sequence represented as [17].
  • FIG. 2 illustrates the T-DNA cassette in the plasmid vector used to transform cotton and produce insertion events.
  • One insertion event when subjected to Cre-recombinase autoexcision of a segment of the inserted DNA containing the selectable marker and Cre-recombinase cassettes, resulted in cotton event GH_BCS246002.
  • SEQ ID NO: 13 represented as [13] illustrates the T- DNA in the plasmid vector before integration into the cotton genome (the “T-DNA Before Integration”).
  • the arrows below [13] represent the individual expression cassettes comprised within the T-DNA before integration, the first three of which are closer to the 5’ end and RB of the insert arc designed to express the three Lcpidoptcran protein toxins.
  • the Cre-recombinase expression cassette (Cre) and the antibiotic selectable marker expression cassette (aadA), which confers resistance to spectinomycin selection agent, are positioned closer to the 3’ end and LB of the insert and between the two LoxP sites [14].
  • Cre-recombinase expression cassette (Cre) and the antibiotic selectable marker expression cassette (aadA) which confers resistance to spectinomycin selection agent, are positioned closer to the 3’ end and LB of the insert and between the two LoxP sites [14].
  • the inserted T-DNA in the Ro generation comprised all five expression cassettes between the 5’ and 3’ flanking genomic DNA (the “Inserted T-DNA After Integration”).
  • the transformed Ro generation events were then allowed to self-pollinate and during this process, autoexcision occurred of the DNA segment bounded by the LoxP sites, causing excision of the Cre-recombinase and selectable marker expression cassettes.
  • the LoxP sites are recognized by Cre-recombinase, which is capable of excising from the insertion event the intervening DNA segment between the two LoxP sites containing the Cre-recombinase cassette and the selectable marker cassette as shown both before and after integration.
  • the resultant Ri generation seeds and progeny comprised only the three pesticidal toxin expression cassettes, without the excised Cre and AadA cassettes, as illustrated in the “Inserted T-DNA After Cre-Excision.”
  • SEQ ID NO: 1 is a 50-nucleotide sequence representing the 5' junction region of cotton genomic DNA and the integrated transgenic expression cassette (25 nucleotides cotton genome DNA at the 5' end of SEQ ID NO: 1, 25 nucleotides transgenic inserted DNA at the 3' end of SEQ ID NO: 1), and can be identified within SEQ ID NO: 10 at nucleotide positions 976-1,025.
  • SEQ ID NO: 2 is a 50-nucleotide sequence representing the 3' junction region of integrated transgenic expression cassette (25 nucleotides at the 5' end of SEQ ID NO: 2, 25 nucleotides of cotton genome DNA at the 3' end of SEQ ID NO: 2) and can be identified within SEQ ID NO: 10 at nucleotide positions 16,105-16,154.
  • SEQ ID NO: 3 is a 100-nuclcotidc sequence representing the 5' junction region of cotton genomic DNA and the integrated transgenic expression cassette (50 nucleotides cotton genome DNA at the 5' end of SEQ ID NO: 3, 50 nucleotides transgenic inserted DNA at the 3' end of SEQ ID NO: 3), and can be identified within SEQ ID NO: 10 at nucleotide positions 951-1,050.
  • SEQ ID NO: 4 is a 100-nucleotide sequence representing the 3' junction region of integrated transgenic expression cassette (50 nucleotides at the 5' end of SEQ ID NO: 4, 50 nucleotides of cotton genome DNA at the 3' end of SEQ ID NO: 4) and can be identified within SEQ ID NO: 10 at nucleotide positions 16,080-16,179.
  • SEQ ID NO: 5 is a 200-nucleotide sequence representing the 5' junction region of cotton genomic DNA and the integrated transgenic expression cassette (100 nucleotides cotton genome DNA at the 5' end of SEQ ID NO: 5, 100 nucleotides transgenic inserted DNA at the 3' end of SEQ ID NO: 5), and can be identified within SEQ ID NO: 10 at nucleotide positions 901-1,100.
  • SEQ ID NO: 6 is a 200-nucleotide sequence representing the 3' junction region of integrated transgenic expression cassette (100 nucleotides at the 5" end of SEQ ID NO: 6, 100 nucleotides of cotton genome DNA at the 3' end of SEQ ID NO: 6) and can be identified within SEQ ID NO: 10 at nucleotide positions 16,030-16,229.
  • SEQ ID NO: 7 is a 1, 200-nucleotide sequence representing the 5' junction region of cotton genomic DNA and the integrated transgenic expression cassette (1,000 nucleotides cotton genome DNA at the 5' end of SEQ ID NO: 7, 200 nucleotides transgenic inserted DNA at the 3' end of SEQ ID NO: 7), and can be identified within SEQ ID NO: 10 at nucleotide positions 1-1,200.
  • SEQ ID NO: 8 is a 1 , 200-nucleotide sequence representing the 3' junction region of integrated transgenic expression cassette (200 nucleotides at the 5' end of SEQ ID NO: 8, 1,000 nucleotides of cotton genome DNA at the 3' end of SEQ ID NO: 8) and can be identified within SEQ ID NO: 10 at nucleotide positions 15,930-17,129.
  • SEQ ID NO: 9 is a 15,129-nucleotide sequence corresponding to the transgenic inserted T- DNA of cotton event GH_BCS246002 and can be identified within SEQ ID NO: 10 at nucleotide positions 1,001-16,129.
  • SEQ ID NO: 10 is a 17,129-nucleotide sequence corresponding to the contig nucleotide sequence of the 5' genomic flanking DNA nucleotide sequence, the inserted T-DNA nucleotide sequence in cotton event GH_BCS246002, and the 3' genomic flanking DNA nucleotide sequence; and includes SEQ ID NO: 11 (nucleotides 1-1,1000), SEQ ID NO: 9 (nucleotides 1,001-16,129), and SEQ ID NO: 12 (nucleotides 16,130-17,129).
  • SEQ ID NO: 11 is a 1 ,000-nucleotide sequence representing the 5' flanking cotton genomic DNA up to the inserted T-DNA and can be identified within SEQ ID NO: 10 at nucleotide positions 1-1,000.
  • SEQ ID NO: 12 is a 1,000-nucleotide sequence representing the 3' flanking cotton genomic DNA after the inserted T-DNA and can be identified within SEQ ID NO: 10 at nucleotide positions 16,130-17,129.
  • SEQ ID NO: 13 is a 21,986-nucleotide sequence representing the transgene cassette comprised within the binary plasmid transformation vector used to transform cotton to produce cotton event GH_BCS246002.
  • SEQ ID NO: 14 is a 35-nucleotide sequence representing a LoxP Cre-recombinase recognition sequence used for Cre-mediated autoexcision and recombination, and the residual sequence can be identified within SEQ ID NO: 10 at nucleotide positions 15,825-15,859.
  • SEQ ID NO: 15 is 24-nucleotide sequence corresponding to a thermal amplification primer referred to as SQ51960 used to identify cotton event GH_BCS246002 in a sample or which can be used to detect an insertion event that, when subjected to Cre-recombinase autoexcision, results in cotton event GH_BCS246002 DNA.
  • SEQ ID NO: 15 is identical to the nucleotide sequence corresponding to positions 941-964 of SEQ ID NO: 10.
  • SEQ ID NO: 16 is a 22-nucleotide sequence corresponding to a thermal amplification primer referred to SQ51702 used to identify cotton event GH_BCS246002 in a sample or which can be used to detect an insertion event that, when subjected to Cre-recombinase autoexcision, results in cotton event GH_BCS246002 DNA.
  • SEQ ID NO: 16 is identical to the reverse complement of the nucleotide sequence corresponding to positions 1,066-1,087 of SEQ ID NO: 10.
  • SEQ ID NO: 17 is an 18-nucleotide sequence corresponding to a probe referred to as PB5O3O8 used to identify cotton event GH_BCS246002 DNA in a sample or which can be used to detect an insertion event that, when subjected to Cre-recombinase autoexcision, results in cotton event GH_BCS246002 DNA.
  • SEQ ID NO: 17 is identical to the reverse complement of the nucleotide sequence corresponding to positions 1,043-1,060 of SEQ ID NO: 10.
  • SEQ ID NO: 18 is a 23-nucleotide sequence corresponding to a thermal amplification primer referred to as SQ22496 used as an internal control for the event and zygosity assay for cotton event GH_BCS246002 and hybridizes to a region of the cotton genome.
  • SEQ ID NO: 19 is a 19-nucleotide sequence corresponding to a thermal amplification primer referred to as SQ22497 used as an internal control for the event and zygosity assay for cotton event GH_BCS246002 and hybridizes to a region of the cotton genome.
  • SEQ ID NO: 20 is a 14-nucleotide sequence corresponding to a probe referred to as PB50562 used as an internal control for the event and zygosity assay for cotton event GH_BCS246002 and hybridizes to a region of the cotton genome.
  • SEQ ID NO: 21 is a 23-nucleotide sequence corresponding to a thermal amplification primer referred to as WTDP393R used in the zygosity assay for cotton event GH_BCS246002 and hybridizes to a region in the 3' flanking cotton genomic DNA.
  • WTDP393R thermal amplification primer
  • SQ51960 primer SQ51960 with conventional cotton DNA as template lacking or devoid of cotton event GH_BCS246002 DNA
  • SEQ ID NO: 22 is a 22-nucleotide sequence corresponding to a probe referred to as WTDP393PR used in the zygosity assay for cotton event GH_BCS246002.
  • Probe WTDP393PR hybridizes to the 3 ' terminal 12 nucleotides of the 3' genomic flanking DNA and the 10 nucleotides of the wild-type allelic DNA that was deleted during insertion of the T-DNA in cotton event GH_BCS246002.
  • SEQ ID NO: 23 is a 27-nucleotide sequence corresponding to an originator guide RNA recognition site (OgRRS), OgRRS_5-l comprised of a Casl2a protospacer adjacent motif (PAM) site operably linked to a guide-RNA hybridization site.
  • OgRRS originator guide RNA recognition site
  • PAM Casl2a protospacer adjacent motif
  • SEQ ID NO: 24 is a 27-nucleotide sequence corresponding to an OgRRS, OgRRS_5-2.
  • SEQ ID NO: 25 is a 27-nucleotide sequence corresponding to an OgRRS, OgRRS_In-l.
  • SEQ ID NO: 26 is a 27-nucleotide sequence corresponding to an OgRRS, OgRRS_In-2.
  • SEQ ID NO: 27 is a 27-nuclcotidc sequence corresponding to an OgRRS, OgRRS_3-l.
  • SEQ ID NO: 28 is a 27-nucleotide sequence corresponding to an OgRRS, OgRRS_3-2.
  • SEQ ID NO: 29 is a 1 -nucleotide sequence corresponding to a guide-RNA (gRNA), gRNA_OgRRS_5-l.
  • gRNA guide-RNA
  • SEQ ID NO: 30 is a 51 -nucleotide sequence corresponding to a guide-RNA (gRNA), gRNA_OgRRS_5-2.
  • gRNA guide-RNA
  • SEQ ID NO: 31 is a 51 -nucleotide sequence corresponding to a guide-RNA (gRNA), gRNA_OgRRS_In- 1.
  • SEQ ID NO: 32 is a 51-nucleotide sequence corresponding to a guide-RNA (gRNA), gRNA_OgRRS_In-2.
  • gRNA guide-RNA
  • SEQ ID NO: 33 is a 51-nucleotide sequence corresponding to a guide-RNA (gRNA), gRNA_OgRRS_3-l.
  • SEQ ID NO: 34 is a 51-nucleotide sequence corresponding to a guide-RNA (gRNA), gRNA_OgRRS_3-2.
  • SEQ ID NO: 35 is a sequence of a synthetic DNA coding sequence designed for expression in a plant cell encoding a nuclear targeted LbCasl2a CRIS PR-associated protein.
  • SEQ ID NO: 36 is an amino acid sequence of a nuclear targeted LbCasl2a CRISPR- associated protein encoded by SEQ ID NO: 34.
  • SEQ ID NO: 37 is a sequence of a synthetic DNA coding sequence designed for expression in a plant cell encoding a nuclear targeted LbCasl 2a-TYCV CRISPR-associated protein.
  • SEQ ID NO: 38 is an amino acid sequence of a nuclear targeted LbCasl2a-TYCV CRISPR-associated protein encoded by SEQ ID NO: 36.
  • SEQ ID NO: 39 is a sequence of a synthetic DNA coding sequence designed for expression in a plant cell encoding a nuclear targeted LbCasl 2a-TATV CRISPR-associated protein.
  • SEQ ID NO: 40 is an amino acid sequence of a nuclear targeted LbCasl2a-TATV CRISPR-associated protein encoded by SEQ ID NO: 38.
  • SEQ ID NO: 41 is a sequence of a synthetic DNA coding sequence designed for expression in a plant cell encoding a nuclear targeted FnCasl2a CRISPR-associated protein.
  • SEQ ID NO: 42 is an amino acid sequence of a nuclear targeted FnCasl2a CRISPR- associated protein encoded by SEQ ID NO: 40.
  • SEQ ID NO: 43 is a 5,000-nucleotide sequence representing cotton genomic DNA that flanks the transgenic insert at the 5 'end of the insert. Nucleotides 4,001-5,000 of SEQ ID NO: 43 are identical to nucleotides 1-1,000 of SEQ ID NO: 11. Nucleotides 1-4,000 are based on the genomic sequence of the TM-1 cotton cultivar.
  • SEQ ID NO: 44 is a 5,000-nucleotide sequence representing cotton genomic DNA that flanks the transgenic insert at the 5 'end of the insert. Nucleotides 1-1,000 of SEQ ID NO: 44 are identical to nucleotides 1-1,000 of SEQ ID NO: 12. The remaining nucleotides (1,001-5,000) a e based on the genomic sequence of the TM- 1 cotton cultivar.
  • SEQ ID NOs: 45-144 are 50-nucleotide sequences in the 5' flank genomic sequence of cotton event GH_BCS246002.
  • SEQ ID NOs: 145-244 are 50-nucleotide sequences in the 3' flank genomic sequence of cotton event GH_BCS246002.
  • SEQ ID NO: 245 is 22-nucleotide sequence corresponding to a thermal amplification primer referred to as SQ51253 used in the zygosity assay for cotton event GH_BCS246002 and hybridizes to a region within the 3’ portion of the Vip3Cbl.l coding sequence.
  • SEQ ID NO: 245 is identical to the nucleotide sequence corresponding to positions 14,048-14,069 of SEQ ID NO: 10.
  • SEQ ID NO: 246 is a 25-nucleotide sequence corresponding to a thermal amplification primer referred to SQ51254 used in the zygosity assay for cotton event GH_BCS246002 and hybridizes to a region within the 3’ portion of the Vip3Cbl.l coding sequence.
  • SEQ ID NO: 246 is identical to the reverse complement of the nucleotide sequence corresponding to positions 15,061-15,085 of SEQ ID NO: 10.
  • SEQ ID NO: 247 is an 18-nucleotide sequence corresponding to a probe referred to as PB50494 used in the zygosity assay for cotton event GH_BCS246002 and hybridizes to a region within the 3’ portion of the Vip3Cbl.l coding sequence.
  • SEQ ID NO: 247 is identical to the nucleotide sequence corresponding to positions 15,041-15,058 of SEQ ID NO: 10.
  • the present disclosure provides a transgenic cotton event GH_BCS246002 that provides insecticidal control over certain Lepidopteran larval pests of cotton by expression of the insecticidal toxins Cry1B.3, CrylDa_7, and Vip3Cbl .l, which are each expressed in tissues of a cotton plant containing this event and presented to Lcpidoptcran pests when they consume the plant tissues.
  • the expression of the CrylB.3, CrylDa_7, and Vip3Cbl.l insect inhibitory proteins in cotton event GH_BCS246002 provides resistance to the larval forms of Lepidopteran insect pests including, but not limited to, Cotton bollworm (Helicoverpa zea), Tobacco budworm (Helio thi virescens). and Fall armyworm (Spodoptera frugiperdd).
  • Cotton event GH_BCS246002 also referred to as event GH_BCS246002 provides an unsolved need in the art for control of these insects in the field of cotton agriculture, because Lepidopteran species have developed, or are expected to develop, resistance to the pest control proteins used in earlier versions of transgenic cotton plants expressing Lepidopteran control proteins.
  • Chemical insecticides have not provided adequate control of these insects, and many of the chemical insecticides have encountered resistance development as well. At times, multiple applications of chemistries are required during the growing season, which increases the input and persistence of chemical pesticides in the environment, increases the carbon footprint with each application of such chemicals, and adds significant cost to the production of the cotton crop.
  • the three insecticidal proteins encoded by the transgenic inserted DNA in the cotton event GH_BCS246002 are (i) Cry IB.3 (see, e.g., United States Patent 10,611,806, the amino acid sequence being referenced therein as SEQ ID NO: 21, and the coding sequence as SEQ ID NO: 20), (ii) CrylDa _7 (see, e.g., United States Patent 10,059,959, the amino acid sequence being referenced therein as SEQ ID NO: 40, and the coding sequence as SEQ ID NO: 39), and (iii) Vip3Cbl.l (see, e.g., United States Patent 10,155,960, the amino acid sequence being referred to therein as SEQ ID NO: 4, and the coding sequence as SEQ ID NO: 3). These three insecticidal proteins are described herein as being expressed from three different but linked expression cassettes within the inserted transgenic DNA construct as set forth herein in SEQ ID NO: 9 and as illustrated in FIG. 1.
  • the DNA sequence encoding the Cry 1 B.3 protein in cotton event GH_BCS246002 is operably linked to Cucumis melo Chlorophyl a/b binding protein promoter and leader (see, e.g., United States Patent 10,550,401, referenced therein as SEQ ID NO: 29) and StMedicago truncatula hypothetical protein 3' UTR (or transcription termination sequence, see, e.g., United States Patent 10,501,749, referenced therein as SEQ ID NO: 3).
  • the DNA sequence encoding the CrylDa_7 protein in cotton event GH_BCS246002 is operably linked to a synthetic promoter, leader, and intron (see, e.g., United States Patent Application 2018/0216129 Al, referenced therein as SEQ ID NOs: 5, 6, and 9) and a Medicago truncatula expansin-related protein 1 precursor protein 3' UTR (or transcription termination sequence, see, e.g., United States Patent Application 2014/0283200 Al, referenced therein as SEQ ID NO: 7).
  • the DNA sequence encoding the Vip3Cbl.l protein cotton event GH_BCS246002 is operably linked to an Arabidopsis thaliana Actin-2 promoter, leader, and intron, and a Medicago truncatula seed maturation protein PM21 3' UTR (or transcription termination sequence, United States Patent Application 2014/0283200 Al, referenced therein as SEQ ID NO: 16).
  • FIG. 1 shows the relative positions of each element as promoter (P), 5' UTR or leader (L), intron (I), toxin coding sequences or ORFs (open reading frames), and 3' UTR (T).
  • the open reading frames are provided in the event in the following order in the 5’ to 3’ direction: the ORF for CrylB.3, the ORF for CrylDa_7, and the ORF for Vip3Cbl.l, and as specified above, these three ORFs are comprised within SEQ ID NO: 9 and SEQ ID NO: 10.
  • cotton event GH_BCS246002 is free of the markers used for selection of the transformed plant cell as a result of the method of autoexcision after transformation (see FIG. 2).
  • the transgene cassette used for the expression of Cre- recombinase comprised a germline-preferred promoter that, when expressed in cotton during the breeding process, caused the excision of the Crc-rccombinasc and Spcctinomycin selection cassettes between flanking LoxP sites, thus removing the Cre and aadA expression cassettes from the transgcnc locus resulting in cotton event GH_BCS246002.
  • Cotton event GH_BCS246002 was created through plant transformation techniques used to insert heterologous DNA (also known as transgenic DNA) randomly into a chromosome of the genome of a cotton cell to produce a genetically engineered cotton cell, also referred to as a “transgenic” or “recombinant” cotton cell.
  • heterologous DNA also known as transgenic DNA
  • many individual cells are transformed, each resulting in a unique “transgenic event” or “event” due to the random insertion of the foreign DNA into the genome.
  • a transgenic plant can then be regenerated from each individual transgenic cell. This results in every cell of the transgenic plant containing the uniquely inserted transgenic event as a stable pail of its genome. This transgenic plant can then be used to produce seed which are then planted and grown into progeny plants, each containing the unique transgenic event.
  • Cotton event GH_BCS246002 was produced by an Agrobacterium-mediated transformation process using the binary transformation plasmid construct pGH_BCS246002 and dry excised cotton embryo explants.
  • the transformed cotton cells were regenerated into intact cotton plants and individual plants were selected from the population of plants that had a fully intact T-DNA insertion comprising expression cassettes encoding the CrylB.3, CrylDa_7, and Vip3Cbl. l proteins, and the absence of (i) the portion of the T-DNA segment encoding the selectable/scorable marker cassettes, which was removed by Cre-Lox excision as described herein, and (ii) any transformation plasmid vector backbone sequence.
  • T-DNA segment encoding the CrylB.3, CrylDa_7, and Vip3Cbl.l protein in plasmid construct pGH_BCS246002 is presented as SEQ ID NO: 13.
  • SEQ ID NO: 13 For cotton event GH_BCS246002, during integration of the T-DNA insertion, two hundred seventeen (217) and one hundred eighty-two (182) contiguous base pairs (bp) were deleted from the right and left borders, respectively, and thirty-five (35) contiguous base pairs (bp) of the wild-type genomic DNA at the point of insertion of the transgenic DNA was deleted during the integration process.
  • cotton event GH_BCS246002 was produced by a complex research and development process in which: (1) over one hundred plasmid vector constructs - which varied with respect to the coding sequences for the insecticidal proteins, the sequences for the transcriptional regulatory elements, and number and orientation of the cassettes within the constructs - were developed and transformed into cotton cells to create thousands of events that were tested and analyzed, resulting in the selection of the construct used to generate cotton event GH_BCS246002; (2) thousands of cotton cells were transformed with the construct used to generate cotton event GH_BCS246002 and regenerated into plants to create a population of transgenic plants in which each plant contained a unique transgenic event that was tested; (3) the final cotton event GH_BCS246002 was selected after a rigorous multi-year event selection process involving the testing and analysis of molecular characteristics, efficacy, protein expression, and agronomic properties in a variety of cotton genetic backgrounds. Thus, cotton event GH_BCS246002 was produced by a complex research and development process
  • the transgenic DNA from the transformation plasmid vector and inserted into the genome of cotton event GH_BCS246002 was characterized by detailed molecular analysis. This analysis included: the insert number (number of integration sites within the cotton genome), the genomic insert location (the specific site in the cotton genome where the insertion occurred), the copy number (the number of copies of the T-DNA within one locus), and the integrity (absence of any rearrangements) of the transgenic inserted DNA.
  • an “expression cassette” or “cassette” or “transgene” is a recombinant DNA molecule comprising a combination of distinct elements that are to be expressed by a transformed cell.
  • the inserted DNA of cotton event GH_BCS246002 as described herein has multiple expression cassettes for three different insecticidal toxins.
  • Table 1 provides a list of the elements contained in SEQ ID NOTO, the DNA sequence that corresponds to cotton event GH_BCS246002.
  • Cry IB.3, CrylDa_7, and Vip3Cbl.l are each different unique toxins, exhibiting less than 60% amino acid sequence identity to each other, each arc toxic to Lcpidoptcran pests, and each toxin exhibits a different mode of action relative to the other.
  • the position and orientation within the transgenic inserted DNA of each of the genetic elements and cassettes involved in expression of these respective toxin proteins are specified and shown in Table 1 and FIG. 1.
  • a recombinant DNA molecule comprises a nucleotide sequence selected from the group consisting of, or comprising one or more of, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, and a complete complement of any of the foregoing.
  • a recombinant DNA molecule is derived from all or part of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002.
  • a recombinant DNA molecule comprises (i) all or part of chromosome 4 of a cotton plant and (ii) all or part of the cotton event GH_BCS246002 or a modified cotton event GH_BCS246002, and/or (iii) a nucleotide sequence comprising one or more of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, and a complete complement of any of the foregoing.
  • Cotton event GH_BCS246002 is characterized as an insertion of the intended transgenic DNA into a single locus in the cotton genome, resulting in a new locus with two junction sequences (e.g., sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8) between the inserted DNA and the 5’ and 3’ cotton flanking genomic DNA that are not known to appear naturally in the cotton genome or other transgenic cotton events - they are unique to cotton DNA containing the cotton event GH_BCS246002.
  • two junction sequences e.g., sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8
  • the insertion of the transgenic DNA into the genome of a cotton plant causes a separation of the genomic sequences of the cotton plant at or near the site of insertion of the transgenic DNA, which produces the two junction sequences at the 5’ and 3’ ends of the inserted transgenic DNA, respectively, wherein the 5’ junction sequence comprises a sequence spanning the 5’ end of the inserted transgenic DNA and the immediately adjacent 5’ flanking genomic DNA, and the 3’ junction sequence comprises a sequence spanning the 3’ end of the inserted transgenic DNA and the immediately adjacent 3’ flanking genomic DNA.
  • junction sequences are unique to the GH_BCS246002 event, these junction sequences are useful in detecting the presence of the GH_BCS246002 event in cotton cells, cotton tissue, cotton seed, cotton pollen and ova, and cotton plants or cotton plant products, such as cotton commodity products. Based on its site of insertion, the cotton event GH_BCS246002 and any modified cotton event GH_BCS246002 are each located on chromosome 4 of a transgenic cotton plant or a modified cotton plant.
  • DNA molecular probes and primer pairs are described herein that have been developed for use in identifying the presence of these various junction segments in biological samples containing or suspected of containing cotton cells, cotton seed, cotton plant parts, cotton pollen or ova, or cotton plant tissue that contain the cotton event GH_BCS246002.
  • a sample is intended to refer to a composition that is either substantially purified cotton DNA or protein, or a composition that contains cotton DNA or protein.
  • the sample can be any biological sample, i.e., it contains biological material, including but not limited to DNA obtained or derived from, either directly or indirectly, from the genome of a cotton plant, plant part, plant tissue or plant cell that may or may not contain cotton event GH_BCS246002 or a modified cotton event GH_BCS246002.
  • “Directly” refers to the ability of the skilled artisan to directly obtain DNA from the cotton genome by fracturing cotton cells or tissues (or by obtaining samples of cotton that contain fractured cotton cells or tissues) and using or exposing the genomic DNA for the purposes of detection.
  • “Indirectly” refers to the ability of the skilled artisan to obtain the target or specific reference DNA by means other than by direct via fracturing of cotton cells or tissues or obtaining a sample that contains fractured cotton cells or tissues.
  • Such indirect means include, but are not limited to, amplification of a DNA segment that contains the DNA sequence targeted by a particular probe designed to bind with specificity to the target sequence, or amplification of a DNA segment that can be measured and characterized, i.e., measured by separation from other segments of DNA through some efficient matrix such as an agarose or acrylamide gel or the like, or characterized by direct sequence analysis of the amplicons, or cloning of the amplicon into a vector and direct sequencing of the inserted amplicon present within such vector.
  • a sample of pure cotton protein or a composition that contains cotton protein is a biological sample, i.e., it contains biological materials, including but not limited to protein derived from the tissues or cells of a cotton plant, plant pail, plant tissue or plant cell that may or may not contain cotton event GH_BCS246002.
  • the sample can be contacted with antibodies, such as monoclonal antibodies that bind specifically to CrylB.3, CrylDa_7, and Vip3Cbl.l in an immunoassay. Detection of the bound proteins is diagnostic or characteristic of CrylB.3, CrylDa_7, and Vip3Cbl.l.
  • the immunoassay method can be, but is not limited to, an ELISA (Enzyme-Linked Immunosorbent Assay), a Radioimmunoassay, or a Lateral flow immunochromatographic assay.
  • ELISA assays are typically performed in the laboratory using tissue or cell samples obtained from whole plants, or plant parts or tissues thereof. Sandwich ELISA assays are frequently used to detect and quantify protein from transgenic crops.
  • monoclonal antibodies can be used, with a first monoclonal antibody which binds specifically to Cry IB.3, a second antibody which binds specifically to CrylDa_7, and a third monoclonal antibody which binds specifically to Vip3Cbl.l.
  • a known amount of monoclonal antibody can be bound to a fixed surface. Nonspecific sites on the solid surface may be blocked by bovine serum albumin, casein, or another such neutral solution.
  • the sample which may contain protein(s) derived from cotton event GH_BCS246002 is applied to the plate, and any such protein(s) can be captured by the antibody.
  • the unbound antigens may be washed away by a washing solution.
  • a secondary antibody can be added which may also be conjugated to an enzyme.
  • the unbound antibodies may be washed away.
  • a substrate can be added, and the enzyme reacts with the substrate and produces a product, typically a pigment or photons, which can be proportional to the amount of antigen present.
  • Separate ELISA assays can be performed for each of the three toxin proteins in a sample derived from cotton event GH_BCS246002.
  • a positive ELISA reaction is diagnostic for, or characteristic of, cotton event GH_BCS246002.
  • the immunochromatographic test strip is composed of a sample pad, conjugate pad, nitrocellulose membrane, absorbent pad, and a backing card.
  • a first monoclonal antibody which binds specifically to CrylB.3, a second monoclonal antibody which binds specifically to CrylDa_7, a third monoclonal antibody which binds specifically to Vip3Cbl.l, and a IgG antibody which binds to antibodies derived from the organism host cells in which the first, second, and third monoclonal antibodies were derived are transferred onto a nitrocellulose membrane to form a test line 1 (CrylB.3), test line 2 (CrylDa_7), test line 3 (Vip3Cbl.l) and the control line (anti-host IgG antibody).
  • the conjugate pad is coated with the first, second, and third monoclonal antibodies labeled with colloidal gold nanoparticles.
  • the blotted membrane, conjugate pad, sample pad, and absorbent pad are assembled sequentially on the plastic backing board.
  • the Cry IB .3, Cry lDa_7, and Vip3Cb 1.1 proteins present in a sample derived from cotton event GH_BCS246002 will combine with their respective gold-labeled monoclonal antibodies and then bind to capture antibodies coated on the test line 1 , test line 2, and test line 3. Visual detection of test line 1 and test line 2 is diagnostic for, or characteristic of, cotton event GH_BCS246002.
  • cotton event GH_BCS246002 contains a single T-DNA insertion with one copy of each of the Cry IB.3, CrylDa_7, and Vip3Cbl.l expression cassettes. No additional elements from the transformation construct other than portions of the Agrobacterium tumefaciens left and right border regions used for transgenic DNA transfer from the plant transformation plasmid to the cotton genome were identified in cotton event GH_BCS246002.
  • SEQ ID NO: 9 is a sequence representing the one thousand (1,000) base-pair (bp) immediately adjacent to the 5' end of the inserted DNA in cotton variety DP393 genomic DNA sequence flanking the inserted T- DNA of cotton event GH_BCS246002.
  • SEQ ID NO: 12 is a sequence representing the one thousand (1,000) bp immediately adjacent to the 3' end of the inserted DNA in cotton variety DP393 genomic DNA sequence flanking the inserted T-DNA sequence.
  • SEQ ID NO: 7 is SEQ ID NO: 11 plus two hundred (200) bp of the 5' end of the inserted T-DNA sequence added to the 3' end of SEQ ID NO: 11 .
  • SEQ ID NO: 8 is SEQ ID NO: 12 plus two hundred (200) bp of the 3' end of the inserted T-DNA sequence added to the 5' end of SEQ ID NO: 12.
  • SEQ ID NO: 10 corresponds to the DNA sequence that defines the cotton event GH_BCS246002 and contains a contiguous sequence (contig) comprising the 5' DP393 flanking sequence, the transgenic insert of cotton event GH_BCS246002, and the 3' DP393 flanking sequence, and thus contains both of the junction sequences of cotton event GH_BCS246002 specified as SEQ ID NO: 1, 3, 5 and 7 (junction sequence at 5' end of insert), and as SEQ ID NO: 2, 4, 6, and 8 (junction sequence at 3' end of insert).
  • the term “cotton” means species belonging to the genus Gossypium, preferably Gossypium hirsutum L. and Gossypium barbadense L. and includes all plant varieties that can be bred with cotton plants containing cotton event GH_BCS246002, including wild cotton species as well as those plants belonging to the genus Gossypium that permit breeding between species.
  • Cotton event GH_BCS246002 was transformed with a DNA construct that contains expression cassettes expressing toxic amounts of insecticidal proteins CrylB.3, CrylDa_7, and Vip3Cb 1.1.
  • a toxic amount refers to an efficacious amount, an insecticidal amount, an insecticidally effective amount, a target insect suppressive amount, an efficacious pesticidal amount, an amount in the diet of insects in the order of Lepidoptera that is insecticidal, and other similar terms to be understood according to conventional usage by those of ordinary skill in the relevant art.
  • Cotton plants transformed according to the methods and with the DNA constructs disclosed herein are resistant to Lepidopteran insect pests.
  • a transgenic “plant” is produced by transformation of a plant cell with heterologous
  • DNA i.e., a polynucleic acid construct that includes a number of efficacious features of interest, regeneration of a plant resulting from the insertion of the transgene into the genome of the plant cell, and selection of a particular plant characterized by insertion into a particular genome location and the number of efficacious features of the regenerated transgenic plant.
  • event refers to DNA from the original transformant comprising the inserted DNA and flanking genomic sequences immediately adjacent to the inserted DNA.
  • Such DNA is unique and would be expected to be transferred to a progeny that receives the inserted DNA, including the transgcnc of interest, as the result of a sexual cross of parental line that includes the inserted DNA (e.g., the original transformant and progeny resulting from selfing) and a parental line that does not contain the inserted DNA.
  • the present disclosure also provides the original transformant plant and progeny of the transformant that include the heterologous DNA.
  • Such progeny may be produced by a sexual outcross between plants comprising the event and another plant wherein the progeny includes the heterologous DNA.
  • Cotton event GH_BCS246002 is a stable transgenic event that can be stably transmitted through one or more generations to progeny or descendants of plants containing the cotton event GH_BCS246002 and stacked with other events or traits in progeny or descendants through one or more crosses.
  • the transgenic event DNA, and the genome into which the transgenic event DNA is detectable within the plant cell, the plant, the seed, and other parts of the plant, are not pre-existent in nature.
  • DNA and DNA molecule refer to a deoxyribonucleic acid (DNA) molecule.
  • a DNA molecule may be of genomic or synthetic origin and is by convention from the 5' (upstream) end to the 3' (downstream) end.
  • DNA sequence refers to the nucleotide sequence of the DNA molecule. By convention, the DNA sequences of the present disclosure and fragments thereof are disclosed with reference to only one strand of the two-strand complementary DNA sequence strands.
  • the complementary sequences of the sequences provided here are within the scope of the present disclosure and are expressly intended to be within the scope of the subject matter claimed.
  • fragment refers to a smaller piece of the whole.
  • fragments of SEQ ID NO: 10 would include sequences that are at least about 12 consecutive nucleotides, at least about 13 consecutive nucleotides, at least about 14 consecutive nucleotides, at least about 15 consecutive nucleotides, at least about 16 consecutive nucleotides, at least about 17 consecutive nucleotides, at least about 18 consecutive nucleotides, at least about 19 consecutive nucleotides, at least about 20 consecutive nucleotides, at least about 25 consecutive nucleotides, at least about 30 consecutive nucleotides, at least about 35 consecutive nucleotides, at least about 40 consecutive nucleotides, at least about 45 consecutive nucleotides, at least about 50 consecutive nucleotides, at least about 60 consecutive nucleotides, at least about 70 consecutive nucleotides, at least about 80 consecutive nucleotides, at least about 90 consecutive nucleotides, or at
  • a fragment of the 5' flank (SEQ ID NO: 11 or SEQ ID NO: 43) or 3' flank (SEQ ID NO: 12 or SEQ ID NO: 44) of cotton event GH_BCS246002 can comprise at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 150, at least about 200, at least about 250, at least about 300, at least about 400, or at least about 500 consecutive nucleotides of SEQ ID NO: 11 or SEQ ID NO: 43; or SEQ ID NO: 12 or SEQ ID NO: 44.
  • nucleotide sequences that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% identical to SEQ ID NO: 11 or 12, or SEQ ID NO: 43 or 44, or any fragment of either thereof.
  • references in this disclosure to an “isolated DNA molecule” or an equivalent term or phrase is intended to mean that the DNA molecule is one that is present alone or in combination with other compositions, but not within its natural environment.
  • nucleic acid elements such as a coding sequence, intron sequence, untranslated leader sequence, promoter sequence, transcriptional termination sequence, and the like, that are naturally found within the DNA of the genome of an organism are not considered to be “isolated” so long as the element is within the genome of the organism and at the location within the genome in which it is naturally found.
  • each of these elements, and subparts of these elements would be “isolated” within the scope of this disclosure so long as the element is not within the genome of the organism and at the location within the genome in which it is naturally found.
  • a nucleotide sequence encoding an insecticidal protein or any naturally occurring insecticidal variant of that protein would be an isolated nucleotide sequence so long as the nucleotide sequence was not within the DNA of the bacterium from which the sequence encoding the protein is naturally found.
  • a synthetic nucleotide sequence encoding the amino acid sequence of the naturally occurring insecticidal protein would be considered to be isolated for the purposes of this disclosure.
  • any transgenic nucleotide sequence i.e., the nucleotide sequence of the DNA inserted into the genome of the cells of a plant or bacterium, or present in an extrachromosomal vector, would be considered to be an isolated nucleotide sequence whether it is present within the plasmid or similar structure used to transform the cells, within the genome of the plant or bacterium, or present in detectable amounts in tissues, progeny, biological samples or commodity products derived from the plant or bacterium.
  • the isolated DNA molecule is a chemical molecule, regardless of whether it is referred to as a nucleic acid, a nucleic acid sequence, a polynucleotide sequence, and the like. It is a novel, inventive molecule that exhibits industrial applicability both when present in a plant cell or in a plant genome, and when present outside of a plant cell, and therefore, exhibits and is intended to exhibit such utility regardless of where the molecule is located.
  • references in this disclosure to an “isolated protein molecule” or an equivalent term or phrase is intended to mean that the protein molecule is one that is present alone or in combination with other compositions but not within its natural environment.
  • the insecticidal protein molecules expressed by cotton event GH_BCS246002 are not naturally found in native cotton plant samples.
  • the CrylB.3, CrylDa_7, and Vip3Cbl.l insecticidal proteins of cotton event GH_BCS246002 are isolated protein molecules so long as the insecticidal proteins or variants thereof was not within the protein of the bacterium from which the protein is naturally found.
  • junction sequence refers to a DNA sequence of any length that spans the 5' or 3' junction of an event. Junction sequences of cotton event GH_BCS246002 are provided as SEQ ID NOs: 1-8.
  • junction sequences of cotton event GH_BCS246002 may be present as part of the genome of a plant, seed, or cell containing cotton event GH_BCS246002.
  • the identification of any one or more of the junction sequences in a sample from a plant, plant part, seed, or cell indicates that the DNA was obtained from cotton containing cotton event GH_BCS246002 and is diagnostic for, or characteristic of, the presence of cotton event GH_BCS246002.
  • junction sequences for cotton event GH_BCS246002 may be represented by a sequence from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 10.
  • the junction sequences may be arbitrarily represented by the nucleotide sequences provided as SEQ ID NO: 1 (5' junction sequence) and SEQ ID NO: 2 (3' junction sequence).
  • the junction sequences may be arbitrarily represented by the nucleotide sequences provided as SEQ ID NO: 3 (5' junction sequence) and SEQ ID NO: 4 (3' junction sequence).
  • junction sequences may be arbitrarily represented by the nucleotide sequences provided as SEQ ID NO: 5 (5' junction sequence) and SEQ ID NO: 6 (3' junction sequence).
  • the junction sequences may be arbitrarily represented by the nucleotide sequences provided as SEQ ID NO: 7 (5" junction sequence) and SEQ ID NO: 8 (3' junction sequence). These nucleotide sequences are connected by phosphodiester linkage, and in cotton event GH_BCS246002 are present as part of the recombinant plant cell genome.
  • junction sequences are diagnostic for, or characteristic of, the presence of cotton event GH_BCS246002, or the construct comprised therein.
  • the identification of one or more of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 10 in a sample derived from a cotton plant, cotton seed, or cotton plant part is diagnostic that the DNA was obtained from cotton event GH_BCS246002.
  • the present disclosure thus provides a DNA molecule that contains at least one of the nucleotide sequences provided as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10.
  • Any segment of DNA derived from transgenic cotton event GH_BCS246002 that is sufficient to include at least one of the sequence provided as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10 is within the scope of the present disclosure.
  • any polynucleotide comprising a sequence complementary to any of the sequences described within this paragraph is within the scope of the present disclosure.
  • the present disclosure provides exemplary DNA molecules that can be used either as primers or probes for detecting the presence of DNA derived from a cotton plant comprising cotton event GH_BCS246002 DNA in a sample.
  • primers or probes are specific for a target nucleic acid sequence and, as such, are useful for the identification of cotton event GH_BCS246002 nucleic acid sequence by the methods of the present disclosure described herein.
  • Capable of being detected refers to the ability of a particular DNA segment to be amplified and its size or sequence characterized or elucidated by DNA sequence analysis, i.e., the target DNA segment, and the subsequent ability to detect the binding of the prove to the target.
  • the particular DNA segment or target DNA segment of the present disclosure is present with cotton that contains the insertion of cotton event GH_BCS246002.
  • a “probe” is a nucleic acid molecule that is complementary to a strand of target nucleic acid and is useful in hybridization methods.
  • a probe may be attached a conventional detectable label or reporter molecule, e.g., a radioactive isotope, ligand, chemiluminescent agent, or enzyme.
  • a probe is complementary to a strand of a target nucleic acid and, in the case of the present disclosure, to a strand of DNA from cotton event GH_BCS246002 whether from a cotton event GH_BCS246002 containing plant or from a sample that includes cotton event GH_BCS246002 DNA.
  • the probes for use herein may comprise DNA molecules or polynucleotide segments of sufficient length to function under stringent hybridization conditions as defined herein to bind to a particular unique segment of DNA present within and diagnostic for, or characteristic of, cotton event GH_BCS246002 in a sample.
  • a probe can be designed to bind only to a single junction or other novel sequence present only in the cotton event GH_BCS246002, or two or more such single junction segments.
  • Probes according to the present disclosure include not only deoxyribonucleic or ribonucleic acids, but also polyamides and other probe materials that bind specifically to a target DNA sequence and can be used to detect the presence of that target DNA sequence.
  • a “primer” is typically a DNA molecule that is designed for use in specific annealing or hybridization methods that involve thermal amplification. Primers may comprise pairs of different oligonucleotides or polynucleotide segments for use in a thermal amplification reaction which amplifies a particular DNA target segment. Each primer in the pair is designed to bind to a rather specific segment of DNA within or near a segment DNA of interest for amplification.
  • primers bind in such a way that these then act as localized regions of nucleic acid sequence polymerization resulting in the production of one or more amplicons (amplified target segments of DNA).
  • the amplicon produced from such reaction would have a DNA sequence corresponding to sequence of the template DNA located between the two sites where the primers hybridized to the template.
  • use of primers designed to bind to unique segments of cotton event GH_BCS246002 and that amplify particular amplicons containing one or more of the junction sequences described herein, and the detection and/or characterization of such amplicons upon completion or termination of polymerase reaction is diagnostic for, or characteristic of, the present of cotton event GH_BCS246002 in a particular sample.
  • the skilled artisan is well familiar with this amplification method and no recitation of the specifics of amplification is necessary here.
  • a primer is typically designed to hybridize to a complementary target DNA strand to form a hybrid between the primer and target DNA strand, and the presence of the primer is a point of recognition by a polymerase to begin extension of the primer (i.e., polymerization of additional nucleotides into a lengthening nucleotide molecule) using as a template the target DNA strand.
  • Primer pairs refer to use of two primers binding opposite strands of a double stranded nucleotide segment for the purpose of amplifying linearly the polynucleotide segment between the positions targeted for binding by the individual members of the primer pair, typically in a thermal amplification reaction or other conventional nucleic-acid amplification methods.
  • Exemplary DNA molecules useful as primers are provided as SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 21.
  • the primer pair SEQ ID NO: 15 and SEQ ID NO: 16 are useful as a first DNA molecule and a second DNA molecule that is different from the first DNA molecule, and both are each of sufficient length of contiguous nucleotides of SEQ ID NO: 10 to function as DNA primers that, when used together in a thermal amplification reaction with template DNA derived from cotton event GH_BCS246002, to produce an amplicon diagnostic for, or characteristic of, cotton event GH_BCS246002 in a sample.
  • the primer pair SEQ ID NO: 18 and SEQ ID NO: 19 are useful as a first DNA molecule and a second DNA molecule that is different from the first DNA molecule, and both are each of sufficient length of contiguous nucleotides of a locus within the cotton genome to function as DNA primers that, when used together in a thermal amplification reaction with template DNA derived cotton event GH_BCS246002, to produce an amplicon that serves as an internal control for both the diagnosis of cotton event GH_BCS246002, as well as the zygosity of cotton event GH_BCS246002 DNA in a sample.
  • the primer pair SEQ ID NO: 20 and SEQ ID NO: 15 are useful as a first DNA molecule and a second DNA molecule that is different from the first DNA molecule, and both are each of sufficient length of contiguous nucleotides of a locus within the cotton genome to function as DNA primers that, when used together in a thermal amplification reaction with template DNA derived from cotton event GH_BCS246002, to produce an amplicon diagnostic for, or characteristic of, non-inserted wild-type cotton genomic DNA not comprising cotton event GH_BCS246002.
  • DNA probes and DNA primers are generally eleven (11) polynucleotides or more in length, often eighteen (18) polynucleotides or more, twenty-four (24) polynucleotides or more, or thirty (30) polynucleotides or more.
  • Such probes and primers arc selected to be of sufficient length to hybridize specifically to a target sequence under high stringency hybridization conditions.
  • probes and primers according to the present disclosure have complete sequence similarity with the target sequence, although probes differing from the target sequence that retain the ability to hybridize to target sequences may be designed by conventional methods.
  • nucleic acid probes and primers of the present disclosure hybridize under stringent conditions to a target DNA molecule. Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of DNA from a transgenic plant in a sample. Polynucleic acid molecules also referred to as nucleic acid segments or fragments thereof are capable of specifically hybridizing to other nucleic acid molecules under certain circumstances. [00133] As used herein, two polynucleic acid molecules are said to be capable of specifically hybridizing to one another if the two molecules arc capable of forming an anti-parallel, double-stranded nucleic acid structure.
  • a nucleic acid molecule is said to be the “complement” of another nucleic acid molecule if they exhibit complete complementarity.
  • molecules are said to exhibit “complete complementarity” when every nucleotide of one of the molecules is complementary to a nucleotide of the other.
  • Two molecules are said to be “minimally complementary” if they can hybridize to one another with sufficient stability to permit them to remain annealed to one another under at least conventional "low-stringency” conditions.
  • the molecules are said to be “complementary” if they can hybridize to one another with sufficient stability to permit them to remain annealed to one another under conventional "high- stringency” conditions.
  • a substantially homologous sequence is a nucleic acid sequence that will specifically hybridize to the complement of the nucleic acid sequence to which it is being compared under high stringency conditions.
  • Appropriate stringency conditions that promote DNA hybridization for example, 6.0 x sodium chloride/sodium citrate (SSC) at about 45°C, followed by a wash of 2.0 x SSC at 50°C, are known to those skilled in the art or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
  • the salt concentration in the wash step can be selected from a low stringency of about 2.0 x SSC at 50°C to a high stringency of about 0.2 x SSC at 50°C.
  • the temperature in the wash step can be increased from low stringency conditions at room temperature, about 22°C, to high stringency conditions at about 65°C. Both temperature and salt may be varied, or either the temperature or the salt concentration may be held constant while the other variable is changed.
  • a polynucleic acid of the present disclosure will specifically hybridize to one or more of the nucleic acid molecules set forth in SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10, or complements thereof or fragments of either under moderately stringent conditions, for example at about 2.0 x SSC and about 65°C.
  • a nucleic acid of the present disclosure will specifically hybridize to one or more of the nucleic acid molecules set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10, or complements or fragments of either under high stringency conditions.
  • a preferred marker nucleic acid molecule of the present disclosure has the nucleic acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10, or complements thereof, or fragments of either.
  • the hybridization of the probe to the target DNA molecule can be detected by any number of methods known to those skilled in the art, these can include, but are not limited to, fluorescent tags, radioactive tags, antibody-based tags, and chemiluminescent tags.
  • stringent conditions are conditions that permit the primer pair to hybridize only to the target nucleic acid sequence to which a primer having the corresponding wild-type sequence (or its complement) would bind and preferably to produce a unique amplification product, the amplicon, in a DNA thermal amplification reaction.
  • the term "specific for (a target sequence)" indicates that a probe or primer hybridizes under stringent hybridization conditions only to the target sequence in a sample comprising the target sequence.
  • amplified DNA refers to the product of polynucleic acid amplification method directed to a target polynucleic acid molecule that is part of a polynucleic acid template.
  • DNA that is extracted from a cotton plant tissue sample may be subjected to a polynucleotide acid amplification method using a primer pair that includes a first primer derived from a genomic DNA sequence in the region flanking the heterologous inserted DNA of cotton event GH_BCS246002 and is elongated by polymerase 5' to 3' in the direction of the inserted DNA.
  • the second primer is derived from the heterologous inserted DNA molecule is elongated by the polymerase 5' to 3' in the direction of the flanking genomic DNA from which the first primer is derived.
  • the amplicon may range in length from the combined length of the primer pair plus one nucleotide base pair, or plus about fifty nucleotide base pairs, or plus about two hundred- fifty nucleotide base pairs, or plus about four hundred-fifty nucleotide base pairs or more.
  • a primer pair can be derived from genomic sequence on both sides of the inserted heterologous DNA so as to produce an amplicon that includes the entire insert polynucleotide sequence (e.g., a forward primer isolated from the genomic portion on the 5' end of SEQ ID NO: 10 and a reverse primer isolated from the genomic portion on the 3' end of SEQ ID NO: 10 that amplifies a DNA molecule comprising the inserted DNA sequence (SEQ ID NO: 9) identified herein in the cotton event GH_BCS246002 genome).
  • a forward primer isolated from the genomic portion on the 5' end of SEQ ID NO: 10 and a reverse primer isolated from the genomic portion on the 3' end of SEQ ID NO: 10 that amplifies a DNA molecule comprising the inserted DNA sequence (SEQ ID NO: 9) identified herein in the cotton event GH_BCS246002 genome.
  • a member of a primer pair derived from the plant genomic sequence adjacent to the inserted transgenic DNA is located a distance from the inserted DNA sequence, this distance can range from one nucleotide base pair up to about twenty thousand nucleotide base pairs.
  • the use of the term “amplicon” specifically excludes primer dimers that may be formed in the DNA thermal amplification reaction.
  • primers which produce amplicons of a limited size range, for example, between 100 to 1000 bases. Smaller (shorter polynucleotide length) sized amplicons in general are more reliably produced in thermal amplification reactions, allow for shorter cycle times, and can be easily separated and visualized on agarose gels or adapted for use in endpoint TAQMAN®-like assays. Smaller amplicons can be produced and detected by methods known in the art of DNA amplicon detection. In addition, amplicons produced using the primer pairs can be cloned into vectors, propagated, isolated, and sequenced or can be sequenced directly with methods well established in the art.
  • Any primer pair derived from the combination of SEQ ID NO: 11 and SEQ ID NO: 9 or the combination of SEQ ID NO: 12 and SEQ ID NO: 9 that are useful in a DNA amplification method to produce an amplicon diagnostic for, or characteristic of, cotton event GH_BCS246002 or progeny thereof is an aspect of the present disclosure.
  • Any single isolated DNA polynucleotide primer molecule comprising at least 15 contiguous nucleotides of SEQ ID NO: 11, or its complement that is useful in a DNA amplification method to produce an amplicon diagnostic for, or characteristic of, cotton event GH_BCS246002 or progeny thereof is an aspect of the present disclosure.
  • Any single isolated DNA polynucleotide primer molecule comprising at least 15 contiguous nucleotides of SEQ ID NO: 12, or its complement that is useful in a DNA amplification method to produce an amplicon diagnostic for, or characteristic of, plants comprising cotton event GH_BCS246002 or progeny thereof is an aspect of the present disclosure.
  • Any single isolated DNA polynucleotide primer molecule comprising at least 15 contiguous nucleotides of SEQ ID NO: 9, or its complement that is useful in a DNA amplification method to produce an amplicon diagnostic for, or characteristic of, cotton event GH_BCS246002 or progeny thereof is an aspect of the present disclosure.
  • Polynucleic acid amplification can be accomplished by any of the various polynucleic acid amplification methods known in the art, including the polymerase chain reaction (PCR). Amplification methods are known in the ail and are described, inter alia, in U.S. Patent Nos. 4,683,195 and 4,683,202 and in PCR Protocols: A Guide to Methods and Applications, ed. Innis et al., Academic Press, San Diego, 1990. PCR amplification methods have been developed to amplify up to 22 kb (kilobase) of genomic DNA and up to 42 kb of bacteriophage DNA (Cheng et al., Proc. Natl. Acad. Sci.
  • PCR polymerase chain reaction
  • the sequence of the heterologous DNA insert or flanking genomic DNA sequence from cotton event GH_BCS246002 can be verified (and corrected if necessary) by amplifying such DNA molecules from cotton seed containing cotton event GH_BCS246002 DNA deposited with the ATCC having accession No. PTA- 127733, using primers derived from the sequences provided herein, followed by standard DNA sequencing of the PCR amplicon or cloned DNA fragments thereof.
  • the diagnostic amplicon produced by these methods may be detected by a plurality of techniques.
  • One such method is Genetic Bit Analysis (Nikiforov, et al. Nucleic Acid Res. 22:4167-4175, 1994) where a DNA oligonucleotide is designed that overlaps both the adjacent flanking genomic DNA sequence and the inserted DNA sequence.
  • the oligonucleotide is immobilized in wells of a microtiter plate.
  • a singlestranded PCR product can be hybridized to the immobilized oligonucleotide and serve as a template for a single base extension reaction using a DNA polymerase and labeled dideoxynucleotide triphosphates (ddNTPs) specific for the expected next base.
  • ddNTPs dideoxynucleotide triphosphates
  • Readout may be fluorescent or ELISA-based. A signal indicates presence of the transgene/genomic sequence due to successful amplification, hybridization, and single base extension.
  • Another method is the Pyro sequencing technique as described by Winge (Innov. Pharma. Tech. 00:18-24, 2000).
  • an oligonucleotide is designed that overlaps the adjacent genomic DNA and insert DNA junction.
  • the oligonucleotide is hybridized to single- stranded PCR product from the region of interest (one primer in the inserted sequence and one in the flanking genomic sequence) and incubated in the presence of a DNA polymerase, ATP, sulfurylase, luciferase, apyrase, adenosine 5' phosphosulfate and luciferin.
  • dNTPs are added individually and the enzymatic reaction of luciferase with these reagents and substrates results in the release of photons (a signal of light) which are then measured or observed.
  • a light signal indicates the presence of the transgene/genomic sequence due to successful amplification, hybridization, and single or multi-base extension.
  • Fluorescence Polarization as described by Chen, et al., (Genome Res. 9:492-498, 1999) is a method that can be used to detect the amplicon of the present disclosure.
  • an oligonucleotide is designed that overlaps the genomic flanking and inserted DNA junction.
  • the oligonucleotide is hybridized to single-stranded PCR product from the region of interest (one primer in the inserted DNA and one in the flanking genomic DNA sequence) and incubated in the presence of a DNA polymerase and a fluorescent-labeled ddNTP. Single base extension results in incorporation of the ddNTP.
  • Incorporation can be measured as a change in polarization using a fluorometer. A change in polarization indicates the presence of the transgene/genomic sequence due to successful amplification, hybridization, and single base extension.
  • PCR Real-time Polymerase Chain Reaction
  • Ct value is defined as the number of cycles required for the fluorescent signal to cross the threshold (i.e., exceeds background level). Ct levels are inversely proportional to the amount of target nucleic acid in the sample (i.e., the lower the Ct value, the greater the amount of target nucleic acid in the sample).
  • Taqman® PE Applied Biosystems, Foster City, CA
  • a FRET oligonucleotide probe is designed that overlaps the genomic flanking and insert DNA junction.
  • the FRET probe and PCR primers are cycled in the presence of a thermostable polymerase and dNTPs.
  • Hybridization of the FRET probe results in cleavage and release of the fluorescent moiety away from the quenching moiety on the FRET probe.
  • a fluorescent signal indicates the presence of the transgene/genomic sequence due to successful amplification and hybridization.
  • Molecular’ Beacons have been described for use in sequence detection as described in Tyangi, et al. (Nature Biotech.14:303-308, 1996). Briefly, a FRET oligonucleotide probe is designed that overlaps the flanking genomic and insert DNA junction. The unique structure of the FRET probe results in it containing secondary structure that keeps the fluorescent and quenching moieties in close proximity.
  • the FRET probe and PCR primers (one primer in the insert DNA sequence and one in the flanking genomic sequence) arc cycled in the presence of a thermostable polymerase and dNTPs.
  • hybridization of the FRET probe to the target sequence results in the removal of the probe secondary structure and spatial separation of the fluorescent and quenching moieties.
  • a fluorescent signal results.
  • a fluorescent signal indicates the presence of the flanking/transgene insert sequence due to successful amplification and hybridization.
  • microfluidics see, e.g., U.S. Patent Publication No. 2006/068398; U.S. Patent No. 6,544,734.
  • Optical dyes can be used to detect and measure specific DNA molecules (see, e.g., WO/05017181).
  • Nanotube devices see, e.g., WO/06024023 that comprise an electronic sensor for the detection of DNA molecules or nanobeads that bind specific DNA molecules can then be detected.
  • DNA detection kits that are based on DNA amplification methods contain DNA primer molecules that hybridize specifically to a target DNA and amplify a diagnostic amplicon under the appropriate reaction conditions.
  • the kit may provide an agarose gel-based detection method or any number of methods of detecting the diagnostic amplicon that are known in the art.
  • DNA detection kits can be developed using the compositions disclosed herein and are useful for identification of cotton event GH_BCS246002 DNA or DNA from a modified cotton event GH_BCS246002 in a sample and can be applied to methods for breeding cotton plants containing cotton event GH_BCS246002 DNA or DNA from a modified cotton event GH_BCS246002.
  • a kit that contains DNA primers that are homologous or complementary to any portion of the cotton genomic region as set forth in SEQ ID NO: 10 and to any portion of the inserted transgenic DNA as set forth in SEQ ID NO: 10 is an object of the present disclosure.
  • the DNA molecules can be used in DNA amplification methods (PCR) or as probes in polynucleic acid hybridization methods, i.e., southern analysis, northern analysis.
  • Probes and primers according to the present disclosure may have complete sequence identity with the target sequence, although primers and probes differing from the target sequence that retain the ability to hybridize preferentially to target sequences may be designed by conventional methods.
  • primers and probes differing from the target sequence that retain the ability to hybridize preferentially to target sequences may be designed by conventional methods.
  • a nucleic acid molecule In order for a nucleic acid molecule to serve as a primer or probe it need only be sufficiently complementary in sequence to be able to form a stable double- stranded structure under the particular solvent and salt concentrations employed. Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of transgenic DNA from cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 in a sample.
  • Probes and primers are generally at least about 11 nucleotides, at least about 18 nucleotides, at least about 24 nucleotides, or at least about 30 nucleotides or more in length. Such probes and primers hybridize specifically to a target DNA sequence under stringent hybridization conditions. Conventional stringency conditions are described by Sambrook et al., 1989, and by Haymes et al., In: Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, DC (1985).
  • DNA molecules, or fragments thereof can also be obtained by other techniques such as by directly synthesizing the fragment by chemical means, as is commonly practiced by using an automated oligonucleotide synthesizer.
  • the DNA molecules and corresponding nucleotide sequences provided herein are therefore useful for, among other things, identifying cotton event GH_BCS246002 or a modified cotton event GH_BCS246002, detecting the presence of DNA derived from the transgenic cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 in a sample, and monitoring samples for the presence and/or absence of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 or plant parts derived from cotton plants comprising cotton event GH_BCS246002 or a modified cotton event GH_BCS246002.
  • cotton plants, cotton plant cells, cotton seed, cotton pollen and ova, cotton plant parts, cotton progeny plants, and cotton commodity products are within the scope of the present disclosure, so long as each embodiment contains a detectable amount of DNA corresponding to any one, two, or more of the segments described herein as being diagnostic for, or characteristic of, the presence of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002, such as a polynucleotide having at least one of the sequences provided as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10.
  • a detectable amount of DNA corresponding to any one, two, or more of the segments described herein as being diagnostic for, or characteristic of, the presence of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002, such as a polyn
  • Cotton plants, cotton plant cells, cotton seed, cotton pollen and ova, cotton plant parts, cotton progeny plants of the present disclosure may also contain one or more additional transgenes.
  • additional transgene(s) may be any nucleotide sequence encoding a protein or RNA molecule conferring a desirable trait including but not limited to increased insect resistance, increased water use efficiency, increased yield performance, increased drought resistance, increased seed quality, and/or increased herbicide tolerance.
  • the present disclosure provides cotton plants, cotton plant cells, cotton seed, cotton plant parts (such as pollen, ovule, squares, bolls, root tissue, leaf tissue), cotton progeny plants derived from a transgenic cotton plant containing cotton event GH_BCS246002 DNA or DNA from a modified cotton event GH_BCS246002.
  • a representative sample of cotton seed containing cotton event GH_BCS246002 has been deposited according to the Budapest Treaty with the American Type Culture Collection (ATCC®).
  • the ATCC repository has assigned the Patent Deposit Designation PTA- 127733 to the seed containing cotton event GH_BCS246002 DNA.
  • the present disclosure provides a microorganism comprising a DNA molecule having at least one sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10 present in its genome.
  • An example of such a microorganism is a transgenic plant cell.
  • Microorganisms such as a plant cell of the present disclosure, are useful in many industrial applications, including but not limited to: (i) use as research tool for scientific inquiry or industrial research; (ii) use in culture for producing endogenous or recombinant carbohydrate, lipid, nucleic acid, or protein products or small molecules that may be used for subsequent scientific research or as industrial products; and (iii) use with modem plant tissue culture techniques to produce transgenic plants or plant tissue cultures that may then be used for agricultural research or production.
  • the production and use of microorganisms such as transgenic plant cells utilizes modem microbiological techniques and human intervention to produce a man-made, unique microorganism.
  • transgenic plant cell recombinant DNA is inserted into a plant cell’s genome to create a transgenic plant cell that is separate and unique from naturally occurring plant cells.
  • This transgenic plant cell can then be cultured much like bacteria and yeast cells using modern microbiology techniques and may exist in an undifferentiated, unicellular state.
  • the transgenic plant cell’s new genetic composition and phenotype is a technical effect created by the integration of the heterologous DNA into the genome of the cell.
  • Another aspect of the present disclosure is a method of using a microorganism provided herein.
  • Methods of using microorganisms of the present disclosure include (i) methods of producing transgenic cells by integrating recombinant DNA into the genome of the cell and then using this cell to derive additional cells possessing the same heterologous DNA; (ii) methods of culturing cells that contain recombinant DNA using modern microbiology techniques; (iii) methods of producing and purifying endogenous or recombinant carbohydrate, lipid, nucleic acid, or protein products from cultured cells; and (iv) methods of using modern plant tissue culture techniques with transgenic plant cells to produce transgenic plants or transgenic plant tissue cultures.
  • Cotton plants of the present disclosure may pass along the cotton event GH_BCS246002 DNA, including the transgene inserted in cotton event GH_BCS246002 or a modified cotton event GH_BCS246002, to progeny, typically through crosses or conventional breeding and selection.
  • progeny includes any plant, plant cell, seed, and/or regenerable plant part containing the cotton event GH_BCS246002 DNA or DNA from a modified cotton event GH_BCS246002 derived from an ancestor plant and/or comprising a DNA molecule having at least one sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10.
  • Cotton plants, progeny, and seed may be homozygous or heterozygous for the cotton event GH_BCS246002 or a modified cotton event GH_BCS246002.
  • Progeny may be grown from seed produced by a cotton plant containing event GH_BCS246002 or a modified cotton event GH_BCS246002 and/or from seed produced by a plant fertilized with pollen from a cotton plant containing event GH_BCS246002 or a modified cotton event GH_BCS246002.
  • Cotton plants may be bred using any method known in the art.
  • Progeny plants may be self-pollinated (also known as “selfing”) to generate a true breeding line of plants, i.c., plants homozygous for the transgene(s), such as cotton event ZM_BCS216090.
  • selfing can result in progeny known as an “inbred” that can be used to produce cotton inbred lines that are genetically uniform.
  • Selfing of appropriate progeny can produce plants that are homozygous for the exogenous transgene(s), such as cotton event ZM_BCS21609 or a modified cotton event GH_BCS246002.
  • progeny plants may be out-crossed, e.g., bred with another unrelated plant, to produce a varietal or a hybrid seed or plant.
  • the other unrelated plant may be transgenic or non-transgenic.
  • a varietal or hybrid seed or plant of the present disclosure may thus be derived by sexually crossing a first parent that lacks the specific and unique DNA of the cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 with a second parent comprising cotton event GH_BCS246002 or a modified cotton event GH_BCS246002, resulting in a hybrid comprising the specific and unique DNA of the cotton event GH_BCS246002 or the modified cotton event GH_BCS246002.
  • Each parent can be a hybrid or an inbred/varietal, so long as the cross or breeding results in a plant or seed of the present disclosure, i.e., a seed having at least one allele containing the DNA of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 and/or a DNA molecule having at least one sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10.
  • Two different transgenic plants may thus be crossed to produce hybrid offspring that contain two independently segregating transgenes or traits, wherein at least one of those transgenes or events comprise or is contained within cotton event GH_BCS246002 or a modified cotton event GH_BCS246002.
  • transgenic cotton plants comprising cotton event GH_BCS246002 containing CrylB.3, CrylDa_7, and Vip3Cbl.l, conferring Lepidopteran resistance to cotton, or a modified cotton event GH_BCS246002, can be crossed with a plant having one or more additional traits such as herbicide tolerance, insect resistance, or drought tolerance, resulting in a progeny plant or seed that has resistance to Lepidopteran insect pests and has at least one or more additional traits.
  • Back-crossing to a parental plant and out-crossing with a non-transgenic plant are also contemplated, as is vegetative propagation.
  • Cotton plants, progeny, seed, pollen and ova, cells and plant parts of the present disclosure may also contain one or more additional cotton traits(s) or transgenic cvcnt(s), which may be introduced by crossing a cotton plant containing cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 with another cotton plant containing the additional trait(s) or transgenic event(s).
  • Such trait(s) or transgenic event(s) may include, but are not limited to, increased insect resistance, herbicide tolerance, increased water use efficiency, increased yield performance, increased drought resistance, increased seed quality, improved nutritional quality, hybrid seed production, or disease or fungal resistance.
  • a cotton trait may include any transgenic traits or mutant or edited traits or alleles. Mutant traits or alleles of a gene may be created by any mutagenesis technique known in the art, whereas edited traits may be generated by any genome editing technique or method known in the art. Many cotton transgenic events are known to those of skill in the art.
  • transgenic events may thus be combined in a progeny seed or plant by crossing two parent plants each comprising one or more transgenic events, collecting the progeny seed, and selecting for progeny seed or plants that contain the two or more transgenic events. These steps may be repeated until the desired combination of transgenic events in a progeny is achieved.
  • a plant part that is derived from cotton plants comprising cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 is also provided.
  • a “plant part” refers to any pail of a plant which is comprised of plant material derived from a cotton plant that may comprise cotton event GH_BCS246002 or a modified cotton event GH_BCS246002.
  • Plant parts include, but are not limited to, pollen, ovule, square, boll, root tissue, stem tissue, fibers, and leaves. Plant parts may be viable, nonviable, regenerable, and/or nonregenerable.
  • a “commodity product” refers to any composition or product which is comprised of material derived from a cotton plant, whole or processed cotton seed, or one or more plant cells and/or plant parts containing the cotton event GH_BCS246002 DNA or DNA from a modified cotton event GH_BCS246002.
  • Nonviable commodity products include, but arc not limited to, nonviable seed, whole or processed seed, seed parts, and plants parts; whole or processed cotton seeds, cotton fiber, cotton oil and derivatives of cotton oil, cotton protein, cotton meal, animal feed comprising cotton, paper comprising cotton, cotton biomass, candle wicks, cotton string, cotton rope, cotton balls, cotton batting, cotton fuel products, and cotton cellulose products.
  • Viable commodity products include, but are not limited to, seed, plants, and plant cells. The cotton plants comprising cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 can thus be used to manufacture any commodity product typically acquired from cotton.
  • Any such commodity product that is derived from cotton plants comprising cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 may contain at least a detectable amount of the specific and unique DNA corresponding to cotton event GH_BCS246002 or a modified cotton event GH_BCS246002, and specifically may contain a detectable amount of a polynucleotide comprising a DNA molecule having at least one sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10.
  • a commodity product is within the scope of the present disclosure if there is any detectable amount of a DNA molecule having at least one sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10 in the commodity product.
  • the cotton plants, cotton plant cells, cotton seed, cotton plant parts (such as pollen, ovule, anther, root tissue, stalk tissue leaf tissue), cotton progeny plants, and commodity products of the present disclosure are therefore, useful for, among other things, growing plants for the purpose of producing seed and/or plant parts comprising cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 for agricultural purposes, producing progeny comprising cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 for plant breeding and research purposes, use with microbiological techniques for industrial and research application, and sale to consumers.
  • a progeny cotton plant comprising the event GH_BCS246002 or a modified cotton event GH_BCS246002 may be produced, for example, by selfing a parent plant or line comprising the event GH_BCS246002 or a modified cotton event GH_BCS246002, wherein such parent plant or line is homozygous or hemizygous for the event GH_BCS246002 or the modified cotton event GH_BCS246002, or by crossing a first parent plant or line comprising the event GH_BCS246002 or a modified cotton event GH BCS246002, wherein such parent plant or line is homozygous or hemizygous for the event GH_BCS246002 or the modified cotton event GH_BCS246002, with a second parent plant
  • Transgenic plants used in these methods may be homozygous or heterozygous (or hemizygous) for the transgene(s), event GH_BCS246002 or a modified cotton event GH_BCS246002.
  • Progeny plants produced by these methods may be varietal or hybrid plants, may be grown from seeds produced by plants containing cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 and/or from seed produced by a plant fertilized with pollen from a cotton plant containing cotton event GH_BCS246002 or a modified cotton event GH_BCS246002.
  • Progeny plants may be subsequently self-pollinated to generate a true breeding line of plants, i.e., plants homozygous for the transgene, or alternatively may be out-crossed, e.g., bred with another unrelated plant, to produce a varietal or hybrid seed or plant.
  • One method comprises (i) extracting a DNA sample from at least one cotton cell, cotton tissue, cotton seed, or cotton plant; (ii) contacting the DNA sample with at least one primer that is capable of producing DNA sequence specific to cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 under conditions appropriate for DNA sequencing; (iii) performing a DNA sequencing reaction; and then (iv) confirming that the nucleotide sequence comprises a nucleotide sequence specific for cotton event GH_BCS246002 or a modified cotton event GH_BCS246002, of the construct comprised therein, such as one selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10.
  • Another method comprises (i) extracting a DNA sample from at least one cotton cell, cotton tissue, cotton seed, or cotton plant; (ii) contacting the DNA sample with a primer pair that is capable of producing an amplicon from cotton event GH_BCS246002 DNA or DNA from a modified cotton event GH_BCS246002 under conditions appropriate for DNA amplification; (iii) performing a DNA amplification reaction; and then (iv) detecting the amplicon molecule and/or confirming that the nucleotide sequence of the amplicon comprises a nucleotide sequence specific for cotton event GH_BCS246002 or a modified cotton event GH_BCS246002, such as one selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.
  • the amplicon should be one that is specific for cotton event GH_BCS246002, such as an amplicon that comprises SEQ ID NO: 1, or SEQ ID NO: 2, or SEQ ID NO: 3, or SEQ ID NO: 4, or SEQ ID NO: 5, or SEQ ID NO: 6.
  • the detection of a nucleotide sequence specific for cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 in the amplicon is determinative and/or diagnostic for, or characteristic of, the presence of DNA specific for the cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 in the sample.
  • SEQ ID NO: 15 and SEQ ID NO: 16 An example of a primer pair that is capable of producing an amplicon from cotton event GH_BCS246002 DNA or DNA from a modified cotton event GH_BCS246002 under conditions appropriate for DNA amplification is provided as SEQ ID NO: 15 and SEQ ID NO: 16.
  • Other primer pairs may be readily designed by one of skill in the art and would produce an amplicon comprising SEQ ID NO: 1, or SEQ ID NO: 2, or SEQ ID NO: 3, or SEQ ID NO: 4, or SEQ ID NO: 5, or SEQ ID NO: 6, wherein such a primer pair comprises at least one primer within the genomic region flanking the insert and a second primer within the insert.
  • Another method of detecting the presence of DNA derived from a cotton cell, cotton tissue, cotton seed, or cotton plant comprising cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 in a sample comprises (i) extracting a DNA sample from at least one cotton cell, cotton tissue, cotton seed, or cotton plant; (ii) contacting the DNA sample with a DNA probe specific for cotton event GH_BCS246002 DNA or DNA specific for a modified cotton event GH_BCS246002; (iii) allowing the probe and the DNA sample to hybridize under stringent hybridization conditions and then (iv) detecting hybridization between the probe and the target DNA sample.
