WO2017214074A1 - Événement d'élite de maïs mzhg0jg - Google Patents

Événement d'élite de maïs mzhg0jg Download PDF

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WO2017214074A1
WO2017214074A1 PCT/US2017/036035 US2017036035W WO2017214074A1 WO 2017214074 A1 WO2017214074 A1 WO 2017214074A1 US 2017036035 W US2017036035 W US 2017036035W WO 2017214074 A1 WO2017214074 A1 WO 2017214074A1
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corn
seq
mzhgojg
event
plant
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PCT/US2017/036035
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Volker Mittendorf
Weining Gu
Shawn BEANBLOSSOM
Andrew NOE
Ryan William Carlin
Yaping JIANG
Wenjin Yu
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Syngenta Participations Ag
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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
    • C12N15/8274—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 herbicide resistance
    • C12N15/8277—Phosphinotricin
    • 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
    • C12N15/8274—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 herbicide resistance
    • C12N15/8275—Glyphosate
    • 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
    • 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
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the invention relates generally to the field of plant molecular biology, plant transformation, and plant breeding. More specifically, the invention relates to herbicide tolerant transgenic corn plants comprising a novel transgenic genotype and to methods of detecting the presence of the corn plant DNA in a sample and compositions thereof.
  • Herbicide tolerant transgenic plants are widely commercially available. Over 90% of
  • transgenic plants commercially grown maize and soybean in the United States are herbicide tolerant transgenic plants. Widespread usage of a single herbicide tolerant trait can result in breakdown of the effectiveness of the herbicide. To address this, transgenic plants can be created which carry herbicide tolerance against multiple classes of herbicide.
  • Glutamine synthetase constitutes in most plants one of the essential enzymes for the development and life of plant cells. It is known that GS converts glutamate into glutamine. GS is involved in an efficient pathway in most plants for the detoxification of ammonia released by nitrate reduction, amino acid degradation or photorespiration. Therefore potent inhibitors of GS are very toxic to plant cells and can be used as broad-spectrum herbicides.
  • a class of herbicides, which include phosphinothricin and glufosinate, are GS inhibitors.
  • Transgenic plants have been made tolerant to this class of herbicides through the introduction of a gene encoding a phosphinothricin acetyltransferase (PAT).
  • PA T phosphinothricin acetyltransferase
  • the PA T gene is derived from Streptomyces viridochromogenes and confers tolerance to glufosinate. (U.S. Patent Nos. 5,531,236, 5,646,024, 5,648,477, and 5,276,268).
  • a second broad-spectrum herbicide is N-phosphonomethyl-glycine, commonly referred to as glyphosate.
  • Glyphosate inhibits the shikimic acid pathway, which is responsible for the biosynthesis of aromatic compounds including amino acids and vitamins.
  • glyphosate inhibits the conversion of phosphoenolpyruvic acid and 3-phosphoshikimic acid to 5-enolpyruvyl-3- phosphoshikimic acid by inhibiting the enzyme 5-enolpyruvyl-3-phosphoshikimic acid synthase (EPSP synthase or EPSPS).
  • EBP synthase enzyme 5-enolpyruvyl-3-phosphoshikimic acid synthase
  • Glyphosate tolerant plants can be produced by transgenically introducing an EPSPS which is tolerant to glyphosate.
  • Glyphosate is a foliar applied, post-emergence herbicide. It is phloem-mobile, and is known to localize in the meristems and in young, actively growing tissues, including roots, leaves, and male reproductive tissues (Hetherington et al., 1999, J of Experimental Botany, 50: 1567-1576). In transgenic plants expressing an EPSPS gene to confer glyphosate tolerance, there can be issues in recovering fully male -fertile transgenic events at commercial rates of glyphosate application (Heck et al, 2005,Crop Science, 44: 329-339; Green, 2009, Weed Science, 57: 108-117). The creation of a commercially viable transgenic event which possesses an acceptable level of herbicide tolerance without compromising male fertility is unpredictable and requires substantial testing and
  • Transgenic plants which carry multiple herbicide tolerance traits would provide the grower with more options to manage weeds and crop cycles.
  • a strategy to provide growers with such plants is to "stack" herbicide tolerance traits, so that multiple traits are in a single germplasm.
  • transgenic traits are frequently stacked through breeding and subsequent screening to get multiple transgenic traits into a single commercial germplasm. These breeding and screening steps are required for every variety of germplasm into which introduction of these two traits is desirable. For many agronomically important crops, such as corn, these two traits need to be maintained as hybrids for dozens of germplasm varieties.
  • factors such as the genetic linkage of undesirable traits or genetic recombination may complicate the introduction of two traits from two distinct loci into a single germplasm variety.
  • transgenic event which comprises a single transgene that carries multiple herbicide tolerance traits at a single locus in the genome of the transgenic plant.
  • the expression of foreign genes in plants can be influenced by their chromosomal position, for example due to chromatin structure and/or the proximity of transcriptional regulation elements close to the integration site (See for example, Weising et al , 1988, "Foreign Genes in Plants," Ann. Rev. Genet. 22:421-477).
  • a high-quality transgenic event is preferred to not be in a promoter or gene region of the genome.
  • a high-quality transgenic event also must not have negative effects on the agronomic performance of the transgenic plant.
  • a high-quality transgenic event is the result of a single, intact, transgene insertion, with little or no transgene rearrangement, and without contamination by extraneous heterologous DNA, such as DNA from the backbone of a vector used during the transformation process.
  • a high-quality transgenic event also is preferred to lack introduced ORFs, which potentially may be expressed in the transgenic plant.
  • the identified event which satisfies all criteria required for a high-quality event which may be used for commercial purposes is considered an elite event.
  • the elite event is characterized by its exact genomic location, as it is that location which is responsible for the molecular qualities, transgene expression levels, and agronomic performance of the event.
  • This elite event is useful for introgressing its transgene into other genetic backgrounds by sexual outcrossing using conventional breeding methods. Progeny of such crosses maintain the transgene expression characteristics of the original transformant.
  • This strategy may be used to generate an infinite number of hybrids and varieties comprising the elite event, and used to ensure reliable transgene expression in each variety and hybrid.
  • a method for detecting a particular elite event would be helpful for complying with regulations requiring the pre-market approval and labeling of foods derived from recombinant crop plants, for example. It is possible to detect the presence of a transgene by any well- known nucleic acid detection method, including but not limited to thermal amplification (polymerase chain reaction (PCR)) using polynucleotide primers or DNA hybridization using nucleic acid probes.
  • PCR polymerase chain reaction
  • these detection methods generally focus on frequently used genetic elements, for example, promoters, terminators, and marker genes, because for many DNA constructs, the coding sequence region is interchangeable. As a result, such methods may not be useful for discriminating between constructs that differ only with reference to the coding sequence. In addition, such methods are not useful for discriminating between different events, particularly those produced using the same DNA construct.
  • sequence of genomic DNA adjacent to the inserted heterologous DNA, the "flanking DNA” needs to be known. In particular, the junction between the flanking genomic sequence and the inserted transgene needs to be known.
  • the present invention includes herbicide tolerant transgenic elite event MZHGOJG corn, which has incorporated into its genome a transgene comprising a PAT gene (U.S. Patent Nos.
  • Elite event MZHGOJG corn also comprises the elite event MZHGOJG junction sequences in its genome.
  • the invention further includes novel isolated nucleic acid sequences, the junction sequences, which are unique to elite event MZHGOJG and are useful for identifying the transgenic corn comprising elite event MZHGOJG and for detecting nucleic acids from transgenic elite event MZHGOJG corn in a biological sample.
  • the present invention also includes kits comprising the reagents necessary for use in detecting these nucleic acids in a biological sample.
  • the invention is drawn to an elite event, designated MZHGOJG, comprising a novel
  • transgenic genotype that comprises a PAT gene and a ZmEPSPS gene, which confer herbicide tolerance to glutamine synthetase (GS) inhibitor herbicides and to glyphosate herbicide to the MZHGOJG corn elite event and progeny thereof.
  • the invention also provides transgenic MZHGOJG corn plants comprising the genotype of the invention, seed, cells, and tissues from transgenic corn plants comprising the genotype of the invention, and methods for producing a transgenic MZHGOJG corn plant comprising the genotype of the invention by crossing a corn inbred comprising the MZHGOJG genotype of the invention with itself or another corn line of a different genotype.
  • the transgenic MZHGOJG corn plants of the invention may have essentially all of the morphological and physiological characteristics of the corresponding isogenic non-transgenic corn plant in addition to those conferred upon the corn plant by the novel MZHGOJG genotype of the invention.
  • the invention also provides compositions and methods for detecting the presence of nucleic acids from elite event MZHGOJG based on the DNA sequence of the recombinant expression cassettes inserted into the corn genome that resulted in the elite event MZHGOJG, and of genomic sequences flanking the insertion site.
  • the elite event MZHGOJG can be further characterized by analyzing gene expression levels or protein levels of PAT and ZmEPSPS, as well as by testing efficacy against GS inhibitor herbicides, such as glufosinate or bialaphos, and glyphosate.
  • the invention provides an optionally isolated nucleic acid molecule comprising at least 10 contiguous nucleotides of a heterologous DNA sequence inserted into the corn plant genome of the elite event MZHGOJG initial corn transformant and at least 10 contiguous nucleotides of a corn plant genome DNA flanking the point of insertion of a heterologous DNA sequence inserted into the corn plant genome of the elite event MZHGOJG initial corn transformant.
  • the optionally isolated nucleic acid molecule according to this aspect may comprise at least 20 or at least 50 contiguous nucleotides of a heterologous DNA sequence inserted into the corn plant genome of the elite event MZHGOJG initial corn transformant and at least 20 or at least 50 contiguous nucleotides of a corn plant genome DNA flanking the point of insertion of a heterologous DNA sequence inserted into the corn plant genome of the elite event MZHGOJG initial corn transformant.
  • the invention provides a optionally isolated nucleic acid
  • a junction sequence spans the junction between the heterologous DNA comprising the expression cassettes inserted into the corn genome and DNA from the corn genome flanking the insertion site and is diagnostic for the elite event MZHGOJG.
  • the invention provides an optionally isolated nucleic acid
  • heterologous DNA molecule to the corn plant genome in corn elite event MZHGOJG comprising a sequence of from about 11 to about 20 contiguous nucleotides selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and complements thereof.
  • the invention provides an optionally isolated nucleic acid
  • SEQ ID NO: 1 SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and complements thereof.
  • an amplicon comprising a nucleic acid
  • flanking sequence primers for detecting elite event MZHGOJG are provided.
  • Such flanking sequence primers comprise a preferably isolated nucleic acid sequence comprising at least 10-15 contiguous nucleotides from nucleotides 1-471 as set forth in SEQ ID NO: 8 (designated herein as the 5' flanking sequence), or the complements thereof.
  • the flanking sequence primers are selected from the group consisting of SEQ ID NO: 25, 56, 57, 60, and complements thereof.
  • flanking sequences primers comprise a preferably isolated nucleic acid sequence comprising at least 10-15 contiguous nucleotides from nucleotides 1- 519 as set forth in SEQ ID NO: 9 (designated herein as the 3' flanking sequence), or the complements thereof.
  • the flanking sequence primers are selected from the group consisting of SEQ ID NO: 11, 28, 29, 58, 61, and complements thereof.
  • primer pairs that are useful for nucleic acid amplification, for example, are provided.
  • Such primer pairs comprise a first primer comprising a nucleotide sequence of at least 10-15 or 15-20 contiguous nucleotides in length which is or is complementary to one of the above -described genomic flanking sequences (SEQ ID NO: 8 or SEQ ID NO: 9) and a second primer comprising a nucleotide sequence of at least 10-15 or 15-20 contiguous nucleotides of heterologous DNA inserted into the elite event MZHG0JG genome.
  • the second primer preferably comprises a nucleotide sequence which is or is complementary to the insert sequence adjacent to the plant genomic flanking DNA sequence as set forth in SEQ ID NO: 7.
  • the insert sequence primers are selected from the group consisting of SEQ ID NO: 10, 12, 17, 18, 19, 20, 21, 22, 23, 24, 27, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, and complements thereof.
  • methods of detecting the presence of DNA corresponding to corn elite event MZHG0JG in a biological sample comprise: (a) contacting the sample comprising DNA with a pair of primers that, when used in a nucleic acid amplification reaction with genomic DNA from corn elite event MZHG0JG; produces an amplicon that is diagnostic for corn elite event MZHG0JG; (b) performing a nucleic acid
  • 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, and complements thereof.
  • the invention provides methods of detecting the presence of a DNA corresponding to the elite event MZHG0JG in a biological sample.
  • Such methods comprise: (a) contacting the sample comprising DNA with a probe that hybridizes under high stringency conditions with genomic DNA from corn elite event MZHG0JG and does not hybridize under high stringency conditions with DNA of a control corn plant; (b) subjecting the sample and probe to high stringency hybridization conditions; and (c) detecting hybridization of the probe to the DNA.
  • the detected hybridized DNA sequence includes at least one ploynucleotide sequence comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and complements thereof.
  • a kit for the detection of elite event MZHG0JG nucleic acids in a biological sample.
  • the kit includes at least one DNA sequence comprising a sufficient length of polynucleotides which is or is complementary to 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, wherein the DNA sequences are useful as primers or probes that hybridize to isolated DNA from elite event MZHGOJG, and which, upon amplification of or hybridization to a nucleic acid sequence in a sample followed by detection of the amplicon or hybridization to the target sequence, are diagnostic for the presence of nucleic acid sequences from corn elite event MZHGOJG in the sample.
  • the kit further includes other materials necessary to enable nucleic acid hybridization or amplification methods.
  • the invention provides a method of detecting protein expressed from corn elite event MZHGOJG in a biological sample comprising: (a) extracting protein from a sample of corn elite event MZHGOJG tissue; (b) assaying the extracted protein using an immunological method comprising antibody specific for the PAT or ZmEPSPS protein produced by the corn elite event MZHGOJG event; and (c) detecting the binding of said antibody to the PAT or ZmEPSPS protein.
  • the invention provides a biological sample derived from an elite event MZHGOJG corn plant, tissue, or seed, wherein the sample comprises a nucleic acid comprising a nucleotide sequence which is or is complementary to a sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2, and wherein the sequence is detectable in the sample using a nucleic acid amplification or nucleic acid hybridization method.
  • the sample is selected from the group consisting of corn flour, corn meal, corn syrup, corn oil, cornstarch, and cereals manufactured in whole or in part to contain corn by-products.
  • the invention provides an extract derived from an elite event MZHGOJG corn plant, tissue, or seed comprising a nucleotide sequence which is or is complementary to a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2.
  • the sequence is detectable in the extract using a nucleic acid amplification or nucleic acid hybridization method.
  • the sample is selected from the group consisting of corn flour, corn meal, corn syrup, corn oil, cornstarch, and cereals manufactured in whole or in part to contain corn by-products.
  • corn plants and seeds comprising the nucleic acid molecules of the invention are provided.
  • a deposit of elite event MZHGOJG corn seed was made to the American Type Culture Collection (ATCC) in accordance with the Budapest Treaty on 11 February 2016. An example of said seed being deposited as ATCC Accession No: PTA-122835.
  • the invention provides a method for producing a corn plant with herbicide tolerance to GS inhibitor herbicides and to glyphosate comprising: (a) sexually crossing a first parent corn plant with a second parent corn plant, wherein first or second parent corn plant comprises corn elite event MZHGOJG DNA, thereby producing a plurality of first generation progeny plants; (b) selecting a first generation progeny plant that has herbicide tolerance to GS inhibitor herbicides and to glyphosate; (c) selfing the first generation progeny plant, thereby producing a plurality of second generation progeny plants; (d) selecting from the second generation progeny plants, a plant that has herbicide tolerance to GS inhibitor herbicides and to glyphosate; wherein the second generation progeny plants comprise a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4.
