BR112012012494A2 - aad-12 416 soy event detection - Google Patents

Abstract

SOY EVENT DETECTION AAD-12 416. The present invention relates, in part, to a method of detecting an AAD-12 soy event. The invention provides tests to detect the presence of the object event in a sample (of soybeans, for example). Kits and conditions useful in conducting the tests are also provided. More specifically, the present invention relates, in part, to an endpoint Taq-Man PCR assay for the AAD-12 soy event. Some embodiments are directed to tests that are capable of high production zygosity analysis. The invention further relates, in part, to the discovery of a preferred reference gene for use in determining zygosity. This invention also relates, in part, to plant generation using any of the object methods. In some embodiments, said event / polynucleotide sequence can be "stacked" with other traits. The object procedures can be used to identify only soybean lines comprising the event of the invention.

Description

. 1/32 Invention Patent Descriptive Report for "SOY EVENT DETECTION AAD-12 416".

BACKGROUND OF THE INVENTION The aad-12 gene (originally from Delftia acidovorans) encodes the aryloxyalkanoate dioxigenase protein (AAD-12). The trait confers tolerance to 2,4-dichlorophenoxyacetic acid, for example, and pyridyloxyacetate herbicides. The aad-12 gene itself, for herbicide tolerance in plants, was first disclosed in WO 2007/053482.

The expression of heterologous or foreign genes in plants is influenced, where the foreign gene is inserted into the chromosome. This may be due to the chromatic structure (eg, heterochromatin) or the proximity of elements of transcriptional regulation (eg, intensifier) close to the integration site (Weising et al. Ann. Rev. Genet 22: 421-477 ', 1988), for example. The same gene in the same type of transgenic plant (or another organism) can exhibit a wide range of expression levels against different events. There may also be differences in spatial or temporal patterns of expression here. For example, differences in the relative expression of a transgene in various plant tissues cannot correspond to the expected patterns of transcriptional regulatory elements present in the introduced gene construct.

In this way, large numbers of events are often created and classified in order to identify an event that expresses an introduced gene of interest at a satisfactory level for a given proposal. For commercial purposes, it is common to produce hundreds to thousands of different events, and to classify those events into a simple event that has desired levels and patterns of transgene expression. An event that has desired levels and / or patterns of transgene expression is useful for the transgene's introgression into other genetic antecedents by sexual crossing using conventional generation methods. The progeny of such crossings maintain the transgene expression characteristics of the original transformant. This strategy is used to ensure safe gene expression in a number of varieties that are well adapted to the conditions.

R 2/32 local growth tions.

Several previous methods can be used to detect the presence of an event in a plant tissue sample. An example is the Pyrosequencing technique, as described by Winge (Innov.

Pharma. Tech. 00: 18-24, 2000). In this method, an oligonucleotide is called that overlaps with genomic DNA and junction of insert DNA. The oligonucleotide is hybridized to a single stranded PCR product from the region of interest (one primer in the inserted sequence and one in the flanking genomic sequence), and incubated in the presence of a poly- DNA. 10 merase, ATP, sulfurylase, luciferase, apyrase, adenosine 5 'phosphosulfate e. luciferin. DNTPs are added individually, and the incorporation results in a light signal that is measured. A light signal indicates the presence of a transgene insert / flanking sequence due to successful amplification, hybridization, and multiple or simple base extension. (This technique is usually used for initial sequencing, not for detecting a specific gene when it is known).

Fluorescence polarization is another method that can be used to detect an amplicon. Following this method, an oligonucleotide is designed to overlap the genomic flanking and joining of inserted DNA. The oligonucleotide is hybridized to the single-stranded PCR product of the region of interest (a primer in the inserted DNA and one in the flanking genomic DNA sequence), and incubated in the presence of a DNA polymerase and a fluorescent-labeled ddNTP. The simple base extension results in the incorporation of ddNTP. Incorporation can be measured as a change in polarization using a fluorometer. A change in polarization indicates the presence of the transgenic insert / flanking sequence due to successful amplification, hybridization, and simple base extension.

Molecular signals have been described for use in sequence detection. Soon, a FRET oligonucleotide probe is designed that overlaps the flanking and junction genome of insert DNA. The FRET probe's unique structure results in it containing a secondary structure

R 3/32 daily that keeps the fluorescent and extinction portions in close proximity. The FRET probe and PCR primers (one primer in the insert DNA sequence and one in the flanking genomic sequence) are cyclized in the presence of a thermostable polymerase and dnTPs. Following successful PCR amplification, hybridization of the FRET probe to the sequence results in removal of the probe's secondary structure and spatial separation of the fluorescent and extinction portions. A fluorescent signal indicates the presence of the flanking genome / transgene insert sequence due to successful amplification and hybridization. á 10 Hydrolysis probe assay, otherwise known as J TAQMAN (Life Technologies, Foster City, Calif), is a method of detecting and quantifying the presence of a DNA sequence. Soon, a FRET oligonucleotide probe is assigned with an oligo within the transgene and one in the flanking genomic sequence for specific event detection. The FRET probe and PCR primers (one primer in the insert DNA sequence and one in the flanking genomic sequence) are cyclized in the presence of a thermostable polymerase and dNTPs. Hybridization of the FRET probe results in the cleavage and release of the fluorescent portion from the extinction portion in the FRET probe. A fluorescent signal indicates the presence of the flanking / transgene insert sequence due to successful amplification and hybridization.