  • SEQ ID NO: 17 An example of the sequence of a DNA probe that is specific for cotton event GH_BCS246002 is provided as SEQ ID NO: 17.
  • Other probes may be readily designed by one of skill in the art and would comprise at least one fragment of genomic DNA flanking the insert and at least on fragment of the insert DNA such as sequence provided in, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 10.
  • Detection of probe hybridization to the DNA sample is diagnostic for, or characteristic of, the presence of cotton event GH_BCS246002 specific DNA in the sample. Absence of hybridization is alternatively diagnostic for, or characteristic of, the absence of cotton event GH_BCS246002 specific DNA in the sample.
  • DNA detection kits are provided that are useful for the identification of cotton event GH_BCS246002 DNA or DNA from a modified cotton event GH_BCS246002 in a sample and can also be applied to methods for breeding cotton plants containing the appropriate event DNA.
  • Such kits contain DNA primers and/or probes comprising fragments of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10.
  • kits comprises at least one DNA molecule of sufficient length of continuous nucleotides of SEQ ID NO: 10 to function as a DNA probe useful for detecting the presence and/or absence of DNA derived from transgenic cotton plants comprising cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 in a sample.
  • the DNA derived from transgenic cotton plants comprising cotton event GH_BCS246002 would comprise a DNA molecule having at least one sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10.
  • a DNA molecule sufficient for use as a DNA probe is provided that is useful for determining, detecting, or diagnosing the presence and/or absence of cotton event GH_BCS246002 in a sample is provided as SEQ ID NO: 17.
  • Other probes may be readily designed by one of skill in the art and should comprise at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, or at least 40 contiguous nucleotides of SEQ ID NO: 10 and be sufficiently unique to cotton event GH_BCS246002 DNA in order to identify DNA derived from the event.
  • kits comprises a primer pair useful for producing an amplicon useful for detecting the presence and/or absence of DNA derived from transgenic cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 in a sample.
  • kit would employ a method comprising contacting a target DNA sample with a primer pair as described herein, then performing a nucleic acid amplification reaction sufficient to produce an amplicon comprising a DNA molecule having at least one sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10 and then detecting the presence and/or absence of the amplicon.
  • Such a method may also include sequencing the amplicon or a fragment thereof, which would be determinative of, i.e., diagnostic for, or characteristic of, the presence of the cotton event GH_BCS246002 specific DNA or DNA specific for a modified cotton event GH_BCS246002 in the target DNA sample.
  • primer pairs may be readily designed by one of skill in the art and should comprise at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 contiguous nucleotides of sequences provided in, but not limited to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, and be sufficiently unique to cotton event GH_BCS246002 DNA in order to identify DNA derived from the event.
  • kits and detection methods of the present disclosure are useful for, among other things, identifying or detecting cotton event GH_BCS246002 or a modified cotton event GH_BCS246002, selecting plant varieties or hybrids comprising cotton event GH_BCS246002 or a modified cotton event GH_BCS246002, detecting the presence of DNA derived from the transgenic cotton plant comprising cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 in a sample, and monitoring samples for the presence and/or absence of cotton plants comprising cotton event GH_BCS246002 or a modified cotton event GH_BCS246002, or plant parts derived from cotton plants comprising cotton event GH_BCS246002 or a modified cotton event GH_BCS246002.
  • sequences of the heterologous DNA insert, junction sequences, or flanking sequences from cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 can be verified (and corrected if necessary) by amplifying such sequences from the event using primers derived from the sequence provided herein followed by standard DNA sequencing of the amplicon or of the cloned DNA.
  • Methods of detecting the zygosity of the transgene DNA from a cotton cell, cotton tissue, cotton seed, or cotton plant or plant pail comprising cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 in a sample are provided.
  • One method comprises (i) extracting a DNA sample from at least one cotton cell, cotton tissue, cotton seed, or cotton plant or plant part; (ii) contacting the DNA sample with a primer pair that is capable of producing a first amplicon diagnostic for, or characteristic of, cotton event GH_BCS246002 or a modified cotton event GH_BCS246002; (iii) contacting the DNA sample with a primer pair that is capable of producing a second amplicon diagnostic for, or characteristic of, native cotton genomic DNA that does not comprise cotton event GH_BCS246002 or a modified cotton event GH_BCS246002; (iv) performing a DNA amplification reaction(s); and then (v) detecting the amplicons, wherein the presence of only the first amplicon is diagnostic for cotton cell, cotton tissue, cotton seed, or cotton plant or plant part being homozygous for the cotton event GH_BCS246002 or the modified cotton event GH_BCS246002, and the presence of both the first amplicon and the second
  • An exemplary set of primer pairs are presented as SEQ ID NO: 15 and SEQ ID NO: 16 which produce an amplicon diagnostic for, or characteristic of, cotton event GH_BCS246002; and SEQ ID NO: 15 and SEQ ID NO: 21 which produces an amplicon for, or characteristic of, the wild-type cotton genomic DNA not comprising cotton event GH_BCS246002.
  • a set of probes can also be incorporated into such an amplification method to be used in real-time PCR format using the primer pair sets described above.
  • An exemplary set of probes are presented as SEQ ID NO: 17 (diagnostic for, or characteristic of, the amplicon for the cotton event GH_BCS246002) and SEQ ID NO: 22 (diagnostic for, or characteristic of, the amplicon for wild-type cotton genomic DNA not comprising cotton event GH_BCS246002).
  • Another method for determining zygosity comprises (i) extracting a DNA sample from at least one cotton cell, cotton tissue, cotton seed, or cotton plant or plant pail; (ii) contacting the DNA sample with a probe set which contains at least a first probe that specifically hybridizes to cotton event GH_BCS246002 DNA or a modified cotton event GH_BCS246002 DNA and at least a second probe that specifically hybridizes to wild-type cotton genomic DNA that was disrupted by insertion of the heterologous DNA of cotton event GH_BCS246002 and does not hybridize to cotton event GH_BCS246002 DNA or a modified cotton event GH_BCS246002 DNA; (iii) hybridizing the probe set with the sample under stringent hybridization conditions, wherein detecting hybridization of only the first probe under the hybridization conditions is diagnostic for, or characteristic of, the sample being homozygous for the cotton event GH_BCS246002 DNA or the modified cotton event GH_BCS246002 DNA,
  • Yet another method for determining zygosity comprises (i) extracting a DNA sample from at least one cotton cell, cotton tissue, cotton seed, or cotton plant; (ii) contacting the DNA sample with a primer pair that is capable of producing an amplicon diagnostic for, or characteristic of, cotton event GH_BCS246002 or a modified cotton event GH_BCS246002; (iii) contacting the DNA sample with a primer pair that is capable of producing an amplicon of an internal standard known to be single-copy and homozygous in the cotton plant; (iv) contacting the DNA sample with a probe set which contains at least a first probe that specifically hybridizes to DNA of the cotton event GH_BCS246002 or the modified cotton event GH_BCS246002, and at least a second probe the specifically hybridizes to the internal standard genomic DNA known to be single-copy and homozygous in the cotton plant; (v) performing a DNA amplification reaction using real-time PCR and determining the cycle thresholds (C
  • Heterozygous and homozygous events are differentiated by a ACt value unit of approximately one (1). Given the normal variability observed in real-time PCR due to multiple factors such as amplification efficiency and ideal annealing temperatures, the range of “about one (1)” is defined as a ACt of 0.75 to 1.25.
  • Primer pairs and probes for the above method for determining zygosity can amplify and detect amplicons from DNA of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 and an internal standard.
  • Exemplary primer pairs for the detection of amplicons corresponding to cotton event GH_BCS246002 and internal standard are presented as SEQ ID NO: 15 combined with SEQ ID NO: 16 (allele for cotton event GH_BCS246002) and SEQ ID NO: 18 combined with SEQ ID NO: 19 (internal standard).
  • the accompanying exemplary probes are presented as SEQ ID NO: 17 (cotton event GH_BCS246002) and SEQ ID NO: 20 (internal standard). Modification of cotton event GH_BCS246002
  • a “cotton event GH_BCS246002 locus” refers to the genomic locus of the cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 or a further modified cotton event GH_BCS246002, wherein the cotton event GH_BCS246002 locus includes the flanking, junction, and insertion sequences of the cotton event GH_BCS246002 or the modified cotton event GH_BCS246002 or further modified cotton event GH_BCS246002.
  • a modified cotton event GH_BCS246002 or a further modified cotton event GH_BCS246002 comprises one or more mutations, edits and/or genetic modifications in the cotton event GH_BCS246002 locus, such as one or more mutations, edits and/or genetic modifications in a flanking, junction and/or insertion sequence(s) of the cotton event GH_BCS246002 locus, relative to cotton event GH_BCS246002.
  • a modified cotton event GH_BCS246002 and methods of making a modified cotton event GH_BCS246002 are provided.
  • various mutagenesis or targeted genome editing techniques and related tools are known and could be made or engineered to permit genetic modification or mutation of the transgenic insert, junction and/or the flanking genomic DNA of cotton event GH_BCS246002, such as by deletion, insertion, transposition, inversion, and/or substitution of nucleic acid sequence(s), and/or by insertion or introduction of a guide RNA target site or a cognate target site or CgRRS, and the transgenic event as modified may still be uniquely characterized by the presence of heterologous DNA and/or one or more sequences of the insertion, junction(s) and/or flanking sequence(s) of cotton event GH_BCS246002 described herein at the same position or location in the genome previously occupied by the unmodified cotton event GH_BCS246002 relative to flanking portions or sequence
  • a modified transgenic event derived from cotton event GH_BCS246002 may comprise all or part of the insertion sequence and/or transgene cassette of cotton event GH_BCS246002, one or more of the junction sequence(s) of cotton event GH_BCS246002, and/or one or more flanking sequence(s) of cotton event GH_BCS246002 as described herein.
  • a “modified cotton event GH_BCS246002” refers to any genomic DNA or sequence of the cotton event GH_BCS246002 locus comprising one or more mutations, edits or genetic modifications relative to the genomic DNA or sequence of the cotton event GH_BCS246002, wherein such mutations or edits are introduced or made by a mutagenesis or targeted genome editing technique of a cotton plant, plant part, tissue or cell comprising the cotton event GH_BCS246002.
  • a “modified cotton event GH_BCS246002” includes, as a type of modified cotton event GH_BCS246002, a “further modified cotton event GH_BCS246002” made by first inserting a target site or cognate target site or CgRRS into the cotton event GH_BCS246002 locus and then further modifying the cotton event GH_BCS246002 locus as described herein.
  • a “modified cotton event GH BCS246002” includes genomic DNA or sequences of the cotton event GH_BCS246002 locus comprising one or more mutations, edits or genetic modifications relative to the genomic DNA or sequence of the cotton event GH_BCS246002, wherein such mutations or edits are introduced or made by a mutagenesis or targeted genome editing technique of a cotton plant, plant part, tissue or cell comprising the cotton event GH_BCS246002, wherein such modified cotton event GH_BCS246002 is not a further modified cotton event GH_BCS246002.
  • Methods and techniques of mutagenesis are known in the art and include, for example, chemical mutagenesis (z.e.
  • treatment with a chemical mutagen such as an azide, hydroxylamine, nitrous acid, acridine, nucleotide base analog, or alkylating agent - e.g. , EMS (ethylmethane sulfonate), MNU (N-methyl-N-nitrosourea), etc.), physical mutagenesis e.g., gamma rays, X-rays, UV, ion beam, other forms of radiation, etc.), and insertional mutagenesis (e.g., transposon or T-DNA insertion).
  • a chemical mutagen such as an azide, hydroxylamine, nitrous acid, acridine, nucleotide base analog, or alkylating agent - e.g. , EMS (ethylmethane sulfonate), MNU (N-methyl-N-nitrosourea), etc.
  • physical mutagenesis e.g., gamma
  • a “modified cotton plant” refers to a cotton plant comprising a modified cotton event GH_BCS246002 or a further modified cotton event GH_BCS246002.
  • a modified cotton plant part, plant seed, plant tissue, or plant cell comprising a modified cotton event GH_BCS246002 or a further modified cotton event GH_BCS246002 that is derived, taken or descended from a modified cotton plant and/or created by genetic modification, mutation or editing of the transgenic insert, junction and/or the flanking genomic DNA of cotton event GH_BCS246002 in a cotton plant part, plant seed, plant tissue, or plant cell using a mutagenesis or targeted genome editing technique.
  • a modified cotton event GH BCS246002 may have one or both of the junction sequences of cotton event GH_BCS246002 altered by a targeted genome editing technique, may have one or more of the expression elements or cassettes altered or removed, or may have additional expression cassettes or sequences inserted or included within the border or junction sequences or near either end of the inserted event DNA, and may have polynucleotide or expression element sequences altered to change expression or to encode a different amino acid sequence of one or more of the respective toxins present in event GH_BCS246002, which may be designed to overcome the development of resistance by one or more target pests to one or more of the respective toxin proteins described herein, wherein the altered expression or different amino acid sequence confers improved toxin properties to the respective protein and/or a different host range of toxicity due to the slightly different amino acid sequence in the modified cotton plant comprising the modified cotton event GH_BCS246002.
  • a “target site” for genome editing refers to the location of a polynucleotide sequence within a plant genome, which may be within or near cotton event GH_BCS246002, that is bound and cleaved by a site-specific nuclease introducing a double stranded break (or single- stranded nick) into the nucleic acid backbone of the polynucleotide sequence and/or its complementary DNA strand.
  • a target site may comprise at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 29, or at least 30 consecutive nucleotides.
  • a “target site” for an RNA-guidcd nuclease may comprise the sequence of either complementary strand of a double-stranded nucleic acid (DNA) molecule or chromosome at the target site that is bound or hybridized to a guide RNA of a ribonucleoprotein complex comprising the RNA-guided nuclease.
  • a site-specific nuclease may bind to a target site, such as via a non-coding guide RNA (e.g., without being limiting, a CRISPR RNA (crRNA) or a single-guide RNA (sgRNA) as described further below).
  • a non-coding guide RNA e.g., without being limiting, a CRISPR RNA (crRNA) or a single-guide RNA (sgRNA) as described further below.
  • gRNA non-coding guide RNA
  • a target site e.g., complementary to either strand of a double- stranded nucleic acid molecule or chromosome at the target site. It will be appreciated that perfect identity or complementarity may not be required for a non-coding guide RNA to bind or hybridize to a target site.
  • target site also refers to the location of a polynucleotide sequence within a plant genome, which may be within or near cotton event GH_BCS246002 or a modified cotton event GH_BCS246002, that is bound and cleaved by another site-specific nuclease that may not be guided by a non-coding RNA molecule, such as a meganuclease, zinc finger nuclease (ZFN), or a transcription activator-like effector nuclease (TALEN), to introduce a double stranded break (or single- stranded nick) into the polynucleotide sequence and/or its complementary DNA strand.
  • a meganuclease zinc finger nuclease (ZFN), or a transcription activator-like effector nuclease (TALEN)
  • TALEN transcription activator-like effector nuclease
  • a “target region” or a “targeted region” refers to a polynucleotide sequence or region that is flanked by two or more target sites. Without being limiting, in some embodiments a target region may be subjected to a mutation, deletion, insertion or inversion. As used herein to describe a target region of a polynucleotide sequence or molecule, “flanked” refers to two or more target sites of the polynucleotide sequence or molecule surrounding the target region, with one target site on each side of the target region.
  • a “donor molecule”, “donor template”, or “donor template molecule” (collectively a “donor template”), which may be a recombinant DNA donor template, is defined as a nucleic acid molecule having a nucleic acid template or insertion sequence for site- directed, targeted insertion or recombination into the genome of a plant cell via repair of a nick or double-stranded DNA break in the genome of a plant cell.
  • a “donor template” may be used for site-directed integration of a guide RNA target site or a cognate target site or CgRRS into a target site within the genome of a plant.
  • a targeted genome editing technique provided herein may comprise the use of one or more, two or more, three or more, four or more, or five or more donor molecules or templates.
  • a “donor template” may be a single-stranded or doublestranded DNA or RNA molecule or plasmid.
  • An “insertion sequence” of a donor template is a sequence designed for targeted insertion into the genome of a plant cell, which may be of any suitable length, such as to include a guide RNA target site or a cognate target site or CgRRS. Such an insertion sequence of a donor template is distinct and different from the transgenic insertion or insert of cotton event GH_BCS246002, although they may potentially have sequence(s) in common.
  • a donor template may also have at least one homology sequence or homology arm, such as two homology arms, to direct the integration of a mutation or insertion sequence into a target site within the genome of a plant via homologous recombination, wherein the homology sequence or homology arm(s) are identical or complementary, or have a percent identity or percent complementarity, to a sequence at or near the target site within the genome of the plant.
  • the homology arm(s) will flank or surround the insertion sequence of the donor template.
  • targeted genome editing technique refers to any method, protocol, or technique that can be used to make a targeted mutation or edit, such as one or more insertions, deletions, substitutions, inversions, transpositions, mutations and/or other genetic modifications at or near a target site in the genome of a plant, and/or a deletion or excision of a target region between two target sites in the genome of a plant, using a site-specific nuclease, such as a meganuclease, a zine-finger nuclease (ZFN), an RNA-guided endonuclease (e.g., the CRISPR/Cas9 system), a TALE-endonuclease (TALEN), a recombinase, or a transposase.
  • a site-specific nuclease such as a meganuclease, a zine-finger nuclease (ZFN), an RNA-guided
  • a site-specific nuclease may introduce a double stranded break (or single-stranded nick) into the nucleic acid backbone of the polynucleotide sequence and/or its complementary DNA strand.
  • the genomic sequence can be repaired via a double strand break repair pathway, which may include, for example, non-homologous end-joining (NHEJ), microhomology-mediated end joining (MMEJ), homologous recombination, synthesisdependent strand annealing (SDSA), single-strand annealing (SSA), or a combination of any thereof, at or near the target site(s).
  • NHEJ non-homologous end-joining
  • MMEJ microhomology-mediated end joining
  • SDSA synthesisdependent strand annealing
  • SSA single-strand annealing
  • a mutation or edit may be introduced at or near the target site(s), and a target region between two or more target sites may be deleted or excised.
  • a “site-specific nuclease” provided herein may be selected from the group consisting of a zine-finger nuclease (ZFN), a mcganuclcasc, an RNA-guidcd endonuclease, a TALE-endonuclease (TALEN), a recombinase, a transposase, or any combination thereof.
  • ZFN zine-finger nuclease
  • mcganuclcasc an RNA-guidcd endonuclease
  • TALEN TALE-endonuclease
  • a recombinase may be a serine recombinase attached to a DNA recognition motif, a tyrosine recombinase attached to a DNA recognition motif or other recombinase enzyme known in the art.
  • a recombinase or transposase may be a DNA transposase or recombinase attached to a DNA binding domain.
  • a tyrosine recombinase attached to a DNA recognition motif may be selected from the group consisting of a Cre recombinase, a Flp recombinase, and a Tnpl recombinase.
  • a Cre recombinase or a Gin recombinase provided herein is tethered to a zine-finger DNA binding domain.
  • a serine recombinase attached to a DNA recognition motif provided herein is selected from the group consisting of a PhiC31 integrase, an R4 integrase, and a TP-901 integrase.
  • a DNA transposase attached to a DNA binding domain provided herein is selected from the group consisting of a TALE-piggyBac and TALE-Mutator.
  • an RNA-guided endonuclease may be selected from the group consisting of Casl , CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, CmiT, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl 7, Csxl 4, Csx lO, Csx16, CsaX, Csx3, Csx l , Csxl 5, Csfl, Csf2, Csf3, Csf4, Cas l2a, Cpfl, Cas
  • a site-specific nuclease provided herein is selected from the group consisting of a zinc-finger nuclease, a meganuclease, an RNA-guided nuclease, a TALE-nuclease, a recombinase, a transposase, or any combination thereof.
  • a site-specific nuclease provided herein is selected from the group consisting of a Cas9 or a Casl2a or Cpfl.
  • a site-specific nuclease provided herein is selected from the group consisting of a Casl, a CaslB, a Cas2, a Cas3, a Cas4, a Cas5, a Cas6, a Cas7, a Cas8, a Cas9, a CaslO, a Casl2a, a Csyl, a Csy2, a Csy3, a Csel, a Cse2, a Cscl, a Csc2, a Csa5, a Csn2, a Csm2, a Csm3, a Csm4, a Csm5, a Csm6, a Cmrl, a Cmr3, a Cmr4, a Cmr5, a Cmr6, a Csbl, a Csb2, a Csb3, a Csxl7, a Csxl4, a CsxlO,
  • an RNA-guided nuclease provided herein is selected from the group consisting of a Cas9 or a Casl 2a or Cpfl.
  • an RNA guided nuclease provided herein is selected from the group consisting of a Casl, a CaslB, a Cas2, a Cas3, a Cas4, a Cas5, a Cas6, a Cas7, a Cas8, a Cas9, a CaslO, a Casl2a, a Csyl, a Csy2, a Csy3, a Csel, a Cse2, a Cscl, a Csc2, a Csa5, a Csn2, a Csm2, a Csm3, a Csm4, a Csm5, a Csm6, a Cmrl, a Cmr3, a Cmr4, a Cmr5, a Cmr6,
  • a method and/or a composition provided herein comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten sitespecific nucleases.
  • a method and/or a composition provided herein comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten polynucleotides encoding at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten site-specific nucleases.
  • a guide RNA (gRNA) molecule is further provided to direct the endonuclease to a target site in the genome of the plant via base-pairing or hybridization to cause a DSB or nick at or near the target site.
  • the gRNA may be transformed or introduced into a plant cell or tissue (perhaps along with a nuclease, or nuclease-encoding DNA molecule, construct or vector) as a gRNA molecule, or as a recombinant DNA molecule, construct or vector comprising a polynucleotide or transcribable DNA sequence encoding the guide RNA operably linked to a plant-expressible promoter.
  • a “guide RNA” may comprise, for example, a CRISPR RNA (crRNA), a single-chain guide RNA (sgRNA), or any other RNA molecule that may guide or direct an endonuclease to a specific target site in the genome.
  • crRNA CRISPR RNA
  • sgRNA single-chain guide RNA
  • a “single-chain guide RNA” is an RNA molecule comprising a crRNA covalently linked a tracrRNA by a linker sequence, which may be expressed as a single RNA transcript or molecule.
  • the guide RNA comprises a guide or targeting sequence that is identical or complementary to a target site within the plant genome, such as within or near cotton event GH_BCS246002 or a modified cotton event GH_BCS246002.
  • a protospacer-adjacent motif may be present in the genome immediately adjacent and upstream or downstream of the genomic target site sequence complementary to the targeting sequence of the guide RNA as known in the art. See, e.g., Wu, X. et al., “Target specificity of the CRISPR-Cas9 system,” Quant Biol. 2(2): 59-70 (2014), the content and disclosure of which is incorporated herein by reference.
  • the guide RNA may typically be a non-coding RNA molecule that does not encode a protein.
  • the guide sequence of the guide RNA may be at least 10 nucleotides in length, such as 12-40 nucleotides, 12-30 nucleotides, 12-20 nucleotides, 12-35 nucleotides, 12-30 nucleotides, 15-30 nucleotides, 17-30 nucleotides, or 17-25 nucleotides in length, or about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more nucleotides in length.
  • the guide sequence may be at least 95%, at least 96%, at least 97%, at least 99% or 100% identical or complementary to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, or more consecutive nucleotides of a DNA sequence at the genomic target site.
  • the term “consecutive” in reference to a polynucleotide or protein sequence means without deletions or gaps in the sequence.
  • a guide RNA may further comprise one or more other structural or scaffold sequence(s), which may bind or interact with an RNA-guided endonuclease.
  • Such scaffold or structural sequences may further interact with other RNA molecules (e.g., tracrRNA).
  • recombinant DNA molecules, constructs and vectors comprising a polynucleotide or transcribable DNA sequence encoding a sitespecific nuclease, such as a zinc-finger nuclease (ZFN), a meganuclease, an RNA-guided endonuclease, a TALE-endonuclease (TALEN), a recombinase, or a transposase, wherein the coding sequence is operably linked to a plant expressible promoter.
  • ZFN zinc-finger nuclease
  • TALEN TALE-endonuclease
  • recombinant DNA molecules, constructs and vectors are further provided comprising a polynucleotide or transcribable DNA sequence encoding a guide RNA, wherein the guide RNA comprises a guide sequence of sufficient length having a percent identity or complementarity to a target site within the genome of a plant.
  • recombinant DNA molecules, constructs and vectors are provided comprising a first polynucleotide or transcribable DNA sequence encoding a site-specific nuclease and a second polynucleotide or transcribable DNA sequence encoding one or more gRNAs.
  • each polynucleotide or transcribable DNA sequence of a recombinant DNA molecule, construct and vector that encodes a site- specific nuclease and/or a guide RNA may be operably linked to a plant expressible promoter, such as an inducible promoter, a constitutive promoter, a tissue-specific promoter, etc.
  • recombinant DNA molecules, constructs and vectors comprising a polynucleotide or transcribable DNA sequence encoding a sitespecific nuclease, such as a zinc-finger nuclease (ZFN), a meganuclease, an RNA-guided endonuclease, a TALE-endonuclease (TALEN), a recombinase, or a transposase, wherein the coding sequence is operably linked to a plant expressible promoter.
  • ZFN zinc-finger nuclease
  • TALEN TALE-endonuclease
  • recombinant DNA molecules, constructs and vectors are further provided comprising a polynucleotide or transcribable DNA sequence encoding a guide RNA, wherein the guide RNA comprises a guide sequence of sufficient length having a percent identity or complementarity to a target site within the genome of a plant.
  • recombinant DNA molecules, constructs and vectors are provided comprising a first polynucleotide or transcribable DNA sequence encoding a site-specific nuclease and a second polynucleotide or transcribable DNA sequence encoding one or more gRNAs.
  • each polynucleotide or transcribable DNA sequence of a recombinant DNA molecule, construct and vector that encodes a site-specific nuclease and/or a guide RNA may be operably linked to a plant expressible promoter, such as an inducible promoter, a constitutive promoter, a tissue-specific promoter, etc.
  • a recombinant DNA molecule, construct or vector may comprise a first polynucleotide sequence encoding a site- specific nuclease and a second polynucleotide sequence encoding a guide RNA(s) that may be introduced into a plant cell together via plant transformation techniques.
  • two recombinant DNA molecules, constructs or vectors may be provided including a first recombinant DNA molecule, construct or vector and a second DNA molecule, construct or vector that may be introduced into a plant cell together or sequentially via plant transformation techniques, wherein the first recombinant DNA molecule, construct or vector comprises a polynucleotide sequence encoding a site-specific nuclease and the second recombinant DNA molecule, construct or vector comprises a polynucleotide sequence encoding a guide RNA(s).
  • a recombinant DNA molecule, construct or vector comprising a polynucleotide sequence encoding a site-specific nuclease may be introduced via plant transformation techniques into a plant cell that already comprises (or is transformed with) a recombinant DNA construct or vector comprising a polynucleotide sequence encoding a guide RNA(s).
  • a recombinant DNA molecule, construct or vector comprising a polynucleotide sequence encoding a guide RNA may be introduced via plant transformation techniques into a plant cell that already comprises (or is transformed with) a recombinant DNA construct or vector comprising a polynucleotide sequence encoding a site-specific nuclease.
  • a first plant comprising (or transformed with) a recombinant DNA construct or vector comprising a polynucleotide sequence encoding a site-specific nuclease may be crossed with a second plant comprising (or transformed with) a recombinant DNA construct or vector comprising a polynucleotide sequence encoding a guide RNA(s).
  • a second plant comprising (or transformed with) a recombinant DNA construct or vector comprising a polynucleotide sequence encoding a guide RNA(s).
  • Such recombinant DNA molecules, constructs or vectors may be transiently transformed into a plant cell or stably transformed or more preferably integrated into the genome of a plant cell.
  • molecules or vectors comprising polynucleotides encoding a sitespecific nuclease, and optionally one or more, two or more, three or more, or four or more gRNAs are provided to a plant cell by transformation methods known in the art (e. ., without being
  • molecules or vectors comprising polynucleotides encoding a Cas9 nuclease, and optionally one or more, two or more, three or more, or four or more gRNAs are provided to a plant cell by transformation methods known in the art (e.g., without being limiting, particle bombardment, PEG-mediated protoplast transfection or Agrobacterium- mediated transformation).
  • vectors comprising polynucleotides encoding a Cpfl and, optionally one or more, two or more, three or more, or four or more crRNAs are provided to a cell by transformation methods known in the art (e.g., without being limiting, viral transfection, particle bombardment, PEG-mediated protoplast transfection or Agrobacterium-mediated transformation).
  • site-specific nucleases such as recombinases, zinc finger nucleases (ZFNs), mcganuclcascs, and TALENs, arc not RNA-guidcd and instead rely on their protein structure to determine their target site for causing the DSB or nick, or they are fused, tethered or attached to a DNA-binding protein domain or motif.
  • the protein structure of the site-specific nuclease (or the fused/attached/tethered DNA binding domain) may target the site-specific nuclease to the target site.
  • non-RNA-guided sitespecific nucleases such as recombinases, zinc finger nucleases (ZFNs), meganucleases, and TALENs, may be designed, engineered and constructed according to known methods to target and bind to a target site in the genome of a plant, to create a DSB or nick at or near such genomic target site or locus.
  • ZFNs zinc finger nucleases
  • TALENs TALENs
  • an engineered site-specific nuclease such as a recombinase, zinc finger nuclease (ZFN), meganuclease, or TALEN, may be designed to target and bind to a genomic target site within the genome of a plant to create a DSB or nick at the genomic target site.
  • ZFN zinc finger nuclease
  • TALEN TALEN
  • a targeted genome editing technique described herein may comprise the use of a zinc finger nuclease (ZFN).
  • ZFNs are synthetic proteins consisting of an engineered zinc finger DNA-binding domain fused to a cleavage domain (or a cleavage half-domain), which may be derived from a restriction endonuclease (e.g., Fold).
  • the DNA binding domain may be canonical (C2H2) or non-canonical (e.g., C3H or C4).
  • the DNA-binding domain can comprise one or more zinc fingers (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or more zinc fingers) depending on the target site. Multiple zinc fingers in a DNA-binding domain may be separated by linker sequence(s).
  • ZFNs can be designed to cleave almost any stretch of double-stranded DNA by modification of the zinc finger DNA-binding domain.
  • ZFNs form dimers from monomers composed of a non-specific DNA cleavage domain (e.g., derived from the FokI nuclease) fused to a DNA-binding domain comprising a zinc finger array engineered to bind a target site DNA sequence.
  • the DNA- binding domain of a ZFN may typically be composed of 3-4 (or more) zinc-fingers.
  • the amino acids at positions -1, +2, +3, and +6 relative to the start of the zinc finger a-helix, which contribute to site- specific binding to the target site, can be changed and customized to fit specific target sequences.
  • the other amino acids may form a consensus backbone to generate ZFNs with different sequence specificities.
  • Methods and rules for designing ZFNs for targeting and binding to specific target sequences are known in the ail. See, e.g., US Patent App. Nos. 2005/0064474, 2009/0117617, and 2012/0142062, the contents and disclosures of which are incorporated herein by reference.
  • the FokI nuclease domain may require dimerization to cleave DNA and therefore two ZFNs with their C-terminal regions are needed to bind opposite DNA strands of the cleavage site (separated by 5-7 bp).
  • the ZFN monomer can cut the target site if the two-ZF-binding sites are palindromic.
  • a ZFN as used herein, is broad and includes a monomeric ZFN that can cleave double stranded DNA without assistance from another ZFN.
  • the term ZFN may also be used to refer to one or both members of a pair of ZFNs that are engineered to work together to cleave DNA at the same site.
  • a method and/or composition provided herein comprises one or more, two or more, three or more, four or more, or five or more ZFNs.
  • a ZFN provided herein is capable of generating a targeted DSB or nick.
  • vectors comprising polynucleotides encoding one or more, two or more, three or more, four or more, or five or more ZFNs are provided to a cell by transformation methods known in the ail (e.g., without being limiting, viral transfection, particle bombardment, PEG-mediated protoplast transfection, or Agrobacterium-mediated transformation).
  • the ZFNs may be introduced as ZFN proteins, as polynucleotides encoding ZFN proteins, and/or as combinations of proteins and protein-encoding polynucleotides.
  • a targeted genome editing technique described herein may comprise the use of a mcganuclcasc.
  • Mcganuclcascs which arc commonly identified in microbes, such as the LAGLIDADG family of homing endonucleases, are unique enzymes with high activity and long recognition sequences (> 14 bp) resulting in site-specific digestion of target DNA.
  • Engineered versions of naturally occurring meganucleases typically have extended DNA recognition sequences (for example, 14 to 40 bp).
  • a meganuclease may comprise a scaffold or base enzyme selected from the group consisting of I- Crel, I-Ceul, I-Msol, I-Scel, I-Anil, and I-Dmol.
  • a meganuclease may be selected or engineered to bind to a genomic target site or sequence in a plant.
  • a method and/or composition provided herein comprises one or more, two or more, three or more, four or more, or five or more meganucleases.
  • a meganuclease provided herein is capable of generating a targeted DSB.
  • vectors comprising polynucleotides encoding one or more, two or more, three or more, four or more, or five or more meganucleases are provided to a cell by transformation methods known in the art (e.g., without being limiting, viral transfection, particle bombardment, PEG-mediated protoplast transfection or Agrobacterium-mediated transformation).
  • a targeted genome editing technique described herein may comprise the use of a transcription activator-like effector nuclease (TALEN).
  • TALENs are artificial restriction enzymes generated by fusing the transcription activator-like effector (TALE) DNA binding domain to a nuclease domain (e.g., FokF).
  • TALE transcription activator-like effector
  • the nuclease is selected from a group consisting of PvuII, MutH, TevI, FokI, Alwl, Mlyl, Sbfl, Sdal, StsI, CleDORF, Clo051, and PeptO7 .
  • FokI nuclease when each member of a TALEN pair binds to the DNA sites flanking a target site, the FokI monomers dimerize and cause a double-stranded DNA break at the target site.
  • FokI cleavage domain variants of the FokI cleavage domain with mutations have been designed to improve cleavage specificity and cleavage activity.
  • the FokI domain functions as a dimer, requiring two constructs with unique DNA binding domains for sites in the target genome with proper orientation and spacing.
  • TALEN Both the number of amino acid residues between the TALEN DNA binding domain and the FokI cleavage domain and the number of bases between the two individual TALEN binding sites arc parameters for achieving high levels of activity.
  • the term TALEN is broad and includes a monomeric TALEN that can cleave double stranded DNA without assistance from another TALEN.
  • the term TALEN also refers to one or both members of a pair of TALENs that work together to cleave DNA at the same site.
  • FokI domains Besides the wild-type FokI cleavage domain, variants of the FokI cleavage domain with mutations have been designed to improve cleavage specificity and cleavage activity.
  • the FokI domain functions as a dimer, requiring two constructs with unique DNA binding domains for sites in the target genome with proper orientation and spacing. Both the number of amino acid residues between the TALEN DNA binding domain and the FokI cleavage domain and the number of bases between the two individual TALEN binding sites arc parameters for achieving high levels of activity.
  • PvuII, MutH, and TevI cleavage domains are useful alternatives to FokI and FokI variants for use with TALEs.
  • PvuII functions as a highly specific cleavage domain when coupled to a TALE (see Yank et al. 2013. PLoS One. 8: e82539). MutH is capable of introducing strand- specific nicks in DNA (see Gabsalilow et al. 2013. Nucleic Acids Research. 41: e83). TevI introduces doublestranded breaks in DNA at targeted sites (see Beurdeley et al., 2013. Nature Communications. 4: 1762).
  • Transcription activator-like effectors can be engineered to bind practically any DNA sequence, such as at or near the genomic locus of cotton event GH_BCS246002 in a plant.
  • TALE has a central DNA-binding domain composed of 13-28 repeat monomers of 33-34 amino acids. The amino acids of each monomer are highly conserved, except for hypervariable amino acid residues at positions 12 and 13. The two variable amino acids are called repeat-variable diresidues (RVDs).
  • the amino acid pairs NI, NG, HD, and NN of RVDs preferentially recognize adenine, thymine, cytosine, and guanine/adenine, respectively, and modulation of RVDs can recognize consecutive DNA bases.
  • This simple relationship between amino acid sequence and DNA recognition has allowed for the engineering of specific DNA binding domains by selecting a combination of repeat segments containing the appropriate RVDs.
  • the relationship between amino acid sequence and DNA recognition of the TALE binding domain allows for designable proteins.
  • Software programs such as DNA Works can be used to design TALE constructs. Other methods of designing TALE constructs are known to those of skill in the art.
  • a method and/or composition provided herein comprises one or more, two or more, three or more, four or more, or five or more TALENs.
  • a TALEN provided herein is capable of generating a targeted DSB.
  • vectors comprising polynucleotides encoding one or more, two or more, three or more, four or more, or five or more TALENs are provided to a cell by transformation methods known in the art (e.g., without being limiting, viral transfection, particle bombardment, PEG-mediated protoplast transfection or Agrobacterium-mediated transformation). See, e.g., US Patent App. Nos. 2011/0145940, 2011/0301073, and 2013/0117869, the contents and disclosures of which are incorporated herein by reference.
  • a targeted genome editing technique described herein may comprise the use of a recombinase.
  • a tyrosine recombinase attached, etc., to a DNA recognition domain or motif may be selected from the group consisting of a Cre recombinase, a Flp recombinase, and a Tnpl recombinase.
  • a Cre recombinase or a Gin recombinase provided herein may be tethered to a zinc-finger DNA binding domain.
  • the Flp-FRT site-directed recombination system may come from the 2p plasmid from the baker’s yeast Saccharomyces cerevisiae. hi this system, Flp recombinase (flippase) may recombine sequences between flippase recognition target (FRT) sites. FRT sites comprise 34 nucleotides. Flp may bind to the “arms” of the FRT sites (one arm is in reverse orientation) and cleaves the FRT site at either end of an intervening nucleic acid sequence. After cleavage, Flp may recombine nucleic acid sequences between two FRT sites.
  • Flp recombinase flippase recognition target
  • Cre-lox is a site-directed recombination system derived from the bacteriophage Pl that is similar to the Flp-FRT recombination system. Cre-lox can be used to invert a nucleic acid sequence, delete a nucleic acid sequence, or translocate a nucleic acid sequence. In this system, Cre recombinase may recombine a pair of lox nucleic acid sequences. Lox sites comprise 34 nucleotides, with the first and last 13 nucleotides (arms) being palindromic. During recombination, Cre recombinase protein binds to two lox sites on different nucleic acids and cleaves at the lox sites.
  • a lox site provided herein is a loxP, lox 2272, loxN, lox 511, lox 5171, lox71, lox66, M2, M3, M7, or Mil site.
  • the term “derived” or “derived from” in reference to a cotton plant product, such as a cotton commodity product, in relation to a cotton cell, cotton tissue, cotton seed, cotton plant, and/or cotton plant part means that the cotton plant product is taken, purified, isolated, or made, directly or indirectly, from such cotton cell, cotton tissue, cotton seed, cotton plant, and/or cotton plant part, as the case may be.
  • “Capable of being detected” refers to the ability of a particular DNA molecule, segment or sequence to be detected in a sample, such as by amplification and determining its presence, size or sequence such as by DNA sequence analysis, and/or binding of a probe to the target DNA molecule, segment or sequence.
  • sample is intended to refer to any composition comprising or derived from, either directly or indirectly, a biological sample, source or material.
  • the sample may generally comprise cotton DNA and/or substantially or completely pure, purified or isolated cotton DNA.
  • a “biological sample” contains biological materials, including but not limited to DNA obtained or derived from, either directly or indirectly, the genome of a cotton cell(s), cotton tissue(s), cotton seed(s), cotton plant(s), cotton plant part(s) and/or cotton plant product(s), such as a cotton commodity product(s).
  • Such cotton cell(s), cotton tissue(s), cotton seed(s), cotton plant(s), cotton plant part(s) and/or cotton plant product(s), such as a cotton commodity product(s), may comprise cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 or DNA molecule(s) and/or DNA segment(s) comprising cotton event GH_BCS246002 or a modified cotton event GH_BCS246002.
  • a sample or biological sample may comprise cotton cell(s), cotton tissue(s), cotton seed(s), cotton plant(s), cotton plant part(s), and/or cotton plant product(s), whose cells or cellular membranes have been fractured (e.g., disrupted or opened) to release the contents of the cotton cell(s) including genomic DNA and/or make the contents of the cotton cell(s) including genomic DNA accessible or usable for assays or testing.
  • “Directly” refers to directly obtaining DNA by a skilled artisan from the cotton genome by fracturing cotton cells (or by obtaining samples of cotton that contain fractured cotton cells) and exposing or using the genomic DNA from cotton cells for the purposes of detection.
  • “Indirectly” refers to obtaining by a skilled artisan a target or specific reference DNA (i.e., a novel and unique junction scgmcnt(s) described herein as being diagnostic for the presence of the cotton event GH_BCS246002 or a modified cotton event GH_BCS246002) in a particular sample, by means other than by obtaining directly via fracturing of cotton cells or obtaining a sample of cotton that contains fractured cotton cells.
  • a target or specific reference DNA i.e., a novel and unique junction scgmcnt(s) described herein as being diagnostic for the presence of the cotton event GH_BCS246002 or a modified cotton event GH_BCS246002
  • Such indirect means include, but are not limited to, amplification of a DNA segment that contains a DNA sequence targeted by a particular probe(s) and/or primer set(s) designed to bind with specificity to or near the target sequence, or amplification of a DNA segment comprising all or part of a target sequence that can be measured and characterized (e.g., measured by migration or separation from other segments of DNA and/or identification in an effective matrix, such as an agarose or acrylamide gel or the like, or characterized by direct sequence analysis of the amplicon(s), or cloning of the amplicon(s) into a vector(s) and direct sequencing of the inserted amplicon(s) present within such vector(s).
  • a transgenic plant is produced by transformation of a plant cell with heterologous DNA, i.e., a polynucleic acid construct that includes a number of efficacious features of interest, regeneration of a plant resulting from the insertion of the transgene into the genome of the plant cell, and selection of a particular plant characterized by insertion into a particular genome location and the number of efficacious features of the regenerated transgenic plant.
  • heterologous DNA i.e., a polynucleic acid construct that includes a number of efficacious features of interest
  • regeneration of a plant resulting from the insertion of the transgene into the genome of the plant cell and selection of a particular plant characterized by insertion into a particular genome location and the number of efficacious features of the regenerated transgenic plant.
  • the term “transgenic event” or “event” refers to the inserted transgenic DNA in the plant genome and flanking genomic sequences immediately adjacent to the inserted transgenic DNA in the genome of the transformed plant, but also refers to
  • Each event is unique and would be expected to be transferred to progeny plants that receive the transgenic DNA and event through genetic inheritance and/or segregation from a parent as the result of a sexual or self-cross of a first parental line that includes the inserted transgenic DNA and event either with itself or a second parental line that may or may not contain the same transgenic DNA and event.
  • the parental line that includes the inserted transgenic DNA and event may itself be the original transformant or a progeny plant of said original transformant that may have been generated by “selfing” the transformant with itself or crossing the transformant or a progeny plant of the transformant that includes the inserted transgenic DNA and event with another plant.
  • the “event” refers to cotton event GH_BCS246002 or a modified cotton event GH_BCS246002.
  • the term “flanking” in reference to a transgenic event refers to the plant genomic sequence(s) immediately adjacent to the transgenic DNA insertion in the genome of a transformed plant, plant part, plant tissue, or plant cell comprising the transgenic event on the 5' and/or 3' sidc(s) or cnd(s) of the transgenic event insertion (i.c., the transgenic insertion of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002).
  • flank DNA means the cotton genomic DNA sequence adjacent to and upstream (or on the 5' end) of the transgenic DNA insertion.
  • a “5" flank” can include the cotton genomic DNA sequence immediately adjacent to and upstream (on the 5' end) of the transgenic insertion, or any cotton genomic DNA sequence upstream (on the 5' end) of the transgenic insertion that is not immediately adjacent to the transgenic insertion but is within about 5000 nucleotides, within about 4000 nucleotides, within about 3000 nucleotides, within about 2000 nucleotides, or within about 1000 nucleotides upstream of the transgenic insertion.
  • a “3" flank” means the cotton genomic DNA sequence adjacent to and downstream (or on the 3' end) of the transgenic insert.
  • a “3" flank” can include the cotton genomic DNA sequence immediately adjacent to and downstream (on the 3' end) of the transgenic insertion, or any cotton genomic DNA sequence downstream (on the 3' end) of the transgenic insertion that is not immediately adjacent to the transgenic insertion but is within about 5000 nucleotides, within about 4000 nucleotides, within about 3000 nucleotides, within about 2000 nucleotides, or within about 1000 nucleotides downstream of the transgenic insertion.
  • SEQ ID NOs: 11 and 12 are 1,000 nucleotide sequences representing cotton Gossypium hirsutum) genomic DNA that flanks the transgenic insert of the 5' and 3' ends of the insert in cotton event GH_BCS246002, respectively.
  • SEQ ID NOs: 43 and 44 are 5,000 nucleotide sequences representing cotton (Gossypium hirsutum) genomic DNA that flanks the transgenic insert of the 5' and 3' ends of the insert in cotton event GH_BCS246002, respectively.
  • Nucleotides 4,001-5,000 of SEQ ID NO: 43 are identical to nucleotides 1-1,000 of SEQ ID NO: 11.
  • Nucleotides 1-4,000 are based on the genomic sequence of the TM-1 cotton cultivar (GenBank: LBLM00000000.1, Gossypium hirsutum cultivar TM-1, whole genome shotgun sequencing). Nucleotides 1-1,000 of SEQ ID NO: 44 are identical to nucleotides 1-1,000 of SEQ ID NO: 12. The remaining nucleotides (1 ,001-5,000) are based on the genomic sequence of the TM-1 cotton cultivar. [00196] The present disclosure provides the original transformant plant and progeny of the transformant that include the transgenic DNA and event.
  • Such progeny may be produced by a sexual cross or outcross between plants comprising the same transgenic DNA and event, or between a plant comprising the transgenic DNA and event with another plant, or by any other method known in the art including any cell or tissue culture method, wherein the progeny includes the transgenic DNA and event.
  • Such other plant may be a transgenic plant comprising the same and/or a different transgene or may be a non-transgenic plant, and each parental plant in a cross or outcross may be the same or different germplasm or breeding line. Even after repeated back- crossing to a recurrent parent, the transgenic DNA and event is present in progeny of the cross at the same chromosomal location.
  • a “transgenic plant” can be the original transformant plant regenerated from the transformed plant cell and comprising the transgenic DNA and event, or a progeny plant of the original transformant plant, which may be separated from the transformant by one or more generations, that retains the transgenic DNA and event at the same specific location and sequence context in the plant’s genome.
  • the transformant or progeny plant may be homozygous or heterozygous for cotton event GH_BCS246002 or a modified cotton event GH_BCS246002.
  • a “transgenic plant” can include a plant produced from a transformed plant cell or tissue, or from another transgenic plant or plant part, by or using cell or tissue culture methods known in the art.
  • a “transgenic plant” may comprise a plant having a transgene or transgenic event stably inserted into the genome of at least one cell of the plant or a modified cotton event GH_BCS246002 (i.e., cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 in at least one cell of the plant), and the plant may be chimeric or nonchimeric with respect to the transgene and/or event or modified event.
  • a transgenic plant is chimeric with respect to a transgene, event or modified event if not all cells of the plant comprise the transgene, event or modified event.
  • the term “recombinant” refers to a non-natural DNA, protein, or combination that would not normally be found in nature, such as a combination of DNA sequences, proteins that would not naturally occur together, and is the result of human intervention.
  • a “recombinant DNA molecule” is a DNA molecule comprising a combination of DNA sequences that would not naturally occur together and is the result of human intervention, such as a combination of DNA segments or sequences that would not naturally occur together in nature in the same relative positions and/or orientation. Two or more elements of such combination of DNA sequences may be operably linked to one another.
  • a recombinant DNA molecule may comprise a combination of at least two DNA sequences that arc heterologous with respect to each other, such as a DNA molecule that comprises a coding or transcribable DNA sequence operably linked to a heterologous promoter and/or other regulatory expression element(s), and/or a plant genomic DNA sequence comprising all or part of a transgene and a heterologous and flanking genomic sequence(s) adjacent to the transgene, and/or a DNA molecule that is artificially synthesized and comprises a polynucleotide sequence that deviates from any polynucleotide sequence that would normally exist in nature.
  • a recombinant DNA molecule may comprise all or part of a junction sequence of the genome of a plant and all or part of the transgene insertion into the genome of the plant, and/or may comprise a recombinant or heterologous DNA fragment of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002.
  • An example of a recombinant DNA molecule is a DNA molecule comprising at least one of SEQ ID NOs: 1-10.
  • a recombinant plant, plant part, plant cell or plant tissue is a plant, plant part, plant cell or plant tissue that would not normally exist in nature, is the result of human intervention, and contains a transgene incorporated into the genome of the plant, plant part, plant cell or plant tissue.
  • the recombinant plant is something new and distinctly different from any related wild-type or naturally occurring plant, plant part, plant cell or plant tissue.
  • An example of a recombinant plant, plant part, plant cell or plant tissue is a cotton plant, plant part, plant cell or plant tissue containing the cotton event GH_BCS246002 or a modified cotton event GH_BCS246002.
  • the term “heterologous” in reference to a combination of two or more DNA sequences or elements means that the two or more DNA sequences or elements do not normally exist together as such combination in nature without human intervention.
  • the term “heterologous” in reference to a DNA molecule, construct or sequence in relation to a plant, microorganism, plant cell or plant genome means that the DNA molecule, construct or sequence does not exist in nature as pail of such plant, microorganism, plant cell or plant genome, and/or does not exist in the same physical or genomic location, context or orientation as part of such plant, microorganism, plant cell or plant genome in nature, without human intervention.
  • DNA molecules and fragments and their corresponding DNA sequences refer to a deoxyribonucleic acid (DNA) molecule.
  • a DNA molecule may be of genomic or synthetic origin and/or comprise a recombinant or heterologous DNA molecule or sequence.
  • a DNA molecule may be described in reference to its 5 " (upstream) end and 3 " (downstream) end.
  • DNA sequence refers to the polynucleotide sequence of the DNA molecule - i.e., the sequence of consecutive nucleotides in the DNA molecule.
  • nucleotides of a polynucleotide or DNA sequence or molecule As used herein in reference to nucleotides of a polynucleotide or DNA sequence or molecule, the terms “consecutive” and “contiguous” are interchangeable and synonymous and refer to the 5' to 3' order of nucleotides in a polynucleotide or DNA sequence, strand or molecule without any gap or interruption between them.
  • DNA sequences of the disclosure and fragments thereof are disclosed with reference to the 5' to 3' direction of only one strand of the two, anti-parallel and complementary DNA strands of a DNA molecule.
  • the complementary sequences of the sequences provided here i.e., the sequences of the complementary, opposing, or antiparallel strand
  • the reverse complementary or reverse complement sequences are within the scope of the present disclosure and are expressly intended to be within the potential scope of the subject matter as claimed.
  • a DNA molecule, or a fragment derived therefrom can also be extracted from plant part(s), plant cell(s) and/or tissue(s) or a homogenate, extract or lysate from plant part(s), plant cell(s) and/or tissue(s), or can be produced as an amplicon from extracted, purified or isolated DNA from plant part(s), plant cell(s) and/or tissue(s), or a homogenate, extract or lysate from plant part(s), plant cell(s) and/or tissue(s), which may further comprise cotton event GH_BCS246002.
  • fragment refers to a smaller piece or sequence of a larger or whole DNA molecule or sequence.
  • a fragment of SEQ ID NO: 9 or 10 may include a sequence that is at least about 12 consecutive nucleotides, at least about 13 consecutive nucleotides, at least about 14 consecutive nucleotides, at least about 15 consecutive nucleotides, at least about 16 consecutive nucleotides, at least about 17 consecutive nucleotides, at least about 18 consecutive nucleotides, at least about 19 consecutive nucleotides, at least about 20 consecutive nucleotides, at least about 21 consecutive nucleotides, at least about 22 consecutive nucleotides, at least about 23 consecutive nucleotides, at least about 24 consecutive nucleotides, at least about 25 consecutive nucleotides, at least about 30 consecutive nucleotides, at least about 35 consecutive nucleotides, at least about 40 consecutive nucleotides, at least about 45 consecutive nucleotides
  • a fragment of the DNA sequence of the 5 ' flank (SEQ ID NO: 11 or SEQ ID NO: 43) or the 3' flank (SEQ ID NO: 12 or SEQ ID NO: 44) of cotton event GH_BCS246002 can comprise at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, or at least 500 consecutive nucleotides of SEQ ID NO: 11 or SEQ ID NO: 43 or SEQ ID NO: 12 or SEQ ID NO: 44.
  • different cotton germplasms may have differences in their genomic sequences, which may include differences in the flanking sequence(s), 5 ' flank and/or 3' flank of cotton event GH_BCS246002. These differences may result from introgression of the cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 into a different germplasm and/or spontaneous, mutagenic or genome editing changes that occur in a given germplasm or line.
  • DNA molecules, constructs or polynucleotides comprising a sequence or flanking sequence, or a 5' flank or 3' flank, that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% identical to SEQ ID NO: 11 or 43 or SEQ ID NO: 12 or 44, or a fragment of either thereof.
  • DNA molecules, constructs or polynucleotides comprising a sequence or flanking sequence, or a 5 ' flank or 3' flank, that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% identical to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400
  • the term “isolated” in reference to a molecule means that the molecule is at least partially separated from other molecules or sequences that are normally associated with the molecule in its native or natural state.
  • the term “isolated” refers to a DNA molecule that is at least partially separated from the nucleic acids or polynucleotide or DNA sequence(s) that normally flank and are covalently linked to the sequence of the DNA molecule in its native or natural state.
  • An “isolated” DNA molecule may have a DNA sequence corresponding to a portion of the genome of a plant cell without other genomic DNA sequence(s) that normally flank and are covalently linked to the DNA sequence in nature.
  • Such an “isolated” DNA molecule may comprise all or part of a transgene and/or transgenic event, which may comprise all or part of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 or the transgene or expression cassette described herein.
  • Nucleic acid sequences or elements such as a coding sequence, intron sequence, untranslated leader sequence, promoter sequence, transcriptional termination sequence, and the like, that are naturally found within the DNA of the genome of an organism are not considered to be “isolated” so long as the element is within the genome of the organism and at the location within the genome in which it is naturally found.
  • an “isolated” DNA molecule may be any recombinant DNA molecule or amplification product or amplicon, and/or may comprise any DNA sequence removed from its natural or biological state and covalently fused to another DNA molecule or sequence with which it is not associated in nature.
  • Such an isolated DNA molecule could be created by the use of biotechnology techniques, such as by making a recombinant DNA or integrating a foreign or heterologous DNA molecule into the chromosome of a cell, plant, or seed.
  • any DNA molecule comprising a transgenic, recombinant, chimeric or artificial nucleotide sequence, transgene or expression cassette would be considered to be an “isolated” DNA molecule since these sequences are not naturally occurring, regardless of whether the sequence, transgene or expression cassette is present within a plasmid, vector or construct used to transform plant cells, within the genome of a plant, plant part, plant tissue, or plant cell, or is present in detectable amounts in tissues, progeny, biological samples or commodity products derived from a plant, plant part, plant tissue, or plant cell.
  • a recombinant DNA molecule or sequence, or any fragment derived therefrom, comprising all or part of a transgene or junction sequence of cotton event would therefore also be considered to be “isolated.”
  • An “isolated” DNA molecule may be extracted or purified from a transgenic plant(s), plant part(s), plant cell(s) and/or tissue(s), or may be present in a homogenate, extract or lysate from any such transgenic plant(s), plant part(s), plant cell(s) and/or tissue(s), or may be produced as an amplicon or amplification product from plant genomic DNA and/or extracted or purified DNA from transgenic plant(s), plant part(s), plant cell(s) and/or tissue(s), or a homogenate, extract or lysate from plant(s), plant part(s), plant cell(s) and/or tissue(s).
  • any transgenic polynucleotide or DNA sequence i.e., the nucleotide sequence of the DNA inserted into the genome of a plant or bacterium, or present in an extrachromosomal vector, would be considered to be an “isolated” nucleotide or DNA sequence whether it is present within the plasmid or similar structure used to transform the cells, within the genome of the plant or bacterium, or present in detectable amounts in tissues, progeny, biological samples or commodity products derived from the plant or bacterium.
  • An “isolated” DNA molecule is a chemical or biochemical molecule, regardless of whether the molecule is referred to as a nucleic acid, a nucleic acid sequence, a polynucleotide sequence, a DNA sequence, a nucleic acid molecule, a polynucleotide molecule, a DNA molecule, or the like.
  • An “isolated” molecule can provide industrial applicability when present in a plant cell or in a plant genome or when present outside of a plant cell, and therefore, provides and exhibits (and is intended to provide and exhibit) utility regardless of where the molecule is located.
  • junction The phosphodiester bond linkage between one end of a transgenic insert (or insertion) into the genome of a plant and the flanking cotton genomic DNA is referred to as a “junction.”
  • a “junction’ is the connection point or covalent linkage of one end of a transgenic insert and the flanking genomic DNA.
  • One junction is found at the 5' end of the transgenic insertion and the other is found at the 3 " end of the transgenic insert, referred to herein as the 5' and 3' junctions, respectively.
  • a “junction sequence” refers to a DNA sequence of any length of consecutive nucleotides that spans the 5 ' or 3' junction of a transgenic event in the plant genome.
  • junction sequence to be specific to a junction between a transgenic event and a flanking genomic sequence, the junction sequence will generally comprise a sufficient number of consecutive nucleotides at one end of the insertion and a sufficient number of consecutive nucleotides of the flanking genomic sequence.
  • a “junction sequence” may comprise (i) at least five (5) consecutive nucleotides, at least ten (10) consecutive nucleotides, at least fifteen (15) consecutive nucleotides, at least twenty (20) consecutive nucleotides, or at least thirty (30) consecutive nucleotides at one end of the insertion and (ii) at least five (5) consecutive nucleotides, at least ten (10) consecutive nucleotides, at least fifteen (15) consecutive nucleotides, at least twenty (20) consecutive nucleotides, or at least thirty (30) consecutive nucleotides of flanking genomic DNA sequence, although it is understood that any length of consecutive nucleotides spanning a junction of a transgenic event in a plant genome may be a junction sequence.
  • junction sequences of cotton event GH_BCS246002 can be determined by one of skill in the art using SEQ ID NO: 10. Examples of junction sequences of cotton event GH_BCS246002 are provided as SEQ ID NOs: 1-8.
  • FIG. 1 illustrates the physical arrangement and locations of the junction sequences, arranged from 5 Go 3' (left to right), relative to SEQ ID NO: 10.
  • the junction sequence(s) of a modified cotton event GH_BCS246002 may be modified, mutated or edited relative to such junction sequence(s) of cotton event GH_BCS246002.
  • junction sequences of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 may be present as part of the genome of a cotton plant, plant part, plant seed, or plant tissue or cell containing cotton event GH_BCS246002 or a modified cotton event GH_BCS246002, a DNA molecule containing all or part of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002, or a microorganism containing cotton event GH_BCS246002 or a modified cotton event GH_BCS246002.
  • any one or more of the junction sequences in a DNA molecule or sample from a plant, plant part, plant seed, or plant tissue or cell indicates that the plant, plant part, plant seed, or plant tissue or cell contains or comprises cotton event GH_BCS246002 or a modified cotton event GH_BCS246002, or the DNA molecule contains or comprises cotton event GH_BCS246002 or a modified cotton event GH BCS246002 or was obtained from a cotton plant, plant part, plant seed, or plant tissue or cell containing or comprising cotton event GH_BCS246002 or a modified cotton event GH_BCS246002, and is diagnostic in each case for the presence of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002.
  • junction sequences described herein are diagnostic for the presence of all or part of cotton event GH_BCS246002, and if unmodified may be diagnostic for the presence of all or part of or a modified cotton event GH_BCS246002, and/or a DNA molecule comprising all, or part of the construct or expression cassettes described herein.
  • the identification or detection, directly or indirectly, of a 5' junction sequence and a 3' junction sequence (each as provided or described herein) in a sample or DNA molecule derived from a cotton plant, cotton plant part, cotton seed, or cotton tissue or cell, or a commodity product from a cotton plant, cotton plant part, cotton seed, or cotton tissue or cell, is diagnostic that the cotton plant, cotton plant part, cotton seed, or cotton tissue or cell, or a commodity product from a cotton plant, cotton plant part, cotton seed, or cotton tissue or cell has or comprises cotton event GH_BCS246002 or a modified cotton event GH_BCS246002.
  • the present disclosure thus provides a DNA molecule that contains at least one of the nucleotide sequences provided as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10.
  • Any segment of DNA derived from transgenic cotton event GH_BCS246002 that is sufficient to include at least one of the sequences provided as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10 is within the scope of the present disclosure.
  • any DNA or polynucleotide molecule or sequence comprising a sequence complementary to any of the sequences described herein is also within the scope of the present disclosure.
  • the disclosure provides DNA, polynucleotide or nucleic acid molecules, which may be single or double stranded, that can be used either as primers or probes for detecting the presence of DNA comprising all or part of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 in a sample derived from a cotton plant, cotton plant part, cotton seed, or cotton tissue or cell, or a commodity product from a cotton plant, cotton plant part, cotton seed, or cotton tissue or cell.
  • primers or probes are specific for a target nucleic acid, polynucleotide or DNA sequence and, as such, arc useful for the identification of cotton event GH_BCS246002 nucleic acid, polynucleotide or DNA sequence, or a nucleic acid, polynucleotide or DNA sequence of a modified cotton event GH_BCS246002, by the methods described herein.
  • a primer or probe can hybridize to a target nucleic acid, polynucleotide or DNA sequence to allow for specific detection or amplification of a nucleic acid, polynucleotide or DNA molecule or sequence that comprises, or is covalently linked and associated with, the target nucleic acid, polynucleotide or DNA sequence.
  • the primers and/or probe may be chosen to identify and distinguish detection of a particular transgenic event and not only the presence of a transgene in a plant genome.
  • the target nucleic acid, polynucleotide or DNA molecule or sequence may comprise all or part of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002, a junction sequence and/or flanking genomic DNA.
  • Probes and primers according to the present disclosure may have (i) complete or 100% sequence complementarity (i.e., 100% complementary) to a target DNA sequence or (ii) incomplete sequence complementarity to a target DNA sequence, such as at least 60% complementary, at least 65% complementary, at least 70% complementary, at least 75% complementary, at least 80% complementary, at least 85% complementary, at least 90% complementary, at least 95% complementary, or at least 99% complementary to the target DNA sequence as long as the probe or primer has sufficient complementarity to the target DNA sequence to hybridize to the target DNA sequence under stringent hybridization conditions that are suitable and necessary for use of the probe or primer in the relevant amplification or detection assay, reaction or method.
  • the percentage complementarity of a primer or probe may be lower if the length of the primer or probe is longer and depends on the stringency and use.
  • a “probe” is a nucleic acid molecule that is complementary to a strand of target nucleic acid and is useful in hybridization methods.
  • a probe may be attached a conventional detectable label or reporter molecule, e.g., a radioactive isotope, ligand, chemiluminescent agent, or enzyme.
  • Such a probe is complementary to a strand of a target nucleic acid and, in the case of the present disclosure, to a strand of DNA from cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 whether from an cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 containing plant or from a sample that includes cotton event GH_BCS246002 DNA or DNA from a modified cotton event GH_BCS246002.
  • Probes according to the present disclosure include not only deoxyribonucleic or ribonucleic acids, but also polyamides and other probe materials that bind specifically to a target DNA sequence and can be used to detect the presence of that target DNA sequence.
  • Exemplary DNA sequences useful as a probe for detecting cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 arc provided as: SEQ ID NO: 17 (PB5O3O8), SEQ ID NO: 20 (PB50562), and SEQ ID NO: 22 (WTDP393PR).
  • a “probe” may also be used to bind a template DNA in a sample comprising all or part of a DNA or nucleotide sequence of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 to purify the template DNA from the remainder of the sample using purification methods or techniques known in the art, for example, if the probe is bound or can be bound to a substrate or a particle or bead that can be purified or separated.
  • Such a template DNA may comprise all or part of a DNA or nucleotide sequence of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002, or a portion or fragment thereof, such as a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, or a complement thereof.
  • a “primer” is typically a DNA molecule that is designed for use in specific annealing or hybridization methods that involve thermal amplification.
  • a pair of primers may be used with template DNA (such as a sample of cotton genomic DNA) in a thermal amplification (such as polymerase chain reaction (PCR)) to produce an amplicon, where the amplicon produced from such reaction would have a DNA sequence corresponding to sequence of the template DNA located between the two sites where the primers hybridized to the template.
  • amplification product” or “amplicon” is a DNA molecule or segment produced by an amplification reaction. Amplification or amplifying refers to making multiple copies of a target DNA molecule or segment from a template DNA.
  • a single “primer” may also be used to initiate a sequencing reaction to determine a DNA sequence of a template DNA according to sequencing methods known in the art. Such a sequencing reaction may be used to determine the presence or absence of a DNA molecule or nucleotide sequence, or a portion or fragment thereof, from cotton event GH_BCS246002 or a modified cotton event GH_BCS246002.
  • Such a template DNA may comprise all or part of a DNA or nucleotide sequence of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002, or a portion or fragment thereof, such as a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, or a complement thereof.
  • DNA amplification reactions, methods and techniques are known to those skilled in art.
  • DNA amplification can be accomplished by any of the various nucleic acid amplification methods known in the art, including thermal and isothermal amplification methods including the polymerase chain reaction or PCR. Amplification methods are known in the art and are described, inter alia, in U.S. Patent Nos. 4,683, 195 and 4,683,202 and in PCR Protocols: A Guide to Methods and Applications, ed. Innis et al., Academic Press, San Diego, 1990. PCR amplification methods have been developed to amplify up to 22 kb (kilobase) of genomic DNA and up to 42 kb of bacteriophage DNA (Cheng et al., Proc. Natl. Acad. Sci.
  • DNA amplification methods include PCR, Recombinase Polymerase Amplification (RPA) (see for example U.S. Pat No. 7,485,428), Strand Displacement Amplification (SDA) (see for example, U.S. Pat. Nos. 5,455,166 and 5,470,723), Transcription-Mediated Amplification (TMA) (see for example, Guatelli et al., Proc. Natl. Acad. Sci. USA 87:1874-1878, 1990), Rolling Circle Amplification (RCA) (see for example, Fire and Xu, Proc. Natl.
  • RPA Recombinase Polymerase Amplification
  • SDA Strand Displacement Amplification
  • TMA Transcription-Mediated Amplification
  • RCA Rolling Circle Amplification
  • a sequence of the heterologous DNA insert and/or flanking genomic DNA sequence from cotton event GH_BCS246002 can be verified or tested by amplifying such DNA molecules from cotton seed containing cotton event GH_BCS246002 DNA or cotton plants grown from the cotton seed containing cotton event GH_BCS246002 DNA, using primers derived from the sequences provided herein, followed by standard DNA sequencing of the PCR amplicon or a cloned DNA fragment thereof.
  • the sequence of an amplicon of an amplification reaction may comprise one or more of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, or a fragment thereof.
  • the sequence of an amplicon comprises at least one junction sequence or two junction sequences, such as a 5' junction sequence and/or a 3' junction sequence for cotton event GH_BCS246002 or a modified cotton event GH_BCS246002.
  • a primer is typically designed to hybridize in a sequence- specific manner to a complementary target DNA strand to form a hybrid between the primer and target DNA strand, and the primer hybridized or bound to the complementary target DNA strand is a point of recognition for a polymerase to begin extension of the primer (i.e., polymerization of additional nucleotides into a lengthening nucleotide molecule) using as a template the target DNA strand.
  • Primer pairs refer to use of two primers binding opposite strands of a double stranded DNA or polynucleotide segment for the purpose of amplifying the polynucleotide or DNA segment between the positions targeted for binding by the individual primers of the primer pair to the original template DNA or an amplicon of the amplification reaction, typically in a thermal cycling amplification reaction or other conventional DNA amplification method.
  • Primer pairs arc typically designed to hybridize to different nearby target positions of a template DNA molecule on opposing strands of the template DNA molecule such that the intervening region or sequence between the two primers can be specifically amplified for use or detection through multiple rounds of amplification.
  • Exemplary DNA molecules useful as primers are provided as SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 21.
  • primer pair SEQ ID NO: 15 and SEQ ID NO: 16 are useful as a first DNA molecule and a second DNA molecule that is different from the first DNA molecule, and both are each of sufficient length of contiguous nucleotides of SEQ ID NO: 10 to function as DNA primers that, when used together in a thermal amplification reaction with template DNA derived from cotton event GH_BCS246002, to produce an amplicon diagnostic for, or characteristic of, cotton event GH_BCS246002 in a sample.
  • the primer pair SEQ ID NO: 18 and SEQ ID NO: 19 are useful as a first DNA molecule and a second DNA molecule that is different from the first DNA molecule, and both are each of sufficient length of contiguous nucleotides of a locus within the cotton genome to function as DNA primers that, when used together in a thermal amplification reaction with template DNA derived cotton event GH_BCS246002, to produce an amplicon that serves as an internal control for both the diagnosis of cotton event GH_BCS246002, as well as the zygosity of cotton event GH_BCS246002 DNA in a sample.
  • the primer pair SEQ ID NO: 20 and SEQ ID NO: 15 are useful as a first DNA molecule and a second DNA molecule that is different from the first DNA molecule, and both are each of sufficient length of contiguous nucleotides of a locus within the cotton genome to function as DNA primers that, when used together in a thermal amplification reaction with template DNA derived from cotton event GH_BCS246002, to produce an amplicon diagnostic for, or characteristic of, non-inserted wildtype cotton genomic DNA not comprising cotton event GH_BCS246002.
  • a DNA molecule, construct, segment, amplicon, fragment or polynucleotide contains or comprises at least one of the nucleotide sequences provided as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10.
  • a DNA molecule, construct, segment, amplicon, fragment or polynucleotide contains or comprises at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, or at least 90 consecutive nucleotides of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.
  • a DNA molecule, construct, segment, amplicon, fragment or polynucleotide contains or comprises at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1,000, at least 1,500, or at least 2,000 consecutive nucleotides of any one of SEQ ID NOs: 1-10, (ii) is at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at
  • a DNA molecule, construct, segment, amplicon, fragment or polynucleotide contains or comprises (i) three expression cassettes that encode the insect pesticidal insect toxins CrylB.3, CrylDa_7, and Vip3Cbl.l, and (ii) a junction sequence.
  • a DNA molecule, construct, segment, amplicon, fragment or polynucleotide contains or comprises (i) expression cassettes that encodes the pesticidal insect toxins CrylB.3, CrylDa_7, and Vip3Cbl.l, and (ii) a 5' flank sequence and/or a 3' flank sequence.
  • the expression cassette may comprise in operable linkage: (a) a promoter sequence (b) a transcribable DNA sequence encoding a pesticidal insect toxins CrylB.3, CrylDa_7, and Vip3Cbl.l sequence that is toxic to Lepidopteran insect pest species, and (c) a transcription termination or 3' UTR sequence.
  • the expression cassette may further comprise any of the elements described in Table 1, which may be operably linked.
  • a modified cotton event GH_BCS246002 may contain or comprise one or more expression cassette(s) that encode one or more of the insect pesticidal insect toxins CrylB.3, CrylDa_7, and/or Vip3Cbl.l (depending on the modification of cotton event GH_BCS246002).
  • a DNA molecule, construct, segment, amplicon, fragment or polynucleotide may not comprise all three expression cassettes that encode the insect pesticidal insect toxins Cry IB.3, CrylDa_7, and Vip3Cbl.l if comprising only a portion of cotton event GH_BCS246002.
  • a DNA molecule, construct, segment, amplicon, fragment or polynucleotide from a modified cotton event GH_BCS246002 contains or comprises (i) one or more expression cassette(s) that encode the insect pesticidal insect toxins CrylB.3, CrylDa_7, and/or Vip3Cbl.l, and (ii) a junction sequence, such as a 5' flank sequence and/or a 3' flank sequence.
  • a DNA molecule, construct, segment, amplicon, fragment or polynucleotide contains or comprises (i) one or more of the expression cassettes that encode Cry IB.3, CrylDa_7, and/or Vip3Cbl.l, and (ii) a polynucleotide sequence comprising one or more of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10.
  • a DNA molecule, construct, segment, amplicon, fragment or polynucleotide contains or comprises (i) one or more of the expression cassettes that encode the pesticidal insect toxins CrylB.3, CrylDa_7, and/or Vip3Cbl.l, and (ii) a polynucleotide sequence comprising at least 10, at least 11 , at least 12, at least 13, at least 1 , at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1,000, at least 1,500, or at least 2,000 consecutive nucleotides of one or more of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3,
  • a DNA molecule, construct, segment, amplicon, fragment or polynucleotide contains or comprises (i) one or more of the expression cassettes that encode the pesticidal insect toxins Cry IB.3, CrylDa_7, and/or Vip3Cbl.l, and (ii) a polynucleotide sequence comprising at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1,000, at least 1,500, or at least 2,000 consecutive nucleotides of SEQ ID NO: 11 or SEQ ID NO: 12 or SEQ ID NO: 43
  • a DNA molecule, construct, segment, amplicon, fragment or polynucleotide contains or comprises (i) one or more of the expression cassettes that encode the pesticidal insect toxins Cry IB.3, CrylDa_7, and/or Vip3Cbl.l, and (ii) a sequence or flanking sequence, or a 5' flank or 3' flank, that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% identical to SEQ ID NO: 11 or 43 or SEQ ID NO: 12 or 44, or a fragment of either thereof.