  • the invention provides a method for producing corn seed comprising crossing a first parent corn plant with a second parent corn plant and harvesting the resultant first generation corn seed, wherein the first or second parent corn plant is an inbred corn plant of the invention.
  • the invention provides a method of producing hybrid corn seeds comprising the steps of: (a) planting seeds of a first inbred corn line according to the invention and seeds of a second inbred corn line having a different genotype; (b) cultivating corn plants resulting from said planting until time of flowering; (c) emasculating flowers of corn plants of one of the corn inbred lines; (d) allowing pollination of the other inbred line to occur, and (e) harvesting the hybrid seed produced thereby.
  • the invention provides a method of selecting corn plants and seeds comprising the nucleic acid molecules of elite event MZHG0JG on chromosome 9.
  • polymorphic markers are used to select or track the sequences specific to the elite event MZHG0JG.
  • the invention provides a method of selecting sequences specific to the elite event MZHG0JG comprising the steps of: (a) detecting a polymorphic marker sequence; (b) designing an assay for the purposes of detecting the marker; (c) running the assay on corn nucleic acid sequences from many corn lines, and (d) selecting corn lines based upon the sequences with nucleotides specific to elite event MZHG0JG.
  • the invention provides a method of determining the zygosity of a corn plant comprising elite event MZHG0JG comprising (a) obtaining a DNA sample of genomic DNA from said corn plant; (b) producing a contacted sample by contacting said DNA sample with (i) a first event primer and a second event primer, wherein said first event primer specifically binds said transgene construct, said second event primer specifically binds said 5' corn genomic flanking DNA or said 3' com genomic flanking DNA, and wherein said first event primer and said second event primer produce an event amplicon which is unique to event MZHG0JG, when subjected to quantitative PCR conditions, (ii) a native insertion site first primer and a native insertion site second primer that produce an amplicon from the native MZHG0JG insertion site when elite event MZHG0JG is not present in the genome, when subjected to quantitative PCR conditions, (iii) a fluorescent event probe that hybridizes with
  • the event primer set and probe and native insertion site primer set and probe may be mixed with the same DNA sample, or they may be separate with different DNA samples derived from the same corn plant.
  • the quantification of the fluorescence from the event probe and the fluorescence from the native insertion site probe may be sequentially or simultaneously. Zygosity determination may be made using data analysis software, such as SDS software on the ABI 7900HT, as described in Example 10 and shown in Fig. 2.
  • the invention provides a site on chromosome 9 for targeted integration of a heterologous nucleic acid.
  • the invention provides a method of selecting sequences specific to the elite event MZHGOJG for targeted integration comprising the steps of: (a) designing homologous sequences based on the insertion site or transgene sequence; (b) using these homologous sequences at a target locus; (c) using a targeted endonuclease, such as a zinc finger nuclease, a meganuclease, a TALEN, or a Cas9 nuclease, to create a break in the target locus, and (d) inserting a heterologous donor molecule within nucleotides specific to elite event MZHGOJG.
  • a targeted endonuclease such as a zinc finger nuclease, a meganuclease, a TALEN, or a Cas9 nuclease
  • SEQ ID NO: 1 is a nucleic acid sequence of the 5' genome-insert junction, unique to event MZHGOJG.
  • SEQ ID NO: 2 is a nucleic acid sequence of the 3' genome-insert junction, unique to event MZHGOJG
  • SEQ ID NO: 3 is a nucleic acid sequence of the 5' genome-insert junction, sequence, plus 5' flanking genomic sequence.
  • SEQ ID NO: 4 is a nucleic acid sequence of the 3' genome-insert junction, sequence, plus additional 3' flanking genomic sequence.
  • SEQ ID NO: 5 is a nucleic acid sequence of the transgene insertion plus the 5' and 3' junction sequences, unique to event MZHGOJG.
  • SEQ ID NO: 6 is a nucleic acid sequence of the transgene insertion plus the 5' and 3' junction sequences, unique to event MZHGOJG, plus additional 5' and 3' flanking genomic sequence.
  • SEQ ID NO: 7 is a nucleic acid sequence of the full-length transgene insertion of event MZHGOJG.
  • SEQ ID NO: 8 is a nucleic acid sequence of the 5' flanking genomic sequence of the MZHGOJG transgene insertion
  • SEQ ID NO: 9 is a nucleic acid sequence of the 3' flanking genomic sequence of the MZHGOJG transgene insertion
  • SEQ ID NO: 10-12 are primer and probe sequences useful for detection of the MZHGOJG event
  • SEQ ID NO: 13 is a nucleic acid sequence of an amplicon produced using primers represented by SEQ ID NO: 10-11.
  • SEQ ID NO: 14-16 are probe sequences useful for hybridization in Southern blot detection of MZHGOJG
  • SEQ ID NO: 17-42 are primer sequences for the elite event MZHGOJG detection and transgene sequencing.
  • SEQ ID NO: 43 is a nucleic acid sequence of the eFMV-05 enhancer of the ZmEPSPS expression cassette within the MZHGOJG transgene.
  • SEQ ID NO: 44 is a nucleic acid sequence of the e35S-05 enhancer of the ZmEPSPS expression cassette within the MZHGOJG transgene.
  • SEQ ID NO: 45 is a nucleic acid sequence of the prZmUbil58-02 promoter of the ZmEPSPS expression cassette within the MZHGOJG transgene.
  • SEQ ID NO: 46 is a nucleic acid sequence of the eTMV-03 translational enhancer of the ZmEPSPS expression cassette within the MZHGOJG transgene.
  • SEQ ID NO: 47 is a nucleic acid sequence of the cZmEPSPSct-02 coding sequence, which encodes for a EPSPS protein that confers tolerance to glyphosate, of the ZmEPSPS expression cassette within the MZHGOJG transgene.
  • SEQ ID NO: 48 is a nucleic acid sequence of the tZmUbil258-02 terminator of the ZmEPSPS expression cassette within the MZHGOJG transgene.
  • SEQ ID NO: 49 is a nucleic acid sequence of the ZmEPSPS expression cassette within the MZHGOJG transgene.
  • SEQ ID NO: 50 is a nucleic acid sequence of the pr35S-19 promoter of the PAT expression cassette within the MZHGOJG transgene.
  • SEQ ID NO: 51 is a nucleic acid sequence of the pr35S-19 promoter of the PAT expression cassette within the MZHGOJG transgene.
  • SEQ ID NO: 51 is a nucleic acid sequence of the cPAT-09 coding sequence, which encodes for a PAT protein that confers tolerance to GS inhibitor herbicides, of the PAT expression cassette within the MZHGOJG transgene.
  • SEQ ID NO: 52 is a nucleic acid sequence of the tNOS-05-01 terminator of the PAT expression cassette within the MZHGOJG transgene.
  • SEQ ID NO: 53 is a nucleic acid sequence of the PAT expression cassette within the MZHGOJG transgene.
  • SEQ ID NO: 54 is AC204677.4 Chromosome 9 sequence, where N is any base "A”, “T”, “G”or “C”. This sequence includes the chromosomal location of the MZHGOJG insertion site.
  • SEQ ID NO: 55 is the reverse complement of SEQ ID NO: 54, AC204677.4 Chromosome 9 Sequence, where N is any base "A", “T”, “G”or "C”.
  • SEQ ID NO: 56 is a primer sequence useful for the detection of the MZHGOJG event.
  • SEQ ID NO: 57-62 are primer and probe sequences useful for the detection of the MZHGOJG insertion site when MZHGOJG is not present, also referred to as the native insertion site.
  • Figure 1 is a graphical map illustrating the organization of the elements comprising the heterologous nucleic acid sequences inserted into the genome of corn to create elite event MZHGOJG and sets forth the relative positions at which the inserted nucleic acid sequences are linked to corn genomic DNA sequences which flank the ends of the inserted heterologous DNA sequences.
  • Figure 2 is a plot of the intensities of the signals from FAM (Y axis) which is the signal from the Event Specific Allele (ESA), which is the transgene insertion, and TET (X axis) which is the signal from the native insertion allele (WT allele; genomic location of transgene, but no transgene present) assay.
  • ESA Event Specific Allele
  • X axis the signal from the native insertion allele
  • WT allele native insertion allele
  • the points or dots are from the final read of each sample of about 90 samples, after 40 cycles of PCR.
  • Clusters in the upper left quadrant are from homozygous plants (HOM) because no TET (WT allele) signal is present.
  • Clusters in the lower right are from null plants (NULL) because no FAM (ESA) signal is present.
  • heterozygotes are located in between in the upper right, because they have signal from both assays.
  • the term "amplified” means the construction of multiple copies of a nucleic acid molecule or multiple copies complementary to the nucleic acid molecule using at least one of the nucleic acid molecules as a template.
  • Amplification systems include the polymerase chain reaction (PCR) system, ligase chain reaction (LCR) system, nucleic acid sequence based amplification (NASBA, Cangene, Mississauga, Ontario), Q-Beta Replicase systems, transcription-based amplification system (TAS), and strand displacement amplification (SDA). See, e.g., Diagnostic Molecular Microbiology: Principles and Applications, D. H. Persing et al., Ed., American Society for Microbiology, Washington, D.C. (1993). The product of amplification is termed an amplicon.
  • a “biological sample” is a plant, plant material or products comprising plant material.
  • plant is intended to encompass corn (Zea mays) plant tissues, at any stage of maturity, as well as cells, tissues, organs taken from or derived from any such plant, including without limitation, any seeds, leaves, stems, flowers, roots, single cells, gametes, cell cultures, tissue cultures or protoplasts.
  • Plant material refers to material which is obtained or derived from a plant. Products comprising plant material relate to food, feed or other products which are produced using plant material or can be contaminated by plant material.
  • a biological sample may be crushed, non-viable material.
  • a biological sample may be derived from a commodity product, such as a corn commodity product.
  • Corn also known as maize, is used as human food, livestock feed, and as raw material in industry.
  • the principal products of maize dry milling are grits, meal and flour.
  • the maize wet-milling industry can provide maize starch, maize syrups and dextrose for food use.
  • Maize oil is recovered from maize germ, which is a by-product of both dry- and wet- milling industries.
  • a corn commodity product is typically derived from the grain, from the ear of the corn. Corn commodity products may also be derived from non-grain parts of the corn plant.
  • Corn commodity products include corn flour, corn meal, corn syrup, corn oil, corn starch, and cereals manufactured in whole or in part to contain corn by-products. Corn commodity products may be crushed, non-viable material derived from corn seeds but which are no longer capable of germination. Corn, including both grain and non-grain portions of the plant, is also used extensively as livestock feed, primarily for beef cattle, dairy cattle, hogs, and poultry. Industrial uses of maize, include production of ethanol, maize starch in the wet-milling industry and maize flour in the dry-milling industry.
  • maize starch and flour are based on functional properties, such as viscosity, film formation, adhesive properties and ability to suspend particles.
  • the maize starch and flour have application in the paper and textile industries.
  • Other industrial uses include applications in adhesives, building materials, foundry binders, laundry starches, explosives, oil-well muds, and other mining applications.
  • Plant parts other than the grain of maize are also used in industry: for example, stalks and husks are made into paper and wallboard and cobs are used for fuel and to make charcoal.
  • a biological extract or "extract” may be derived from a biological sample or from a corn
  • a biological extract is from crushed biological material, and is no longer viable or capable of germination. It is understood that, in the context of the invention, such biological samples or extracts are tested for the presence of nucleic acids specific to corn elite event MZHGOJG, implying the presence of nucleic acids in the samples.
  • the methods referred to herein for identifying elite event MZHGOJG in biological samples or extracts relate to the identification in biological samples or extracts of nucleic acids which are from an elite event MZHGOJG corn plant and are diagnostic for elite event MZHGOJG.
  • a "coding sequence” is a nucleic acid sequence that is transcribed into RNA such as mRNA, rRNA, tRNA, snRNA, sense RNA or antisense RNA. Preferably the RNA is then translated in an organism to produce a protein.
  • Detection kit refers to a kit used to detect the presence or absence of DNA from corn elite event MZHGOJG plants in a sample comprising nucleic acid probes and primers of the invention, which hybridize specifically under high stringency conditions to a target DNA sequence, and other materials necessary to enable nucleic acid hybridization or amplification methods.
  • “Expression cassette” as used herein means a nucleic acid molecule capable of directing expression of a particular nucleotide sequence in an appropriate host cell, comprising a promoter operably linked to the nucleotide sequence of interest which is operably linked to termination signals. It also typically comprises sequences required for proper translation of the nucleotide sequence.
  • the expression cassette may also comprise sequences not necessary in the direct expression of a nucleotide sequence of interest but which are present due to convenient restriction sites for removal of the cassette from an expression vector.
  • the expression cassette comprising the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components.
  • the expression cassette may also be one that is naturally occurring but has been obtained in a recombinant form useful for heterologous expression. Typically, however, the expression cassette is heterologous with respect to the host, i.e., the particular nucleic acid sequence of the expression cassette does not occur naturally in the host cell and must have been introduced into the host cell or an ancestor of the host cell by a transformation process known in the art.
  • the expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or of an inducible promoter that initiates transcription only when the host cell is exposed to some particular external stimulus. In the case of a multicellular organism, such as a plant, the promoter can also be specific to a particular tissue, or organ, or stage of development.
  • An expression cassette, or fragment thereof can also be referred to as "inserted sequence" or "insertion sequence” when transformed into a plant.
  • a "gene” is a defined region that is located within a genome and that, besides the
  • aforementioned coding nucleic acid sequence comprises other, primarily regulatory, nucleic acid sequences responsible for the control of the expression, that is to say the transcription and translation, of the coding portion.
  • a gene may also comprise other 5' and 3' untranslated sequences and termination sequences. Further elements that may be present are, for example, introns.
  • Gene of interest refers to any gene which, when transferred to a plant, confers upon the plant a desired characteristic such as antibiotic resistance, virus resistance, insect resistance, disease resistance, or resistance to other pests, herbicide tolerance, improved nutritional value, improved performance in an industrial process or altered reproductive capability.
  • the "gene of interest” may also be one that is transferred to plants for the production of commercially valuable enzymes or metabolites in the plant.
  • MZHG0JG genotype refers to the heterologous genetic material transformed into the genome of a plant as well as the genetic material flanking the inserted sequence.
  • MZHGOJG-specific refers to a nucleotide sequence which is suitable for discriminatively identifying elite event MZHGOJG in plants, plant material, or in products such as, but not limited to, food or feed products (fresh or processed) comprising or derived from plant material.
  • Insert DNA or "insert sequence” refers to the heterologous DNA within the expression cassettes used to transform the plant material.
  • Fusion DNA or “flanking sequence” can exist of either genomic DNA naturally present in an organism such as a plant, or foreign (heterologous) DNA introduced via the transformation process which is extraneous to the original insert DNA molecule, e.g. fragments associated with the transformation event.
  • Flanking sequence as used herein refers to a sequence of at least 10 bp, at least 20 bp, at least 50 bp, at least 100 bp, at least 150 bp, at least 200 bp, at least 250 bp, at least 300 bp, at least 400 bp, at least 500 bp, at least 1000 bp, at least 2000 bp, at least 3000 bp, at least 4000 bp, and at least 5000 bp, which is located either immediately upstream of and contiguous with or immediately downstream of and contiguous with the original foreign insert DNA molecule. Transformation procedures leading to random integration of the foreign DNA will result in transformants containing different flanking regions characteristic of and likely unique to each transformant.