Another challenge, among many, is to find a suitable reference gene for a given test. For example, as cited in the summary by Czechowski et al, "An exceptionally large set of data from GeneChip studies of total genome Affymetrix ATH1 provided the means to identify a new generation of reference genes with very stable levels of expression in species of Arabidopsis plant model (Arabidopsis thaliana). Hundreds of Arabidopsis genes have been found that carry out traditional reference genes in terms of expression stability throughout development, and under a range of environmental conditions ". (Czechowski et al. (2005) Comprehensive genome identification and testing of superior reference genes for normalization transcribed in Arabidopsis.

. 4/32 Physiol. 139, 5-17).

Brodmann et al. (2002) refers to a real-time quantitative PCR detection of transgenic corn content in food for four different corn varieties approved in the European Union. Brodmann, PD ,, P.D., IMgEC., Berthoud H., and Herrmann, A. Real-time Quantitative Polymerase Chain Reaction Methods for Four Genetically Modified Maize Varieties and Maize DNA Content in Food. J. of AOAC international 2002 85 (3) Hernandez et al. (2004) mention four possible genes for use with real-time PCR. '10 Hernandez et al. (2004) mentions four possible genes for 'use with real-time PCR. Hernandez, M., Duplan, M - N., Berthier, G., Vaitiingom, M., Hauser, W., Freyer, R., Pla, M., and Bertheau, Y. Development and comparison of systems real-time polymerase chain reaction for specific detection and quantification of Zea mays LJ Agric. Food Chem. 2004,52,4632-4637.

Costa et al. (2007) reached these four genes (also in the context of real-time PCR) and concluded that alcohol dehydrogenase and zein genes were the best reference genes to detect a sample "event" (a lectin gene) for sources of inter-mixing of transgenic food. Costa, L. D., and Martinelli L. Development of a Real Time PCR Method Based on Double Target Plasmids for Determining an Unexpected Genetically Modified Soy Intermix with Food Components. J. Agric. Food Chem. 2007, 55, 1264-1273.

Huang et al. (2004) used plasmid pMulM2 as reference molecules to detect MON810 and NK603 transgenes in corn. Huang and Pan, "Genetically Modified MON810 and NK603 Maize Detection by Multiplex and Real Time Polymerase Chain Reaction Methods", J. Agric. Food Chem., 2004, 52 (11), pp 3264-3268.

Gasparic et al. (2008) suggest LNA technology, from a comparison to cyclization of TaqMan probe technology, and various real-time PCR chemicals, to quantitatively analyze corn events (such as MONB810). Gasparic, Cankar, Zel, and Gruden, "Comparison of

: 5/32 different real-time PCR cases and any suitability for the detection and quantification of genetically modified organisms ", BMC Biotechnol. 2008; 8: 26. US 20070148646 refers to an initiator extension method for quantification that requires controlled dispensing of individual nucleotides that can be detected and quantified by the amount of nucleotides incorporated.This is different from the TaqMan PCR method using an internal reference gene.To distinguish between homozygous and hemizygote genotypes of 1 10 TCI 507, an Invasive assay was used successfully for this event. 'Gupta, M., Nirunsuksiri, W., Schulenberg, G., Hartl, T., Novak, S., Bryan, J., Vanopdorp, N., Bing, J. and Thompson, S. An Inder Assay based on Non-PCR-Quantitatively Detects Single Copy Genes in Complex Plant Genomes, Mol. Breeding 2008,21, 173-181.

Huabang (2009) refers to a zygosity test based on transgenic corn PCR. However, no reference gene appears to be used. Huabang, "An Accurate and Fast PCR Zygism Test Method for Genetically Modified Maize", Molecular Plant Generation, 2009, Vol.7, No.3, 619-623.

BRIEF SUMMARY OF THE INVENTION The present invention is related, in part, to methods of detecting the AAD-12 soybean event (Max glycine) designated DAS-68416-4 having seed deposited with American Type Culture Collection (ATCC) with No. Access. PTA-10442.

More specifically, the present invention relates, in part, to the TagMan PCR endpoint assay for the ADD-12 soy event. Some embodiments are directed to tests that are capable of high production zygosity analysis. The invention additionally relates, in part, to the discovery of a preferred lectin reference gene for the determination of zygosity. These and other related procedures can be used to identify only soybean lines comprising the event of the invention.

'6/32 This invention also relates, in part, to plant generation using any of the object methods. In some embodiments, said event / polynucleotide sequence can be "stacked" with other traits, including, for example, other tolerances to the herbicidal gene (s) and insect inhibitory proteins. However, the invention includes plants having the simple event, as described herein.

Additionally, the invention provides tests to detect the presence of the object event in a sample (of soybeans, for example). Kits and conditions useful in conducting the tests are also provided.

10 BRIEF DESCRIPTION OF THE FIGURES: Figure 1. Genomic DNA from the DAS-68416-4 soy event was distributed with EcoRV, or Pvu Il, and used to generate corresponding GENOME-WALKER libraries, which were used as templates for amplifying - stay the target DNA sequences.

Figure 2. The schematic diagram represents the primer locations and cloning strategy for full-length sequencing of the DAS-68416-4 soybean event from the 5 'to 3' limits.