  • a DNA molecule, construct, segment, amplicon, fragment or polynucleotide contains or comprises (i) one or more of the expression cassettes that encode the pesticidal insect toxins CrylB.3, CrylDa_7, and/or Vip3Cb 1.1, and (ii) a sequence or flanking sequence, or a 5 ' flank or 3 ' flank, that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% identical to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21 , at least 22, at least 23,
  • a DNA molecule, construct, segment, amplicon, fragment or polynucleotide contains or comprises (i) one or more of the expression cassettes that encode the pesticidal insect toxins CrylB.3, CrylDa_7, and/or Vip3Cbl.l, and (ii) a polynucleotide sequence comprising one or more polynucleotide sequences selected from SEQ ID NOs: 45-144.
  • a DNA molecule, construct, segment, amplicon, fragment or polynucleotide contains or comprises (i) one or more of the expression cassettes that encode the pesticidal insect toxins Cry IB.3, CrylDa_7, and/or Vip3Cbl.l, and (ii) a polynucleotide sequence comprising one or more polynucleotide sequences selected from SEQ ID NOs: 145-244.
  • a DNA molecule, construct, segment, amplicon, fragment or polynucleotide contains or comprises (i) one or more of the expression cassettes that encode the pesticidal insect toxins Cry IB.3, CrylDa_7, and/or Vip3Cbl.
  • a polynucleotide sequence comprising at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1,000, at least 1,500, or at least 2,000 consecutive nucleotides of SEQ ID NO: 11 or SEQ ID NO: 43, and (iii) a polynucleotide sequence comprising at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at
  • a DNA molecule, construct, segment, amplicon, fragment or polynucleotide contains or comprises (i) one or more of the expression cassettes that encode the pesticidal insect toxins Cry I B.3, CrylDa_7, and/or Vip3Cbl .l , (ii) a sequence or flanking sequence, or a 5 ' flank, that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1 %, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% identical to SEQ ID NO: 11 or 43, or a fragment of either thereof, and (iii) a sequence or flanking sequence, or a 3' flank, that is at least 90%,
  • a DNA molecule, construct, segment, amplicon, fragment or polynucleotide contains or comprises (i) one or more of the expression cassettes that encode the pesticidal insect toxins Cry IB.3, CrylDa_7, and/or Vip3Cbl.l, and (ii) a sequence or flanking sequence, or a 5' flank, that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% identical to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25,
  • a DNA molecule, construct, segment, amplicon, fragment or polynucleotide contains or comprises (i) one or more of the expression cassettes that encode the pesticidal insect toxins Cry IB.3, CrylDa_7, and/or Vip3Cbl.l, (ii) a polynucleotide sequence comprising one or more polynucleotide sequences selected from SEQ ID NOs: 45-144, and (iii) a polynucleotide sequence comprising one or more polynucleotide sequences selected from SEQ ID NOs: 145-244.
  • a DNA molecule, construct, segment, amplicon, fragment or polynucleotide contains or comprises one or more of the expression cassettes that encode the pesticidal insect toxins Cry IB.3, CrylDa_7, and/or Vip3Cbl.l, such as all three of the expression cassettes that encode the pesticidal insect toxins CrylB.3, CrylDa_7, and/or Vip3Cbl.l, wherein the DNA molecule, construct, segment, amplicon, fragment or polynucleotide comprises at its 5' end at least 50 consecutive nucleotides of SEQ ID NO: 11 or 43.
  • a DNA molecule, construct, segment, amplicon, fragment or polynucleotide contains or comprises one or more of the expression cassettes that encode the pesticidal insect toxins CrylB.3, CrylDa_7, and/or Vip3Cb 1.1, wherein the DNA molecule, construct, segment, amplicon, fragment or polynucleotide comprises at its 3 ' end at least 50 consecutive nucleotides of SEQ ID NO: 12 or 44.
  • a DNA molecule, construct, segment, amplicon, fragment or polynucleotide contains or comprises one or more of the expression cassettes that encode the pesticidal insect toxins CrylB.3, CrylDa_7, and/or Vip3Cbl.l, wherein the DNA molecule, construct, segment, amplicon, fragment or polynucleotide comprises at its 5 " end a polynucleotide sequence selected from SEQ ID NOs: 45-144.
  • a DNA molecule, construct, segment, amplicon, fragment or polynucleotide contains or comprises one or more of the expression cassettes that encode the pesticidal insect toxins Cry IB.3, CrylDa_7, and/or Vip3Cbl.l, wherein the DNA molecule, construct, segment, amplicon, fragment or polynucleotide comprises at its 3 " end a polynucleotide sequence selected from SEQ ID NOs: 145-244.
  • a DNA molecule, construct, segment, amplicon, fragment or polynucleotide contains or comprises one or more of the expression cassettes that encode the pesticidal insect toxins Cry IB.3, CrylDa_7, and/or Vip3Cbl.l, such as all three of the expression cassettes that encode the pesticidal insect toxins CrylB.3, CrylDa_7, and/or Vip3Cbl.l, wherein the DNA molecule, construct, segment, amplicon, fragment or polynucleotide comprises at its 5' end at least 50 consecutive nucleotides of SEQ ID NO: 11 or 43, wherein the DNA molecule, construct, segment, amplicon, fragment or polynucleotide comprises at its 3' end at least 50 consecutive nucleotides of SEQ ID NO: 12 or 44, wherein the DNA molecule, construct, segment, amplicon, fragment or polynucleotide comprises at its 5' end
  • a DNA molecule, construct, segment, amplicon, fragment or polynucleotide can comprise at the 5 " and/or 3 " end of the construct (i) at least 50 consecutive nucleotides of SEQ ID NO: 11 or SEQ ID NO: 43; and/or (ii) at least 50 consecutive nucleotides of SEQ ID NO: 12 or SEQ ID NO: 44, respectively.
  • a DNA molecule, construct, segment, amplicon, fragment or polynucleotide contains or comprises (i) SEQ ID NO: 9 or a polynucleotide sequence comprising at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1,000, at least 1,500, or at least 2,000 consecutive nucleotides of SEQ ID NO: 9, and (ii) a junction sequence.
  • a DNA molecule, construct, segment, amplicon, fragment or polynucleotide contains or comprises (i) SEQ ID NO: 9 or a polynucleotide sequence comprising at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1,000, at least 1,500, or at least 2,000 consecutive nucleotides of SEQ ID NO: 9, and (ii) a 5' flank and/or a 3 ' flank sequence.
  • a DNA molecule, construct, segment, amplicon, fragment or polynucleotide contains or comprises (i) SEQ ID NO: 9 or a polynucleotide sequence comprising at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1,000, at least 1,500, or at least 2,000 consecutive nucleotides of SEQ ID NO: 9, and (ii) a polynucleotide sequence comprising at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least
  • a DNA molecule, construct, segment, amplicon, fragment or polynucleotide contains or comprises (i) SEQ ID NO: 9 or a polynucleotide sequence comprising at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21 , at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1,000, at least 1,500, or at least 2,000 consecutive nucleotides of SEQ ID NO: 9, and (ii) a sequence or flanking sequence, or a 5' flank or 3' flank, that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 9
  • a DNA molecule, construct, segment, amplicon, fragment or polynucleotide contains or comprises (i) SEQ ID NO: 9 or a polynucleotide sequence comprising at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1,000, at least 1,500, or at least 2,000 consecutive nucleotides of SEQ ID NO: 9, and (ii) a sequence or flanking sequence, or a 5' flank or 3' flank, that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at
  • a DNA molecule, construct, segment, amplicon, fragment or polynucleotide contains or comprises (i) SEQ ID NO: 9 or a polynucleotide sequence comprising at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1 ,000, at least 1 ,500, or at least 2,000 consecutive nucleotides of SEQ TD NO: 9, and (ii) a polynucleotide sequence comprising one or more polynucleotide sequences selected from SEQ ID NOs: 45-144.
  • a DNA molecule, construct, segment, amplicon, fragment or polynucleotide contains or comprises (i) SEQ ID NO: 9 or a polynucleotide sequence comprising at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1,000, at least 1,500, or at least 2,000 consecutive nucleotides of SEQ ID NO: 9, and (ii) a polynucleotide sequence comprising one or more polynucleotide sequences selected from SEQ ID NOs: 145-244.
  • a DNA molecule, construct, segment, amplicon, fragment or polynucleotide contains or comprises (i) SEQ ID NO: 9 or a polynucleotide sequence comprising at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1,000, at least 1,500, or at least 2,000 consecutive nucleotides of SEQ ID NO: 9, (ii) a polynucleotide sequence comprising at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at
  • a DNA molecule, construct, segment, amplicon, fragment or polynucleotide contains or comprises (i) SEQ ID NO: 9 or a polynucleotide sequence comprising at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1,000, at least 1,500, or at least 2,000 consecutive nucleotides of SEQ ID NO: 9, (ii) a sequence or flanking sequence, or a 5' flank, that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
  • a DNA molecule, construct, segment, amplicon, fragment or polynucleotide contains or comprises (i) SEQ ID NO: 9 or a polynucleotide sequence comprising at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1,000, at least 1,500, or at least 2,000 consecutive nucleotides of SEQ ID NO: 9, and (ii) a sequence or flanking sequence, or a 5' flank, that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%
  • a DNA molecule, construct, segment, amplicon, fragment or polynucleotide contains or comprises (i) SEQ ID NO: 9 or a polynucleotide sequence comprising at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1,000, at least 1,500, or at least 2,000 consecutive nucleotides of SEQ ID NO: 9, (ii) a polynucleotide sequence comprising one or more polynucleotide sequences selected from SEQ ID NOs: 45-148, and (iii) a polynucleotide sequence
  • a DNA molecule, construct, segment, amplicon, fragment or polynucleotide contains or comprises SEQ ID NO: 9 or a polynucleotide sequence comprising at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1,000, at least 1,500, or at least 2,000 consecutive nucleotides of SEQ ID NO: 9, wherein the DNA molecule, construct, segment, amplicon, fragment or polynucleotide comprises at its 5" end at least 50 consecutive nucleotides of SEQ ID NO: 11 or 43.
  • a DNA molecule, construct, segment, amplicon, fragment or polynucleotide contains or comprises SEQ ID NO: 9 or a polynucleotide sequence comprising at least 10, at least 1 1 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1,000, at least 1,500, or at least 2,000 consecutive nucleotides of SEQ ID NO: 9, wherein the DNA molecule, construct, segment, amplicon, fragment or polynucleotide comprises at its 3' end at least 50 consecutive nucleotides of SEQ ID NO: 12 or 44.
  • a DNA molecule, construct, segment, amplicon, fragment or polynucleotide contains or comprises one or more of the expression cassettes that encode the pesticidal insect toxins Cry IB.3, CrylDa_7, and/or Vip3Cbl.l, wherein the DNA molecule, construct, segment, amplicon, fragment or polynucleotide comprises at its 5 " end a polynucleotide sequence selected from SEQ ID NOs: 45-148.
  • a DNA molecule, construct, segment, amplicon, fragment or polynucleotide contains or comprises SEQ ID NO: 9 or a polynucleotide sequence comprising at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1,000, at least 1,500, or at least 2,000 consecutive nucleotides of SEQ ID NO: 9, wherein the DNA molecule, construct, segment, amplicon, fragment or polynucleotide comprises at its 3' end a polynucleotide sequence selected from SEQ ID NOs: 145-244.
  • a DNA molecule, construct, segment, amplicon, fragment or polynucleotide contains or comprises SEQ ID NO: 9 or a polynucleotide sequence comprising at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1,000, at least 1,500, or at least 2,000 consecutive nucleotides of SEQ ID NO: 9, wherein the DNA molecule, construct, segment, amplicon, fragment or polynucleotide comprises at its 5' end at least 50 consecutive nucleotides of SEQ ID NO: 11 or 43, wherein the DNA molecule, construct,
  • the target positions and/or the intervening region or sequence of a template DNA molecule may comprise at least one junction sequence and/or at least a portion of the insert of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002.
  • the target positions and/or the intervening region or sequence of a template DNA molecule may comprise cotton genomic DNA that does not include a junction sequence or any portion of the insert of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002.
  • the presence or absence of an amplicon with a primer pair may be diagnostic of the presence or absence, respectively, of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 in a DNA molecule or sample, or vice versa.
  • This may also be possible with more than one primer pair.
  • a first primer pair may produce a first amplicon if cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 is present
  • a second primer pair may produce a second amplicon if cotton event is absent or not present.
  • the size of an amplicon produced in an amplification reaction may also be diagnostic of the presence or absence of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 in a DNA molecule or sample - e.g., a primer pair may produce a first amplicon of a first size if cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 is present or a second amplicon of a second size if cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 is absent and not present; or a first primer pair may produce a first amplicon of a first size if cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 is present, and a second primer pair may produce a second amplicon of a second size if cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 is absent or not present.
  • a primer pair to detect the presence of all or part of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 in a DNA molecule or sample comprises a first primer and a second primer, wherein the first primer is complementary to a 5' flanking genomic DNA sequence and the second primer is complementary to a sequence within the transgenic insert; or wherein the first primer is complementary to a 5' flanking genomic DNA sequence and the second primer is complementary to a 3' flanking genomic DNA sequence; or wherein the first primer is complementary to a 3' flanking genomic DNA sequence and the second primer is complementary to a 5' flanking genomic DNA sequence; or wherein the first primer is complementary to a sequence within the transgenic insert and the second primer is complementary to a 3' flanking genomic DNA sequence; or wherein the first primer is complementary to a sequence within the transgenic insert and the second primer is complementary to a 3' flanking genomic DNA sequence; or wherein the first primer is complementary to a sequence within the transgenic insert and the second primer is complementary to a 5
  • a primer complementary to a 5' flanking genomic DNA sequence, a 3' flanking genomic DNA sequence, or a sequence within the transgenic insert of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 is also intended to potentially include a primer complementary to the reverse complement or opposing strand of the respective 5' flanking genomic DNA sequence, 3' flanking genomic DNA sequence, or sequence within the transgenic insert of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002.
  • Exemplary DNA molecules useful as primers are provided as SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 19.
  • the primer pair SEQ ID NO: 15 and SEQ ID NO: 16 can be useful as a first DNA molecule or primer and a second DNA molecule or primer, wherein each primer has sufficient length of consecutive nucleotides of SEQ ID NO: 10 or a sequence complementary to SEQ ID NO: 10 to function as DNA primers that, when used together in an amplification reaction with template DNA derived from cotton event GH_BCS246002 or a modified cotton event GH_BCS246002, hybridize to opposite strands of the template DNA and produce an amplicon diagnostic for cotton event GH_BCS246002 DNA or a modified cotton event GH_BCS246002 in a sample.
  • the primer pair SEQ ID NO: 18 and SEQ ID NO: 19 are useful as a first DNA molecule or primer and a second DNA molecule or primer, wherein each primer has sufficient length of consecutive nucleotides of a locus within the cotton genome to function as DNA primers that, when used together in a thermal amplification reaction with template DNA derived from cotton event GH_BCS246002 or a modified cotton event GH_BCS246002, to produce an amplicon that serves as an internal control for both the diagnosis of cotton event
  • DNA probes and DNA primers are generally eleven (11) polynucleotides or more in length, often eighteen (18) polynucleotides or more, twenty-four (24) polynucleotides or more, or thirty (30) polynucleotides or more. Such probes and primers are selected to be of sufficient length to hybridize specifically to a target sequence under high stringency hybridization conditions.
  • probes and primers according to the present disclosure have complete sequence similarity with the target sequence, although probes differing from the target sequence that retain the ability to hybridize to target sequences may be designed by conventional methods.
  • the nucleic acid probes and primers of the present disclosure hybridize under stringent conditions to a target DNA molecule. Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of DNA from a transgenic plant in a sample. Polynucleic acid molecules also referred to as nucleic acid segments or fragments thereof are capable of specifically hybridizing to other nucleic acid molecules under certain circumstances.
  • two polynucleic acid molecules are said to be capable of specifically hybridizing to one another if the two molecules are capable of forming an anti-parallel, double-stranded nucleic acid structure.
  • a nucleic acid molecule is said to be the “complement” of another nucleic acid molecule if they exhibit complete complementarity.
  • molecules are said to exhibit “complete complementarity” when every nucleotide of one of the molecules is complementary to a nucleotide of the other.
  • Two molecules are said to be “minimally complementary” if they can hybridize to one another with sufficient stability to permit them to remain annealed to one another under at least conventional "low- stringency" conditions.
  • the molecules are said to be “complementary” if they can hybridize to one another with sufficient stability to permit them to remain annealed to one another under conventional "high- stringency" conditions.
  • Conventional stringency conditions are described by Sambrook et al., 1989, and by Haymes et al., In: Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, DC (1985). Departures from complete complementarity are therefore permissible, as long as such departures do not completely preclude the capacity of the molecules to form a double-stranded structure.
  • a substantially homologous sequence is a nucleic acid sequence that will specifically hybridize to the complement of the nucleic acid sequence to which it is being compared under high stringency conditions.
  • Appropriate stringency conditions that promote DNA hybridization for example, 6.0 x sodium chloride/sodium citrate (SSC) at about 45 °C, followed by a wash of 2.0 x SSC at 50°C, are known to those skilled in the art or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
  • the salt concentration in the wash step can be selected from a low stringency of about 2.0 x SSC at 50°C to a high stringency of about 0.2 x SSC at 50°C.
  • the temperature in the wash step can be increased from low stringency conditions at room temperature, about 22°C, to high stringency conditions at about 65°C.
  • a polynucleic acid of the present disclosure will specifically hybridize to one or more of the nucleic acid molecules set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10, or complements thereof or fragments thereof under moderately stringent conditions, for example at about 2.0 x SSC and about 65°C.
  • a nucleic acid of the present disclosure will specifically hybridize to one or more of the nucleic acid molecules set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10, or complements or fragments thereof under high stringency conditions.
  • a preferred marker nucleic acid molecule of the present disclosure has the nucleic acid sequence set forth in SEQ ID NO: 1, or SEQ ID NO: 2, or SEQ ID NO: 3, or SEQ ID NO: 4, or SEQ ID NO: 5, or SEQ ID NO: 6, or SEQ ID NO: 7, or SEQ ID NO: 8, or SEQ ID NO: 9, or SEQ ID NO: 10, or complements thereof, or fragments thereof.
  • the hybridization of the probe to the target DNA molecule can be detected by any number of methods known to those skilled in the art, these can include, but are not limited to, fluorescent tags, radioactive tags, antibody based tags, colorimetric tags, and chemiluminescent tags.
  • stringent conditions are conditions that pennit the primer pair to hybridize only to the target nucleic acid sequence to which a primer having the corresponding wild-type sequence (or its complement) would bind and preferably to produce a unique amplification product, the amplicon, in a DNA thermal amplification reaction.
  • the term "specific for (a target sequence)" indicates that a probe or primer hybridizes under stringent hybridization conditions only to the target sequence in a sample comprising the target sequence.
  • amplified DNA refers to the product of polynucleic acid amplification method directed to a target polynucleic acid molecule that is part of a polynucleic acid template.
  • DNA that is extracted from a cotton plant tissue sample may be subjected to a polynucleic acid amplification method using a primer pair that includes a first primer derived from a genomic DNA sequence in the region flanking the heterologous inserted DNA of cotton event GH_BCS246002 and is elongated by polymerase 5 ' to 3' in the direction of the inserted DNA.
  • the second primer is derived from the heterologous inserted DNA molecule is elongated by the polymerase 5' to 3' in the direction of the flanking genomic DNA from which the first primer is derived.
  • the amplicon may range in length from the combined length of the primer pair plus one nucleotide base pair, or plus about fifty nucleotide base pairs, or plus about two hundred- fifty nucleotide base pairs, or plus about four hundred-fifty nucleotide base pairs or more.
  • a primer pair can be derived from genomic sequence on both sides of the inserted heterologous DNA so as to produce an amplicon that includes the entire insert polynucleotide sequence (e.g., a forward primer isolated from the genomic portion on the 5' end of SEQ ID NO: 10 and a reverse primer isolated from the genomic portion on the 3' end of SEQ ID NO: 10 that amplifies a DNA molecule comprising the inserted DNA sequence (SEQ ID NO: 9) identified herein in the cotton event GH_BCS246002 genome).
  • a forward primer isolated from the genomic portion on the 5' end of SEQ ID NO: 10 and a reverse primer isolated from the genomic portion on the 3' end of SEQ ID NO: 10 that amplifies a DNA molecule comprising the inserted DNA sequence (SEQ ID NO: 9) identified herein in the cotton event GH_BCS246002 genome.
  • a member of a primer pair derived from the plant genomic sequence adjacent to the inserted transgenic DNA is located a distance from the inserted DNA sequence, this distance can range from one nucleotide base pair up to about twenty thousand nucleotide base pairs.
  • the use of the term “amplicon” specifically excludes primer dimers that may be formed in the DNA thermal amplification reaction.
  • primers which produce amplicons of a limited size range, for example, between 100 to 1000 bases.
  • Smaller (shorter polynucleotide length) sized amplicons in general are more reliably produced in thermal amplification reactions, allow for shorter cycle times, and can be easily separated and visualized on agarose gels or adapted for use in endpoint TaqMan®-likc assays.
  • Smaller amplicons can be produced and detected by methods known in the ail of DNA amplicon detection.
  • amplicons produced using the primer pairs can be cloned into vectors, propagated, isolated, and sequenced or can be sequenced directly with methods well established in the art.
  • Any primer pair derived from the combination of SEQ ID NO: 11 and SEQ ID NO: 9 or the combination of SEQ ID NO: 12 and SEQ ID NO: 9 that are useful in a DNA amplification method to produce an amplicon diagnostic for cotton event GH_BCS246002, or progeny thereof is an aspect of the present disclosure.
  • Any single isolated DNA polynucleotide primer molecule comprising at least 15 contiguous nucleotides of SEQ ID NO: 11, or its complement that is useful in a DNA amplification method to produce an amplicon diagnostic for cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 or progeny thereof is an aspect of the present disclosure.
  • Any single isolated DNA polynucleotide primer molecule comprising at least 15 contiguous nucleotides of SEQ ID NO: 12, or its complement that is useful in a DNA amplification method to produce an amplicon diagnostic for plants comprising cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 or progeny thereof is an aspect of the present disclosure.
  • Any single isolated DNA polynucleotide primer molecule comprising at least 15 contiguous nucleotides of SEQ ID NO: 9, or its complement that is useful in a DNA amplification method to produce an amplicon diagnostic for cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 or progeny thereof is an aspect of the present disclosure.
  • Probes and primers as provided herein may have complete sequence identity with the target sequence, although primers and probes differing from the target sequence that retain the ability to hybridize preferentially to target sequences may be designed by conventional methods.
  • primers and probes differing from the target sequence that retain the ability to hybridize preferentially to target sequences may be designed by conventional methods.
  • a nucleic acid molecule In order for a nucleic acid molecule to serve as a primer or probe it need only be sufficiently complementary in sequence to be able to form a stable double- stranded structure under the particular solvent and salt concentrations employed. Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of transgenic DNA from cotton event GH_BCS246002 in a sample.
  • DNA molecule, or fragment thereof, disclosed herein Any number of methods well known to those skilled in the art can be used to isolate and manipulate a DNA molecule, or fragment thereof, disclosed herein, including DNA isolation or thermal amplification or PCR methods. Such DNA molecule or fragment may be inserted or placed into any suitable vector or plasmid or combined with other elements, sequences or fragments using molecular or recombinant techniques.
  • the DNA molecules and corresponding nucleotide sequences provided herein are therefore useful for, among other things, identifying cotton event GH_BCS246002, detecting the presence of DNA derived from the transgenic cotton event GH_BCS246002 in a sample, and monitoring samples for the presence and/or absence of cotton event GH_BCS246002 or plant parts derived from cotton plants comprising cotton event GH_BCS246002.
  • a transgenic cotton plant or plant part, one or more transgenic cotton plants or plant parts or a plurality transgenic cotton plants or plant parts as provided herein, or an agricultural field or soil in which a transgenic cotton plant or plant part, one or more transgenic cotton plants or plant parts or a plurality of transgenic cotton plants or plant parts as provided herein are planted or grown can be treated with an agricultural composition comprising one or more active ingredients or other agents, such as, for example and without limitation, an herbicide or one or more herbicides, a fungicide or one or more fungicides, an insecticide or one or more insecticides, a plant growth regulator or plant stimulant or one or more plant growth regulators and/or plant stimulants, and/or a safener or one or more safeners.
  • an agricultural composition comprising one or more active ingredients or other agents, such as, for example and without limitation, an herbicide or one or more herbicides, a fungicide or one or more fungicides, an insecticide or one or more insecticides, a plant growth regulator
  • an agricultural composition may comprise one or any combination or multiplicity of these actives, agents or compounds.
  • Such an agricultural composition may be applied, for example, as a foliar, soil or in-furrow treatment, as a pre-emergent, pre-sowing and/or post- emergent treatment, and/or in some cases, may be applied to a transgenic plant part or seed provided herein.
  • An agricultural composition may be formulated according to its intended use and application.
  • the appropriate formulation of the agricultural composition may be chosen to have different physicochemical parameters, components and stabilities of the respective compound(s).
  • Possible types of formulations for an agricultural composition can include, for example: wettable powders (WP), water-soluble powders (SP), water-soluble concentrates, emulsifiable concentrates (EC), emulsions (EW), such as oil-in-water and water-in-oil emulsions, sprayable solutions, suspension concentrates (SC), dispersions based on oil or water, oil-miscible solutions, capsule suspensions (CS), dusting products (DP), dressings, granules for scattering and soil application, granules (GR) in the form of microgranules, spray granules, absorption and adsorption granules, water-dispersible granules (WG), water-soluble granules (SG), ULV formulations, microcapsule
  • Plants, progeny, plant parts, plant seeds, plant tissues, and plant cells may contain or comprise one or more additional desirable trait(s).
  • desirable traits may be transgenic traits, native traits, or traits produced by other methods, such as genome editing, base editing, prime editing or other conventional mutagenesis methods.
  • Such desirable trait(s) may provide an agronomic, agricultural or commodity benefit to a plant, plant part, plant seed or plant product.
  • Desirable traits may be combined with cotton event GH_BCS246002 by, for example, crossing a cotton plant comprising cotton event GH_BCS246002 with another cotton plant containing the additional trait(s).
  • a trait may be created by mutagenesis, editing or site-directed integration of or into a plant, plant part or plant cell comprising cotton event GH_BCS246002.
  • Such traits may include, but are not limited to, increased insect resistance, increased water use efficiency, increased nitrogen use efficiency, increased yield performance, increased drought resistance, increased disease resistance, increased seed quality, improved nutritional quality, hybrid seed production, and/or increase herbicide tolerance, in which the trait is measured with respect to a cotton plant lacking such transgenic trait.
  • the cotton event GH_BCS246002 could be stacked by breeding or introgression with another event(s), or a combination of events, known in the art including, but not limited to:
  • BXN10211 also known as 10211, BXN-10211-9, BXNTM Cotton for herbicide tolerance, described in USDA-APHIS Petition 93-196-01p, the entire content and disclosure of which are incorporated herein by reference in their entirety
  • BXN10215 also known as 10215, BXN-10215-4, BXNTM Cotton for herbicide tolerance, described in USDA-APHIS Petition 93- 196-01p, the entire content and disclosure of which are incorporated herein by reference in their entirety
  • BXN10222 also known as 10222, BXN- 10222-2, BXNTM Cotton for herbicide tolerance, described in USDA-APHIS Petition 93- 196-0 Ip, the entire content and disclosure of which are incorporated herein by reference in their entirety
  • BXN10224 also known as IR102, for herbicide tolerance, described in USDA-APHIS Petition 93-196-01p, the entire content and disclosure of which are incorporated herein by reference in their entirety
  • COT102 also known as IR102, SYN-IR 102-7 for insect resistance, described in US Patent No. 7,371,940, and in USDA-APHIS Petition 03-155-01p, the entire contents and disclosure of which are incorporated herein by reference in their entirety
  • COT67B also known as IR67B, SYN-IR67B-1 for insect resistance, described in USDA- APHIS Petition 07-108-01p, the entire content and disclosure of which are incorporated herein by reference in their entirety
  • DAS81910 also known as DAS-81910-7 for herbicide tolerance, deposited as ATCC PTA-12456, and described in US Patent Nos. 9,551,024 and 9,896,718, and in USDA-APHIS Petition 13-262-0 Ip, the entire contents and disclosure of each of which are incorporated herein by reference in their entirety
  • GHB 119 also known as BCS-GHOO5-8, EE-GH6 for insect resistance, deposited as ATCC PTA-8398, and described in US Patent Nos. 8,309,818 and 9,328390, the entire contents and disclosure of each of which are incorporated herein by reference in their entirety
  • GHB614 also known as BCS-GH002-5, EE-GH3, and GlyTolTM for herbicide tolerance, deposited as ATCC PTA-6878, and described in US Patent Nos. 7,932,439, 8,501,411 and 9,394,566, and in USDA-APHIS Petition 06-332-01p, the entire contents and disclosure of each of which are incorporated herein by reference in their entirety
  • GHB811 also known as BCS-GH811-4 for herbicide tolerance, described in USDA- APHIS Petition 17- 138-0 lp, the entire content and disclosure of which are incorporated herein by reference in their entirety
  • LLCotton25 also known as ACS-GH001-3, EE-GH1, and FibermaxTM Liberty LinkTM for herbicide tolerance, deposited as ATCC PTA-3343, and described in US Patent Nos. 6,818,807, 7,442,504 and 7,834,168, the entire contents and disclosure of each of which are incorporated herein by reference in their entirety
  • T303-3 also known as BCS-GH003-6 for herbicide tolerance and insect resistance, described in USDA- APHIS Petition 12-033-01p (Extension of 08-340-01p), the entire content and disclosure of which are incorporated herein by reference in their entirety
  • T304-40 also known as BCS-GH004-7 and EE-GH5 for herbicide tolerance and insect resistance, deposited as ATCC PTA-8171, and described in US Patent Nos. 8,247,654, 9,382,550 and 10,356,996, and in USDA-APHIS Petition 08-340-01p, the entire contents and disclosure of each of which are incorporated herein by reference in their entirety
  • MON1076 also known as 1076, MON-89924-2 and BollgardTM Cotton for insect resistance, described in USDA-APHIS Petition 94-308-01p, the entire content and disclosure of which are incorporated herein by reference in their entirety
  • MON1445 also known as 1445, MON-01445-2, and Roundup ReadyTM Cotton for herbicide tolerance, described in US Patent Nos. 6,740,488, 7,189,514, 7,807,357 and 7,820,392, and in USDA-APHIS Petition 95-045-01p, the entire contents and disclosure of each of which are incorporated herein by reference in their entirety
  • MON15985 also known as MON-15985-7, and Bollgard IITM Cotton for insect resistance, deposited as ATCC PTA-2516, described in US Patent Nos. 7,223,907, 7,858,764 and 9,133,473, and in USDA-APHIS Petition 00-342-01p, the entire contents and disclosure of each of which are incorporated herein by reference in their entirety
  • MON 1698 also known as MON-89383-1, and Roundup ReadyTM Cotton for herbicide tolerance, described in USDA-APHIS Petition 95-045-01p, the entire content and disclosure of which are incorporated herein by reference in their entirety
  • MON531 also known as MON-00531-6, and BollgardTM Cotton, IngardTM for insect tolerance, described in USDA-APHIS Petition 94-308-0 Ip, the entire content and disclosure of which are incorporated herein by reference in their entirety
  • MON531 also known as MON-00531-6, and BollgardTM Cotton, IngardTM for insect tolerance, described in USDA-APHIS Petition 94-308-0 Ip, the entire content and disclosure of which are incorporated herein by reference in their entirety
  • MON757 also known as MON-00757-7, and BollgardTM Cotton for insect tolerance, described in USDA-APHIS Petition 94-308-0 Ip, the entire content and disclosure of which are incorporated herein by reference in their entirety
  • MON88701 also known as MON-88701-3, and Dicamba-Glufosinate Tolerant Cotton for herbicide tolerance, deposited as ATCC PTA-11754, described in US Patent Nos. 8,735,661, 9,024,115, 10,030,277 and 10,774,341, and in USDA-APHIS Petition 12-185-01p, the entire contents and disclosure of each of which are incorporated herein by reference in their entirety
  • ATCC PTA-11754 described in US Patent Nos. 8,735,661, 9,024,115, 10,030,277 and 10,774,341, and in USDA-APHIS Petition 12-185-01p, the entire contents and disclosure of each of which are incorporated herein by reference in their entirety
  • MON88702 also known as MON-88702-4 for insect resistance, deposited as ATCC PTA- 122520, described in US Patent Nos. 10,604,769 and 11,286,499, the entire contents and disclosure of each of which are incorporated herein by reference in their entirety
  • MON88913 also known as MON-88913-8, and Roundup ReadyTM FlexTM Cotton for herbicide tolerance, deposited as ATCC PTA-4854, described in US Patent Nos. 7,381,861, 8,071,735 and 8,435,743, and in USDA-APHIS Petition 04-086-01p, the entire contents and disclosure of each of which arc incorporated herein by reference in their entirety
  • TAM66274 also known as TAM-66274-5 for modified product quality, deposited as ATCC PTA-124218, described in US Patent No. 10,604,764, and in USDA-APHIS Petition 17-292-01p, the entire contents and disclosure of each of which are incorporated herein by reference in their entirety
  • 3006-210-23 x 281-24-236 x MON88913 x COT102 also known as DAS-21023-5 x DAS- 24236-5 x MON-88913-8 x SYN-IR102-7, and WidestrikeTM x Roundup Ready FlexTM x VIPCOTTM Cotton for insect resistance and herbicide tolerance
  • 3006-210-23 x 281-24-236 x MON88913 x COT102 x DAS8191O also known as DAS- 21023-5 x DAS-24236-5 x MON-88913-8 x SYN-IR102-7 x DAS-81910-7 for insect resistance and herbicide tolerance
  • COT102 x COT67B also known as SYN-IR102-7 x SYN-IR67B- 1 and VIPCOTTM Cotton for insect resistance
  • COT102 x COT67B x MON88913 also known as SYN-IR102-7 x SYN-IR67B-1 x MON- 88913-8 and VIPCOTTM Roundup Ready FlexTM Cotton for insect resistance and herbicide tolerance
  • COT102 x MON15985 also known as SYN-IR102-7 x MON- 15985-7 and Bollgard® III for insect resistance
  • COT102 x MON15985 x MON88913 also known as SYN-IR102-7 x MON-15985-7 x MON-88913-8 and Bollgard® III x Roundup ReadyTM FlexTM for insect resistance and herbicide tolerance
  • COT102 x MON15985 x MON88913 x MON88701 also known as SYN-IR102-7 x MON-15985-7 x MON-88913-8 x MON 88701-3 for insect resistance and herbicide tolerance
  • GHB614 x LLCotton25 also known as BCS-GH002-5 x ACS-GH001-3 and GlyTolTM Liberty LinkTM for herbicide tolerance
  • GHB614 x LLCotton25 x MON15985 also known as BCS-GH002-5 x ACS-GH001-3 x MON- 15985-7 for insect resistance and herbicide tolerance
  • GHB614 x MON15985 also known as BCS-GH002-5 x MON-15985-7 for insect resistance and herbicide tolerance
  • GHB614 x T304-40 x GHB 119 also known as BCS-GH002-5 x BCS-GH004-7 x BCS- GH005-8 and GlytolTM x TwinlinkTM for insect resistance and herbicide tolerance
  • GHB614 x T304-40 x GHB119 x COT102 (also known as BCS-GH002-5 x BCS-GH004-
  • GHB811 x T304-40 x GHB 119 x COT102 also known as BCS-GHB811-4 x BCS- GH004-7 x BCS-GHOO5-8 x SYN-IR102-7 for insect resistance and herbicide tolerance
  • LLCotton25 x MONL5985 also known as ACS-GH001-3 x MON-15985-7 and FibermaxTM Liberty LinkTM Bollgard IITM for insect resistance and herbicide tolerance
  • MON15985 x MON1445 also known as MON- 15985-7 x MON-01445-2 and Roundup ReadyTM Bollgard IITM Cotton for insect resistance and herbicide tolerance
  • MON531 x MON1445 also known as MON-00531-6 x MON-01445-2 and Roundup ReadyTM BollgardTM Cotton for insect resistance and herbicide tolerance
  • MON88701 x MON88913 also known as MON 88701-3 x MON-88913-8 for herbicide tolerance
  • MON88701 x MON88913 x MON15985 also known as MON 88701-3 x MON-88913-8 x MON-15985-7 for insect resistance and herbicide tolerance
  • MON88913 x MON15985 also known as MON-88913-8 x MON-15985-7 and Roundup ReadyTM FlexTM Bollgard IITM Cotton for insect resistance and herbicide tolerance
  • T304-40 x GHB 119 also known as BCS-GH004-7 x BCS-GHOO5-8 and TwinLinkTM Cotton for insect resistance and herbicide tolerance, described in in USDA- APHIS Petition 08- 340-01p, the entire content and disclosure of which are incorporated herein by reference in their entirety
  • GHB 119 also known as BCS-GH004-7 x BCS-GHOO5-8 and TwinLinkTM Cotton for insect resistance and herbicide tolerance, described in in USDA- APHIS Petition 08- 340-01p, the entire content and disclosure of which are incorporated herein by reference in their entirety
  • T304-40 x GHB 119 x COT102 also known as BCS-GH004-7 x BCS-GHOO5-8 x SYN-
  • MON15947 corresponding to event MON15985 without MON531 for insect resistance, an event developed by ballistic methods into germplasm containing event MON531 , and which was determined to be unlinked to MON531, as described in USDA- APHIS Confirmation Request 21-211-Olcr and subsequent USDA confirmation, the entire content and disclosure of which are incorporated herein by reference in their entirety).
  • transgenic cotton plant comprising insecticidal proteins that were active against Lepidopteran pests without any observable negative effects on agronomics, yield, or stacking viability required extensive research, development, and analysis. Specifically, over a 10 year period, approximately 4,498 proof of concept and commercial transgenic events derived from 169 different plasmid vector constructs were developed, tested, and analyzed.
  • This example describes the design and testing in cotton plants of 169 different constructs, to identify the preferred construct for event creation.
  • Each construct varied with respect to the coding sequences for the insecticidal proteins and the transcriptional regulatory elements, and these were tested to select the preferred construct for use in expressing the insecticidal proteins in plants.
  • Each construct had a unique configuration, varying by expression cassette composition (both insecticidal proteins and expression elements), orientation, and whether or not proteins were targeted for insertion into the chloroplast.
  • the events were further evaluated with respect to molecular characteristics and performance, as well as selection for “Gold Standard Seed” (indicated as “GSS” in Table 2) wherein seed is selected to be self-bred for multiple generations to purify the event line.
  • GSS Gold Standard Seed
  • plants are evaluated for molecular, performance, and seed production ability.
  • 39 event were eliminated.
  • 13 events were eliminated from the GSS3 collection of events.
  • R4 generation 58 GSS4 event were eliminated based upon performance, 56 of which were derived from transformations using Construct-4.
  • 3 events remained, one each transformed by Construct- 1, Construct-4, pGH_BCS246002.
  • the remaining 3 events were compared with each other, and the remaining event derived from pGH_BCS246002 was selected as the lead event and is herein referred to as cotton event GH_BCS246002.
  • Cotton event GH_BCS246002 Demonstrates Resistance to the Lepidopteran Insect Pest Species Cotton bollworm, Tobacco budworm, and Fall army worm.
  • This Example describes the insecticidal activity of the cotton event GH_BCS246002 against several Lepidopteran insect pests of cotton.
  • the insect toxin proteins CrylB.3, CrylDa_7, and Vip3Cbl.l when expressed together in cotton containing cotton event GH_BCS246002, provide resistance to Lepidopteran pests of cotton including, but not limited to, Cotton bollworm (CBW, Helicoverpa zed), Tobacco budworm (TBW, Heliothis virescens), and Fall army worm (FAW, Spodoptera frugiperda).
  • Table 3 shows the average total and injured squares, the average total and injured bolls, the average total and injured fruits, and the average percent fruit injury of cotton containing cotton event GH_BCS246002 and the negative control.
  • Table 3 Average total and injured squares and bolls of cotton event GH_BCS246002 compared to negative control (conventional cotton) infested with CBW and TBW in screenhouse trials.
  • Table 4 Average total and injured squares and bolls of cotton plants containing cotton event GH_BCS246002 compared to negative control (conventional cotton) under natural field infestations.
  • Table 5 Average total fruit and average percent fruit injury of cotton containing event GH_BCS246002 compared to the negative control (conventional cotton) when infested with CBW, TBW and FAW in screenhouse trials.
  • Table 6 Average total and injured squares and bolls of cotton containing event GH_BCS246002 compared to the negative control (conventional cotton) under natural field infestations. [00265] The data presented in Table 6 shows that in each case cotton containing cotton event
  • GH_BCS246002 exhibited significantly less average percent fruit damage than the control plants. [00266] From the screenhouse data presented in Table 5, cotton containing event GH_BCS246002 provides resistance against the Lepidopteran insect pest species Cotton boll worm (Helicoverpa zea), Tobacco budworm (Heliothis virescens), and Fall armyworm (Spodoptera frugiperdd). The field studies presented above demonstrates cotton containing event GH_BCS246002 provides Lepidopteran insect infestation resistance under natural infestation conditions.
  • Cotton containing event GH_BCS246002 provides consistent yield, similar agronomics, and similar fiber quality in the field when compared to DP393 conventional cotton
  • transgenic cotton containing cotton event GH_BCS246002 provides consistent yields and agronomics in the field that arc similar to untransformed DP393 cotton plants.
  • Cotton containing cotton event GH_BCS246002 was compared to the untransformed DP393 control plants in the field over three seasons in multiple locations. Two (2) row plots of cotton were grown using one hundred sixty (160) seeds of cotton transgenic event GH_BCS246002 or the untransformed DP393 negative control per forty (40) foot long row (four (4) seeds per row foot). At the end of the growing season, the growth was terminated by using commercially available defoliants and boll openers. Defoliants used in this manner are often referred to as “harvest aids.” Removing the leaves prior to harvest provides several advantages. For example, removing the leaves before harvest increases the air movement through the crop canopy which facilitates quicker drying and prevents boll rot.
  • Table 7 Yield of seed cotton in pounds per acre over 3 growing season field harvests.
  • Table 8 Plant height and early vigor score for cotton containing event GH_BCS246002 compared to DP393 (conventional cotton) averaged over 3 growing seasons.
  • Fiber properties can be determined through a combination of instrument evaluations using High Volume Instrument (HVI) technology. Fiber length is measured in inches. Fiber strength measurements are conducted on the same beard of cotton used by the HVI to measure length and uniformity. Fiber strength measurement is made by clamping and breaking a bundle of fibers with a 1/8-inch spacing between the clamp jaws. Results are reported in terms of grams per tex to the nearest tenth. A “tex” unit is equal to the weight in grams of one thousand meters of fiber. Therefore, the strength reported is the force in grams required to break a bundle of fibers one tex unit in size. Table 9 shows a general description and corresponding strength measurements in grams per tex.
  • HVI High Volume Instrument
  • Fiber length uniformity is a measure of the degree of uniformity of the fibers in a sample to the nearest tenth.
  • the ratio between the “mean length” of fibers and the “uppcr-half- mean length” of fibers is referred to as the “length uniformity index.” Both the mean length and upper-half-mean length measurements are taken when the fiber is passed through the length sensor. There is a natural distribution in the length of cotton fibers, but the lower the variation in this length distribution, the higher the length uniformity index.
  • Micronaire is a measure of fiber fineness and maturity. An airflow instrument can be used to measure the air permeability of a constant mass of cotton fibers compressed to a fixed volume.
  • the volume of airflow through a specimen of cotton fibers is expressed as a micronaire.
  • Cotton fiber with micronaire measurements between 3.7 and 4.2 are considered in the premium range of micronaire.
  • Cotton fiber within the micronaire ranges of 3.5-3.6 or 4.3-4.9 are considered base quality, while cotton fiber above 4.9 or below 3.5 are in the discount ranges.
  • Micronaire measurements can be influenced during the growing period by environmental conditions such as moisture, temperature, sunlight, plant nutrients, and extremes in plant or boll population.
  • Favorable growing conditions result in fully mature fibers with premium range micronaire readings.
  • Unfavorable conditions such as lack of moisture, early freeze, or any other conditions that interrupt plant processes, will result in immature fibers and low micronaire measurements.
  • High micronaire cotton is caused by such things as abnormally warm temperatures during boll maturation, or poor boll set leading to excessive availability of carbohydrates and over-maturing of fibers.
  • Fiber fineness affects processing performance and the quality of the end-product in several ways. In the opening, cleaning, and carding processes, low micronaire or fine-fiber cottons require slower processing speeds to prevent damage to the fibers. Yams made from finer fiber result in more fibers per cross section, which in turn produces stronger yarns. High micronaire or coarse fibers are not suitable for fine yams since the result would be fewer fibers per cross section, which would reduce the yarn strength. Micronaire and maturity are highly correlated within a cotton variety. Dye absorbency and retention varies with the maturity of the fibers. Low maturity fibers have poor dye absorbency and retention while higher micronaire fibers have good absorbency and retention. Table 10 below shows the range of values and classification for Fiber Length, Fiber Strength, Length Uniformity, and Micronaire.
  • Lint percentage is one of the most important fiber yield components and is commonly used as a proxy for lint yield during breeding because of its high heritability (Sun et al. (2024) Dissecting the major genetic components underlying cotton lint development. Genetics. 226(2), iyad219). Lint percentage can be determined by dividing lint weight by the total seed cotton weight.
  • Short fiber content is defined as the percentage of fibers less than Yi inch long, relative to the total fiber length distribution.
  • Table 11 shows the fiber quality characteristics such as Fiber Strength, Fiber Length, Length Uniformity, Micronaire, Lint Percentage, and Short Fiber Content determined from the harvested bolls from cotton containing cotton event GH_BCS246002 compared to DP393 control plants.
  • Cotton plants containing transgenic event GH_BCS246002 provides excellent yield and similar agronomic and fiber characteristics as the DP393 non-transgenic control plants. Expression of the insect toxins Cry IB.3, CrylDa_7, and Vip3Cbl.l does not cause a yield drag or compromise the growth and fiber characteristics of cotton containing the cotton event GH_BCS246002 when compared to the conventional cotton DP393 control.
  • This Example describes methods useful in identifying the presence of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 in a cotton sample.
  • a pair of PCR primers and a probe were designed for the purpose of identifying the unique junction formed between the cotton genomic DNA and the inserted DNA of cotton event GH_BCS246002 in an event- specific endpoint TAQMAN® PCR assay. Examples of conditions utilized for identifying the presence of cotton event GH_BCS246002 in a cotton sample in an event- specific endpoint TAQMAN® PCR assay are described in Tables 12 and 13.
  • the sequence of the oligonucleotide forward primer SQ51960 (SEQ ID NO: 15) is identical to the nucleotide sequence corresponding to positions 941-964 of SEQ ID NO: 10.
  • the sequence of the oligonucleotide reverse primer SQ51702 (SEQ ID NO: 16) is identical to the reverse complement of the nucleotide sequence corresponding to positions 1,066-1,087 of SEQ ID NO: 10.
  • sequence of the oligonucleotide probe PB5O3O8 (SEQ ID NO: 17) is identical to the reverse complement of the nucleotide sequence corresponding to positions 1,043-1,060 of SEQ ID NO: 10 which may be fluorescently labeled (e.g., a 6-FAMTM fluorescent label), can be used in an endpoint TAQMAN® PCR assay to identify the presence of DNA derived from cotton event GH_BCS246002.
  • SQ51960 SEQ ID NO: 15
  • SQ51702 SEQ ID NO: 16
  • PB5O3O8 SEQ ID NO: 17
  • other primers and/or probes can be designed to either amplify or hybridize to sequences within SEQ ID NO: 10 which are unique to, and useful for, detecting the presence of DNA derived from cotton event GH_BCS246002 or a modified cotton event GH_BCS246002.
  • PCR assays for event identification were developed for detection of cotton event GH_BCS246002 in a sample. Parameters of either a standard PCR assay or a TAQMAN® PCR assay were optimized with each set of primer pairs and probes (e.g., probes labeled with a fluorescent tag such as 6-FAMTM ) used to detect the presence of DNA derived from cotton event GH_BCS246002 in a sample.
  • probes e.g., probes labeled with a fluorescent tag such as 6-FAMTM
  • a control for the PCR reaction can include internal control primers and an internal control probe (e.g., VIC®- labeled) specific to a region within the cotton genome that is used as an internal control, and are primers SQ22496 (SEQ ID NO: 18), SQ22497 (SEQ ID NO: 19), and VIC® labeled probe PB50562 (SEQ ID NO: 19).
  • an internal control probe e.g., VIC®- labeled
  • the parameters which were optimized for detection of cotton event GH_BCS246002 in a sample included primer and probe concentration, amount of templated DNA, and PCR amplification cycling parameters.
  • the controls for this analysis include a positive control from cotton containing cotton event GH_BCS246002, a negative control from non-transgenic cotton, and a negative control that contains no template DNA.
  • This Example describes methods useful in identifying the zygosity of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 (e.g., homozygous, hemizygous or null). Pairs of PCR primers and a probe arc designed for the purpose of identifying properties of alleles positive for the T-DNA insertion that gave rise to cotton event GH_BCS246002 and pairs of PCR primers and a probe are designed as an internal control probe specific to a regions within the cotton genome that is used as an internal control which is represented in the cotton genome as homozygous.
  • PCR primers SQ51960 SEQ ID NO: 15
  • SQ51702 SEQ ID NO: 16
  • 6-FAMTM labeled probe PB50308 SEQ ID NO: 17
  • primers SQ22496 SEQ ID NO: 18
  • SQ22497 SEQ ID NO: 19
  • VIC® labeled probe PB50562 SEQ ID NO: 19
  • the cycle thresholds are determined for the amplicon corresponding to cotton event GH_BCS246002 inserted allele and the single-copy, homozygous internal standard.
  • the difference (ACt) between the Ct value of the single-copy, homozygous internal standard amplicon, and the Ct value of the cotton event GH_BCS246002 inserted allele amplicon are determined.
  • zygosity a ACt of around zero (0) indicates homozygosity of the inserted cotton event GH_BCS246002 T-DNA and ACt of around one (1) indicated heterozygosity of the inserted cotton event GH_BCS246002 T-DNA.
  • This Example describes a method useful in identifying the zygosity of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002.
  • Pairs of PCR primers and a probe are designed for the purpose of identifying specific properties of alleles positive and negative for the T-DNA insertion that gave rise to cotton event GH_BCS246002.
  • Examples and conditions that may be used in an event-specific zygosity TAQMAN® PCR are provided in Tables 14 and 15.
  • the DNA primer pairs used in the zygosity assay are primers SQ51960 (SEQ ID NO: 15) and SQ51702 (SEQ ID NO: 16); and SQ51960 (SEQ ID NO: 15) and WTDP393R (SEQ ID NO: 21).
  • the probes used in the zygosity assay are 6FAMTM-labeled probe PB5O3O8 (SEQ ID NO: 17) and VIC®-labeled probe WTDP393PR (SEQ ID NO: 22).
  • SQ51960 (SEQ ID NO: 15) and SQ51702 (SEQ ID NO: 16) and the 6FAMTM-labeled probe PB5O3O8 (SEQ ID NO: 17) are diagnostic for, or characteristic of, cotton event GH_BCS246002 DNA.
  • SQ51960 SEQ ID NO: 15
  • WTDP393R SEQ ID NO: 21
  • VIC®-labeled probe WTDP393PR SEQ ID NO: 22
  • the template DNA samples and controls for this analysis are a positive control from cotton containing cotton event GH_BCS246002 (from both a known homozygous and known heterozygous sample), a negative control from non-transgenic cotton, and a negative control that contains no template DNA.
  • This Example describes a method for identifying the cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 within progeny of any breeding activity using cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 in breeding or crossing activities with cotton plants having other event(s) or trait(s) to produce progeny plants having a stack of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 with the other event(s) or trait(s).
  • the cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 could be stacked by breeding or by site directed integration with other events known in the art to control insect pest species or to provide tolerance to herbicides or to provide other agronomic traits or characteristics, including for example, but not limited to, 19-5 la (DD- 01951A-7 for herbicide tolerance, described in USDA-APHIS Petition 95-256-01p, the entire content and disclosure of which are incorporated herein by reference in their entirety), 281-24-236 (also known as DAS-24236-5 for herbicide tolerance and insect resistance, deposited as ATCC PTA-6233, and described in US Patent Nos.
  • 19-5 la DD- 01951A-7 for herbicide tolerance, described in USDA-APHIS Petition 95-256-01p, the entire content and disclosure of which are incorporated herein by reference in their entirety
  • 281-24-236 also known as DAS-24236-5 for herbicide tolerance and insect resistance, deposited as ATCC PTA-6233, and described
  • 3006-210-23 also known as DAS-21023-5 for herbicide tolerance and insect resistance, deposited as ATCC PTA- 6233, and described in US Patent Nos.
  • 31707, 31803, 31807, 31808 and 42317 also known as BXNTM Plus BollgardTM Cotton for herbicide tolerance and insect resistance, described in USDA-APHIS Petition 97-013-01p, the entire content and disclosure of which are incorporated herein by reference in their entirety
  • BXN10211 also known as 10211, BXN-10211-9, BXNTM Cotton for herbicide tolerance, described in USDA-APHIS Petition 93- 196-0 Ip, the entire content and disclosure of which are incorporated herein by reference in their entirety
  • BXN10215 also known as 10215, BXN- 10215-4, BXNTM Cotton for herbicide tolerance, described in USDA-APHIS Petition 93-196-01p, the entire content and disclosure of which are incorporated herein by reference in their entirety
  • BXN10222 also known as 10222, BXN- 10222-2,
  • COT202 for insect resistance, described in US Patent No. 7,521,550, the entire content and disclosure of which are incorporated herein by reference in their entirety
  • COT203 for insect resistance, described in PCT Patent Publication No.
  • COT67B also known as IR67B, SYN-IR67B-1 for insect resistance, described in USDA-APHIS Petition 07-108-01p, the entire content and disclosure of which are incorporated herein by reference in their entirety
  • 1143-14A for insect resistance, described in PCT Patent Publication No. W02006128570, the entire content and disclosure of which are incorporated herein by reference in their entirety
  • 1143-5 IB for insect resistance, described in PCT Patent Publication No. WO2006128569, the entire content and disclosure of which are incorporated herein by reference in their entirety
  • CE43-67B for insect resistance, described in US Patent Nos.
  • CE44-69D for insect resistance, described in US Patent Publication No. 20100024077, the entire content and disclosure of which are incorporated herein by reference in their entirety
  • CE46-02A for insect resistance, described in PCT Patent Publication No. WO2006128572, the entire content and disclosure of which are incorporated herein by reference in their entirety
  • DAS81910 also known as DAS- 81910-7 for herbicide tolerance, deposited as ATCC PTA-12456, and described in US Patent Nos.
  • GHB 119 also known as BCS-GH005-8, EE-GH6 for insect resistance, deposited as ATCC PTA-8398, and described in US Patent Nos. 8,309,818 and 9,328390, the entire contents and disclosure of each of which arc incorporated herein by reference in their entirety
  • GHB614 also known as BCS- GH002-5, EE-GH3, and GlyTolTM for herbicide tolerance, deposited as ATCC PTA-6878, and described in US Patent Nos.
  • GHB811 also known as BCS-GH811-4 for herbicide tolerance, described in USDA-APHIS Petition 17-138-01p, the entire content and disclosure of which are incorporated herein by reference in their entirety
  • LLCotton25 also known as ACS-GH001-3, EE-GH1, and FibermaxTM Liberty LinkTM for herbicide tolerance, deposited as ATCC PTA-3343, and described in US Patent Nos.
  • T303-3 also known as BCS-GH003-6 for herbicide tolerance and insect resistance, described in USDA-APHIS Petition 12-033-01p (Extension of 08-340-01p), the entire content and disclosure of which are incorporated herein by reference in their entirety
  • T304-40 also known as BCS-GH004-7 and EE-GH5 for herbicide tolerance and insect resistance, deposited as ATCC PTA-8171, and described in US Patent Nos.
  • MON1698 also known as MON- 89383-1, and Roundup ReadyTM Cotton for herbicide tolerance, described in USDA-APHIS Petition 95-045-01p, the entire content and disclosure of which are incorporated herein by reference in their entirety
  • MON531 also known as MON-00531 -6, and BollgardTM Cotton, IngardTM for insect tolerance, described in USDA- APHIS Petition 94-308-0 Ip, the entire content and disclosure of which are incorporated herein by reference in their entirety
  • MON757 also known as MON-00757-7, and BollgardTM Cotton for insect tolerance, described in USDA- APHIS Petition 94-308-0 Ip, the entire content and disclosure of which are incorporated herein by reference in their entirety
  • MON88701 also known as MON-8870
  • MON88702 also known as MON-88702-4 for insect resistance, deposited as ATCC PTA-122520, described in US Patent Nos. 10,604,769 and 11,286,499, the entire contents and disclosure of each of which are incorporated herein by reference in their entirety
  • MON88913 also known as MON-88913-8, and Roundup ReadyTM FlexTM Cotton for herbicide tolerance, deposited as ATCC PTA-4854, described in US Patent Nos.
  • TAM66274 also known as TAM-66274-5 for modified product quality, deposited as ATCC PTA- 124218, described in US Patent No.
  • 281-24-236 x 3006-210-23 also known as MXB-13, DAS-24236-5 x DAS-21023-5, and WideStrikeTM Cotton for insect resistance
  • 281-24-236 x 3006-210-23 x COT102 also known as DAS-24236-5 x DAS-21023-5 x SYN-IR102-7 for insect resistance
  • 281-24-236 x 3006-210-23 x COT102 x 81910 also known as DAS-24236-5 x DAS-21023-5 x SYN-IR102-7 x DAS-81910-7 for insect resistance and herbicide tolerance
  • 3006-210-23 x 281-24-236 x MON1445 also known as DAS-21023-5 x DAS-24236-5 x MON-01445-2, and WideStrikeTM Roundup ReadyTM Cotton for insect resistance and herbicide
  • DNA primer pairs are used to produce an amplicon diagnostic for, or characteristic of, cotton event GH_BCS246002.
  • An amplicon diagnostic for, or characteristic of, cotton event GH_BCS246002 may comprise at least one junction sequence.
  • the junction sequences for cotton event GH_BCS246002 are SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6 ([1], [2], [3], [4], [5], and [6], respectively in FIG. 1).
  • [15] represents SEQ ID NO: 15 or primer SQ51960
  • [16] represents SEQ ID NO: 16 or primer SQ51702.
  • the arrows show the approximate hybridization position within [10] of FIG. 1 and the direction in which synthesis proceeds during the amplification cycles.
  • These two primers may be used together as a primer pair that can be applied to a sample containing cotton event GH_BCS246002 DNA and that, when used in a thermal amplification reaction, produces an amplicon of one hundred forty-seven (147) nucleotides containing a junction sequence comprising the 5' end of the insert and a flanking genomic DNA. [17] in FIG.
  • 1 represents SEQ ID NO: 17 or probe PB50308, which can bind or hybridize to an amplicon produced using, for example, primers [15] and [16] together in an amplification reaction with cotton event GH_BCS246002 DNA as template, for detecting the presence of the cotton event GH_BCS246002 DNA in a sample. [21] in FIG.
  • 1 represents SEQ ID NO: 21 or primer WTDP393R that binds or hybridizes to a position within the 3’ flanking cotton genomic DNA, and when combined with primer [15] in a thermal amplification reaction together with conventional cotton DNA as template lacking or devoid of cotton event GH_BCS246002 DNA, produces an amplicon of one hundred fifty-one (151) nucleotides containing undisrupted cotton genomic DNA, and detection of that amplicon is representative of a sample which does not contains the cotton transgenic event GH_BCS246002 DNA at that chromosomal locus.
  • Probe WTDP393PR (SEQ ID NO: 22) is a probe that could be used to bind or hybridize to an amplicon produced using primers [21] and [15] for detecting an allele lacking, or devoid of, cotton event GH_BCS246002 DNA.
  • Probe WTDP393PR hybridizes to the 3' terminal 12 nucleotides of the 3' genomic flanking DNA and the 10 nucleotides of the wild-type allelic DNA that was deleted during insertion of the T-DNA in cotton event GH_BCS246002.
  • Primer pairs that will produce an amplicon diagnostic for, or characteristic of, cotton event GH_BCS246002 include primer pairs based upon the flanking sequences (SEQ ID NO: 11 and SEQ ID NO: 12) and the inserted T-DNA (SEQ ID NO: 9).
  • SEQ ID NO: 1 SEQ ID NO: 3 or SEQ ID NO: 5 is found
  • a forward primer molecule based on the 5' flanking cotton genomic DNA (SEQ ID NO:11) from bases 1-1,000 and a reverse primer molecule based upon the inserted T-DNA (SEQ ID NO:9) from positions 1,001-16,129 in which the primer molecules are of sufficient length of contiguous nucleotides to specifically hybridize to SEQ ID NO: 11 and SEQ ID NO: 9.
  • SEQ ID NO: 2 SEQ ID NO: 4
  • SEQ ID NO: 6 SEQ ID NO: 6
  • a forward primer molecule based upon the inserted T-DNA (e.g., within SEQ ID NO: 9) from positions 1,001-16,129 and a reverse primer molecule based upon the 3' flanking cotton genomic DNA (e.g., within SEQ ID NO: 12) from positions 16,130-17,129 in which the primer molecules are of sufficient length of contiguous nucleotides to specifically hybridize to those regions (e.g., SEQ ID NO: 9 and SEQ ID NO: 12).
  • primers which produce amplicons of a limited size range, preferably between 200 to 1,000 bases. Smaller sized amplicons in general are more reliably produced in PCR reactions, allow for shorter cycle times, and can be easily separated and visualized on agarose gels or adapted for use in in endpoint TAQMAN®-like assays.
  • amplicons produced using said primer pairs can be cloned into vectors, propagated, isolated and sequenced, or can be sequenced directly with methods well established in the art.
  • Any primer pair derived from the combinations of SEQ ID NO: 11 and SEQ ID NO: 9 or SEQ ID NO: 12 and SEQ ID NO: 9 that are useful in a DNA amplification method to produce an amplicon diagnostic for, or characteristic of, cotton event GH_BCS246002 or progeny thereof is an aspect of the present disclosure.
  • Any single isolated DNA polynucleotide primer molecule comprising, for example, at least eleven (11), at least fifteen (15) or at least eighteen (18) contiguous nucleotides of SEQ ID NO: 11 , SEQ ID NO: 9 or SEQ ID NO: 12 or their complements can be useful in a DNA amplification method to produce an amplicon diagnostic for, or characteristic of, cotton event GH_BCS246002 or progeny thereof.
  • An example of amplification conditions for this analysis is illustrated in Tables 12 and 13.
  • a diagnostic amplicon comprises a DNA molecule homologous or complementary to at least one transgene/genomic junction DNA sequence (or genomic/transgene junction DNA sequence based on orientation), or a substantial portion thereof.
  • An analysis for a cotton event GH_BCS246002 plant tissue sample should include a positive tissue control from a plant that contains cotton event GH_BCS246002, a negative control from a cotton plant that does not contain cotton event GH_BCS246002 (e.g., DP393), and possibly a negative control that contains no cotton genomic DNA.
  • a primer pair will amplify an endogenous cotton DNA molecule and will serve as an internal control for the DNA amplification conditions. Additional primer sequences can be selected from SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 9 by those skilled in the art of DNA amplification methods.
  • Conditions selected for the production of an amplicon by the methods shown in Table 12 and Table 13 may differ but result in an amplicon diagnostic for, or characteristic of, cotton event GH_BCS246002 DNA.
  • the use of different DNA primer and/or probe sequences within (or with modifications to) the methods of Table 12 and Table 14 for detection or determination of presence or absence of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 are within the scope of the present disclosure.
  • An amplicon produced by at least one DNA primer sequence derived from SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 9 that is diagnostic for, or characteristic of, cotton event GH_BCS246002 is an aspect of the present disclosure.
  • DNA detection kits that contain at least one DNA primer of sufficient length of contiguous nucleotides derived from SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 9 that, when used in a DNA amplification method, produces a diagnostic amplicon for cotton event GH_BCS246002 or its progeny is an aspect of the present disclosure.
  • a cotton plant or seed, wherein its genome will produce an amplicon diagnostic for, or characteristic of, cotton event GH_BCS246002, when tested in a DNA amplification method is an aspect of the present disclosure.
  • the assay for the cotton event GH_BCS246002 amplicon can be performed by using an Applied Biosystems GeneAmpTM PCR System 9700, Stratagene Robocycler®, Eppendorf® Mastercycler® Gradient thermocycler or any other amplification system that can be used to produce an amplicon diagnostic of, or characteristic of, cotton event GH_BCS246002 as shown in Table 12.
  • This Example describes how all, or part of a transgenic insertion present in cotton event GH_BCS246002, including any portion of the flanking genomic DNA adjacent to the transgenic DNA insert, may be excised using targeted genome editing techniques. Sequences useful in excision of the cotton event GH_BCS246002 transgene insertion or expression cassettes within SEQ ID NO: 10 can be introduced through genomic editing using a variety of methods, particularly through the use of Clustered Regularly Interspersed Short Palindromic Repeats (CR1SPR) editing systems.
  • C1SPR Clustered Regularly Interspersed Short Palindromic Repeats
  • a CRISPR-associated protein can be selected from a Type 1 CR1SPR- associated protein, a Type II CRISPR-associated protein, a Type III CRISPR-associated protein, a Type IV CRISPR-associated protein, Type V CRISPR-associated protein, or a Type VI CRISPR- associated protein, such as but not limited to, Casl , CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Casl2a (also known as Cpfl), Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csx
  • the CRISPR-associated protein and one or more guide RNAs can be introduced into a plant cell comprising cotton event GH_BCS246002 to target a specific sequence within the transgenic insertion via a double strand break repair pathway, which may include, for example, non-homologous end-joining (NHEJ), microhomology-mediated end joining (MMEJ), homologous recombination, synthesis-dependent strand annealing (SDSA), single-strand annealing (SSA), or a combination thereof, at the genomic target site.
  • NHEJ non-homologous end-joining
  • MMEJ microhomology-mediated end joining
  • SDSA synthesis-dependent strand annealing
  • SSA single-strand annealing
  • One or more nucleotides or nucleotide sequences can be inserted within the cotton event GH_BCS246002 transgene insertion locus which can allow for the excision of all or part of the transgenic insertion from cotton event GH_BCS246002 or all or part of the specific expression cassettes within cotton event GH_BCS246002 or SEQ ID NO: 10.
  • Cas9 recognizes a G-rich protospacer-adjacent motif (PAM) that is 3 z to its guide RNA binding site whereas Casl2a systems recognize a T-rich protospacer-adjacent motif (PAM) that is 5' to its guide RNA binding site.
  • PAM G-rich protospacer-adjacent motif
  • PAM T-rich protospacer-adjacent motif
  • the OgRRS sequence is then used to define a cognate guide RNA recognition site (CgRRS) which is inserted into the transgene insertion locus of cotton event GH_BCS246002 using a CRISPR editing system.
  • the CgRRS comprises the same gRNA target sequence as the selected OgRRS.
  • the CgRRS is inserted in a region within the transgcnc insertion locus of cotton event GH_BCS246002 that is on the opposite side of the transgene insertion, relative to the OgRRS in a manner that will permit the excision of a fragment of DNA corresponding to either the entire transgene insertion of cotton event GH_BCS246002, or a fragment within the transgene insertion of cotton event GH_BCS246002 such as an expression cassette or genetic element within the transgene cassette, using a single gRNA.
  • the CgRRS will be inserted within the 5' genomic flanking sequence, or the 5' junction region, or within the transgene insert such as between expression cassettes or genetic elements within an expression cassette. Insertion of the CgRRS on the opposite side of the transgene insertion or within the region between expression cassettes, relative to the OgRRS allows for excision of the transgene insertion or specific expression cassettes to be excised using a single gRNA.
  • An OgRRS located between the expression cassettes of cotton event GH_BCS246002 can be used to design a CgRRS that can be inserted in either the 5' or 3' genomic flanking sequence to permit excision of one or the other expression cassette using a single gRNA.
  • Table 16 shows exemplary OgRRS sequences located within the 5' and 3' genomic flanking sequences and between the two expression cassettes of cotton event GH_BCS246002 that can be used in a CRISPR editing system employing FnCasl2a, a Type V CRISPR-associated protein (coding sequence presented as SEQ ID NO:41; protein sequence presented as SEQ ID NO:42). Table 16. Exemplary OgRRS sequences within cotton event GH_BCS246002.
  • Table 17 below shows gRNAs which include a poly-T transcript termination region that can be used to target the FnCasl2a nuclease to cut within both the OgRRS and CgRRS sequences.
  • any of the OgRRS sequences presented in Table 16 above can be used alternatively as a site to insert a CgRRS that was designed using a different OgRRS.
  • a CgRRS can be inserted into a flanking sequence to allow for the excision of the entire transgene insertion of cotton event GH_BCS246002.
  • OgRRS_3_l is selected as the OgRRS that will be used to design a corresponding CgRRS comprising DNA fragment
  • OgRRS_5_2 is selected as the target site in which the CgRRS comprising DNA fragment is inserted.
  • the OgRRS_5_2 site is targeted using the gRNA, gRNA_OgRRS_5_2 presented in Table 17 to cut within the OgRRS_5_2 site.
  • the CgRRS comprising DNA fragment that comprises the OgRRS_3_l target site is then inserted within the cut site that was introduced into the OgRRS_5_2 sequence.
  • the event can be bred into another germplasm.
  • the transgene insert of cotton event GH_BCS246002 can be excised from the plant using an FnCasl2a editing system and the gRNA, gRNA_OgRRS_3_l as presented in Table 17.
  • any of the OgRRS sequence presented in Table 16 that arc within the 5' and 3' genomic flanking sequences of cotton event GH_BCS246002 can be used as a site to insert a CgRRS comprising DNA fragment, comprising an OgRRS sequence that is between expression cassettes, to permit the excision of a specific expression cassette using a single gRNA.
  • OgRRS_In_l is selected as the OgRRS that will be used to design a corresponding CgRRS comprising DNA fragment
  • OgRRS_5_2 is selected as the target site in which the CgRRS comprising DNA fragment is inserted.
  • the OgRRS_5_2 site is targeted using the gRNA, gRNA_OgRRS_5_2 presented in Table 17 to cut within the OgRRS_5_2 site.
  • the CgRRS comprising DNA fragment that comprises the OgRRS_In_l target site is then inserted within the cut site that was introduced into the OgRRS_5_2 sequence. After selection of a transgenic event comprising the introduced CgRRS site, the event can be bred into another germplasm.
  • the first expression cassette which expresses the Cry IB.3 toxin protein can be excised from the plant using an FnCasl2a editing system and the gRNA, gRNA_OgRRS_In_l as presented in Table 17.
  • the CgRRS can be introduced into the transgene insertion locus through multiple methods using a CRISPR system.
  • a CRISPR system can be utilized for targeting 5' insertion of a blunt-end double-stranded DNA fragment into a genomic target site of interest such as an OgRRS that is not the OgRRS that has been selected for the design of the CgRRS.
  • the CRIS PR-mediated endonuclease activity can introduce a double stand break (DSB) in the selected genomic target site and DNA repair, such as microhomology-driven nonhomologous end-joining DNA repair, results in insertion of the blunt-end double-stranded DNA fragment into the DSB.
  • DSB double stand break
  • Blunt-cnd double- stranded DNA fragments can be designed with 1-10 bp of microhomology, on both the 5 ' and 3' ends of the DNA fragment that correspond to the 5' and 3' flanking sequence at the cut site of the protospacer in the genomic target site.
  • the CRISPR system can be introduced into cotton event GH_BCS246002 by several methods.
  • One or more expression cassettes encoding the gRNA and/or CRISPR associated protein components of a Type I, Type II, Type III, Type IV, Type V, or Type VI CRISPR-Cas system is transiently introduced into a cell.
  • the introduced one or more expression cassettes encoding the gRNA and/or CRISPR associated protein, along with a DNA fragment comprising the CgRRS is provided in sufficient quantity to modify the cell but does not persist after a contemplated period of time has passed or after one or more cell divisions. In such embodiments, no further steps arc needed to remove or segregate the one or more expression cassettes encoding the gRNA and/or CRISPR associated protein from the modified cell.
  • an expression construct comprising one or more expression cassette for the expression of a gRNA, and an expression construct encoding a Type I, Type II, Type III, Type IV, Type V, or Type VI CRISPR associated protein is stably transformed into cotton event GH_BCS246002 to introduce the CgRRS within the desired target locus.
  • the gRNA will direct the nuclease to cut within the target locus which can be an OgRRS different from the selected OgRRS.
  • the expression construct would also comprise a CgRRS DNA fragment which is flanked 5' and 3' with the PAM/gRNA sequence of the desired locus (i.e., an OgRRS different from the selected OgRRS) which will permit the excision of the CgRRS DNA fragment that can then be introduced into the target locus via a double strand break repair pathway.
  • a CgRRS DNA fragment which is flanked 5' and 3' with the PAM/gRNA sequence of the desired locus (i.e., an OgRRS different from the selected OgRRS) which will permit the excision of the CgRRS DNA fragment that can then be introduced into the target locus via a double strand break repair pathway.