  • Transformants When recombinant DNA is introduced into a plant through traditional crossing, its flanking sequences will generally not be changed. Transformants will also contain unique junctions between a piece of heterologous insert DNA and genomic DNA, or two pieces of genomic DNA, or two pieces of heterologous DNA.
  • a "junction” is a point where two specific DNA fragments join. For example, a junction exists where insert DNA joins flanking DNA. A junction point also exists in a transformed organism where two DNA fragments join together in a manner that is modified from that found in the native organism.
  • “Junction DNA” or “junction sequence” refers to DNA that comprises a junction point.
  • junction sequences set forth in this disclosure are the junction point between the maize genomic DNA and the 5' end of the insert as set forth in SEQ ID NO: 1, and the junction point between the 3' end of the insert and maize genomic DNA as set forth in SEQ ID NO: 2.
  • An event may be defined by its junction sequences. These junction sequences can be transmitted to progeny and introgressed into other germplasms via traditional crossing.
  • vent refers to the original transformant that includes the heterologous DNA, and/or progeny of said event. More generally, the term “event” refers to an artificial genetic locus or genotype that, as a result of genetic engineering such as transformation, carries a foreign inserted DNA or transgene comprising at least one copy of at least one gene of interest and also comprises flanking genomic sequence immediately adjacent to the inserted DNA that would be expected to be transferred to a progeny that receives the inserted DNA, including the transgene of interest, as a result of a sexual cross of one parental line which comprises 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 term “event” refers to the original transformant that includes the heterologous DNA, and/or progeny of said event. More generally, the term “event” refers to an artificial genetic locus or genotype that, as a result of genetic engineering such as transformation, carries a foreign inserted DNA
  • the event comprises the junction sequences and the foreign inserted DNA, which may be referred to as the transgene or the T-DNA insertion.
  • the presence of an event in a cell may be identified genotypically by its junction sequences.
  • an event is part of the genetic makeup of a plant.
  • vent MZHGOJG plant refers to a corn plant that comprises the MZHGOJG event.
  • An event MZHGOJG plant may refer to progeny of the original transformant.
  • event MZHGOJG plant may refer to progeny of the original transformant.
  • MZHGOJG plant also refers to progeny produced by a sexual outcross between an event MZHGOJG plant and another corn line. Even after repeated backcrossing to a recurrent parent, the inserted DNA, genomic flanking DNA, and junction sequences from the originally transformed plant are present in the progeny of the cross at the same chromosomal location.
  • an "MZHGOJG seed” refers to a seed which comprises the MZHGOJG event.
  • An "elite event” comprises all of the desirable characteristics of an event required for commercial utility.
  • An elite event comprises one and only one complete copy of the transgene, with an absence of vector backbone sequence.
  • the transgene is inserted at a desirable location in the genome, which, among other characteristics, allows easy introgression into desired commercial genetic backgrounds.
  • the genomic location of the transgene of an elite event also allows for proper expression of the traits comprising the transgene.
  • the expression of the traits of the transgene in an elite event is correct, appropriate, and stable spatially and temporally, both in heterozygous (or hemizygous) and homozygous conditions; is at a commercially acceptable level for a range of environmental conditions in which the plants carrying the event are likely to be exposed in normal agronomic use; and is stable through multiple generations of progeny.
  • the transgene of the elite event also shows normal Mendelian segregation.
  • An elite event has desirable agronomic characteristics, such as yield, vigor, fertility, and the like, which are not negatively impacted by the presence of the event in the genome of the plant.
  • An elite event has a superior combination of efficacy, including herbicide tolerance and agronomic performance in broad genotype backgrounds and across multiple environmental locations. The status of an event as an elite event is confirmed by introgression of the elite event in different relevant genetic backgrounds and observing compliance with the criteria described above.
  • An "elite event” may refer to a genetic locus comprising a foreign DNA as a transgene, which meets the above- described criteria.
  • a plant, plant material or progeny such as seeds can comprise one or more elite events in its genome. The likelihood of having all of these characteristics in an event is small, such that an elite event is non-obvious and atypical of events recovered from the transformation process. An elite event may only be found by an extensive selection procedure.
  • corn elite event refers to an elite event of corn, for example corn elite event MZHGOJG.
  • Plants harboring elite event MZHGOJG are characterized by their glyphosate tolerance, as well as by their tolerance to GS inhibitors such as glufosinate or bialaphos. Plants harboring elite event MZHGOJG are also characterized by having agronomical characteristics that are comparable to commercially available varieties of corn, in the absence of herbicide application. Thus, plants comprising elite event MZHGOJG can tolerate the application of glyphosate and glufosinate, either simultaneously or separately, without negatively affecting the yield of said plants compared to isogenic lines lacking event MZHGOJG.
  • corn plants comprising elite event MZHGOJG have no statistically significant difference in their disease susceptibility, or lodging compared to isogenic corn plants without the MZHGOJG event. These characteristics make the elite event MZHGOJG very useful for control of glyphosate-resistant weeds in corn fields, and can also be used in approaches to prevent or delay further glyphosate resistance development in corn fields (e.g., by application of glyphosate and glufosinate).
  • a "heterologous" nucleic acid sequence is a nucleic acid sequence not naturally associated with a host cell into which it is introduced, including non-naturally occurring multiple copies of a naturally occurring nucleic acid sequence.
  • a "homologous" nucleic acid sequence is a nucleic acid sequence naturally associated with a host cell into which it is introduced.
  • isolated when used in relation to a nucleic acid refers to a nucleic acid sequence that is identified and separated from at least one contaminant nucleic acid with which it is ordinarily associated in its natural source. An isolated nucleic acid is present in a form or setting that is different from that in which it is found in nature. In contrast, a non-isolated nucleic acids such as DNA and RNA found in the state they exist in nature. An isolated nucleic acid may be in a transgenic plant or biological sample and still be considered “isolated”.
  • operably-linked refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one affects the function of the other.
  • a promoter is operably-linked with a coding sequence or functional RNA when it is capable of affecting the expression of that coding sequence or functional RNA (i.e., that the coding sequence or functional RNA is under the transcriptional control of the promoter). Coding sequences in sense or antisense orientation can be operably-linked to regulatory sequences.
  • the tools developed to identify an elite event or the plant or plant material comprising an elite event, or products which comprise plant material comprising the elite event are based on the specific genomic characteristics of the elite event, such as, a specific restriction map of the genomic region comprising the inserted DNA, molecular markers, or the sequence of the flanking region(s) of the inserted DNA.
  • primers and probes can be developed which specifically recognize this (these) sequence(s) in the nucleic acid (DNA or RNA) of a sample by way of a molecular biological technique.
  • a PCR method can be developed to identify the elite event in biological samples (such as samples of plants, plant material or products comprising plant material).
  • biological samples such as samples of plants, plant material or products comprising plant material.
  • Such a PCR is based on at least two specific "primers", one recognizing a sequence within the 5' or 3' flanking region of the elite event and the other recognizing a sequence within the foreign DNA.
  • the primers preferably have a sequence of between 15 and 35 nucleotides which under optimized PCR conditions "specifically recognize” a sequence within the 5' or 3' flanking region of the elite event and the foreign DNA of the elite event respectively, so that a specific fragment (“integration fragment” or discriminating amplicon) is amplified from a nucleic acid sample comprising the elite event. This means that only the targeted integration fragment, and no other sequence in the plant genome or foreign DNA, is amplified under optimized PCR conditions.
  • Primer pairs or sets can be used for amplification of a nucleic acid molecule, for example, by the polymerase chain reaction (PCR) or other conventional nucleic-acid amplification methods.
  • PCR polymerase chain reaction
  • PCR primers suitable for identification of elite corn event MGHZ0JG may be the following:
  • oligonucleotides ranging in length from 17 bp to about 200 bp, comprising a nucleotide
  • oligonucleotides ranging in length from 17 bp to about 200 bp, comprising a nucleotide
  • the primers may of course be longer than the mentioned 17 consecutive nucleotides, and may, e.g., be 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 50, 75, 100, 150, 200 bp long or even longer.
  • the primers may entirely consist of nucleotide sequence selected from the mentioned nucleotide sequences of flanking sequences and transgene DNA sequences. However, the nucleotide sequence of the primers at their 5' end (i.e. outside of the 3'-located 17 consecutive nucleotides) is less critical.
  • the 5' sequence of the primers may comprise or consist of a nucleotide sequence selected from the flanking sequences or foreign DNA, as appropriate, but may contain several (e.g., 1,2, 5, or 10) mismatches.
  • the 5' sequence of the primers may even entirely be a nucleotide sequence unrelated to the flanking sequences or foreign DNA, such as, e.g., a nucleotide sequence representing one or more restriction enzyme recognition sites.
  • Such unrelated sequences or flanking DNA sequences with mismatches should preferably be not longer than 100, more preferably not longer than 50 or 25 nucleotides.
  • suitable primers may comprise, consist or consist essentially of a nucleotide sequence spanning the junction region between the plant DNA derived sequences and the inserted DNA sequences (SEQ ID NO: 1 and 2). It will also be immediately clear to the skilled artisan that properly selected PCR primer pairs should also not comprise sequences complementary to each other.
  • Examples of suitable primers for detection or identification of elite event MZHG0JG include SEQ ID NOs: 10 through 12 and SEQ ID NOs: 17 through 58, and SEQ ID NO: 60-61, and complements thereof.
  • Other examples of suitable oligonucleotide primers for the detection or identification of elite event MZHG0JG comprise at least 10 contiguous nucleotides of SEQ ID NO: 6, 8, and 9, and complements thereof.
  • the resulting amplicon must comprise SEQ ID NO: 1 or SEQ ID NO: 2.
  • a "probe” is an isolated nucleic acid to which is attached a conventional detectable label or reporter molecule, such as a radioactive isotope, ligand, chemiluminescent agent, or enzyme.
  • a conventional detectable label or reporter molecule such as a radioactive isotope, ligand, chemiluminescent agent, or enzyme.
  • Such a probe is complimentary to a strand of a target nucleic acid, in the case of the invention, to a strand of genomic DNA from corn elite event, MZHG0JG.
  • the genomic DNA of elite event MZHG0JG can be from a corn plant or from a sample that includes DNA from the event.
  • Probes according to the invention 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.
  • Probes and primers are generally 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117
  • Primers and probes according to the invention may have complete sequence complementarity with the target sequence, although probes differing from the target sequence and which retain the ability to hybridize to target sequences may be designed by conventional methods.
  • Stringent conditions or “stringent hybridization conditions” include reference to conditions under which a probe will hybridize to its target sequence, to a detectably greater degree than to other sequences. Stringent conditions are target-sequence-dependent and will differ depending on the structure of the polynucleotide. By controlling the stringency of the hybridization and/or wash conditions, target sequences can be identified which are 100% complementary to the probe
  • Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution.
  • high stringency hybridization and wash conditions are selected to be about 5°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
  • T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • a probe will hybridize to its target subsequence, but to no other sequences.
  • An example of high stringency hybridization conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on a filter in a Southern or northern blot is 50% formamide with 1 mg of heparin at 42°C, with the hybridization being carried out overnight.
  • An example of very high stringency wash conditions is 0.15M NaCl at 72°C for about 15 minutes.
  • An example of high stringency wash conditions is a 0.2x SSC wash at 65°C for 15 minutes (see, Sambrook, infra, for a description of SSC buffer).
  • Exemplary hybridization conditions for the invention include hybridization in 7% SDS, 0.25 M NaP0 4 pH 7.2 at 67°C overnight, followed by two washings in 5% SDS, 0.20 M NaP0 4 pH7.2 at 65°C for 30 minutes each wash, and two washings in 1% SDS, 0.20 M NaP0 4 pH7.2 at 65°C for 30 minutes each wash.
  • An exemplary medium stringency wash for a duplex of, e.g., more than 100 nucleotides, is lx SSC at 45°C for 15 minutes.
  • An exemplary low stringency wash for a duplex of, e.g., more than 100 nucleotides is 4-6x SSC at 40°C for 15 minutes.
  • high stringency conditions typically involve salt concentrations of less than about 1.0 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is typically at least about 30°C.
  • High stringency conditions can also be achieved with the addition of destabilizing agents such as formamide.
  • a signal to noise ratio of 2x (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization.
  • Nucleic acids that do not hybridize to each other under high stringency conditions are still substantially identical if the proteins that they encode are substantially identical. This occurs, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.
  • a reference nucleotide sequence preferably hybridizes to the reference nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaP0 4 , 1 mM EDTA at 50°C with washing in 2X SSC, 0.1% SDS at 50°C, more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaP0 4 , 1 mM EDTA at 50°C with washing in IX SSC, 0.1% SDS at 50°C, more desirably still in 7% sodium dodecyl sulfate (SDS), 0.5 M NaP0 4 , 1 mM EDTA at 50°C with washing in 0.5X SSC, 0.1% SDS at 50°C, preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaP0 4 ,
  • amplified DNA refers to the product of nucleic acid amplification of a target nucleic acid sequence that is part of a nucleic acid template.
  • DNA extracted from the com plant tissue sample may be subjected to a nucleic acid amplification method using a DNA primer pair that includes a first primer derived from flanking sequence adjacent to the insertion site of inserted heterologous DNA, and a second primer derived from the inserted heterologous DNA to produce an amplicon that is diagnostic for the presence of the event DNA.
  • the second primer may be derived from the flanking sequence.
  • the amplicon is of a length and has a sequence that is also diagnostic for the event.
  • the amplicon may range in length from the combined length of the primer pairs plus one nucleotide base pair to any length of amplicon producible by a DNA amplification protocol, and/or the combined length of the primer pairs plus about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91
  • primer pairs can be derived from flanking sequence on both sides of the inserted DNA so as to produce an amplicon that includes the entire insert nucleotide sequence of the transgene as well as the sequence flanking the transgenic insert.
  • a member of a primer pair derived from the flanking sequence may be located a distance from the inserted DNA sequence, this distance can range from one nucleotide base pair up to the limits of the amplification reaction, or about 20,000 bp.
  • the use of the term "amplicon" specifically excludes primer dimers.
  • Taqman® PE Applied Biosystems, Foster City, Calif.
  • a FRET oligonucleotide probe is designed which overlaps the flanking and insert DNA junction.
  • the FRET probe and PCR primers are cycled in the presence of a thermostable polymerase and dNTPs. Flybridization 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 flanking/transgene insert sequence due to successful amplification and hybridization.
  • Transformation is a process for introducing heterologous nucleic acid into a host cell or
  • transformation means the stable integration of a DNA molecule into the genome of an organism of interest.
  • Transformed refers to a host organism such as a bacterium or a plant into which a heterologous nucleic acid molecule has been introduced.
  • the nucleic acid molecule can be stably integrated into the genome of the host or the nucleic acid molecule can also be as an extrachromosomal molecule. Such an extrachromosomal molecule can be auto-replicating.
  • Transformed cells, tissues, or plants are understood to encompass not only the end product of a transformation process, but also transgenic progeny thereof.
  • a “non-transformed”, “non-transgenic”, or “non- recombinant” host refers to a wild-type organism, e.g., a bacterium or plant, which does not contain the heterologous nucleic acid molecule.
  • transgenic refers to a plant, plant cell, or multitude of structured or unstructured plant cells having integrated, via well known techniques of genetic manipulation and gene insertion, a nucleic acid representing a gene of interest into the plant genome, and typically into a chromosome of a cell nucleus, mitochondria or other organelle containing chromosomes, at a locus different to, or in a number of copies greater than, that normally present in the native plant or plant cell.