Figure 3. The schematic diagram represents the primer locations to confirm the full length sequence of the DAS-68416-4 soybean event from the 5'a3 limits.

Figure 4. The schematic diagram represents the primer locations to confirm the insertion site sequence for the soybean event AAD-12 DAS-68416-4.

BRIEF DESCRIPTION OF SEQUENCES SEQ ID NO: 1 provides a sequence of 5 'and 3' genomic flanking sequences on either side of the AAD-12 insert, including the insert. The flanking strings are underlined.

SEQ ID NOs: 2-7 provides sequences for primers and probes for use according to the invention.

DETAILED DESCRIPTION OF THE INVENTION AAD-12 transgenic soybean event (providing tolerance to the herbicide) pDAB4468-416 was generated by Agrobacterium transformation.

- 7/32 Both 5 'and 3' terminal flanking sequences of this AAD-12 transgenic insert were cloned, sequenced and characterized as detailed in USSN 61 / 263,950 (deposited on November 24, 2009). In some specific embodiments, AAD-12 is present in soybeans according to the event designated DAS-68416-4 having seed deposited with American Type Culture Collection (ATCC) with Accession No. PTA-10442, and progeny derived from it. 2500 seeds were deposited according to the Budapest Treaty on 22 October 2009. The deposit was tested on 2 November 2009, and on that date, the seeds were viable. Í 10 Specific primers TAQMAN and probe have been designated, as detailed here, in part, according to the DNA sequences located in the junction region between the transgene and the host genomic DNA. The event specificity of the primers and probe has been successfully tested in duplex format with Lectin soybean as a reference gene in real-time PCR against different AAD-12 soybean events and Maverick non-GM soybean variety. The procedures for specific endpoint event TAQMAN trials for the AAD-12 soy event were developed, as detailed here.

The sequence extension of the integration junction region between host plant DNA and the gene construct integrated in this AAD-12 soy event is a unique sequence. It was used to develop specific event tests (conventional PCR or real-time PCR) to detect the presence of soybean AAD-12 pDAB4468-416 event for GMO testing, and to determine plant zygosity status in generation populations . The specific event TAQMAN test reported here can be used for both applications.

The invention provides assays to detect the presence of the transgenic soybean DAS-68416-4 in a sample. Aspects of the invention include methods of designating and / or producing any diagnostic nucleic acid molecules exemplified or suggested here. Plant lines comprising this event can be detected using sequences revealed and suggested here.

: 8/32 Thus, in some embodiments, this invention refers to the identification of herbicide-tolerant soybean lines. The invention relates, in part, to detecting the presence of the object event in order to determine whether the progeny of a sexual crossing contains the event of interest. In addition, a method for detecting the event is included, and is useful, for example, to comply with regulations requiring pre-marketed approval and labeling of foods derived from recombinant crop plants, for example.

More specifically, the event is an AAD-12 event also 7 10 called pDAB4468-0416. This invention can be used for its selection and characterization for stability and expression in the total plant and molecular levels from generation to generation.

The synthetic object gene (aad-12) used in accordance with the invention was derived from Delftia acidovorans and encodes an enzyme capable of deactivating various herbicides with a portion of aryloxyalkanoate, including phoxy auxin (for example, 2, 4-D, MCPA), as well as pyridyloxy auxins (for example, fluroxypyr, triciopir). Thegene aad-12, powered by actuating promoters, was introduced in the Maverick soybean line, using Agrobacterium tumefaciens techniques.

The invention relates, in part, to a fluorescent-based TaqMan endpoint PCR assay using an endogenous gene as a reference control (copy number) for high production zygidity analysis of the AAD- soy event 12. The invention further relates to, in part, the discovery of a preferred reference gene, invertase. Several reference genes have been identified as possible options.

The invention also relates, in part, to the development of a biplex endpoint TaqMan PCT for analysis of the specific zytosity of soybean AAD-12. Additionally, the invention relates, in part, to the development of AAD-12 generation test kits.

The TaqgMan endpoint assays are based on a plus / minus strategy, whereby "more" means which sample is positive for

: 9/32 the tested gene and "less" means that the sample is negative for the tested gene. These assays typically use two sets of oligonucleotides to identify the AAD-12 transgene sequence and the wild type gene sequence respectively, as well as double labeled probes for the transgene content and wild type sequence.

Although the Invasor assay was a robust technique for characterization events, it is very sensitive to DNA quality. In addition, the assay requires a high amount of DNA. Invasor also requires an additional denaturation step which, if not handled correctly, can make the Invasor assay unsuccessful. In addition, the longest test time of the Invasor test is limited in its flexibility to efficiently large handling numbers of 416 AAD-12 wind samples for analysis in a commercial setting. A major advantage of the invention is that it saves time and eliminates the denaturation step.

The analysis of Endpoint TagMan object for the detection of 416 AAD-12 events offers surprising advantages over Invaders, particularly in the analysis of a larger number of samples.

Definitions and examples are provided here to assist in describing the present invention and to guide those skilled in the art in practicing the invention. Unless otherwise noted, the terms are to be understood in accordance with conventional usage by those skilled in the art. The nomenclature for DNA bases as placed at 37 CFR 8 1,822 is used.