  • Casl2a PAM/gRNA sites can be found within SEQ ID NO: 10 that can be used as potential OgRRS sequences, depending upon the desired outcome after genomic editing.
  • Table 18 below shows the coordinates of each of 416 potential OgRRS sequences within SEQ ID NO: 10 and the element in which they can be found. Those indicated in bold were previously presented in Table 16.
  • the table presents coordinates for four Casl2a endonucleases, LbCasl2a (coding sequence SEQ ID NO: 35, protein sequence SEQ ID NO: 36) derived from Lachnospiraceae bacterium ND2006 (also known as LbCpfl); two engineered variants of Lachnospiraceae bacterium ND2006 LbCasl2a, LbCasl2a-TYCV (coding sequence SEQ ID NO: 37, protein sequence SEQ ID NO: 38) and LbCasl2a-TATV (coding sequence SEQ ID NO: 39, protein sequence SEQ ID NO: 40), and FnCasl2a (coding sequence SEQ ID NO: 41 , protein sequence SEQ ID NO: 42) derived from Francisella novicida U112.
  • This Example describes the excision of all or any portion of the transgenic inserted
  • CRISPR Clustered Regularly Interspersed Short Palindromic Repeats
  • the CRISPR-associated protein is an RNA guided nuclease and can be selected from a Type I CRISPR-associated protein, a Type II CRISPR-associated protein, a Type III CRISPR-associated protein, a Type IV CRISPR-associated protein, Type V CRISPR-associated protein, or a Type VI CRISPR-associated protein, such as but not limited to, Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Casl2a, Cpfl, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl
  • the CRISPR-associated protein and two guide RNAs may be introduced into a plant cell comprising the cotton event GH_BCS246002 to target a specific sequence within the transgene insertion locus.
  • the CRISPR nuclease system cleaves at two distinct guide RNA hybridization sites thereby permitting the excision of the intervening sequence.
  • the genomic sequence may be repaired via a double strand break repair pathway, which may include, for example, non-homologous end-joining (NHEJ), microhomology-mediated end joining (MMEJ), homologous recombination, synthesis-dependent strand annealing (SDSA), single-strand annealing (SSA), or a combination thereof, at the genomic target site.
  • NHEJ non-homologous end-joining
  • MMEJ microhomology-mediated end joining
  • SDSA synthesis-dependent strand annealing
  • SSA single-strand annealing
  • the guide RNAs presented in Table 17 from Example 8 are used to excise the entire transgene cassette, or alternatively, are used to remove one or two of the three expression cassettes in cotton event GH_BCS246002.
  • a gRNA selected from the group consisting of SEQ ID NOs: 29 and 30 and a gRNA selected from the group consisting of SEQ ID NOs: 33 and 34 are used to guide an FnCasl2a nuclease to cut within regions of the 5' and 3' genomic flanking sequence of cotton event GH_BCS246002, causing the excision of the entire transgene insert.
  • a gRNA is selected from the group consisting of SEQ ID NOs: 29 and 30 and a gRNA presented as SEQ ID NO: 31 is used to guide an FnCasl2a nuclease to cut with the region of the 5 " genomic flanking sequence and a region between the first two transgcnc expression cassettes causing the excision of the Cry1B.3 expression cassette.
  • the third expression cassette used to express Vip3Cbl.1 a gRNA presented as SEQ ID NO: 32 and a gRNA selected from the group consisting of SEQ ID NOs: 33 and 34 is used to guide FnCasl2a nuclease to cut with the region between the CrylDa_7 and Vip3Cbl.l expression cassettes, and within the region of the 3' flanking genomic sequence causing excision of the Vip3Cbl.l expression cassette.
  • the gRNAs presented as SEQ ID NOs: 31 and 32 are used to guide an FnCasl2a nuclease to cut within the 3' UTR of the Cry IB.3 expression cassette and the promoter of the Vip3Cbl.l expression cassette, respectively allowing excision of the CrylDa_7 expression cassette.
  • the gRNAs presented above can also be used to replace one or more expression cassettes with expression cassettes that drive transgenes for the expression of any gene of agronomic interest.
  • the product of a gene of agronomic interest may act within the plant in order to cause an effect upon the plant morphology, physiology, growth, development, yield, grain composition, nutritional profile, disease or pest resistance, and/or environmental or chemical tolerance or may act as a pesticidal agent in the diet of a pest that feeds on the plant.
  • a beneficial agronomic trait may include, for example, but is not limited to, herbicide tolerance, insect control, modified yield, disease resistance, pathogen resistance, modified plant growth and development, modified starch content, modified oil content, modified fatty acid content, modified protein content, modified fruit ripening, enhanced animal and human nutrition, biopolymer productions, environmental stress resistance, pharmaceutical peptides, improved processing qualities, improved flavor, hybrid seed production utility, improved fiber production, augmented carbon sequestration, and/or desirable biofuel production.
  • This Example describes methods useful in identifying the zygosity of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 (e.g., homozygous, hemizygous or null).
  • Pairs of PCR primers and a probe are designed which hybridize to the 3’ portion of the Vip3Cbl.l coding sequence within the inserted T-DNA of cotton event GH_BCS246002 and for the purpose of identifying properties of alleles positive for the T-DNA insertion that gave rise to cotton event GH_BCS246002; and pairs of PCR primers and a probe arc designed as an internal control probe specific to a regions within the cotton genome that is used as an internal control which is represented in the cotton genome as homozygous. Examples of conditions utilized for determining the zygosity of cotton event GH_BCS246002 in a cotton sample are described in Tables 19 and 20 below.
  • PCR primers and probe specific to the Vip3Cbl.l coding sequence PCR primers SQ51253 (SEQ ID NO:245) and SQ51254 (SEQ ID NO:246), and 6-FAMTM labeled probe PB50494 (SEQ ID NO: 247) and the pairs of PCR primers and probe specific to the internal control, primers SQ22496 (SEQ ID NO: 18), SQ22497 (SEQ ID NO: 19), and VIC® labeled probe PB50562 (SEQ ID NO: 19) are used in a real-time PCR reaction similar to that as that described in Example 4 above.
  • the sequence of the oligonucleotide forward primer SQ51253 (SEQ ID NO:245) is identical to the nucleotide sequence corresponding to positions 14,048-14,069 of SEQ ID NO: 10.
  • the sequence of the oligonucleotide reverse primer SQ51254 (SEQ ID NO:246) is identical to the reverse complement of the nucleotide sequence corresponding to positions 15,0561-15,085 of SEQ ID NO: 10.
  • the sequence of the oligonucleotide probe (SEQ ID NO: 247) is identical to the nucleotide sequence corresponding to positions 15,041-15,058 of SEQ ID NO: 10 which may be fluorescently labeled (e.g., a 6-FAMTM fluorescent label), can be used in an TAQMAN® PCR assay to identify the zygosity of DNA derived from cotton event GH_BCS246002 or a modified cotton event GH_BCS246002.
  • PCR assays for zygosity determination were developed for cotton event GH_BCS246002 in a sample. Parameters of either a standard PCR assay or a TAQMAN® PCR assay were optimized with each set of primer pairs and probes (e.g., probes labeled with a fluorescent tag such as 6-FAMTM ) used to detect the presence of DNA derived from cotton event GH_BCS246002 in a sample.
  • probes e.g., probes labeled with a fluorescent tag such as 6-FAMTM
  • a control for the PCR reaction can include internal control primers and an internal control probe (e.g., VlC®-labeled) specific to a region within the cotton genome that is used as an internal control and are primers SQ22496 (SEQ ID NO: 18), SQ22497 (SEQ ID NO: 19), and VIC® labeled probe PB50562 (SEQ ID NO: 19).
  • an internal control probe e.g., VlC®-labeled
  • the parameters which were optimized for determination of zygosity for cotton event GH_BCS246002 in a sample included primer and probe concentration, amount of templated DNA, and PCR amplification cycling parameters.
  • the controls for this analysis include a positive control from cotton containing cotton event GH_BCS246002 which is homozygous, a negative control from non-transgenic cotton, and a negative control that contains no template DNA.
  • the cycle thresholds are determined for the amplicon corresponding to the Vip3Cbl.l coding sequence within the cotton event GH_BCS246002 inserted allele and the single-copy, homozygous internal standard.
  • the difference (ACt) between the Ct value of the single-copy, homozygous internal standard amplicon, and the Ct value of the Vip3Cbl.l amplicon derived from the cotton event GH_BCS246002 inserted allele are determined.
  • a ACt of around zero (0) indicates homozygosity of the inserted cotton event GH_BCS246002 T-DNA and ACt of around one (1) indicated heterozygosity of the inserted cotton event GH_BCS246002 T-DNA.
  • Lack of an amplicon corresponding to the cotton event GH_BCS246002 inserted allele indicates the sample is null for the inserted cotton event GH_BCS246002 T-DNA.
  • the Ct values in the TAQMAN® thermal amplification method will have some variability due to multiple factors such as amplification efficiency and ideal annealing temperatures. Therefore, the range of “about one (1)” is defined as a ACt of 0.75 to 1.25.
  • Similar methods could be used to detect or determine the zygosity of a modified cotton event GH_BCS246002, although a different primer(s), probe and/or other parameters may need to be used depending on the genetic modification(s) present with the modified cotton event GH_BCS246002.

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Abstract

The present disclosure provides a transgenic cotton event GH_BCS246002, plants, plant cells, seed, plant parts, progeny plants, commodity products comprising event GH_BCS246002, polynucleotides specific for defining and detecting event GH_BCS246002 and plants, plant cells, seed, plant parts, progeny plants, and commodity products comprising event GH_BCS246002; and methods related to detection, characterization, and selection of event GH_BCS246002. Polynucleotides and sequences specific for a modified cotton event GH_BCS246002 and methods for making and using plants, plant cells, seeds, plant parts, progeny plants, and commodity products comprising a modified cotton event GH_BCS246002 and detecting a modified cotton event GH_BCS246002, or a polynucleotide or DNA sequence specific for a modified cotton event GH_BCS246002, in a DNA molecule or sample, are also provided.

Description

COTTON TRANSGENIC EVENT GH_BCS246002 AND METHODS FOR DETECTION AND USES THEREOF
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Application Ser. No. 63/645,146, filed May 10, 2024, which is incorporated herein by reference in its entirety.
INCORPORATION OF SEQUENCE LISTING
[0002] The sequence listing contained in the file named MONS594WO_ST26.xml is 314,503 bytes (measured in Microsoft Windows®), was created on February 25, 2025, is filed herewith by electronic submission, and is incorporated by reference.
FIELD OF THE INVENTION
[0003] The present invention relates to transgenic cotton plants, plant parts, seeds, cells and agricultural products containing cotton event GH_BCS246002 or a modified cotton event GH_BCS246002, recombinant DNA molecules present in and/or isolated from cotton event GH_BCS246002 or a modified cotton event GH_BCS246002, as well as methods of using the same and detecting the presence of DNA for a cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 in a sample. The present invention also relates to transgenic cotton plants, plant parts, seed and cells containing cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 DNA that exhibit resistance to Lepidopteran insect infestations. The present invention also relates to methods for modifying the cotton event GH_BCS246002 using genome editing techniques to produce a modified cotton event GH_BCS246002.
BACKGROUND
[0004] Cotton is an important crop in many areas of the world. The methods of biotechnology have been applied to cotton for improvement of the agronomic traits and quality of the product. One such agronomic trait is insect resistance, which is accomplished through the expression of heterologous insect toxins, also known as transgenes, inserted into the genome of the cotton plant. [0005] There arc a number of different transgenic events in cotton that have been described in the art that provide various types of insect resistance, particularly to Lepidopteran species, and these include, MON531, MON 15985, DAS-24236 (281 -24-236), DAS-21023 (3006-210-23), COT102, GHB 119, and T304-40. The Lcpidoptcran resistance trait conferred by these transgenic events have been in use commercially in a variety of geographies across the globe for an extended period of time, and resistance to many of the expressed toxins in these events by targeted insect pests has developed in many of the geographic regions where these transgenic traits have been deployed.
[0006] Thus, there is a continuing need in the art to provide novel transgenic events in cotton that confer novel traits for controlling Lepidopteran target pest species, and preferably the novel transgenic events confer resistance to the target insect pests, including those races that have evolved resistance to the existing commercially deployed traits. Such transgenic traits will preferably use modes of action that are different than the modes of action previously deployed in existing commercial embodiments. Described herein is an example of a novel transgenic event in cotton that confers resistance to Lcpidoptcran infestations, including resistance to Lcpidoptcrans that have evolved resistance to commercial embodiments that have been previously deployed.
SUMMARY
[0007] In one aspect, a novel transgenic cotton event, GH_BCS246002 or a modified cotton event GH_BCS246002, that provides insecticidal control over Lcpidoptcran pests of cotton is provided. In a further aspect, transgenic plants, plant cells, seed, plant parts, and commodity products are provided comprising cotton event GH_BCS246002 DNA or a modified cotton event GH_BCS246002 DNA. Novel DNA molecules and constructs that are specific and unique to the event GH_BCS246002 or a modified cotton event GH_BCS246002 event and comprise the inserted transgenic DNA segment and the novel DNA segments that are adjacent to the inserted transgenic DNA segment. Adjacent DNA segments are described herein as the junction sequences which correspond to the DNA sequences spanning the flanking chromosomal or genomic DNA and a portion of the adjacent transgenic DNA insert at or near a chromosomal breakpoint where the transgenic inserted DNA has been introduced into the genome of a cotton plant. The orientation of the inserted DNA (SEQ ID NO: 9) is specified herein in reference to the adjacent flanking chromosomal or genomic DNA. Because the transgenic DNA insert is a linear arrangement of elements with a 5’ end and 3’ end relative to the orientation of the insert and expression cassettes and the surrounding genome as depicted, for example, in FIGs. 1 and 2. In some embodiments, the 5’ junction is represented herein by the sequence as set forth in SEQ ID NO: 1 , while the 3’ junction is represented herein by the sequence as set forth in SEQ ID NO: 2, but longer sequences described herein arc also examples of both junction sequences, although SEQ ID NO: 1 or SEQ ID NO: 2, as applicable, are included within the respective longer sequences. For example, 5’ junction sequences containing SEQ ID NO: 1 include but are not limited to SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 7, and 3’ junction sequences containing SEQ ID NO: 2 include but are not limited to SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8.
[0008] In another aspect, polynucleotides and molecules are provided that are specific and unique to, and capable of use for identifying and detecting the presence of cotton event GH_BCS246002 DNA or a modified cotton event GH_BCS246002 DNA in a biological sample containing or derived from a cotton plant, plant part, tissue, cell or other plant material, such progeny plants and commodity products, comprising cotton event GH_BCS246002 DNA or a modified cotton event GH_BCS246002 DNA. In yet another aspect, methods related to selecting a plant, plant part, plant cells and/or seed comprising the cotton event GH_BCS246002 DNA or a modified cotton event GH_BCS246002 DNA, and detection of the presence (or absence) of cotton event GH_BCS246002 DNA or a modified cotton event GH_BCS246002 DNA in a sample are provided, such methods provided for the purpose of confirming that the event GH_BCS246002 DNA is, or is not, present in a particular sample subjected to the method or to methods reliant upon polynucleotide sequences that are the subject of this disclosure. For example, detection of a polynucleotide sequence in a sample from cotton tissue, cells, seed, plants, or plant parts that contain cotton genomic DNA and one or both of SEQ ID NO: 1 or SEQ ID NO: 2, is determinative of the presence of the cotton event GH_BCS246002 DNA in said sample.
[0009] In an aspect, the present disclosure provides recombinant DNA molecules comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, and a complete complements thereof. It is understood that a homologous or complementary RNA sequence corresponding to any of the foregoing polynucleotide sequences could also be used for similar purposes and would include a uridine residue in place of any thymidine residue depicted in the applicable DNA provided herein and complements thereof, and references to DNA herein may also be applied to a corresponding RNA sequence and complements thereof. [0010] In an aspect, the recombinant DNA molecules provided herein may be derived from cotton event GH_BCS246002 in a sample of seed containing the event and containing the unique and specific DNA segments as provided herein corresponding to cotton event GH_BCS246002 seed which has been deposited as ATCC Accession No. PTA-127733. room Another aspect of the present disclosure provides a DNA molecule comprising a polynucleotide segment of sufficient length to function as a nucleic acid probe that hybridizes specifically under stringent hybridization conditions with cotton event GH_BCS246002 DNA in a sample, wherein detecting hybridization of the probe to the event DNA in the sample under the stringent hybridization conditions is diagnostic for, or characteristic of, confirming the presence of cotton event GH_BCS246002 DNA in that sample. Similarly, provided that the cotton DNA in the sample does not contain the event GH_BCS246002 DNA, an absence of detection of hybridization of the probe to the cotton DNA could be used to demonstrate the absence of the event in the sample. In certain embodiments, the sample may comprise whole or process cotton seeds, cotton fiber, cotton oil and derivatives of cotton oil, cotton protein, cotton meal, animal feed comprising cotton, paper comprising cotton, cotton biomass, candle wicks, cotton string, cotton rope, cotton balls, cotton batting, cotton fuel products, and cotton cellulose products, wherein such cotton and cotton products contain detectable amounts of cotton event GH_BCS246002 DNA or detectable amounts of the novel toxin proteins produced by such transgenic cotton plants, cells and the like, to contain the cotton event GH_BCS246002.
[0012] Yet another aspect of the invention provides a pair of DNA molecules comprising a first DNA molecule and a second DNA molecule different from the first DNA molecule, wherein the pair of DNA molecules can be used together as DNA primers in an amplification reaction containing the appropriate and necessary reagents with a sample that may or may not contain cotton event GH_BCS246002 template DNA to produce an amplicon diagnostic for, or characteristic of, the presence of said cotton event GH_BCS246002 DNA if present in said sample. According to many embodiments, the amplicon produced will contain at least the nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, and the complete complements thereof.
[0013] Another aspect of the present disclosure provides a method of detecting or determining the presence or absence of a DNA segment diagnostic for confirming the presence or absence of cotton event GH_BCS246002 DNA in a sample. In certain embodiments, methods are provided that may be conducted by contacting the sample with a probe DNA molecule that hybridizes specifically to DNA uniquely associated with cotton event GH_BCS246002, then subjecting the sample and the probe DNA molecule to stringent hybridization conditions to allow the probe to bind to the appropriate complement segment of cotton event GH_BCS246002 specific DNA. Detecting hybridization of the probe DNA molecule to the DNA in the sample is conclusive, diagnostic, or determinative that the DNA in the sample contains the cotton event GH_BCS246002 DNA. In other embodiments, methods are provided that may comprise the steps of contacting a biological sample with a pair of DNA molecules that function as nucleic acid primers specific for amplification of a DNA segment specific for the cotton event GH_BCS246002 DNA, performing an amplification reaction sufficient to produce the DNA amplicon, and detecting the presence or absence of the DNA amplicon in the reaction. Detection of the DNA amplicon is diagnostic for, or characteristic of, the presence of a detectable amount of the cotton event GH_BCS246002 DNA in the sample, and the amplicon should contain all or a portion of the nucleotide sequence targeted for amplification that lies between the primer hybridization positions, which may include a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, and the complete complements thereof. On the other hand, the lack of detection of the DNA amplicon would be diagnostic for, or characteristic of, the absence of at least a detectable amount of the cotton event GH_BCS246002 DNA in the sample.
[0014] Another embodiment of the invention is a method of detecting the presence of protein diagnostic for or characteristic of cotton event GH_BCS246002 in a sample, said method comprising an immunoassay in which one or more of the antibodies specific for the respective toxin proteins encoded by one or more of the cassettes of cotton event GH_BCS246002 to detect the presence or absence of one or more of the toxins produced by the cotton event, the method comprising: (a) contacting said sample with a first, second, and/or third monoclonal antibody, wherein the first monoclonal antibody binds specifically to one or more epitopes of Cry IB.3 toxin protein, the second monoclonal antibody binds specifically to one or more epitopes of CrylDa_7 toxin protein, and the third monoclonal antibody binds specifically to one or more epitopes of Vip3Cbl .1 toxin protein; (2) incubating the immunoassay for a sufficient amount of time to allow for binding of the one or more monoclonal antibodies; and (3) detecting the presence of the Cry IB.3, CrylDa_7, and/or Vip3Cbl .l toxin proteins in said immunoassay, wherein said detection is diagnostic for, or characteristic of, the presence of said cotton event GH_BCS246002 DNA in said sample. The assay can be selected from the group consisting of an Enzyme-linked Immunosorbent Assay (ELISA), a Radioimmunoassay, and a Lateral flow immunochromatographic assay. The detection step can be colorimetric, radiologic, or fluorescent relative to a control samples, such as a negative control and/or a positive control for the detecting the presence of the respective toxin protein.
[0015] Another embodiment of the invention is a cotton plant, cotton plant part, cotton cell, or part thereof comprising a recombinant polynucleotide molecule comprising the nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, and the complete complement thereof. These SEQ ID NOs arc each the specific and selective DNA that defines the cotton event GH_BCS246002. This cotton plant, cotton plant part, cotton cell, or part thereof is insecticidal when provided in the diet of a Lepidopteran insect pest. Lepidopteran insect target pests intended to be controlled include Cotton bollworm (Helicoverpa zed), Tobacco budworm (Heliothis virescens), and/or Fall armyworm (Spodopiera frugiperdd), and any combination of any two or all three of the foregoing pest species. In addition, the cotton plant can be further defined as progeny of any generation of a cotton plant comprising the cotton event GH_BCS246002, provided that the progeny also contains the specific and selective DNA that defines the cotton event GH_BCS246002.
[0016] Yet another aspect of the present disclosure provides methods for protecting a cotton plant from insect infestation, wherein said method comprises providing in the diet of a Lepidopteran insect pest an insecticidally effective amount of cotton cells or tissue of a cotton plant comprising cotton event GH_BCS246002. Contemplated Lepidopteran insect pests include Cotton bollworm (Helicoverpa zea), Tobacco budworm (Heliothis virescens), and Fall armyworm (Spodoptera frugiperdd).
[0017] Another aspect of the present disclosure provides methods of producing an insect resistant cotton plant comprising: a) manually crossing by conventional breeding, two different cotton plants to produce progeny, wherein at least one of the two different cotton plants contains the cotton event GH_BCS246002 DNA or DNA from a modified cotton event GH_BCS246002; b) confirming in the seed arising from the breeding activity, and in the progeny plants and tissue grown from such seed, the presence of a DNA segment diagnostic for, or characteristic of, cotton event GH_BCS246002 or a modified cotton event GH_BCS246002; and c) selecting the progeny comprising the cotton event GH_BCS246002 DNA or DNA from a modified cotton event GH_BCS246002. Such seed and progeny are Lepidopteran resistant cotton plants.
[00181 A further aspect of the present disclosure provides cotton seeds, nonliving plant materials, or microorganisms comprising a detectable amount of the polynucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, and complete complements thereof.
[0019] Yet another aspect of the present disclosure is a commodity cotton product comprising a detectable amount of a recombinant DNA molecule unique to cotton event GH_BCS246002, wherein the recombinant DNA molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, and complete complements thereof. Commodity cotton products may include, but are not limited to, whole or process cotton seeds, cotton fiber, cotton oil and derivatives of cotton oil, cotton protein, cotton meal, animal feed comprising cotton, paper comprising cotton, cotton biomass, candle wicks, cotton string, cotton rope, cotton balls, cotton batting, cotton fuel products, and cotton cellulose products.
[0020] Another aspect of the present disclosure is a cotton plant, cotton plant part, cotton seed, cotton tissue or cell comprising a DNA molecule functional as a template in a DNA amplification assay to produce an amplicon diagnostic for, or characteristic of, the presence of cotton event GH_BCS246002 DNA.
[0021] Yet another aspect of the present disclosure are methods of determining the zygosity of a cotton plant, plant part or seed comprising DNA specific to the cotton event GH_BCS246002 or a modified cotton event GH_BCS246002. The zygosity can be determined in a series of consecutive steps. In a first step, a sample comprising cotton DNA can be contacted with a first primer pair that is capable of producing an amplicon in a DNA amplification assay that is diagnostic for, or characteristic of, DNA that is specific for and uniquely present in cotton event GH_BCS246002. The sample comprising the cotton DNA can be contacted with a second primer pair that is designed to produce an amplicon of an internal standard known to be single-copy and homozygous in the cotton plant. Such methods may additionally include contacting the DNA sample with a probe set which contains at least a first probe that specifically hybridizes to a cotton event GH_BCS246002 DNA, and a second probe that specifically hybridizes to an internal genomic DNA standard known to be single-copy and homozygous in the cotton plant. The DNA amplification reaction in these methods may be performed using real-time PCR to determine the cycle thresholds (Ct values) of the amplicon corresponding the allele of cotton event GH_BCS246002 and the single-copy, homozygous internal standard. After the amplification, the difference (delta-Ct) between the Ct value of the single-copy, homozygous internal standard amplicon, and the Ct value of the allele for cotton event GH_BCS246002 amplicon may be calculated. In one embodiment, zygosity is determined wherein a delta-Ct of about zero (0) indicates homozygosity of the inserted T-DNA of cotton event GH_BCS246002, and a delta-Ct of about one (1) indicates heterozygosity of the inserted T-DNA of cotton event GH_BCS246002. In certain embodiments, the primer pairs are selected from the group consisting of SEQ ID NO: 15 combined with SEQ ID NO: 16, and SEQ ID NO: 18 combined with SEQ ID NO: 19; and wherein the probes are SEQ ID NO: 17 and SEQ ID NO: 20. In yet other embodiments the delta-Ct of about one (1) indicating heterozygosity of the inserted T-DNA of cotton event GH_BCS246002 is in the range of 0.75 to 1.25. In certain embodiments, a delta-Ct of about zero (0) may be about 0, 0.05, 0.1, 0.15, 0.2, or 0.25, in other embodiments, a delta-Ct of about one (1) may be about 0.75, 0.8, 0.85, 0.9, 0.95, 1.0, 1.05, 1.1, 1.15, 1.2, or 1.25. In a further embodiment, a delta-Ct of about one (1) may be in the range of 0.75 to 1.25, 0.8 to 1.25, 0.85 to 1.25, 0.9 to 1.25, 0.95 to 1.25, 1.0 to 1.25, 1.05 to 1.25, 1.1 to 1.25, 1.15 to 1.25, 1.2 to 1.25, 0.75 to 1.2, 0.8 to 1.2, 0.85 to 1.2, 0.9 to 1.2, 0.95 to 1.2, 1.0 to 1.2, 1.05 to 1.2, 1.1 to 1.2, 1.15 to 1.2, 0.75 to 1.15, 0.8 to 1.15, 0.85 to 1.15, 0.9 to 1.15, 0.95 to 1.15, 1.0 to 1.15, 1.05 to 1.15, 1.1 to 1.15, 0.75 to 1.1, 0.8 to 1.1, 0.85 to 1.1, 0.9 to 1.1, 0.95 to 1.1, 1.0 to 1.1, 1.05 to 1.1, 0.75 to 1.05, 0.8 to 1.05, 0.85 to 1.05, 0.9 to 1.05, 0.95 to 1.05, 1.0 to 1.05, 0.75 to 1.0, 0.8 to 1.0, 0.85 to 1.0, 0.9 to 1.0, 0.95 to 1.0, 0.75 to 0.95, 0.8 to 0.95, 0.85 to 0.95, 0.9 to 0.95, 0.75 to 0.9, 0.75 to 0.85, 0.75 to 0.8, 0.8 to 0.9, 0.8 to 0.85, or 0.85 to 0.9.
[0022] A further aspect of the present disclosure provides methods of determining the zygosity of a cotton plant, plant part, seed, pollen, ovum, tissue or cell, in which such biological material from cotton suspected of containing cotton event GH_BCS246002 DNA may be subjected to the following method: (a) contacting a sample containing cotton DNA obtained from the biological material with at least two different sets of primers, in which (i) a first primer pair consisting of a first primer and a second primer (different from the first primer) that, when used in an amplification reaction with the cotton DNA containing sample, arc capable of producing a first amplicon diagnostic for, or characteristic of, the presence of cotton event GH_BCS246002 DNA in the sample, and (ii) a second primer pair consisting of the first primer and a third primer (different from the first primer and from the second primer) that, when used in a DNA amplification reaction with the cotton DNA, are capable of producing a second amplicon diagnostic for, or characteristic of, native cotton genomic DNA which does not contain or include DNA specific for the cotton event GH_BCS246002; (b) performing a nucleic acid amplification reaction with the sample and the two primer pairs; (c) detecting in the amplification reaction the first amplicon diagnostic for, or characteristic of, cotton event GH_BCS246002, and/or the second amplicon diagnostic for, or characteristic of, native cotton genomic DNA not comprising cotton event GH_BCS246002, wherein the presence of only the first amplicon is diagnostic of, or characteristic of, a homozygous cotton event GH_BCS246002 DNA in a sample, and the presence of both the first amplicon and the second amplicon is diagnostic of, or characteristic of, a cotton plant heterozygous for cotton event GH_BCS246002 allele. Alternatively, step (b) may comprise contacting a sample comprising cotton DNA from a cotton biological material with a probe set which contains at least a first probe that specifically hybridizes to DNA specific for cotton event GH_BCS246002 and at least a second probe that specifically hybridizes to cotton genomic DNA spanning the segment of chromosomal DNA that was disrupted by insertion of the heterologous transgenic DNA insert (SEQ ID NO: 9) of cotton event GH_BCS246002 and which does not hybridize to DNA from cotton event GH_BCS246002 DNA, and then hybridizing the probe set with a sample under stringent hybridization conditions, wherein detecting the hybridization of only the first probe under the hybridization conditions is diagnostic for, or characteristic of, a homozygous allele of cotton event GH_BCS246002 DNA in the sample, and wherein detecting hybridization of both the first probe and the second probe under the hybridization conditions is diagnostic for, or characteristic of, a heterozygous allele of cotton event GH_BCS246002 in said sample. In some embodiments, the set of primer pairs may comprise SEQ ID NO: 15 combined with SEQ ID NO: 16, and SEQ ID NO: 15 combined with SEQ ID NO: 21. In other embodiments, the probe set may comprise SEQ ID NO: 17 and SEQ ID NO: 22.
[0023] Another aspect of the present disclosure provides a recombinant DNA molecule comprising: (a) a first nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8; and (b) a second nucleotide sequence that (i) comprises at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1,000, at least 1,500, or at least 2,000 consecutive nucleotides of SEQ ID NO: 11 or 43, or (ii) is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% identical to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1,000, at least 1,500, or at least 2,000 consecutive nucleotides of SEQ ID NO: 11 or 43, or (iii) is selected from the group consisting of SEQ ID NOs: 44-144. The recombinant DNA molecule can further comprise: (c) a third nucleotide sequence that (i) comprises at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1,000, at least 1,500, or at least 2,000 consecutive nucleotides of SEQ ID NO: 12 or 44, or (ii) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% identical to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1,000, at least 1,500, or at least 2,000 consecutive nucleotides of SEQ ID NO: 12 or 44, or (iii) is selected from the group consisting of SEQ ID NOs: 145-244. The recombinant DNA molecule can have a deletion of one or more consecutive nucleotides between the second nucleotide sequence and the nucleotide sequence relative to SEQ ID NO: 10, 12, or 44. The second recombinant DNA molecule is selected from the group consisting of SEQ ID NOs: 45-144. The recombinant DNA molecule can have a deletion of one or more consecutive nucleotides between the first nucleotide sequence and the second nucleotide sequence relative to SEQ ID NO: 10, 11, or 43.
[00241 Another aspect of the present disclosure provides a recombinant DNA molecule comprising: (a) a first nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9; and (b) a second nucleotide sequence that (i) comprises at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least
18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least
100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1,000, at least 1,500, or at least 2,000 consecutive nucleotides of SEQ ID NO: 12 or 49, or (ii) is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% identical to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least
19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1,000, at least 1,500, or at least 2,000 consecutive nucleotides of SEQ ID NO: 12 or 44, or (iii) is selected from the group consisting of SEQ ID NOs: 45-144. The recombinant DNA molecule can have a deletion of one or more consecutive nucleotides between the first nucleotide sequence and the second nucleotide sequence relative to SEQ ID NO: 10, 12, or 44. The recombinant DNA molecule further comprises nucleotides 1-1,000 or 16,130-17,129 of SEQ ID NO: 10. The recombinant DNA molecule is comprised in a cotton plant, cotton plant part, cotton plant cell, cotton plant seed, cotton progeny plant, or commodity product made from cotton and cotton plant parts. The recombinant further comprises an amplicon diagnostic for the presence of DNA comprising a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9. The recombinant DNA molecule is derived from a cotton plant, cotton plant part, cotton seed, processed cotton seed, cotton plant cell or tissue, cotton fiber, cotton oil and derivatives of cotton oil, cotton protein, cotton meal, animal feed comprising cotton, paper comprising cotton, cotton biomass, candle wicks, cotton string, cotton rope, cotton balls, cotton batting, cotton fuel products, and cotton cellulose products.
[00251 Another aspect of the present disclosure is a recombinant DNA molecule comprising a polynucleotide segment of sufficient length to function as a DNA probe that hybridizes specifically under stringent hybridization conditions with a polynucleotide having a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 43, and SEQ ID NO: 44.
[0026] A further embodiment of the invention are a pair of DNA molecules that can function as DNA primers when used together in an amplification reaction comprising a first DNA molecule and a second DNA molecule, wherein the first DNA molecule and the second DNA molecule are different, (a) wherein the first DNA molecule is: (i) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% identical or 100% identical to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 consecutive nucleotides of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, or a complement thereof, or (ii) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% identical or 100% identical to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 consecutive nucleotides of SEQ ID NO: 11 or 43, or a complement thereof; and (b) wherein the second DNA molecule is: (i) at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% identical or 100% identical to at least 10, at least 1 1 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 consecutive nucleotides of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, or a complement thereof, or (ii) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% identical or 100% identical to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 consecutive nucleotides of SEQ ID NO: 11 or 43, or a complement thereof, or (iii) at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% identical or 100% identical to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 consecutive nucleotides of SEQ ID NO: 12 or 44, or a complement thereof. The first DNA molecule and the second DNA molecule can be used together in an amplification reaction with a sample from a plant, plant part, plant seed, plant cell, food or animal feed, or commodity or fuel product made from a plant or plant part to produce an amplicon diagnostic for the presence of a modified cotton event GH_BCS246002 DNA in said sample. The resulting amplicon comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10.
[0027] A further aspect of the present disclosure provides methods of detecting or determining the presence or absence of a DNA segment diagnostic for a modified cotton event GH_BCS246002 DNA in a sample, which may be unique to the modified cotton event GH_BCS246002 DNA, said method comprising: (a) contacting said sample with a DNA probe that binds or hybridizes specifically to the modified cotton event DNA; (b) subjecting said sample and said DNA probe to stringent hybridization conditions; and (c) detecting hybridization of said DNA probe to said DNA segment in said sample, wherein said detection is diagnostic for the presence of said modified cotton event GH_BCS246002 DNA in said sample.
[0028] Another aspect of the present disclosure provides methods for detecting or determining the presence or absence of a DNA segment diagnostic for a modified cotton event GH_BCS246002 DNA in a sample, said method comprising: (a) contacting said sample with the pair of DNA molecules that can function as DNA primers when used together in an amplification reaction comprising a first DNA molecule and a second DNA molecule, wherein the first DNA molecule and the second DNA molecule are different (b) performing an amplification reaction sufficient to produce a DNA amplicon; and (c) detecting the presence of said DNA amplicon in said reaction, wherein the presence of said DNA amplicon is diagnostic for the presence of said modified cotton event GH_BCS246002 DNA in said sample.
[0029] A further aspect of the present disclosure provides methods of detecting the presence of a DNA segment diagnostic for a modified cotton event GH_BCS246002 DNA in a sample, said method comprising performing a sequencing reaction with a sample, wherein the production in the sequencing reaction of a target nucleotide sequence comprises at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 consecutive nucleotides of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:
4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, or a complement thereof, is diagnostic for the modified cotton event GH_BCS246002 DNA in a sample.
[0030] Another aspect of the present disclosure provides a modified cotton plant, cotton plant part, cotton seed, cotton tissue or cotton cell comprising a modified cotton event GH_BCS246002. The modified cotton plant, cotton plant part, cotton seed, cotton tissue or cotton cell comprises (a) a recombinant DNA molecule or DNA segment comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO:
5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, or a complement thereof; or (b) a recombinant DNA molecule or DNA segment comprising a nucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical to at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1,000, at least 1,500, or at least 2,000 consecutive nucleotides of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 43, or SEQ ID NO: 44, or a complement thereof. The modified cotton plant, cotton plant part, cotton seed, cotton tissue or cotton cell may be further defined as a progeny plant of any generation of a cotton plant comprising a modified cotton event GH_BCS246002, or a cotton plant part, cotton seed, or cotton cell derived therefrom. The modified cotton plant, cotton plant part, cotton seed, cotton tissue or cotton cell may exhibit resistance to a Lepidopteran insect pest species. [0031] Another aspect of the present disclosure provides a DNA kit comprising: (a) the DNA probes as described herein; and (b) the pair(s) of DNA primers as described herein.
[0032] A further aspect of the present disclosure provides methods of producing a progeny cotton plant comprising a modified cotton event GH_BCS246002 comprising: (a) sexually crossing a first modified cotton plant that comprises a modified cotton event GH_BCS246002 with itself or a second cotton plant; (b) collecting one or more seeds produced from said cross; (c) growing said seed to produce one or more progeny plants; and (d) selecting at least a first progeny plant or seed comprising a modified cotton event GH_BCS246002. The method can be used to make a hybrid modified cotton plant or seed comprising a modified cotton event GH_BCS246002. The method may further comprise: (e) collecting seed from said at least first progeny plant comprising a modified cotton event GH_BCS246002.
[0033] Another aspect of the present disclosure provides a nonliving, non-viable or non- regenerable cotton plant material comprising a detectable amount of the recombinant DNA molecule of a modified cotton event GH_BCS246002 as described herein.
[0034] In yet another aspect of the present disclosure, a recombinant DNA molecule of a modified cotton event GH_BCS246002 is provided as described herein, which may be comprised within a host cell, such as a cotton cell or a microorganism (e.g., a bacterial cell). Inn present embodiments, the microorganism can be a plant cell. [0035] In a further aspect of the present disclosure a commodity product is provided comprising a recombinant DNA molecule of a modified cotton event GH_BCS246002 as described herein. The commodity product is produced from a modified cotton plant or a modified cotton plant part, cotton seed, or cotton tissue or cell comprising a modified cotton event GH_BCS246002. The commodity product may be further selected from the group consisting of whole or processed cotton seed, animal feed comprising cotton, cotton oil, cotton meal, cotton flour, cotton flakes, cotton bran, cotton biomass, and fuel products produced using cotton and cotton plant parts.
[0036] Another aspect of the present disclosure provides methods of producing a commodity product, said method comprising: (a) obtaining a modified cotton plant, cotton plant part, or cotton seed comprising a modified cotton event GH_BCS246002; and (b) producing a commodity product from the transgenic cotton plant, cotton plant part, or cotton seed.
[0037] In a further aspect, the present disclosure provides a cotton plant, cotton plant pail, or cotton seed comprising a DNA molecule or segment functional as a template in a DNA amplification method to produce an amplicon diagnostic for the presence of a modified cotton event GH BCS246002 DNA.
[0038] A further aspect of the present disclosure provides methods of determining the zygosity of a cotton plant, cotton plant part, or cotton seed comprising a modified cotton event GH_BCS246002 comprising: (a) contacting a sample comprising DNA from the cotton plant, cotton plant pail, or cotton seed with a primer pair capable of producing a first amplicon diagnostic for the modified cotton event GH_BCS246002 and a second amplicon diagnostic for native cotton genomic DNA not comprising the modified cotton event GH_BCS246002; (b) performing a nucleic acid amplification reaction with the sample and the set of primer pairs; and (c) detecting the first amplicon and the second amplicon, wherein the presence of only the first amplicon is diagnostic of a cotton plant, cotton plant part, or cotton seed homozygous for the modified cotton event GH_BCS246002, the presence of only the second amplicon is diagnostic of a cotton plant, cotton plant part, or cotton seed homozygous for native cotton genomic DNA not comprising the modified cotton event GH_BCS246002, and the presence of both the first amplicon and the second amplicon is diagnostic of a cotton plant, cotton plant part, or cotton seed heterozygous for the modified cotton event GH_BCS246002.
[0039] Another aspect of the present disclosure provides methods for determining the zygosity of a cotton plant, cotton plant part, or cotton seed comprising a modified cotton event GH_BCS246002 comprising: (a) contacting a sample comprising DNA from the cotton plant, cotton plant part, or cotton seed with a probe set which contains at least a first probe that specifically hybridizes to the modified cotton event GH_BCS246002 and at least a second probe that specifically hybridizes to cotton genomic DNA that was disrupted by insertion of the heterologous DNA of cotton event GH_BCS246002 and is disrupted by the modified cotton event GH BCS246002 DNA, wherein the second probe does not hybridize to the modified cotton event GH_BCS246002 DNA; and (b) hybridizing the probe set with the sample under stringent hybridization conditions, wherein detecting hybridization of only the first probe under the hybridization conditions is diagnostic for a cotton plant, cotton plant part, or cotton seed homozygous for the modified cotton event GH_BCS246002, and wherein detecting hybridization of both the first probe and the second probe under the hybridization conditions is diagnostic for a cotton plant, cotton plant part, or cotton seed heterozygous for the modified cotton event GH_BCS246002. The probe set comprises SEQ ID NO: 17 and SEQ ID NO: 22.
[0040] Another aspect of the present disclosure provides a population of transgenic cotton plants, wherein each transgenic cotton plant comprises a modified cotton event GH_BCS246002. Each of the transgenic cotton plants of the population may exhibit resistance to one or more Lepidopteran insect pest species.
[0041] A further aspect of the present disclosure provides a method of modifying a cotton plant, the method comprising: (a) introducing a site-specific nuclease or a recombinant DNA construct comprising an expression cassette encoding a sit-specific nuclease into at least one cell of an explant of a cotton plant comprising cotton event GH_BCS246002, or a plant part thereof, to produce a modified cotton event GH_BCS246002 via a genome editing technique; and (b) developing or regenerating a modified cotton plant from the explant, wherein the modified cotton plant comprises the modified cotton event GH_BCS246002. The site-specific nuclease is a zinc- finger nuclease (ZFN), a meganuclease, an RNA-guided endonuclease, a TALE-endonuclease (TALEN), a recombinase, or a transposase. In an embodiment of the method, the site-specific nuclease is an RNA-guided endonuclease or a CRISPR/Cas nuclease. In an embodiment of the method the introducing step (a) comprises introducing the recombinant DNA construct into the at least one cell of the explant, and wherein the recombinant DNA construct further comprises an expression cassette encoding a first guide RNA (gRNA). The recombinant DNA construct further comprises an expression cassette encoding a second guide RNA (gRNA). In another embodiment of the method the introducing step (a) further comprises introducing a first guide RNA (gRNA) or a second recombinant DNA construct comprising an expression cassette encoding a first guide RNA (gRNA) into the at least one cell of the explant. In another embodiment of the method the introducing step (a) comprises introducing at least two guide RNAs (gRNAs) comprising the first gRNA and a second gRNA into the at least one cell of the explant. The second recombinant DNA construct further comprises an expression cassette encoding a second guide RNA (gRNA). In a further aspect of the method, the introducing step (a) comprises introducing a first gRNA and second recombinant DNA construct comprising an expression cassette encoding a second guide RNA (gRNA) into the at least one cell of the explant. The site-specific nuclease has a first target site in the genome of the cotton plant at or near cotton event GH_BCS246002. The site-specific nuclease has a second target site in the genome of the cotton plant at or near cotton event GH_BCS246002. In another embodiment of the method, the introducing step (a) comprises introducing a second site-specific nuclease or a recombinant DNA construct comprising an expression cassette encoding a second site specific nuclease into at least one cell of the explant, and wherein the second site-specific nuclease has a second target site in the genome of the cotton plant at or near cotton event GH_BCS246002. In one embodiment of the method, the first gRNA has a first target site in a flanking DNA sequence, 5 ' flank, 3 ' flank, junction sequence, or insertion sequence of cotton event GH_BCS246002, or a complement thereof. In a further embodiment of the method, the first gRNA has a first target site comprising a target sequence that is: (i) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 consecutive nucleotides of SEQ ID NO: 11 or 43, or a complement thereof; or (ii) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 consecutive nucleotides of SEQ ID NO: 12 or 44, or a complement thereof; or (iii) at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 consecutive nucleotides of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, or a complement thereof. In another aspect of the method, the first gRNA has a second target site comprising a target sequence that is: (i) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 consecutive nucleotides of SEQ ID NO: 11 or 43, or a complement thereof; or (ii) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 consecutive nucleotides of SEQ ID NO: 12 or 44, or a complement thereof; or (iii) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 consecutive nucleotides of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, or a complement thereof. In a further embodiment of the method, (i) wherein the first gRNA has a first target site comprising a target sequence that is: (1 ) at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 consecutive nucleotides of SEQ ID NO: 11 or 43, or a complement thereof; or (2) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 consecutive nucleotides of SEQ ID NO: 12 or 44, or a complement thereof; or (3) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 consecutive nucleotides of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, or a complement thereof; and (ii) wherein the second gRNA has a second target site comprising a target sequence that is: (1) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 consecutive nucleotides of SEQ ID NO: 11 or 43, or a complement thereof; or (2) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical to at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 consecutive nucleotides of SEQ ID NO: 12 or 44, or a complement thereof; or (3) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 consecutive nucleotides of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, or a complement thereof. In a further aspect of the method, the modified cotton event GH_BCS246002 comprises a deletion or excision of intervening genomic DNA between the first target site and the second target site, relative to the cotton event GH_BCS246002. In another aspect of the method, the method further comprises: (c) selecting the modified cotton plant comprising the modified cotton event GH_BCS246002; and (d) sexually crossing the modified cotton plant with itself or a second cotton plant to produce one or more modified progeny cotton plants.
[0042] Another aspect of the present disclosure provides methods of introducing a target site into a cotton plant, the method comprising: (a) introducing a cognate target site into the cotton event GH_BCS246002 locus of at least one cell of a cotton plant or cotton plant part comprising the cotton event GH_BCS246002 or an explant thereof via a targeted genome editing technique, wherein the cognate target site is identical or similar to an originator target site for a site-specific nuclease present in the cotton event GH_BCS246002 locus; and (b) developing or regenerating a modified cotton plant comprising a modified cotton event GH_BCS246002 comprising the cognate target site. In an aspect of the method, the method further comprises: (c) introducing a site-specific nuclease or a recombinant DNA construct comprising an expression cassette encoding a site-specific nuclease into at least one cell of an explant of a cotton plant comprising the modified cotton event GH_BCS246002 or a plant part thereof, to produce a further modified cotton event GH_BCS246002 via a targeted genome editing technique, wherein the target site of the sitespecific nuclease includes the cognate target site and the originator target site; and (d) developing or regenerating a second modified cotton plant comprising the further modified cotton event GH_BCS246002.
[0043] A further aspect of the present disclosure provides methods of introducing a target site into a cotton plant, the method comprising: (a) introducing a cognate target site into the cotton event GH_BCS246002 locus of at least one cell of a cotton plant or cotton plant part comprising the cotton event GH_BCS246002 or an explant thereof via a targeted genome editing technique to produce a modified cotton event GH_BCS246002 comprising the cognate target site, wherein the cognate target site is identical or similar to an originator target site for a site- specific nuclease present in the cotton event GH_BCS246002 locus; (b) introducing a site-specific nuclease or a recombinant DNA construct comprising an expression cassette encoding a site specific nuclease into at least one cell of an explant of a modified cotton plant comprising the modified cotton event GH_BCS246002, or a plant part thereof, to produce a further modified cotton event GH_BCS246002 via a targeted genome editing technique, wherein the target site of the sitespecific nuclease includes the cognate target site and the originator target site; and (c) developing or regenerating a second modified cotton plant comprising the further modified cotton event GH_BCS246002. The further modified cotton event GH_BCS246002 of the second modified cotton plant comprises a deletion or excision of intervening genomic DNA between the originator target site and the cognate target site, relative to the cotton event GH_BCS246002 or modified cotton event GH_BCS246002. The method further comprises: selecting the second modified cotton plant or a progeny plant of the second modified cotton plant comprising the further modified cotton event GH_BCS246002, and sexually crossing the second modified cotton plant or the progeny plant with itself or another cotton plant to produce one or more modified progeny cotton plants comprising the further modified cotton event GH_BCS246002.
[0044] Another aspect of the present disclosure provides methods of producing a progeny cotton plant comprising a modified cotton event GH_BCS246002 comprising: (a) sexually crossing a first modified cotton plant that comprises a modified cotton event GH_BCS246002 with itself or a second cotton plant; (b) collecting one or more seeds produced from said cross; (c) growing said seed to produce one or more progeny plants; and (d) selecting at least a first progeny plant or seed comprising a modified cotton event GH_BCS246002. [0045] Another aspect of the present disclosure provides methods for insertion of a supplemental expression cassette into a sequence within cotton event GH_BCS246002 or a modified cotton event GH_BCS246002, wherein said supplemental expression cassette comprising at least a heterologous plant expressible promoter operably linked to an open reading frame that encodes a RNA or protein conferring a trait upon a cotton plant or plant part when expressed in the plant or plant part, which is further operably linked to a plant functional transcription termination sequence. Such a trait may be selected from the group consisting of an insect control trait, an herbicide tolerance trait, a nematode resistance trait, a trait that improves yield of cotton fiber, a stay-green trait, a drought tolerance trait, and a trait that confers a unique phenotype to the cotton fiber of the cotton plant. According to some embodiments, the method for insertion of the transgenic DNA may rely upon a lox site for Cre-recombinase integration, or site directed integration of the supplemental expression cassette using a targeted genome editing technique.
[0046] Another aspect of the present disclosure provides methods for editing of an inserted DNA and/or junction sequence of cotton event GH_BCS246002 or a modified cotton event GH BCS246002. In some embodiments, the protoxin domain of the Cry IB or the Cry ID expression cassettes may be deleted from the coding sequence or open reading frame of the respective expression cassette, or a translation termination codon, whether amber, opal or ocher, is inserted or engineered into the coding sequence or open reading frame of the respective toxin coding sequence, to eliminate the expression of the protoxin coding domain, resulting in a modified cotton event GH_BCS246002.
[0047] Yet another aspect of the present disclosure are methods of determining the zygosity of a cotton plant, plant part or seed comprising DNA specific to the cotton event GH_BCS246002 or a modified cotton event GH_BCS246002. The zygosity can be determined in a series of consecutive steps. In a first step, a sample comprising cotton DNA can be contacted with a first primer pair that is capable of producing an amplicon in a DNA amplification assay that is diagnostic for, or characteristic of, the presence of a coding sequence or expression cassette encoding the CrylB.3, CrylDa_7, or Vip3Cbl.l protein within the allele corresponding to cotton event GH_BCS246002. The sample comprising the cotton DNA can be contacted with a second primer pair that is designed to produce an amplicon of an internal standard known to be single-copy and homozygous in the cotton plant. Such methods may additionally include contacting the DNA sample with a probe set which contains at least a first probe that specifically hybridizes to the coding sequence or expression cassette encoding the Cry IB.3, CrylDa_7, or Vip3Cbl .l protein within cotton event GH_BCS246002 DNA, and a second probe that specifically hybridizes to an internal genomic DNA standard known to be single-copy and homozygous in the cotton plant. The DNA amplification reaction in these methods may be performed using real-time PCR to determine the cycle thresholds (Ct values) of the amplicon corresponding the allele of cotton event GH BCS246002 and the single-copy, homozygous internal standard. After the amplification, the difference (delta-Ct) between the Ct value of the single-copy, homozygous internal standard amplicon, and the Ct value of the allele for cotton event GH_BCS246002 amplicon may be calculated. In one embodiment, zygosity is determined wherein a delta-Ct of about zero (0) indicates homozygosity of the inserted T-DNA of cotton event GH_BCS246002, and a delta-Ct of about one (1) indicates heterozygosity of the inserted T-DNA of cotton event GH_BCS246002. In certain embodiments, the DNA amplification assay is diagnostic for, or characteristic of, the presence of a coding sequence or expression cassette encoding the Vip3Cbl.l protein within the allele corresponding to cotton event GH_BCS246002. In certain embodiments, the primer pairs are selected from the group consisting of SEQ ID NO: 245 combined with SEQ ID NO: 246, and SEQ ID NO: 18 combined with SEQ ID NO: 19; and wherein the probes are SEQ ID NO: 247 and SEQ ID NO: 20. In yet other embodiments the delta-Ct of about one (1) indicating heterozygosity of the inserted T-DNA of cotton event GH_BCS246002 is in the range of 0.75 to 1.25. In certain embodiments, a delta-Ct of about zero (0) may be about 0, 0.05, 0.1, 0.15, 0.2, or 0.25, in other embodiments, a delta-Ct of about one (1) may be about 0.75, 0.8, 0.85, 0.9, 0.95, 1.0, 1.05, 1.1, 1.15, 1.2, or 1.25. In a further embodiment, a delta-Ct of about one (1) may be in the range of 0.75 to 1.25, 0.8 to 1.25, 0.85 to 1.25, 0.9 to 1.25, 0.95 to 1.25, 1.0 to 1.25, 1.05 to 1.25, 1.1 to 1.25, 1.15 to 1.25, 1.2 to 1.25, 0.75 to 1.2, 0.8 to 1.2, 0.85 to 1.2, 0.9 to 1.2, 0.95 to 1.2, 1.0 to 1.2, 1.05 to 1.2, 1.1 to 1.2, 1.15 to 1.2, 0.75 to 1.15, 0.8 to 1.15, 0.85 to 1.15, 0.9 to 1.15, 0.95 to 1.15, 1.0 to 1.15, 1.05 to 1.15, 1.1 to 1.15, 0.75 to 1.1, 0.8 to 1.1, 0.85 to 1.1, 0.9 to 1.1, 0.95 to 1.1, 1.0 to 1.1, 1.05 to 1.1, 0.75 to 1.05, 0.8 to 1.05, 0.85 to 1.05, 0.9 to 1.05, 0.95 to 1.05, 1.0 to 1.05, 0.75 to 1.0, 0.8 to 1.0, 0.85 to 1.0, 0.9 to 1.0, 0.95 to 1.0, 0.75 to 0.95, 0.8 to 0.95, 0.85 to 0.95, 0.9 to 0.95, 0.75 to 0.9, 0.75 to 0.85, 0.75 to 0.8, 0.8 to 0.9, 0.8 to 0.85, or 0.85 to 0.9. BRIEF DESCRIPTION OF THE DRAWINGS
[0048] FIG. 1 is a graphical depiction of the orientation and alignment of the DNA elements, segments and sequences that are present within the nucleotide sequence of SEQ ID NO: 10, represented in the drawing as [10], which is the sequence of the inserted transgenic DNA and adjacent 5' and 3' genomic sequences of the cotton event GH_BCS246002. SEQ ID NO: 1 is represented by [1] and is a fifty (50) nucleotide junction sequence or segment spanning twenty- five (25) nucleotides of the 5’ end of the inserted DNA and twenty-five (25) nucleotides of the flanking cotton chromosomal or genomic DNA sequence immediately adjacent to the 5’ end of the inserted DNA. SEQ ID NO: 2 is represented by [2] and is a fifty (50) nucleotide junction sequence or segment spanning twenty-five (25) nucleotides of the 3’ end of the inserted DNA and twenty five (25) nucleotides of the flanking cotton chromosomal or genomic DNA sequence immediately adjacent to the 3’ end of the inserted DNA. SEQ ID NO: 1 is embedded within SEQ ID NO: 3 represented by [3], which is a one hundred (100) nucleotide junction sequence or segment spanning fifty (50) nucleotides of the 5’ end of the inserted DNA and fifty (50) nucleotides of the flanking cotton chromosomal or genomic DNA sequence immediately adjacent to the 5’ end of the inserted DNA. SEQ ID NO: 2 is embedded within SEQ ID NO: 4 represented by [4], which is a one hundred (100) nucleotide junction sequence or segment spanning fifty (50) nucleotides of the 3’ end of the inserted DNA and fifty (50) nucleotides of the flanking cotton chromosomal or genomic DNA segment immediately adjacent to the 3’ end of the inserted DNA. SEQ ID NO: 3 is embedded within SEQ ID NO: 5 represented by [5], which is a two hundred (200) nucleotide junction sequence or segment spanning one hundred (100) nucleotides of the 5’ end of the inserted DNA and one hundred (100) nucleotides of the flanking cotton chromosomal or genomic DNA sequence immediately adjacent to the 5’ end of the inserted DNA. SEQ ID NO: 4 is embedded within SEQ ID NO: 6 represented by [6], which is a two hundred (200) nucleotide junction sequence or segment spanning one hundred (100) nucleotides of the 3’ end of the inserted DNA and one hundred (100) nucleotides of the flanking cotton chromosomal or genomic DNA sequence immediately adjacent to the 3’ end of the inserted DNA. SEQ ID NO:7 represented as [7] is one thousand (1,000) consecutive nucleotides of the flanking cotton chromosomal or genomic DNA sequence immediately adjacent to the 5' end of the inserted DNA and two hundred (200) consecutive nucleotides of the 5’ end of the inserted DNA. SEQ ID NO: 8 represented as [8] is one thousand (1,000) consecutive nucleotides of the flanking cotton chromosomal or genomic DNA sequence immediately adjacent to the 3' end of the inserted DNA and two hundred (200) consecutive nucleotides of the 3' end of the inserted DNA. SEQ ID NO: 9 represented as [9] is the full length nucleotide sequence or segment of the inserted transgenic DNA. The arrows, and the labels below each arrow, represent the orientation of the direction of transcription and translation, as applicable, from the applicable expression elements that are positioned within each of the cassettes within the inserted DNA. RB and LB represent the positions of the right and left borders of the Agrobacterium T-DNA transformation vector, letter P represents the positions of the promoter elements in the respective constructs or cassettes, letter L represents the positions of leader sequences (5' untranslated regions, 5' UTR) in the respective constructs or cassettes, letter I represents the position of the intron sequence in the respective constructs or cassettes, the letter T represents the positions of the transcription termination sequences (3' untranslated regions, 3' UTR) in the respective constructs or cassettes. Each series of elements from a P to the immediately following T in the graphic depiction of [9] and [10] represents a single expression cassette or construct. The construct or cassette closer to the 5’ end of [9] and [10] encodes a Cry IB.3 pesticidal toxin, the middle construct or cassette in the center of [9] and [10] encodes a CrylDa_7 pesticidal toxin, and the construct or cassette closer to the 3’ end of [9] and [10] encodes a Vip3Cbl. l pesticidal toxin. SEQ ID NO: 11 represented as [11] is the flanking cotton chromosomal or genomic DNA sequence immediately adjacent to the 5' end of the inserted DNA. SEQ ID NO: 12 represented as [12] is the flanking cotton chromosomal or genomic DNA sequence immediately adjacent to the 3' end of the inserted DNA. SEQ ID NO: 14 represented as [14] is a LoxP Cre-recombinase recognition sequence which remained in the cotton event GH_BCS246002 after Cre/Lox autoexcision was performed to remove the antibiotic resistance selection marker and the Cre-recombinase expression cassette at the Ri generation (see FIG. 2). SEQ ID NOs: 15, 16 and 21 are primer sequences represented as [15], [16] and [21], respectively, and SEQ ID NO: 17 is a probe sequence represented as [17].
[0049] FIG. 2 illustrates the T-DNA cassette in the plasmid vector used to transform cotton and produce insertion events. One insertion event, when subjected to Cre-recombinase autoexcision of a segment of the inserted DNA containing the selectable marker and Cre-recombinase cassettes, resulted in cotton event GH_BCS246002. SEQ ID NO: 13 represented as [13] illustrates the T- DNA in the plasmid vector before integration into the cotton genome (the “T-DNA Before Integration”). The arrows below [13] represent the individual expression cassettes comprised within the T-DNA before integration, the first three of which are closer to the 5’ end and RB of the insert arc designed to express the three Lcpidoptcran protein toxins. As shown in [13] and after integration (but before Cre-Excision), the Cre-recombinase expression cassette (Cre) and the antibiotic selectable marker expression cassette (aadA), which confers resistance to spectinomycin selection agent, are positioned closer to the 3’ end and LB of the insert and between the two LoxP sites [14]. After insertion of the T-DNA, a portion of the right and left borders were deleted during integration, and the inserted T-DNA in the Ro generation comprised all five expression cassettes between the 5’ and 3’ flanking genomic DNA (the “Inserted T-DNA After Integration”). The transformed Ro generation events were then allowed to self-pollinate and during this process, autoexcision occurred of the DNA segment bounded by the LoxP sites, causing excision of the Cre-recombinase and selectable marker expression cassettes. The LoxP sites are recognized by Cre-recombinase, which is capable of excising from the insertion event the intervening DNA segment between the two LoxP sites containing the Cre-recombinase cassette and the selectable marker cassette as shown both before and after integration. The resultant Ri generation seeds and progeny comprised only the three pesticidal toxin expression cassettes, without the excised Cre and AadA cassettes, as illustrated in the “Inserted T-DNA After Cre-Excision.”
BRIEF DESCRIPTION OF THE SEQUENCES
[0050] SEQ ID NO: 1 is a 50-nucleotide sequence representing the 5' junction region of cotton genomic DNA and the integrated transgenic expression cassette (25 nucleotides cotton genome DNA at the 5' end of SEQ ID NO: 1, 25 nucleotides transgenic inserted DNA at the 3' end of SEQ ID NO: 1), and can be identified within SEQ ID NO: 10 at nucleotide positions 976-1,025.
[0051] SEQ ID NO: 2 is a 50-nucleotide sequence representing the 3' junction region of integrated transgenic expression cassette (25 nucleotides at the 5' end of SEQ ID NO: 2, 25 nucleotides of cotton genome DNA at the 3' end of SEQ ID NO: 2) and can be identified within SEQ ID NO: 10 at nucleotide positions 16,105-16,154.
[0052] SEQ ID NO: 3 is a 100-nuclcotidc sequence representing the 5' junction region of cotton genomic DNA and the integrated transgenic expression cassette (50 nucleotides cotton genome DNA at the 5' end of SEQ ID NO: 3, 50 nucleotides transgenic inserted DNA at the 3' end of SEQ ID NO: 3), and can be identified within SEQ ID NO: 10 at nucleotide positions 951-1,050. [0053] SEQ ID NO: 4 is a 100-nucleotide sequence representing the 3' junction region of integrated transgenic expression cassette (50 nucleotides at the 5' end of SEQ ID NO: 4, 50 nucleotides of cotton genome DNA at the 3' end of SEQ ID NO: 4) and can be identified within SEQ ID NO: 10 at nucleotide positions 16,080-16,179.
[0054] SEQ ID NO: 5 is a 200-nucleotide sequence representing the 5' junction region of cotton genomic DNA and the integrated transgenic expression cassette (100 nucleotides cotton genome DNA at the 5' end of SEQ ID NO: 5, 100 nucleotides transgenic inserted DNA at the 3' end of SEQ ID NO: 5), and can be identified within SEQ ID NO: 10 at nucleotide positions 901-1,100.
[0055] SEQ ID NO: 6 is a 200-nucleotide sequence representing the 3' junction region of integrated transgenic expression cassette (100 nucleotides at the 5" end of SEQ ID NO: 6, 100 nucleotides of cotton genome DNA at the 3' end of SEQ ID NO: 6) and can be identified within SEQ ID NO: 10 at nucleotide positions 16,030-16,229.
[0056] SEQ ID NO: 7 is a 1, 200-nucleotide sequence representing the 5' junction region of cotton genomic DNA and the integrated transgenic expression cassette (1,000 nucleotides cotton genome DNA at the 5' end of SEQ ID NO: 7, 200 nucleotides transgenic inserted DNA at the 3' end of SEQ ID NO: 7), and can be identified within SEQ ID NO: 10 at nucleotide positions 1-1,200.
[0057] SEQ ID NO: 8 is a 1 , 200-nucleotide sequence representing the 3' junction region of integrated transgenic expression cassette (200 nucleotides at the 5' end of SEQ ID NO: 8, 1,000 nucleotides of cotton genome DNA at the 3' end of SEQ ID NO: 8) and can be identified within SEQ ID NO: 10 at nucleotide positions 15,930-17,129.
[0058] SEQ ID NO: 9 is a 15,129-nucleotide sequence corresponding to the transgenic inserted T- DNA of cotton event GH_BCS246002 and can be identified within SEQ ID NO: 10 at nucleotide positions 1,001-16,129.
[0059] SEQ ID NO: 10 is a 17,129-nucleotide sequence corresponding to the contig nucleotide sequence of the 5' genomic flanking DNA nucleotide sequence, the inserted T-DNA nucleotide sequence in cotton event GH_BCS246002, and the 3' genomic flanking DNA nucleotide sequence; and includes SEQ ID NO: 11 (nucleotides 1-1,1000), SEQ ID NO: 9 (nucleotides 1,001-16,129), and SEQ ID NO: 12 (nucleotides 16,130-17,129). [0060] SEQ ID NO: 11 is a 1 ,000-nucleotide sequence representing the 5' flanking cotton genomic DNA up to the inserted T-DNA and can be identified within SEQ ID NO: 10 at nucleotide positions 1-1,000.
[0061] SEQ ID NO: 12 is a 1,000-nucleotide sequence representing the 3' flanking cotton genomic DNA after the inserted T-DNA and can be identified within SEQ ID NO: 10 at nucleotide positions 16,130-17,129.
[0062] SEQ ID NO: 13 is a 21,986-nucleotide sequence representing the transgene cassette comprised within the binary plasmid transformation vector used to transform cotton to produce cotton event GH_BCS246002.
[0063] SEQ ID NO: 14 is a 35-nucleotide sequence representing a LoxP Cre-recombinase recognition sequence used for Cre-mediated autoexcision and recombination, and the residual sequence can be identified within SEQ ID NO: 10 at nucleotide positions 15,825-15,859.
[0064] SEQ ID NO: 15 is 24-nucleotide sequence corresponding to a thermal amplification primer referred to as SQ51960 used to identify cotton event GH_BCS246002 in a sample or which can be used to detect an insertion event that, when subjected to Cre-recombinase autoexcision, results in cotton event GH_BCS246002 DNA. SEQ ID NO: 15 is identical to the nucleotide sequence corresponding to positions 941-964 of SEQ ID NO: 10.
[0065] SEQ ID NO: 16 is a 22-nucleotide sequence corresponding to a thermal amplification primer referred to SQ51702 used to identify cotton event GH_BCS246002 in a sample or which can be used to detect an insertion event that, when subjected to Cre-recombinase autoexcision, results in cotton event GH_BCS246002 DNA. SEQ ID NO: 16 is identical to the reverse complement of the nucleotide sequence corresponding to positions 1,066-1,087 of SEQ ID NO: 10.
[0066] SEQ ID NO: 17 is an 18-nucleotide sequence corresponding to a probe referred to as PB5O3O8 used to identify cotton event GH_BCS246002 DNA in a sample or which can be used to detect an insertion event that, when subjected to Cre-recombinase autoexcision, results in cotton event GH_BCS246002 DNA. SEQ ID NO: 17 is identical to the reverse complement of the nucleotide sequence corresponding to positions 1,043-1,060 of SEQ ID NO: 10. [0067] SEQ ID NO: 18 is a 23-nucleotide sequence corresponding to a thermal amplification primer referred to as SQ22496 used as an internal control for the event and zygosity assay for cotton event GH_BCS246002 and hybridizes to a region of the cotton genome.
[0068] SEQ ID NO: 19 is a 19-nucleotide sequence corresponding to a thermal amplification primer referred to as SQ22497 used as an internal control for the event and zygosity assay for cotton event GH_BCS246002 and hybridizes to a region of the cotton genome.
[0069] SEQ ID NO: 20 is a 14-nucleotide sequence corresponding to a probe referred to as PB50562 used as an internal control for the event and zygosity assay for cotton event GH_BCS246002 and hybridizes to a region of the cotton genome.
[0070] SEQ ID NO: 21 is a 23-nucleotide sequence corresponding to a thermal amplification primer referred to as WTDP393R used in the zygosity assay for cotton event GH_BCS246002 and hybridizes to a region in the 3' flanking cotton genomic DNA. When combined in a thermal amplification reaction with primer SQ51960 with conventional cotton DNA as template lacking or devoid of cotton event GH_BCS246002 DNA, produces an amplicon of one hundred fifty-one (151) nucleotides containing undisrupted cotton genomic DNA. Detection of that amplicon is representative of a sample which does not contain the cotton transgenic event GH_BCS246002 DNA at that chromosomal locus.
[0071] SEQ ID NO: 22 is a 22-nucleotide sequence corresponding to a probe referred to as WTDP393PR used in the zygosity assay for cotton event GH_BCS246002. Probe WTDP393PR hybridizes to the 3 ' terminal 12 nucleotides of the 3' genomic flanking DNA and the 10 nucleotides of the wild-type allelic DNA that was deleted during insertion of the T-DNA in cotton event GH_BCS246002.
[0072] SEQ ID NO: 23 is a 27-nucleotide sequence corresponding to an originator guide RNA recognition site (OgRRS), OgRRS_5-l comprised of a Casl2a protospacer adjacent motif (PAM) site operably linked to a guide-RNA hybridization site.
[0073] SEQ ID NO: 24 is a 27-nucleotide sequence corresponding to an OgRRS, OgRRS_5-2.
[0074] SEQ ID NO: 25 is a 27-nucleotide sequence corresponding to an OgRRS, OgRRS_In-l. [0075] SEQ ID NO: 26 is a 27-nucleotide sequence corresponding to an OgRRS, OgRRS_In-2. [0076] SEQ ID NO: 27 is a 27-nuclcotidc sequence corresponding to an OgRRS, OgRRS_3-l. [0077] SEQ ID NO: 28 is a 27-nucleotide sequence corresponding to an OgRRS, OgRRS_3-2. [0078] SEQ ID NO: 29 is a 1 -nucleotide sequence corresponding to a guide-RNA (gRNA), gRNA_OgRRS_5-l.
[0079] SEQ ID NO: 30 is a 51 -nucleotide sequence corresponding to a guide-RNA (gRNA), gRNA_OgRRS_5-2.
[0080] SEQ ID NO: 31 is a 51 -nucleotide sequence corresponding to a guide-RNA (gRNA), gRNA_OgRRS_In- 1.
[0081] SEQ ID NO: 32 is a 51-nucleotide sequence corresponding to a guide-RNA (gRNA), gRNA_OgRRS_In-2.
[0082] SEQ ID NO: 33 is a 51-nucleotide sequence corresponding to a guide-RNA (gRNA), gRNA_OgRRS_3-l.
[0083] SEQ ID NO: 34 is a 51-nucleotide sequence corresponding to a guide-RNA (gRNA), gRNA_OgRRS_3-2.
[0084] SEQ ID NO: 35 is a sequence of a synthetic DNA coding sequence designed for expression in a plant cell encoding a nuclear targeted LbCasl2a CRIS PR-associated protein.
[0085] SEQ ID NO: 36 is an amino acid sequence of a nuclear targeted LbCasl2a CRISPR- associated protein encoded by SEQ ID NO: 34.
[0086] SEQ ID NO: 37 is a sequence of a synthetic DNA coding sequence designed for expression in a plant cell encoding a nuclear targeted LbCasl 2a-TYCV CRISPR-associated protein.
[0087] SEQ ID NO: 38 is an amino acid sequence of a nuclear targeted LbCasl2a-TYCV CRISPR-associated protein encoded by SEQ ID NO: 36.
[0088] SEQ ID NO: 39 is a sequence of a synthetic DNA coding sequence designed for expression in a plant cell encoding a nuclear targeted LbCasl 2a-TATV CRISPR-associated protein.
[0089] SEQ ID NO: 40 is an amino acid sequence of a nuclear targeted LbCasl2a-TATV CRISPR-associated protein encoded by SEQ ID NO: 38.
[0090] SEQ ID NO: 41 is a sequence of a synthetic DNA coding sequence designed for expression in a plant cell encoding a nuclear targeted FnCasl2a CRISPR-associated protein.
[0091] SEQ ID NO: 42 is an amino acid sequence of a nuclear targeted FnCasl2a CRISPR- associated protein encoded by SEQ ID NO: 40. [0092] SEQ ID NO: 43 is a 5,000-nucleotide sequence representing cotton genomic DNA that flanks the transgenic insert at the 5 'end of the insert. Nucleotides 4,001-5,000 of SEQ ID NO: 43 are identical to nucleotides 1-1,000 of SEQ ID NO: 11. Nucleotides 1-4,000 are based on the genomic sequence of the TM-1 cotton cultivar.
[0093] SEQ ID NO: 44 is a 5,000-nucleotide sequence representing cotton genomic DNA that flanks the transgenic insert at the 5 'end of the insert. Nucleotides 1-1,000 of SEQ ID NO: 44 are identical to nucleotides 1-1,000 of SEQ ID NO: 12. The remaining nucleotides (1,001-5,000) a e based on the genomic sequence of the TM- 1 cotton cultivar.
[0094] SEQ ID NOs: 45-144 are 50-nucleotide sequences in the 5' flank genomic sequence of cotton event GH_BCS246002.
[0095] SEQ ID NOs: 145-244 are 50-nucleotide sequences in the 3' flank genomic sequence of cotton event GH_BCS246002.