  • Transgenic plants result from the manipulation and insertion of such nucleic acid sequences, as opposed to naturally occurring mutations, to produce a non-naturally occurring plant or a plant with a non-naturally occurring genotype.
  • Techniques for transformation of plants and plant cells are well known in the art and may comprise for example electroporation, microinjection, Agrobacterium-mediated transformation, and ballistic transformation.
  • This invention relates to corn plants comprising elite event MZHGOJG, which produce the PAT and ZmEPSPS proteins to confer herbicide tolerance to the GS inhibitor herbicides and to glyphosate.
  • the invention is particularly drawn to the novel genotype of elite event MZHGOJG, which describes a unique genomic insertion of a transgene into a corn plant genome, as well as to compositions and methods for detecting nucleic acids of this event in a biological sample.
  • the invention is further drawn to corn plants comprising the elite event MZHGOJG genotype, to transgenic seed from the corn plants, and to methods for producing a corn plant comprising the elite event MZHGOJG genotype by crossing a corn inbred comprising the elite event MZHGOJG genotype with itself or another corn line.
  • Corn plants comprising the elite event MZHGOJG of the invention are useful as part of a weed control program because they possess herbicide tolerance to GS inhibitor herbicides in addition to glyphosate.
  • the present invention embodies a novel genotype of corn, which is a result of the random insertion of a transgene in the genome of a corn plant by Agrobacterium-mediates transformation. It is recognized in the art that the genomic location of such an insertion cannot be predicted.
  • the present invention encompasses the particular novel genotype and novel sequences, namely 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, and complements thereof, which are novel and diagnostic for elite event MZHGOJG.
  • the present invention discloses a corn plant comprising elite event MZHGOJG, an example of which is deposited as ATCC Accession No: PTA-122835.
  • the present invention encompasses specific tools in the form of specific polynucleotide molecules that are capable of identifying the transgene insertion at its specific insertion site in the corn genome.
  • the invention encompasses a transgenic corn seed of an elite event
  • a transgenic seed, a transgenic plant, transgenic cell, and transgenic tissue of elite event MZHGOJG comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or complements thereof. These sequences define a point of insertion of a heterologous DNA sequence inserted into the corn plant genome of the corn elite event MZHGOJG original transformant.
  • the invention further comprises a transgenic corn plant, transgenic seed, transgenic cell, and transgenic tissue comprising elite event MZHGOJG and capable of producing an elite event
  • MZHGOJG diagnostic amplicon wherein said diagnostic amplicon hybridizes under stringent conditions to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.
  • the invention further encompasses a corn plant comprising the genotype of corn elite event MZHGOJG deposited as ATCC Accession No. PTA-122835, wherein said genotype comprises SEQ ID NOs: 1 through 6.
  • the invention further comprises a transgenic herbicide tolerant plant, cell, seed, tissue, or progeny thereof , comprising: a DNA construct comprising: a first and a second expression cassette, wherein said first expression cassette in operable linkage comprises (i) a FMV enhancer represented by SEQ ID NO: 43; (ii) a CaMV 35S enhancer represented by SEQ ID NO: 44; (iii) a maize ZmUbil58 promoter represented by SEQ ID NO: 45; (iv) a TMV enhancer represented by SEQ ID NO: 46; (v) a glyphosate tolerant EPSPS encoding DNA molecule represented by SEQ ID NO: 47; and (vi) a maize ZmUbil58 transcriptional terminator DNA molecule represented by SEQ ID NO: 48; and wherein said second expression cassette in operable linkage comprises (a) a CaMV 35S promoter represented by SEQ ID NO: 50; (b) a PAT encoding DNA molecule represented by SEQ ID NO:
  • the present invention comprises a molecular stack comprising expression cassettes of the ZmEPSPS and PAT coding sequences.
  • the present invention possesses many advantages over a breeding stack which may comprise the same two herbicide tolerance traits.
  • a plant comprising a breeding stack would comprise the two herbicide tolerance traits present as two single events, such that the genome of the plant has two separate transgene insertions.
  • the creation of such a plant technically can be complicated, as two parents each containing at least one copy of an event would need to be crossed, and the progeny would need to be screened for the presence of each trait.
  • the resultant plant would need to be evaluated for agronomic and trait performance, to ensure that the presence of two insertions in the genome had no deleterious effects.
  • the progeny of a plant comprising a breeding stack would need to be screened for the presence of each trait.
  • such a procedure would need to be performed for every corn variety, or germplasm, into which the breeding stack was desired.
  • the present invention solves these problems by having both traits as a molecular stack, so that they are singly inserted into the genome together.
  • the molecular stack also provides greater stability in the expression of the traits in multiple germplasms, as the traits are inserted together at a single genomic locus and therefore have the same position effects regardless of the germplasm into which they have been introduced.
  • the invention encompasses a preferably isolated nucleic acid molecule comprising 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, and complements thereof.
  • the invention encompasses an optionally isolated nucleic acid molecule, wherein the nucleic acid molecule is comprised in a corn seed deposited as ATCC Accession No. PTA-122835.
  • the invention comprises an isolated nucleic acid molecule comprising: a) SEQ ID NO: 1; b) operably linked at the 3' end of said sequence of step (a) an expression cassette comprising SEQ ID NO: 49 (prZmUbil58- ZmEPSPS- -tZmUbil58); c) operably linked at the 3' end of said cassette of step (b) and in the same orientation, an expression cassette comprising SEQ ID NO: 53 (pr35S-cPAT-tNOS); and d) operably linked at the 3' end of said cassette of step (c) SEQ ID NO: 2.
  • the invention encompasses a nucleic acid molecule relating to the corn elite event MZHGOJG, characterized in that it consists of the nucleic acid sequence of SEQ ID NO: 1 and/or SEQ ID NO: 2.
  • the invention encompasses a nucleic acid molecule, optionally isolated, comprising at least 10 or more (for example 15, 20, 25, or 50) contiguous nucleotides of a heterologous DNA sequence inserted into the corn plant genome of corn elite event MZHGOJG and at least 10 or more (for example 15, 20, 25, or 50) contiguous nucleotides of a corn plant genome DNA flanking the point of insertion of a heterologous DNA sequence inserted into the corn plant genome of corn elite event MZHGOJG.
  • nucleotide sequences that comprise 10 or more nucleotides of contiguous insert sequence from elite event MZHGOJG and at lease one nucleotide of flanking DNA from elite event MZHGOJG adjacent to the insert sequence.
  • Such nucleotide sequences are diagnostic for elite event MZHGOJG.
  • Nucleic acid amplification of genomic DNA from the elite event MZHGOJG may produce an amplicon of the invention, which comprises such diagnostic nucleotide sequences (namely, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and the complements thereof).
  • the invention encompasses a nucleic acid molecule, preferably
  • nucleotide sequence which comprises at least one junction sequence of elite event MZHGOJG selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and complements thereof, wherein a junction sequence spans the junction between a heterologous expression cassette inserted into the corn genome and DNA from the corn genome flanking the insertion site and is diagnostic for the event.
  • the invention comprises a nucleic acid molecule relating to the corn elite event MZHGOJG, characterized in that it consists of the sequence of SEQ ID NO: 1.
  • the invention comprises a nucleic acid molecule relating to the corn elite event MZHGOJG, characterized in that it consists of the sequence of SEQ ID NO: 2. These nucleic acid molecules of the invention may be amplicons.
  • the invention comprises the use of SEQ ID NO: 1 or SEQ ID NO: 2 to identify corn elite event MZHGOJG in a plant. The invention also embodies the use of SEQ ID NO: 1 and/or SEQ ID NO: 2 to identify elite event MZHGOJG.
  • the invention encompasses a preferably isolated nucleic acid linking a heterologous DNA molecule to the corn plant genome in corn elite event MZHGOJG comprising a sequence of from about 11 to about 20 contiguous nucleotides selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and the complements thereof.
  • the invention encompasses a nucleic acid molecule, preferably
  • nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and complements thereof.
  • flanking sequence primers for detecting elite event MZHGOJG.
  • flanking sequence primers comprise an isolated nucleic acid sequence comprising at least 10-15 contiguous nucleotides from SEQ ID NO: 8 (designated herein as the 5' flanking sequence), SEQ ID NO: 9 (designated herein as the 3' flanking sequence) or the complements thereof.
  • the flanking sequence primers are selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 61, and complements thereof.
  • flanking sequences can be extended to include additional chromosome 9 sequence, with specific emphasis on nucleotide comprised within SEQ ID NO: 54 or SEQ ID NO: 55, useful in detecting sequences associated with the corn elite event MZHGOJG.
  • an "N” is defined as any base "A”, “T”, “G”, or "C”.
  • SEQ ID NO: 55 is the reverse complement of SEQ ID NO: 54.
  • the invention encompasses a pair of polynucleotide primers comprising a first polynucleotide primer and a second polynucleotide primer which function together in the presence of a corn elite event MZHGOJG DNA template in a sample to produce an amplicon diagnostic for the corn elite event MZHGOJG.
  • the first primer sequence is or is complementary to a corn plant genomic sequence flanking the point of insertion of a heterologous DNA sequence inserted into the corn plant genome of corn elite event MZHGOJG
  • the second polynucleotide primer sequence is or is complementary to the heterologous DNA sequence inserted into the corn plant genome of the corn elite event MZHGOJG.
  • Another embodiment of the invention is the use of these polynucleotide primers to identify corn elite event MZHGOJG in a plant.
  • the first polynucleotide primer comprises at least 10 contiguous nucleotides from position 1-481 of SEQ ID NO: 8, or at least 10 contiguous nucleotides from position 1-529 of SEQ ID NO: 9, or complements thereof.
  • the first polynucleotide primer comprises the nucleotide sequence set forth in SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 60, or complements thereof.
  • the second polynucleotide primer comprises at least 10 contiguous nucleotides of SEQ ID NO: 7, or the complements thereof.
  • the second polynucleotide primer comprises the nucleotide sequence set forth in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 17 through SEQ ID NO: 42, or the complements thereof.
  • the first polynucleotide primer comprises the nucleotide sequence of SEQ ID NO: 25, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 60
  • the second polynucleotide primer comprises the nucleotide sequence of SEQ ID NO: 17-20, 26, 30-32, or the complement thereof, such that the pair function together in the presence of a corn elite event MZHGOJG DNA template in a sample to produce an amplicon diagnostic for the corn elite event MZHGOJG.
  • the first polynucleotide primer comprises the nucleotide sequence of SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 58, or SEQ ID NO: 61
  • the second polynucleotide primer comprises the nucleotide sequence of SEQ ID NO: 10, 12, 21-24, 27, 38, 39, 40-42, or the complement thereof, such that the pair function together in the presence of a corn elite event MZHGOJG DNA template in a sample to produce an amplicon diagnostic for the corn elite event MZHGOJG.
  • Another embodiment of the invention is the use of these polynucleotide primers to identify corn elite event MZHGOJG in a plant.
  • the first polynucleotide primer comprises SEQ ID NO:
  • the second polynucleotide primer comprises SEQ ID NO: 11, and the pair function together in the presence of a corn elite event MZHGOJG DNA template in a sample to produce an amplicon diagnostic for the corn event MZHGOJG which can be detected by a probe comprising SEQ ID NO: 12, as described in Example 2.
  • a primer sequence corresponding to or complementary to a part of the insert sequence should prime the transcriptional extension of a nascent strand of DNA or RNA toward the nearest flanking sequence junction. Consequently, a primer sequence corresponding to or complementary to a part of the genomic flanking sequence should prime the transcriptional extension of a nascent strand of DNA or RNA toward the nearest flanking sequence junction.
  • a primer sequence can be, or can be complementary to, a heterologous DNA sequence inserted into the chromosome of the plant, or a genomic flanking sequence.
  • primer sequence would need to be, or would need to be complementary to, the sequence as set forth within the inserted heterologous DNA sequence or as set forth in SEQ ID NO: 1 or SEQ ID NO: 2 depending upon the nature of the product desired to be obtained through the use of the nested set of primers intended for use in amplifying a particular flanking sequence containing the junction between the genomic DNA sequence and the inserted heterologous DNA sequence.
  • primers for a multitude of native corn genes for the purposes of designing a positive control.
  • One such example is the corn Adhl gene, where examples of suitable primers for producing an amplicon by nucleic acid amplification are well known in the art (see, for example, U.S. Patent No. 8,466,346, incorporated by reference herein).
  • the invention encompasses a method of detecting the presence of a nucleic acid molecule that is unique to event MZHGOJG in a sample comprising corn nucleic acids, the method comprising: a) obtaining a DNA sample of genomic DNA from said corn plant; b) combining the nucleic acid molecule with a pair of polynucleotide primers of the invention; c) performing a nucleic acid amplification reaction which results in an amplicon diagnostic for the corn elite event MZHGOJG; and d) detecting the amplicon.
  • the invention encompasses a method of confirming the absence of a nucleic acid molecule that is unique to event MZHGOJG in a sample comprising corn nucleic acids, the method comprising: a) obtaining a DNA sample of genomic DNA from said corn plant; b) combining the nucleic acid molecule with a pair of polynucleotide primers of the invention and with a pair of polynucleotide primers to a corn native gene, for example to the corn Adhl gene, as a positive control; c) performing a nucleic acid amplification reaction which results in no amplicon specific to elite event MZHGOJG and results in an amplicon specific to the corn native gene positive control; and d) detecting an amplicon specific to the corn native gene positive control.
  • the invention encompasses the amplicon produced by the method described above.
  • the invention encompasses a method of detecting the presence of a
  • nucleic acid molecule that is unique to event MZHGOJG in a sample comprising corn nucleic acids, for example a biological sample
  • the method comprising: a) isolating a nucleic acid molecule from corn; b) combining the nucleic acid molecule with a pair of polynucleotide primers of the invention and with a polynucleotide probe comprising a nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 10 through 12, SEQ ID NO: 17 through SEQ ID NO: 42, or a complement thereof; c) performing a nucleic acid amplification reaction which results in an amplicon which can be detected by the probe; and d) detecting the probe.
  • the invention encompasses a DNA molecule comprising the amplicon produced by the methods of the invention.
  • 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, and complements thereof.
  • the invention encompasses a method of detecting the presence of DNA corresponding to the corn elite event MZHGOJG in a biological sample, wherein the method comprises: (a) contacting the sample comprising DNA with a probe that hybridizes under high stringency conditions with genomic DNA from corn elite event MZHGOJG and does not hybridize under high stringency conditions with DNA of a control corn plant; (b) subjecting the sample and probe to high stringency hybridization conditions; and (c) detecting hybridization of the probe to the DNA.
  • the probe 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, and complements thereof.
  • the invention encompasses a method of detecting the presence of a DNA corresponding to the corn elite event MZHGOJG event in a biological sample, wherein the method comprises: (a) contacting the sample comprising DNA with a probe that hybridizes under high stringency conditions with genomic DNA from corn event corn elite event MZHGOJG and does not hybridize under high stringency conditions with DNA of a control corn plant; (b) subjecting the sample and probe to high stringency hybridization conditions; and (c) detecting hybridization of the probe to the DNA. Detection can be by any means well known in the art including but not limited to fluorescent, chemiluminescent, radiological, immunological, or otherwise.
  • hybridization is intended to be used as a means for amplification of a particular sequence to produce an amplicon which is diagnostic for the corn elite event MZHGOJG corn event
  • the production and detection by any means well known in the art of the amplicon is intended to be indicative of the intended hybridization to the target sequence where one probe or primer is utilized, or sequences where two or more probes or primers are utilized.