As used herein, the term "progeny" denotes the offspring of any generation of a plant of origin that comprises soybean event AAD-12 DAS-68416-4.

A transgenic "event" is produced by transforming plant cells with heterologous DNA, that is, a nucleic acid construct that includes a transgene of interest, regeneration of a plant population resulting from the insertion of the transgene into the plant's genome, and selection of a particular plant characterized by the insertion of a location of

: 10/32 particular genome. The term "event" refers to the original transformant and progeny of the transformant that includes the heterologous DNA. The term "event" also refers to the progeny produced by a sexual cross between the transformant and another variety that includes genomic / transgene DNA. Even after repeated posterior crossing to a recurrent origin, the inserted transgene DNA and genomic flanking DNA (genomic / transgene DNA) from the transformed origin are present in the progeny of the crossing at the same chromosomal location. The term "event" also refers to DNA from the original transformant and progeny of this gene comprising the inserted DNA and flanking genomic sequence immediately adjacent to the inserted DNA that would be expected to be transferred to a progeny that receives inserted DNA including the transgene of interest as the result of a sexual crossing of a source line that includes the inserted DNA (for example, the original transformant and progeny resulting from individuality) and a source line that does not contain the inserted DNA.

A "junction sequence" reaches the point where the DNA inserted into the genome is linked to DNA from the native soybean genome that flanks the insertion point, the identification or detection of one or the other junction sequences in the plant's genetic material being sufficient to be diagnostic for the event. Included are the DNA sequences that reach insertions in the soy events described here and similar lengths of flanking DNA. Specific examples of such diagnostic sequences are provided here; however, other sequences that overlap the insertion junctions, or the insertion junctions and the genomic sequence, are also diagnostic and can be used according to the invention.

The invention relates to the identification of such flanking, joining, and insertion sequences. Related PCR primers and amplicons are included in the invention. According to the invention, PCR analysis methods using amplicons that reach through the inserted DNA and six limits can be used to detect or identify soybean varieties

. 11/32 transgenic sold or lines derived from the proprietary transgenic soybean lines.

The total sequences of each of these inserts, together with portions of the respective flanking sequences, are provided here as SEQIDNO: 1. The coordinates of the insert and flanking sequences for this event with respect to SEQ ID NO: 1 (10,212 base pairs total) are indicated below.

[| Tranqueamento 5 Tinserto - [fianqueamento 3 | | : LH Ng O O IO The components of the insert and flanking sequences are further illustrated in figures 1 to 4.

Detection techniques of the invention are especially useful in conjunction with plant generation, to determine whether progeny plants comprise a given event, after a source plant comprising an event of interest is crossed with another plant line in an effort to grant one or more additional traits of interest in the progeny.

These methods of analysis benefit soybean generation programs as well as quality control, especially for commercialized transgenic soybean seeds. Detection kits for these transgenic soy lines can now also be produced and used. This can also benefit product registration and product waiver. These can be used for accelerated generation strategies and to establish articulation data.

The sequences provided here can be used to study and characterize transgene integration processes, characteristics of genomic integration site, event classification, transgenic stability and two flanking sequences, and gene expression (especially related to silencing gene, transgene methylation patterns, position effects, and potential elements related to expression such as MARS [matrix fixation regions], and the like).

. 12/32 Still further, the invention includes selection of descending progeny and / or plant, preferably a herbicide resistant soy plant in which said plant has a genome comprising a detectable DNA insert as described herein.

As used herein, the term "soy" means glycine max and includes all varieties of it that can be generated with a soy plant.

This invention further includes processes for producing crosses using a plant of the invention as at least one origin.

This invention includes a method for producing a hybrid Fi '10 seed by crossing an exemplified plant with a different plant (for example, origin in generation) and collecting the resulting hybrid seed.

The characteristics of the resulting plants (or a female) can be improved by careful consideration of the original plants.

A herbicide-tolerant soy plant can be generated by the first sexually crossing of a first source soy plant consisting of a soy plant grown from seed of any of the lines referred to here, and a second source soy plant, produced thus, from a plurality of first progeny plants; and then selecting a first progeny plant that is resistant to an herbicide (or that has at least one of the events of the invention); and individuality of the first progeny plant, thus producing a plurality of second progeny plants; and then select from the second progeny plants a plant that is resistant to a herbicide (or that has at least one of the events of the invention). These steps may additionally include crossing after the first progeny plant, or from the second progeny plant to the second soybean plant of origin, or a third soybean plant of origin.

A soybean crop comprising soybean seeds of the invention, or progeny thereof, can then be planted.

It is also to be understood that two different transgenic plants can also be combined to produce offspring that contain two segregations independently added by genes

: 13/32 exogenous. The appropriate progeny individuality can produce plants that are homozygous for both added exogenous genes. The subsequent crossing to a plant of origin and crossing with a non-transgenic plant are also contemplated, as is vegetative propagation. Other generation methods commonly used for different traits and harvests are known in the art. The generation of posterior crossing was used to transfer genes to a highly inherited trait simply inherited in a desirable homozygous cultivar, or generation line, which is the recurrent origin. The source of the trait to be transferred is called "10 donor origin. The resulting plant is expected to have the attributes of the origin: recurrent (for example, cultivar), and the desirable trait transferred from the donor origin. After the initial crossing, individuals who have the phenotype of the donor origin are selected and repeatedly crossed (subsequently crossed) to the recurrent origin.The resulting origin is expected to have attributes of the recurrent origin (eg cultivar) and the desirable trait transferred from the origin donor.