[0096] SEQ ID NO: 245 is 22-nucleotide sequence corresponding to a thermal amplification primer referred to as SQ51253 used in the zygosity assay for cotton event GH_BCS246002 and hybridizes to a region within the 3’ portion of the Vip3Cbl.l coding sequence. SEQ ID NO: 245 is identical to the nucleotide sequence corresponding to positions 14,048-14,069 of SEQ ID NO: 10.
[0097] SEQ ID NO: 246 is a 25-nucleotide sequence corresponding to a thermal amplification primer referred to SQ51254 used in the zygosity assay for cotton event GH_BCS246002 and hybridizes to a region within the 3’ portion of the Vip3Cbl.l coding sequence. SEQ ID NO: 246 is identical to the reverse complement of the nucleotide sequence corresponding to positions 15,061-15,085 of SEQ ID NO: 10.
[0098] SEQ ID NO: 247 is an 18-nucleotide sequence corresponding to a probe referred to as PB50494 used in the zygosity assay for cotton event GH_BCS246002 and hybridizes to a region within the 3’ portion of the Vip3Cbl.l coding sequence. SEQ ID NO: 247 is identical to the nucleotide sequence corresponding to positions 15,041-15,058 of SEQ ID NO: 10.
DETAILED DESCRIPTION
[0099] The present disclosure provides a transgenic cotton event GH_BCS246002 that provides insecticidal control over certain Lepidopteran larval pests of cotton by expression of the insecticidal toxins Cry1B.3, CrylDa_7, and Vip3Cbl .l, which are each expressed in tissues of a cotton plant containing this event and presented to Lcpidoptcran pests when they consume the plant tissues. Specifically, the expression of the CrylB.3, CrylDa_7, and Vip3Cbl.l insect inhibitory proteins in cotton event GH_BCS246002 provides resistance to the larval forms of Lepidopteran insect pests including, but not limited to, Cotton bollworm (Helicoverpa zea), Tobacco budworm (Helio thi virescens). and Fall armyworm (Spodoptera frugiperdd). Cotton event GH_BCS246002, also referred to as event GH_BCS246002, provides an unsolved need in the art for control of these insects in the field of cotton agriculture, because Lepidopteran species have developed, or are expected to develop, resistance to the pest control proteins used in earlier versions of transgenic cotton plants expressing Lepidopteran control proteins. Chemical insecticides have not provided adequate control of these insects, and many of the chemical insecticides have encountered resistance development as well. At times, multiple applications of chemistries are required during the growing season, which increases the input and persistence of chemical pesticides in the environment, increases the carbon footprint with each application of such chemicals, and adds significant cost to the production of the cotton crop.
[00100] Protection against infestation by Lepidopteran species is provided by cotton event GH_BCS246002 with the expression of a DNA segment encoding three different Lepidopteran specific insecticidal proteins that are covalently linked within the inserted transgenic DNA that in part defines the cotton event GH_BCS246002. The three insecticidal proteins encoded by the transgenic inserted DNA in the cotton event GH_BCS246002 are (i) Cry IB.3 (see, e.g., United States Patent 10,611,806, the amino acid sequence being referenced therein as SEQ ID NO: 21, and the coding sequence as SEQ ID NO: 20), (ii) CrylDa _7 (see, e.g., United States Patent 10,059,959, the amino acid sequence being referenced therein as SEQ ID NO: 40, and the coding sequence as SEQ ID NO: 39), and (iii) Vip3Cbl.l (see, e.g., United States Patent 10,155,960, the amino acid sequence being referred to therein as SEQ ID NO: 4, and the coding sequence as SEQ ID NO: 3). These three insecticidal proteins are described herein as being expressed from three different but linked expression cassettes within the inserted transgenic DNA construct as set forth herein in SEQ ID NO: 9 and as illustrated in FIG. 1.
[00101] The DNA sequence encoding the Cry 1 B.3 protein in cotton event GH_BCS246002 is operably linked to Cucumis melo Chlorophyl a/b binding protein promoter and leader (see, e.g., United States Patent 10,550,401, referenced therein as SEQ ID NO: 29) and StMedicago truncatula hypothetical protein 3' UTR (or transcription termination sequence, see, e.g., United States Patent 10,501,749, referenced therein as SEQ ID NO: 3). The DNA sequence encoding the CrylDa_7 protein in cotton event GH_BCS246002 is operably linked to a synthetic promoter, leader, and intron (see, e.g., United States Patent Application 2018/0216129 Al, referenced therein as SEQ ID NOs: 5, 6, and 9) and a Medicago truncatula expansin-related protein 1 precursor protein 3' UTR (or transcription termination sequence, see, e.g., United States Patent Application 2014/0283200 Al, referenced therein as SEQ ID NO: 7). The DNA sequence encoding the Vip3Cbl.l protein cotton event GH_BCS246002 is operably linked to an Arabidopsis thaliana Actin-2 promoter, leader, and intron, and a Medicago truncatula seed maturation protein PM21 3' UTR (or transcription termination sequence, United States Patent Application 2014/0283200 Al, referenced therein as SEQ ID NO: 16). Expression (transcription into the mRNAs coding for the toxin amino acid sequences, and translation of the mRNAs into the toxin proteins) of the toxin proteins CrylB.3, CrylDa_7, and Vip3Cbl.l from their respective transgene cassettes is oriented in the same direction (head to tail/head to tail/ head to tail). FIG. 1 shows the relative positions of each element as promoter (P), 5' UTR or leader (L), intron (I), toxin coding sequences or ORFs (open reading frames), and 3' UTR (T). The open reading frames are provided in the event in the following order in the 5’ to 3’ direction: the ORF for CrylB.3, the ORF for CrylDa_7, and the ORF for Vip3Cbl.l, and as specified above, these three ORFs are comprised within SEQ ID NO: 9 and SEQ ID NO: 10.
[00102] As described herein, numerous constructs which varied with respect to the use of expression elements, toxin coding sequences, and orientation of transcription and translation were evaluated. One hundred seventy-five (175) constructs, comprising one or more of twenty-three (23) different insect toxin coding sequences, were used to generate events for assay, leading to the selection of cotton event GH_BCS246002. The construct used to create cotton event GH_BCS246002, presented herein as SEQ ID NO: 13, provided a superior performance relative to other constructs when evaluated for the corresponding transgenic cotton plants’ resistance to Lepidopteran insect pest infestation. In addition, cotton event GH_BCS246002 is free of the markers used for selection of the transformed plant cell as a result of the method of autoexcision after transformation (see FIG. 2). The transgene cassette used for the expression of Cre- recombinase comprised a germline-preferred promoter that, when expressed in cotton during the breeding process, caused the excision of the Crc-rccombinasc and Spcctinomycin selection cassettes between flanking LoxP sites, thus removing the Cre and aadA expression cassettes from the transgcnc locus resulting in cotton event GH_BCS246002.
[00103] Cotton event GH_BCS246002 was created through plant transformation techniques used to insert heterologous DNA (also known as transgenic DNA) randomly into a chromosome of the genome of a cotton cell to produce a genetically engineered cotton cell, also referred to as a “transgenic” or “recombinant” cotton cell. Using these non-targeted transformation techniques, many individual cells are transformed, each resulting in a unique “transgenic event” or “event” due to the random insertion of the foreign DNA into the genome. A transgenic plant can then be regenerated from each individual transgenic cell. This results in every cell of the transgenic plant containing the uniquely inserted transgenic event as a stable pail of its genome. This transgenic plant can then be used to produce seed which are then planted and grown into progeny plants, each containing the unique transgenic event.
[00104] Cotton event GH_BCS246002 was produced by an Agrobacterium-mediated transformation process using the binary transformation plasmid construct pGH_BCS246002 and dry excised cotton embryo explants. The transformed cotton cells were regenerated into intact cotton plants and individual plants were selected from the population of plants that had a fully intact T-DNA insertion comprising expression cassettes encoding the CrylB.3, CrylDa_7, and Vip3Cbl. l proteins, and the absence of (i) the portion of the T-DNA segment encoding the selectable/scorable marker cassettes, which was removed by Cre-Lox excision as described herein, and (ii) any transformation plasmid vector backbone sequence. Further selection was also made based upon the location of the T-DNA insertion with expression cassettes encoding the CrylB.3, CrylDa_7, and Vip3Cbl.l proteins within the cotton genome and other characteristics such as efficacy and agronomics. The expression of the CrylB.3, CrylDa_7, and Vip3Cbl.l insecticidal toxin proteins in a cotton cell comprising the cotton event GH_BCS246002 confers resistance to Lepidopteran insect pests when the cotton cells comprising the cotton event GH_BCS246002 are provided in the diet of the Lepidopteran insects.
[00105] The T-DNA segment encoding the CrylB.3, CrylDa_7, and Vip3Cbl.l protein in plasmid construct pGH_BCS246002 is presented as SEQ ID NO: 13. For cotton event GH_BCS246002, during integration of the T-DNA insertion, two hundred seventeen (217) and one hundred eighty-two (182) contiguous base pairs (bp) were deleted from the right and left borders, respectively, and thirty-five (35) contiguous base pairs (bp) of the wild-type genomic DNA at the point of insertion of the transgenic DNA was deleted during the integration process.
[00106] As specifically described herein, cotton event GH_BCS246002 was produced by a complex research and development process in which: (1) over one hundred plasmid vector constructs - which varied with respect to the coding sequences for the insecticidal proteins, the sequences for the transcriptional regulatory elements, and number and orientation of the cassettes within the constructs - were developed and transformed into cotton cells to create thousands of events that were tested and analyzed, resulting in the selection of the construct used to generate cotton event GH_BCS246002; (2) thousands of cotton cells were transformed with the construct used to generate cotton event GH_BCS246002 and regenerated into plants to create a population of transgenic plants in which each plant contained a unique transgenic event that was tested; (3) the final cotton event GH_BCS246002 was selected after a rigorous multi-year event selection process involving the testing and analysis of molecular characteristics, efficacy, protein expression, and agronomic properties in a variety of cotton genetic backgrounds. Thus, cotton event GH_BCS246002 was produced from a large number of transformants and selected as a uniquely superior event useful for broad-scale agronomic purposes.
[00107] The transgenic DNA from the transformation plasmid vector and inserted into the genome of cotton event GH_BCS246002 was characterized by detailed molecular analysis. This analysis included: the insert number (number of integration sites within the cotton genome), the genomic insert location (the specific site in the cotton genome where the insertion occurred), the copy number (the number of copies of the T-DNA within one locus), and the integrity (absence of any rearrangements) of the transgenic inserted DNA. The detailed molecular analysis demonstrated that the integrated T-DNA containing the CrylB.3, CrylDa_7, and Vip3Cbl.l expression cassettes remained intact after integration and the T-DNA segment comprising the selectable/scoreable marker and Cre-recombinase cassettes flanked by the LoxP recognition sequences was excised from the transgenic progeny. As used herein, an “expression cassette” or “cassette” or “transgene” is a recombinant DNA molecule comprising a combination of distinct elements that are to be expressed by a transformed cell. The inserted DNA of cotton event GH_BCS246002 as described herein has multiple expression cassettes for three different insecticidal toxins. Table 1 provides a list of the elements contained in SEQ ID NOTO, the DNA sequence that corresponds to cotton event GH_BCS246002. Cry IB.3, CrylDa_7, and Vip3Cbl.l are each different unique toxins, exhibiting less than 60% amino acid sequence identity to each other, each arc toxic to Lcpidoptcran pests, and each toxin exhibits a different mode of action relative to the other. The position and orientation within the transgenic inserted DNA of each of the genetic elements and cassettes involved in expression of these respective toxin proteins are specified and shown in Table 1 and FIG. 1.
Table 1. Description of cotton event GH_BCS246002.
[00108] According to present embodiments, a recombinant DNA molecule comprises a nucleotide sequence selected from the group consisting of, or comprising one or more of, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, and a complete complement of any of the foregoing. According to some embodiments, a recombinant DNA molecule is derived from all or part of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002. According to some embodiments, a recombinant DNA molecule comprises (i) all or part of chromosome 4 of a cotton plant and (ii) all or part of the cotton event GH_BCS246002 or a modified cotton event GH_BCS246002, and/or (iii) a nucleotide sequence comprising one or more of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, and a complete complement of any of the foregoing.
[00109] Cotton event GH_BCS246002 is characterized as an insertion of the intended transgenic DNA into a single locus in the cotton genome, resulting in a new locus with two junction sequences (e.g., sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8) between the inserted DNA and the 5’ and 3’ cotton flanking genomic DNA that are not known to appear naturally in the cotton genome or other transgenic cotton events - they are unique to cotton DNA containing the cotton event GH_BCS246002. The insertion of the transgenic DNA into the genome of a cotton plant causes a separation of the genomic sequences of the cotton plant at or near the site of insertion of the transgenic DNA, which produces the two junction sequences at the 5’ and 3’ ends of the inserted transgenic DNA, respectively, wherein the 5’ junction sequence comprises a sequence spanning the 5’ end of the inserted transgenic DNA and the immediately adjacent 5’ flanking genomic DNA, and the 3’ junction sequence comprises a sequence spanning the 3’ end of the inserted transgenic DNA and the immediately adjacent 3’ flanking genomic DNA. Since these junction sequences are unique to the GH_BCS246002 event, these junction sequences are useful in detecting the presence of the GH_BCS246002 event in cotton cells, cotton tissue, cotton seed, cotton pollen and ova, and cotton plants or cotton plant products, such as cotton commodity products. Based on its site of insertion, the cotton event GH_BCS246002 and any modified cotton event GH_BCS246002 are each located on chromosome 4 of a transgenic cotton plant or a modified cotton plant. DNA molecular probes and primer pairs are described herein that have been developed for use in identifying the presence of these various junction segments in biological samples containing or suspected of containing cotton cells, cotton seed, cotton plant parts, cotton pollen or ova, or cotton plant tissue that contain the cotton event GH_BCS246002.
[00110] A sample is intended to refer to a composition that is either substantially purified cotton DNA or protein, or a composition that contains cotton DNA or protein. With respect to a sample containing DNA, the sample can be any biological sample, i.e., it contains biological material, including but not limited to DNA obtained or derived from, either directly or indirectly, from the genome of a cotton plant, plant part, plant tissue or plant cell that may or may not contain cotton event GH_BCS246002 or a modified cotton event GH_BCS246002. “Directly” refers to the ability of the skilled artisan to directly obtain DNA from the cotton genome by fracturing cotton cells or tissues (or by obtaining samples of cotton that contain fractured cotton cells or tissues) and using or exposing the genomic DNA for the purposes of detection. “Indirectly” refers to the ability of the skilled artisan to obtain the target or specific reference DNA by means other than by direct via fracturing of cotton cells or tissues or obtaining a sample that contains fractured cotton cells or tissues. Such indirect means include, but are not limited to, amplification of a DNA segment that contains the DNA sequence targeted by a particular probe designed to bind with specificity to the target sequence, or amplification of a DNA segment that can be measured and characterized, i.e., measured by separation from other segments of DNA through some efficient matrix such as an agarose or acrylamide gel or the like, or characterized by direct sequence analysis of the amplicons, or cloning of the amplicon into a vector and direct sequencing of the inserted amplicon present within such vector. room] A sample of pure cotton protein or a composition that contains cotton protein is a biological sample, i.e., it contains biological materials, including but not limited to protein derived from the tissues or cells of a cotton plant, plant pail, plant tissue or plant cell that may or may not contain cotton event GH_BCS246002. The sample can be contacted with antibodies, such as monoclonal antibodies that bind specifically to CrylB.3, CrylDa_7, and Vip3Cbl.l in an immunoassay. Detection of the bound proteins is diagnostic or characteristic of CrylB.3, CrylDa_7, and Vip3Cbl.l. The immunoassay method can be, but is not limited to, an ELISA (Enzyme-Linked Immunosorbent Assay), a Radioimmunoassay, or a Lateral flow immunochromatographic assay. ELISA assays are typically performed in the laboratory using tissue or cell samples obtained from whole plants, or plant parts or tissues thereof. Sandwich ELISA assays are frequently used to detect and quantify protein from transgenic crops. To detect the presence or absence of cotton event GH_BCS246002 in a sample using a sandwich ELISA assay, monoclonal antibodies can be used, with a first monoclonal antibody which binds specifically to Cry IB.3, a second antibody which binds specifically to CrylDa_7, and a third monoclonal antibody which binds specifically to Vip3Cbl.l. A known amount of monoclonal antibody can be bound to a fixed surface. Nonspecific sites on the solid surface may be blocked by bovine serum albumin, casein, or another such neutral solution. The sample which may contain protein(s) derived from cotton event GH_BCS246002 is applied to the plate, and any such protein(s) can be captured by the antibody. The unbound antigens may be washed away by a washing solution. A secondary antibody can be added which may also be conjugated to an enzyme. The unbound antibodies may be washed away. A substrate can be added, and the enzyme reacts with the substrate and produces a product, typically a pigment or photons, which can be proportional to the amount of antigen present. Separate ELISA assays can be performed for each of the three toxin proteins in a sample derived from cotton event GH_BCS246002. A positive ELISA reaction is diagnostic for, or characteristic of, cotton event GH_BCS246002.
[00112] Lateral flow immunochromatographic assays, also known as immunochromatographic strips (ICS) or dipstick tests, can be used in the field where laboratory facilities are not available for more complex immunoassays. The immunochromatographic test strip is composed of a sample pad, conjugate pad, nitrocellulose membrane, absorbent pad, and a backing card. A first monoclonal antibody which binds specifically to CrylB.3, a second monoclonal antibody which binds specifically to CrylDa_7, a third monoclonal antibody which binds specifically to Vip3Cbl.l, and a IgG antibody which binds to antibodies derived from the organism host cells in which the first, second, and third monoclonal antibodies were derived are transferred onto a nitrocellulose membrane to form a test line 1 (CrylB.3), test line 2 (CrylDa_7), test line 3 (Vip3Cbl.l) and the control line (anti-host IgG antibody). The conjugate pad is coated with the first, second, and third monoclonal antibodies labeled with colloidal gold nanoparticles. The blotted membrane, conjugate pad, sample pad, and absorbent pad are assembled sequentially on the plastic backing board. The Cry IB .3, Cry lDa_7, and Vip3Cb 1.1 proteins present in a sample derived from cotton event GH_BCS246002 will combine with their respective gold-labeled monoclonal antibodies and then bind to capture antibodies coated on the test line 1 , test line 2, and test line 3. Visual detection of test line 1 and test line 2 is diagnostic for, or characteristic of, cotton event GH_BCS246002.
[00113] Detailed molecular analysis also demonstrated that cotton event GH_BCS246002 contains a single T-DNA insertion with one copy of each of the Cry IB.3, CrylDa_7, and Vip3Cbl.l expression cassettes. No additional elements from the transformation construct other than portions of the Agrobacterium tumefaciens left and right border regions used for transgenic DNA transfer from the plant transformation plasmid to the cotton genome were identified in cotton event GH_BCS246002. Further, thermal amplification producing specific amplicons diagnostic for, or characteristic of, the presence of cotton event GH_BCS246002 in a sample and DNA sequence analyses were performed to determine the arbitrarily assigned 5" and 3" insert-to-plant genome junctions, confirm the organization of the elements within the insert, and determine the complete DNA sequence of the inserted transgenic DNA (SEQ ID NO: 9). SEQ ID NO: 11 is a sequence representing the one thousand (1,000) base-pair (bp) immediately adjacent to the 5' end of the inserted DNA in cotton variety DP393 genomic DNA sequence flanking the inserted T- DNA of cotton event GH_BCS246002. SEQ ID NO: 12 is a sequence representing the one thousand (1,000) bp immediately adjacent to the 3' end of the inserted DNA in cotton variety DP393 genomic DNA sequence flanking the inserted T-DNA sequence. SEQ ID NO: 7 is SEQ ID NO: 11 plus two hundred (200) bp of the 5' end of the inserted T-DNA sequence added to the 3' end of SEQ ID NO: 11 . SEQ ID NO: 8 is SEQ ID NO: 12 plus two hundred (200) bp of the 3' end of the inserted T-DNA sequence added to the 5' end of SEQ ID NO: 12. SEQ ID NO: 10 corresponds to the DNA sequence that defines the cotton event GH_BCS246002 and contains a contiguous sequence (contig) comprising the 5' DP393 flanking sequence, the transgenic insert of cotton event GH_BCS246002, and the 3' DP393 flanking sequence, and thus contains both of the junction sequences of cotton event GH_BCS246002 specified as SEQ ID NO: 1, 3, 5 and 7 (junction sequence at 5' end of insert), and as SEQ ID NO: 2, 4, 6, and 8 (junction sequence at 3' end of insert).
[00114] Unless otherwise noted herein, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art. Definitions of common terms in molecular biology may be found in Rieger et al., Glossary of Genetics: Classical and Molecular, 5th edition, Springer- Vcrlag: New York, 1991; and Lewin, Genes V, Oxford University Press: New York, 1994, along with other sources known to those of ordinary skill in the art. As used herein, the term “cotton” means species belonging to the genus Gossypium, preferably Gossypium hirsutum L. and Gossypium barbadense L. and includes all plant varieties that can be bred with cotton plants containing cotton event GH_BCS246002, including wild cotton species as well as those plants belonging to the genus Gossypium that permit breeding between species.
[00115] Cotton event GH_BCS246002 was transformed with a DNA construct that contains expression cassettes expressing toxic amounts of insecticidal proteins CrylB.3, CrylDa_7, and Vip3Cb 1.1. As used herein, a toxic amount refers to an efficacious amount, an insecticidal amount, an insecticidally effective amount, a target insect suppressive amount, an efficacious pesticidal amount, an amount in the diet of insects in the order of Lepidoptera that is insecticidal, and other similar terms to be understood according to conventional usage by those of ordinary skill in the relevant art. Cotton plants transformed according to the methods and with the DNA constructs disclosed herein are resistant to Lepidopteran insect pests.
[00116] A transgenic “plant” is produced by transformation of a plant cell with heterologous
DNA, i.e., a polynucleic acid construct that includes a number of efficacious features of interest, regeneration of a plant resulting from the insertion of the transgene into the genome of the plant cell, and selection of a particular plant characterized by insertion into a particular genome location and the number of efficacious features of the regenerated transgenic plant. The term “event” refers to DNA from the original transformant comprising the inserted DNA and flanking genomic sequences immediately adjacent to the inserted DNA. Such DNA is unique and would be expected to be transferred to a progeny that receives the inserted DNA, including the transgcnc of interest, as the result of a sexual cross of parental line that includes the inserted DNA (e.g., the original transformant and progeny resulting from selfing) and a parental line that does not contain the inserted DNA. The present disclosure also provides the original transformant plant and progeny of the transformant that include the heterologous DNA. Such progeny may be produced by a sexual outcross between plants comprising the event and another plant wherein the progeny includes the heterologous DNA. As evidence of the stability of the event in the position in which it has been introduced into the cotton genome, the event is present in the progeny of a cross at the same chromosomal location after repeated back-crossing to a recurrent parent. Cotton event GH_BCS246002 is a stable transgenic event that can be stably transmitted through one or more generations to progeny or descendants of plants containing the cotton event GH_BCS246002 and stacked with other events or traits in progeny or descendants through one or more crosses. The transgenic event DNA, and the genome into which the transgenic event DNA is detectable within the plant cell, the plant, the seed, and other parts of the plant, are not pre-existent in nature.
[00117] The terms “DNA” and “DNA molecule” referred to herein refer to a deoxyribonucleic acid (DNA) molecule. A DNA molecule may be of genomic or synthetic origin and is by convention from the 5' (upstream) end to the 3' (downstream) end. As used herein, the term “DNA sequence” refers to the nucleotide sequence of the DNA molecule. By convention, the DNA sequences of the present disclosure and fragments thereof are disclosed with reference to only one strand of the two-strand complementary DNA sequence strands. By implication and intent, the complementary sequences of the sequences provided here (the sequences of the complementary strand), also referred to in the art as the reverse complementary sequences, are within the scope of the present disclosure and are expressly intended to be within the scope of the subject matter claimed.
[00118] As used herein, the term “fragment” refers to a smaller piece of the whole. For example, fragments of SEQ ID NO: 10 would include sequences that are at least about 12 consecutive nucleotides, at least about 13 consecutive nucleotides, at least about 14 consecutive nucleotides, at least about 15 consecutive nucleotides, at least about 16 consecutive nucleotides, at least about 17 consecutive nucleotides, at least about 18 consecutive nucleotides, at least about 19 consecutive nucleotides, at least about 20 consecutive nucleotides, at least about 25 consecutive nucleotides, at least about 30 consecutive nucleotides, at least about 35 consecutive nucleotides, at least about 40 consecutive nucleotides, at least about 45 consecutive nucleotides, at least about 50 consecutive nucleotides, at least about 60 consecutive nucleotides, at least about 70 consecutive nucleotides, at least about 80 consecutive nucleotides, at least about 90 consecutive nucleotides, or at least about 100 consecutive nucleotides of the complete sequence of SEQ ID NO: 10.
[00119] Similarly, a fragment of the 5' flank (SEQ ID NO: 11 or SEQ ID NO: 43) or 3' flank (SEQ ID NO: 12 or SEQ ID NO: 44) of cotton event GH_BCS246002 can comprise at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 150, at least about 200, at least about 250, at least about 300, at least about 400, or at least about 500 consecutive nucleotides of SEQ ID NO: 11 or SEQ ID NO: 43; or SEQ ID NO: 12 or SEQ ID NO: 44. In addition, the present disclosure encompasses nucleotide sequences that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% identical to SEQ ID NO: 11 or 12, or SEQ ID NO: 43 or 44, or any fragment of either thereof.
[00120] Reference in this disclosure to an “isolated DNA molecule” or an equivalent term or phrase is intended to mean that the DNA molecule is one that is present alone or in combination with other compositions, but not within its natural environment. For example, nucleic acid elements such as a coding sequence, intron sequence, untranslated leader sequence, promoter sequence, transcriptional termination sequence, and the like, that are naturally found within the DNA of the genome of an organism are not considered to be “isolated” so long as the element is within the genome of the organism and at the location within the genome in which it is naturally found. However, each of these elements, and subparts of these elements, would be “isolated” within the scope of this disclosure so long as the element is not within the genome of the organism and at the location within the genome in which it is naturally found. Similarly, a nucleotide sequence encoding an insecticidal protein or any naturally occurring insecticidal variant of that protein would be an isolated nucleotide sequence so long as the nucleotide sequence was not within the DNA of the bacterium from which the sequence encoding the protein is naturally found. A synthetic nucleotide sequence encoding the amino acid sequence of the naturally occurring insecticidal protein would be considered to be isolated for the purposes of this disclosure. For the purposes of this disclosure, any transgenic nucleotide sequence, i.e., the nucleotide sequence of the DNA inserted into the genome of the cells of a plant or bacterium, or present in an extrachromosomal vector, would be considered to be an isolated nucleotide sequence whether it is present within the plasmid or similar structure used to transform the cells, within the genome of the plant or bacterium, or present in detectable amounts in tissues, progeny, biological samples or commodity products derived from the plant or bacterium. In any circumstance, the isolated DNA molecule is a chemical molecule, regardless of whether it is referred to as a nucleic acid, a nucleic acid sequence, a polynucleotide sequence, and the like. It is a novel, inventive molecule that exhibits industrial applicability both when present in a plant cell or in a plant genome, and when present outside of a plant cell, and therefore, exhibits and is intended to exhibit such utility regardless of where the molecule is located.
[00121] Reference in this disclosure to an “isolated protein molecule” or an equivalent term or phrase is intended to mean that the protein molecule is one that is present alone or in combination with other compositions but not within its natural environment. For example, the insecticidal protein molecules expressed by cotton event GH_BCS246002 are not naturally found in native cotton plant samples. The CrylB.3, CrylDa_7, and Vip3Cbl.l insecticidal proteins of cotton event GH_BCS246002 are isolated protein molecules so long as the insecticidal proteins or variants thereof was not within the protein of the bacterium from which the protein is naturally found.
[00122] The DNA sequence of the region spanning the connection by phosphodiester bond linkage of one end of the transgenic insert to the flanking cotton genomic DNA is referred to as a “junction.” A junction is the connection point of the transgenic insert and flanking DNA as one contiguous molecule. One junction is found at the 5' end of the transgenic insert and the other is found at the 3' end of the transgenic insert, referred to herein as the 5' and 3' junctions, respectively. A “junction sequence” refers to a DNA sequence of any length that spans the 5' or 3' junction of an event. Junction sequences of cotton event GH_BCS246002 are provided as SEQ ID NOs: 1-8. FIG. 1 illustrates the physical arrangement of the junction sequences, arranged from 5' to 32 relative to SEQ ID NO: 10. The junction sequences of cotton event GH_BCS246002 may be present as part of the genome of a plant, seed, or cell containing cotton event GH_BCS246002. The identification of any one or more of the junction sequences in a sample from a plant, plant part, seed, or cell indicates that the DNA was obtained from cotton containing cotton event GH_BCS246002 and is diagnostic for, or characteristic of, the presence of cotton event GH_BCS246002.
[00123] The junction sequences for cotton event GH_BCS246002 may be represented by a sequence from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 10. For example, the junction sequences may be arbitrarily represented by the nucleotide sequences provided as SEQ ID NO: 1 (5' junction sequence) and SEQ ID NO: 2 (3' junction sequence). Alternatively, the junction sequences may be arbitrarily represented by the nucleotide sequences provided as SEQ ID NO: 3 (5' junction sequence) and SEQ ID NO: 4 (3' junction sequence). Alternatively, the junction sequences may be arbitrarily represented by the nucleotide sequences provided as SEQ ID NO: 5 (5' junction sequence) and SEQ ID NO: 6 (3' junction sequence). Alternatively, the junction sequences may be arbitrarily represented by the nucleotide sequences provided as SEQ ID NO: 7 (5" junction sequence) and SEQ ID NO: 8 (3' junction sequence). These nucleotide sequences are connected by phosphodiester linkage, and in cotton event GH_BCS246002 are present as part of the recombinant plant cell genome.
[00124] These junction sequences are diagnostic for, or characteristic of, the presence of cotton event GH_BCS246002, or the construct comprised therein. Thus, the identification of one or more of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 10 in a sample derived from a cotton plant, cotton seed, or cotton plant part is diagnostic that the DNA was obtained from cotton event GH_BCS246002. The present disclosure thus provides a DNA molecule that contains at least one of the nucleotide sequences provided as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10. Any segment of DNA derived from transgenic cotton event GH_BCS246002 that is sufficient to include at least one of the sequence provided as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10 is within the scope of the present disclosure. In addition, any polynucleotide comprising a sequence complementary to any of the sequences described within this paragraph is within the scope of the present disclosure.
[00125] The present disclosure provides exemplary DNA molecules that can be used either as primers or probes for detecting the presence of DNA derived from a cotton plant comprising cotton event GH_BCS246002 DNA in a sample. Such primers or probes are specific for a target nucleic acid sequence and, as such, are useful for the identification of cotton event GH_BCS246002 nucleic acid sequence by the methods of the present disclosure described herein. [00126] It is intended by the use of the word “derived” that a particular DNA molecule is in the cotton plant genome or is capable of being detected in cotton plant DNA. “Capable of being detected” refers to the ability of a particular DNA segment to be amplified and its size or sequence characterized or elucidated by DNA sequence analysis, i.e., the target DNA segment, and the subsequent ability to detect the binding of the prove to the target. The particular DNA segment or target DNA segment of the present disclosure is present with cotton that contains the insertion of cotton event GH_BCS246002.
[00127] A “probe” is a nucleic acid molecule that is complementary to a strand of target nucleic acid and is useful in hybridization methods. A probe may be attached a conventional detectable label or reporter molecule, e.g., a radioactive isotope, ligand, chemiluminescent agent, or enzyme. Such a probe is complementary to a strand of a target nucleic acid and, in the case of the present disclosure, to a strand of DNA from cotton event GH_BCS246002 whether from a cotton event GH_BCS246002 containing plant or from a sample that includes cotton event GH_BCS246002 DNA. Thus, the probes for use herein may comprise DNA molecules or polynucleotide segments of sufficient length to function under stringent hybridization conditions as defined herein to bind to a particular unique segment of DNA present within and diagnostic for, or characteristic of, cotton event GH_BCS246002 in a sample. Such a probe can be designed to bind only to a single junction or other novel sequence present only in the cotton event GH_BCS246002, or two or more such single junction segments. Probes according to the present disclosure include not only deoxyribonucleic or ribonucleic acids, but also polyamides and other probe materials that bind specifically to a target DNA sequence and can be used to detect the presence of that target DNA sequence. An exemplary DNA sequence useful as a probe for detecting cotton event GH_BCS246002 is provided as SEQ ID NO: 17 (PB5O3O8). [00128] A “primer” is typically a DNA molecule that is designed for use in specific annealing or hybridization methods that involve thermal amplification. Primers may comprise pairs of different oligonucleotides or polynucleotide segments for use in a thermal amplification reaction which amplifies a particular DNA target segment. Each primer in the pair is designed to bind to a rather specific segment of DNA within or near a segment DNA of interest for amplification. The primers bind in such a way that these then act as localized regions of nucleic acid sequence polymerization resulting in the production of one or more amplicons (amplified target segments of DNA). The amplicon produced from such reaction would have a DNA sequence corresponding to sequence of the template DNA located between the two sites where the primers hybridized to the template. In certain embodiments, use of primers designed to bind to unique segments of cotton event GH_BCS246002 and that amplify particular amplicons containing one or more of the junction sequences described herein, and the detection and/or characterization of such amplicons upon completion or termination of polymerase reaction, is diagnostic for, or characteristic of, the present of cotton event GH_BCS246002 in a particular sample. The skilled artisan is well familiar with this amplification method and no recitation of the specifics of amplification is necessary here.
[00129] A primer is typically designed to hybridize to a complementary target DNA strand to form a hybrid between the primer and target DNA strand, and the presence of the primer is a point of recognition by a polymerase to begin extension of the primer (i.e., polymerization of additional nucleotides into a lengthening nucleotide molecule) using as a template the target DNA strand. Primer pairs refer to use of two primers binding opposite strands of a double stranded nucleotide segment for the purpose of amplifying linearly the polynucleotide segment between the positions targeted for binding by the individual members of the primer pair, typically in a thermal amplification reaction or other conventional nucleic-acid amplification methods. Exemplary DNA molecules useful as primers are provided as SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 21.
[00130] The primer pair SEQ ID NO: 15 and SEQ ID NO: 16 are useful as a first DNA molecule and a second DNA molecule that is different from the first DNA molecule, and both are each of sufficient length of contiguous nucleotides of SEQ ID NO: 10 to function as DNA primers that, when used together in a thermal amplification reaction with template DNA derived from cotton event GH_BCS246002, to produce an amplicon diagnostic for, or characteristic of, cotton event GH_BCS246002 in a sample. The primer pair SEQ ID NO: 18 and SEQ ID NO: 19 are useful as a first DNA molecule and a second DNA molecule that is different from the first DNA molecule, and both are each of sufficient length of contiguous nucleotides of a locus within the cotton genome to function as DNA primers that, when used together in a thermal amplification reaction with template DNA derived cotton event GH_BCS246002, to produce an amplicon that serves as an internal control for both the diagnosis of cotton event GH_BCS246002, as well as the zygosity of cotton event GH_BCS246002 DNA in a sample. The primer pair SEQ ID NO: 20 and SEQ ID NO: 15 are useful as a first DNA molecule and a second DNA molecule that is different from the first DNA molecule, and both are each of sufficient length of contiguous nucleotides of a locus within the cotton genome to function as DNA primers that, when used together in a thermal amplification reaction with template DNA derived from cotton event GH_BCS246002, to produce an amplicon diagnostic for, or characteristic of, non-inserted wild-type cotton genomic DNA not comprising cotton event GH_BCS246002. It is within the skill of the art to determine for any particular desired amplification parameters, which probes and primers would be optimum for inclusion in the thermal amplification reaction to detect the presence or absence of the transgenic event DNA of the present disclosure based on the DNA sequences provided in the inserted DNA (SEQ ID NO: 9) and the full segment of DNA set forth herein as SEQ ID NO: 10 which defines the transgenic cotton event of this disclosure, GH_BCS246002.
[00131] DNA probes and DNA primers are generally eleven (11) polynucleotides or more in length, often eighteen (18) polynucleotides or more, twenty-four (24) polynucleotides or more, or thirty (30) polynucleotides or more. Such probes and primers arc selected to be of sufficient length to hybridize specifically to a target sequence under high stringency hybridization conditions. Preferably, probes and primers according to the present disclosure have complete sequence similarity with the target sequence, although probes differing from the target sequence that retain the ability to hybridize to target sequences may be designed by conventional methods.
[00132] The nucleic acid probes and primers of the present disclosure hybridize under stringent conditions to a target DNA molecule. Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of DNA from a transgenic plant in a sample. Polynucleic acid molecules also referred to as nucleic acid segments or fragments thereof are capable of specifically hybridizing to other nucleic acid molecules under certain circumstances. [00133] As used herein, two polynucleic acid molecules are said to be capable of specifically hybridizing to one another if the two molecules arc capable of forming an anti-parallel, double-stranded nucleic acid structure. A nucleic acid molecule is said to be the “complement” of another nucleic acid molecule if they exhibit complete complementarity. As used herein, molecules are said to exhibit “complete complementarity” when every nucleotide of one of the molecules is complementary to a nucleotide of the other. Two molecules are said to be “minimally complementary” if they can hybridize to one another with sufficient stability to permit them to remain annealed to one another under at least conventional "low-stringency" conditions. Similarly, the molecules are said to be “complementary” if they can hybridize to one another with sufficient stability to permit them to remain annealed to one another under conventional "high- stringency" conditions. Conventional stringency conditions are described by Sambrook et al., 1989, and by Haymes et al., In: Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, DC (1985). Departures from complete complementarity are therefore permissible, as long as such departures do not completely preclude the capacity of the molecules to form a double-stranded structure. In order for a nucleic acid molecule to serve as a primer or probe it need only be sufficiently complementary in sequence to be able to form a stable double-stranded structure under the particular solvent and salt concentrations employed.
[00134] As used herein, a substantially homologous sequence is a nucleic acid sequence that will specifically hybridize to the complement of the nucleic acid sequence to which it is being compared under high stringency conditions. Appropriate stringency conditions that promote DNA hybridization, for example, 6.0 x sodium chloride/sodium citrate (SSC) at about 45°C, followed by a wash of 2.0 x SSC at 50°C, are known to those skilled in the art or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. For example, the salt concentration in the wash step can be selected from a low stringency of about 2.0 x SSC at 50°C to a high stringency of about 0.2 x SSC at 50°C. In addition, the temperature in the wash step can be increased from low stringency conditions at room temperature, about 22°C, to high stringency conditions at about 65°C. Both temperature and salt may be varied, or either the temperature or the salt concentration may be held constant while the other variable is changed. In a preferred embodiment, a polynucleic acid of the present disclosure will specifically hybridize to one or more of the nucleic acid molecules set forth in SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10, or complements thereof or fragments of either under moderately stringent conditions, for example at about 2.0 x SSC and about 65°C. In a particularly preferred embodiment, a nucleic acid of the present disclosure will specifically hybridize to one or more of the nucleic acid molecules set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10, or complements or fragments of either under high stringency conditions. In one aspect of the present disclosure, a preferred marker nucleic acid molecule of the present disclosure has the nucleic acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10, or complements thereof, or fragments of either. The hybridization of the probe to the target DNA molecule can be detected by any number of methods known to those skilled in the art, these can include, but are not limited to, fluorescent tags, radioactive tags, antibody-based tags, and chemiluminescent tags.
[00135] Regarding the amplification of a target nucleic acid sequence (e.g., by PCR) using a particular amplification primer pair, "stringent conditions" are conditions that permit the primer pair to hybridize only to the target nucleic acid sequence to which a primer having the corresponding wild-type sequence (or its complement) would bind and preferably to produce a unique amplification product, the amplicon, in a DNA thermal amplification reaction.
[00136] The term "specific for (a target sequence)" indicates that a probe or primer hybridizes under stringent hybridization conditions only to the target sequence in a sample comprising the target sequence.
[00137] As used herein, “amplified DNA” or “amplicon” refers to the product of polynucleic acid amplification method directed to a target polynucleic acid molecule that is part of a polynucleic acid template. For example, to determine whether cotton plant resulting from a sexual cross contains transgenic plant genomic DNA from a cotton plant comprising cotton event GH_BCS246002 of the present disclosure, DNA that is extracted from a cotton plant tissue sample may be subjected to a polynucleotide acid amplification method using a primer pair that includes a first primer derived from a genomic DNA sequence in the region flanking the heterologous inserted DNA of cotton event GH_BCS246002 and is elongated by polymerase 5' to 3' in the direction of the inserted DNA. The second primer is derived from the heterologous inserted DNA molecule is elongated by the polymerase 5' to 3' in the direction of the flanking genomic DNA from which the first primer is derived. The amplicon may range in length from the combined length of the primer pair plus one nucleotide base pair, or plus about fifty nucleotide base pairs, or plus about two hundred- fifty nucleotide base pairs, or plus about four hundred-fifty nucleotide base pairs or more. Alternatively, a primer pair can be derived from genomic sequence on both sides of the inserted heterologous DNA so as to produce an amplicon that includes the entire insert polynucleotide sequence (e.g., a forward primer isolated from the genomic portion on the 5' end of SEQ ID NO: 10 and a reverse primer isolated from the genomic portion on the 3' end of SEQ ID NO: 10 that amplifies a DNA molecule comprising the inserted DNA sequence (SEQ ID NO: 9) identified herein in the cotton event GH_BCS246002 genome). A member of a primer pair derived from the plant genomic sequence adjacent to the inserted transgenic DNA is located a distance from the inserted DNA sequence, this distance can range from one nucleotide base pair up to about twenty thousand nucleotide base pairs. The use of the term “amplicon” specifically excludes primer dimers that may be formed in the DNA thermal amplification reaction.
[00138] For practical purposes, one should design primers which produce amplicons of a limited size range, for example, between 100 to 1000 bases. Smaller (shorter polynucleotide length) sized amplicons in general are more reliably produced in thermal amplification reactions, allow for shorter cycle times, and can be easily separated and visualized on agarose gels or adapted for use in endpoint TAQMAN®-like assays. Smaller amplicons can be produced and detected by methods known in the art of DNA amplicon detection. In addition, amplicons produced using the primer pairs can be cloned into vectors, propagated, isolated, and sequenced or can be sequenced directly with methods well established in the art. Any primer pair derived from the combination of SEQ ID NO: 11 and SEQ ID NO: 9 or the combination of SEQ ID NO: 12 and SEQ ID NO: 9 that are useful in a DNA amplification method to produce an amplicon diagnostic for, or characteristic of, cotton event GH_BCS246002 or progeny thereof is an aspect of the present disclosure. Any single isolated DNA polynucleotide primer molecule comprising at least 15 contiguous nucleotides of SEQ ID NO: 11, or its complement that is useful in a DNA amplification method to produce an amplicon diagnostic for, or characteristic of, cotton event GH_BCS246002 or progeny thereof is an aspect of the present disclosure. Any single isolated DNA polynucleotide primer molecule comprising at least 15 contiguous nucleotides of SEQ ID NO: 12, or its complement that is useful in a DNA amplification method to produce an amplicon diagnostic for, or characteristic of, plants comprising cotton event GH_BCS246002 or progeny thereof is an aspect of the present disclosure. Any single isolated DNA polynucleotide primer molecule comprising at least 15 contiguous nucleotides of SEQ ID NO: 9, or its complement that is useful in a DNA amplification method to produce an amplicon diagnostic for, or characteristic of, cotton event GH_BCS246002 or progeny thereof is an aspect of the present disclosure.
[001391 Polynucleic acid amplification can be accomplished by any of the various polynucleic acid amplification methods known in the art, including the polymerase chain reaction (PCR). Amplification methods are known in the ail and are described, inter alia, in U.S. Patent Nos. 4,683,195 and 4,683,202 and in PCR Protocols: A Guide to Methods and Applications, ed. Innis et al., Academic Press, San Diego, 1990. PCR amplification methods have been developed to amplify up to 22 kb (kilobase) of genomic DNA and up to 42 kb of bacteriophage DNA (Cheng et al., Proc. Natl. Acad. Sci. USA 91:5695-5699, 1994). These methods as well as other methods known in the art of DNA amplification may be used in the practice of the present disclosure. The sequence of the heterologous DNA insert or flanking genomic DNA sequence from cotton event GH_BCS246002 can be verified (and corrected if necessary) by amplifying such DNA molecules from cotton seed containing cotton event GH_BCS246002 DNA deposited with the ATCC having accession No. PTA- 127733, using primers derived from the sequences provided herein, followed by standard DNA sequencing of the PCR amplicon or cloned DNA fragments thereof.
[00140] The diagnostic amplicon produced by these methods may be detected by a plurality of techniques. One such method is Genetic Bit Analysis (Nikiforov, et al. Nucleic Acid Res. 22:4167-4175, 1994) where a DNA oligonucleotide is designed that overlaps both the adjacent flanking genomic DNA sequence and the inserted DNA sequence. The oligonucleotide is immobilized in wells of a microtiter plate. Following PCR of the region of interest (using one primer in the inserted sequence and one in the adjacent flanking genomic sequence), a singlestranded PCR product can be hybridized to the immobilized oligonucleotide and serve as a template for a single base extension reaction using a DNA polymerase and labeled dideoxynucleotide triphosphates (ddNTPs) specific for the expected next base. Readout may be fluorescent or ELISA-based. A signal indicates presence of the transgene/genomic sequence due to successful amplification, hybridization, and single base extension.
[00141] Another method is the Pyro sequencing technique as described by Winge (Innov. Pharma. Tech. 00:18-24, 2000). In this method, an oligonucleotide is designed that overlaps the adjacent genomic DNA and insert DNA junction. The oligonucleotide is hybridized to single- stranded PCR product from the region of interest (one primer in the inserted sequence and one in the flanking genomic sequence) and incubated in the presence of a DNA polymerase, ATP, sulfurylase, luciferase, apyrase, adenosine 5' phosphosulfate and luciferin. dNTPs are added individually and the enzymatic reaction of luciferase with these reagents and substrates results in the release of photons (a signal of light) which are then measured or observed. A light signal indicates the presence of the transgene/genomic sequence due to successful amplification, hybridization, and single or multi-base extension.
[00142] Fluorescence Polarization as described by Chen, et al., (Genome Res. 9:492-498, 1999) is a method that can be used to detect the amplicon of the present disclosure. Using this method an oligonucleotide is designed that overlaps the genomic flanking and inserted DNA junction. The oligonucleotide is hybridized to single-stranded PCR product from the region of interest (one primer in the inserted DNA and one in the flanking genomic DNA sequence) and incubated in the presence of a DNA polymerase and a fluorescent-labeled ddNTP. Single base extension results in incorporation of the ddNTP. Incorporation can be measured as a change in polarization using a fluorometer. A change in polarization indicates the presence of the transgene/genomic sequence due to successful amplification, hybridization, and single base extension.
[00143] Real-time Polymerase Chain Reaction (PCR) is the ability to monitor the progress of the PCR as it occurs (i.e., in real time). Data is collected throughout the PCR process, rather than at the end of the PCR. In real-time PCR, reactions are characterized by the point in time during cycling when amplification of a target is first detected rather than the amount of target accumulated after a fixed number of cycles. In a real-time PCR assay, a positive reaction is detected by accumulation of a fluorescent signal. The higher the starting copy number of the nucleic acid target, the sooner a significant increase in fluorescence is observed. The cycle threshold (Ct value) is defined as the number of cycles required for the fluorescent signal to cross the threshold (i.e., exceeds background level). Ct levels are inversely proportional to the amount of target nucleic acid in the sample (i.e., the lower the Ct value, the greater the amount of target nucleic acid in the sample).
[00144] Taqman® (PE Applied Biosystems, Foster City, CA) is described as a method of detecting and quantifying the presence of a DNA sequence using real-time PCR and is fully understood in the instructions provided by the manufacturer. Briefly, a FRET oligonucleotide probe is designed that overlaps the genomic flanking and insert DNA junction. The FRET probe and PCR primers (one primer in the insert DNA sequence and one in the flanking genomic sequence) are cycled in the presence of a thermostable polymerase and dNTPs. Hybridization of the FRET probe results in cleavage and release of the fluorescent moiety away from the quenching moiety on the FRET probe. A fluorescent signal indicates the presence of the transgene/genomic sequence due to successful amplification and hybridization.
[00145] Molecular’ Beacons have been described for use in sequence detection as described in Tyangi, et al. (Nature Biotech.14:303-308, 1996). Briefly, a FRET oligonucleotide probe is designed that overlaps the flanking genomic and insert DNA junction. The unique structure of the FRET probe results in it containing secondary structure that keeps the fluorescent and quenching moieties in close proximity. The FRET probe and PCR primers (one primer in the insert DNA sequence and one in the flanking genomic sequence) arc cycled in the presence of a thermostable polymerase and dNTPs. Following successful PCR amplification, hybridization of the FRET probe to the target sequence results in the removal of the probe secondary structure and spatial separation of the fluorescent and quenching moieties. A fluorescent signal results. A fluorescent signal indicates the presence of the flanking/transgene insert sequence due to successful amplification and hybridization.
[00146] Other detection methods known in the art may be used. For example, microfluidics (see, e.g., U.S. Patent Publication No. 2006/068398; U.S. Patent No. 6,544,734) provide methods and devices that can be used to separate and amplify DNA samples or molecules. Optical dyes can be used to detect and measure specific DNA molecules (see, e.g., WO/05017181). Nanotube devices (see, e.g., WO/06024023) that comprise an electronic sensor for the detection of DNA molecules or nanobeads that bind specific DNA molecules can then be detected.
[00147] DNA detection kits that are based on DNA amplification methods contain DNA primer molecules that hybridize specifically to a target DNA and amplify a diagnostic amplicon under the appropriate reaction conditions. The kit may provide an agarose gel-based detection method or any number of methods of detecting the diagnostic amplicon that are known in the art. DNA detection kits can be developed using the compositions disclosed herein and are useful for identification of cotton event GH_BCS246002 DNA or DNA from a modified cotton event GH_BCS246002 in a sample and can be applied to methods for breeding cotton plants containing cotton event GH_BCS246002 DNA or DNA from a modified cotton event GH_BCS246002. A kit that contains DNA primers that are homologous or complementary to any portion of the cotton genomic region as set forth in SEQ ID NO: 10 and to any portion of the inserted transgenic DNA as set forth in SEQ ID NO: 10 is an object of the present disclosure. The DNA molecules can be used in DNA amplification methods (PCR) or as probes in polynucleic acid hybridization methods, i.e., southern analysis, northern analysis.
[00148] Probes and primers according to the present disclosure may have complete sequence identity with the target sequence, although primers and probes differing from the target sequence that retain the ability to hybridize preferentially to target sequences may be designed by conventional methods. In order for a nucleic acid molecule to serve as a primer or probe it need only be sufficiently complementary in sequence to be able to form a stable double- stranded structure under the particular solvent and salt concentrations employed. Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of transgenic DNA from cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 in a sample. Probes and primers are generally at least about 11 nucleotides, at least about 18 nucleotides, at least about 24 nucleotides, or at least about 30 nucleotides or more in length. Such probes and primers hybridize specifically to a target DNA sequence under stringent hybridization conditions. Conventional stringency conditions are described by Sambrook et al., 1989, and by Haymes et al., In: Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, DC (1985).
[00149] Any number of methods well known to those skilled in the art can be used to isolate and manipulate a DNA molecule, or fragment thereof, that is disclosed herein, including thermal amplification methods. DNA molecules, or fragments thereof, can also be obtained by other techniques such as by directly synthesizing the fragment by chemical means, as is commonly practiced by using an automated oligonucleotide synthesizer.
[00150] The DNA molecules and corresponding nucleotide sequences provided herein are therefore useful for, among other things, identifying cotton event GH_BCS246002 or a modified cotton event GH_BCS246002, detecting the presence of DNA derived from the transgenic cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 in a sample, and monitoring samples for the presence and/or absence of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 or plant parts derived from cotton plants comprising cotton event GH_BCS246002 or a modified cotton event GH_BCS246002. [00151] By reference to cotton it is intended that cotton plants, cotton plant cells, cotton seed, cotton pollen and ova, cotton plant parts, cotton progeny plants, and cotton commodity products are within the scope of the present disclosure, so long as each embodiment contains a detectable amount of DNA corresponding to any one, two, or more of the segments described herein as being diagnostic for, or characteristic of, the presence of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002, such as a polynucleotide having at least one of the sequences provided as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10. Cotton plants, cotton plant cells, cotton seed, cotton pollen and ova, cotton plant parts, cotton progeny plants of the present disclosure may also contain one or more additional transgenes. Such additional transgene(s) may be any nucleotide sequence encoding a protein or RNA molecule conferring a desirable trait including but not limited to increased insect resistance, increased water use efficiency, increased yield performance, increased drought resistance, increased seed quality, and/or increased herbicide tolerance.
[00152] The present disclosure provides cotton plants, cotton plant cells, cotton seed, cotton plant parts (such as pollen, ovule, squares, bolls, root tissue, leaf tissue), cotton progeny plants derived from a transgenic cotton plant containing cotton event GH_BCS246002 DNA or DNA from a modified cotton event GH_BCS246002. A representative sample of cotton seed containing cotton event GH_BCS246002 has been deposited according to the Budapest Treaty with the American Type Culture Collection (ATCC®). The ATCC repository has assigned the Patent Deposit Designation PTA- 127733 to the seed containing cotton event GH_BCS246002 DNA.
[00153] The present disclosure provides a microorganism comprising a DNA molecule having at least one sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10 present in its genome. An example of such a microorganism is a transgenic plant cell. Microorganisms, such as a plant cell of the present disclosure, are useful in many industrial applications, including but not limited to: (i) use as research tool for scientific inquiry or industrial research; (ii) use in culture for producing endogenous or recombinant carbohydrate, lipid, nucleic acid, or protein products or small molecules that may be used for subsequent scientific research or as industrial products; and (iii) use with modem plant tissue culture techniques to produce transgenic plants or plant tissue cultures that may then be used for agricultural research or production. The production and use of microorganisms such as transgenic plant cells utilizes modem microbiological techniques and human intervention to produce a man-made, unique microorganism. In this process, recombinant DNA is inserted into a plant cell’s genome to create a transgenic plant cell that is separate and unique from naturally occurring plant cells. This transgenic plant cell can then be cultured much like bacteria and yeast cells using modern microbiology techniques and may exist in an undifferentiated, unicellular state. The transgenic plant cell’s new genetic composition and phenotype is a technical effect created by the integration of the heterologous DNA into the genome of the cell. Another aspect of the present disclosure is a method of using a microorganism provided herein. Methods of using microorganisms of the present disclosure, such as transgenic plant cells, include (i) methods of producing transgenic cells by integrating recombinant DNA into the genome of the cell and then using this cell to derive additional cells possessing the same heterologous DNA; (ii) methods of culturing cells that contain recombinant DNA using modern microbiology techniques; (iii) methods of producing and purifying endogenous or recombinant carbohydrate, lipid, nucleic acid, or protein products from cultured cells; and (iv) methods of using modern plant tissue culture techniques with transgenic plant cells to produce transgenic plants or transgenic plant tissue cultures.
[00154] Cotton plants of the present disclosure may pass along the cotton event GH_BCS246002 DNA, including the transgene inserted in cotton event GH_BCS246002 or a modified cotton event GH_BCS246002, to progeny, typically through crosses or conventional breeding and selection. As used herein, “progeny” includes any plant, plant cell, seed, and/or regenerable plant part containing the cotton event GH_BCS246002 DNA or DNA from a modified cotton event GH_BCS246002 derived from an ancestor plant and/or comprising a DNA molecule having at least one sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10. Cotton plants, progeny, and seed may be homozygous or heterozygous for the cotton event GH_BCS246002 or a modified cotton event GH_BCS246002. Progeny may be grown from seed produced by a cotton plant containing event GH_BCS246002 or a modified cotton event GH_BCS246002 and/or from seed produced by a plant fertilized with pollen from a cotton plant containing event GH_BCS246002 or a modified cotton event GH_BCS246002.
[00155] Methods for producing cotton plants and seeds containing or comprising cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 are provided. Cotton plants may be bred using any method known in the art. Progeny plants may be self-pollinated (also known as “selfing”) to generate a true breeding line of plants, i.c., plants homozygous for the transgene(s), such as cotton event ZM_BCS216090. Indeed, selfing can result in progeny known as an “inbred” that can be used to produce cotton inbred lines that are genetically uniform. Selfing of appropriate progeny can produce plants that are homozygous for the exogenous transgene(s), such as cotton event ZM_BCS21609 or a modified cotton event GH_BCS246002. Alternatively, progeny plants may be out-crossed, e.g., bred with another unrelated plant, to produce a varietal or a hybrid seed or plant. The other unrelated plant may be transgenic or non-transgenic. A varietal or hybrid seed or plant of the present disclosure may thus be derived by sexually crossing a first parent that lacks the specific and unique DNA of the cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 with a second parent comprising cotton event GH_BCS246002 or a modified cotton event GH_BCS246002, resulting in a hybrid comprising the specific and unique DNA of the cotton event GH_BCS246002 or the modified cotton event GH_BCS246002. Each parent can be a hybrid or an inbred/varietal, so long as the cross or breeding results in a plant or seed of the present disclosure, i.e., a seed having at least one allele containing the DNA of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 and/or a DNA molecule having at least one sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10. Two different transgenic plants may thus be crossed to produce hybrid offspring that contain two independently segregating transgenes or traits, wherein at least one of those transgenes or events comprise or is contained within cotton event GH_BCS246002 or a modified cotton event GH_BCS246002. For example, transgenic cotton plants comprising cotton event GH_BCS246002 containing CrylB.3, CrylDa_7, and Vip3Cbl.l, conferring Lepidopteran resistance to cotton, or a modified cotton event GH_BCS246002, can be crossed with a plant having one or more additional traits such as herbicide tolerance, insect resistance, or drought tolerance, resulting in a progeny plant or seed that has resistance to Lepidopteran insect pests and has at least one or more additional traits. Back-crossing to a parental plant and out-crossing with a non-transgenic plant are also contemplated, as is vegetative propagation. Descriptions of other breeding methods that are commonly used for different traits and crops can be found in one of several references, e.g., Fehr, in Breeding Methods for Cultivar Development, Wilcox J. ed., American Society of Agronomy, Madison WI (1987). [00156] Cotton plants, progeny, seed, pollen and ova, cells and plant parts of the present disclosure may also contain one or more additional cotton traits(s) or transgenic cvcnt(s), which may be introduced by crossing a cotton plant containing cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 with another cotton plant containing the additional trait(s) or transgenic event(s). Such trait(s) or transgenic event(s) may include, but are not limited to, increased insect resistance, herbicide tolerance, increased water use efficiency, increased yield performance, increased drought resistance, increased seed quality, improved nutritional quality, hybrid seed production, or disease or fungal resistance. A cotton trait may include any transgenic traits or mutant or edited traits or alleles. Mutant traits or alleles of a gene may be created by any mutagenesis technique known in the art, whereas edited traits may be generated by any genome editing technique or method known in the art. Many cotton transgenic events are known to those of skill in the art. For example, a list of such traits is provided by the United States Department of Agriculture’s (USDA) Animal and Plant Health Inspection Service (APHIS) and can be found on their website www.aphis.usda.gov on the worldwide web. Two or more transgenic events may thus be combined in a progeny seed or plant by crossing two parent plants each comprising one or more transgenic events, collecting the progeny seed, and selecting for progeny seed or plants that contain the two or more transgenic events. These steps may be repeated until the desired combination of transgenic events in a progeny is achieved.
[00157] A plant part that is derived from cotton plants comprising cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 is also provided. As used herein, a “plant part” refers to any pail of a plant which is comprised of plant material derived from a cotton plant that may comprise cotton event GH_BCS246002 or a modified cotton event GH_BCS246002. Plant parts include, but are not limited to, pollen, ovule, square, boll, root tissue, stem tissue, fibers, and leaves. Plant parts may be viable, nonviable, regenerable, and/or nonregenerable.
[00158] Further provided is a commodity product that is derived from cotton plants comprising cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 and that contains a detectable amount of a nucleic acid specific for cotton event GH_BCS246002 or a modified cotton event GH_BCS246002. As used herein, a “commodity product” refers to any composition or product which is comprised of material derived from a cotton plant, whole or processed cotton seed, or one or more plant cells and/or plant parts containing the cotton event GH_BCS246002 DNA or DNA from a modified cotton event GH_BCS246002. Nonviable commodity products include, but arc not limited to, nonviable seed, whole or processed seed, seed parts, and plants parts; whole or processed cotton seeds, cotton fiber, cotton oil and derivatives of cotton oil, cotton protein, cotton meal, animal feed comprising cotton, paper comprising cotton, cotton biomass, candle wicks, cotton string, cotton rope, cotton balls, cotton batting, cotton fuel products, and cotton cellulose products. Viable commodity products include, but are not limited to, seed, plants, and plant cells. The cotton plants comprising cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 can thus be used to manufacture any commodity product typically acquired from cotton. Any such commodity product that is derived from cotton plants comprising cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 may contain at least a detectable amount of the specific and unique DNA corresponding to cotton event GH_BCS246002 or a modified cotton event GH_BCS246002, and specifically may contain a detectable amount of a polynucleotide comprising a DNA molecule having at least one sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10. Any standard method of detection for nucleotide molecules may be used, including methods of detection disclosed herein. A commodity product is within the scope of the present disclosure if there is any detectable amount of a DNA molecule having at least one sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10 in the commodity product.
[00159] The cotton plants, cotton plant cells, cotton seed, cotton plant parts (such as pollen, ovule, anther, root tissue, stalk tissue leaf tissue), cotton progeny plants, and commodity products of the present disclosure are therefore, useful for, among other things, growing plants for the purpose of producing seed and/or plant parts comprising cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 for agricultural purposes, producing progeny comprising cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 for plant breeding and research purposes, use with microbiological techniques for industrial and research application, and sale to consumers.
[00160] Methods for producing an insect resistant cotton plant comprising the DNA sequences specific and unique to cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 of the present disclosure are provided. A progeny cotton plant comprising the event GH_BCS246002 or a modified cotton event GH_BCS246002 may be produced, for example, by selfing a parent plant or line comprising the event GH_BCS246002 or a modified cotton event GH_BCS246002, wherein such parent plant or line is homozygous or hemizygous for the event GH_BCS246002 or the modified cotton event GH_BCS246002, or by crossing a first parent plant or line comprising the event GH_BCS246002 or a modified cotton event GH BCS246002, wherein such parent plant or line is homozygous or hemizygous for the event GH_BCS246002 or the modified cotton event GH_BCS246002, with a second parent plant or line having a different genotype or germplasm than the first parent line, wherein the second parent plant or line may or may not contain or comprise the event GH_BCS246002 or a modified cotton event GH_BCS246002. Transgenic plants used in these methods may be homozygous or heterozygous (or hemizygous) for the transgene(s), event GH_BCS246002 or a modified cotton event GH_BCS246002. Progeny plants produced by these methods may be varietal or hybrid plants, may be grown from seeds produced by plants containing cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 and/or from seed produced by a plant fertilized with pollen from a cotton plant containing cotton event GH_BCS246002 or a modified cotton event GH_BCS246002. Progeny plants may be subsequently self-pollinated to generate a true breeding line of plants, i.e., plants homozygous for the transgene, or alternatively may be out-crossed, e.g., bred with another unrelated plant, to produce a varietal or hybrid seed or plant.
[00161] Methods of detecting the presence of DNA derived from a cotton cell, cotton tissue, cotton seed, or cotton plant comprising cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 in a sample arc provided. One method comprises (i) extracting a DNA sample from at least one cotton cell, cotton tissue, cotton seed, or cotton plant; (ii) contacting the DNA sample with at least one primer that is capable of producing DNA sequence specific to cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 under conditions appropriate for DNA sequencing; (iii) performing a DNA sequencing reaction; and then (iv) confirming that the nucleotide sequence comprises a nucleotide sequence specific for cotton event GH_BCS246002 or a modified cotton event GH_BCS246002, of the construct comprised therein, such as one selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10. Another method comprises (i) extracting a DNA sample from at least one cotton cell, cotton tissue, cotton seed, or cotton plant; (ii) contacting the DNA sample with a primer pair that is capable of producing an amplicon from cotton event GH_BCS246002 DNA or DNA from a modified cotton event GH_BCS246002 under conditions appropriate for DNA amplification; (iii) performing a DNA amplification reaction; and then (iv) detecting the amplicon molecule and/or confirming that the nucleotide sequence of the amplicon comprises a nucleotide sequence specific for cotton event GH_BCS246002 or a modified cotton event GH_BCS246002, such as one selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6. The amplicon should be one that is specific for cotton event GH_BCS246002, such as an amplicon that comprises SEQ ID NO: 1, or SEQ ID NO: 2, or SEQ ID NO: 3, or SEQ ID NO: 4, or SEQ ID NO: 5, or SEQ ID NO: 6. The detection of a nucleotide sequence specific for cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 in the amplicon is determinative and/or diagnostic for, or characteristic of, the presence of DNA specific for the cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 in the sample. An example of a primer pair that is capable of producing an amplicon from cotton event GH_BCS246002 DNA or DNA from a modified cotton event GH_BCS246002 under conditions appropriate for DNA amplification is provided as SEQ ID NO: 15 and SEQ ID NO: 16. Other primer pairs may be readily designed by one of skill in the art and would produce an amplicon comprising SEQ ID NO: 1, or SEQ ID NO: 2, or SEQ ID NO: 3, or SEQ ID NO: 4, or SEQ ID NO: 5, or SEQ ID NO: 6, wherein such a primer pair comprises at least one primer within the genomic region flanking the insert and a second primer within the insert. Another method of detecting the presence of DNA derived from a cotton cell, cotton tissue, cotton seed, or cotton plant comprising cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 in a sample comprises (i) extracting a DNA sample from at least one cotton cell, cotton tissue, cotton seed, or cotton plant; (ii) contacting the DNA sample with a DNA probe specific for cotton event GH_BCS246002 DNA or DNA specific for a modified cotton event GH_BCS246002; (iii) allowing the probe and the DNA sample to hybridize under stringent hybridization conditions and then (iv) detecting hybridization between the probe and the target DNA sample. An example of the sequence of a DNA probe that is specific for cotton event GH_BCS246002 is provided as SEQ ID NO: 17. Other probes may be readily designed by one of skill in the art and would comprise at least one fragment of genomic DNA flanking the insert and at least on fragment of the insert DNA such as sequence provided in, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 10. Detection of probe hybridization to the DNA sample is diagnostic for, or characteristic of, the presence of cotton event GH_BCS246002 specific DNA in the sample. Absence of hybridization is alternatively diagnostic for, or characteristic of, the absence of cotton event GH_BCS246002 specific DNA in the sample.
[001621 DNA detection kits are provided that are useful for the identification of cotton event GH_BCS246002 DNA or DNA from a modified cotton event GH_BCS246002 in a sample and can also be applied to methods for breeding cotton plants containing the appropriate event DNA. Such kits contain DNA primers and/or probes comprising fragments of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10. One example of such a kit comprises at least one DNA molecule of sufficient length of continuous nucleotides of SEQ ID NO: 10 to function as a DNA probe useful for detecting the presence and/or absence of DNA derived from transgenic cotton plants comprising cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 in a sample. The DNA derived from transgenic cotton plants comprising cotton event GH_BCS246002 would comprise a DNA molecule having at least one sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10. A DNA molecule sufficient for use as a DNA probe is provided that is useful for determining, detecting, or diagnosing the presence and/or absence of cotton event GH_BCS246002 in a sample is provided as SEQ ID NO: 17. Other probes may be readily designed by one of skill in the art and should comprise at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, or at least 40 contiguous nucleotides of SEQ ID NO: 10 and be sufficiently unique to cotton event GH_BCS246002 DNA in order to identify DNA derived from the event.
[00163] Another type of kit comprises a primer pair useful for producing an amplicon useful for detecting the presence and/or absence of DNA derived from transgenic cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 in a sample. Such a kit would employ a method comprising contacting a target DNA sample with a primer pair as described herein, then performing a nucleic acid amplification reaction sufficient to produce an amplicon comprising a DNA molecule having at least one sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10 and then detecting the presence and/or absence of the amplicon. Such a method may also include sequencing the amplicon or a fragment thereof, which would be determinative of, i.e., diagnostic for, or characteristic of, the presence of the cotton event GH_BCS246002 specific DNA or DNA specific for a modified cotton event GH_BCS246002 in the target DNA sample. Other primer pairs may be readily designed by one of skill in the art and should comprise at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 contiguous nucleotides of sequences provided in, but not limited to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, and be sufficiently unique to cotton event GH_BCS246002 DNA in order to identify DNA derived from the event.
[00164] The kits and detection methods of the present disclosure are useful for, among other things, identifying or detecting cotton event GH_BCS246002 or a modified cotton event GH_BCS246002, selecting plant varieties or hybrids comprising cotton event GH_BCS246002 or a modified cotton event GH_BCS246002, detecting the presence of DNA derived from the transgenic cotton plant comprising cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 in a sample, and monitoring samples for the presence and/or absence of cotton plants comprising cotton event GH_BCS246002 or a modified cotton event GH_BCS246002, or plant parts derived from cotton plants comprising cotton event GH_BCS246002 or a modified cotton event GH_BCS246002.
[00165] The sequences of the heterologous DNA insert, junction sequences, or flanking sequences from cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 can be verified (and corrected if necessary) by amplifying such sequences from the event using primers derived from the sequence provided herein followed by standard DNA sequencing of the amplicon or of the cloned DNA.
[00166] Methods of detecting the zygosity of the transgene DNA from a cotton cell, cotton tissue, cotton seed, or cotton plant or plant pail comprising cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 in a sample are provided. One method comprises (i) extracting a DNA sample from at least one cotton cell, cotton tissue, cotton seed, or cotton plant or plant part; (ii) contacting the DNA sample with a primer pair that is capable of producing a first amplicon diagnostic for, or characteristic of, cotton event GH_BCS246002 or a modified cotton event GH_BCS246002; (iii) contacting the DNA sample with a primer pair that is capable of producing a second amplicon diagnostic for, or characteristic of, native cotton genomic DNA that does not comprise cotton event GH_BCS246002 or a modified cotton event GH_BCS246002; (iv) performing a DNA amplification reaction(s); and then (v) detecting the amplicons, wherein the presence of only the first amplicon is diagnostic for cotton cell, cotton tissue, cotton seed, or cotton plant or plant part being homozygous for the cotton event GH_BCS246002 or the modified cotton event GH_BCS246002, and the presence of both the first amplicon and the second amplicon is diagnostic for the cotton cell, cotton tissue, cotton seed, or cotton plant or plant part being heterozygous for the cotton event GH_BCS246002 or the modified cotton event GH_BCS246002. An exemplary set of primer pairs are presented as SEQ ID NO: 15 and SEQ ID NO: 16 which produce an amplicon diagnostic for, or characteristic of, cotton event GH_BCS246002; and SEQ ID NO: 15 and SEQ ID NO: 21 which produces an amplicon for, or characteristic of, the wild-type cotton genomic DNA not comprising cotton event GH_BCS246002. A set of probes can also be incorporated into such an amplification method to be used in real-time PCR format using the primer pair sets described above. An exemplary set of probes are presented as SEQ ID NO: 17 (diagnostic for, or characteristic of, the amplicon for the cotton event GH_BCS246002) and SEQ ID NO: 22 (diagnostic for, or characteristic of, the amplicon for wild-type cotton genomic DNA not comprising cotton event GH_BCS246002).
[00167] Another method for determining zygosity comprises (i) extracting a DNA sample from at least one cotton cell, cotton tissue, cotton seed, or cotton plant or plant pail; (ii) contacting the DNA sample with a probe set which contains at least a first probe that specifically hybridizes to cotton event GH_BCS246002 DNA or a modified cotton event GH_BCS246002 DNA and at least a second probe that specifically hybridizes to wild-type cotton genomic DNA that was disrupted by insertion of the heterologous DNA of cotton event GH_BCS246002 and does not hybridize to cotton event GH_BCS246002 DNA or a modified cotton event GH_BCS246002 DNA; (iii) hybridizing the probe set with the sample under stringent hybridization conditions, wherein detecting hybridization of only the first probe under the hybridization conditions is diagnostic for, or characteristic of, the sample being homozygous for the cotton event GH_BCS246002 DNA or the modified cotton event GH_BCS246002 DNA, and wherein detecting hybridization of both the first probe and the second probe under the hybridization conditions is diagnostic for, or characteristic of, the sample being heterozygous for the cotton event GH_BCS246002 DNA or the modified cotton event GH_BCS246002 DNA.