  • the term "biological sample” is intended to comprise a sample that contains or is suspected of containing a nucleic acid comprising from between five and ten nucleotides either side of the point at which one or the other of the two terminal ends of the inserted heterologous DNA sequence contacts the genomic DNA sequence within the
  • junction sequences comprises as little as two nucleotides: those being the first nucleotide within the flanking genomic DNA adjacent to and covalently linked to the first nucleotide within the inserted heterologous DNA sequence.
  • the invention encompasses a kit for detecting the presence of corn elite event MZHGOJG nucleic acids in a biological sample, wherein the kit comprises at least one nucleic acid molecule of sufficient length of contiguous nucleotides homologous or
  • telomere sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, that functions as a DNA primer or probe specific for corn elite event MZHGOJG, and other materials necessary to enable nucleic acid hybridization or amplification.
  • detection methods can be used including TAQMAN (Perkin Elmer), thermal amplification, ligase chain reaction, southern hybridization, ELISA methods, and colorimetric and fluorescent detection methods.
  • kits for detecting the presence of the target sequence i.e., at least one of the junctions of the insert DNA with the genomic DNA of the corn plant in corn elite event MZHGOJG, in a sample containing genomic nucleic acid from event corn elite event MZHGOJG.
  • the kit is comprised of at least one
  • the detecting means can be fluorescent, chemiluminescent, colorimetric, or isotopic and can be coupled at least with immunological methods for detecting the binding.
  • a kit is also envisioned which can detect the presence of the target site in a sample, i.e., at least one of the junctions of the insert DNA with the genomic DNA of the corn plant in corn elite event MZHGOJG, taking advantage of two or more polynucleotide sequences which together are capable of binding to nucleotide sequences adjacent to or within about 100 base pairs, or within about 200 base pairs, or within about 500 base pairs or within about 1000 base pairs of the target sequence and which can be extended toward each other to form an amplicon which contains at least the target site
  • the invention encompasses a method for detecting corn elite event MZHG0JG protein in a biological sample, the method comprising: (a) extracting protein from a sample of corn elite event MZHG0JG tissue; (b) assaying the extracted protein using an
  • immunological method comprising antibody specific for the ZmEPSPS and/or PAT protein produced by the corn elite event MZHG0JG event; and (c) detecting the binding of said antibody to the ZmEPSPS and/or PAT protein.
  • Another embodiment of the invention encompasses a corn plant, or parts thereof, comprising the genotype of the transgenic corn elite event MZHG0JG, wherein the genotype comprises the nucleotide sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or the complements thereof.
  • the corn plant is from the inbred corn lines CG5NA58, CG5NA58A, CG3ND97, CG5NA01, CG5NF22, CG4NU15, CG00685, CG00526, CG00716, NP904, NP948, NP934, NP982, NP991, NP993, NP2010, NP2013, NP2015, NP2017, NP2029, NP2031, NP2034, NP2045, NP2052, NP2138, NP2151, NP2166, NP2161, NP2171, NP2174, NP2208, NP2213, NP2222, NP2275, NP2276, NP2316, BCTT609, AF031, H8431, 894, BUTT201, R327H, 2044BT, and 2070BT.
  • corn elite event MZHG0JG genotype can be introgressed into any plant variety that can be bred with corn, including wild maize species, and thus the preferred inbred lines of this embodiment are not meant to be limiting.
  • the invention encompasses a corn plant comprising at least a first and a second DNA sequence linked together to form a contiguous nucleotide sequence, wherein the first DNA sequence is within a junction sequence and comprises at least about 10-15 contiguous nucleotides selected from the group consisting of nucleotides SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 11, and complements thereof, wherein the second DNA sequence is within the heterologous insert DNA sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 17 through SEQ ID NO: 42, and complements thereof; and wherein the first and the second DNA sequences are useful as nucleotide primers or probes for detecting the presence of corn elite event MZHG0JG nucleic acid sequences in a biological sample.
  • the nucleotide primers are used in a DNA amplification method to amplify a target DNA sequence from template DNA extracted from the corn plant and the corn plant is identifiable from other corn plants by the production of an amplicon corresponding to a DNA sequence comprising SEQ ID NO: 1 or SEQ ID NO: 2.
  • the invention provides a corn plant, wherein the event corn elite event MZHGOJG genotype confers upon the corn plant tolerance to the herbicides glyphosate and to GS inhibitor herbicides.
  • the genotype conferring glyphosate tolerance upon the corn plant comprises a ZmEPSPS gene.
  • the genotype conferring upon the corn plant tolerance to GS inhibitor herbicides comprises a PAT gene.
  • the invention provides a biological sample derived from an elite event MZHGOJG corn plant, tissue, or seed, wherein the sample comprises a nucleotide sequence which is or is complementary to 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 and SEQ ID NO: 6, and wherein the sequence is detectable in the sample using a nucleic acid amplification or nucleic acid hybridization method.
  • the genetic sequence functions a means of detection.
  • the sample is selected from a corn commodity product, for example and not limited to corn flour, corn meal, corn syrup, corn oil, corn starch, and cereals manufactured in whole or in part to contain corn products.
  • the invention provides an extract derived from an elite event
  • MZHGOJG corn plant, tissue, or seed comprising a nucleotide sequence which is or is complementary to 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, and SEQ ID NO: 6.
  • An example of such seed is deposited at the ATCC under Accession No. PTA-122835.
  • the sequence is detected in the extract using a nucleic acid amplification or nucleic acid hybridization method.
  • the sample is selected from a corn commodity product, such as corn flour, corn meal, corn syrup, corn oil, cornstarch, and cereals manufactured in whole or in part to contain corn products.
  • the invention provides a method of producing a corn commodity product, comprising the steps of: a) obtaining transgenic elite event MZHGOJG corn plant, cells or tissues thereof; and b) producing a corn commodity product from the said transgenic corn plant, cells, or tissue thereof, wherein the commodity product comprises protein concentrate, protein isolate, starch, meal, flour or oil therefrom.
  • the invention provides a corn commodity product comprising a detectable amount of a DNA molecule unique for corn elite event MZHGOJG, wherein said molecule comprises 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.
  • the invention provides a non-living plant material comprising a detectable amount of a nucleic acid molecule comprising 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, wherein the nucleic acid molecule is comprised in a corn seed deposited at the ATCC under the Accession No. PTA-122835.
  • the invention provides a method for determining zygosity of a corn plant comprising corn elite event MZHG0JG, said method comprising: (a) obtaining a DNA sample of genomic DNA from said corn plant; (b) producing a contacted sample by contacting said DNA sample with (i) a first event primer and a second event primer, wherein said first event primer specifically binds to at least a fragment of the transgene of event MZHG0JG, and said second event primer specifically binds to at least a fragment of the 5' corn genomic flanking DNA or 3' com genomic flanking DNA of event MZHG0JG, and wherein said first event primer and said second event primer produce an event amplicon which is unique to event MZHG0JG, when subjected to quantitative PCR conditions, (ii) a native insertion site first primer and a native insertion site second primer that produce an amplicon from the native MZHG0JG insertion site when elite event
  • MZHG0JG is not present in the genome, when subjected to quantitative PCR conditions, (iii) a fluorescent event probe capable of hybridization with said event amplicon, and (iv) a fluorescent native insertion site probe capable of hybridization with said native insertion site amplicon; (c) subjecting said contacted sample to fluorescence -based endpoint quantitative PCR conditions; (d) quantitating said fluorescent event probe that hybridized to said event amplicon and quantitating said fluorescent native insertion site probe that hybridized to said native insertion site amplicon; (e) comparing amounts of hybridized fluorescent event probe to hybridized fluorescent native insertion site probe; and (f) determining zygosity of said corn plant comprising corn elite event MZHG0JG by comparing fluorescence ratios of hybridized fluorescent event probe and hybridized fluorescent native insertion site probe.
  • the event primer set and probe and native insertion site primer set and probe may be mixed with the same DNA sample, or they may be separate with different DNA samples derived from the same corn plant.
  • the quantification of the fluorescence from the event probe and the fluorescence from the native insertion site probe may be sequentially or simultaneously. Zygosity determination may be made using data analysis software, such as SDS software on the ABI 7900HT, as described in Example 10 and shown in Fig. 2.
  • a native insertion site primer set may need to be identified so that an amplicon is produced when elite event MZHGOJG is not present in the genome, when subjected to quantitative PCF conditions.
  • Multiple native insertion site primer sets and probes may be needed to properly determine the zygosity of event MZHGOJG in a variety of corn germplasm.
  • Multiple native insertion site primer sets and/or probes may be included in a single reaction to produce a native insertion site amplicon that hybridizes with a native insertion site probe.
  • event primers which specifically bind to the 5' or 3' flanking sequence of the MZHGOJG may need to be identified for successful zygosity determination in novel corn germplasms, as the 5' and/or 3' flanking sequences may be diverse among a variety of germplams.
  • multiple event primer sets may be included in a single reaction to produce an event amplicon unique to event MZHGOJG.
  • the event and/or native insertion site amplicon may consists of 50-200 residues.
  • the amplicon for the event and for the native insertion site is 50- 150 nucleic acid residues.
  • the first event primer comprises at least 10 contiguous nucleotides from position 1-8900 as set forth in SEQ ID NO: 7, or a complement thereof
  • the second event primer comprises at least 10 contiguous nucleotides from position 1-471 as set forth in SEQ ID NO: 8 or from position 1- 519 as set forth in SEQ ID NO: 9, or a complement thereof.
  • the first event primer is selected from SEQ ID NO: 10, 12, 17 through 24, 26, 27, 30 through 42, or a complement thereof.
  • the second event primer is selected from SEQ ID NO: 11, 25, 28, 29, 56, 57, 58, 60, 61, or a complement thereof.
  • the first native insertion site primer comprises at least 10 contiguous nucleotides from position 1-471 as set forth in SEQ ID NO: 8, or a complement thereof, and the second native insertion site primer comprises at least 10 contiguous nucleotides from position 1-519 as set forth in SEQ ID NO: 9, or a complement thereof.
  • the first native insertion site primer is selected from SEQ ID NO: 25, 56, 57, 60, or a complement thereof.
  • the second native insertion site primer is selected from SEQ ID NO: 11, 28, 29, 58, 61, or a complement thereof.
  • the fluorescent native insertion site probe comprises SEQ ID NO: 59 or SEQ ID NO: 62.
  • the results of the method for determining zygosity described above are read directly in a plate reader.
  • the present invention also encompasses a kit for performing the method of determing zygosity described above.
  • the kit comprises all primers and probes needed for performing the zygosity assay on a DNA sample, including a first event primer, a second event primer, a native insertion site primer, a native insertion site reverse primer, an event probe, and a native insertion site probe.
  • the kit may include more than one primer set/probe for the event, for the native insertion site, or both.
  • the present invention encompasses a method of breeding a corn plant comprising herbicide tolerant corn elite event MZHGOJG wherein the zygosity of a corn plant comprising corn elite event MZHGOJG is determined by the method described above.
  • the zygosity determination method may be used in a breeding program to determine the zygosity of the event MZHGOJG in a segregating progeny population.
  • Corn plants may then be selected which are homozygous for event MZHGOJG based on the results of the zygosity determination method described above. Corn plants may also be discarded if they are found to be heterozygous for event MZHGOJG based on the results of the zygosity determination method.
  • the invention provides a method for producing a corn plant
  • tolerant to herbicides comprising: (a) sexually crossing a first parent corn plant with a second parent corn plant, wherein said first or second parent corn plant comprises corn elite event MZHGOJG, thereby producing a plurality of first generation progeny plants; (b) selecting a first generation progeny plant that has herbicide tolerance to glyphosate and to GS inhibitors; (c) selfing the first generation progeny plant, thereby producing a plurality of second generation progeny plants; and (d) selecting from the second generation progeny plants a plant that has herbicide tolerance to glyphosate and to GS inhibitors; wherein the second generation progeny plants comprise 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, and SEQ ID NO: 6.
  • the invention provides a use of corn elite event MZHGOJG to confer herbicide tolerance to a plant lacking said event.
  • This usage may comprise, for example, sexually crossing a parent corn plant comprising event MZHGOJG with a second parent corn plant which does not comprise event MZHGOJG and selecting for progeny which comprise event
  • the progeny may further be backcrossed to the second parent, optionally multiple times, or crossed with additional corn plants as part of a breeding program to produce at least one variety of corn comprising elite event MZHGOJG which previously did not comprise said event.
  • the invention provides a method of asexually propagating corn elite event MZHGOJG.
  • Asexual propagation of a corn plant may be performed using methods well-known in the art, for example by anther culture or by microspore -derived plant tissue culture, in vitro plant regeneration may be performed by micropropagation, which involves the suppression of apical dominance resulting in the activation and multiplication of auxiliary buds, or by somatic embryogenesis, where for example cotyledon containing embryos are formed from somatic cells .
  • Asexual propagation and in vitro plant regeneration are needed for asexual reproduction.
  • the invention provides a corn elite event MZHGOJG produced by asexual propagation.
  • the invention also provides use of a corn elite event MZGHOJG plant, cells, or tissues to produce a corn elite event MZHGOJG plant. This plant may be produced by asexual propagation.
  • the invention provides a method of producing hybrid corn seeds comprising: (a) planting seeds of a first inbred corn line, an example of said seed deposited as ATCC accession No. PTA-122835, wherein said seed 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, and SEQ ID NO: 6, and seeds of a second inbred line having a different genotype than the first inbred corn plant; (b) cultivating corn plants resulting from said planting until time of flowering and the production of flowers; (c) emasculating said flowers of plants of either the first or the second corn inbred line; (d) sexually crossing the two different inbred lines with each other by pollinating the non- emasculated plant with pollen of the emasculated plant; and (e) allowing hybrid seed to be produced and harvesting the hybrid seed produced thereby.
  • a first inbred corn line an example of
  • the invention provides a method of selecting markers associated with corn elite event MZHGOJG comprising: (a) screening corn elite event MZHGOJG chromosome 9 sequences, (b) comparing these with a non-transgenic NP2222 sequences, (c) comparing the sequences for the purpose of detecting sequence variations, (d) using these sequence variations as a means to develop markers associated with corn elite event MZHGOJG, (e) using the markers to screen lines, and (f) detecting marker confirming the presence of corn elite event MZHGOJG sequences on chromosome 9.
  • the invention provides a method of breeding a corn plant comprising herbicide tolerant elite event MZHGOJG which is genetically linked to or a complement of a nucleic acid marker, wherein said marker is identified using SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 10 through SEQ ID NO: 12, SEQ ID NO: 17 through SEQ ID NO: 42, SEQ ID NO: 56 through SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 61, or their complements.
  • the invention provides a method of marker assisted selection for herbicide tolerant corn elite event MZHGOJG comprising: (a) isolating nucleic acid molecule(s), or preparing a nucleic acid sample, from corn; (b) combining the nucleic acid molecule(s) with a pair of polynucleotide primers and probes, selected from the group comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 10 through SEQ ID NO: 12, SEQ ID NO: 17 through SEQ ID NO: 42, SEQ ID NO: 56 through SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 61, or their complements; (c) performing a nucleic acid amplification reaction which results in an amplicon; (d) detecting the amplicon; and (e) selecting the plant for the purposes of breeding herbicide tolerant corn comprising corn elite event MZHGOJG.
  • the invention comprises a transgenic corn plant, cells, or tissues comprising elite event MZHGOJG, characterized by the 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, wherein the transgenic corn plant, cells, or tissues are further defined as a progeny or derived from a progeny of any generation of a corn plant comprising elite event MZHGOJG.
  • the transgenic corn plant, cells, or tissues are or are derived from a hybrid bred from at least one parent comprising elite event
  • transgenic genotype of the invention can be introgressed by breeding into other corn lines comprising different transgenic genotypes.