The present invention can be used with molecular markers in a marker generation (MAB) method. The DNA molecules of the present invention can be used with other methods (such as AFLP markers, RFLP markers, RAPD markers, SNP's, and SSRs) that identify genetically linked agronomically useful traits, as is known in the art. The progeny-resistant trait can be traced to the progeny of a cross with a soybean plant of the invention (or progeny thereof, and any soybean variety or variety) using MAB methods. DNA molecules are markers for this trait, and MAB methods that are well known in the art can be used to trace the herbicide-resistant trait (s) in soybean plants where at least one soybean line of the invention, or progeny of this, was an origin or predecessor. The methods of the present invention can be used to identify any soybean variety having the object event.

The methods of the invention include a method of producing a herbicide-tolerant soy plant in which said method comprises

: 14/32 generation with an invention plant. More specifically, said methods may comprise crossing two plants of the invention, or one plant of the invention and any other plant. The preferred methods additionally include selecting progeny from said cross by analyzing said progeny for a detectable event according to the invention. For example, the invention can be used to trace the object event through generation cycles with plants comprising other desirable traits, such as agronomic traits, such as those tested here in various Examples. Plants comprising the object event and the desired trace 10 can be detected, identified, selected and quickly used in additional generation steps, for example. The object / trait event can also be combined through generation, and tracked according to the invention, with an insect-resistant trait (s) and / or with additional herbicide tolerance traits. A preferred embodiment of the latter is a plant comprising the object event combined with a gene that encodes resistance to the herbicide dicamba.

The object event can be combined with, for example, traits encoding glyphosate resistance (eg, plant resistant or bacterial EPSPS, GOX, GAT), glyphosate resistance (eg, Pat, bar), acetolatate synthase, herbicide resistance inhibition (ALS) (eg, imidazolinones [such as imazetapyr], sulfonylureas, triazolopyrimidine sulfonanilide, pyrmidmiltiobenzoates, and other chemicals [Csrl, SurA, et al.]), resistance to bromoxynil (eg, Bxn), resistance to HPPD (4-hydroxylphenyl-pyruvate-dioxigenase) enzyme, resistance to phytene desaturase inhibitors (PDS), resistance to photosystem inhibition herbicides | l (eg psbA), resistance to herbicide inhibition photosynthesis |, resistance to protoporphyrinogen oxidase IX (PPO) inhibiting herbicides (for example, PPO-1), resistance to phenylurea herbicides (for example, CYP76B1), dicamba degradation enzymes (see, for example , US 20030135879), and others can be stacked alone or in multiple combinations to provide the ability to effectively control or prevent weed changes and / or resistance to any herbicides.

: 15/32 of the classes mentioned above.

With respect to additional herbicides, some preferred additional ALS inhibitors (also known as AHAS) include triazolopyrimidine sulfonanilides (such as chloransulam-methyl, dichlossulam, florassulam, flumetsulam, metosulam, and penoxsulam), pyrimidinylthiobenzoates (such as bis piritiobac), and flucarbazone. Some preferred HPPD inhibitors include mesotrione, isoxaflutole, and sulcotrione. Some preferred PPO inhibitors include flumiclorac, flumioxazin, flufenpir, piraflufen, flutiacet, bufenacil, carfentrazone, sulfentrazone, and diphenylethers (such as acifluor- '10 fen, fomesafen, lactofen, and oxifluorfen). In addition, AAD-12 alone or stacked with one or more additional HTC traits can be stacked with one or more additional admissions (for example, insect resistance, fungal resistance, or stress tolerance, et al.) Or discharge ( for example, increased yield, improved oil profile, improved fiber quality, et al.) traces. In this way, the invention can be used to provide complete agronomic packaging of improved crop quality with the ability to flexibly effectively control the cost of any number of agronomic pests.

The enzyme object of AAD-12 enables transgenic expression resulting in tolerance to combinations of herbicides that would control almost all broadleaf and grass weeds. AAD-12 can serve as an excellent herbicide-tolerant crop trait (HTC) to stack with other traits (eg glyphosate resistance, glyphosinate resistance, imidazolinone resistance, bromoxynil resistance, et al), and tra - insect resistance rods (CryIF, CrylAb, Cry 34/45, et al.) for example. Additionally, AAD-12 can serve as a selectable marker to assist in the selection of primary transformants from genetically engineered plants with a second gene, or group of genes.

The HTC traits of the invention can be used in new combinations with other HTC traits (including, but not limited to, glyphosate tolerance). These combinations of traits occur in new methods.

'16/32 of weed species (and similar) control, due to recently acquired resistance, or inherent tolerance to herbicides (for example, glyphosate). Thus, in addition to the HTC traits, new methods for weed control using herbicides, for which tolerance to the herbicide was created by that enzyme in transgenic crops, are within the scope of the invention.

In addition, glyphosate-tolerant crops grown around the world are prevalent. Often in rotation with other glyphosate-tolerant crops, control of volunteers resistant to glyphosate '10 can be difficult in rotational harvests. In this way, the use of traits: transgenic objects, stacked or individually transformed into crops, provides a tool to control other HTC volunteer crops.