[00168] Yet another method for determining zygosity comprises (i) extracting a DNA sample from at least one cotton cell, cotton tissue, cotton seed, or cotton plant; (ii) contacting the DNA sample with a primer pair that is capable of producing an amplicon diagnostic for, or characteristic of, cotton event GH_BCS246002 or a modified cotton event GH_BCS246002; (iii) contacting the DNA sample with a primer pair that is capable of producing an amplicon of an internal standard known to be single-copy and homozygous in the cotton plant; (iv) contacting the DNA sample with a probe set which contains at least a first probe that specifically hybridizes to DNA of the cotton event GH_BCS246002 or the modified cotton event GH_BCS246002, and at least a second probe the specifically hybridizes to the internal standard genomic DNA known to be single-copy and homozygous in the cotton plant; (v) performing a DNA amplification reaction using real-time PCR and determining the cycle thresholds (Ct values) of the amplicon corresponding to the cotton event GH_BCS246002 or the modified cotton event GH_BCS246002 and the single-copy, homozygous internal standard; (vi) calculating the difference (ACt) between the Ct value of the single-copy, homozygous internal standard amplicon and the Ct value of the amplicon for the cotton event GH_BCS246002 or the modified cotton event GH_BCS246002; and (vii) determining zygosity of the cotton event GH_BCS246002 or the modified cotton event GH_BCS246002, wherein a ACt of around zero (0) indicates homozygosity of the inserted T-DNA event and a ACt of around one (1) indicates heterozygosity of the inserted T-DNA event. Heterozygous and homozygous events are differentiated by a ACt value unit of approximately one (1). Given the normal variability observed in real-time PCR due to multiple factors such as amplification efficiency and ideal annealing temperatures, the range of “about one (1)” is defined as a ACt of 0.75 to 1.25. Primer pairs and probes for the above method for determining zygosity can amplify and detect amplicons from DNA of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 and an internal standard. Exemplary primer pairs for the detection of amplicons corresponding to cotton event GH_BCS246002 and internal standard are presented as SEQ ID NO: 15 combined with SEQ ID NO: 16 (allele for cotton event GH_BCS246002) and SEQ ID NO: 18 combined with SEQ ID NO: 19 (internal standard). The accompanying exemplary probes are presented as SEQ ID NO: 17 (cotton event GH_BCS246002) and SEQ ID NO: 20 (internal standard). Modification of cotton event GH_BCS246002
[00169] As used herein, a “cotton event GH_BCS246002 locus” refers to the genomic locus of the cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 or a further modified cotton event GH_BCS246002, wherein the cotton event GH_BCS246002 locus includes the flanking, junction, and insertion sequences of the cotton event GH_BCS246002 or the modified cotton event GH_BCS246002 or further modified cotton event GH_BCS246002. A modified cotton event GH_BCS246002 or a further modified cotton event GH_BCS246002 comprises one or more mutations, edits and/or genetic modifications in the cotton event GH_BCS246002 locus, such as one or more mutations, edits and/or genetic modifications in a flanking, junction and/or insertion sequence(s) of the cotton event GH_BCS246002 locus, relative to cotton event GH_BCS246002.
[00170] According to present embodiments, a modified cotton event GH_BCS246002 and methods of making a modified cotton event GH_BCS246002 are provided. As is described further herein, various mutagenesis or targeted genome editing techniques and related tools are known and could be made or engineered to permit genetic modification or mutation of the transgenic insert, junction and/or the flanking genomic DNA of cotton event GH_BCS246002, such as by deletion, insertion, transposition, inversion, and/or substitution of nucleic acid sequence(s), and/or by insertion or introduction of a guide RNA target site or a cognate target site or CgRRS, and the transgenic event as modified may still be uniquely characterized by the presence of heterologous DNA and/or one or more sequences of the insertion, junction(s) and/or flanking sequence(s) of cotton event GH_BCS246002 described herein at the same position or location in the genome previously occupied by the unmodified cotton event GH_BCS246002 relative to flanking portions or sequences of the native cotton genome. According to present embodiments, a modified transgenic event derived from cotton event GH_BCS246002 may comprise all or part of the insertion sequence and/or transgene cassette of cotton event GH_BCS246002, one or more of the junction sequence(s) of cotton event GH_BCS246002, and/or one or more flanking sequence(s) of cotton event GH_BCS246002 as described herein. As used herein, a “modified cotton event GH_BCS246002” refers to any genomic DNA or sequence of the cotton event GH_BCS246002 locus comprising one or more mutations, edits or genetic modifications relative to the genomic DNA or sequence of the cotton event GH_BCS246002, wherein such mutations or edits are introduced or made by a mutagenesis or targeted genome editing technique of a cotton plant, plant part, tissue or cell comprising the cotton event GH_BCS246002. A “modified cotton event GH_BCS246002” includes, as a type of modified cotton event GH_BCS246002, a “further modified cotton event GH_BCS246002” made by first inserting a target site or cognate target site or CgRRS into the cotton event GH_BCS246002 locus and then further modifying the cotton event GH_BCS246002 locus as described herein. For clarity, a “modified cotton event GH BCS246002” includes genomic DNA or sequences of the cotton event GH_BCS246002 locus comprising one or more mutations, edits or genetic modifications relative to the genomic DNA or sequence of the cotton event GH_BCS246002, wherein such mutations or edits are introduced or made by a mutagenesis or targeted genome editing technique of a cotton plant, plant part, tissue or cell comprising the cotton event GH_BCS246002, wherein such modified cotton event GH_BCS246002 is not a further modified cotton event GH_BCS246002. Methods and techniques of mutagenesis are known in the art and include, for example, chemical mutagenesis (z.e. , treatment with a chemical mutagen, such as an azide, hydroxylamine, nitrous acid, acridine, nucleotide base analog, or alkylating agent - e.g. , EMS (ethylmethane sulfonate), MNU (N-methyl-N-nitrosourea), etc.), physical mutagenesis e.g., gamma rays, X-rays, UV, ion beam, other forms of radiation, etc.), and insertional mutagenesis (e.g., transposon or T-DNA insertion). As used herein, a “modified cotton plant” refers to a cotton plant comprising a modified cotton event GH_BCS246002 or a further modified cotton event GH_BCS246002. Thus, a modified cotton plant part, plant seed, plant tissue, or plant cell comprising a modified cotton event GH_BCS246002 or a further modified cotton event GH_BCS246002 that is derived, taken or descended from a modified cotton plant and/or created by genetic modification, mutation or editing of the transgenic insert, junction and/or the flanking genomic DNA of cotton event GH_BCS246002 in a cotton plant part, plant seed, plant tissue, or plant cell using a mutagenesis or targeted genome editing technique. According to some embodiments, a modified cotton event GH BCS246002 may have one or both of the junction sequences of cotton event GH_BCS246002 altered by a targeted genome editing technique, may have one or more of the expression elements or cassettes altered or removed, or may have additional expression cassettes or sequences inserted or included within the border or junction sequences or near either end of the inserted event DNA, and may have polynucleotide or expression element sequences altered to change expression or to encode a different amino acid sequence of one or more of the respective toxins present in event GH_BCS246002, which may be designed to overcome the development of resistance by one or more target pests to one or more of the respective toxin proteins described herein, wherein the altered expression or different amino acid sequence confers improved toxin properties to the respective protein and/or a different host range of toxicity due to the slightly different amino acid sequence in the modified cotton plant comprising the modified cotton event GH_BCS246002.
[001711 As used herein, a “target site” for genome editing refers to the location of a polynucleotide sequence within a plant genome, which may be within or near cotton event GH_BCS246002, that is bound and cleaved by a site-specific nuclease introducing a double stranded break (or single- stranded nick) into the nucleic acid backbone of the polynucleotide sequence and/or its complementary DNA strand. A target site may comprise at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 29, or at least 30 consecutive nucleotides. A “target site” for an RNA-guidcd nuclease may comprise the sequence of either complementary strand of a double-stranded nucleic acid (DNA) molecule or chromosome at the target site that is bound or hybridized to a guide RNA of a ribonucleoprotein complex comprising the RNA-guided nuclease. A site-specific nuclease may bind to a target site, such as via a non-coding guide RNA (e.g., without being limiting, a CRISPR RNA (crRNA) or a single-guide RNA (sgRNA) as described further below). A non-coding guide RNA (gRNA) provided herein may be complementary to a target site (e.g., complementary to either strand of a double- stranded nucleic acid molecule or chromosome at the target site). It will be appreciated that perfect identity or complementarity may not be required for a non-coding guide RNA to bind or hybridize to a target site. For example, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 mismatches (or more) between a target site and a noncoding RNA may be tolerated. A “target site” also refers to the location of a polynucleotide sequence within a plant genome, which may be within or near cotton event GH_BCS246002 or a modified cotton event GH_BCS246002, that is bound and cleaved by another site-specific nuclease that may not be guided by a non-coding RNA molecule, such as a meganuclease, zinc finger nuclease (ZFN), or a transcription activator-like effector nuclease (TALEN), to introduce a double stranded break (or single- stranded nick) into the polynucleotide sequence and/or its complementary DNA strand. As used herein, a “target region” or a “targeted region” refers to a polynucleotide sequence or region that is flanked by two or more target sites. Without being limiting, in some embodiments a target region may be subjected to a mutation, deletion, insertion or inversion. As used herein to describe a target region of a polynucleotide sequence or molecule, “flanked” refers to two or more target sites of the polynucleotide sequence or molecule surrounding the target region, with one target site on each side of the target region.
[00172] As used herein, a “donor molecule”, “donor template”, or “donor template molecule” (collectively a “donor template”), which may be a recombinant DNA donor template, is defined as a nucleic acid molecule having a nucleic acid template or insertion sequence for site- directed, targeted insertion or recombination into the genome of a plant cell via repair of a nick or double-stranded DNA break in the genome of a plant cell. For example, a “donor template” may be used for site-directed integration of a guide RNA target site or a cognate target site or CgRRS into a target site within the genome of a plant. A targeted genome editing technique provided herein may comprise the use of one or more, two or more, three or more, four or more, or five or more donor molecules or templates. A “donor template” may be a single-stranded or doublestranded DNA or RNA molecule or plasmid. An “insertion sequence” of a donor template is a sequence designed for targeted insertion into the genome of a plant cell, which may be of any suitable length, such as to include a guide RNA target site or a cognate target site or CgRRS. Such an insertion sequence of a donor template is distinct and different from the transgenic insertion or insert of cotton event GH_BCS246002, although they may potentially have sequence(s) in common. A donor template may also have at least one homology sequence or homology arm, such as two homology arms, to direct the integration of a mutation or insertion sequence into a target site within the genome of a plant via homologous recombination, wherein the homology sequence or homology arm(s) are identical or complementary, or have a percent identity or percent complementarity, to a sequence at or near the target site within the genome of the plant. When a donor template comprises homology arm(s) and an insertion sequence, the homology arm(s) will flank or surround the insertion sequence of the donor template.
[00173] As used herein, “targeted genome editing technique” refers to any method, protocol, or technique that can be used to make a targeted mutation or edit, such as one or more insertions, deletions, substitutions, inversions, transpositions, mutations and/or other genetic modifications at or near a target site in the genome of a plant, and/or a deletion or excision of a target region between two target sites in the genome of a plant, using a site-specific nuclease, such as a meganuclease, a zine-finger nuclease (ZFN), an RNA-guided endonuclease (e.g., the CRISPR/Cas9 system), a TALE-endonuclease (TALEN), a recombinase, or a transposase. A site- specific nuclease may introduce a double stranded break (or single-stranded nick) into the nucleic acid backbone of the polynucleotide sequence and/or its complementary DNA strand. Following the introduction of the single or double-stranded break or nick or cleavage of DNA at or near the target site(s) by the site-specific nuclease, the genomic sequence can be repaired via a double strand break repair pathway, which may include, for example, non-homologous end-joining (NHEJ), microhomology-mediated end joining (MMEJ), homologous recombination, synthesisdependent strand annealing (SDSA), single-strand annealing (SSA), or a combination of any thereof, at or near the target site(s). However, if the repair is imperfect, a mutation or edit may be introduced at or near the target site(s), and a target region between two or more target sites may be deleted or excised.
[00174] A “site-specific nuclease” provided herein may be selected from the group consisting of a zine-finger nuclease (ZFN), a mcganuclcasc, an RNA-guidcd endonuclease, a TALE-endonuclease (TALEN), a recombinase, a transposase, or any combination thereof. See, e.g., Khandagale, K. et al., “Genome editing for targeted improvement in plants,” Plant Biotechnol Rep 10: 327-343 (2016); and Gaj, T. et al., “ZFN, TALEN and CRISPR/Cas-based methods for genome engineering,” Trends Biotechnol. 31(7): 397-405 (2013), the contents and disclosures of which are incorporated herein by reference. A recombinase may be a serine recombinase attached to a DNA recognition motif, a tyrosine recombinase attached to a DNA recognition motif or other recombinase enzyme known in the art. A recombinase or transposase may be a DNA transposase or recombinase attached to a DNA binding domain. A tyrosine recombinase attached to a DNA recognition motif may be selected from the group consisting of a Cre recombinase, a Flp recombinase, and a Tnpl recombinase. According to some embodiments, a Cre recombinase or a Gin recombinase provided herein is tethered to a zine-finger DNA binding domain. In another embodiment, a serine recombinase attached to a DNA recognition motif provided herein is selected from the group consisting of a PhiC31 integrase, an R4 integrase, and a TP-901 integrase. In another embodiment, a DNA transposase attached to a DNA binding domain provided herein is selected from the group consisting of a TALE-piggyBac and TALE-Mutator.
[00175] According to embodiments of the present disclosure, an RNA-guided endonuclease may be selected from the group consisting of Casl , CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, CmiT, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl 7, Csxl 4, Csx lO, Csx16, CsaX, Csx3, Csx l , Csxl 5, Csfl, Csf2, Csf3, Csf4, Cas l2a, Cpfl, CasX, CasY, and homologs or modified versions thereof, Argonautc (non-limiting examples of Argonaute proteins include Thermus thermophilus Argonaute (TtAgo), Pyrococcus furiosus Argonaute (PfAgo), Natronobacterium gregoryi Argonaute (NgAgo) and homologs or modified versions thereof. According to some embodiments, an RNA-guided endonuclease may be a Cas9 or Casl2a or Cpfl enzyme.
[00176] In an aspect, a site- specific nuclease provided herein is selected from the group consisting of a zinc-finger nuclease, a meganuclease, an RNA-guided nuclease, a TALE-nuclease, a recombinase, a transposase, or any combination thereof. In another aspect, a site-specific nuclease provided herein is selected from the group consisting of a Cas9 or a Casl2a or Cpfl. In another aspect, a site-specific nuclease provided herein is selected from the group consisting of a Casl, a CaslB, a Cas2, a Cas3, a Cas4, a Cas5, a Cas6, a Cas7, a Cas8, a Cas9, a CaslO, a Casl2a, a Csyl, a Csy2, a Csy3, a Csel, a Cse2, a Cscl, a Csc2, a Csa5, a Csn2, a Csm2, a Csm3, a Csm4, a Csm5, a Csm6, a Cmrl, a Cmr3, a Cmr4, a Cmr5, a Cmr6, a Csbl, a Csb2, a Csb3, a Csxl7, a Csxl4, a CsxlO, a Csxl6, a CsaX, a Csx3, a Csxl, a Csxl5, a Csfl, a Csf2, a Csf3, a Csf4, a Cpfl, CasX, CasY, a homolog thereof, or a modified version thereof. In another aspect, an RNA-guided nuclease provided herein is selected from the group consisting of a Cas9 or a Casl 2a or Cpfl. In another aspect, an RNA guided nuclease provided herein is selected from the group consisting of a Casl, a CaslB, a Cas2, a Cas3, a Cas4, a Cas5, a Cas6, a Cas7, a Cas8, a Cas9, a CaslO, a Casl2a, a Csyl, a Csy2, a Csy3, a Csel, a Cse2, a Cscl, a Csc2, a Csa5, a Csn2, a Csm2, a Csm3, a Csm4, a Csm5, a Csm6, a Cmrl, a Cmr3, a Cmr4, a Cmr5, a Cmr6, a Csbl, a Csb2, a Csb3, a Csxl7, a Csxl4, a CsxlO, a Csxl6, a CsaX, a Csx3, a Csxl, a Csxl5, a Csfl, a Csf2, a Csf3, a Csf4, a Cpfl , CasX, CasY, a homolog thereof, or a modified version thereof. In another aspect, a method and/or a composition provided herein comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten sitespecific nucleases. In yet another aspect, a method and/or a composition provided herein comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten polynucleotides encoding at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten site-specific nucleases. [00177] For RNA-guided endonucleases, a guide RNA (gRNA) molecule is further provided to direct the endonuclease to a target site in the genome of the plant via base-pairing or hybridization to cause a DSB or nick at or near the target site. The gRNA may be transformed or introduced into a plant cell or tissue (perhaps along with a nuclease, or nuclease-encoding DNA molecule, construct or vector) as a gRNA molecule, or as a recombinant DNA molecule, construct or vector comprising a polynucleotide or transcribable DNA sequence encoding the guide RNA operably linked to a plant-expressible promoter. As understood in the art, a “guide RNA” may comprise, for example, a CRISPR RNA (crRNA), a single-chain guide RNA (sgRNA), or any other RNA molecule that may guide or direct an endonuclease to a specific target site in the genome. A “single-chain guide RNA” (or “sgRNA”) is an RNA molecule comprising a crRNA covalently linked a tracrRNA by a linker sequence, which may be expressed as a single RNA transcript or molecule. The guide RNA comprises a guide or targeting sequence that is identical or complementary to a target site within the plant genome, such as within or near cotton event GH_BCS246002 or a modified cotton event GH_BCS246002. A protospacer-adjacent motif (PAM) may be present in the genome immediately adjacent and upstream or downstream of the genomic target site sequence complementary to the targeting sequence of the guide RNA as known in the art. See, e.g., Wu, X. et al., “Target specificity of the CRISPR-Cas9 system,” Quant Biol. 2(2): 59-70 (2014), the content and disclosure of which is incorporated herein by reference. The guide RNA may typically be a non-coding RNA molecule that does not encode a protein. The guide sequence of the guide RNA may be at least 10 nucleotides in length, such as 12-40 nucleotides, 12-30 nucleotides, 12-20 nucleotides, 12-35 nucleotides, 12-30 nucleotides, 15-30 nucleotides, 17-30 nucleotides, or 17-25 nucleotides in length, or about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more nucleotides in length. The guide sequence may be at least 95%, at least 96%, at least 97%, at least 99% or 100% identical or complementary to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, or more consecutive nucleotides of a DNA sequence at the genomic target site. As used herein, the term “consecutive” in reference to a polynucleotide or protein sequence means without deletions or gaps in the sequence.
[00178] In addition to the guide sequence, a guide RNA may further comprise one or more other structural or scaffold sequence(s), which may bind or interact with an RNA-guided endonuclease. Such scaffold or structural sequences may further interact with other RNA molecules (e.g., tracrRNA). Methods and techniques for designing targeting constructs and guide RNAs for genome editing and sitc-dircctcd integration at a target site within the genome of a plant using an RNA-guided endonuclease are known in the art.
[00179] According to some embodiments, recombinant DNA molecules, constructs and vectors are provided comprising a polynucleotide or transcribable DNA sequence encoding a sitespecific nuclease, such as a zinc-finger nuclease (ZFN), a meganuclease, an RNA-guided endonuclease, a TALE-endonuclease (TALEN), a recombinase, or a transposase, wherein the coding sequence is operably linked to a plant expressible promoter. For RNA-guided endonucleases, recombinant DNA molecules, constructs and vectors are further provided comprising a polynucleotide or transcribable DNA sequence encoding a guide RNA, wherein the guide RNA comprises a guide sequence of sufficient length having a percent identity or complementarity to a target site within the genome of a plant. According to some embodiments, recombinant DNA molecules, constructs and vectors are provided comprising a first polynucleotide or transcribable DNA sequence encoding a site-specific nuclease and a second polynucleotide or transcribable DNA sequence encoding one or more gRNAs. According to some embodiments, each polynucleotide or transcribable DNA sequence of a recombinant DNA molecule, construct and vector that encodes a site- specific nuclease and/or a guide RNA may be operably linked to a plant expressible promoter, such as an inducible promoter, a constitutive promoter, a tissue-specific promoter, etc.
[00180] According to some embodiments, recombinant DNA molecules, constructs and vectors are provided comprising a polynucleotide or transcribable DNA sequence encoding a sitespecific nuclease, such as a zinc-finger nuclease (ZFN), a meganuclease, an RNA-guided endonuclease, a TALE-endonuclease (TALEN), a recombinase, or a transposase, wherein the coding sequence is operably linked to a plant expressible promoter. For RNA-guided endonucleases, recombinant DNA molecules, constructs and vectors are further provided comprising a polynucleotide or transcribable DNA sequence encoding a guide RNA, wherein the guide RNA comprises a guide sequence of sufficient length having a percent identity or complementarity to a target site within the genome of a plant. According to some embodiments, recombinant DNA molecules, constructs and vectors are provided comprising a first polynucleotide or transcribable DNA sequence encoding a site-specific nuclease and a second polynucleotide or transcribable DNA sequence encoding one or more gRNAs. According to some embodiments, each polynucleotide or transcribable DNA sequence of a recombinant DNA molecule, construct and vector that encodes a site-specific nuclease and/or a guide RNA may be operably linked to a plant expressible promoter, such as an inducible promoter, a constitutive promoter, a tissue-specific promoter, etc.
[001811 According to some embodiments, a recombinant DNA molecule, construct or vector may comprise a first polynucleotide sequence encoding a site- specific nuclease and a second polynucleotide sequence encoding a guide RNA(s) that may be introduced into a plant cell together via plant transformation techniques. Alternatively, two recombinant DNA molecules, constructs or vectors may be provided including a first recombinant DNA molecule, construct or vector and a second DNA molecule, construct or vector that may be introduced into a plant cell together or sequentially via plant transformation techniques, wherein the first recombinant DNA molecule, construct or vector comprises a polynucleotide sequence encoding a site-specific nuclease and the second recombinant DNA molecule, construct or vector comprises a polynucleotide sequence encoding a guide RNA(s). According to some embodiments, a recombinant DNA molecule, construct or vector comprising a polynucleotide sequence encoding a site-specific nuclease may be introduced via plant transformation techniques into a plant cell that already comprises (or is transformed with) a recombinant DNA construct or vector comprising a polynucleotide sequence encoding a guide RNA(s). Alternatively, a recombinant DNA molecule, construct or vector comprising a polynucleotide sequence encoding a guide RNA may be introduced via plant transformation techniques into a plant cell that already comprises (or is transformed with) a recombinant DNA construct or vector comprising a polynucleotide sequence encoding a site-specific nuclease. According to yet further embodiments, a first plant comprising (or transformed with) a recombinant DNA construct or vector comprising a polynucleotide sequence encoding a site-specific nuclease may be crossed with a second plant comprising (or transformed with) a recombinant DNA construct or vector comprising a polynucleotide sequence encoding a guide RNA(s). Such recombinant DNA molecules, constructs or vectors may be transiently transformed into a plant cell or stably transformed or more preferably integrated into the genome of a plant cell.
[00182] In an aspect, molecules or vectors comprising polynucleotides encoding a sitespecific nuclease, and optionally one or more, two or more, three or more, or four or more gRNAs are provided to a plant cell by transformation methods known in the art (e. ., without being
16 limiting, particle bombardment, PEG-mediated protoplast transfection or Agrobacterium- mcdiatcd transformation). In an aspect, molecules or vectors comprising polynucleotides encoding a Cas9 nuclease, and optionally one or more, two or more, three or more, or four or more gRNAs are provided to a plant cell by transformation methods known in the art (e.g., without being limiting, particle bombardment, PEG-mediated protoplast transfection or Agrobacterium- mediated transformation). In another aspect, vectors comprising polynucleotides encoding a Cpfl and, optionally one or more, two or more, three or more, or four or more crRNAs are provided to a cell by transformation methods known in the art (e.g., without being limiting, viral transfection, particle bombardment, PEG-mediated protoplast transfection or Agrobacterium-mediated transformation).
[00183] Several site-specific nucleases, such as recombinases, zinc finger nucleases (ZFNs), mcganuclcascs, and TALENs, arc not RNA-guidcd and instead rely on their protein structure to determine their target site for causing the DSB or nick, or they are fused, tethered or attached to a DNA-binding protein domain or motif. The protein structure of the site-specific nuclease (or the fused/attached/tethered DNA binding domain) may target the site-specific nuclease to the target site. According to many of these embodiments, non-RNA-guided sitespecific nucleases, such as recombinases, zinc finger nucleases (ZFNs), meganucleases, and TALENs, may be designed, engineered and constructed according to known methods to target and bind to a target site in the genome of a plant, to create a DSB or nick at or near such genomic target site or locus. For example, an engineered site-specific nuclease, such as a recombinase, zinc finger nuclease (ZFN), meganuclease, or TALEN, may be designed to target and bind to a genomic target site within the genome of a plant to create a DSB or nick at the genomic target site.
[00184] In an aspect, a targeted genome editing technique described herein may comprise the use of a zinc finger nuclease (ZFN). ZFNs are synthetic proteins consisting of an engineered zinc finger DNA-binding domain fused to a cleavage domain (or a cleavage half-domain), which may be derived from a restriction endonuclease (e.g., Fold). The DNA binding domain may be canonical (C2H2) or non-canonical (e.g., C3H or C4). The DNA-binding domain can comprise one or more zinc fingers (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or more zinc fingers) depending on the target site. Multiple zinc fingers in a DNA-binding domain may be separated by linker sequence(s). ZFNs can be designed to cleave almost any stretch of double-stranded DNA by modification of the zinc finger DNA-binding domain. ZFNs form dimers from monomers composed of a non- specific DNA cleavage domain (e.g., derived from the FokI nuclease) fused to a DNA-binding domain comprising a zinc finger array engineered to bind a target site DNA sequence. The DNA- binding domain of a ZFN may typically be composed of 3-4 (or more) zinc-fingers. The amino acids at positions -1, +2, +3, and +6 relative to the start of the zinc finger a-helix, which contribute to site- specific binding to the target site, can be changed and customized to fit specific target sequences. The other amino acids may form a consensus backbone to generate ZFNs with different sequence specificities. Methods and rules for designing ZFNs for targeting and binding to specific target sequences are known in the ail. See, e.g., US Patent App. Nos. 2005/0064474, 2009/0117617, and 2012/0142062, the contents and disclosures of which are incorporated herein by reference. The FokI nuclease domain may require dimerization to cleave DNA and therefore two ZFNs with their C-terminal regions are needed to bind opposite DNA strands of the cleavage site (separated by 5-7 bp). The ZFN monomer can cut the target site if the two-ZF-binding sites are palindromic. A ZFN, as used herein, is broad and includes a monomeric ZFN that can cleave double stranded DNA without assistance from another ZFN. The term ZFN may also be used to refer to one or both members of a pair of ZFNs that are engineered to work together to cleave DNA at the same site.
[00185] Without being limited by any scientific theory, because the DNA-binding specificities of zinc finger domains can be re-engineered using one of various methods, customized ZFNs can theoretically be constructed to target nearly any target sequence (e.g., at or near cotton event GH_BCS246002 in a plant genome). Publicly available methods for engineering zinc finger domains include Context-dependent Assembly (CoDA), Oligomerized Pool Engineering (OPEN), and Modular Assembly. In an aspect, a method and/or composition provided herein comprises one or more, two or more, three or more, four or more, or five or more ZFNs. In another aspect, a ZFN provided herein is capable of generating a targeted DSB or nick. In an aspect, vectors comprising polynucleotides encoding one or more, two or more, three or more, four or more, or five or more ZFNs are provided to a cell by transformation methods known in the ail (e.g., without being limiting, viral transfection, particle bombardment, PEG-mediated protoplast transfection, or Agrobacterium-mediated transformation). The ZFNs may be introduced as ZFN proteins, as polynucleotides encoding ZFN proteins, and/or as combinations of proteins and protein-encoding polynucleotides. [00186] In an aspect, a targeted genome editing technique described herein may comprise the use of a mcganuclcasc. Mcganuclcascs, which arc commonly identified in microbes, such as the LAGLIDADG family of homing endonucleases, are unique enzymes with high activity and long recognition sequences (> 14 bp) resulting in site-specific digestion of target DNA. Engineered versions of naturally occurring meganucleases typically have extended DNA recognition sequences (for example, 14 to 40 bp). According to some embodiments, a meganuclease may comprise a scaffold or base enzyme selected from the group consisting of I- Crel, I-Ceul, I-Msol, I-Scel, I-Anil, and I-Dmol. The engineering of meganucleases can be more challenging than ZFNs and TALENs because the DNA recognition and cleavage functions of meganucleases are intertwined in a single domain. Specialized methods of mutagenesis and high- throughput screening have been used to create novel meganuclease variants that recognize unique sequences and possess improved nuclease activity. Thus, a meganuclease may be selected or engineered to bind to a genomic target site or sequence in a plant. In an aspect, a method and/or composition provided herein comprises one or more, two or more, three or more, four or more, or five or more meganucleases. In another aspect, a meganuclease provided herein is capable of generating a targeted DSB. In an aspect, vectors comprising polynucleotides encoding one or more, two or more, three or more, four or more, or five or more meganucleases are provided to a cell by transformation methods known in the art (e.g., without being limiting, viral transfection, particle bombardment, PEG-mediated protoplast transfection or Agrobacterium-mediated transformation).
[00187] In an aspect, a targeted genome editing technique described herein may comprise the use of a transcription activator-like effector nuclease (TALEN). TALENs are artificial restriction enzymes generated by fusing the transcription activator-like effector (TALE) DNA binding domain to a nuclease domain (e.g., FokF). In some aspects, the nuclease is selected from a group consisting of PvuII, MutH, TevI, FokI, Alwl, Mlyl, Sbfl, Sdal, StsI, CleDORF, Clo051, and PeptO7 . For FokI nuclease, when each member of a TALEN pair binds to the DNA sites flanking a target site, the FokI monomers dimerize and cause a double-stranded DNA break at the target site. Besides the wild-type FokI cleavage domain, variants of the FokI cleavage domain with mutations have been designed to improve cleavage specificity and cleavage activity. The FokI domain functions as a dimer, requiring two constructs with unique DNA binding domains for sites in the target genome with proper orientation and spacing. Both the number of amino acid residues between the TALEN DNA binding domain and the FokI cleavage domain and the number of bases between the two individual TALEN binding sites arc parameters for achieving high levels of activity. The term TALEN, as used herein, is broad and includes a monomeric TALEN that can cleave double stranded DNA without assistance from another TALEN. The term TALEN also refers to one or both members of a pair of TALENs that work together to cleave DNA at the same site.
[00188] Besides the wild-type FokI cleavage domain, variants of the FokI cleavage domain with mutations have been designed to improve cleavage specificity and cleavage activity. The FokI domain functions as a dimer, requiring two constructs with unique DNA binding domains for sites in the target genome with proper orientation and spacing. Both the number of amino acid residues between the TALEN DNA binding domain and the FokI cleavage domain and the number of bases between the two individual TALEN binding sites arc parameters for achieving high levels of activity. PvuII, MutH, and TevI cleavage domains are useful alternatives to FokI and FokI variants for use with TALEs. PvuII functions as a highly specific cleavage domain when coupled to a TALE (see Yank et al. 2013. PLoS One. 8: e82539). MutH is capable of introducing strand- specific nicks in DNA (see Gabsalilow et al. 2013. Nucleic Acids Research. 41: e83). TevI introduces doublestranded breaks in DNA at targeted sites (see Beurdeley et al., 2013. Nature Communications. 4: 1762).
[00189] Transcription activator-like effectors (TALEs) can be engineered to bind practically any DNA sequence, such as at or near the genomic locus of cotton event GH_BCS246002 in a plant. TALE has a central DNA-binding domain composed of 13-28 repeat monomers of 33-34 amino acids. The amino acids of each monomer are highly conserved, except for hypervariable amino acid residues at positions 12 and 13. The two variable amino acids are called repeat-variable diresidues (RVDs). The amino acid pairs NI, NG, HD, and NN of RVDs preferentially recognize adenine, thymine, cytosine, and guanine/adenine, respectively, and modulation of RVDs can recognize consecutive DNA bases. This simple relationship between amino acid sequence and DNA recognition has allowed for the engineering of specific DNA binding domains by selecting a combination of repeat segments containing the appropriate RVDs. The relationship between amino acid sequence and DNA recognition of the TALE binding domain allows for designable proteins. Software programs such as DNA Works can be used to design TALE constructs. Other methods of designing TALE constructs are known to those of skill in the art. See Doyle et al., Nucleic Acids Research (2012) 40: W117-122.; Cermak et al., Nucleic Acids Research (2011). 39:c82; and talc-nt.cac.cottoncll.cdu/about. In an aspect, a method and/or composition provided herein comprises one or more, two or more, three or more, four or more, or five or more TALENs. In another aspect, a TALEN provided herein is capable of generating a targeted DSB. In an aspect, vectors comprising polynucleotides encoding one or more, two or more, three or more, four or more, or five or more TALENs are provided to a cell by transformation methods known in the art (e.g., without being limiting, viral transfection, particle bombardment, PEG-mediated protoplast transfection or Agrobacterium-mediated transformation). See, e.g., US Patent App. Nos. 2011/0145940, 2011/0301073, and 2013/0117869, the contents and disclosures of which are incorporated herein by reference.
[00190] In an aspect, a targeted genome editing technique described herein may comprise the use of a recombinase. In some embodiments, a tyrosine recombinase attached, etc., to a DNA recognition domain or motif may be selected from the group consisting of a Cre recombinase, a Flp recombinase, and a Tnpl recombinase. In an aspect, a Cre recombinase or a Gin recombinase provided herein may be tethered to a zinc-finger DNA binding domain. The Flp-FRT site-directed recombination system may come from the 2p plasmid from the baker’s yeast Saccharomyces cerevisiae. hi this system, Flp recombinase (flippase) may recombine sequences between flippase recognition target (FRT) sites. FRT sites comprise 34 nucleotides. Flp may bind to the “arms” of the FRT sites (one arm is in reverse orientation) and cleaves the FRT site at either end of an intervening nucleic acid sequence. After cleavage, Flp may recombine nucleic acid sequences between two FRT sites. Cre-lox is a site-directed recombination system derived from the bacteriophage Pl that is similar to the Flp-FRT recombination system. Cre-lox can be used to invert a nucleic acid sequence, delete a nucleic acid sequence, or translocate a nucleic acid sequence. In this system, Cre recombinase may recombine a pair of lox nucleic acid sequences. Lox sites comprise 34 nucleotides, with the first and last 13 nucleotides (arms) being palindromic. During recombination, Cre recombinase protein binds to two lox sites on different nucleic acids and cleaves at the lox sites. The cleaved nucleic acids are spliced together (reciprocally translocated), and recombination is complete. In another aspect, a lox site provided herein is a loxP, lox 2272, loxN, lox 511, lox 5171, lox71, lox66, M2, M3, M7, or Mil site.
[00191] As used herein, the term “derived” or “derived from” in reference to a particular DNA molecule, amplicon or sequence in relation to a cotton cell, cotton tissue, cotton seed, cotton plant, cotton plant part and/or cotton plant product, such as a cotton commodity product, means that the DNA molecule, amplicon or sequence is taken, purified, isolated, or made, directly or indirectly, from such cotton cell, cotton tissue, cotton seed, cotton plant, cotton plant part and/or cotton plant product, such as a cotton commodity product, as the case may be. Alternatively, the term “derived” or “derived from” in reference to a cotton plant product, such as a cotton commodity product, in relation to a cotton cell, cotton tissue, cotton seed, cotton plant, and/or cotton plant part, means that the cotton plant product is taken, purified, isolated, or made, directly or indirectly, from such cotton cell, cotton tissue, cotton seed, cotton plant, and/or cotton plant part, as the case may be. “Capable of being detected” refers to the ability of a particular DNA molecule, segment or sequence to be detected in a sample, such as by amplification and determining its presence, size or sequence such as by DNA sequence analysis, and/or binding of a probe to the target DNA molecule, segment or sequence.
[00192] A “sample” is intended to refer to any composition comprising or derived from, either directly or indirectly, a biological sample, source or material. The sample may generally comprise cotton DNA and/or substantially or completely pure, purified or isolated cotton DNA. A “biological sample” contains biological materials, including but not limited to DNA obtained or derived from, either directly or indirectly, the genome of a cotton cell(s), cotton tissue(s), cotton seed(s), cotton plant(s), cotton plant part(s) and/or cotton plant product(s), such as a cotton commodity product(s). Such cotton cell(s), cotton tissue(s), cotton seed(s), cotton plant(s), cotton plant part(s) and/or cotton plant product(s), such as a cotton commodity product(s), may comprise cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 or DNA molecule(s) and/or DNA segment(s) comprising cotton event GH_BCS246002 or a modified cotton event GH_BCS246002. In some embodiments, a sample or biological sample may comprise cotton cell(s), cotton tissue(s), cotton seed(s), cotton plant(s), cotton plant part(s), and/or cotton plant product(s), whose cells or cellular membranes have been fractured (e.g., disrupted or opened) to release the contents of the cotton cell(s) including genomic DNA and/or make the contents of the cotton cell(s) including genomic DNA accessible or usable for assays or testing. “Directly” refers to directly obtaining DNA by a skilled artisan from the cotton genome by fracturing cotton cells (or by obtaining samples of cotton that contain fractured cotton cells) and exposing or using the genomic DNA from cotton cells for the purposes of detection. “Indirectly” refers to obtaining by a skilled artisan a target or specific reference DNA (i.e., a novel and unique junction scgmcnt(s) described herein as being diagnostic for the presence of the cotton event GH_BCS246002 or a modified cotton event GH_BCS246002) in a particular sample, by means other than by obtaining directly via fracturing of cotton cells or obtaining a sample of cotton that contains fractured cotton cells. Such indirect means include, but are not limited to, amplification of a DNA segment that contains a DNA sequence targeted by a particular probe(s) and/or primer set(s) designed to bind with specificity to or near the target sequence, or amplification of a DNA segment comprising all or part of a target sequence that can be measured and characterized (e.g., measured by migration or separation from other segments of DNA and/or identification in an effective matrix, such as an agarose or acrylamide gel or the like, or characterized by direct sequence analysis of the amplicon(s), or cloning of the amplicon(s) into a vector(s) and direct sequencing of the inserted amplicon(s) present within such vector(s).
[00193] A transgenic plant is produced by transformation of a plant cell with heterologous DNA, i.e., a polynucleic acid construct that includes a number of efficacious features of interest, regeneration of a plant resulting from the insertion of the transgene into the genome of the plant cell, and selection of a particular plant characterized by insertion into a particular genome location and the number of efficacious features of the regenerated transgenic plant. The term “transgenic event” or “event” refers to the inserted transgenic DNA in the plant genome and flanking genomic sequences immediately adjacent to the inserted transgenic DNA in the genome of the transformed plant, but also refers to a DNA molecule comprising the inserted transgenic DNA in the plant genome and flanking genomic sequences. Each event is unique and would be expected to be transferred to progeny plants that receive the transgenic DNA and event through genetic inheritance and/or segregation from a parent as the result of a sexual or self-cross of a first parental line that includes the inserted transgenic DNA and event either with itself or a second parental line that may or may not contain the same transgenic DNA and event. The parental line that includes the inserted transgenic DNA and event may itself be the original transformant or a progeny plant of said original transformant that may have been generated by “selfing” the transformant with itself or crossing the transformant or a progeny plant of the transformant that includes the inserted transgenic DNA and event with another plant. For purposes of the present disclosure, the “event” refers to cotton event GH_BCS246002 or a modified cotton event GH_BCS246002.
[00194] As used herein, the term “flanking” in reference to a transgenic event refers to the plant genomic sequence(s) immediately adjacent to the transgenic DNA insertion in the genome of a transformed plant, plant part, plant tissue, or plant cell comprising the transgenic event on the 5' and/or 3' sidc(s) or cnd(s) of the transgenic event insertion (i.c., the transgenic insertion of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002). Likewise, “flanking DNA”, “flanking sequence” or “flanking DNA sequence” each refers to a length of genomic DNA sequence immediately adjacent to the transgenic DNA insertion in the genome of the transformed plant on the 5" and/or 3' side(s) or end(s) of the insertion. A “5" flank” means the cotton genomic DNA sequence adjacent to and upstream (or on the 5' end) of the transgenic DNA insertion. For example, a “5" flank” can include the cotton genomic DNA sequence immediately adjacent to and upstream (on the 5' end) of the transgenic insertion, or any cotton genomic DNA sequence upstream (on the 5' end) of the transgenic insertion that is not immediately adjacent to the transgenic insertion but is within about 5000 nucleotides, within about 4000 nucleotides, within about 3000 nucleotides, within about 2000 nucleotides, or within about 1000 nucleotides upstream of the transgenic insertion. Likewise, a “3" flank” means the cotton genomic DNA sequence adjacent to and downstream (or on the 3' end) of the transgenic insert. For example, a “3" flank” can include the cotton genomic DNA sequence immediately adjacent to and downstream (on the 3' end) of the transgenic insertion, or any cotton genomic DNA sequence downstream (on the 3' end) of the transgenic insertion that is not immediately adjacent to the transgenic insertion but is within about 5000 nucleotides, within about 4000 nucleotides, within about 3000 nucleotides, within about 2000 nucleotides, or within about 1000 nucleotides downstream of the transgenic insertion.
[00195] SEQ ID NOs: 11 and 12 are 1,000 nucleotide sequences representing cotton Gossypium hirsutum) genomic DNA that flanks the transgenic insert of the 5' and 3' ends of the insert in cotton event GH_BCS246002, respectively. SEQ ID NOs: 43 and 44 are 5,000 nucleotide sequences representing cotton (Gossypium hirsutum) genomic DNA that flanks the transgenic insert of the 5' and 3' ends of the insert in cotton event GH_BCS246002, respectively. Nucleotides 4,001-5,000 of SEQ ID NO: 43 are identical to nucleotides 1-1,000 of SEQ ID NO: 11. Nucleotides 1-4,000 are based on the genomic sequence of the TM-1 cotton cultivar (GenBank: LBLM00000000.1, Gossypium hirsutum cultivar TM-1, whole genome shotgun sequencing). Nucleotides 1-1,000 of SEQ ID NO: 44 are identical to nucleotides 1-1,000 of SEQ ID NO: 12. The remaining nucleotides (1 ,001-5,000) are based on the genomic sequence of the TM-1 cotton cultivar. [00196] The present disclosure provides the original transformant plant and progeny of the transformant that include the transgenic DNA and event. Such progeny may be produced by a sexual cross or outcross between plants comprising the same transgenic DNA and event, or between a plant comprising the transgenic DNA and event with another plant, or by any other method known in the art including any cell or tissue culture method, wherein the progeny includes the transgenic DNA and event. Such other plant may be a transgenic plant comprising the same and/or a different transgene or may be a non-transgenic plant, and each parental plant in a cross or outcross may be the same or different germplasm or breeding line. Even after repeated back- crossing to a recurrent parent, the transgenic DNA and event is present in progeny of the cross at the same chromosomal location. Thus, a “transgenic plant” can be the original transformant plant regenerated from the transformed plant cell and comprising the transgenic DNA and event, or a progeny plant of the original transformant plant, which may be separated from the transformant by one or more generations, that retains the transgenic DNA and event at the same specific location and sequence context in the plant’s genome. The transformant or progeny plant may be homozygous or heterozygous for cotton event GH_BCS246002 or a modified cotton event GH_BCS246002. In addition, a “transgenic plant” can include a plant produced from a transformed plant cell or tissue, or from another transgenic plant or plant part, by or using cell or tissue culture methods known in the art. A “transgenic plant” may comprise a plant having a transgene or transgenic event stably inserted into the genome of at least one cell of the plant or a modified cotton event GH_BCS246002 (i.e., cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 in at least one cell of the plant), and the plant may be chimeric or nonchimeric with respect to the transgene and/or event or modified event. A transgenic plant is chimeric with respect to a transgene, event or modified event if not all cells of the plant comprise the transgene, event or modified event.
[00197] As used herein, the term “recombinant” refers to a non-natural DNA, protein, or combination that would not normally be found in nature, such as a combination of DNA sequences, proteins that would not naturally occur together, and is the result of human intervention. A “recombinant DNA molecule” is a DNA molecule comprising a combination of DNA sequences that would not naturally occur together and is the result of human intervention, such as a combination of DNA segments or sequences that would not naturally occur together in nature in the same relative positions and/or orientation. Two or more elements of such combination of DNA sequences may be operably linked to one another. For example, a recombinant DNA molecule may comprise a combination of at least two DNA sequences that arc heterologous with respect to each other, such as a DNA molecule that comprises a coding or transcribable DNA sequence operably linked to a heterologous promoter and/or other regulatory expression element(s), and/or a plant genomic DNA sequence comprising all or part of a transgene and a heterologous and flanking genomic sequence(s) adjacent to the transgene, and/or a DNA molecule that is artificially synthesized and comprises a polynucleotide sequence that deviates from any polynucleotide sequence that would normally exist in nature. A recombinant DNA molecule may comprise all or part of a junction sequence of the genome of a plant and all or part of the transgene insertion into the genome of the plant, and/or may comprise a recombinant or heterologous DNA fragment of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002. An example of a recombinant DNA molecule is a DNA molecule comprising at least one of SEQ ID NOs: 1-10. As used herein, a recombinant plant, plant part, plant cell or plant tissue is a plant, plant part, plant cell or plant tissue that would not normally exist in nature, is the result of human intervention, and contains a transgene incorporated into the genome of the plant, plant part, plant cell or plant tissue. As a result of such genomic insertion, the recombinant plant is something new and distinctly different from any related wild-type or naturally occurring plant, plant part, plant cell or plant tissue. An example of a recombinant plant, plant part, plant cell or plant tissue is a cotton plant, plant part, plant cell or plant tissue containing the cotton event GH_BCS246002 or a modified cotton event GH_BCS246002.
[00198] As used herein, the term “heterologous” in reference to a combination of two or more DNA sequences or elements means that the two or more DNA sequences or elements do not normally exist together as such combination in nature without human intervention. As used herein, the term “heterologous” in reference to a DNA molecule, construct or sequence in relation to a plant, microorganism, plant cell or plant genome means that the DNA molecule, construct or sequence does not exist in nature as pail of such plant, microorganism, plant cell or plant genome, and/or does not exist in the same physical or genomic location, context or orientation as part of such plant, microorganism, plant cell or plant genome in nature, without human intervention.
[00199] The present disclosure provides DNA molecules and fragments and their corresponding DNA sequences. The terms “DNA” and “DNA molecule” as used herein refer to a deoxyribonucleic acid (DNA) molecule. A DNA molecule may be of genomic or synthetic origin and/or comprise a recombinant or heterologous DNA molecule or sequence. A DNA molecule may be described in reference to its 5 " (upstream) end and 3 " (downstream) end. As used herein, the term “DNA sequence” refers to the polynucleotide sequence of the DNA molecule - i.e., the sequence of consecutive nucleotides in the DNA molecule. As used herein in reference to nucleotides of a polynucleotide or DNA sequence or molecule, the terms “consecutive” and “contiguous” are interchangeable and synonymous and refer to the 5' to 3' order of nucleotides in a polynucleotide or DNA sequence, strand or molecule without any gap or interruption between them. By convention, DNA sequences of the disclosure and fragments thereof are disclosed with reference to the 5' to 3' direction of only one strand of the two, anti-parallel and complementary DNA strands of a DNA molecule. By implication and intent, the complementary sequences of the sequences provided here (i.e., the sequences of the complementary, opposing, or antiparallel strand), also referred to in the art as the reverse complementary or reverse complement sequences, are within the scope of the present disclosure and are expressly intended to be within the potential scope of the subject matter as claimed. A DNA molecule, or a fragment derived therefrom, can also be extracted from plant part(s), plant cell(s) and/or tissue(s) or a homogenate, extract or lysate from plant part(s), plant cell(s) and/or tissue(s), or can be produced as an amplicon from extracted, purified or isolated DNA from plant part(s), plant cell(s) and/or tissue(s), or a homogenate, extract or lysate from plant part(s), plant cell(s) and/or tissue(s), which may further comprise cotton event GH_BCS246002.
[00200] As used herein, the term “fragment” refers to a smaller piece or sequence of a larger or whole DNA molecule or sequence. For example, a fragment of SEQ ID NO: 9 or 10 may include a sequence that is at least about 12 consecutive nucleotides, at least about 13 consecutive nucleotides, at least about 14 consecutive nucleotides, at least about 15 consecutive nucleotides, at least about 16 consecutive nucleotides, at least about 17 consecutive nucleotides, at least about 18 consecutive nucleotides, at least about 19 consecutive nucleotides, at least about 20 consecutive nucleotides, at least about 21 consecutive nucleotides, at least about 22 consecutive nucleotides, at least about 23 consecutive nucleotides, at least about 24 consecutive nucleotides, at least about 25 consecutive nucleotides, at least about 30 consecutive nucleotides, at least about 35 consecutive nucleotides, at least about 40 consecutive nucleotides, at least about 45 consecutive nucleotides, at least about 50 consecutive nucleotides, at least about 60 consecutive nucleotides, at least about 70 consecutive nucleotides, at least about 80 consecutive nucleotides, at least about 90 consecutive nucleotides, at least about 100 consecutive nucleotides, at least about 200 consecutive nucleotides, at least about 300 consecutive nucleotides, at least about 400 consecutive nucleotides, or at least about 500 consecutive nucleotides of the larger, whole or complete DNA molecule or sequence of SEQ ID NO: 9 or 10.
[002011 According to present embodiments, a fragment of the DNA sequence of the 5 ' flank (SEQ ID NO: 11 or SEQ ID NO: 43) or the 3' flank (SEQ ID NO: 12 or SEQ ID NO: 44) of cotton event GH_BCS246002 can comprise at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, or at least 500 consecutive nucleotides of SEQ ID NO: 11 or SEQ ID NO: 43 or SEQ ID NO: 12 or SEQ ID NO: 44. It is possible that different cotton germplasms may have differences in their genomic sequences, which may include differences in the flanking sequence(s), 5 ' flank and/or 3' flank of cotton event GH_BCS246002. These differences may result from introgression of the cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 into a different germplasm and/or spontaneous, mutagenic or genome editing changes that occur in a given germplasm or line. According to some embodiments, DNA molecules, constructs or polynucleotides are provided comprising a sequence or flanking sequence, or a 5' flank or 3' flank, that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% identical to SEQ ID NO: 11 or 43 or SEQ ID NO: 12 or 44, or a fragment of either thereof. According to some embodiments, DNA molecules, constructs or polynucleotides are provided comprising a sequence or flanking sequence, or a 5 ' flank or 3' flank, that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% identical to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, or at least 500 consecutive nucleotides of SEQ TD NO: 11 or 43 or SEQ ID NO: 12 or 44.
[00202] As used herein, the term “isolated” in reference to a molecule means that the molecule is at least partially separated from other molecules or sequences that are normally associated with the molecule in its native or natural state. In some embodiments, the term “isolated” refers to a DNA molecule that is at least partially separated from the nucleic acids or polynucleotide or DNA sequence(s) that normally flank and are covalently linked to the sequence of the DNA molecule in its native or natural state. An “isolated” DNA molecule may have a DNA sequence corresponding to a portion of the genome of a plant cell without other genomic DNA sequence(s) that normally flank and are covalently linked to the DNA sequence in nature. Such an “isolated” DNA molecule may comprise all or part of a transgene and/or transgenic event, which may comprise all or part of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 or the transgene or expression cassette described herein. Nucleic acid sequences or elements, such as a coding sequence, intron sequence, untranslated leader sequence, promoter sequence, transcriptional termination sequence, and the like, that are naturally found within the DNA of the genome of an organism are not considered to be “isolated” so long as the element is within the genome of the organism and at the location within the genome in which it is naturally found. However, each of these elements, and subparts of these elements, would be “isolated” within the scope of this disclosure so long as the element or subpart is not within the genome of the organism, and at the location within the genome of the organism, in which it is naturally found. An “isolated” DNA molecule may be any recombinant DNA molecule or amplification product or amplicon, and/or may comprise any DNA sequence removed from its natural or biological state and covalently fused to another DNA molecule or sequence with which it is not associated in nature. Such an isolated DNA molecule could be created by the use of biotechnology techniques, such as by making a recombinant DNA or integrating a foreign or heterologous DNA molecule into the chromosome of a cell, plant, or seed. Thus, any DNA molecule comprising a transgenic, recombinant, chimeric or artificial nucleotide sequence, transgene or expression cassette would be considered to be an “isolated” DNA molecule since these sequences are not naturally occurring, regardless of whether the sequence, transgene or expression cassette is present within a plasmid, vector or construct used to transform plant cells, within the genome of a plant, plant part, plant tissue, or plant cell, or is present in detectable amounts in tissues, progeny, biological samples or commodity products derived from a plant, plant part, plant tissue, or plant cell. A recombinant DNA molecule or sequence, or any fragment derived therefrom, comprising all or part of a transgene or junction sequence of cotton event would therefore also be considered to be “isolated.” An “isolated” DNA molecule may be extracted or purified from a transgenic plant(s), plant part(s), plant cell(s) and/or tissue(s), or may be present in a homogenate, extract or lysate from any such transgenic plant(s), plant part(s), plant cell(s) and/or tissue(s), or may be produced as an amplicon or amplification product from plant genomic DNA and/or extracted or purified DNA from transgenic plant(s), plant part(s), plant cell(s) and/or tissue(s), or a homogenate, extract or lysate from plant(s), plant part(s), plant cell(s) and/or tissue(s). For the purposes of this disclosure, any transgenic polynucleotide or DNA sequence, i.e., the nucleotide sequence of the DNA inserted into the genome of a plant or bacterium, or present in an extrachromosomal vector, would be considered to be an “isolated” nucleotide or DNA sequence whether it is present within the plasmid or similar structure used to transform the cells, within the genome of the plant or bacterium, or present in detectable amounts in tissues, progeny, biological samples or commodity products derived from the plant or bacterium. An “isolated” DNA molecule is a chemical or biochemical molecule, regardless of whether the molecule is referred to as a nucleic acid, a nucleic acid sequence, a polynucleotide sequence, a DNA sequence, a nucleic acid molecule, a polynucleotide molecule, a DNA molecule, or the like. An “isolated” molecule can provide industrial applicability when present in a plant cell or in a plant genome or when present outside of a plant cell, and therefore, provides and exhibits (and is intended to provide and exhibit) utility regardless of where the molecule is located.
[00203] The phosphodiester bond linkage between one end of a transgenic insert (or insertion) into the genome of a plant and the flanking cotton genomic DNA is referred to as a “junction.” In other words, a “junction’ is the connection point or covalent linkage of one end of a transgenic insert and the flanking genomic DNA. One junction is found at the 5' end of the transgenic insertion and the other is found at the 3 " end of the transgenic insert, referred to herein as the 5' and 3' junctions, respectively. A “junction sequence” refers to a DNA sequence of any length of consecutive nucleotides that spans the 5 ' or 3' junction of a transgenic event in the plant genome. For a “junction sequence” to be specific to a junction between a transgenic event and a flanking genomic sequence, the junction sequence will generally comprise a sufficient number of consecutive nucleotides at one end of the insertion and a sufficient number of consecutive nucleotides of the flanking genomic sequence. According to some embodiments, a “junction sequence” may comprise (i) at least five (5) consecutive nucleotides, at least ten (10) consecutive nucleotides, at least fifteen (15) consecutive nucleotides, at least twenty (20) consecutive nucleotides, or at least thirty (30) consecutive nucleotides at one end of the insertion and (ii) at least five (5) consecutive nucleotides, at least ten (10) consecutive nucleotides, at least fifteen (15) consecutive nucleotides, at least twenty (20) consecutive nucleotides, or at least thirty (30) consecutive nucleotides of flanking genomic DNA sequence, although it is understood that any length of consecutive nucleotides spanning a junction of a transgenic event in a plant genome may be a junction sequence. A variety of junction sequences of cotton event GH_BCS246002 can be determined by one of skill in the art using SEQ ID NO: 10. Examples of junction sequences of cotton event GH_BCS246002 are provided as SEQ ID NOs: 1-8. FIG. 1 illustrates the physical arrangement and locations of the junction sequences, arranged from 5 Go 3' (left to right), relative to SEQ ID NO: 10. The junction sequence(s) of a modified cotton event GH_BCS246002 may be modified, mutated or edited relative to such junction sequence(s) of cotton event GH_BCS246002. The junction sequences of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 may be present as part of the genome of a cotton plant, plant part, plant seed, or plant tissue or cell containing cotton event GH_BCS246002 or a modified cotton event GH_BCS246002, a DNA molecule containing all or part of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002, or a microorganism containing cotton event GH_BCS246002 or a modified cotton event GH_BCS246002. The identification of any one or more of the junction sequences in a DNA molecule or sample from a plant, plant part, plant seed, or plant tissue or cell indicates that the plant, plant part, plant seed, or plant tissue or cell contains or comprises cotton event GH_BCS246002 or a modified cotton event GH_BCS246002, or the DNA molecule contains or comprises cotton event GH_BCS246002 or a modified cotton event GH BCS246002 or was obtained from a cotton plant, plant part, plant seed, or plant tissue or cell containing or comprising cotton event GH_BCS246002 or a modified cotton event GH_BCS246002, and is diagnostic in each case for the presence of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002.
[00204] The junction sequences described herein are diagnostic for the presence of all or part of cotton event GH_BCS246002, and if unmodified may be diagnostic for the presence of all or part of or a modified cotton event GH_BCS246002, and/or a DNA molecule comprising all, or part of the construct or expression cassettes described herein. Thus, the identification or detection, directly or indirectly, of one or more of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 10 in a sample or DNA molecule derived from a cotton plant, cotton plant part, cotton seed, or cotton tissue or cell, or a commodity product from a cotton plant, cotton plant part, cotton seed, or cotton tissue or cell, is diagnostic that the cotton plant, cotton plant part, cotton seed, or cotton tissue or cell, or a commodity product from a cotton plant, cotton plant part, cotton seed, or cotton tissue or cell has or comprises all or part of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002. The identification or detection, directly or indirectly, of a 5' junction sequence and a 3' junction sequence (each as provided or described herein) in a sample or DNA molecule derived from a cotton plant, cotton plant part, cotton seed, or cotton tissue or cell, or a commodity product from a cotton plant, cotton plant part, cotton seed, or cotton tissue or cell, is diagnostic that the cotton plant, cotton plant part, cotton seed, or cotton tissue or cell, or a commodity product from a cotton plant, cotton plant part, cotton seed, or cotton tissue or cell has or comprises cotton event GH_BCS246002 or a modified cotton event GH_BCS246002. The present disclosure thus provides a DNA molecule that contains at least one of the nucleotide sequences provided as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10. Any segment of DNA derived from transgenic cotton event GH_BCS246002 that is sufficient to include at least one of the sequences provided as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10 is within the scope of the present disclosure. In addition, any DNA or polynucleotide molecule or sequence comprising a sequence complementary to any of the sequences described herein is also within the scope of the present disclosure.
[00205] The disclosure provides DNA, polynucleotide or nucleic acid molecules, which may be single or double stranded, that can be used either as primers or probes for detecting the presence of DNA comprising all or part of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 in a sample derived from a cotton plant, cotton plant part, cotton seed, or cotton tissue or cell, or a commodity product from a cotton plant, cotton plant part, cotton seed, or cotton tissue or cell. Such primers or probes are specific for a target nucleic acid, polynucleotide or DNA sequence and, as such, arc useful for the identification of cotton event GH_BCS246002 nucleic acid, polynucleotide or DNA sequence, or a nucleic acid, polynucleotide or DNA sequence of a modified cotton event GH_BCS246002, by the methods described herein. A primer or probe can hybridize to a target nucleic acid, polynucleotide or DNA sequence to allow for specific detection or amplification of a nucleic acid, polynucleotide or DNA molecule or sequence that comprises, or is covalently linked and associated with, the target nucleic acid, polynucleotide or DNA sequence. According to present embodiments, the primers and/or probe may be chosen to identify and distinguish detection of a particular transgenic event and not only the presence of a transgene in a plant genome. The target nucleic acid, polynucleotide or DNA molecule or sequence may comprise all or part of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002, a junction sequence and/or flanking genomic DNA. Probes and primers according to the present disclosure may have (i) complete or 100% sequence complementarity (i.e., 100% complementary) to a target DNA sequence or (ii) incomplete sequence complementarity to a target DNA sequence, such as at least 60% complementary, at least 65% complementary, at least 70% complementary, at least 75% complementary, at least 80% complementary, at least 85% complementary, at least 90% complementary, at least 95% complementary, or at least 99% complementary to the target DNA sequence as long as the probe or primer has sufficient complementarity to the target DNA sequence to hybridize to the target DNA sequence under stringent hybridization conditions that are suitable and necessary for use of the probe or primer in the relevant amplification or detection assay, reaction or method. As understood in the art, the percentage complementarity of a primer or probe may be lower if the length of the primer or probe is longer and depends on the stringency and use.
[00206] A “probe” is a nucleic acid molecule that is complementary to a strand of target nucleic acid and is useful in hybridization methods. A probe may be attached a conventional detectable label or reporter molecule, e.g., a radioactive isotope, ligand, chemiluminescent agent, or enzyme. Such a probe is complementary to a strand of a target nucleic acid and, in the case of the present disclosure, to a strand of DNA from cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 whether from an cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 containing plant or from a sample that includes cotton event GH_BCS246002 DNA or DNA from a modified cotton event GH_BCS246002. Probes according to the present disclosure include not only deoxyribonucleic or ribonucleic acids, but also polyamides and other probe materials that bind specifically to a target DNA sequence and can be used to detect the presence of that target DNA sequence. Exemplary DNA sequences useful as a probe for detecting cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 arc provided as: SEQ ID NO: 17 (PB5O3O8), SEQ ID NO: 20 (PB50562), and SEQ ID NO: 22 (WTDP393PR). A “probe” may also be used to bind a template DNA in a sample comprising all or part of a DNA or nucleotide sequence of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 to purify the template DNA from the remainder of the sample using purification methods or techniques known in the art, for example, if the probe is bound or can be bound to a substrate or a particle or bead that can be purified or separated. Such a template DNA may comprise all or part of a DNA or nucleotide sequence of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002, or a portion or fragment thereof, such as a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, or a complement thereof.
[00207] A “primer” is typically a DNA molecule that is designed for use in specific annealing or hybridization methods that involve thermal amplification. A pair of primers may be used with template DNA (such as a sample of cotton genomic DNA) in a thermal amplification (such as polymerase chain reaction (PCR)) to produce an amplicon, where the amplicon produced from such reaction would have a DNA sequence corresponding to sequence of the template DNA located between the two sites where the primers hybridized to the template. As understood in the art, an “amplification product” or “amplicon” is a DNA molecule or segment produced by an amplification reaction. Amplification or amplifying refers to making multiple copies of a target DNA molecule or segment from a template DNA. A single “primer” may also be used to initiate a sequencing reaction to determine a DNA sequence of a template DNA according to sequencing methods known in the art. Such a sequencing reaction may be used to determine the presence or absence of a DNA molecule or nucleotide sequence, or a portion or fragment thereof, from cotton event GH_BCS246002 or a modified cotton event GH_BCS246002. Such a template DNA may comprise all or part of a DNA or nucleotide sequence of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002, or a portion or fragment thereof, such as a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, or a complement thereof. [00208] DNA amplification reactions, methods and techniques are known to those skilled in art. DNA amplification can be accomplished by any of the various nucleic acid amplification methods known in the art, including thermal and isothermal amplification methods including the polymerase chain reaction or PCR. Amplification methods are known in the art and are described, inter alia, in U.S. Patent Nos. 4,683, 195 and 4,683,202 and in PCR Protocols: A Guide to Methods and Applications, ed. Innis et al., Academic Press, San Diego, 1990. PCR amplification methods have been developed to amplify up to 22 kb (kilobase) of genomic DNA and up to 42 kb of bacteriophage DNA (Cheng et al., Proc. Natl. Acad. Sci. USA 91:5695-5699, 1994). These methods as well as other methods known in the art of DNA amplification may be used in the practice of the present disclosure. Examples of DNA amplification methods include PCR, Recombinase Polymerase Amplification (RPA) (see for example U.S. Pat No. 7,485,428), Strand Displacement Amplification (SDA) (see for example, U.S. Pat. Nos. 5,455,166 and 5,470,723), Transcription-Mediated Amplification (TMA) (see for example, Guatelli et al., Proc. Natl. Acad. Sci. USA 87:1874-1878, 1990), Rolling Circle Amplification (RCA) (see for example, Fire and Xu, Proc. Natl. Acad Sci. USA 92:4641-4645, 1995; Lui, et al., J. Am. Chem. Soc. 118:1587-1594, 1996; Lizardi, et al., Nature Genetics 19:225-232, 1998; U.S. Pat. Nos. 5,714,320 and 6,235,502), Helicase Dependent Amplification (HDA) (see for example Vincent et al., EMBO Reports 5(8): 795-800, 2004; U.S. Pat. No. 7,282,328), and Multiple Displacement Amplification (MDA) (see for example Dean et al., Proc. Natl. Acad Sci. USA 99:5261-5266, 2002). A sequence of the heterologous DNA insert and/or flanking genomic DNA sequence from cotton event GH_BCS246002 can be verified or tested by amplifying such DNA molecules from cotton seed containing cotton event GH_BCS246002 DNA or cotton plants grown from the cotton seed containing cotton event GH_BCS246002 DNA, using primers derived from the sequences provided herein, followed by standard DNA sequencing of the PCR amplicon or a cloned DNA fragment thereof.
[00209] According to present embodiments, the sequence of an amplicon of an amplification reaction may comprise one or more of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, or a fragment thereof. According to present embodiments, the sequence of an amplicon comprises at least one junction sequence or two junction sequences, such as a 5' junction sequence and/or a 3' junction sequence for cotton event GH_BCS246002 or a modified cotton event GH_BCS246002.
[00210] A primer is typically designed to hybridize in a sequence- specific manner to a complementary target DNA strand to form a hybrid between the primer and target DNA strand, and the primer hybridized or bound to the complementary target DNA strand is a point of recognition for a polymerase to begin extension of the primer (i.e., polymerization of additional nucleotides into a lengthening nucleotide molecule) using as a template the target DNA strand. Primer pairs refer to use of two primers binding opposite strands of a double stranded DNA or polynucleotide segment for the purpose of amplifying the polynucleotide or DNA segment between the positions targeted for binding by the individual primers of the primer pair to the original template DNA or an amplicon of the amplification reaction, typically in a thermal cycling amplification reaction or other conventional DNA amplification method. Primer pairs arc typically designed to hybridize to different nearby target positions of a template DNA molecule on opposing strands of the template DNA molecule such that the intervening region or sequence between the two primers can be specifically amplified for use or detection through multiple rounds of amplification. Exemplary DNA molecules useful as primers are provided as SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 21.
[00211] As described above, primer pair SEQ ID NO: 15 and SEQ ID NO: 16 are useful as a first DNA molecule and a second DNA molecule that is different from the first DNA molecule, and both are each of sufficient length of contiguous nucleotides of SEQ ID NO: 10 to function as DNA primers that, when used together in a thermal amplification reaction with template DNA derived from cotton event GH_BCS246002, to produce an amplicon diagnostic for, or characteristic of, cotton event GH_BCS246002 in a sample. The primer pair SEQ ID NO: 18 and SEQ ID NO: 19 are useful as a first DNA molecule and a second DNA molecule that is different from the first DNA molecule, and both are each of sufficient length of contiguous nucleotides of a locus within the cotton genome to function as DNA primers that, when used together in a thermal amplification reaction with template DNA derived cotton event GH_BCS246002, to produce an amplicon that serves as an internal control for both the diagnosis of cotton event GH_BCS246002, as well as the zygosity of cotton event GH_BCS246002 DNA in a sample. The primer pair SEQ ID NO: 20 and SEQ ID NO: 15 are useful as a first DNA molecule and a second DNA molecule that is different from the first DNA molecule, and both are each of sufficient length of contiguous nucleotides of a locus within the cotton genome to function as DNA primers that, when used together in a thermal amplification reaction with template DNA derived from cotton event GH_BCS246002, to produce an amplicon diagnostic for, or characteristic of, non-inserted wildtype cotton genomic DNA not comprising cotton event GH_BCS246002. It is within the skill of the art to determine for any particular desired amplification parameters, which probes and primers would be optimum for inclusion in the thermal amplification reaction to detect the presence or absence of the transgenic event DNA of the present disclosure based on the DNA sequences provided in the inserted DNA (SEQ ID NO: 9) and the full segment of DNA set forth herein as SEQ ID NO: 10 which defines the transgenic cotton event provided herein, GH_BCS246002.
[00212] According to present embodiments, a DNA molecule, construct, segment, amplicon, fragment or polynucleotide is provided that contains or comprises at least one of the nucleotide sequences provided as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10. According to present embodiments, a DNA molecule, construct, segment, amplicon, fragment or polynucleotide is provided that contains or comprises at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, or at least 90 consecutive nucleotides of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6. According to present embodiments, a DNA molecule, construct, segment, amplicon, fragment or polynucleotide is provided that (i) contains or comprises at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1,000, at least 1,500, or at least 2,000 consecutive nucleotides of any one of SEQ ID NOs: 1-10, (ii) is at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1 ,000, at least 1 ,500, or at least 2,000 nucleotides in length, and (iii) comprises nucleotides 1,000-1,001 and/or 17,128-17,129 of SEQ ID NO: 10. [00213] According to present embodiments, a DNA molecule, construct, segment, amplicon, fragment or polynucleotide is provided that contains or comprises (i) three expression cassettes that encode the insect pesticidal insect toxins CrylB.3, CrylDa_7, and Vip3Cbl.l, and (ii) a junction sequence. According to present embodiments, a DNA molecule, construct, segment, amplicon, fragment or polynucleotide is provided that contains or comprises (i) expression cassettes that encodes the pesticidal insect toxins CrylB.3, CrylDa_7, and Vip3Cbl.l, and (ii) a 5' flank sequence and/or a 3' flank sequence. The expression cassette may comprise in operable linkage: (a) a promoter sequence (b) a transcribable DNA sequence encoding a pesticidal insect toxins CrylB.3, CrylDa_7, and Vip3Cbl.l sequence that is toxic to Lepidopteran insect pest species, and (c) a transcription termination or 3' UTR sequence. The expression cassette may further comprise any of the elements described in Table 1, which may be operably linked. While the cotton event GH_BCS246002 comprises expression cassettes that encode the insect pesticidal insect toxins CrylB.3, CrylDa_7, and Vip3Cbl.l, a modified cotton event GH_BCS246002 may contain or comprise one or more expression cassette(s) that encode one or more of the insect pesticidal insect toxins CrylB.3, CrylDa_7, and/or Vip3Cbl.l (depending on the modification of cotton event GH_BCS246002). However, a DNA molecule, construct, segment, amplicon, fragment or polynucleotide may not comprise all three expression cassettes that encode the insect pesticidal insect toxins Cry IB.3, CrylDa_7, and Vip3Cbl.l if comprising only a portion of cotton event GH_BCS246002. According to some embodiments, a DNA molecule, construct, segment, amplicon, fragment or polynucleotide from a modified cotton event GH_BCS246002 is provided that contains or comprises (i) one or more expression cassette(s) that encode the insect pesticidal insect toxins CrylB.3, CrylDa_7, and/or Vip3Cbl.l, and (ii) a junction sequence, such as a 5' flank sequence and/or a 3' flank sequence.
[00214] According to present embodiments, a DNA molecule, construct, segment, amplicon, fragment or polynucleotide is provided that contains or comprises (i) one or more of the expression cassettes that encode Cry IB.3, CrylDa_7, and/or Vip3Cbl.l, and (ii) a polynucleotide sequence comprising one or more of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10. According to present embodiments, a DNA molecule, construct, segment, amplicon, fragment or polynucleotide is provided that contains or comprises (i) one or more of the expression cassettes that encode the pesticidal insect toxins CrylB.3, CrylDa_7, and/or Vip3Cbl.l, and (ii) a polynucleotide sequence comprising at least 10, at least 11 , at least 12, at least 13, at least 1 , at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1,000, at least 1,500, or at least 2,000 consecutive nucleotides of one or more of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10.
[00215] According to present embodiments, a DNA molecule, construct, segment, amplicon, fragment or polynucleotide is provided that contains or comprises (i) one or more of the expression cassettes that encode the pesticidal insect toxins Cry IB.3, CrylDa_7, and/or Vip3Cbl.l, and (ii) a polynucleotide sequence comprising at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1,000, at least 1,500, or at least 2,000 consecutive nucleotides of SEQ ID NO: 11 or SEQ ID NO: 12 or SEQ ID NO: 43 or SEQ ID NO: 44. According to some embodiments, a DNA molecule, construct, segment, amplicon, fragment or polynucleotide is provided that contains or comprises (i) one or more of the expression cassettes that encode the pesticidal insect toxins Cry IB.3, CrylDa_7, and/or Vip3Cbl.l, and (ii) a sequence or flanking sequence, or a 5' flank or 3' flank, that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% identical to SEQ ID NO: 11 or 43 or SEQ ID NO: 12 or 44, or a fragment of either thereof. According to some embodiments, a DNA molecule, construct, segment, amplicon, fragment or polynucleotide is provided that contains or comprises (i) one or more of the expression cassettes that encode the pesticidal insect toxins CrylB.3, CrylDa_7, and/or Vip3Cb 1.1, and (ii) a sequence or flanking sequence, or a 5 ' flank or 3 ' flank, that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% identical to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21 , at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1,000, at least 1,500, or at least 2,000 consecutive nucleotides of SEQ ID NO: 11 or 43 or SEQ ID NO: 12 or 44. According to present embodiments, a DNA molecule, construct, segment, amplicon, fragment or polynucleotide is provided that contains or comprises (i) one or more of the expression cassettes that encode the pesticidal insect toxins CrylB.3, CrylDa_7, and/or Vip3Cbl.l, and (ii) a polynucleotide sequence comprising one or more polynucleotide sequences selected from SEQ ID NOs: 45-144. According to present embodiments, a DNA molecule, construct, segment, amplicon, fragment or polynucleotide is provided that contains or comprises (i) one or more of the expression cassettes that encode the pesticidal insect toxins Cry IB.3, CrylDa_7, and/or Vip3Cbl.l, and (ii) a polynucleotide sequence comprising one or more polynucleotide sequences selected from SEQ ID NOs: 145-244.