  • a corn inbred comprising the transgenic genotype of the invention can be crossed with a corn inbred comprising the transgenic genotype of the lepidopteran resistant Btl 1 event, which is known in the art, thus producing corn seed that comprises both the transgenic genotype of the invention and the Btl 1 transgenic genotype.
  • Examples of other transgenic events which can be crossed with an inbred of the invention include: the Enogen event 3272, the glyphosate
  • tolerant/lepidopteran insect resistant MON802 event the lepidopteran insect resistant event DBT418, the lepidopteran insect resistant event DAS-06275-8, the lepidopteran insect resistant event MIR162, the male sterile event MS3, the lepidopteran insect resistant event MON 80100, the lepidopteran insect resistant event 176, the coleopteran insect resistant event MIR604, the coleopteran insect resistant event 5307, and the coleopteran insect resistant event MON863, all of which are known in the art. It will be further recognized that other combinations can be made with the transgenic genotype of the invention and thus these examples should not be viewed as limiting.
  • the invention encompasses a process for producing corn elite event MZHGOJG seed.
  • This process comprises crossing a corn elite event MZHGOJG of the invention with a second corn plant.
  • the second corn plant may or may not comprise the MZHGOJG event.
  • the second corn plant does not comprise the MZHGOJG event.
  • the invention further comprises the corn elite event MZHGOJG seed produced by the process described above, as well as the corn elite event MZHGOJG plant produced by germinating the seed.
  • the invention provides a process of introducing an additional trait into a corn elite event MZHGOJG plant, comprising: (a) crossing a corn elite event MZHGOJG plant grown from corn elite event MZHGOJG seed, representative seed deposited under ATCC Accession Number PTA-122835, with another maize plant that comprises an additional trait to produce hybrid progeny plants, (b) selecting hybrid progeny plants that have the additional trait to produce selected hybrid progeny plants; (c) crossing the selected progeny plants with the corn elite event MZHGOJG parental plants to produce backcross progeny plants; (d) selecting for backcross progeny plants that have the additional trait to produce selected backcross progeny plants; and (e) repeating steps (c) and (d) at least three or more times to produce backcross progeny plants that comprise the additional trait and corn elite event MZHGOJG.
  • the invention further comprises a plant produced by the process described above.
  • the invention provides a method for developing a corn elite event MZHGOJG plant germplasm in a corn plant breeding program, comprising applying plant breeding techniques wherein said techniques comprise recurrent selection, backcrossing, pedigree breeding, marker enhanced selection, haploid/double haploid production, or transformation of a corn elite event MZHGOJG plant, or its parts, wherein application of said techniques results in development of a second corn germplasm comprising elite event MZHGOJG.
  • the invention provides a method of producing a corn elite event MZHGOJG plant with doubled haploid chromosomes, the method comprising: (a) crossing the plant of claim 2 with an inducer maize plant to produce a progeny with haploid chromosomes; and (b) doubling the haploid chromosomes in the progeny to produce a corn elite event MZHGOJG plant with doubled haploid chromosomes.
  • Manipulations (such as mutation, further transfection, and further breeding) of plants or seeds, or parts thereof, may lead to the creation of what may be termed "essentially derived” varieties.
  • the International Union for the Protection of New Varieties of Plants (UPOV) has provided the following guideline for determining if a variety has been essentially derived from a protected variety:
  • a "line” is a group of plants that display little or no genetic variation between individuals for at least one trait. Such lines may be created by several generations of self-pollination and selection, or vegetative propagation from a single parent using tissue or cell culture techniques.
  • cultivar and “variety” are synonymous and refer to a line which is used for commercial production.
  • Stability or “stable” means that with respect to the given component, the component is maintained from generation to generation and, preferably, at least three generations at substantially the same level, e.g., preferably ⁇ 15%, more preferably ⁇ 10%, most preferably ⁇ 5%.
  • the stability may be affected by temperature, location, stress and the time of planting. Comparison of subsequent generations under field conditions should produce the component in a similar manner.
  • Communication Utility is defined as having good plant vigor and high fertility, such that the crop can be produced by farmers using conventional farming equipment.
  • the transgenic genotype of the invention can be introgressed in any corn inbred or hybrid using art recognized breeding techniques.
  • the goal of plant breeding is to combine in a single variety or hybrid various desirable traits.
  • these traits may include resistance to insects and diseases, tolerance to herbicides, tolerance to heat and drought, reducing the time to crop maturity, greater yield, and better agronomic quality.
  • uniformity of plant characteristics such as germination and stand establishment, growth rate, maturity, and plant and ear height, is important.
  • a plant is self-pollinated if pollen from one flower is transferred to the same or another flower of the same plant.
  • a plant is cross-pollinated if the pollen comes from a flower on a different plant.
  • Plants that have been self-pollinated and selected for type for many generations become homozygous at almost all gene loci and produce a uniform population of true breeding progeny.
  • a cross between two different homozygous lines produces a uniform population of hybrid plants that may be heterozygous for many gene loci.
  • a cross of two plants each heterozygous at a number of gene loci will produce a population of hybrid plants that differ genetically and will not be uniform.
  • Corn can be bred by both self-pollination and cross-pollination techniques. Corn has separate male and female flowers on the same plant, located on the tassel and the ear, respectively. Natural pollination occurs in corn when wind blows pollen from the tassels to the silks that protrude from the tops of the ears. [00140] A reliable method of controlling male fertility in plants offers the opportunity for improved plant breeding. This is especially true for development of corn hybrids, which relies upon some sort of male sterility system. There are several options for controlling male fertility available to breeders, such as: manual or mechanical emasculation (or detasseling), cytoplasmic male sterility, genetic male sterility, gametocides and the like.
  • Hybrid corn seed is typically produced by a male sterility system incorporating manual or mechanical detasseling. Alternate strips of two corn inbreds are planted in a field, and the pollen- bearing tassels are removed from one of the inbreds (female). Providing that there is sufficient isolation from sources of foreign corn pollen, the ears of the detasseled inbred will be fertilized only from the other inbred (male), and the resulting seed is therefore hybrid and will form hybrid plants.
  • Plant breeding techniques known in the art and used in a corn plant breeding program include, but are not limited to, recurrent selection, backcrossing, pedigree breeding, restriction length polymorphism enhanced selection, marker assisted selection and transformation.
  • the development of corn hybrids in a corn plant breeding program requires, in general, the development of homozygous inbred lines, the crossing of these lines, and the evaluation of the crosses.
  • Pedigree breeding and recurrent selection breeding methods are used to develop inbred lines from breeding populations.
  • Corn plant breeding programs combine the genetic backgrounds from two or more inbred lines or various other germplasm sources into breeding pools from which new inbred lines are developed by selfing and selection of desired phenotypes. The new inbreds are crossed with other inbred lines and the hybrids from these crosses are evaluated to determine which of those have commercial potential. Plant breeding and hybrid development, as practiced in a corn plant-breeding program, are expensive and time-consuming processes. [00144] Pedigree breeding starts with the crossing of two genotypes, each of which may have one or more desirable characteristics that is lacking in the other or which complements the other. If the two original parents do not provide all the desired characteristics, other sources can be included in the breeding population.
  • Recurrent selection breeding can be used to improve an inbred line and a hybrid that is made using those inbreds.
  • Backcrossing can be used to transfer a specific desirable trait from one inbred or source to an inbred that lacks that trait. This can be accomplished, for example, by first crossing a superior inbred (recurrent parent) to a donor inbred (non-recurrent parent), that carries the appropriate gene(s) for the trait in question. The progeny of this cross is then mated back to the superior recurrent parent followed by selection in the resultant progeny for the desired trait to be transferred from the non-recurrent parent.
  • the progeny After five or more backcross generations with selection for the desired trait, the progeny will be homozygous for loci controlling the characteristic being transferred, but will be like the superior parent for essentially all other genes. The last backcross generation is then selfed to give pure breeding progeny for the gene(s) being transferred.
  • a hybrid developed from inbreds containing the transferred gene(s) is essentially the same as a hybrid developed from the same inbreds without the transferred gene(s).
  • An inbred plant could also be produced by applying double haploid methods to the progeny of a cross between a corn plant comprising elite event MZHG0JG and a different plant.
  • Double haploid methods produce substantially homozygous plants without repeated backcrossing steps.
  • the haploid/doubled haploid process of developing inbreds starts with the induction of a haploid by using, for example, KWS inducers lines, Krasnador inducers lines, stock six inducer lines (Coe, 1959, Am. Nat. 93:381-382).
  • the haploid cell is then doubled, and the doubled haploid plant is produced.
  • the invention is a method of producing a corn plant with doubled haploid chromosomes derived from a corn elite event MZHG0JG, the method comprising: (a) crossing a plant, wherein said plant comprises elite event MZHG0JG, with an inducer maize plant to produce a progeny with haploid chromosomes; and (b) doubling the haploid chromosomes in the progeny to produce a maize plant with doubled haploid chromosomes.
  • the progeny may be for example a cell, seed, embryo or plant.
  • the maize plant with doubled haploid chromosomes produced by step (b) above is a maize inbred plant with the characteristics of corn elite event MZHG0JG.
  • the plant crossed with an inducer in step (a) is a hybrid maize plant produced by crossing a corn plant comprising elite event MZHGOJG with a different plant.
  • inbred lines derived from elite inbred lines can be developed using the pedigree breeding and recurrent selection breeding methods described earlier. As an example, when backcross breeding is used to create these derived lines in a corn plant-breeding program, elite inbreds can be used as a parental line or starting material or source population and can serve as either the donor or recurrent parent.
  • Fi The hybrid progeny of the first generation is designated Fi.
  • Preferred Fi hybrids are more vigorous than their inbred parents. This hybrid vigor, or heterosis, can be manifested in many polygenic traits, including increased vegetative growth and increased yield.
  • the development of a corn hybrid in a corn plant breeding program involves three steps: (1) the selection of plants from various germplasm pools for initial breeding crosses; (2) the selfing of the selected plants from the breeding crosses for several generations to produce a series of inbred lines, which, although different from each other, breed true and are highly uniform; and (3) crossing the selected inbred lines with different inbred lines to produce the hybrid progeny (Fi).
  • the vigor of the lines decreases. Vigor is restored when two different inbred lines are crossed to produce the hybrid progeny (Fi).
  • An important consequence of the homozygosity and homogeneity of the inbred lines is that the hybrid between a defined pair of inbreds will always be the same.
  • the hybrid seed can be reproduced indefinitely as long as the homogeneity of the inbred parents is maintained. Much of the hybrid vigor exhibited by Fi hybrids is lost in the next generation (F2). Consequently, seed from hybrids is not used for planting stock.
  • Hybrid seed production requires elimination or inactivation of pollen produced by the female parent. Incomplete removal or inactivation of the pollen provides the potential for self-pollination. This inadvertently self -pollinated seed may be unintentionally harvested and packaged with hybrid seed.
  • transgenic corn seed comprising the transgenic genotype of the invention can be treated with various seed-treatment chemicals, including insecticides.
  • the invention comprises a method for protecting a corn elite event MZHGOJG plant against feeding damage by one or more pests, said method comprising (a) providing a MZHGOJG seed of the corn elite event MZHGOJG plant; and (b) treating the MZHGOJG plant with an insecticide.
  • the insecticide may comprise an active ingredient selected from the group consisting of thiamethoxam, lambda-cyhalothrin, and tefluthrin
  • the transgenic corn seed of the invention can be treated with the commercial insecticide Cruiser ® .
  • the present invention also encompasses a corn elite event MZHGOJG seed treated with an insecticide.
  • the invention comprises a corn elite event MZHGOJG seed treated with an insecticide which comprises an active ingredient selected from the group consisting of thiamethoxam, lambda-cyhalothrin, and tefluthrin.
  • the invention encompasses a corn elite event MZHGOJG plant treated with an insecticide.
  • the invention encompasses a corn elite event MZHGOJG plant treated with an insecticide which comprises an active ingredient selected from the group consisting of thiamethoxam, lambda-cyhalothrin, and tefluthrin.
  • the invention provides a method of controlling weeds, where an herbicide is applied to a field comprising corn elite event MZHGOJG plants.
  • the herbicide is glyphosate or a GS inhibitor, such as glufosinate or bialaphos.
  • a second herbicide is applied.
  • the second herbicide is not the same as the first herbicide and is selected from the group consisting of glyphosate and a GS inhibitor, such as glufosinate or bialaphos.
  • the first herbicide and the second herbicide may be applied sequentially or at the same time. They may be applied at the same time because they are mixed together.
  • Examples of commercially available herbicides comprising a GS inhibitor include Herbiace, Meiji Herbiace, Liberty®, Ignite®, Rely®, Finale®, and Basta®.
  • Examples of commercially available herbicides comprising glyphosate include RoundUp®.
  • the invention provides a method of controlling glyphosate-resistant weeds in an area comprising at least corn elite event MZHGOJG plant, wherein said method comprises applying a GS inhibitor herbicide, such as glufosinate or bialaphos, to at least a portion of said area.
  • a GS inhibitor herbicide such as glufosinate or bialaphos
  • the GS inhibitor herbicide may be applied at the same time or sequentially with glyphosate.
  • the herbicides are applied at the same time because they are mixed together, for example the GS inhibitor herbicide may be applied from a tank mix with glyphosate.
  • the glyphosate -resistance weed is a glyphosate-resistant volunteer of a species different than the plant comprising elite event MZHGOJG.
  • the glyphosate-resistant weeds may be unwanted volunteer Brassica ssp, millet, switchgrass, maize, sorghum, wheat, oat, turf grass, pasture grass, papaya, flax, peppers, potato, sunflower, tomato, crucifers, soybean, common bean, lotus, grape, peach, cacao, cotton, rice, soybean, sugarcane, sugar beet, tobacco, barley, cassava, cucumber, watermelon, melon, orange, Clementine, castor bean, or grapevine.
  • the invention provides a method of controlling weeds in an area under cultivation, said area comprising a plurality of corn elite event MZHGOJG plants, said method comprising applying glyphosate and/or a GS inhibitor herbicide, such as glufosinate or bialaphos, over the top of the plants.
  • glyphosate and/or a GS inhibitor herbicide such as glufosinate or bialaphos
  • Another embodiment of the invention is a recombinant sequence, which comprises a maize chromosomal target site located on chromosome 9 between SNP markers rs 128284356 and rsl31707802, wherein the target site comprises a heterologous nucleic acid.
  • the maize chromosomal target site on chromosome 9 is also located between SNP markers rsl31707685, rsl31707686, rsl31707700, and rsl31707756 (all at the 5' flanking sequence) and SNP markers rsl31707806, rsl31177003, rsl31707863, and rsl31707870 (all at the 3' flanking sequence).
  • the heterologous nucleic acid may be introduced at the maize chromosomal target site by targeted insertion.
  • a further embodiment is a recombinant nucleic acid molecule of chromosome 9 comprising a heterologous nucleic acid sequence inserted on chromosome 9 set forth as nucleotide 1 to nucleotide 123,274 of SEQ ID NO: 54.
  • a preferred embodiment is a recombinant nucleic acid molecule comprising a heterologous nucleic acid sequence inserted on chromosome 9 set forth as nucleotide 88,290 to 88,329 of SEQ ID NO: 54.
  • Another embodiment is a recombinant nucleic acid molecule comprising a heterologous nucleic acid proximal at its 5' end to nucleotides 86,290 to 88,290 of SEQ ID NO: 54 and proximal at its 3' end to nucleotides 88,329 to 90,329 of SEQ ID NO: 54.
  • Another embodiment of the invention is a method of making a transgenic maize plant
  • heterologous nucleic acid comprising inserting a heterologous nucleic acid at a position on chromosome 9 between SNP markers rsl28284356 and rsl31707802.