Unless otherwise indicated, reference to the flanking sequences refers to those identified with respect to SEQ ID NO: | (see Table above). Again, SEQ ID NO!:! | includes the heterologous DNA inserted in the original transformant and illustrative flanking genomic sequences immediately adjacent to the inserted DNA. All or part of these flanking sequences can be expected to be transferred to progeny that receive the inserted DNA as a result of a sexual crossing of a source line that includes the event.

As used herein, a "line" is a group of plants that reveals little or no genetic variation between individuals by at least one trait. Such lines can be created by several generations of self-pollination and selection, or vegetative propagation from a simple source using tissue or cell culture techniques.

As used herein, the terms "cultivar" and "variety" are synonymous and refer to a line that 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, for example, preferably + 15%, more preferably + 10%>, more preferable

: 17/32 + 5%. Stability can be affected by temperature, location, stress and planting time. The comparison of subsequent generations under field conditions should produce the component in a similar way.

"Commercial utility" is defined as having good plant vigor and high fertility, such that the crop can be produced by farmers using conventional farm equipment, and the oil with the components described can be extracted from the seed using conventional crushing. - extraction equipment and equipment. To be commercially useful, yield, as measured by seed weight, oil content, and total oil produced per acre, is within 15% of the average yield of an otherwise commercial soybean variety. comparable without traits of special value grown in the same region. "Agronomic elite" means that a line has desirable agronomic characteristics, such as yield, maturity, disease resistance, and the like, in addition to insect resistance due to the event (s) (s) object (s) Agronomic traits, taken individually in or in any combination, as placed in the Examples below, in a plant comprising an event of the invention, are within the scope of the invention. characteristics and data points can be used to identify such plants, either as a point or at either end or both ends of a range of characteristics used to define such plants. and will recognize in light of this disclosure, preferred embodiments of detection kits, for example, may include probes and / or primers, including polynucleotide probes, and / or amplicons. Initiators (") that touch down" in the flanking sequence are not typically designed to hybridize beyond about 200 bases or beyond the junction. Thus, typical flanking primers would be designed to comprise at least 15 residues of any braid within 200 bases in the flanking sequences from

'18/32 from the beginning of the insert. That is, primers comprising a sequence of an appropriate size (or hybridizing to) 2530-2730 residues and / or 9122-9322 of SEQ ID NO: 1 are within the scope of the invention. Insert starters can likewise be designated in any place, but 2731-2931 and 8921-9121 residues can be used, for example, not exclusively for such an initiator design.

One skilled in the art will also recognize that primers and probes can be assigned to hybridize, under a standard hybridization range and / or PCR conditions, to a segment of SEQ ID NO: 1 (or the complement 1), and complements thereof, wherein the primer or probe is not 'perfectly complementary to the exemplified sequence. That is, some degree of mismatch can be tolerated. For a primer of approximately 20 nucleotides, for example, typically one or two or, therefore, nucleotides, do not need to be linked with the opposite braid if the unequipped base is internal, or at the end of the primer that is opposite the amplicon. Several appropriate hybridization conditions are provided below. Synthetic nucleotide analogs, such as inosine, can also be used in probes. Peptide nucleic acid (PNA) probes, as well as DNA and RNA probes, can also be used. What is important is that such probes and primers are diagnostic for (able to uniquely identify and distinguish) the presence of an event of the invention.

The components of the "insert" are illustrated in the figures. The DNA polynucleotide sequences of these components, or fragments thereof, can be used as DNA primers or probes in the methods of the present invention.

In some embodiments of the invention, compositions and methods are provided for detecting the presence of the transgene / genomic insertion region, in plants and seeds and the like, of a soybean plant.

DNA sequences are provided, as well as segments thereof, and complementary to the exemplified sequences and any segments thereof.

These and other related procedures can be used

: 19/32 to uniquely identify these soybean lines.

In some embodiments, DNA sequences that comprise a contiguous fragment of the new transgene / genomic insertion region are an aspect of this invention. Included are DNA sequences that comprise a sufficient length of transgene insert sequence polynucleotides and a sufficient length of soybean genomic sequence polynucleotides from one or more of the three soybean plants mentioned above and / or sequences that are useful as primer sequences for producing an amplicon product diagnosis for 110 one or more of these soybean plants. : Related embodiments belong to DNA sequences that comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more contiguous nucleotides of a transgene portion of a DNA sequence identified here (as with SEQ ID NO: l and segments thereof), or complements thereof, and a similar length of flanking of the soybean DNA sequence of these sequences, or complements thereof. Such sequences are useful as DNA primers in DNA amplification methods. The amplicons produced using these primers are diagnostic for any of the soy events mentioned here. Therefore, the invention also includes the amplicons produced by such DNA primers and homologous primers.

This invention also includes methods of detecting the presence of DNA in a sample, which corresponds to the soy event referred to here. Such methods may comprise: (a) contacting the sample comprising —DNA with a primer set that, when used in a nucleic acid amplification reaction with DNA from at least one of these soy events, produces an amplicon that is diagnostic for said (s) event (s); (b) carrying out a nucleic acid amplification reaction, thereby producing amplicons; and (c) detecting the amplicon.