[00216] According to present embodiments, a DNA molecule, construct, segment, amplicon, fragment or polynucleotide is provided that contains or comprises (i) one or more of the expression cassettes that encode the pesticidal insect toxins Cry IB.3, CrylDa_7, and/or Vip3Cbl. l, (ii) a polynucleotide sequence comprising at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1,000, at least 1,500, or at least 2,000 consecutive nucleotides of SEQ ID NO: 11 or SEQ ID NO: 43, and (iii) a polynucleotide sequence comprising at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1,000, at least 1,500, or at least 2,000 consecutive nucleotides of SEQ ID NO: 12 or SEQ ID NO: 44. According to some embodiments, a DNA molecule, construct, segment, amplicon, fragment or polynucleotide is provided that contains or comprises (i) one or more of the expression cassettes that encode the pesticidal insect toxins Cry I B.3, CrylDa_7, and/or Vip3Cbl .l , (ii) a sequence or flanking sequence, or a 5 ' flank, that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1 %, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% identical to SEQ ID NO: 11 or 43, or a fragment of either thereof, and (iii) a sequence or flanking sequence, or a 3' flank, that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% identical to SEQ ID NO: 12 or 44, or a fragment of either thereof. According to some embodiments, a DNA molecule, construct, segment, amplicon, fragment or polynucleotide is provided that contains or comprises (i) one or more of the expression cassettes that encode the pesticidal insect toxins Cry IB.3, CrylDa_7, and/or Vip3Cbl.l, and (ii) a sequence or flanking sequence, or a 5' flank, that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% identical to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1,000, at least 1,500, or at least 2,000 consecutive nucleotides of SEQ ID NO: 11 or 43, and (iii) a sequence or flanking sequence, or a 3' flank, that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% identical to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1,000, at least 1,500, or at least 2,000 consecutive nucleotides of SEQ ID NO: 12 or 44. According to present embodiments, a DNA molecule, construct, segment, amplicon, fragment or polynucleotide is provided that contains or comprises (i) one or more of the expression cassettes that encode the pesticidal insect toxins Cry IB.3, CrylDa_7, and/or Vip3Cbl.l, (ii) a polynucleotide sequence comprising one or more polynucleotide sequences selected from SEQ ID NOs: 45-144, and (iii) a polynucleotide sequence comprising one or more polynucleotide sequences selected from SEQ ID NOs: 145-244.
[00217] According to present embodiments, a DNA molecule, construct, segment, amplicon, fragment or polynucleotide is provided that contains or comprises one or more of the expression cassettes that encode the pesticidal insect toxins Cry IB.3, CrylDa_7, and/or Vip3Cbl.l, such as all three of the expression cassettes that encode the pesticidal insect toxins CrylB.3, CrylDa_7, and/or Vip3Cbl.l, wherein the DNA molecule, construct, segment, amplicon, fragment or polynucleotide comprises at its 5' end at least 50 consecutive nucleotides of SEQ ID NO: 11 or 43. According to present embodiments, a DNA molecule, construct, segment, amplicon, fragment or polynucleotide is provided that contains or comprises one or more of the expression cassettes that encode the pesticidal insect toxins CrylB.3, CrylDa_7, and/or Vip3Cb 1.1, wherein the DNA molecule, construct, segment, amplicon, fragment or polynucleotide comprises at its 3 ' end at least 50 consecutive nucleotides of SEQ ID NO: 12 or 44. According to present embodiments, a DNA molecule, construct, segment, amplicon, fragment or polynucleotide is provided that contains or comprises one or more of the expression cassettes that encode the pesticidal insect toxins CrylB.3, CrylDa_7, and/or Vip3Cbl.l, wherein the DNA molecule, construct, segment, amplicon, fragment or polynucleotide comprises at its 5 " end a polynucleotide sequence selected from SEQ ID NOs: 45-144. According to present embodiments, a DNA molecule, construct, segment, amplicon, fragment or polynucleotide is provided that contains or comprises one or more of the expression cassettes that encode the pesticidal insect toxins Cry IB.3, CrylDa_7, and/or Vip3Cbl.l, wherein the DNA molecule, construct, segment, amplicon, fragment or polynucleotide comprises at its 3 " end a polynucleotide sequence selected from SEQ ID NOs: 145-244.
[00218] According to present embodiments, a DNA molecule, construct, segment, amplicon, fragment or polynucleotide is provided that contains or comprises one or more of the expression cassettes that encode the pesticidal insect toxins Cry IB.3, CrylDa_7, and/or Vip3Cbl.l, such as all three of the expression cassettes that encode the pesticidal insect toxins CrylB.3, CrylDa_7, and/or Vip3Cbl.l, wherein the DNA molecule, construct, segment, amplicon, fragment or polynucleotide comprises at its 5' end at least 50 consecutive nucleotides of SEQ ID NO: 11 or 43, wherein the DNA molecule, construct, segment, amplicon, fragment or polynucleotide comprises at its 3' end at least 50 consecutive nucleotides of SEQ ID NO: 12 or 44, wherein the DNA molecule, construct, segment, amplicon, fragment or polynucleotide comprises at its 5' end a polynucleotide sequence selected from SEQ ID NOs: 45-144, and/or wherein the DNA molecule, construct, segment, amplicon, fragment or polynucleotide comprises at its 3' end a polynucleotide sequence selected from SEQ ID NOs: 145-244.
[002191 According to present embodiments, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1,000, at least 1,500, or at least 2,000 consecutive nucleotides of SEQ ID NO: 11 or SEQ ID NO: 43 at the 5' end of a DNA molecule, construct, segment, amplicon, fragment or polynucleotide may be immediately adjacent to and upstream (on the 5' end) of the transgenic insertion, or may not be immediately adjacent to, but further upstream (on the 5' end) and within about 5000 nucleotides, within about 4000 nucleotides, within about 3000 nucleotides, within about 2000 nucleotides, or within about 1000 nucleotides of the transgenic insertion. According to present embodiments, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1,000, at least 1,500, or at least 2,000 consecutive nucleotides of SEQ ID NO: 12 or SEQ ID NO: 44 at the 3' end of a DNA molecule, construct, segment, amplicon, fragment or polynucleotide may be immediately adjacent to and downstream (on the 3' end) of the transgenic insertion, or may not be immediately adjacent to but further downstream (on the 3' end) and within about 5000 nucleotides, within about 4000 nucleotides, within about 3000 nucleotides, within about 2000 nucleotides, or within about 1000 nucleotides of the transgenic insertion. Any sequence comprising at least 50 consecutive nucleotides of SEQ ID NO: 11 or SEQ ID NO:43, or at least 50 consecutive nucleotides of SEQ ID NO: 12 or SEQ ID NO: 44 is within the scope of the present disclosure. A DNA molecule, construct, segment, amplicon, fragment or polynucleotide can comprise at the 5 " and/or 3 " end of the construct (i) at least 50 consecutive nucleotides of SEQ ID NO: 11 or SEQ ID NO: 43; and/or (ii) at least 50 consecutive nucleotides of SEQ ID NO: 12 or SEQ ID NO: 44, respectively. [00220] According to present embodiments, a DNA molecule, construct, segment, amplicon, fragment or polynucleotide is provided that contains or comprises (i) SEQ ID NO: 9 or a polynucleotide sequence comprising at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1,000, at least 1,500, or at least 2,000 consecutive nucleotides of SEQ ID NO: 9, and (ii) a junction sequence. According to present embodiments, a DNA molecule, construct, segment, amplicon, fragment or polynucleotide is provided that contains or comprises (i) SEQ ID NO: 9 or a polynucleotide sequence comprising at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1,000, at least 1,500, or at least 2,000 consecutive nucleotides of SEQ ID NO: 9, and (ii) a 5' flank and/or a 3 ' flank sequence.
[00221] According to present embodiments, a DNA molecule, construct, segment, amplicon, fragment or polynucleotide is provided that contains or comprises (i) SEQ ID NO: 9 or a polynucleotide sequence comprising at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1,000, at least 1,500, or at least 2,000 consecutive nucleotides of SEQ ID NO: 9, and (ii) a polynucleotide sequence comprising at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1,000, at least 1,500, or at least 2,000 consecutive nucleotides of SEQ ID NO: 11 or SEQ ID NO: 43 or SEQ ID NO: 12 or SEQ ID NO: 44. According to some embodiments, a DNA molecule, construct, segment, amplicon, fragment or polynucleotide is provided that contains or comprises (i) SEQ ID NO: 9 or a polynucleotide sequence comprising at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21 , at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1,000, at least 1,500, or at least 2,000 consecutive nucleotides of SEQ ID NO: 9, and (ii) a sequence or flanking sequence, or a 5' flank or 3' flank, that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% identical to SEQ ID NO: 11 or 43 or SEQ ID NO: 12 or 44, or a fragment of either thereof.
[00222] According to some embodiments, a DNA molecule, construct, segment, amplicon, fragment or polynucleotide is provided that contains or comprises (i) SEQ ID NO: 9 or a polynucleotide sequence comprising at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1,000, at least 1,500, or at least 2,000 consecutive nucleotides of SEQ ID NO: 9, and (ii) a sequence or flanking sequence, or a 5' flank or 3' flank, that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% identical to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1,000, at least 1,500, or at least 2,000 consecutive nucleotides of SEQ ID NO: 11 or 43 or SEQ ID NO: 12 or 44. According to present embodiments, a DNA molecule, construct, segment, amplicon, fragment or polynucleotide is provided that contains or comprises (i) SEQ ID NO: 9 or a polynucleotide sequence comprising at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1 ,000, at least 1 ,500, or at least 2,000 consecutive nucleotides of SEQ TD NO: 9, and (ii) a polynucleotide sequence comprising one or more polynucleotide sequences selected from SEQ ID NOs: 45-144.
[00223] According to present embodiments, a DNA molecule, construct, segment, amplicon, fragment or polynucleotide is provided that contains or comprises (i) SEQ ID NO: 9 or a polynucleotide sequence comprising at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1,000, at least 1,500, or at least 2,000 consecutive nucleotides of SEQ ID NO: 9, and (ii) a polynucleotide sequence comprising one or more polynucleotide sequences selected from SEQ ID NOs: 145-244.
[00224] According to present embodiments, a DNA molecule, construct, segment, amplicon, fragment or polynucleotide is provided that contains or comprises (i) SEQ ID NO: 9 or a polynucleotide sequence comprising at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1,000, at least 1,500, or at least 2,000 consecutive nucleotides of SEQ ID NO: 9, (ii) a polynucleotide sequence comprising at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1,000, at least 1,500, or at least 2,000 consecutive nucleotides of SEQ ID NO: 11 or SEQ ID NO: 43, and (iii) a polynucleotide sequence comprising at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1,000, at least 1,500, or at least 2,000 consecutive nucleotides of SEQ ID NO: 12 or SEQ ID NO: 44. According to some embodiments, a DNA molecule, construct, segment, amplicon, fragment or polynucleotide is provided that contains or comprises (i) SEQ ID NO: 9 or a polynucleotide sequence comprising at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1,000, at least 1,500, or at least 2,000 consecutive nucleotides of SEQ ID NO: 9, (ii) a sequence or flanking sequence, or a 5' flank, that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% identical to SEQ ID NO: 11 or 43, or a fragment of either thereof, and (iii) a sequence or flanking sequence, or a 3 ' flank, that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% identical to SEQ ID NO: 12 or 44, or a fragment of either thereof. According to some embodiments, a DNA molecule, construct, segment, amplicon, fragment or polynucleotide is provided that contains or comprises (i) SEQ ID NO: 9 or a polynucleotide sequence comprising at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1,000, at least 1,500, or at least 2,000 consecutive nucleotides of SEQ ID NO: 9, and (ii) a sequence or flanking sequence, or a 5' flank, that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% identical to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1,000, at least 1,500, or at least 2,000 consecutive nucleotides of SEQ ID NO: 11 or 43, and (iii) a sequence or flanking sequence, or a 3' flank, that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1 %, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% identical to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1,000, at least 1,500, or at least 2,000 consecutive nucleotides of SEQ ID NO: 12 or 44.
[00225] According to present embodiments, a DNA molecule, construct, segment, amplicon, fragment or polynucleotide is provided that contains or comprises (i) SEQ ID NO: 9 or a polynucleotide sequence comprising at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1,000, at least 1,500, or at least 2,000 consecutive nucleotides of SEQ ID NO: 9, (ii) a polynucleotide sequence comprising one or more polynucleotide sequences selected from SEQ ID NOs: 45-148, and (iii) a polynucleotide sequence comprising one or more polynucleotide sequences selected from SEQ ID NOs: 145-244.
[00226] According to present embodiments, a DNA molecule, construct, segment, amplicon, fragment or polynucleotide is provided that contains or comprises SEQ ID NO: 9 or a polynucleotide sequence comprising at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1,000, at least 1,500, or at least 2,000 consecutive nucleotides of SEQ ID NO: 9, wherein the DNA molecule, construct, segment, amplicon, fragment or polynucleotide comprises at its 5" end at least 50 consecutive nucleotides of SEQ ID NO: 11 or 43. According to present embodiments, a DNA molecule, construct, segment, amplicon, fragment or polynucleotide is provided that contains or comprises SEQ ID NO: 9 or a polynucleotide sequence comprising at least 10, at least 1 1 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1,000, at least 1,500, or at least 2,000 consecutive nucleotides of SEQ ID NO: 9, wherein the DNA molecule, construct, segment, amplicon, fragment or polynucleotide comprises at its 3' end at least 50 consecutive nucleotides of SEQ ID NO: 12 or 44. According to present embodiments, a DNA molecule, construct, segment, amplicon, fragment or polynucleotide is provided that contains or comprises one or more of the expression cassettes that encode the pesticidal insect toxins Cry IB.3, CrylDa_7, and/or Vip3Cbl.l, wherein the DNA molecule, construct, segment, amplicon, fragment or polynucleotide comprises at its 5 " end a polynucleotide sequence selected from SEQ ID NOs: 45-148. According to present embodiments, a DNA molecule, construct, segment, amplicon, fragment or polynucleotide is provided that contains or comprises SEQ ID NO: 9 or a polynucleotide sequence comprising at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1,000, at least 1,500, or at least 2,000 consecutive nucleotides of SEQ ID NO: 9, wherein the DNA molecule, construct, segment, amplicon, fragment or polynucleotide comprises at its 3' end a polynucleotide sequence selected from SEQ ID NOs: 145-244.
[00227] According to present embodiments, a DNA molecule, construct, segment, amplicon, fragment or polynucleotide is provided that contains or comprises SEQ ID NO: 9 or a polynucleotide sequence comprising at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1,000, at least 1,500, or at least 2,000 consecutive nucleotides of SEQ ID NO: 9, wherein the DNA molecule, construct, segment, amplicon, fragment or polynucleotide comprises at its 5' end at least 50 consecutive nucleotides of SEQ ID NO: 11 or 43, wherein the DNA molecule, construct, segment, amplicon, fragment or polynucleotide comprises at its 3' end at least 50 consecutive nucleotides of SEQ ID NO: 12 or 44, wherein the DNA molecule, construct, segment, amplicon, fragment or polynucleotide comprises at its 5' end a polynucleotide sequence selected from SEQ ID NOs: 45-144, and/or wherein the DNA molecule, construct, segment, amplicon, fragment or polynucleotide comprises at its 3' end a polynucleotide sequence selected from SEQ ID NOs: 145-244.
[00228] To detect the presence or absence of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002, the target positions and/or the intervening region or sequence of a template DNA molecule may comprise at least one junction sequence and/or at least a portion of the insert of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002. To detect the absence of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002, the target positions and/or the intervening region or sequence of a template DNA molecule may comprise cotton genomic DNA that does not include a junction sequence or any portion of the insert of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002. Thus, the presence or absence of an amplicon with a primer pair may be diagnostic of the presence or absence, respectively, of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 in a DNA molecule or sample, or vice versa. This may also be possible with more than one primer pair. For example, a first primer pair may produce a first amplicon if cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 is present, and a second primer pair may produce a second amplicon if cotton event is absent or not present. Alternatively, the size of an amplicon produced in an amplification reaction may also be diagnostic of the presence or absence of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 in a DNA molecule or sample - e.g., a primer pair may produce a first amplicon of a first size if cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 is present or a second amplicon of a second size if cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 is absent and not present; or a first primer pair may produce a first amplicon of a first size if cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 is present, and a second primer pair may produce a second amplicon of a second size if cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 is absent or not present. According to some of these embodiments at least two primer pairs may be used wherein at least one of the primer pairs is used as an internal control and is not associated with cotton event GH_BCS246002 or a modified cotton event GH_BCS246002.
[00229] According to present embodiments, a primer pair to detect the presence of all or part of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 in a DNA molecule or sample comprises a first primer and a second primer, wherein the first primer is complementary to a 5' flanking genomic DNA sequence and the second primer is complementary to a sequence within the transgenic insert; or wherein the first primer is complementary to a 5' flanking genomic DNA sequence and the second primer is complementary to a 3' flanking genomic DNA sequence; or wherein the first primer is complementary to a 3' flanking genomic DNA sequence and the second primer is complementary to a 5' flanking genomic DNA sequence; or wherein the first primer is complementary to a sequence within the transgenic insert and the second primer is complementary to a 3' flanking genomic DNA sequence; or wherein the first primer is complementary to a sequence within the transgenic insert and the second primer is complementary to a 5' flanking genomic DNA sequence; or wherein the first primer is complementary to a 3 ' flanking genomic DNA sequence and the second primer is complementary to a sequence within the transgenic insert. Each reference in this paragraph to a primer complementary to a 5' flanking genomic DNA sequence, a 3' flanking genomic DNA sequence, or a sequence within the transgenic insert of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 is also intended to potentially include a primer complementary to the reverse complement or opposing strand of the respective 5' flanking genomic DNA sequence, 3' flanking genomic DNA sequence, or sequence within the transgenic insert of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002.
[00230] Exemplary DNA molecules useful as primers are provided as SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 19. The primer pair SEQ ID NO: 15 and SEQ ID NO: 16 can be useful as a first DNA molecule or primer and a second DNA molecule or primer, wherein each primer has sufficient length of consecutive nucleotides of SEQ ID NO: 10 or a sequence complementary to SEQ ID NO: 10 to function as DNA primers that, when used together in an amplification reaction with template DNA derived from cotton event GH_BCS246002 or a modified cotton event GH_BCS246002, hybridize to opposite strands of the template DNA and produce an amplicon diagnostic for cotton event GH_BCS246002 DNA or a modified cotton event GH_BCS246002 in a sample. The primer pair SEQ ID NO: 18 and SEQ ID NO: 19 are useful as a first DNA molecule or primer and a second DNA molecule or primer, wherein each primer has sufficient length of consecutive nucleotides of a locus within the cotton genome to function as DNA primers that, when used together in a thermal amplification reaction with template DNA derived from cotton event GH_BCS246002 or a modified cotton event GH_BCS246002, to produce an amplicon that serves as an internal control for both the diagnosis of cotton event
Il l GH_BCS246002 or a modified cotton event GH_BCS246002, as well as the zygosity of cotton event GH_BCS246002 DNA or DNA from a modified cotton event GH_BCS246002 in a sample. [00231] DNA probes and DNA primers are generally eleven (11) polynucleotides or more in length, often eighteen (18) polynucleotides or more, twenty-four (24) polynucleotides or more, or thirty (30) polynucleotides or more. Such probes and primers are selected to be of sufficient length to hybridize specifically to a target sequence under high stringency hybridization conditions. Preferably, probes and primers according to the present disclosure have complete sequence similarity with the target sequence, although probes differing from the target sequence that retain the ability to hybridize to target sequences may be designed by conventional methods. [00232] The nucleic acid probes and primers of the present disclosure hybridize under stringent conditions to a target DNA molecule. Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of DNA from a transgenic plant in a sample. Polynucleic acid molecules also referred to as nucleic acid segments or fragments thereof are capable of specifically hybridizing to other nucleic acid molecules under certain circumstances. [00233] As used herein, two polynucleic acid molecules are said to be capable of specifically hybridizing to one another if the two molecules are capable of forming an anti-parallel, double-stranded nucleic acid structure. A nucleic acid molecule is said to be the “complement” of another nucleic acid molecule if they exhibit complete complementarity. As used herein, molecules are said to exhibit “complete complementarity” when every nucleotide of one of the molecules is complementary to a nucleotide of the other. Two molecules are said to be “minimally complementary” if they can hybridize to one another with sufficient stability to permit them to remain annealed to one another under at least conventional "low- stringency" conditions. Similarly, the molecules are said to be “complementary” if they can hybridize to one another with sufficient stability to permit them to remain annealed to one another under conventional "high- stringency" conditions. Conventional stringency conditions are described by Sambrook et al., 1989, and by Haymes et al., In: Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, DC (1985). Departures from complete complementarity are therefore permissible, as long as such departures do not completely preclude the capacity of the molecules to form a double-stranded structure. In order for a nucleic acid molecule to serve as a primer or probe it need only be sufficiently complementary in sequence to be able to form a stable double-stranded structure under the particular solvent and salt concentrations employed. [00234] As used herein, a substantially homologous sequence is a nucleic acid sequence that will specifically hybridize to the complement of the nucleic acid sequence to which it is being compared under high stringency conditions. Appropriate stringency conditions that promote DNA hybridization, for example, 6.0 x sodium chloride/sodium citrate (SSC) at about 45 °C, followed by a wash of 2.0 x SSC at 50°C, are known to those skilled in the art or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. For example, the salt concentration in the wash step can be selected from a low stringency of about 2.0 x SSC at 50°C to a high stringency of about 0.2 x SSC at 50°C. In addition, the temperature in the wash step can be increased from low stringency conditions at room temperature, about 22°C, to high stringency conditions at about 65°C. Both temperature and salt may be varied, or either the temperature or the salt concentration may be held constant while the other variable is changed. In a preferred embodiment, a polynucleic acid of the present disclosure will specifically hybridize to one or more of the nucleic acid molecules set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10, or complements thereof or fragments thereof under moderately stringent conditions, for example at about 2.0 x SSC and about 65°C. In a particularly preferred embodiment, a nucleic acid of the present disclosure will specifically hybridize to one or more of the nucleic acid molecules set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10, or complements or fragments thereof under high stringency conditions. In one aspect of the present disclosure, a preferred marker nucleic acid molecule of the present disclosure has the nucleic acid sequence set forth in SEQ ID NO: 1, or SEQ ID NO: 2, or SEQ ID NO: 3, or SEQ ID NO: 4, or SEQ ID NO: 5, or SEQ ID NO: 6, or SEQ ID NO: 7, or SEQ ID NO: 8, or SEQ ID NO: 9, or SEQ ID NO: 10, or complements thereof, or fragments thereof. The hybridization of the probe to the target DNA molecule can be detected by any number of methods known to those skilled in the art, these can include, but are not limited to, fluorescent tags, radioactive tags, antibody based tags, colorimetric tags, and chemiluminescent tags.
[00235] Regarding the amplification of a target nucleic acid sequence (e.g., by PCR) using a particular amplification primer pair, "stringent conditions" are conditions that pennit the primer pair to hybridize only to the target nucleic acid sequence to which a primer having the corresponding wild-type sequence (or its complement) would bind and preferably to produce a unique amplification product, the amplicon, in a DNA thermal amplification reaction.
[00236] The term "specific for (a target sequence)" indicates that a probe or primer hybridizes under stringent hybridization conditions only to the target sequence in a sample comprising the target sequence.
[00237] As used herein, “amplified DNA” or “amplicon” refers to the product of polynucleic acid amplification method directed to a target polynucleic acid molecule that is part of a polynucleic acid template. For example, to determine whether a cotton plant resulting from a sexual cross contains transgenic plant genomic DNA from a cotton plant comprising cotton event GH_BCS246002 of the present disclosure, DNA that is extracted from a cotton plant tissue sample may be subjected to a polynucleic acid amplification method using a primer pair that includes a first primer derived from a genomic DNA sequence in the region flanking the heterologous inserted DNA of cotton event GH_BCS246002 and is elongated by polymerase 5 ' to 3' in the direction of the inserted DNA. The second primer is derived from the heterologous inserted DNA molecule is elongated by the polymerase 5' to 3' in the direction of the flanking genomic DNA from which the first primer is derived. The amplicon may range in length from the combined length of the primer pair plus one nucleotide base pair, or plus about fifty nucleotide base pairs, or plus about two hundred- fifty nucleotide base pairs, or plus about four hundred-fifty nucleotide base pairs or more. Alternatively, a primer pair can be derived from genomic sequence on both sides of the inserted heterologous DNA so as to produce an amplicon that includes the entire insert polynucleotide sequence (e.g., a forward primer isolated from the genomic portion on the 5' end of SEQ ID NO: 10 and a reverse primer isolated from the genomic portion on the 3' end of SEQ ID NO: 10 that amplifies a DNA molecule comprising the inserted DNA sequence (SEQ ID NO: 9) identified herein in the cotton event GH_BCS246002 genome). A member of a primer pair derived from the plant genomic sequence adjacent to the inserted transgenic DNA is located a distance from the inserted DNA sequence, this distance can range from one nucleotide base pair up to about twenty thousand nucleotide base pairs. The use of the term “amplicon” specifically excludes primer dimers that may be formed in the DNA thermal amplification reaction.
[00238] For practical purposes, one should design primers which produce amplicons of a limited size range, for example, between 100 to 1000 bases. Smaller (shorter polynucleotide length) sized amplicons in general are more reliably produced in thermal amplification reactions, allow for shorter cycle times, and can be easily separated and visualized on agarose gels or adapted for use in endpoint TaqMan®-likc assays. Smaller amplicons can be produced and detected by methods known in the ail of DNA amplicon detection. In addition, amplicons produced using the primer pairs can be cloned into vectors, propagated, isolated, and sequenced or can be sequenced directly with methods well established in the art. Any primer pair derived from the combination of SEQ ID NO: 11 and SEQ ID NO: 9 or the combination of SEQ ID NO: 12 and SEQ ID NO: 9 that are useful in a DNA amplification method to produce an amplicon diagnostic for cotton event GH_BCS246002, or progeny thereof is an aspect of the present disclosure. Any single isolated DNA polynucleotide primer molecule comprising at least 15 contiguous nucleotides of SEQ ID NO: 11, or its complement that is useful in a DNA amplification method to produce an amplicon diagnostic for cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 or progeny thereof is an aspect of the present disclosure. Any single isolated DNA polynucleotide primer molecule comprising at least 15 contiguous nucleotides of SEQ ID NO: 12, or its complement that is useful in a DNA amplification method to produce an amplicon diagnostic for plants comprising cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 or progeny thereof is an aspect of the present disclosure. Any single isolated DNA polynucleotide primer molecule comprising at least 15 contiguous nucleotides of SEQ ID NO: 9, or its complement that is useful in a DNA amplification method to produce an amplicon diagnostic for cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 or progeny thereof is an aspect of the present disclosure.
[00239] Probes and primers as provided herein may have complete sequence identity with the target sequence, although primers and probes differing from the target sequence that retain the ability to hybridize preferentially to target sequences may be designed by conventional methods. In order for a nucleic acid molecule to serve as a primer or probe it need only be sufficiently complementary in sequence to be able to form a stable double- stranded structure under the particular solvent and salt concentrations employed. Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of transgenic DNA from cotton event GH_BCS246002 in a sample.
[00240] Any number of methods well known to those skilled in the art can be used to isolate and manipulate a DNA molecule, or fragment thereof, disclosed herein, including DNA isolation or thermal amplification or PCR methods. Such DNA molecule or fragment may be inserted or placed into any suitable vector or plasmid or combined with other elements, sequences or fragments using molecular or recombinant techniques.
[00241] The DNA molecules and corresponding nucleotide sequences provided herein are therefore useful for, among other things, identifying cotton event GH_BCS246002, detecting the presence of DNA derived from the transgenic cotton event GH_BCS246002 in a sample, and monitoring samples for the presence and/or absence of cotton event GH_BCS246002 or plant parts derived from cotton plants comprising cotton event GH_BCS246002.
[00242] According to embodiments of the present disclosure, a transgenic cotton plant or plant part, one or more transgenic cotton plants or plant parts or a plurality transgenic cotton plants or plant parts as provided herein, or an agricultural field or soil in which a transgenic cotton plant or plant part, one or more transgenic cotton plants or plant parts or a plurality of transgenic cotton plants or plant parts as provided herein are planted or grown, can be treated with an agricultural composition comprising one or more active ingredients or other agents, such as, for example and without limitation, an herbicide or one or more herbicides, a fungicide or one or more fungicides, an insecticide or one or more insecticides, a plant growth regulator or plant stimulant or one or more plant growth regulators and/or plant stimulants, and/or a safener or one or more safeners. Provided below are lists of possible or representative compounds for each of these types of actives or agents, and an agricultural composition may comprise one or any combination or multiplicity of these actives, agents or compounds. Such an agricultural composition may be applied, for example, as a foliar, soil or in-furrow treatment, as a pre-emergent, pre-sowing and/or post- emergent treatment, and/or in some cases, may be applied to a transgenic plant part or seed provided herein.
[00243] An agricultural composition may be formulated according to its intended use and application. The appropriate formulation of the agricultural composition may be chosen to have different physicochemical parameters, components and stabilities of the respective compound(s). Possible types of formulations for an agricultural composition can include, for example: wettable powders (WP), water-soluble powders (SP), water-soluble concentrates, emulsifiable concentrates (EC), emulsions (EW), such as oil-in-water and water-in-oil emulsions, sprayable solutions, suspension concentrates (SC), dispersions based on oil or water, oil-miscible solutions, capsule suspensions (CS), dusting products (DP), dressings, granules for scattering and soil application, granules (GR) in the form of microgranules, spray granules, absorption and adsorption granules, water-dispersible granules (WG), water-soluble granules (SG), ULV formulations, microcapsules and waxes. If appropriate, some agricultural compositions of a pesticidal compound or one or more pesticidal compounds might be formulated and used as a seed coating applied to a plant part or seed as provided herein.
[002441 Plants, progeny, plant parts, plant seeds, plant tissues, and plant cells may contain or comprise one or more additional desirable trait(s). Such desirable traits may be transgenic traits, native traits, or traits produced by other methods, such as genome editing, base editing, prime editing or other conventional mutagenesis methods. Such desirable trait(s) may provide an agronomic, agricultural or commodity benefit to a plant, plant part, plant seed or plant product. Desirable traits may be combined with cotton event GH_BCS246002 by, for example, crossing a cotton plant comprising cotton event GH_BCS246002 with another cotton plant containing the additional trait(s). Alternatively, a trait may be created by mutagenesis, editing or site-directed integration of or into a plant, plant part or plant cell comprising cotton event GH_BCS246002. Such traits may include, but are not limited to, increased insect resistance, increased water use efficiency, increased nitrogen use efficiency, increased yield performance, increased drought resistance, increased disease resistance, increased seed quality, improved nutritional quality, hybrid seed production, and/or increase herbicide tolerance, in which the trait is measured with respect to a cotton plant lacking such transgenic trait. For example, the cotton event GH_BCS246002 could be stacked by breeding or introgression with another event(s), or a combination of events, known in the art including, but not limited to:
• 19-5 la (DD-01951A-7 for herbicide tolerance, described in USDA- APHIS Petition 95- 256-01p, the entire content and disclosure of which are incorporated herein by reference in their entirety),
• 281-24-236 (also known as DAS-24236-5 for herbicide tolerance and insect resistance, deposited as ATCC PTA-6233, and described in US Patent Nos. 7,179,965 and 7,883,850, the entire contents and disclosure of which are incorporated herein by reference in their entirety),
• 3006-210-23 (also known as DAS-21023-5 for herbicide tolerance and insect resistance, deposited as ATCC PTA-6233, and described in US Patent Nos. 7,179,965 and 7,883,850, the entire contents and disclosure of each of which are incorporated herein by reference in their entirety), • 31707, 31803, 31807, 31808 and 42317 (also known as BXN™ Plus Bollgard™ Cotton for herbicide tolerance and insect resistance, described in USDA-APHIS Petition 97-013-01p, the entire content and disclosure of which are incorporated herein by reference in their entirety),
• BXN10211 (also known as 10211, BXN-10211-9, BXN™ Cotton for herbicide tolerance, described in USDA-APHIS Petition 93-196-01p, the entire content and disclosure of which are incorporated herein by reference in their entirety),
• BXN10215 (also known as 10215, BXN-10215-4, BXN™ Cotton for herbicide tolerance, described in USDA-APHIS Petition 93- 196-01p, the entire content and disclosure of which are incorporated herein by reference in their entirety),
• BXN10222 (also known as 10222, BXN- 10222-2, BXN™ Cotton for herbicide tolerance, described in USDA-APHIS Petition 93- 196-0 Ip, the entire content and disclosure of which are incorporated herein by reference in their entirety),
• BXN10224 (also known as IR102, for herbicide tolerance, described in USDA-APHIS Petition 93-196-01p, the entire content and disclosure of which are incorporated herein by reference in their entirety),
• COT102 (also known as IR102, SYN-IR 102-7 for insect resistance, described in US Patent No. 7,371,940, and in USDA-APHIS Petition 03-155-01p, the entire contents and disclosure of which are incorporated herein by reference in their entirety),
• COT202 (for insect resistance, described in US Patent No. 7,521,550, the entire content and disclosure of which are incorporated herein by reference in their entirety),
• COT203 (for insect resistance, described in PCT Patent Publication No. W02005054480, the entire content and disclosure of which are incorporated herein by reference in their entirety),
• COT67B (also known as IR67B, SYN-IR67B-1 for insect resistance, described in USDA- APHIS Petition 07-108-01p, the entire content and disclosure of which are incorporated herein by reference in their entirety),
• 1143- 14A (for insect resistance, described in PCT Patent Publication No. W02006128570, the entire content and disclosure of which are incorporated herein by reference in their entirety), • 1143-5 IB (for insect resistance, described in PCT Patent Publication No. W02006128569, the entire content and disclosure of which arc incorporated herein by reference in their entirety),
• CE43-67B (for insect resistance, described in US Patent Nos. 7,834,254 and 9,131,651, the entire contents and disclosure of each of which are incorporated herein by reference in their entirety),
• CE44-69D (for insect resistance, described in US Patent Publication No. 20100024077, the entire content and disclosure of which are incorporated herein by reference in their entirety),
• CE46-02A (for insect resistance, described in PCT Patent Publication No. W02006128572, the entire content and disclosure of which are incorporated herein by reference in their entirety),
• DAS81910 (also known as DAS-81910-7 for herbicide tolerance, deposited as ATCC PTA-12456, and described in US Patent Nos. 9,551,024 and 9,896,718, and in USDA-APHIS Petition 13-262-0 Ip, the entire contents and disclosure of each of which are incorporated herein by reference in their entirety),
• GHB 119 (also known as BCS-GHOO5-8, EE-GH6 for insect resistance, deposited as ATCC PTA-8398, and described in US Patent Nos. 8,309,818 and 9,328390, the entire contents and disclosure of each of which are incorporated herein by reference in their entirety),
• GHB614 (also known as BCS-GH002-5, EE-GH3, and GlyTol™ for herbicide tolerance, deposited as ATCC PTA-6878, and described in US Patent Nos. 7,932,439, 8,501,411 and 9,394,566, and in USDA-APHIS Petition 06-332-01p, the entire contents and disclosure of each of which are incorporated herein by reference in their entirety),
• GHB811 (also known as BCS-GH811-4 for herbicide tolerance, described in USDA- APHIS Petition 17- 138-0 lp, the entire content and disclosure of which are incorporated herein by reference in their entirety),
• LLCotton25 (also known as ACS-GH001-3, EE-GH1, and Fibermax™ Liberty Link™ for herbicide tolerance, deposited as ATCC PTA-3343, and described in US Patent Nos. 6,818,807, 7,442,504 and 7,834,168, the entire contents and disclosure of each of which are incorporated herein by reference in their entirety), • T303-3 (also known as BCS-GH003-6 for herbicide tolerance and insect resistance, described in USDA- APHIS Petition 12-033-01p (Extension of 08-340-01p), the entire content and disclosure of which are incorporated herein by reference in their entirety),
• T304-40 (also known as BCS-GH004-7 and EE-GH5 for herbicide tolerance and insect resistance, deposited as ATCC PTA-8171, and described in US Patent Nos. 8,247,654, 9,382,550 and 10,356,996, and in USDA-APHIS Petition 08-340-01p, the entire contents and disclosure of each of which are incorporated herein by reference in their entirety),
• T342-142 (for insect resistance, described in PCT Patent Publication No W02006128568, the entire content and disclosure of which are incorporated herein by reference in their entirety),
• MON1076 (also known as 1076, MON-89924-2 and Bollgard™ Cotton for insect resistance, described in USDA-APHIS Petition 94-308-01p, the entire content and disclosure of which are incorporated herein by reference in their entirety),
• MON1445 (also known as 1445, MON-01445-2, and Roundup Ready™ Cotton for herbicide tolerance, described in US Patent Nos. 6,740,488, 7,189,514, 7,807,357 and 7,820,392, and in USDA-APHIS Petition 95-045-01p, the entire contents and disclosure of each of which are incorporated herein by reference in their entirety),
• MON15985 (also known as MON-15985-7, and Bollgard II™ Cotton for insect resistance, deposited as ATCC PTA-2516, described in US Patent Nos. 7,223,907, 7,858,764 and 9,133,473, and in USDA-APHIS Petition 00-342-01p, the entire contents and disclosure of each of which are incorporated herein by reference in their entirety),
• MON 1698 (also known as MON-89383-1, and Roundup Ready™ Cotton for herbicide tolerance, described in USDA-APHIS Petition 95-045-01p, the entire content and disclosure of which are incorporated herein by reference in their entirety),
• MON531 (also known as MON-00531-6, and Bollgard™ Cotton, Ingard™ for insect tolerance, described in USDA-APHIS Petition 94-308-0 Ip, the entire content and disclosure of which are incorporated herein by reference in their entirety),
• MON757 (also known as MON-00757-7, and Bollgard™ Cotton for insect tolerance, described in USDA-APHIS Petition 94-308-0 Ip, the entire content and disclosure of which are incorporated herein by reference in their entirety), • MON88701 (also known as MON-88701-3, and Dicamba-Glufosinate Tolerant Cotton for herbicide tolerance, deposited as ATCC PTA-11754, described in US Patent Nos. 8,735,661, 9,024,115, 10,030,277 and 10,774,341, and in USDA-APHIS Petition 12-185-01p, the entire contents and disclosure of each of which are incorporated herein by reference in their entirety),
• MON88702 (also known as MON-88702-4 for insect resistance, deposited as ATCC PTA- 122520, described in US Patent Nos. 10,604,769 and 11,286,499, the entire contents and disclosure of each of which are incorporated herein by reference in their entirety),
• MON88913 (also known as MON-88913-8, and Roundup Ready™ Flex™ Cotton for herbicide tolerance, deposited as ATCC PTA-4854, described in US Patent Nos. 7,381,861, 8,071,735 and 8,435,743, and in USDA-APHIS Petition 04-086-01p, the entire contents and disclosure of each of which arc incorporated herein by reference in their entirety),
• TAM66274 (also known as TAM-66274-5 for modified product quality, deposited as ATCC PTA-124218, described in US Patent No. 10,604,764, and in USDA-APHIS Petition 17-292-01p, the entire contents and disclosure of each of which are incorporated herein by reference in their entirety),
• 281-24-236 x 3006-210-23 (also known as MXB-13, DAS-24236-5 x DAS-21023-5, and WideStrike™ Cotton for insect resistance),
• 281-24-236 x 3006-210-23 x COT102 (also known as DAS-24236-5 x DAS-21023-5 x SYN-IR102-7 for insect resistance),
• 281-24-236 x 3006-210-23 x COT102 x 81910 (also known as DAS-24236-5 x DAS- 21023-5 x SYN-IR102-7 x DAS-81910-7 for insect resistance and herbicide tolerance),
• 3006-210-23 x 281-24-236 x MON1445 (also known as DAS-21023-5 x DAS-24236-5 x MON-01445-2, and WideStrike™ Roundup Ready™ Cotton for insect resistance and herbicide tolerance),
• 3006-210-23 x 281-24-236 x MON88913 (also known as DAS-21023-5 x DAS-24236-5 x MON-88913-8, and Widestrike™ Roundup Ready Flex™ Cotton for insect resistance and herbicide tolerance),
• 3006-210-23 x 281-24-236 x MON88913 x COT102 (also known as DAS-21023-5 x DAS- 24236-5 x MON-88913-8 x SYN-IR102-7, and Widestrike™ x Roundup Ready Flex™ x VIPCOT™ Cotton for insect resistance and herbicide tolerance), • 3006-210-23 x 281-24-236 x MON88913 x COT102 x DAS8191O (also known as DAS- 21023-5 x DAS-24236-5 x MON-88913-8 x SYN-IR102-7 x DAS-81910-7 for insect resistance and herbicide tolerance),
• 31807 x 31808 (for insect resistance and herbicide tolerance),
• COT102 x COT67B (also known as SYN-IR102-7 x SYN-IR67B- 1 and VIPCOT™ Cotton for insect resistance),
• COT102 x COT67B x MON88913 (also known as SYN-IR102-7 x SYN-IR67B-1 x MON- 88913-8 and VIPCOT™ Roundup Ready Flex™ Cotton for insect resistance and herbicide tolerance),
• COT102 x MON15985 (also known as SYN-IR102-7 x MON- 15985-7 and Bollgard® III for insect resistance),
• COT102 x MON15985 x MON88913 (also known as SYN-IR102-7 x MON-15985-7 x MON-88913-8 and Bollgard® III x Roundup Ready™ Flex™ for insect resistance and herbicide tolerance),
• COT102 x MON15985 x MON88913 x MON88701 (also known as SYN-IR102-7 x MON-15985-7 x MON-88913-8 x MON 88701-3 for insect resistance and herbicide tolerance),
• GHB614 x LLCotton25 (also known as BCS-GH002-5 x ACS-GH001-3 and GlyTol™ Liberty Link™ for herbicide tolerance),
• GHB614 x LLCotton25 x MON15985 (also known as BCS-GH002-5 x ACS-GH001-3 x MON- 15985-7 for insect resistance and herbicide tolerance),
• GHB614 x MON15985 (also known as BCS-GH002-5 x MON-15985-7 for insect resistance and herbicide tolerance),
• GHB614 x T304-40 x GHB 119 (also known as BCS-GH002-5 x BCS-GH004-7 x BCS- GH005-8 and Glytol™ x Twinlink™ for insect resistance and herbicide tolerance),
• GHB614 x T304-40 x GHB119 x COT102 (also known as BCS-GH002-5 x BCS-GH004-
7 x BCS-GHOO5-8 x SYN-IR102-7 and GlyTol LibertyLink TwinLink® Plus for insect resistance and herbicide tolerance),
• GHB811 x T304-40 x GHB 119 x COT102 (also known as BCS-GHB811-4 x BCS- GH004-7 x BCS-GHOO5-8 x SYN-IR102-7 for insect resistance and herbicide tolerance), • LLCotton25 x MONL5985 (also known as ACS-GH001-3 x MON-15985-7 and Fibermax™ Liberty Link™ Bollgard II™ for insect resistance and herbicide tolerance),
• MON15985 x MON1445 (also known as MON- 15985-7 x MON-01445-2 and Roundup Ready™ Bollgard II™ Cotton for insect resistance and herbicide tolerance),
• MON531 x MON1445 (also known as MON-00531-6 x MON-01445-2 and Roundup Ready™ Bollgard™ Cotton for insect resistance and herbicide tolerance),
• MON88701 x MON88913 (also known as MON 88701-3 x MON-88913-8 for herbicide tolerance),
• MON88701 x MON88913 x MON15985 (also known as MON 88701-3 x MON-88913-8 x MON-15985-7 for insect resistance and herbicide tolerance),
• MON88913 x MON15985 (also known as MON-88913-8 x MON-15985-7 and Roundup Ready™ Flex™ Bollgard II™ Cotton for insect resistance and herbicide tolerance),
. T304-40 x GHB 119 (also known as BCS-GH004-7 x BCS-GHOO5-8 and TwinLink™ Cotton for insect resistance and herbicide tolerance, described in in USDA- APHIS Petition 08- 340-01p, the entire content and disclosure of which are incorporated herein by reference in their entirety),
. T304-40 x GHB 119 x COT102 (also known as BCS-GH004-7 x BCS-GHOO5-8 x SYN-
IR 102-7 for insect resistance and herbicide tolerance).
• MON15947 (corresponding to event MON15985 without MON531 for insect resistance, an event developed by ballistic methods into germplasm containing event MON531 , and which was determined to be unlinked to MON531, as described in USDA- APHIS Confirmation Request 21-211-Olcr and subsequent USDA confirmation, the entire content and disclosure of which are incorporated herein by reference in their entirety).
DEPOSIT INFORMATION
[00245] In accordance with the terms of the Budapest Treaty, a deposit of a representative sample of cotton seed containing cotton event GH_BCS246002 was made on March 5, 2024 with the American Type Culture Collection (ATCC) having an address at 10801 University Boulevard, Manassas, Virginia USA, Zip Code 20110, and assigned ATCC Accession No. PTA-127733. Access to the deposits will be available during the pendency of the application to the Commissioner of Patents and Trademarks and persons determined by the Commissioner to be entitled thereto upon request. Upon issuance of the patent, all restrictions upon availability to the public will be irrevocably removed. The deposit will be maintained in the depository for a period of thirty (30) years, or five (5) years after the last request, or for the effective life of the patent, whichever is longer, and will be replaced as necessary during that period.
EXAMPLES
[00246] The following Examples are included to more fully describe aspects and embodiments of the present disclosure resulting from the construction and testing of 169 constructs, the production of about 4,498 transgenic events, and the analysis of hundreds of thousands of individual plants over 10 years through the rigorous molecular, agronomic, and field testing required for the creation and selection of cotton event GH_BCS246002.
[00247] Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.
EXAMPLE 1
Expression Cassette Testing, Construct Design, Plant Testing, and Construct Selection
[00248] It is often necessary to create and screen a large number of gene expression constructs and transformation events in order to identify a construct, and then an event, which demonstrates optimal expression of the introduced genes of interest, while also not producing agronomic or phenotypic off-types.
[00249] For these reasons, the development of a transgenic cotton plant comprising insecticidal proteins that were active against Lepidopteran pests without any observable negative effects on agronomics, yield, or stacking viability required extensive research, development, and analysis. Specifically, over a 10 year period, approximately 4,498 proof of concept and commercial transgenic events derived from 169 different plasmid vector constructs were developed, tested, and analyzed.
[00250] This example describes the design and testing in cotton plants of 169 different constructs, to identify the preferred construct for event creation. Each construct varied with respect to the coding sequences for the insecticidal proteins and the transcriptional regulatory elements, and these were tested to select the preferred construct for use in expressing the insecticidal proteins in plants. Each construct had a unique configuration, varying by expression cassette composition (both insecticidal proteins and expression elements), orientation, and whether or not proteins were targeted for insertion into the chloroplast.
[00251] In an initial proof of concept and developmental stage, 164 constructs comprising different combinations of 31 distinct promoters, 5 distinct enhancers, 21 distinct introns, 23 distinct insect toxin coding sequences, 9 distinct target signals, and 21 distinct 3' UTRs were used to generate approximately 3,855 transformed events. These events were evaluated for phenotypic or agronomic off-types, the level of expression of the insect toxin proteins, and efficacy against selected Lepidopteran insect pest species. The resulting efficacy and protein expression data, along with any information regarding phenotypic and agronomic off-types was used to eliminate inefficacious proteins, expression elements and combinations, and was used to design a smaller number of binary commercial transformation plasmid constructs to be used in the next phase of event generation, testing and development.
[00252] In this next phase of development, five new commercial constructs were created. These constructs comprised combinations of 2 to 3 insect toxin transgene expression cassettes in different orientations (convergent or divergent). Construct- 1, Construct-2, Construct-3, Construct- 4, and pGH_BCS246002 were used to generate transformed events (also referred to as “transformants”). Table 2 below shows the event selection and elimination process for all five of these commercial construct transformation events.
Table 2. Event election and elimination of stably transformed cotton events, leading to selection of cotton event GH_BCS246002.
[00253] After shoot formation in culture, a subset of the transformed events was selected based upon visual characteristics and early molecular analysis. After transformation, transformants were transferred to culture plates containing selective media. Those transformants that survived this initial selection were transferred to culture plates containing media to permit the development of whole plants. A total of 643 Ro transformants were transplanted to pots and grown in the greenhouse (GH) for further assay. The 643 events were evaluated for molecular characteristics and efficacy, and based upon the studies, 295 Ro were eliminated.
[00254] The remaining 348 Ro event were allowed to self-pollinate, producing Ri seed. The Ri events were further characterized molecularly, assessed for zygosity, performance and maturity. From this analysis, 170 events were eliminated.
[00255] During the Ri generation, the events were further evaluated with respect to molecular characteristics and performance, as well as selection for “Gold Standard Seed” (indicated as “GSS” in Table 2) wherein seed is selected to be self-bred for multiple generations to purify the event line. During each GSS generation, plants are evaluated for molecular, performance, and seed production ability. After evaluation of the R2 generation, 39 event were eliminated. At R3 generation, 13 events were eliminated from the GSS3 collection of events. At R4 generation, 58 GSS4 event were eliminated based upon performance, 56 of which were derived from transformations using Construct-4. At this point in selection, 3 events remained, one each transformed by Construct- 1, Construct-4, pGH_BCS246002. The remaining 3 events were compared with each other, and the remaining event derived from pGH_BCS246002 was selected as the lead event and is herein referred to as cotton event GH_BCS246002.
[00256] Numerous rounds of testing and comparison of various constructs revealed that the transgene cassette provided as SEQ ID NO: 13, Construct pM63770, when compared to events produced with all other constructs that were evaluated, produced events which exhibited superior efficacy against the Lepidopteran pest species Cotton bollworm (Helicoverpa zed), Tobacco budworm (He Hol his virescens), and Fall armyworm (Spodoptera frugiperda). Cotton event GH_BCS246002 was selected from the group of events generated using this pGH_BCS246002 construct, based on its superior characteristics with respect to efficacy, agronomic performance, and yield. EXAMPLE 2
Cotton event GH_BCS246002 Demonstrates Resistance to the Lepidopteran Insect Pest Species Cotton bollworm, Tobacco budworm, and Fall army worm.
[00257] This Example describes the insecticidal activity of the cotton event GH_BCS246002 against several Lepidopteran insect pests of cotton. The insect toxin proteins CrylB.3, CrylDa_7, and Vip3Cbl.l, when expressed together in cotton containing cotton event GH_BCS246002, provide resistance to Lepidopteran pests of cotton including, but not limited to, Cotton bollworm (CBW, Helicoverpa zed), Tobacco budworm (TBW, Heliothis virescens), and Fall army worm (FAW, Spodoptera frugiperda).
[00258] Screenhouse trials were conducted in Union City, Tennessee to assess resistance to Cotton bollworm (CBW, Helicoverpa zed) and Tobacco budworm (TBW, Heliothis virescens). Cotton containing cotton event GH_BCS246002 along with events derived from transformations with Construct- 1, Construct-2, Construct-3, Construct-4 and pGH_BCS246002 were evaluated using a randomized complete block design. Each event plot contained 2-4 foot rows of 4 seeds per foot spaced every 3 inches after emergence. Each plot was separated by an alley of 2 feet. Each screenhouse contained 74 plots which also included untransformed, negative control plants from cotton variety DP393 (indicated in the tables herein as “DP393”). Six screenhouses were used, 3 of which were infested with CBW and the other 3 scrccnhouscs were infested with TBW. For both CBW and TBW, plants were infested by releasing approximately 3,000 CBW or TBW adults into each greenhouse at approximately 50-55 days after planting and before cotton flowers bloom when there are several matchhead to precandle squares on plants. If a second infest was required, 3,000 CBW or TBW adults were released in the early boll stage at approximately 65-70 days after planting. Data with respect to fruit damage was taken in early mid-bloom and optionally late-bloom. The trigger for data collection in each screenhouse began when damage was first observed in that screenhouse on plants that were part of the negative control plots. Table 3 shows the average total and injured squares, the average total and injured bolls, the average total and injured fruits, and the average percent fruit injury of cotton containing cotton event GH_BCS246002 and the negative control. Table 3. Average total and injured squares and bolls of cotton event GH_BCS246002 compared to negative control (conventional cotton) infested with CBW and TBW in screenhouse trials.
[00259] As can be seen in Table 3 above, the plants containing cotton event GH_BCS246002 had more squares and bolls when compared to the negative control in plants infested with CBW and TBW. Injury was not observed in the bolls and squares of cotton containing cotton event GH_BCS246002, whereas the average fruit injury percent was between 30% and 57% in the DP393 controls.
[00260] Field efficacy trials were also performed in the same year as the screenhouse trials presented above. Field efficacy trials were conducted in Glendora, Mississippi; Snook, Texas; Stoneville, Mississippi; and Tillar, Arkansas. Cotton event GH_BCS246002 along with events derived from transformations from Construct- 1, Construct-2, Construct-3, and Construct-4, and other events from pGH_BCS246002 were evaluated using a randomized complete block design. Each plot consisted of four 30-foot rows with 3 to 4 plants for every foot, each row having between ninety and one hundred twenty plants. Data was collected at early mid-bloom stage with an optional late-bloom data collection as needed. Table 4 shows the average total and injured squares, the average total and injured bolls, the average total and injured fruits, and the average percent fruit injury of plants corresponding to cotton event GH_BCS246002 and the negative control.
Table 4. Average total and injured squares and bolls of cotton plants containing cotton event GH_BCS246002 compared to negative control (conventional cotton) under natural field infestations.
[00261] The data in Table 4 shows that cotton containing cotton event GH_BCS246002 had little to no damage to the squares and bolls when compared to the damage observed on negative control plants. In addition, cotton containing cotton event GH_BCS246002 had much less average percent fruit damage than the control plants.
[00262] In the year following the data collected in Table 4, additional screenhouse and field efficacy trials were conducted. Screenhouse trials in this subsequent year were performed in two different locations, Scott, Mississippi and Union City, Tennessee in a similar manner as described above for Table 3 data to assess the resistance of cotton containing event GH_BCS246002 to infestations of Cotton bollworm (CBW, Helicoverpa zed), Tobacco budworm (TBW, Heliothis virescens), and Fall army worm (FAW, Spodoptera frugiperdd). CBW, TBW, and FAW trials were conducted in Scott, Mississippi. CBW and FAW trials were conducted in Union City, Tennessee. Table 5 below shows the average total number of fruit and average fruit injury for cotton containing event GH_BCS246002 compared to the negative control, DP393.
Table 5. Average total fruit and average percent fruit injury of cotton containing event GH_BCS246002 compared to the negative control (conventional cotton) when infested with CBW, TBW and FAW in screenhouse trials.
[00263] The data in in Table 5 shows that the average total amount of fruit and average percent fruit injury was higher for cotton containing cotton event GH_BCS246002 when compared to the negative control.
[00264] Field efficacy trials were conducted in Glendora, Mississippi; Rocky Mount, North Carolina; Sommerville, Tennessee, Stoneville, Mississippi; and Tillar, Arkansas as previously described above. Table 6 shows the average total and injured squares, the average total and injured bolls, the average total and injured fruits, and the average percent fruit injury of cotton containing cotton event GH_BCS246002 compared to the negative control.
Table 6. Average total and injured squares and bolls of cotton containing event GH_BCS246002 compared to the negative control (conventional cotton) under natural field infestations. [00265] The data presented in Table 6 shows that in each case cotton containing cotton event
GH_BCS246002 exhibited significantly less average percent fruit damage than the control plants. [00266] From the screenhouse data presented in Table 5, cotton containing event GH_BCS246002 provides resistance against the Lepidopteran insect pest species Cotton boll worm (Helicoverpa zea), Tobacco budworm (Heliothis virescens), and Fall armyworm (Spodoptera frugiperdd). The field studies presented above demonstrates cotton containing event GH_BCS246002 provides Lepidopteran insect infestation resistance under natural infestation conditions.
EXAMPLE 3
Cotton containing event GH_BCS246002 provides consistent yield, similar agronomics, and similar fiber quality in the field when compared to DP393 conventional cotton
[00267] This example demonstrates that transgenic cotton containing cotton event GH_BCS246002 provides consistent yields and agronomics in the field that arc similar to untransformed DP393 cotton plants.
[00268] Cotton containing cotton event GH_BCS246002 was compared to the untransformed DP393 control plants in the field over three seasons in multiple locations. Two (2) row plots of cotton were grown using one hundred sixty (160) seeds of cotton transgenic event GH_BCS246002 or the untransformed DP393 negative control per forty (40) foot long row (four (4) seeds per row foot). At the end of the growing season, the growth was terminated by using commercially available defoliants and boll openers. Defoliants used in this manner are often referred to as “harvest aids.” Removing the leaves prior to harvest provides several advantages. For example, removing the leaves before harvest increases the air movement through the crop canopy which facilitates quicker drying and prevents boll rot. This process also allows the picker to begin earlier in the day and provides for a faster and more efficient picker operation. By reducing moisture more effectively, the storage of the bolls in modules is greatly improved. Removing the leaves also eliminates a main source of stain and trash which provides a better lint grade. Boll openers facilitate the opening of mature bolls which permits harvesting operations to start several days earlier, increasing the percentage of the crop harvested during the first picking, and makes picking an once-over operation in many fields. Defoliants and boll openers were applied when sixty (60) percent of the bolls were open in the field. [00269] To assess yield, both rows of each two (2) row plot were harvested and the yield recorded as “seed cotton in pounds per acre”. The yield results arc presented in Table 7.
Table 7. Yield of seed cotton in pounds per acre over 3 growing season field harvests.
[00270] The data presented in Table 7 shows that cotton plants containing cotton event GH_BCS246002 had similar yield as the non-transgenic control plants, DP393. This data demonstrates that cotton containing cotton event GH_BCS246002 does not experience a yield drag due to expression of the insect toxin proteins Cry IB.3, CrylDa_7, and Vip3Cbl.l. The mean yield data is supportive of cotton containing event GH_BCS246002 providing at least a ten percent yield advantage in terms of seed cotton yield relative to DP393 control plants.
[00271] For the experiments in this Example, the plants were assessed for any phenotypic differences over three growing seasons, such as plant height and early vigor score. Early vigor score is determined at approximately ten (10) to fourteen (14) days after the plants have emerged. The score is a visual rating used to determine if the plot has full yield potential based upon the emergence of the plants in the plot. The plots are rated using a scale of one to five. A rating of four or five indicates low vigor, while a rating of one corresponds to high vigor. Table 8 below presents the average plant height and early vigor scores for cotton plants containing event GH_BCS246002 compared to the DP393 untransformed control plants.
Table 8. Plant height and early vigor score for cotton containing event GH_BCS246002 compared to DP393 (conventional cotton) averaged over 3 growing seasons.
[00272] The data presented in Table 8 illustrates that cotton containing cotton event GH BCS246002 and DP393 control plants had similar plant heights and were in acceptable ranges. Cotton containing event GH_BCS246002 demonstrated better early vigor relative to DP393 control. [00273] Twenty-five bolls per plot were collected prior to harvest for assessment of fiber quality and other characteristics. Bolls were collected from a representative spot in the planting row. All bolls were harvested from the selected plants to prevent the introduction of any bias in the collection due to size or other characteristics. Fiber quality characteristics such as Fiber Strength, Fiber Length, Length Uniformity, Micronaire, Lint Percentage, and Short Fiber Content were determined from the harvested bolls.
[00274] Fiber properties can be determined through a combination of instrument evaluations using High Volume Instrument (HVI) technology. Fiber length is measured in inches. Fiber strength measurements are conducted on the same beard of cotton used by the HVI to measure length and uniformity. Fiber strength measurement is made by clamping and breaking a bundle of fibers with a 1/8-inch spacing between the clamp jaws. Results are reported in terms of grams per tex to the nearest tenth. A “tex” unit is equal to the weight in grams of one thousand meters of fiber. Therefore, the strength reported is the force in grams required to break a bundle of fibers one tex unit in size. Table 9 shows a general description and corresponding strength measurements in grams per tex.
Table 9. Fiber strength description and corresponding strength measure.
[00275] Fiber length uniformity is a measure of the degree of uniformity of the fibers in a sample to the nearest tenth. The ratio between the “mean length” of fibers and the “uppcr-half- mean length” of fibers is referred to as the “length uniformity index.” Both the mean length and upper-half-mean length measurements are taken when the fiber is passed through the length sensor. There is a natural distribution in the length of cotton fibers, but the lower the variation in this length distribution, the higher the length uniformity index. [00276] Micronaire is a measure of fiber fineness and maturity. An airflow instrument can be used to measure the air permeability of a constant mass of cotton fibers compressed to a fixed volume. The volume of airflow through a specimen of cotton fibers is expressed as a micronaire. Cotton fiber with micronaire measurements between 3.7 and 4.2 are considered in the premium range of micronaire. Cotton fiber within the micronaire ranges of 3.5-3.6 or 4.3-4.9 are considered base quality, while cotton fiber above 4.9 or below 3.5 are in the discount ranges. Micronaire measurements can be influenced during the growing period by environmental conditions such as moisture, temperature, sunlight, plant nutrients, and extremes in plant or boll population. Favorable growing conditions result in fully mature fibers with premium range micronaire readings. Unfavorable conditions, such as lack of moisture, early freeze, or any other conditions that interrupt plant processes, will result in immature fibers and low micronaire measurements. High micronaire cotton is caused by such things as abnormally warm temperatures during boll maturation, or poor boll set leading to excessive availability of carbohydrates and over-maturing of fibers. Fiber fineness affects processing performance and the quality of the end-product in several ways. In the opening, cleaning, and carding processes, low micronaire or fine-fiber cottons require slower processing speeds to prevent damage to the fibers. Yams made from finer fiber result in more fibers per cross section, which in turn produces stronger yarns. High micronaire or coarse fibers are not suitable for fine yams since the result would be fewer fibers per cross section, which would reduce the yarn strength. Micronaire and maturity are highly correlated within a cotton variety. Dye absorbency and retention varies with the maturity of the fibers. Low maturity fibers have poor dye absorbency and retention while higher micronaire fibers have good absorbency and retention. Table 10 below shows the range of values and classification for Fiber Length, Fiber Strength, Length Uniformity, and Micronaire.
Table 10. Fiber Length, Fiber Strength, Length Uniformity, Micronaire description and score.
[00277] Lint percentage is one of the most important fiber yield components and is commonly used as a proxy for lint yield during breeding because of its high heritability (Sun et al. (2024) Dissecting the major genetic components underlying cotton lint development. Genetics. 226(2), iyad219). Lint percentage can be determined by dividing lint weight by the total seed cotton weight.
[00278] Short fiber content is defined as the percentage of fibers less than Yi inch long, relative to the total fiber length distribution. Table 11 shows the fiber quality characteristics such as Fiber Strength, Fiber Length, Length Uniformity, Micronaire, Lint Percentage, and Short Fiber Content determined from the harvested bolls from cotton containing cotton event GH_BCS246002 compared to DP393 control plants.
Table 11. Fiber Strength, Fiber Length, Length Uniformity, Micronaire, Lint Percentage, and Short Fiber Content of cotton event GH BCS246002 and DP393. [00279] The data presented in Table 11 shows that each of the characteristics of Fiber Strength, Fiber Length, Length Uniformity, Micronaire, Lint Percentage, and Short Fiber Content were similar between cotton containing event GH_BCS246002 and the DP393 control.
[00280] Cotton plants containing transgenic event GH_BCS246002 provides excellent yield and similar agronomic and fiber characteristics as the DP393 non-transgenic control plants. Expression of the insect toxins Cry IB.3, CrylDa_7, and Vip3Cbl.l does not cause a yield drag or compromise the growth and fiber characteristics of cotton containing the cotton event GH_BCS246002 when compared to the conventional cotton DP393 control.
EXAMPLE 4
Cotton event GH_BCS246002 Event-Specific Endpoint TAQMAN® assays
[00281] This Example describes methods useful in identifying the presence of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 in a cotton sample. A pair of PCR primers and a probe were designed for the purpose of identifying the unique junction formed between the cotton genomic DNA and the inserted DNA of cotton event GH_BCS246002 in an event- specific endpoint TAQMAN® PCR assay. Examples of conditions utilized for identifying the presence of cotton event GH_BCS246002 in a cotton sample in an event- specific endpoint TAQMAN® PCR assay are described in Tables 12 and 13.
[00282] The sequence of the oligonucleotide forward primer SQ51960 (SEQ ID NO: 15) is identical to the nucleotide sequence corresponding to positions 941-964 of SEQ ID NO: 10. The sequence of the oligonucleotide reverse primer SQ51702 (SEQ ID NO: 16) is identical to the reverse complement of the nucleotide sequence corresponding to positions 1,066-1,087 of SEQ ID NO: 10. The sequence of the oligonucleotide probe PB5O3O8 (SEQ ID NO: 17) is identical to the reverse complement of the nucleotide sequence corresponding to positions 1,043-1,060 of SEQ ID NO: 10 which may be fluorescently labeled (e.g., a 6-FAM™ fluorescent label), can be used in an endpoint TAQMAN® PCR assay to identify the presence of DNA derived from cotton event GH_BCS246002.
[00283] In addition, to SQ51960 (SEQ ID NO: 15), SQ51702 (SEQ ID NO: 16), and PB5O3O8 (SEQ ID NO: 17), it should be apparent to persons skilled in the ail that other primers and/or probes can be designed to either amplify or hybridize to sequences within SEQ ID NO: 10 which are unique to, and useful for, detecting the presence of DNA derived from cotton event GH_BCS246002 or a modified cotton event GH_BCS246002.
[00284] Following standard molecular biology laboratory practices, PCR assays for event identification were developed for detection of cotton event GH_BCS246002 in a sample. Parameters of either a standard PCR assay or a TAQMAN® PCR assay were optimized with each set of primer pairs and probes (e.g., probes labeled with a fluorescent tag such as 6-FAM™ ) used to detect the presence of DNA derived from cotton event GH_BCS246002 in a sample. A control for the PCR reaction can include internal control primers and an internal control probe (e.g., VIC®- labeled) specific to a region within the cotton genome that is used as an internal control, and are primers SQ22496 (SEQ ID NO: 18), SQ22497 (SEQ ID NO: 19), and VIC® labeled probe PB50562 (SEQ ID NO: 19).
[00285] Generally, the parameters which were optimized for detection of cotton event GH_BCS246002 in a sample included primer and probe concentration, amount of templated DNA, and PCR amplification cycling parameters. The controls for this analysis include a positive control from cotton containing cotton event GH_BCS246002, a negative control from non-transgenic cotton, and a negative control that contains no template DNA.
Table 12. Cotton event GH_BCS246002 event-specific endpoint TAQMAN® PCR reaction components. Table 13. Endpoint TAQMAN® thermocycler conditions.
EXAMPLE 5
Assays for Determining Zygosity for Cotton Event GH_BCS246002 using TAQMAN®
[00286] This Example describes methods useful in identifying the zygosity of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 (e.g., homozygous, hemizygous or null). Pairs of PCR primers and a probe arc designed for the purpose of identifying properties of alleles positive for the T-DNA insertion that gave rise to cotton event GH_BCS246002 and pairs of PCR primers and a probe are designed as an internal control probe specific to a regions within the cotton genome that is used as an internal control which is represented in the cotton genome as homozygous.
[00287] The pairs of PCR primers and probe specific to the cotton event GH_BCS246002 transgenic allele, described in Example 4, PCR primers SQ51960 (SEQ ID NO: 15), SQ51702 (SEQ ID NO: 16), and 6-FAM™ labeled probe PB50308 (SEQ ID NO: 17) and the pairs of PCR primers and probe specific to the internal control, primers SQ22496 (SEQ ID NO: 18), SQ22497 (SEQ ID NO: 19), and VIC® labeled probe PB50562 (SEQ ID NO: 19) are used in a real-time PCR reaction such as that described in Example 4 above.
[00288] After amplification, the cycle thresholds (Ct values) are determined for the amplicon corresponding to cotton event GH_BCS246002 inserted allele and the single-copy, homozygous internal standard. The difference (ACt) between the Ct value of the single-copy, homozygous internal standard amplicon, and the Ct value of the cotton event GH_BCS246002 inserted allele amplicon are determined. With respect to zygosity, a ACt of around zero (0) indicates homozygosity of the inserted cotton event GH_BCS246002 T-DNA and ACt of around one (1) indicated heterozygosity of the inserted cotton event GH_BCS246002 T-DNA. Lack of an amplicon corresponding to the cotton event GH_BCS246002 inserted allele indicates the sample is null for the inserted cotton event GH_BCS246002 T-DNA. The Ct values in the TAQMAN® thermal amplification method will have some variability due to multiple factors such as amplification efficiency and ideal annealing temperatures. Therefore, the range of “about one (1)” is defined as a ACt of 0.75 to 1.25. Similar methods could be used to detect or determine the zygosity of a modified cotton event GH_BCS246002, although a different primer(s), probe and/or other parameters may need to be used depending on the genetic modification(s) present with the modified cotton event GH_BCS246002.
EXAMPLE 6
Assays for Determining Zygosity for Cotton Event GH_BCS246002 using TAQMAN®
[00289] This Example describes a method useful in identifying the zygosity of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002.
[00290] Pairs of PCR primers and a probe are designed for the purpose of identifying specific properties of alleles positive and negative for the T-DNA insertion that gave rise to cotton event GH_BCS246002. Examples and conditions that may be used in an event-specific zygosity TAQMAN® PCR are provided in Tables 14 and 15. For this assay, four primers and two probes are mixed together with the sample. The DNA primer pairs used in the zygosity assay are primers SQ51960 (SEQ ID NO: 15) and SQ51702 (SEQ ID NO: 16); and SQ51960 (SEQ ID NO: 15) and WTDP393R (SEQ ID NO: 21). The probes used in the zygosity assay are 6FAM™-labeled probe PB5O3O8 (SEQ ID NO: 17) and VIC®-labeled probe WTDP393PR (SEQ ID NO: 22). SQ51960 (SEQ ID NO: 15) and SQ51702 (SEQ ID NO: 16) and the 6FAM™-labeled probe PB5O3O8 (SEQ ID NO: 17) are diagnostic for, or characteristic of, cotton event GH_BCS246002 DNA. There primers SQ51960 (SEQ ID NO: 15) and WTDP393R (SEQ ID NO: 21) and the VIC®-labeled probe WTDP393PR (SEQ ID NO: 22) are diagnostic when there is no copy of cotton event GH_BCS246002; i.e., they are diagnostic for, or characteristic of, the wild type allele.
[00291] When the three primers and two probes are mixed together in a PCR reaction with DNA extracted from a plant heterozygous for cotton event GH_BCS246002, there is a fluorescent signal from both the 6FAM™-labeled probe PB50308 (SEQ ID NO: 17) and the VIC®-labeled probe WTDP393PR (SEQ ID NO: 22) which is indicative of and diagnostic for, or characteristic of, a plant heterozygous for cotton event GH_BCS246002. When the three primers and two probes are mixed together in a PCR reaction with DNA extracted from a plant homozygous for cotton event GH_BCS246002, there is a fluorescent signal from only the 6FAM™-labclcd probe PB50308 (SEQ ID NO: 17) and not the VIC®-labeled probe WTDP393PR (SEQ ID NO: 22). When the three primers and the two probes are mixed together in a PCR reaction with DNA extracted from a plant which is null for cotton event GH_BCS246002 (i.e., the wild-type), there is a fluorescent signal from only the VIC®-labeled probe WTDP393PR (SEQ ID NO: 22). The template DNA samples and controls for this analysis are a positive control from cotton containing cotton event GH_BCS246002 (from both a known homozygous and known heterozygous sample), a negative control from non-transgenic cotton, and a negative control that contains no template DNA.
Table 14. Cotton event GH_BCS246002 zygosity TAQMAN® PCR reaction components. Table 15. Zygosity TAQMAN® thermocycler conditions.