  • the heterologous nucleic acid is inserted at a position on chromosome 9 set forth as nucleotide 1 to nucleotide 123,274 of SEQ ID NO: 54.
  • a preferred embodiment is a method of making a transgenic maize plant, wherein the heterologous nucleic acid is inserted on chromosome 9 set forth as between nucleotide 88,290 to nucleotide 88,329 of SEQ ID NO: 54.
  • a further embodiment is a method of making a transgenic maize plant comprising inserting a heterologous nucleic acid at a position on chromosome 9, wherein the heterologous nucleic acid is proximal at its 5' end to nucleotides 86,290 to 88,290 of SEQ ID NO: 54 and proximal at its 3' end to nucleotides 88,329 to 90,329 of SEQ ID NO: 54.
  • the MZHG0JG event was produced by Agrobacterium-mediated transformation of the inbred corn (Zea mays) line NP2222. Immature embyos were transformed essentially as described in Negrotto et al. (Plant Cell Reports 19: 798-803, 2000), incorporated herein by reference, using a DNA fragment from binary vector 18857 (PCT Application No. PCT/US 16/018432, incorporated by reference herein). Vector 18857 contains a nucleotide sequence comprising PAT and mEPSPS tandem expression cassettes as part of its T-DNA sequence.
  • the first expression cassette comprises a Figwort mosaic virus (FMV) enhancer region (eFMV-05; SEQ ID NO: 43) operably linked to a Cauliflower mosaic virus (CaMV) 35S enhancer region (e35S-05; SEQ ID NO: 44), operably linked to a corn constitutive promoter based on the corn ubiquitin ZmUbil58-3 gene (Christensen et al., 1992, PMB 18: 675-689) (prZmUbil 58-02; SEQ ID NO: 45), operably linked to a Tobacco Mosaic virus (TMV) enhancer (eTMV-03; SEQ ID NO: 46), operably linked to a modified corn mEPSPS coding sequence (cZmEPSPSct-02 (niEPSPS-02); SEQ ID NO: 47), operably linked to a terminator based on the corn ubiquitin ZmUbil58-3 gene (Christensen et al., 1992) (tZmUbil58
  • the second cassette comprises a CaMV 35S promoter (pr35S-19; SEQ ID NO: 50) operably linked to a PAT coding sequence (cPAT-09 (pat-09); SEQ ID NO: 51), operably linked to a terminator sequence from the nopaline synthase (NOS)gene from A. tumefaciens (NOS-05-01 ; SEQ ID NO: 52).
  • the T-DNA sequence comprising these two expression cassettes is SEQ ID NO: 7.
  • Immature embryos were excised from 8 - 12 day old ears and rinsed with fresh medium in preparation for transformation. Embryos were mixed with the suspension of Agrobacterium cells harboring the binary vector 18857, vortexed for 30 seconds, and allowed to incubate for an additional 5 minutes. Excess Agrobacterium solution was aspirated and embryos were then moved to plates containing a non-selective culture medium. Embryos were co-cultured with the remaining
  • the PAT gene was used as a selectable marker during the transformation process (Negrotto et al. 2000).
  • the embryos producing embryogenic calli were transferred to a series of cell culture selection media containing bialaphos as selection agent and cultured for 10-11 weeks in total.
  • the selection media contained 200mg/ml timentin and/or lOml/1 PPM (Plant Preservative Mix) to ensure that the Agrobacterium was cleared from the transformed tissue.
  • Agrobacterium-mediated transformation From these, only 841 events were produced, and of these, 68 were found to have the necessary molecular characteristics to be considered for further agronomic analysis. This represents a 92% attrition rate, and demonstrates the unpredictability of identifying a transgenic event that has all the molecular attributes required to be considered for commercial application.
  • junction ORF is a putative Open Reading Frame that crosses the T-DNA and genomic DNA junction.
  • a junction ORF is initiated by an "ATG" nucleic acid sequence, which may be created as a result of the random insertion of the T-DNA into the genome, where genomic sequence breaks as well as small insertion, deletions, and/or rearrangements of nucleic acid sequence can occur.
  • junction ORF may comprise a sequence which could, if expressed, contain an amino acid sequence similar to amino acid sequences of known allergens. Therefore, junction ORFs are highly undesirable and cannot occur in a high quality genomic insertion, or in an elite transgenic event. Of the five events, one had the transgene insertion proximal to a putative uncharacterized protein. A second event had a right border flanking sequence which is highly repetitive in the maize genome throughout all the chromosomes, so that the exact location of the genomic insertion could not be identified. Of the remaining three events, two had significantly poor agronomic performance in at least two of the hybrid combinations across at least 5 locations, as described above.
  • Fig. 1 illustrates the transgene of MZHG0JG inserted into the corn genome.
  • a real-time, MZHGOJG-specific polymerase chain reaction (PCR) method was developed to detect and quantify MZHG0JG deoxyribonucleic acid (DNA) extracted from seed, grain and other plant material samples.
  • the method consists of a maize-specific PCR method as a reference and an event-specific PCR method for detection and quantification of MZHGOJG maize DNA. This method can be used to determine the relative content of event MZHGOJG maize DNA in proportion to total maize DNA in samples.
  • binding sites for one of the primers and the probe are located in the MZHGOJG insert, and the binding site for the other primer is located in the maize genomic DNA sequence
  • TAQMAN analysis was essentially carried out as described in Ingham et al. (Biotechniques, 31 : 132-140, 2001) herein incorporated by reference. Briefly, genomic DNA was isolated from leaves of transgenic and non-transgenic corn plants using the Puregene® Genomic DNA Extraction kit (Gentra Systems, Minneapolis, MN) essentially according to the manufacturer's instruction, except all steps were conducted in 1.2 ml 96-well plates. The dried DNA pellet was resuspended in TE buffer (10 Mm Tris-HCl, pH 8.0, lmM EDTA).
  • TAQMAN PCR reactions were carried out in 96-well plates.
  • primers and probes were designed specific to the Zea mays alcohol dehydrogenase (Adh) gene (Genbank accession no. AF044295). It will be recognized by the skilled person that other corn genes can be used as endogenous controls.
  • PCR was performed in the ABI Prism 7700 instrument using the following amplification parameters: 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 1 min at 60°C.
  • the PCR may be run on an ABI 7900HT, a GeneAmp PCR system 9700, or any other appropriate system.
  • the data was analyzed using the SDS software on the ABI 7900HT. Results validated the above methodology, verifying it successfully identified the MZHGOJG event in a biological sample derived from corn tissue, including seed, grain, and other plant material samples.
  • NP2222/NP2391 corn All material was grown in a greenhouse.
  • the genomic DNA used for Southern blot analyses was isolated from leaf tissue by a method modified from that described by Murray and Thompson (1980, Nucleic Acids Research, 8: 4321-4325).
  • the number of integration sites within the MZHG0JG corn genome and number of copies of the T-DNA at each location within the MZHG0JG corn genome were determined through the use of three T-DNA-specific probes that together covered every base pair of the vector 18857 T-DNA expected to be transferred and integrated into the corn genome.
  • the templates for the probes were segments of the vector 18857 T-DNA corresponding to (1) the right border sequence to the end of TMV enhancer (SEQ ID NO: 14), (2) the OTP-02 and the mEPSPS-02 coding sequence (SEQ ID NO: 15), and (3) the Ubil58-02 terminator sequence to the left border (SEQ ID NO: 16).
  • the left border and right border are categorized as "border regions" because only a portion of each border was expected to be integrated into the corn genome (Tzfira et al. 2004, Trends Genet. 20: 375-383).
  • Plasmid backbone the region outside of the T-DNA.
  • Southern blot analyses the presence or absence of plasmid backbone was determined through the use of two backbone-specific probes that together covered every base pair of vector 18857 outside of the T- DNA. These elements were not expected to be transferred to the plant cell or integrated into the plant genome during T-DNA transfer.
  • each Southern blot analysis was performed with genomic DNA extracted from MZHG0JG corn and from nontransgenic, near-isogenic corn, which was used as a negative control to identify any endogenous corn DNA sequences that hybridized with the probes.
  • each analysis also included two positive assay controls representing 1 copy and 1/7 copy per genome of a DNA fragment of known size in the corn genome.
  • the positive assay controls were PCR-amplified fragments that corresponded to each of the five probes used in characterization of the MZHG0JG insert.
  • T-DNA-specific probes 2 SEQ ID NO: 15
  • 3 SEQ ID NO: 16
  • backbone-specific probes 1 and 2 which together cover every base pair of vector 18857 outside of the T-DNA
  • nontransgenic, near-isogenic NP2222/NP2391 corn in order to more accurately reflect their migration speeds in the corn genome matrix.
  • the positive assay control for T-DNA-specific probe 1 was analyzed in the absence of nontransgenic corn genomic DNA, so that endogenous bands would not obscure the positive assay control.
  • Corn genomic DNA was analyzed via two restriction enzyme digestion strategies.
  • the genomic DNA was digested with an enzyme that cut within the MZHGOJG insert and in the corn genome flanking the MZHGOJG insert.
  • This first strategy was used twice, with two different enzymes, to determine the numbers of vector 18857 T-DNA inserts within the MZHGOJG corn genome and the presence or absence of extraneous DNA fragments of the insert in other regions of the event MZHGOJG corn genome.
  • the enzymes used were PpuMl, EcoRV, Dralll, Sspl, Eagl, Seal, and Noil.
  • the genomic DNA was digested with restriction enzymes that cut within the insert to release DNA fragments of predictable size.
  • This strategy was used to determine the number of copies of the T-DNA at each location within the event MZHGOJG corn genome, the intactness of the insert, and the presence or absence of any closely linked extraneous T- DNA fragments.
  • the enzymes used were Ascl, Pad, and Kpnl.
  • Genomic DNA samples at about 7.5 ⁇ g/lane, were cut with restriction enzymes and run overnight on an agarose gel in IX TBE buffer at about 32 volts. Gels were photographed, washed, and blotted onto nylon membrane with 10X SSC as the transfer solution. They were linked to the membrane with UV light and pre -hybridized with calf thymus DNA at 65°C. The probes were labeled with radioactive Phosphorus 32. Probes were added and hybridized at 65°C, 3 hrs to overnight. Blots were washed several times and exposed in a phosphorimager cassette. Images were developed and scored.
  • the MZHGOJG maize event is an elite event, comprising a fully-intact, single copy T- DNA, with no extraneous DNA fragments, either from the vector 18857 backbone or from partial secondary T-DNA insertions, were detected.
  • the Southern blot analyses demonstrated that the hybridization bands specific to the MZHGOJG insert were identical in all lanes containing genomic DNA extracted from MZHGOJG corn plants of generation T2 (ear 4), T2 (ear 35), T3, T4, T5, or Fl.
  • MZHGOJG corn plants from the BCiFi generation were backcrossed two more times with the nontransgenic recurrent parent (NP2681) to yield the BC 2 F 1 and BC 3 F 1 generations analyzed in this study.
  • the expected segregation ratio for each gene was 1 : 1 in each generation (i.e., 50% of the plants in each generation were expected to carry the gene).
  • Chi-square analysis of the segregation data was performed to test the hypothesis that the MZHGOJG insert is inherited in a predictable manner according to Mendelian principles and consistent with insertion into a chromosome within the corn nuclear genome.
  • Genome WalkerTM Universal Kit (Clontech, Cat No.638904) was used to recover the genome sequence flanking the transgene insert of the event MZHGOJG. Restriction digestion was completed by combing 8 ⁇ 1 genomic DNA (20 to 100 ng/ ⁇ ), lul blunt restriction enzyme (Pvull, EcoKV or Stul in separate reactions) and ⁇ digestion buffer specific to selected enzyme, followed by incubating at 37°C overnight. Ligation of digested genomic DNA to Genome Walker Adaptor was completed by combining the restriction digestion product with 0.475ul Genome Walker Adaptor, l.lul 10X T4 DNA ligase buffer and 0.125ul T4 DNA ligase, followed by incubating at 16°C overnight. Several insert specific primers (listed below in the table) were designed based on either the right or left TDNA border sequence of vector 18857, and were used along with the adaptor primers provided by the manufacturer in primary PCR and secondary (nested) PCR.
  • the 5' and 3' flanking sequences and junction sequences were confirmed using standard PCR procedures.
  • the 5' flanking and junction sequence was confirmed using a first polynucleotide primer set forth in SEQ ID NO: 25 combined with a second polynucleotide primer of SEQ ID NO: 26 (see table below).
  • the 3' flanking and junction sequence were confirmed using a first polynucleotide primer set forth in SEQ ID NO: 28 through SEQ ID NO: 29 combined with a second polynucleotide primer of SEQ ID NO: 27 (see table below). It will be recognized by the skilled person that other primer sequences can be used to confirm the flanking and junction sequences.
  • flanking sequence and 3' flanking sequence of the event MZHGOJG were used to search maize genome databases. Identical matches to both flanking sequences were found on Chromosome 9 (M AIZE_REF_3_GENOME) , and on a BAC clone, NCBI Accession No.
  • the integration site of the 18857 vector transgene is comprised within SEQ ID NO: 54 and its complement SEQ ID NO: 55.
  • event MZHGOJG The nucleotide sequence of the entire transgene DNA insert present in event MZHGOJG was determined to demonstrate overall integrity of the insert, contiguousness of the functional elements and to detect any individual basepair changes.
  • event MZHGOJG insert was PCR amplified from genomic DNA derived from individual plants as overlapping fragments to cover the entire TDNA insert which is linked to its 5' flanking sequence and 3' flanking sequence on each side.
  • PCR amplification was carried out using high fidelity enzyme (Sigma, Cat. No.D1313) with PCR parameters adjusted for different target regions.
  • PCR was carried out using the following parameters: 30 sec at 96°C for 1 cycle, followed by 35 cycles of 30s at 94°C, 30s at 60°C and 3 min at 68C, followed by 1 cycle of 7 min at 68°C.
  • PCR was carried out using the following parameters: 5 min at 95°C for 1 cycle, followed by 35 cycles of 30s at 94°C, 30s at 59°C and 1 to 7 min at 68°C, followed by 1 cycle of 10 min at 68°C.
  • EXO-SAP master mix was prepared by combining Exonuclease I (USB, Cat No.72073), Shrimp Alkaline Phosphatase (USB, Cat No.70092Z) and EX- SAP buffer (20mM Tris-HCl (pH8.0), lOmM MgC12) at 1 :1 :2. To each PCR product, 1/10 volume of EXO-SAP master mix was added. The reaction was carried out for 30 min at 37°C followed by 20 min at 80°C to inactivate the enzymes.
  • the final consensus sequence was determined by combining the sequence data from different PCR amplicons to generate consensus sequence of the event MZHG0JG insert (SEQ ID NO: 7) linked to its 5' genome sequence and 3' genome sequence at each side (SEQ ID NO: 8 and SEQ ID NO: 9).
  • SEQ ID NO: 6 This final sequence, illustrating the insertion of the MZHG0JG event in the maize genome, is SEQ ID NO: 6.
  • the consensus sequence data for the event MZHG0JG insert demonstrates that the overall integrity of the insert and contiguousness of the functional elements within the insert as intended in pSYN18857 have been maintained.
  • the nucleotide sequence analysis thus demonstrated that the MZHGOJG insert contains a single copy of each of the functional elements (mepsps-02, pat-09, the FMV-05 enhancer, the 35S-05 enhancer, OTP-02, the Ubil58-02 promoter, the TMV-03 enhancer, the Ubil58-02 terminator, the 35S-19 promoter, and the NOS-05-01 terminator).