Additional detection methods of the invention include a method of detecting the presence of a DNA, in a sample, corresponding to at least one of said events, wherein said method comprises:

: 20/32 (a) contact the sample comprising DNA with a probe that hybridizes under stringent hybridization conditions with DNA from at least one of said soybean events, and that does not hybridize under stringent hybridization conditions with a control soy (DNA of event of no interest); (b) subjecting the sample and probe to stringent hybridization conditions; and (c) detecting probe hybridization to DNA.

In still further embodiments, the invention includes methods of producing a soybean plant comprising the aad-12 event of the invention, in which said method comprises the steps of: (a) sexually crossing a first soybean line of origin (comprising expression cassettes of the present invention, which confer that trait of resistance to the herbicide to plants of that line) and a second line of soy of origin (that lacks this trait of tolerance to the herbicide) thus producing , a plurality of progeny plants; and (b) selecting a progeny plant using the invention. Such methods may optionally comprise the additional step of posterior crossing of the progeny plant to the second soybean line of origin for the production of a true-generation soybean plant that comprises said trait of tolerance to the herbicide.

According to some embodiments of the invention, methods of determining progeny zygness of a cross are provided. Said methods may comprise contacting a sample, comprising soybean DNA, with a primer set of the invention. Said primers, when used in a nucleic acid amplification reaction with genomic bDNA from at least one of said soybean events, produce a first amplicon that is diagnostic for at least one of said soybean events. Such methods further comprise carrying out a nucleic acid amplification reaction, thereby producing the first amplicon; detection of the first amplicon; and contacting the sample comprising soybean DNA with said primer set, when used in a nucleic acid amplification reaction with soybean plant genomic DNA, produces a second amplicon comprising the native genomic DNA of soybean homologous to the genomic region of soybean, and carrying out a nucleic acid amplification reaction, thereby producing the second amplicon.

The methods further comprise detecting the second amplicon, and comparing the first and second amplicons in a sample, in which the presence of both amplicons indicates that the sample is heterozygous for transgene insertion.

DNA detection kits using the compositions disclosed herein and methods well known in the DNA detection technique.

The kits are useful for identifying the soybean DNA object event in a sample, and can be applied to methods for generating soybean plants containing this DNA.

The kits contain homologous DNA sequences or complementary to amplicons, for example, disclosed here, or the homologous DNA sequences or complementary to DNA contained in the transgenic genetic elements of the object events.

These DNA sequences can be used in DNA amplification reactions, or as probes in a DNA hybridization method.

The kits can also contain the reagents and materials necessary for the performance of the detection method.

A "probe" is an isolated nucleic acid molecule that is attached to a conventional detectable tag, or reporter molecule (such as one: radioactive isotope, ligand, chemiluminescent agent, or enzyme). Such a probe is complementary to a strand of a target nucleic acid, in the case of the present invention, to a strand of genomic DNA from one of said soybean events, whether from a soybean plant or from a sample that includes DNA from the event.

The probes according to the present invention include not only deoxyribonucleic or ribonucleic acids, but also polyamides and other probe materials that specifically bind to a target DNA sequence and can be used to detect the presence of that target DNA sequence. "Primers" are isolated / synthesized nucleic acids that are reinforced to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, then extended along the target DNA strand by a

: 22/32 polymerase, for example, a DNA polymerase. The primer pairs of the present invention refer to their use for amplification of a target nucleic acid sequence, for example, by polymerase chain reaction (PCR), or other methods of nucleic acid amplification. 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, n 10 94,95,96,97,98,99,100,101, 102, 103 , 104, 105, 106, 107, 108, 109, 110,, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171,

15. 172,173, 174, 175, 176, 177, 178. 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237,238,239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305m 306, 307, 308, 309, 310, 311, 312,313,314,315,316, 317, 318 , 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343 , 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362 , 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387,388,389,390,391,392 , 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417 , 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431,

432, 433, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, or 500 polynucleotides or more in length. Such probes and primers hybridize specifically to a target sequence under high stringency hybridization conditions. Preferably, probes and primers according to the present invention have complete sequence similarity to the target sequence, although these probes differ from the target sequence and which retain the ability to hybridize to the target sequences can be referred to as conventional methods.

Methods for preparing and using probes and primers are described, for example, in Molecular Cloning: A Laboratory Manual, 2nd ed., Vol. 1-3, ed. Sambrook et ah, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1989. PCR primer pairs can be derived from a known sequence, for example, by using the intended computer programs for this proposal.

Primers and probes based on the flanking DNA and insert sequences disclosed herein can be used to confirm (and, if necessary, to correct) the sequences revealed by conventional methods, for example, by recloning and sequencing such sequences.

The nucleic acid probes and primers of the present invention hybridize under stringent conditions to a target DNA sequence. Any conventional nucleic acid hybridization, or amplification method, can be used to identify the presence of DNA from a transgenic event in a sample. Nucleic acid molecules, or fragments thereof, are able to specifically hybridize to other nucleic acid molecules under certain circumstances. As used herein, two nucleic acid molecules are said to be able to specifically hybridize to one another if the molecules are capable of forming an antiparallel double stranded nucleic acid structure. One nucleic acid molecule is said to be the "complement" to another molecule of

. 24/32 nucleic acid if they exhibit complete complementarity.