EXAMPLE 7
Stacking of Cotton Event GH_BCS246002 with other Cotton Traits
[00292] This Example describes a method for identifying the cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 within progeny of any breeding activity using cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 in breeding or crossing activities with cotton plants having other event(s) or trait(s) to produce progeny plants having a stack of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 with the other event(s) or trait(s). For example, the cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 could be stacked by breeding or by site directed integration with other events known in the art to control insect pest species or to provide tolerance to herbicides or to provide other agronomic traits or characteristics, including for example, but not limited to, 19-5 la (DD- 01951A-7 for herbicide tolerance, described in USDA-APHIS Petition 95-256-01p, the entire content and disclosure of which are incorporated herein by reference in their entirety), 281-24-236 (also known as DAS-24236-5 for herbicide tolerance and insect resistance, deposited as ATCC PTA-6233, and described in US Patent Nos. 7,179,965 and 7,883,850, the entire contents and disclosure of which are incorporated herein by reference in their entirety), 3006-210-23 (also known as DAS-21023-5 for herbicide tolerance and insect resistance, deposited as ATCC PTA- 6233, and described in US Patent Nos. 7,179,965 and 7,883,850, the entire contents and disclosure of each of which are incorporated herein by reference in their entirety), 31707, 31803, 31807, 31808 and 42317 (also known as BXN™ Plus Bollgard™ Cotton for herbicide tolerance and insect resistance, described in USDA-APHIS Petition 97-013-01p, the entire content and disclosure of which are incorporated herein by reference in their entirety), BXN10211 (also known as 10211, BXN-10211-9, BXN™ Cotton for herbicide tolerance, described in USDA-APHIS Petition 93- 196-0 Ip, the entire content and disclosure of which are incorporated herein by reference in their entirety), BXN10215 (also known as 10215, BXN- 10215-4, BXN™ Cotton for herbicide tolerance, described in USDA-APHIS Petition 93-196-01p, the entire content and disclosure of which are incorporated herein by reference in their entirety), BXN10222 (also known as 10222, BXN- 10222-2, BXN™ Cotton for herbicide tolerance, described in USDA-APHIS Petition 93- 196-0 Ip, the entire content and disclosure of which are incorporated herein by reference in their entirety), BXN10224 (also known as IR102, for herbicide tolerance, described in USDA-APHIS Petition 93- 196-0 Ip, the entire content and disclosure of which are incorporated herein by reference in their entirety), COT102 (also known as IR102, SYN-IR102-7 for insect resistance, described in US Patent No. 7,371,940, and in USDA-APHIS Petition 03-155-01p, the entire contents and disclosure of which are incorporated herein by reference in their entirety), COT202 (for insect resistance, described in US Patent No. 7,521,550, the entire content and disclosure of which are incorporated herein by reference in their entirety), COT203 (for insect resistance, described in PCT Patent Publication No. W02005054480, the entire content and disclosure of which are incorporated herein by reference in their entirety), COT67B (also known as IR67B, SYN-IR67B-1 for insect resistance, described in USDA-APHIS Petition 07-108-01p, the entire content and disclosure of which are incorporated herein by reference in their entirety), 1143-14A (for insect resistance, described in PCT Patent Publication No. W02006128570, the entire content and disclosure of which are incorporated herein by reference in their entirety), 1143-5 IB (for insect resistance, described in PCT Patent Publication No. WO2006128569, the entire content and disclosure of which are incorporated herein by reference in their entirety), CE43-67B (for insect resistance, described in US Patent Nos. 7,834,254 and 9, 131,651, the entire contents and disclosure of each of which are incorporated herein by reference in their entirety), CE44-69D (for insect resistance, described in US Patent Publication No. 20100024077, the entire content and disclosure of which are incorporated herein by reference in their entirety), CE46-02A (for insect resistance, described in PCT Patent Publication No. WO2006128572, the entire content and disclosure of which are incorporated herein by reference in their entirety), DAS81910 (also known as DAS- 81910-7 for herbicide tolerance, deposited as ATCC PTA-12456, and described in US Patent Nos. 9,551,024 and 9,896,718, and in USDA-APHIS Petition 13-262-01p, the entire contents and disclosure of each of which arc incorporated herein by reference in their entirety), GHB 119 (also known as BCS-GH005-8, EE-GH6 for insect resistance, deposited as ATCC PTA-8398, and described in US Patent Nos. 8,309,818 and 9,328390, the entire contents and disclosure of each of which arc incorporated herein by reference in their entirety), GHB614 (also known as BCS- GH002-5, EE-GH3, and GlyTol™ for herbicide tolerance, deposited as ATCC PTA-6878, and described in US Patent Nos. 7,932,439, 8,501,411 and 9,394,566, and in USDA-APHIS Petition 06-332-0 Ip, the entire contents and disclosure of each of which are incorporated herein by reference in their entirety), GHB811 (also known as BCS-GH811-4 for herbicide tolerance, described in USDA-APHIS Petition 17-138-01p, the entire content and disclosure of which are incorporated herein by reference in their entirety), LLCotton25 (also known as ACS-GH001-3, EE-GH1, and Fibermax™ Liberty Link™ for herbicide tolerance, deposited as ATCC PTA-3343, and described in US Patent Nos. 6,818,807, 7,442,504 and 7,834,168, the entire contents and disclosure of each of which are incorporated herein by reference in their entirety), T303-3 (also known as BCS-GH003-6 for herbicide tolerance and insect resistance, described in USDA-APHIS Petition 12-033-01p (Extension of 08-340-01p), the entire content and disclosure of which are incorporated herein by reference in their entirety), T304-40 (also known as BCS-GH004-7 and EE-GH5 for herbicide tolerance and insect resistance, deposited as ATCC PTA-8171, and described in US Patent Nos. 8,247,654, 9,382,550 and 10,356,996, and in USDA-APHIS Petition 08-340-0 Ip, the entire contents and disclosure of each of which are incorporated herein by reference in their entirety), T342-142 (for insect resistance, described in PCT Patent Publication No W02006128568, the entire content and disclosure of which are incorporated herein by reference in their entirety), MON1076 (also known as 1076, MON-89924-2 and Bollgard™ Cotton for insect resistance, described in USDA-APHIS Petition 94-308-01p, the entire content and disclosure of which are incorporated herein by reference in their entirety), MON1445 (also known as 1445, MON-01445-2, and Roundup Ready™ Cotton for herbicide tolerance, described in US Patent Nos. 6,740,488, 7,189,514, 7,807,357 and 7,820,392, and in USDA-APHIS Petition 95- 045-01p, the entire contents and disclosure of each of which arc incorporated herein by reference in their entirety), MON15985 (also known as MON-15985-7, and Bollgard II™ Cotton for insect resistance, deposited as ATCC PTA-2516, described in US Patent Nos. 7,223907, 7,858,764 and 9,133,473, and in USDA-APHIS Petition 00-342-01p, the entire contents and disclosure of each of which are incorporated herein by reference in their entirety), MON1698 (also known as MON- 89383-1, and Roundup Ready™ Cotton for herbicide tolerance, described in USDA-APHIS Petition 95-045-01p, the entire content and disclosure of which are incorporated herein by reference in their entirety), MON531 (also known as MON-00531 -6, and Bollgard™ Cotton, Ingard™ for insect tolerance, described in USDA- APHIS Petition 94-308-0 Ip, the entire content and disclosure of which are incorporated herein by reference in their entirety), MON757 (also known as MON-00757-7, and Bollgard™ Cotton for insect tolerance, described in USDA- APHIS Petition 94-308-0 Ip, the entire content and disclosure of which are incorporated herein by reference in their entirety), MON88701 (also known as MON-88701-3, and Dicamba-Glufosinate Tolerant Cotton for herbicide tolerance, deposited as ATCC PTA- 11754, described in US Patent Nos. 8,735,661, 9,024,115, 10,030,277 and 10,774,341, and in USDA-APHIS Petition 12-185- Olp, the entire contents and disclosure of each of which are incorporated herein by reference in their entirety), MON88702 (also known as MON-88702-4 for insect resistance, deposited as ATCC PTA-122520, described in US Patent Nos. 10,604,769 and 11,286,499, the entire contents and disclosure of each of which are incorporated herein by reference in their entirety), MON88913 (also known as MON-88913-8, and Roundup Ready™ Flex™ Cotton for herbicide tolerance, deposited as ATCC PTA-4854, described in US Patent Nos. 7,381,861, 8,071,735 and 8,435,743, and in USDA-APHIS Petition 04-086-0 Ip, the entire contents and disclosure of each of which are incorporated herein by reference in their entirety), TAM66274 (also known as TAM-66274-5 for modified product quality, deposited as ATCC PTA- 124218, described in US Patent No. 10,604,764, and in USDA-APHIS Petition 17-292-01p, the entire contents and disclosure of each of which are incorporated herein by reference in their entirety), 281-24-236 x 3006-210-23 (also known as MXB-13, DAS-24236-5 x DAS-21023-5, and WideStrike™ Cotton for insect resistance), 281-24-236 x 3006-210-23 x COT102 (also known as DAS-24236-5 x DAS-21023-5 x SYN-IR102-7 for insect resistance), 281-24-236 x 3006-210-23 x COT102 x 81910 (also known as DAS-24236-5 x DAS-21023-5 x SYN-IR102-7 x DAS-81910-7 for insect resistance and herbicide tolerance), 3006-210-23 x 281-24-236 x MON1445 (also known as DAS-21023-5 x DAS-24236-5 x MON-01445-2, and WideStrike™ Roundup Ready™ Cotton for insect resistance and herbicide tolerance), 3006-210-23 x 281-24-236 x MON88913 (also known as DAS-21023-5 x DAS-24236-5 x MON-88913-8, and Widestrike™ Roundup Ready Flex™ Cotton for insect resistance and herbicide tolerance), 3006-210-23 x 281-24-236 x MON88913 x COT102 (also known as DAS-21023-5 x DAS-24236-5 x MON-88913-8 x SYN-IR102-7, and Widestrike™ x Roundup Ready Flex™ x VIPCOT™ Cotton for insect resistance and herbicide tolerance), 3006- 210-23 x 281-24-236 x MON88913 x COT102 x DAS81910 (also known as DAS-21023-5 x DAS- 24236-5 x MON-88913-8 x SYN-IR102-7 x DAS-81910-7 for insect resistance and herbicide tolerance), 31807 x 31808 (for insect resistance and herbicide tolerance), COT102 x COT67B (also known as SYN-IR102-7 x SYN-IR67B-1 and VIPCOT™ Cotton for insect resistance), COT102 x COT67B x MON88913 (also known as SYN-IR102-7 x SYN-IR67B-1 x MON-88913- 8 and VIPCOT™ Roundup Ready Flex™ Cotton for insect resistance and herbicide tolerance), COT102 x MON15985 (also known as SYN-IR102-7 x MON- 15985-7 and Bollgard® III for insect resistance), COT102 x MON15985 x MON88913 (also known as SYN-IR102-7 x MON- 15985-7 x MON-88913-8 and Bollgard® III x Roundup Ready™ Flex™ for insect resistance and herbicide tolerance), COT102 x MON15985 x MON88913 x MON88701 (also known as SYN- IR102-7 x MON-15985-7 x MON-88913-8 x MON 88701-3 for insect resistance and herbicide tolerance), GHB614 x LLCotton25 (also known as BCS-GH002-5 x ACS-GH001-3 and GlyTol™ Liberty Link™ for herbicide tolerance), GHB614 x LLCotton25 x MON15985 (also known as BCS-GH002-5 x ACS-GH001-3 x MON-15985-7 for insect resistance and herbicide tolerance), GHB614 x MON15985 (also known as BCS-GH002-5 x MON-15985-7 for insect resistance and herbicide tolerance), GHB614 x T304-40 x GHB119 (also known as BCS-GH002-5 x BCS- GH004-7 x BCS-GHOO5-8 and Glytol™ x Twinlink™ for insect resistance and herbicide tolerance), GHB614 x T304-40 x GHB 119 x COT102 (also known as BCS-GH002-5 x BCS- GH004-7 x BCS-GHOO5-8 x SYN-IR102-7 and GlyTol LibertyLink TwinLink® Plus for insect resistance and herbicide tolerance), GHB811 x T304-40 x GHB 119 x COT102 (also known as BCS-GHB811-4 x BCS-GH004-7 x BCS-GH005-8 x SYN-IR102-7 for insect resistance and herbicide tolerance), LLCotton25 x MON15985 (also known as ACS-GH001-3 x MON- 15985-7 and Fibermax™ Liberty Link™ Bollgard II™ for insect resistance and herbicide tolerance), MON15985 x MON1445 (also known as MON-15985-7 x MON-01445-2 and Roundup Ready™ Bollgard II™ Cotton for insect resistance and herbicide tolerance), MON531 x MON1445 (also known as MON-00531-6 x MON-01445-2 and Roundup Ready™ Bollgard™ Cotton for insect resistance and herbicide tolerance), MON88701 x MON88913 (also known as MON 88701-3 x MON-88913-8 for herbicide tolerance), MON88701 x MON88913 x MON15985 (also known as MON 88701-3 x MON-88913-8 x MON-15985-7 for insect resistance and herbicide tolerance), MON889L3 x MON15985 (also known as MON-88913-8 x MON-15985-7 and Roundup Ready™ Flex™ Bollgard II™ Cotton for insect resistance and herbicide tolerance), T304-40 x GHB 119 (also known as BCS-GH004-7 x BCS-GH005-8 and TwinLink™ Cotton for insect resistance and herbicide tolerance, described in in USDA-APHIS Petition 08-340-01p, the entire content and disclosure of which arc incorporated herein by reference in their entirety), T304-40 x GHB 119 x COT 102 (also known as BCS-GH004-7 x BCS-GH005-8 x SYN-IR102-7 for insect resistance and herbicide tolerance), and/or MON15947 (corresponding to MON15985 without MON531 for insect resistance, described in USDA-APHIS Confirmation Request 21-21 l-01cr and subsequent USDA confirmation, the entire content and disclosure of which are incorporated herein by reference in their entirety).
[00293] DNA primer pairs are used to produce an amplicon diagnostic for, or characteristic of, cotton event GH_BCS246002. An amplicon diagnostic for, or characteristic of, cotton event GH_BCS246002 may comprise at least one junction sequence. The junction sequences for cotton event GH_BCS246002 are SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6 ([1], [2], [3], [4], [5], and [6], respectively in FIG. 1). As shown in FIG. 1, [15] represents SEQ ID NO: 15 or primer SQ51960, and [16] represents SEQ ID NO: 16 or primer SQ51702. The arrows show the approximate hybridization position within [10] of FIG. 1 and the direction in which synthesis proceeds during the amplification cycles. These two primers may be used together as a primer pair that can be applied to a sample containing cotton event GH_BCS246002 DNA and that, when used in a thermal amplification reaction, produces an amplicon of one hundred forty-seven (147) nucleotides containing a junction sequence comprising the 5' end of the insert and a flanking genomic DNA. [17] in FIG. 1 represents SEQ ID NO: 17 or probe PB50308, which can bind or hybridize to an amplicon produced using, for example, primers [15] and [16] together in an amplification reaction with cotton event GH_BCS246002 DNA as template, for detecting the presence of the cotton event GH_BCS246002 DNA in a sample. [21] in FIG. 1 represents SEQ ID NO: 21 or primer WTDP393R that binds or hybridizes to a position within the 3’ flanking cotton genomic DNA, and when combined with primer [15] in a thermal amplification reaction together with conventional cotton DNA as template lacking or devoid of cotton event GH_BCS246002 DNA, produces an amplicon of one hundred fifty-one (151) nucleotides containing undisrupted cotton genomic DNA, and detection of that amplicon is representative of a sample which does not contains the cotton transgenic event GH_BCS246002 DNA at that chromosomal locus. Probe WTDP393PR (SEQ ID NO: 22) is a probe that could be used to bind or hybridize to an amplicon produced using primers [21] and [15] for detecting an allele lacking, or devoid of, cotton event GH_BCS246002 DNA. Probe WTDP393PR hybridizes to the 3' terminal 12 nucleotides of the 3' genomic flanking DNA and the 10 nucleotides of the wild-type allelic DNA that was deleted during insertion of the T-DNA in cotton event GH_BCS246002.
[00294] Primer pairs that will produce an amplicon diagnostic for, or characteristic of, cotton event GH_BCS246002 include primer pairs based upon the flanking sequences (SEQ ID NO: 11 and SEQ ID NO: 12) and the inserted T-DNA (SEQ ID NO: 9). To acquire a diagnostic amplicon in which SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5 is found, one would design a forward primer molecule based on the 5' flanking cotton genomic DNA (SEQ ID NO:11) from bases 1-1,000 and a reverse primer molecule based upon the inserted T-DNA (SEQ ID NO:9) from positions 1,001-16,129 in which the primer molecules are of sufficient length of contiguous nucleotides to specifically hybridize to SEQ ID NO: 11 and SEQ ID NO: 9. To acquire a diagnostic amplicon in which SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6 is found, one would design a forward primer molecule based upon the inserted T-DNA (e.g., within SEQ ID NO: 9) from positions 1,001-16,129 and a reverse primer molecule based upon the 3' flanking cotton genomic DNA (e.g., within SEQ ID NO: 12) from positions 16,130-17,129 in which the primer molecules are of sufficient length of contiguous nucleotides to specifically hybridize to those regions (e.g., SEQ ID NO: 9 and SEQ ID NO: 12).
[00295] For practical purposes, one should design primers which produce amplicons of a limited size range, preferably between 200 to 1,000 bases. Smaller sized amplicons in general are more reliably produced in PCR reactions, allow for shorter cycle times, and can be easily separated and visualized on agarose gels or adapted for use in in endpoint TAQMAN®-like assays. In addition, amplicons produced using said primer pairs can be cloned into vectors, propagated, isolated and sequenced, or can be sequenced directly with methods well established in the art. Any primer pair derived from the combinations of SEQ ID NO: 11 and SEQ ID NO: 9 or SEQ ID NO: 12 and SEQ ID NO: 9 that are useful in a DNA amplification method to produce an amplicon diagnostic for, or characteristic of, cotton event GH_BCS246002 or progeny thereof is an aspect of the present disclosure. Any single isolated DNA polynucleotide primer molecule comprising, for example, at least eleven (11), at least fifteen (15) or at least eighteen (18) contiguous nucleotides of SEQ ID NO: 11 , SEQ ID NO: 9 or SEQ ID NO: 12 or their complements can be useful in a DNA amplification method to produce an amplicon diagnostic for, or characteristic of, cotton event GH_BCS246002 or progeny thereof. [00296] An example of amplification conditions for this analysis is illustrated in Tables 12 and 13. Any modification of these methods or the use of DNA primers homologous or complementary to SEQ ID NO: 11 or SEQ ID NO: 12, or DNA sequences of the genetic elements contained in the transgene insert (SEQ ID NO: 9) of cotton event GH_BCS246002, that produce an amplicon diagnostic for, or characteristic of, cotton event GH_BCS246002 is within the art. A diagnostic amplicon comprises a DNA molecule homologous or complementary to at least one transgene/genomic junction DNA sequence (or genomic/transgene junction DNA sequence based on orientation), or a substantial portion thereof.
[00297] An analysis for a cotton event GH_BCS246002 plant tissue sample should include a positive tissue control from a plant that contains cotton event GH_BCS246002, a negative control from a cotton plant that does not contain cotton event GH_BCS246002 (e.g., DP393), and possibly a negative control that contains no cotton genomic DNA. A primer pair will amplify an endogenous cotton DNA molecule and will serve as an internal control for the DNA amplification conditions. Additional primer sequences can be selected from SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 9 by those skilled in the art of DNA amplification methods. Conditions selected for the production of an amplicon by the methods shown in Table 12 and Table 13 may differ but result in an amplicon diagnostic for, or characteristic of, cotton event GH_BCS246002 DNA. The use of different DNA primer and/or probe sequences within (or with modifications to) the methods of Table 12 and Table 14 for detection or determination of presence or absence of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 are within the scope of the present disclosure. An amplicon produced by at least one DNA primer sequence derived from SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 9 that is diagnostic for, or characteristic of, cotton event GH_BCS246002 is an aspect of the present disclosure.
[00298] DNA detection kits that contain at least one DNA primer of sufficient length of contiguous nucleotides derived from SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 9 that, when used in a DNA amplification method, produces a diagnostic amplicon for cotton event GH_BCS246002 or its progeny is an aspect of the present disclosure. A cotton plant or seed, wherein its genome will produce an amplicon diagnostic for, or characteristic of, cotton event GH_BCS246002, when tested in a DNA amplification method is an aspect of the present disclosure. The assay for the cotton event GH_BCS246002 amplicon can be performed by using an Applied Biosystems GeneAmp™ PCR System 9700, Stratagene Robocycler®, Eppendorf® Mastercycler® Gradient thermocycler or any other amplification system that can be used to produce an amplicon diagnostic of, or characteristic of, cotton event GH_BCS246002 as shown in Table 12.
EXAMPLE 8
Insertion of Sequences into Cotton event GH BCS246002 to Facilitate Removal of a Transgene or Sequence of Insertion using a Single Guide RNA
[00299] This Example describes how all, or part of a transgenic insertion present in cotton event GH_BCS246002, including any portion of the flanking genomic DNA adjacent to the transgenic DNA insert, may be excised using targeted genome editing techniques. Sequences useful in excision of the cotton event GH_BCS246002 transgene insertion or expression cassettes within SEQ ID NO: 10 can be introduced through genomic editing using a variety of methods, particularly through the use of Clustered Regularly Interspersed Short Palindromic Repeats (CR1SPR) editing systems. A CRISPR-associated protein can be selected from a Type 1 CR1SPR- associated protein, a Type II CRISPR-associated protein, a Type III CRISPR-associated protein, a Type IV CRISPR-associated protein, Type V CRISPR-associated protein, or a Type VI CRISPR- associated protein, such as but not limited to, Casl , CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Casl2a (also known as Cpfl), Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, CasX, CasY, and Mad7. The CRISPR-associated protein and one or more guide RNAs (gRNA) can be introduced into a plant cell comprising cotton event GH_BCS246002 to target a specific sequence within the transgenic insertion via a double strand break repair pathway, which may include, for example, non-homologous end-joining (NHEJ), microhomology-mediated end joining (MMEJ), homologous recombination, synthesis-dependent strand annealing (SDSA), single-strand annealing (SSA), or a combination thereof, at the genomic target site. One or more nucleotides or nucleotide sequences can be inserted within the cotton event GH_BCS246002 transgene insertion locus which can allow for the excision of all or part of the transgenic insertion from cotton event GH_BCS246002 or all or part of the specific expression cassettes within cotton event GH_BCS246002 or SEQ ID NO: 10.
[00300] Sequences corresponding to the 5' and 3' genomic flanking sequences of cotton event GH_BCS246002 (presented as SEQ ID NOs: 11 and 12), the 5' and 3' junction regions (presented as SEQ ID NOs: 1-6), the inserted T-DNA are scanned for potential originator guide RNA recognition sites (OgRRS) which comprises a protospaccr adjacent motif (PAM) site operably linked to a guide RNA hybridization site. The OgRRS can be located within the flanking 5' or 3' genomic sequence, or within the 5' or 3' junction regions, or within the inserted T-DNA. The OgRRS will be determined based upon the specific CRISPR editing system chosen. For example, Cas9 recognizes a G-rich protospacer-adjacent motif (PAM) that is 3 z to its guide RNA binding site whereas Casl2a systems recognize a T-rich protospacer-adjacent motif (PAM) that is 5' to its guide RNA binding site.
[00301] The OgRRS sequence is then used to define a cognate guide RNA recognition site (CgRRS) which is inserted into the transgene insertion locus of cotton event GH_BCS246002 using a CRISPR editing system. The CgRRS comprises the same gRNA target sequence as the selected OgRRS. The CgRRS is inserted in a region within the transgcnc insertion locus of cotton event GH_BCS246002 that is on the opposite side of the transgene insertion, relative to the OgRRS in a manner that will permit the excision of a fragment of DNA corresponding to either the entire transgene insertion of cotton event GH_BCS246002, or a fragment within the transgene insertion of cotton event GH_BCS246002 such as an expression cassette or genetic element within the transgene cassette, using a single gRNA. For example, to the extent that the OgRRS is located within the 3' genomic flanking sequence or the 3' junction region, then the CgRRS will be inserted within the 5' genomic flanking sequence, or the 5' junction region, or within the transgene insert such as between expression cassettes or genetic elements within an expression cassette. Insertion of the CgRRS on the opposite side of the transgene insertion or within the region between expression cassettes, relative to the OgRRS allows for excision of the transgene insertion or specific expression cassettes to be excised using a single gRNA. An OgRRS located between the expression cassettes of cotton event GH_BCS246002 can be used to design a CgRRS that can be inserted in either the 5' or 3' genomic flanking sequence to permit excision of one or the other expression cassette using a single gRNA.
[00302] Table 16 shows exemplary OgRRS sequences located within the 5' and 3' genomic flanking sequences and between the two expression cassettes of cotton event GH_BCS246002 that can be used in a CRISPR editing system employing FnCasl2a, a Type V CRISPR-associated protein (coding sequence presented as SEQ ID NO:41; protein sequence presented as SEQ ID NO:42). Table 16. Exemplary OgRRS sequences within cotton event GH_BCS246002.
[00303] Table 17 below shows gRNAs which include a poly-T transcript termination region that can be used to target the FnCasl2a nuclease to cut within both the OgRRS and CgRRS sequences.
Table 17. gRNAs useful in targeting FnCasl2a nuclease.
[00304] Any of the OgRRS sequences presented in Table 16 above can be used alternatively as a site to insert a CgRRS that was designed using a different OgRRS. For example, a CgRRS can be inserted into a flanking sequence to allow for the excision of the entire transgene insertion of cotton event GH_BCS246002. To illustrate this approach, OgRRS_3_l is selected as the OgRRS that will be used to design a corresponding CgRRS comprising DNA fragment, and OgRRS_5_2 is selected as the target site in which the CgRRS comprising DNA fragment is inserted. Using an FnCasl2a editing system, the OgRRS_5_2 site is targeted using the gRNA, gRNA_OgRRS_5_2 presented in Table 17 to cut within the OgRRS_5_2 site. The CgRRS comprising DNA fragment that comprises the OgRRS_3_l target site is then inserted within the cut site that was introduced into the OgRRS_5_2 sequence. After selection of a transgenic event comprising the introduced CgRRS site, the event can be bred into another germplasm. When desired, the transgene insert of cotton event GH_BCS246002 can be excised from the plant using an FnCasl2a editing system and the gRNA, gRNA_OgRRS_3_l as presented in Table 17.
[00305] Any of the OgRRS sequence presented in Table 16 that arc within the 5' and 3' genomic flanking sequences of cotton event GH_BCS246002 can be used as a site to insert a CgRRS comprising DNA fragment, comprising an OgRRS sequence that is between expression cassettes, to permit the excision of a specific expression cassette using a single gRNA. To illustrate this approach, OgRRS_In_l is selected as the OgRRS that will be used to design a corresponding CgRRS comprising DNA fragment, and OgRRS_5_2 is selected as the target site in which the CgRRS comprising DNA fragment is inserted. Using an FnCasl2a editing system, the OgRRS_5_2 site is targeted using the gRNA, gRNA_OgRRS_5_2 presented in Table 17 to cut within the OgRRS_5_2 site. The CgRRS comprising DNA fragment that comprises the OgRRS_In_l target site is then inserted within the cut site that was introduced into the OgRRS_5_2 sequence. After selection of a transgenic event comprising the introduced CgRRS site, the event can be bred into another germplasm. When desired, the first expression cassette which expresses the Cry IB.3 toxin protein can be excised from the plant using an FnCasl2a editing system and the gRNA, gRNA_OgRRS_In_l as presented in Table 17.
[00306] The CgRRS can be introduced into the transgene insertion locus through multiple methods using a CRISPR system. For example, a CRISPR system can be utilized for targeting 5' insertion of a blunt-end double-stranded DNA fragment into a genomic target site of interest such as an OgRRS that is not the OgRRS that has been selected for the design of the CgRRS. The CRIS PR-mediated endonuclease activity can introduce a double stand break (DSB) in the selected genomic target site and DNA repair, such as microhomology-driven nonhomologous end-joining DNA repair, results in insertion of the blunt-end double-stranded DNA fragment into the DSB. Blunt-cnd double- stranded DNA fragments can be designed with 1-10 bp of microhomology, on both the 5 ' and 3' ends of the DNA fragment that correspond to the 5' and 3' flanking sequence at the cut site of the protospacer in the genomic target site.
[003071 The CRISPR system can be introduced into cotton event GH_BCS246002 by several methods. One or more expression cassettes encoding the gRNA and/or CRISPR associated protein components of a Type I, Type II, Type III, Type IV, Type V, or Type VI CRISPR-Cas system is transiently introduced into a cell. The introduced one or more expression cassettes encoding the gRNA and/or CRISPR associated protein, along with a DNA fragment comprising the CgRRS is provided in sufficient quantity to modify the cell but does not persist after a contemplated period of time has passed or after one or more cell divisions. In such embodiments, no further steps arc needed to remove or segregate the one or more expression cassettes encoding the gRNA and/or CRISPR associated protein from the modified cell.
[00308] Alternatively, an expression construct comprising one or more expression cassette for the expression of a gRNA, and an expression construct encoding a Type I, Type II, Type III, Type IV, Type V, or Type VI CRISPR associated protein is stably transformed into cotton event GH_BCS246002 to introduce the CgRRS within the desired target locus. The gRNA will direct the nuclease to cut within the target locus which can be an OgRRS different from the selected OgRRS. The expression construct would also comprise a CgRRS DNA fragment which is flanked 5' and 3' with the PAM/gRNA sequence of the desired locus (i.e., an OgRRS different from the selected OgRRS) which will permit the excision of the CgRRS DNA fragment that can then be introduced into the target locus via a double strand break repair pathway.
[00309] Other Casl2a PAM/gRNA sites can be found within SEQ ID NO: 10 that can be used as potential OgRRS sequences, depending upon the desired outcome after genomic editing. Table 18 below shows the coordinates of each of 416 potential OgRRS sequences within SEQ ID NO: 10 and the element in which they can be found. Those indicated in bold were previously presented in Table 16. The table presents coordinates for four Casl2a endonucleases, LbCasl2a (coding sequence SEQ ID NO: 35, protein sequence SEQ ID NO: 36) derived from Lachnospiraceae bacterium ND2006 (also known as LbCpfl); two engineered variants of Lachnospiraceae bacterium ND2006 LbCasl2a, LbCasl2a-TYCV (coding sequence SEQ ID NO: 37, protein sequence SEQ ID NO: 38) and LbCasl2a-TATV (coding sequence SEQ ID NO: 39, protein sequence SEQ ID NO: 40), and FnCasl2a (coding sequence SEQ ID NO: 41 , protein sequence SEQ ID NO: 42) derived from Francisella novicida U112.
Table 18. Potential OgRRS sequences within SEQ ID NO: 10 and Elements.
EXAMPLE 9
Removal of Transgene(s), Cassette(s) or Sequence of Cotton Event GH_BCS246002 using Two Guide RNAs
[00310] This Example describes the excision of all or any portion of the transgenic inserted
DNA or an expression cassette within the transgenic inserted DNA defining and present in cotton event GH_BCS246002, using CRTSPR editing systems comprising two guide RNAs by genomic editing methods. Excision of the cotton event GH_BCS246002 transgenic insertion or expression cassettes within SEQ ID NO: 9 or SEQ ID NO: 10 can be performed through genomic editing using a variety of methods. In one embodiment, Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) editing systems comprising a CRISPR associated protein and two cognate guide RNAs may be used for targeted excision. The CRISPR-associated protein is an RNA guided nuclease and can be selected from a Type I CRISPR-associated protein, a Type II CRISPR-associated protein, a Type III CRISPR-associated protein, a Type IV CRISPR-associated protein, Type V CRISPR-associated protein, or a Type VI CRISPR-associated protein, such as but not limited to, Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Casl2a, Cpfl, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, CasX, CasY, and Mad7. The CRISPR-associated protein and two guide RNAs (gRNA) may be introduced into a plant cell comprising the cotton event GH_BCS246002 to target a specific sequence within the transgene insertion locus. In one embodiment, the CRISPR nuclease system cleaves at two distinct guide RNA hybridization sites thereby permitting the excision of the intervening sequence. Following DNA cleavage, the genomic sequence may be repaired via a double strand break repair pathway, which may include, for example, non-homologous end-joining (NHEJ), microhomology-mediated end joining (MMEJ), homologous recombination, synthesis-dependent strand annealing (SDSA), single-strand annealing (SSA), or a combination thereof, at the genomic target site.
[00311] The guide RNAs presented in Table 17 from Example 8 are used to excise the entire transgene cassette, or alternatively, are used to remove one or two of the three expression cassettes in cotton event GH_BCS246002. For example, a gRNA selected from the group consisting of SEQ ID NOs: 29 and 30 and a gRNA selected from the group consisting of SEQ ID NOs: 33 and 34 are used to guide an FnCasl2a nuclease to cut within regions of the 5' and 3' genomic flanking sequence of cotton event GH_BCS246002, causing the excision of the entire transgene insert. Alternatively, to excise the first transgene expression cassette used for expression of Cry IB.3 from cotton event GH_BCS246002 a gRNA is selected from the group consisting of SEQ ID NOs: 29 and 30 and a gRNA presented as SEQ ID NO: 31 is used to guide an FnCasl2a nuclease to cut with the region of the 5 " genomic flanking sequence and a region between the first two transgcnc expression cassettes causing the excision of the Cry1B.3 expression cassette. Likewise, to excise the third expression cassette used to express Vip3Cbl.1 a gRNA presented as SEQ ID NO: 32 and a gRNA selected from the group consisting of SEQ ID NOs: 33 and 34 is used to guide FnCasl2a nuclease to cut with the region between the CrylDa_7 and Vip3Cbl.l expression cassettes, and within the region of the 3' flanking genomic sequence causing excision of the Vip3Cbl.l expression cassette. In a similar manner, to excise the middle CrylDa_7 expression cassette from cotton event GH_BCS246002 the gRNAs presented as SEQ ID NOs: 31 and 32 are used to guide an FnCasl2a nuclease to cut within the 3' UTR of the Cry IB.3 expression cassette and the promoter of the Vip3Cbl.l expression cassette, respectively allowing excision of the CrylDa_7 expression cassette. It should also be noted that the gRNAs presented above can also be used to replace one or more expression cassettes with expression cassettes that drive transgenes for the expression of any gene of agronomic interest. The product of a gene of agronomic interest may act within the plant in order to cause an effect upon the plant morphology, physiology, growth, development, yield, grain composition, nutritional profile, disease or pest resistance, and/or environmental or chemical tolerance or may act as a pesticidal agent in the diet of a pest that feeds on the plant. A beneficial agronomic trait may include, for example, but is not limited to, herbicide tolerance, insect control, modified yield, disease resistance, pathogen resistance, modified plant growth and development, modified starch content, modified oil content, modified fatty acid content, modified protein content, modified fruit ripening, enhanced animal and human nutrition, biopolymer productions, environmental stress resistance, pharmaceutical peptides, improved processing qualities, improved flavor, hybrid seed production utility, improved fiber production, augmented carbon sequestration, and/or desirable biofuel production.
EXAMPLE 10
Assay for Determining Zygosity for Cotton Event GH_BCS246002 using TAQMAN® and Primers and Probe that Hybridize to the Vip3CbLl Coding Sequence
[00312] This Example describes methods useful in identifying the zygosity of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 (e.g., homozygous, hemizygous or null). Pairs of PCR primers and a probe are designed which hybridize to the 3’ portion of the Vip3Cbl.l coding sequence within the inserted T-DNA of cotton event GH_BCS246002 and for the purpose of identifying properties of alleles positive for the T-DNA insertion that gave rise to cotton event GH_BCS246002; and pairs of PCR primers and a probe arc designed as an internal control probe specific to a regions within the cotton genome that is used as an internal control which is represented in the cotton genome as homozygous. Examples of conditions utilized for determining the zygosity of cotton event GH_BCS246002 in a cotton sample are described in Tables 19 and 20 below.
[00313] The pairs of PCR primers and probe specific to the Vip3Cbl.l coding sequence, PCR primers SQ51253 (SEQ ID NO:245) and SQ51254 (SEQ ID NO:246), and 6-FAM™ labeled probe PB50494 (SEQ ID NO: 247) and the pairs of PCR primers and probe specific to the internal control, primers SQ22496 (SEQ ID NO: 18), SQ22497 (SEQ ID NO: 19), and VIC® labeled probe PB50562 (SEQ ID NO: 19) are used in a real-time PCR reaction similar to that as that described in Example 4 above. The sequence of the oligonucleotide forward primer SQ51253 (SEQ ID NO:245) is identical to the nucleotide sequence corresponding to positions 14,048-14,069 of SEQ ID NO: 10. The sequence of the oligonucleotide reverse primer SQ51254 (SEQ ID NO:246) is identical to the reverse complement of the nucleotide sequence corresponding to positions 15,0561-15,085 of SEQ ID NO: 10. The sequence of the oligonucleotide probe (SEQ ID NO: 247) is identical to the nucleotide sequence corresponding to positions 15,041-15,058 of SEQ ID NO: 10 which may be fluorescently labeled (e.g., a 6-FAM™ fluorescent label), can be used in an TAQMAN® PCR assay to identify the zygosity of DNA derived from cotton event GH_BCS246002 or a modified cotton event GH_BCS246002.
[00314] Following standard molecular biology laboratory practices, PCR assays for zygosity determination were developed for cotton event GH_BCS246002 in a sample. Parameters of either a standard PCR assay or a TAQMAN® PCR assay were optimized with each set of primer pairs and probes (e.g., probes labeled with a fluorescent tag such as 6-FAM™ ) used to detect the presence of DNA derived from cotton event GH_BCS246002 in a sample. A control for the PCR reaction can include internal control primers and an internal control probe (e.g., VlC®-labeled) specific to a region within the cotton genome that is used as an internal control and are primers SQ22496 (SEQ ID NO: 18), SQ22497 (SEQ ID NO: 19), and VIC® labeled probe PB50562 (SEQ ID NO: 19).
[00315] Generally, the parameters which were optimized for determination of zygosity for cotton event GH_BCS246002 in a sample included primer and probe concentration, amount of templated DNA, and PCR amplification cycling parameters. The controls for this analysis include a positive control from cotton containing cotton event GH_BCS246002 which is homozygous, a negative control from non-transgenic cotton, and a negative control that contains no template DNA.
Table 19. Cotton event GH_BCS246002 zygosity TAQMAN® PCR reaction components. Table 20. TAQMAN® thermocycler conditions.
[00316] After amplification, the cycle thresholds (Ct values) are determined for the amplicon corresponding to the Vip3Cbl.l coding sequence within the cotton event GH_BCS246002 inserted allele and the single-copy, homozygous internal standard. The difference (ACt) between the Ct value of the single-copy, homozygous internal standard amplicon, and the Ct value of the Vip3Cbl.l amplicon derived from the cotton event GH_BCS246002 inserted allele are determined. With respect to zygosity, a ACt of around zero (0) indicates homozygosity of the inserted cotton event GH_BCS246002 T-DNA and ACt of around one (1) indicated heterozygosity of the inserted cotton event GH_BCS246002 T-DNA. Lack of an amplicon corresponding to the cotton event GH_BCS246002 inserted allele indicates the sample is null for the inserted cotton event GH_BCS246002 T-DNA. The Ct values in the TAQMAN® thermal amplification method will have some variability due to multiple factors such as amplification efficiency and ideal annealing temperatures. Therefore, the range of “about one (1)” is defined as a ACt of 0.75 to 1.25. Similar methods could be used to detect or determine the zygosity of a modified cotton event GH_BCS246002, although a different primer(s), probe and/or other parameters may need to be used depending on the genetic modification(s) present with the modified cotton event GH_BCS246002.
[00317] All publications and published patent documents cited in this specification, and which are material to the present disclosure, are incorporated herein by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. Having illustrated and described the principles of the present disclosure and inventions, it should be apparent to persons skilled in the art that the invention can be modified in arrangement and detail without departing from such principles. All modifications that are within the spirit and scope of the appended claims are included and claimed.

Claims

What is claimed is:
1. A recombinant DNA molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10; and a complete complement thereof of any of the foregoing.
2. The recombinant DNA molecule of claim 1, wherein said molecule is derived from cotton event GH_BCS246002, a representative sample of cotton seed comprising said event having been deposited as ATCC Accession No. PTA- 127733.
3. The recombinant DNA molecule of claim 1 or 2, further comprising a first expression cassette encoding a Cry IB.3 protein, a second expression cassette encoding a CrylDa_7 protein, and/or a third expression cassette encoding a Vip3Cbl.l protein.
4. A DNA molecule comprising a polynucleotide segment of sufficient length to function as a DNA probe that hybridizes specifically under stringent hybridization conditions with cotton event GH_BCS246002 DNA in a sample, wherein detecting hybridization of said DNA molecule under said stringent hybridization conditions is diagnostic for the presence of cotton event GH_BCS246002 DNA in said sample.
5. The DNA molecule of claim 4, wherein said sample comprises a cotton plant, cotton plant cell, cotton seed, cotton plant part, cotton progeny plant, processed cotton seed, animal feed comprising cotton, cotton oil, cotton meal, cotton flour, cotton flakes, cotton bran, cotton biomass, and fuel products produced using cotton and cotton parts.
6. A pair of DNA molecules, comprising a first DNA molecule and a second DNA molecule different from the first DNA molecule, that function as DNA primers when used together in an amplification reaction with a sample containing cotton event GH_BCS246002 template DNA to produce an amplicon diagnostic for the presence of said cotton event GH_BCS246002 DNA in said sample, wherein said amplicon comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10.
7. A method of detecting the presence of a DNA segment diagnostic for or present in cotton event GH_BCS246002 DNA in a sample, said method comprising: a. contacting said sample with the DNA molecule of claim 4; b. subjecting said sample and said DNA molecule to stringent hybridization conditions; and c. detecting hybridization of said DNA molecule to said DNA in said sample, wherein said detection is diagnostic for the presence of said cotton event GH_BCS246002 DNA in said sample.
8. A method of detecting the presence of a DNA segment diagnostic for or present in cotton event GH_BCS246002 DNA in a sample, said method comprising: a. contacting said sample with the pair of DNA molecules of claim 6; b. performing an amplification reaction sufficient to produce a DNA amplicon; and c. detecting the presence of said DNA amplicon in said reaction, wherein said DNA amplicon comprises the nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, wherein said detection is diagnostic for the presence of said cotton event GH_BCS246002 DNA in said sample.
9. A method of detecting the presence of protein diagnostic for or present in cotton event
GH_BCS246002 in a sample, said method comprising: a. contacting said sample with a first monoclonal antibody, second monoclonal antibody, and/or third monoclonal antibody, wherein the first monoclonal antibody binds specifically to CrylB.3 protein, the second monoclonal antibody binds specifically to CrylDa_7 protein, and the third monoclonal antibody binds specifically to Vip3Cbl.l protein; b. incubating said first, second and/or third monoclonal antibody and said sample for a sufficient amount of time to allow for binding of the monoclonal antibodies to their respective target protein to form a respective antibody-protein complex; and c. detecting the presence of the respective antibody-protein complex for one or more of the Cry IB.3 protein, CrylDa_7 protein, and Vip3Cbl.l protein in said sample, wherein said detection of the respective antibody-protein complex for one or more of the CrylB.3 protein, CrylDa_7 protein, and Vip3Cbl.l protein is diagnostic for the presence of cotton containing said cotton event GH_BCS246002 in said sample.
10. The method of claim 9, wherein said sample contains cotton cells, cotton tissue, cotton fiber, cotton oil, cotton meal, plant material or commodity product in which a detectable amount of said protein diagnostic for or present in cotton event GH_BCS246002 is suspected of being present.
11. The method of claim 9, wherein the assay is performed as an Enzyme-linked Immunosorbent Assay (ELISA), a Radioimmunoassay, or a Lateral flow immunochromatographic assay.
12. A cotton plant, cotton plant part, or cotton cell comprising cotton event GH_BCS246002 DNA characterized by the presence of a detectable amount of a recombinant polynucleotide molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, wherein said cotton plant, cotton plant part, or cotton plant cell exhibits insecticidal activity against a Lepidopteran insect pest when provided in the diet of the Lepidopteran insect pest.
13. A cotton plant, cotton plant part, or cotton cell comprising the recombinant DNA molecule of claim 1, 2 or 3, wherein said cotton plant, cotton plant part, or cotton cell exhibits insecticidal activity against a Lepidopteran insect pest when provided in the diet of the Lepidopteran insect pest.
14. The cotton plant, cotton plant pail, or cotton cell of claim 12 or 13, wherein the Lepidopteran insect pest is selected from the group consisting of Cotton bollworm (Helicoverpa zea). Tobacco budworm (Heliothis virescens), and Fall armyworm (Spodoptera frugiperdci) .
15. The cotton plant, cotton plant pail, or cotton cell of any one of claims 12, 13 or 14, wherein the cotton plant, cotton plant part, or cotton cell is further defined as progeny of any generation of a cotton plant comprising the cotton event GH_BCS246002 DNA.
16. The cotton plant, cotton plant part, or cotton cell of any one of claims 12-15, wherein the cotton plant, cotton plant part, or cotton cell further comprises event MON 15947 or event MON15947 DNA.
17. A method for protecting a cotton plant from insect infestation, wherein said method comprises providing in the diet of a Lepidopteran insect pest an insecticidally effective amount of cells or tissue of a cotton plant comprising cotton event GH_BCS246002.
18. The method of claim 17, wherein said Lepidopteran insect pest is selected from the group consisting of Cotton bollworm (Helicoverpa zed), Tobacco budworm (Heliothis virescens), and Fall army worm (Spodoptera frugiperdd).
19. A method of producing a Lepidopteran resistant cotton plant comprising: a. breeding two different cotton plants, wherein at least one of the two different cotton plants comprises cotton event GH_BCS246002, to produce progeny plants; b. detecting in at least one of said progeny plants the presence of a DNA segment diagnostic for cotton event GH_BCS246002 DNA; and c. selecting said progeny plant comprising cotton event GH_BCS246002 DNA; wherein said progeny plant selected in step (c) is Lepidopteran resistant.
20. A method of producing a Lepidopteran resistant cotton plant comprising: a. breeding two different cotton plants, wherein one of the two different cotton plants comprises cotton event GH_BCS246002, and wherein the other of the two different cotton plants comprises cotton event MON15947, to produce progeny plants; b. detecting in at least one of said progeny plants the presence of a DNA segment diagnostic for cotton event GH_BCS246002 DNA and the presence of a DNA segment diagnostic for cotton event MON15947; and c. selecting said progeny plant comprising cotton event GH_BCS246002 DNA and cotton event MON15947 DNA; wherein said progeny plant selected in step (c) is Lepidopteran resistant.
21 . A cotton seed comprising a detectable amount of a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, or complete complements thereof.
22. A nonliving cotton plant material comprising a detectable amount of the recombinant DNA molecule of claim 1, 2 or 3.
23. A microorganism comprising a detectable amount of the recombinant DNA molecule of claim 1, 2 or 3.
24. The microorganism of claim 23, wherein the microorganism is selected from the group consisting of a bacterial cell and a plant cell.
25. A commodity product comprising a detectable amount of a DNA molecule unique to cotton event GH_BCS246002, wherein the DNA molecule comprises the recombinant DNA molecule of claim 1 , 2 or 3.
26. The commodity product of claim 25, further selected from the group consisting of whole or processed cotton seeds, cotton fiber, cotton oil and derivatives of cotton oil, cotton protein, cotton meal, animal feed comprising cotton, paper comprising cotton, cotton biomass, candle wicks, cotton string, cotton rope, cotton balls, cotton batting, cotton fuel products, and cotton cellulose products.
27. A cotton plant, cotton plant pail, or cotton seed comprising DNA functional as a template in a DNA amplification method producing an amplicon diagnostic for cotton event GH_BCS246002 DNA.
28. A method of determining the zygosity of a cotton plant, cotton plant part or cotton seed comprising cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 comprising: a. contacting a sample comprising cotton DNA with a primer pair that is capable of producing an amplicon diagnostic for the allele corresponding to cotton event GH_BCS246002 DNA or a modified cotton event GH_BCS246002 DNA; b. contacting said sample with a second primer pair that is capable of producing, using a thermal amplification reaction, an amplicon of an internal standard cotton genomic DNA known to be single-copy and homozygous in the cotton plant; c. contacting said sample with a probe set which contains at least a first probe that specifically hybridizes to (or with) the allele DNA of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002, and a second probe that specifically hybridizes to the internal standard cotton genomic DNA known to be single-copy and homozygous in the cotton plant; d. performing a DNA amplification reaction using real-time PCR and determining the cycle thresholds (Ct values) of the amplicon corresponding to the allele DNA of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 and the single-copy, homozygous internal standard; e. calculating the difference (ACt) between the Ct value of the single-copy, homozygous internal standard amplicon and the Ct value of the amplicon corresponding to the allele DNA of cotton event GH_BCS246002 sequence amplicon; and f. determining zygosity, wherein a ACt of about zero (0) indicates homozygosity of the inserted T-DNA of cotton event GH_BCS246002 or a modified cotton event GH BCS246002 and a ACt of about one (1) indicates heterozygosity of the inserted T-DNA of cotton event GH_BCS246002.
29. The method of claim 28, wherein the primer pairs are selected from the group consisting of SEQ ID NO: 15 combined with SEQ ID NO: 16, and SEQ ID NO: 18 combined with SEQ ID NO: 19; and wherein the probes are SEQ ID NO: 17 and SEQ ID NO: 20.
30. The method of claim 28, wherein the ACt of about one (1) indicating heterozygosity of the inserted T-DNA of cotton event GH_BCS246002 is in the range of 0.75 to 1.25.
31. A method of determining the zygosity of a cotton plant or cotton seed comprising cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 comprising: a. contacting a sample comprising cotton DNA with a set of primer pairs comprising at least two different primer pairs capable of producing a first amplicon diagnostic for cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 and a second amplicon diagnostic for native cotton genomic DNA devoid of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002; b. performing a nucleic acid amplification reaction with the sample and the set of primer pairs; and c. detecting in the nucleic acid amplification reaction the first amplicon diagnostic for cotton event GH_BCS246002 DNA or a modified cotton event GH_BCS246002 DNA, or the second amplicon diagnostic for native cotton genomic DNA devoid cotton event GH_BCS246002 or a modified cotton event GH_BCS246002, wherein the presence of only the first amplicon is diagnostic of a cotton plant or cotton seed homozygous for cotton event GH_BCS246002 DNA or a modified cotton event GH_BCS246002 DNA, and the presence of both the first amplicon and the second amplicon is diagnostic of a cotton plant or cotton seed heterozygous for cotton event GH_BCS246002 DNA.
32. The method of claim 31, wherein the set of primer pairs comprises SEQ ID NO: 15 combined with SEQ ID NO: 16, and SEQ ID NO: 15 combined with SEQ ID NO: 21.
33. A recombinant DNA molecule comprising: a. a first nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8; and b. a second nucleotide sequence that i. comprises at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1,000, at least 1,500, or at least 2,000 consecutive nucleotides of SEQ ID NO: 11 or 43, or ii. is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% identical to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1,000, at least 1 ,500, or at least 2,000 consecutive nucleotides of SEQ TD NO: 1 1 or 43, or iii. is selected from the group consisting of SEQ ID NOs: 44-144.
34. The recombinant DNA molecule of claim 33, further comprising: c) a third nucleotide sequence that i. comprises at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1,000, at least 1,500, or at least 2,000 consecutive nucleotides of SEQ ID NO: 12 or 44, or ii. at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% identical to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1,000, at least 1,500, or at least 2,000 consecutive nucleotides of SEQ ID NO: 12 or 44, or iii. is selected from the group consisting of SEQ ID NOs: 145-244.
35. The recombinant DNA molecule of claim 34, wherein the third nucleotide sequence is selected from the group consisting of SEQ ID NOs: 145-244.
36. The recombinant DNA molecule of any one of claims 33-35, wherein the recombinant DNA molecule has a deletion of one or more consecutive nucleotides between the second nucleotide sequence and the nucleotide sequence relative to SEQ ID NO: 10, 12, or 44.
37. The recombinant DNA molecule of any one of claims 33-36, wherein the second nucleotide sequence is selected from the group consisting of SEQ ID NOs: 45-144.
38. The recombinant DNA molecule of any one of claims 33-37, wherein the recombinant DNA molecule has a deletion of one or more consecutive nucleotides between the first nucleotide sequence and the second nucleotide sequence relative to SEQ ID NO: 10, 11, or- 43.
39. The recombinant DNA molecule of any one of claims 33-38, further comprising a first expression cassette encoding a CrylB.3 protein, a second expression cassette encoding a CrylDa_7 protein, and/or a third expression cassette encoding a Vip3Cbl. l protein.
40. A recombinant DNA molecule comprising: a. a first nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9; and b. a second nucleotide sequence that i. comprises at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1,000, at least 1,500, or at least 2,000 consecutive nucleotides of SEQ ID NO: 12 or 49, or ii. is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% identical to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1,000, at least 1 ,500, or at least 2,000 consecutive nucleotides of SEQ TD NO: 1 or 44, or iii. is selected from the group consisting of SEQ ID NOs: 45-144.
41. The recombinant DNA molecule of claim 40, wherein the second nucleotide sequence is selected from the group consisting of SEQ ID NOs: 45-144.
42. The recombinant DNA molecule of claim 40 or 41, wherein the recombinant DNA molecule has a deletion of one or more consecutive nucleotides between the first nucleotide sequence and the second nucleotide sequence relative to SEQ ID NO: 10, 12, or 44.
43. The recombinant DNA molecule of any one of claims 33-42, further comprising nucleotides 1-1,000 or 16,130-17,129 of SEQ ID NO: 10.
44. The recombinant DNA molecule of any one of claims 33-43, wherein said recombinant DNA molecule is comprised in a cotton plant, cotton plant part, cotton plant cell, cotton plant seed, cotton progeny plant, or commodity product made from cotton and cotton plant parts.
45. The recombinant DNA molecule of any one of claims 33-44, wherein said recombinant DNA molecule comprises an amplicon diagnostic for the presence of DNA comprising a sequence selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9.
46. The recombinant DNA molecule of any one of claims 33-45, wherein the recombinant DNA molecule is derived from a cotton plant, cotton plant part, cotton seed, processed cotton seed, cotton plant cell or tissue, cotton fiber, cotton oil and derivatives of cotton oil, cotton protein, cotton meal, animal feed comprising cotton, paper comprising cotton, cotton biomass, candle wicks, cotton string, cotton rope, cotton balls, cotton batting, cotton fuel products, and cotton cellulose products.
47. A recombinant DNA molecule comprising a polynucleotide segment of sufficient length to function as a DNA probe that hybridizes specifically under stringent hybridization conditions with a polynucleotide having a sequence selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 43, and SEQ ID NO: 44.
48. A pair of DNA molecules that can function as DNA primers when used together in an amplification reaction comprising a first DNA molecule and a second DNA molecule, wherein the first DNA molecule and the second DNA molecule are different, a. wherein the first DNA molecule is: i. at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% identical or 100% identical to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 consecutive nucleotides of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, or a complement thereof, or ii. at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% identical or 100% identical to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 consecutive nucleotides of SEQ ID NO: 11 or 43, or a complement thereof; and b. wherein the second DNA molecule is: i. at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1 %, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% identical or 100% identical to at least 10, at least 1 1 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 consecutive nucleotides of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, or a complement thereof, or ii. at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% identical or 100% identical to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 consecutive nucleotides of SEQ ID NO: 11 or 43, or a complement thereof, or iii. at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% identical or 100% identical to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 consecutive nucleotides of SEQ ID NO: 12 or 44, or a complement thereof.
49. The pair of DNA molecules of claim 48, wherein the first DNA molecule and the second DNA molecule can be used together in an amplification reaction with a sample from a plant, plant pail, plant seed, plant cell, food or animal feed, or commodity or fuel product made from a plant or plant part to produce an amplicon diagnostic for the presence of a modified cotton event GH_BCS246002 DNA in said sample.
50. The pair of DNA molecules of claim 49, wherein the amplicon comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10.
51. A method of detecting the presence of a DNA segment diagnostic for a modified cotton event GH_BCS246002 DNA in a sample, said method comprising: a. contacting said sample with the DNA molecule of claim 47; b. subjecting said sample and said DNA molecule to stringent hybridization conditions; and c. detecting hybridization of said DNA molecule to said DNA segment in said sample, wherein said detection is diagnostic for the presence of said modified cotton event
GH_BCS246002 DNA in said sample.
52. A method of detecting the presence of a DNA segment diagnostic for a modified cotton event GH_BCS246002 DNA in a sample, said method comprising: a. contacting said sample with the pair of DNA molecules of claim 48, 49 or 50; b. performing an amplification reaction sufficient to produce a DNA amplicon; and c. detecting the presence of said DNA amplicon in said reaction, wherein the present of said DNA amplicon is diagnostic for the presence of said modified cotton event GH_BCS246002 DNA in said sample.
53. A method of detecting the presence of a DNA segment diagnostic for a modified cotton event GH_BCS246002 DNA in a sample, said method comprising performing a sequencing reaction with a sample, wherein the production in the sequencing reaction of a target nucleotide sequence comprises at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 consecutive nucleotides of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, or a complement thereof, is diagnostic for the modified cotton event GH_BCS246002 DNA in a sample.
54. A modified cotton plant, cotton plant part, cotton seed, or cotton cell comprising a modified cotton event GH_BCS 246002.
55. The modified cotton plant, cotton plant part, cotton seed, or cotton cell of claim 54 comprising: a. the recombinant DNA molecule of any one of claims 33-43; or b. a recombinant DNA molecule or DNA segment comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, or a complement thereof; or c. a recombinant DNA molecule or DNA segment comprising a nucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1,000, at least 1,500, or at least 2,000 consecutive nucleotides of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 43, or SEQ ID NO: 44, or a complement thereof.
56. The modified cotton plant, cotton plant part, cotton seed, or cotton cell of claim 54 or 55, wherein the cotton plant, cotton plant part, cotton seed, or cotton cell is further defined as a progeny plant of any generation of a cotton plant comprising a modified cotton event GH_BCS246002, or a cotton plant pail, cotton seed, or cotton cell derived therefrom.
57. The modified cotton plant, cotton plant pail, cotton seed, or cotton cell of any of claims 54-56, wherein the cotton plant, cotton plant part, cotton seed, or cotton cell exhibits resistance to a Lepidopteran insect pest species.
58. A DNA detection kit comprising: a. the recombinant DNA molecule of claim 47; and b. the pair of DNA molecules of claim 48, 49 or 50.
59. A method of producing a progeny cotton plant comprising a modified cotton event GH_BCS246002 comprising: a. sexually crossing a first modified cotton plant that comprises a modified cotton event GH_BCS246002 with itself or a second cotton plant; b. collecting one or more seeds produced from said cross; c. growing said seed to produce one or more progeny plants; and d. selecting at least a first progeny plant or seed comprising a modified cotton event GH_BCS246002.
60. A hybrid modified cotton plant or seed comprising a modified cotton event GH_BCS246002 produced by the method of claim 59.
61. The method of claims 59 or 60, further comprising: e) collecting seed from said at least first progeny plant comprising a modified cotton event GH_BCS246002.
62. A nonliving cotton plant material comprising a detectable amount of the recombinant DNA molecule of any one of claims 33-43.
63. A microorganism comprising the recombinant DNA molecule of any one of claims 33- 43.
64. The microorganism of claim 63, wherein the microorganism is a plant cell.
65. A commodity product comprising the recombinant DNA molecule of any one of claims 33-43.
66. The commodity product of claim 65, wherein said commodity product is produced from a modified cotton plant, cotton plant part, cotton seed, or cotton tissue or cell comprising a modified cotton event GH_BCS246002.
67. The commodity product of claims 65 or 66, further selected from the group consisting of whole or processed cotton seed, animal feed comprising cotton, cotton oil, cotton meal, cotton flour, cotton flakes, cotton bran, cotton biomass, and fuel products produced using cotton and cotton plant parts.
68. A method of producing a commodity product, said method comprising: a. obtaining a modified cotton plant, cotton plant part, or cotton seed comprising a modified cotton event GH_BCS246002; and b. producing a commodity product from the transgenic cotton plant, cotton plant part, or cotton seed.
69. A cotton plant, cotton plant part, or cotton seed comprising a DNA molecule or segment functional as a template when tested in a DNA amplification method to produce an amplicon diagnostic for the presence of a modified cotton event GH_BCS246002 DNA.
70. A method of determining the zygosity of a cotton plant, cotton plant part, or cotton seed comprising a modified cotton event GH_BCS246002 comprising: a. contacting a sample comprising DNA from the cotton plant, cotton plant part, or cotton seed with a primer pair capable of producing a first amplicon diagnostic for the modified cotton event GH_BCS246002 and a second amplicon diagnostic for native cotton genomic DNA not comprising the modified cotton event GH_BCS246002; b. performing a nucleic acid amplification reaction with the sample and the set of primer pairs; and c. detecting the first amplicon and the second amplicon, wherein the presence of only the first amplicon is diagnostic of a cotton plant, cotton plant part, or cotton seed homozygous for the modified cotton event GH_BCS246002, the presence of only the second amplicon is diagnostic of a cotton plant, cotton plant part, or cotton seed homozygous for native cotton genomic DNA not comprising the modified cotton event GH_BCS246002, and the presence of both the first amplicon and the second amplicon is diagnostic of a cotton plant, cotton plant part, or cotton seed heterozygous for the modified cotton event GH_BCS246002.
71. A method for determining the zygosity of a cotton plant, cotton plant part, or cotton seed comprising a modified cotton event GH_BCS246002 comprising: a. contacting a sample comprising DNA from the cotton plant, cotton plant part, or cotton seed with a probe set which contains at least a first probe that specifically hybridizes to the modified cotton event GH_BCS246002 and at least a second probe that specifically hybridizes to cotton genomic DNA that was disrupted by insertion of the heterologous DNA of cotton event GH_BCS246002 and is disrupted by the modified cotton event GH_BCS246002 DNA, wherein the second probe does not hybridize to the modified cotton event GH_BCS246002 DNA; and b. hybridizing the probe set with the sample under stringent hybridization conditions, wherein detecting hybridization of only the first probe under the hybridization conditions is diagnostic for a cotton plant, cotton plant part, or cotton seed homozygous for the modified cotton event GH_BCS246002, and wherein detecting hybridization of both the first probe and the second probe under the hybridization conditions is diagnostic for a cotton plant, cotton plant part, or cotton seed heterozygous for the modified cotton event GH_BCS246002.
72. The method of claim 71, wherein the probe set comprises SEQ ID NO: 17 and SEQ ID NO: 22.
73. A method of determining the zygosity of a cotton plant or cotton seed comprising cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 comprising: a. contacting a sample comprising cotton DNA with a probe set which contains at least a first probe that specifically hybridizes to cotton event GH_BCS246002 DNA or a modified cotton event GH_BCS246002 DNA and at least a second probe that specifically hybridizes to cotton genomic DNA that was disrupted by insertion of the heterologous DNA of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 and does not hybridize to cotton event GH_BCS246002 DNA or a modified cotton event GH_BCS246002 DNA; b. hybridizing the probe set with the sample under stringent hybridization conditions, wherein detecting hybridization of only the first probe under the hybridization conditions is diagnostic for a homozygous allele of cotton event GH_BCS246002 DNA, and wherein detecting hybridization of both the first probe and the second probe under the hybridization conditions is diagnostic for a cotton plant or cotton seed heterozygous for cotton event GH_BCS246002 in said sample.
74. The method of claim 73, wherein the probe set comprises SEQ ID NO: 17 and SEQ ID NO: 22.
75. A population of transgenic cotton plants, wherein each transgenic cotton plant comprises a modified cotton event GH_BCS246002.
76. The population of transgenic cotton plants of claim 75, wherein each transgenic cotton plant of the population exhibits resistance to a Lepidopteran insect pest species.
77. A method of modifying a cotton plant, the method comprising: a. introducing a site-specific nuclease or a recombinant DNA construct comprising an expression cassette encoding a site- specific nuclease into at least one cell of an explant of a cotton plant comprising cotton event GH_BCS246002, or a plant part thereof, to produce a modified cotton event GH_BCS246002 via a genome editing technique; and b. developing or regenerating a modified cotton plant from the explant, wherein the modified cotton plant comprises the modified cotton event GH_BCS246002.
78. The method of claim 77, wherein the site-specific nuclease is a zine-finger nuclease (ZFN), a meganuclease, an RNA-guided endonuclease, a TALE-endonuclease (TALEN), a recombinase, or a transposase.
79. The method of claim 77 or 78, wherein the site-specific nuclease is an RNA-guided endonuclease or a CRISPR/Cas nuclease.
80. The method of claim 78 or 79, wherein the introducing step (a) comprises introducing the recombinant DNA construct into the at least one cell of the explant, and wherein the recombinant DNA construct further comprises an expression cassette encoding a first guide RNA (gRNA).
81. The method of claim 80, wherein the recombinant DNA construct further comprises an expression cassette encoding a second guide RNA (gRNA).
82. The method of claim 77, 78 or 79, wherein the introducing step (a) further comprises introducing a first guide RNA (gRNA) or a second recombinant DNA construct comprising an expression cassette encoding a first guide RNA (gRNA) into the at least one cell of the explant.
83. The method of claim 78 or 79, wherein the introducing step (a) comprises introducing at least two guide RNAs (gRNAs) comprising the first gRNA and a second gRNA into the at least one cell of the explant.
84. The method of claim 83, wherein the second recombinant DNA construct further comprises an expression cassette encoding a second guide RNA (gRNA).
85. The method of claim 83, wherein the introducing step (a) comprises introducing a first gRNA and second recombinant DNA construct comprising an expression cassette encoding a second guide RNA (gRNA) into the at least one cell of the explant.
86. The method of any one of claims 77-85, wherein the site-specific nuclease has a first target site in the genome of the cotton plant at or near cotton event GH_BCS246002.
87. The method of claim 86, wherein the site-specific nuclease has a second target site in the genome of the cotton plant at or near cotton event GH_BCS246002.
88. The method of any one of claims 77-87, wherein the introducing step (a) comprises introducing a second site- specific nuclease or a recombinant DNA construct comprising an expression cassette encoding a second site specific nuclease into at least one cell of the explant, and wherein the second site- specific nuclease has a second target site in the genome of the cotton plant at or near cotton event GH_BCS246002.
89. The method of any one of claims 80-85, wherein the first gRNA has a first target site in a flanking DNA sequence, 5 ' flank, 3' flank, junction sequence, or insertion sequence of cotton event GH_BCS246002, or a complement thereof.
90. The method of any one of claims 80-85 and 89, wherein the first gRNA has a first target site comprising a target sequence that is; i. at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 consecutive nucleotides of SEQ ID NO: 1 1 or 43, or a complement thereof; or ii. at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 consecutive nucleotides of SEQ ID NO: 12 or 44, or a complement thereof; or iii. at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 consecutive nucleotides of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, or a complement thereof.
91. The method of claim 90, wherein the first gRNA has a second target site comprising a target sequence that is: i. at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21 , at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 consecutive nucleotides of SEQ ID NO: 1 1 or 43, or a complement thereof; or ii. at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 consecutive nucleotides of SEQ ID NO: 12 or 44, or a complement thereof; or iii. at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 consecutive nucleotides of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, or a complement thereof.
92. The method of any one of claims 81 and 83-91, i. wherein the first gRNA has a first target site comprising a target sequence that is:
1. at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical to at least 10, at least 1 1 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 consecutive nucleotides of SEQ ID NO: 11 or 43, or a complement thereof; or
2. at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 consecutive nucleotides of SEQ ID NO: 12 or 44, or a complement thereof; or
3. at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 consecutive nucleotides of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, or a complement thereof; and ii. wherein the second gRNA has a second target site comprising a target sequence that is:
1. at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 consecutive nucleotides of SEQ ID NO: 11 or 43, or a complement thereof; or
2. at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 consecutive nucleotides of SEQ ID NO: 12 or 44, or a complement thereof; or
3. at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 consecutive nucleotides of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, or a complement thereof.
93. The method of any one of claims 87, 88, 91, or 92, wherein the modified cotton event GH_BCS246002 comprises a deletion or excision of intervening genomic DNA between the first target site and the second target site, relative to the cotton event GH_BCS246002.
94. The method of any one of claims 77-93, further comprising: c. selecting the modified cotton plant comprising the modified cotton event GH_BCS246002; and d. sexually crossing the modified cotton plant with itself or a second cotton plant to produce one or more modified progeny cotton plants.
95. A method of introducing a target site into a cotton plant, the method comprising: a. introducing a cognate target site into the cotton event GH_BCS246002 locus of at least one cell of a cotton plant or cotton plant part comprising the cotton event GH_BCS246002 or an explant thereof via a targeted genome editing technique, wherein the cognate target site is identical or similar to an originator target site for a site-specific nuclease present in the cotton event GH_BCS246002 locus; and b. developing or regenerating a modified cotton plant comprising a modified cotton event GH_BCS246002 comprising the cognate target site.
96. The method of claim 95, further comprising: c. introducing a site-specific nuclease or a recombinant DNA construct comprising an expression cassette encoding a site- specific nuclease into at least one cell of an explant of a cotton plant comprising the modified cotton event GH_BCS246002 or a plant part thereof, to produce a further modified cotton event GH_BCS246002 via a targeted genome editing technique, wherein the target site of the site-specific nuclease includes the cognate target site and the originator target site; and d. developing or regenerating a second modified cotton plant comprising the further modified cotton event GH_BCS246002.
97. A method of introducing a target site into a cotton plant, the method comprising: a. introducing a cognate target site into the cotton event GH_BCS246002 locus of at least one cell of a cotton plant or cotton plant part comprising the cotton event GH_BCS246002 or an explant thereof via a targeted genome editing technique to produce a modified cotton event GH_BCS246002 comprising the cognate target site, wherein the cognate target site is identical or similar to an originator target site for a site-specific nuclease present in the cotton event GH_BCS246002 locus; b. introducing a site-specific nuclease or a recombinant DNA construct comprising an expression cassette encoding a site specific nuclease into at least one cell of an explant of a modified cotton plant comprising the modified cotton event GH_BCS246002, or a plant part thereof, to produce a further modified cotton event GH_BCS246002 via a targeted genome editing technique, wherein the target site of the site- specific nuclease includes the cognate target site and the originator target site; and c. developing or regenerating a second modified cotton plant comprising the further modified cotton event GH_BCS246002.
98. The method of claim 96 or 97, wherein the further modified cotton event GH_BCS246002 of the second modified cotton plant comprises a deletion or excision of intervening genomic DNA between the originator target site and the cognate target site, relative to the cotton event GH_BCS246002 or modified cotton event GH_BCS246002.
99. The method of any one of claims 96-98, further comprising: selecting the second modified cotton plant or a progeny plant of the second modified cotton plant comprising the further modified cotton event GH_BCS246002, and sexually crossing the second modified cotton plant or the progeny plant with itself or another cotton plant to produce one or more modified progeny cotton plants comprising the further modified cotton event GH_BCS246002.
100. A method of producing a progeny cotton plant comprising a modified cotton event GH BCS246002 comprising: a. sexually crossing a first modified cotton plant that comprises a modified cotton event GH_BCS246002 with itself or a second cotton plant; b. collecting one or more seeds produced from said cross; c. growing said seed to produce one or more progeny plants; and d. selecting at least a first progeny plant or seed comprising a modified cotton event GH_BCS246002.
101. A method of determining the zygosity of a cotton plant, cotton plant part or cotton seed comprising cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 comprising: a. contacting a sample comprising cotton DNA with a primer pair that is capable of producing, using a thermal amplification reaction, an amplicon diagnostic for the presence of a coding sequence or expression cassette encoding the Cry IB.3, CrylDa_7, or Vip3Cbl.l protein within the allele corresponding to cotton event GH_BCS246002 DNA or a modified cotton event GH_BCS246002 DNA; b. contacting said sample with a second primer pair that is capable of producing, using a thermal amplification reaction, an amplicon of an internal standard cotton genomic DNA known to be single-copy and homozygous in the cotton plant; c. contacting said sample with a probe set which contains at least a first probe that specifically hybridizes to (or with) the coding sequence or expression cassette encoding the CrylB.3, CrylDa_7, or Vip3Cbl.l protein within the allele DNA of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002, and a second probe that specifically hybridizes to the internal standard cotton genomic DNA known to be single-copy and homozygous in the cotton plant; d. performing a DNA amplification reaction using real-time PCR and determining the cycle thresholds (Ct values) of the amplicon corresponding to the coding sequence or expression cassette encoding the Cry IB.3, CrylDa_7, or Vip3Cbl.l protein within the allele DNA of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 and the single-copy, homozygous internal standard; e. calculating the difference (ACt) between the Ct value of the single-copy, homozygous internal standard amplicon and the Ct value of the amplicon corresponding to the coding sequence or expression cassette encoding the Cry IB.3, CrylDa_7, or Vip3Cbl.l protein within the allele DNA of cotton event GH_BCS246002 sequence amplicon; and f. determining zygosity, wherein a ACt of about zero (0) indicates homozygosity of the inserted T-DNA of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 and a ACt of about one (1) indicates heterozygosity of the inserted T-DNA of cotton event GH_BCS246002.
102. The method of claim 101, wherein contacting the sample comprising cotton DNA with the primer pair is capable of producing an amplicon diagnostic for the presence of an expression cassette or coding sequence encoding a Vip3Cbl.l protein within the allele corresponding to cotton event GH_BCS246002 DNA or a modified cotton event GH_BCS246002 DNA; wherein contacting said sample with the probe set contains at least a first probe that specifically hybridizes to (or with) an expression cassette or coding sequence encoding a Vip3Cbl.l protein within the allele DNA of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002; wherein performing the DNA amplification reaction using real-time PCR and determining the cycle thresholds (Ct values) of the amplicon corresponds to an expression cassette or coding sequence encoding a Vip3Cbl.l protein within the allele DNA of cotton event GH_BCS246002 or a modified cotton event GH_BCS246002 and the single-copy, homozygous internal standard; and wherein calculating the difference (ACt) between the Ct value of the singlecopy, homozygous internal standard amplicon and the Ct value of the amplicon corresponds to the an expression cassette or coding sequence encoding a Vip3Cbl.l protein within the allele DNA of cotton event GH_BCS246002 sequence amplicon.
103. The method of claim 101 or 102, wherein the primer pairs are selected from the group consisting of SEQ ID NO: 245 combined with SEQ ID NO: 246, and SEQ ID NO: 18 combined with SEQ ID NO: 19; and wherein the probes are SEQ ID NO: 247 and SEQ ID NO: 20.
104. The method of claim 101, 102 or 103, wherein the ACt of about one (1) indicating heterozygosity of the inserted T-DNA of cotton event GH_BCS246002 is in the range of 0.75 to 1.25.
PCT/US2025/028295 2024-05-10 2025-05-08 Cotton transgenic event gh_bcs246002 and methods for detection and uses thereof Pending WO2025235697A1 (en)

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