  • the concentrations of mEPSPS and PAT in various MZHGOJG corn tissues were quantified by enzyme -linked immunosorbent assay (ELISA) to establish an expression profile for these proteins as produced in MZHGOJG corn.
  • the tissues analyzed were leaves and roots at four growth stages (V6, Rl, R6, and senescence), whole plants at three stages (V6, Rl, and R6), kernels at two stages (R6 and senescence), and pollen (stage Rl).
  • the tissues were collected from MZHGOJG corn and a nontransgenic, near-isogenic control corn grown concurrently according to local agronomic practices at four U.S. locations in 2013.
  • the genotypes of the plants used in these studies were NP2391 x NP2222(MZHG0JG) and NP2391 x NP2222.
  • Protein was extracted from representative aliquots of the lyophilized tissue samples. Protein extract samples were prepared from two 1 ⁇ 4 inch maize leaf punches sampled into 96-well blocks, which were then macerated, clarified, and diluted in ELISA diluent (PBS containing 1% BSA, 0.05% Tween-20). The sample extracts were analyzed by ELISA in duplicate or triplicate, and a standard curve was generated for each ELISA plate with known amounts of the corresponding reference protein. Concurrent analysis of tissues from the nontransgenic corn confirmed the absence of plant- matrix effects on the analysis methods. All protein concentrations were adjusted for extraction efficiency.
  • Table 8 shows the ranges of protein concentrations observed in each tissue type across all growth stages and all four locations on a fresh-weight (FW) basis for MZHGOJG corn.
  • Table 8 Ranges of concentrations of mEPSPS and PAT in tissues of MZHGOJG corn No. of Fresh-weight concentration ⁇ g/g
  • ⁇ OD for mEPSPS in leaves 2.00 ⁇ g/g DW
  • LOQ for mEPSPS in pollen 75.0 ⁇ g/g DW.
  • Example 8 Glyphosate Field Efficacy of Elite Event MZHG0.TG
  • the treatment consisted of an application of 2x or 4x the maximum labeled rate applied at the V4 developmental stage of the corn plant.
  • Phytotoxicity was assessed at 7 days and 14 days after treatment (7 DAT and 14 DAT). Factors of phytotoxicity that are taken into account when rating phytotoxicity include leaf discoloration (for example yellowing of leaf tips or margin) , leaf damage (for example burning of leaf tips or margin), and plant growth stunting (for example inadequate elongation between internodes).
  • Phytotoxicity was quantified as the percentage of plants showing phytotoxicity at 7 or 14 days after V4 application. Comparisons were made using Least Square Mean analysis (Table 10)
  • This protocol describes a procedure for determination of zygosity status of event MZHGOJG present in individual plants comprising elite event MZHGOJG.
  • This method uses duplex end-point TaqManTM PCR, where one reaction is specific for the event MZHGOJG insertion and other is specific for the corresponding wild type allele sequence where the event MZHGOJG transgenic DNA integrated. This wild type allele sequence may be referred to as the native insertion site. Because event MZHGOJG is introgressed into a large number of corn germplasms, a primer/probe set for the native insertion site which is suitable for as many varieties as possible needed to be identified.
  • DNA samples from biological samples are prepared using methods known in the art.
  • Primers/probe master mixes are prepared as 50x stocks, with primers at a concentration of 15 ⁇ and the probe at a concentration of 5 ⁇ .
  • Table 11 indicates each reagent and amount for a 10 ⁇ L ⁇ reaction:
  • sequences of the primers used for detection of event MZHGOJG were 5'- CAACTAGCTAGATTAATTAACGCAATCTG-3' (SEQ ID NO: 10) and 5'-
  • ATTTGTTTGC AAGGTGTGGGA-3 ' (SEQ ID NO: 56).
  • the sequence of the MZHGOJG probe was 5 ' -TT AAGTTGTCT AAGCGTC AATTTG-3 ' (SEQ ID NO: 12).
  • the primer/probe set may be 5 ' -TTTGTTTGCAAGGTGTGGGAC-3 ' (SEQ ID NO: 57) and 5'- TCACCGTGAC ATGCTTAGC A-3 ' (SEQ ID NO: 58), with a native insertion site probe of 5'- TCCC ATC AAATT ATGCT-3 ' (SEQ ID NO: 59).
  • the primer/probe set for the native insertion site may be 5' TCATTTGTTTGCAAGGTGTGG-3 ' (SEQ ID NO: 60), and 5'- TT AATCACCGTGAC ATGCTT AGC-3 ' (SEQ ID NO: 61), with a native insertion site probe of 5'- TT AGGTCCCATC AAATT-3 ' (SEQ ID NO: 62).
  • PCR was performed in the ABI 7900HT instrument using the following amplification parameters: 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 1 min at 60°C. The PCR may be run on an ABI Prism 7700, a GeneAmp PCR system 9700, or any other appropriate system.
  • results indicate if the biological sample was taken from a corn plant which is "homozygous", or contains one copy of the MZHGOJG event on each Chromosome 9, for a total of two copies of the MZHGOJG event, "heterozygous", where the plant comprises event MZHGOJG in one Chromosome 9 but the second Chromosome 9 does not have the event, or "null", where event MZHGOJG is not present on either Chromosome 9 of the corn plant evaluated. Determining how many copies of event MZHGOJG are present in a maize plant is useful for breeding purposes, so that the segregation of event MZHGOJG in the resulting progeny of a cross may be determined.
  • Example 11 Use of elite event MZHGOJG insertion site for targeted integration in maize.
  • the elite event MZHGOJG flanking sequences are disclosed in SEQ ID NO: 8 (5' flanking sequence) and SEQ ID NO: 9 (3' flanking sequence) and were used to search maize genome databases. Matches to both flanking sequences were found on a BAC clone, ZMMBBb-513K6, of chromosome 9 (NCBI Accession No. AC204677.4; SEQ ID NO: 54). Using this information, it was determined that the elite event MZHGOJG insertion is in the corn genome on chromosome 9 between nucleotides 88,290-88,329 of NCBI Accession No. AC204677.4 (SEQ ID NO: 54).
  • flanking sequences were used to determine the physical position of the MZHGOJG insertion site on the publicly available maize reference assembly of Maize B73, using version 5a.57 / RefGen_v2 (static) of the maize_ref_3_genome.
  • This reference assembly was created by the Arizona Genomics Institute. The assembly was for corn_v3 in Chado, and chromosome pseudo-assemblies of component BAC clones were guided by the physical map, also known as the maize accessioned golden path (AGP).
  • SEQ ID NO: 9 aligns to physical position 21,684,922 - 21,685,410 on chromosome 9, and the SEQ ID NO: 8 aligns to 21,685,433 - 21,685,910 on chromosome 9.
  • the MZHGOJG insertion is at about 21,685,410-21,685,433 on chromosome 9. This location can be found on-line at the Maize Genetics and Genomics Database (maizegdb.org), using the Maize B73 RefGen_v3 data source.
  • the 50k SNP Illumina Infinium chip is an Illumina BeadChip array of 56,110 maize SNPs developed from B73 genes and initially validated on a variety of germplasm (Americas, Europe,and wild relatives; Ganal et al 2011, Plos One 6(12): e28334. doi: 10.1371). Approximately 50% (28,156) of these SNP markers have been mapped onto the very high resolution IBM and LHRF mapping panels (Ganal et al 2011).
  • the MZHGOJG insertion site is between markers rsl28284356 and rsl31707802. These markers are described with respect to the positions of marker loci in the genome of the maize B73 variety (version 3) at the Maize Genetics and Genomics Database internet resource (maizegdb.org).
  • Such targeted integration overcomes the problems with so-called "positions effects," where a transgene may insert into a transcriptionally silent region of the genome, or may disrupt the function or expression pattern of a native gene by insertion either into or in proximity to a native gene. Further advantages of such targeted integration include, but are not limited to, reducing the extremely large amount of resources required for the screening and testing of thousands of randomly inserted transgenic events before obtaining a transgenic plant that exhibits the desired level of transgene expression without also exhibiting abnormalities resulting from the inadvertent insertion of the transgene into an important locus in the host genome. Moreover, such targeted integration allows for stacking transgenes at a single genomic location, rendering introgression of the stacked traits into desirable germplasm significantly more efficient.
  • CRISPR/Cas Repeats/CRISPR-associated nuclease (CRISPR/Cas) with an engineered crRNA/tracr RNA, and methods of using this nucleases to target known genomic locations, such as the genomic insertion site of elite event MZHGOJG, are well-known in the art (see reviews by Bortesi and Fischer, 2015, Biotechnology Advances 33: 41-52; and by Chen and Gao, 2014, Plant Cell Rep 33: 575-583, and references within).
  • Patent publication WO2016106121 (hereby incorporated by reference in its entirety) exemplifies using the insertion site of the corn event MIR604 for targeted insertion. This application is incorporated in its entirety herein.
  • the MIR604 insertion site in untransformed corn is used as a site for targeted insertion for some examples.
  • the MIR604 event is also used as a site for targeted insertion, to add additional transgenes.
  • the disclosure of the present application, as well as the teachings known in the art and taught, for example, in the WO2016106121 publication provide sufficient disclosure for a position of ordinary skill in the art to perform targeted insertion at the genomic insertion site of elite event MZHGOJG, either the genomic location in the absence of the event, or the genomic location of the event itself with targeted integration into part of the MZHGOJG transgene or proximal to the MZHGOJG transgene.
  • Applicants have made a deposit of corn seed comprising elite event MZHGOJG on February 11, 2016 in accordance with the Budapest Treaty at the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA 20110-2209 under ATCC Accession No. PTA-122835. The seed was tested on February 25, 2016 and found to be viable. The deposit will be maintained in the depositary for a period of 30 years, or 5 years after the last request, or the effective life of the patent, whichever is longer, and will be replaced as necessary during that period. Applicants impose no restrictions on the availability of the deposited material from the ATCC; however, applicants have no authority to waive any restrictions imposed by law on the transfer of biological material or its transportation in commerce.

Abstract

L'invention concerne un nouvel événement d'élite de maïs transgénique appelé MZHG0JG. L'invention concerne des séquences d'acides nucléiques des constructions recombinantes insérées dans le génome de maïs, et les séquences génomiques flanquant le site d'insertion, ce qui a pour résultat l'événement d'élite MZHG0JG qui comprend des molécules d'acide nucléique contenant de nouvelles séquences d'acides nucléiques. L'invention concerne en outre des tests pour détecter la présence des nouvelles séquences d'acides nucléiques de l'événement d'élite MZHG0JG, des plants de maïs et des graines de maïs comprenant le génotype de ce dernier, et des procédés de production d'un plant de maïs par croisement d'un plant de maïs comprenant le génotype d'événement d'élite MZHG0JG avec lui-même ou une autre variété de maïs.
PCT/US2017/036035 2016-06-07 2017-06-06 Événement d'élite de maïs mzhg0jg WO2017214074A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109234431A (zh) * 2018-09-27 2019-01-18 北京大北农生物技术有限公司 玉米抗茎腐病qtl的分子标记及其应用
CN110157833A (zh) * 2019-07-01 2019-08-23 北京市农林科学院 检测高赖氨酸玉米的方法及其专用分子标记
CN110195122A (zh) * 2019-01-02 2019-09-03 四川省农业科学院生物技术核技术研究所 一种用于检测玉米植物t抗-4的核酸序列及其检测方法
CN113201531A (zh) * 2021-04-27 2021-08-03 隆平生物技术(海南)有限公司 转基因玉米事件lw2-1及其检测方法
CN116694627A (zh) * 2023-07-26 2023-09-05 隆平生物技术(海南)有限公司 转基因玉米事件lp035-1及其检测方法

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6951929B2 (en) * 2000-01-11 2005-10-04 Bayer Bioscience N.V. Methods and kits for identifying elite event GAT-ZM1 in biological samples
US20070130645A1 (en) * 2005-12-07 2007-06-07 Wei Wu Genome-wide identification and characterization of gene expression regulatory elements in zea mays for use in plants
US20100197503A1 (en) * 2009-01-22 2010-08-05 Syngenta Participations Ag Mutant Hydroxyphenylpyruvate Dioxygenase Polypeptides and Methods of Use
US20110231946A1 (en) * 2008-09-02 2011-09-22 Syngenta Participations Ag Engineering of bolting resistance in sugar beet by means of the transgenic expression of the beet homologue of flowering time control gene ft
US20150315605A1 (en) * 2014-02-21 2015-11-05 E I Du Pont De Nemours And Company Novel transcripts and uses thereof for improvement of agronomic characteristics in crop plants
US20160145586A1 (en) * 2009-08-19 2016-05-26 Dow Agrosciences Llc Aad-1 event das-40278-9, related transgenic corn lines, and event-specific identification thereof

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6951929B2 (en) * 2000-01-11 2005-10-04 Bayer Bioscience N.V. Methods and kits for identifying elite event GAT-ZM1 in biological samples
US20070130645A1 (en) * 2005-12-07 2007-06-07 Wei Wu Genome-wide identification and characterization of gene expression regulatory elements in zea mays for use in plants
US20110231946A1 (en) * 2008-09-02 2011-09-22 Syngenta Participations Ag Engineering of bolting resistance in sugar beet by means of the transgenic expression of the beet homologue of flowering time control gene ft
US20100197503A1 (en) * 2009-01-22 2010-08-05 Syngenta Participations Ag Mutant Hydroxyphenylpyruvate Dioxygenase Polypeptides and Methods of Use
US20160145586A1 (en) * 2009-08-19 2016-05-26 Dow Agrosciences Llc Aad-1 event das-40278-9, related transgenic corn lines, and event-specific identification thereof
US20150315605A1 (en) * 2014-02-21 2015-11-05 E I Du Pont De Nemours And Company Novel transcripts and uses thereof for improvement of agronomic characteristics in crop plants

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
DATABASE GenBank [O] 19 August 2015 (2015-08-19), XP055455216, Database accession no. LGUP01000058 *
DATABASE GenBank [O] 22 September 2014 (2014-09-22), XP055455228, Database accession no. LK872025 *
DATABASE GenBank 30 March 2015 (2015-03-30), Database accession no. 012026471 *
DATABASE NCBI [O] 3 March 2010 (2010-03-03), Database accession no. 131707802 *
DATABASE NCBI 3 March 2010 (2010-03-03), Database accession no. 128284356 *
FEDERAL REGISTER OF THE NATIONAL ARCHIVES AND RECORDS ADMINISTRATION, vol. 80, no. 207, 27 October 2015 (2015-10-27), pages 65605 - 65880 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109234431A (zh) * 2018-09-27 2019-01-18 北京大北农生物技术有限公司 玉米抗茎腐病qtl的分子标记及其应用
CN110195122A (zh) * 2019-01-02 2019-09-03 四川省农业科学院生物技术核技术研究所 一种用于检测玉米植物t抗-4的核酸序列及其检测方法
CN110157833A (zh) * 2019-07-01 2019-08-23 北京市农林科学院 检测高赖氨酸玉米的方法及其专用分子标记
CN113201531A (zh) * 2021-04-27 2021-08-03 隆平生物技术(海南)有限公司 转基因玉米事件lw2-1及其检测方法
CN113201531B (zh) * 2021-04-27 2023-06-27 隆平生物技术(海南)有限公司 转基因玉米事件lw2-1及其检测方法
CN116694627A (zh) * 2023-07-26 2023-09-05 隆平生物技术(海南)有限公司 转基因玉米事件lp035-1及其检测方法
CN116694627B (zh) * 2023-07-26 2023-09-29 隆平生物技术(海南)有限公司 转基因玉米事件lp035-1及其检测方法

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