As used herein, molecules are said to exhibit "complete complementarity" when many nucleotides in one molecule are complementary to one nucleotide in the other.

Two molecules are said to be "minimally complementary" if they can hybridize to each other with sufficient stability to allow them to remain reinforced with each other under at least conventional "low stringency" conditions.

Similarly, molecules are said to be "complementary" if they can hybridize to each other with sufficient stability to allow them to remain reinforced with each other under conventional "high stringency" conditions.

Conventional stringency conditions are described by Sambrook et al., 1989. Deviations from complete complementarity are therefore permissible, considering that such deviations do not completely impede the ability of molecules to form a double stranded structure.

In order for a nucleic acid molecule to serve as a primer or probe, it needs to be sufficiently complementary in sequence to be able to form a stable double-stranded structure under the particular solvent and salt concentrations employed.

As used herein, a substantially homologous sequence is a nucleic acid sequence that will hybridize specifically to the complement of the nucleic acid sequence to which it is being compared under high stringent conditions.

The term "stringent conditions" is functionally defined with respect to hybridization of a nucleic acid probe to a target nucleic acid (ie, to a particular nucleic acid sequence of interest) by the specific hybridization procedure discussed in Sambrook et a / l., 1989, until 9.52-9.55. See also, Sambrook et al., 1989 to 9.47-9.52 and 9.56-9.58. Consequently, the nucleic acid sequences of the invention can be used for their ability to selectively form duplex molecules with complementary stretches of DNA fragments.

Depending on the application considered, different hybridization conditions can be used to achieve varying degrees of

: 25/32 probe for target sequence. For applications requiring high selectivity, relatively stringent conditions will typically be employed to form hybrids, for example, relatively low salt and / or high temperature conditions will be selected, as provided by about 0.02 M to about 0.15 M NaClem temperatures from about 50º C to about 70º C. Strict conditions, for example, may involve washing the hybridization filter at least twice with high stringency wash buffer (0.2X SSC, 0.1% SDS , 65 ° C). The appropriate stringency conditions that promote DNA hybridization, for example, 6.0X sodium chloride / 10 sodium citrate (SSC) at about 45 ° C, followed by a 2.0X SSC wash a. 50º C are known to those technicians in the signature. For example, the concentration of salt in the washing step can be selected at low stringency of about 2.0X SSC at 50º C to a high stringency of about 0.2X SSC at 50º C. In addition, the temperature in the washing step can be increased from low stringency conditions to room temperature, about 22º C, to high stringency conditions to around 65º C. Both temperature and salt can be varied, or the temperature or the salt concentration can be kept constant, while another variable is changed. Such selective conditions, poorly tolerated, if any, are unbalanced between the probe and the target template or braid. The detection of DNA sequences, via hybridization, is well known to those skilled in the art, and the teachings of United States Patent Nos. 4,965,188 and 5,176,995 are exemplary of hybridization analysis methods. In a particularly preferred embodiment, a nucleic acid of the present invention will specifically hybridize to one or more of the primers (or amplicons or other sequences) exemplified or suggested herein, including complements and fragments thereof, under conditions of high stringency. In one aspect of the present invention, a marker nucleic acid molecule of the present invention has the nucleic acid sequence as placed here in one of the exemplified sequences, or supplements and / or fragments thereof.

In another aspect of the present invention, a marker nucleic acid molecule of the present invention divides between 80% and 100% or 90% and 100% sequence identity with such nucleic acid sequences. In a further aspect of the present invention, a marker nucleic acid molecule of the present invention divides between 95% and 100% sequence identity with such a sequence. Such sequences can be used as markers in plant generation methods to identify the progeny of genetic crosses. Hybridization of the probe to the target DNA molecule can be detected by any number of methods known to those skilled in the art, these may include, but are not limited to, 10 fluorescent tags, radioactive tags, antibody-based tags. powder, and chemiluminescent labels.

With respect to amplification of a target nucleic acid sequence (for example, by PCR) using a particular amplification primer pair, "stringent conditions" are conditions that allow the primer pair to hybridize only to the target nucleic acid sequence to which primer having the corresponding wild-type sequence (or its complement) would bind and, preferably, produce a unique amplification product, amplicon.

The term "specific for (a target sequence)" indicates that a probe or primer hybridizes under stringent hybridization conditions only to the target sequence in a sample comprising the target sequence.

As used herein, "amplified DNA" or "amplicon" refers to the nucleic acid amplification product of a target nucleic acid sequence that is part of a nucleic acid template. For example, to determine whether the soybean resulting from a sexual crossing contains genomic DNA from transgenic event from the soybean plant of the present invention, DNA extracted from a sample of soybean tissue can be subjected to the amplification method nucleic acid using a primer pair that includes a primer derived from flanking sequence in the plant genome adjacent to the inserted heterologous DNA insertion site, and a second primer derived from heterologous DNA

: 27/32 inserted to produce an amplicon that is diagnostic for the presence of the DNA event.

The amplicon is of a length and has a sequence that is also diagnostic for the event.

The amplicon can vary in length from the combined length of the primer seas, plus a pair of nucleotide bases, 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, E 10 81.82 , 83.84.85, 86.87, 88.89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, U 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162,163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178. 179, 180, 181, 182, 183, 184 , 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209 , 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224 , 225, 226, 227,228,229,230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252 , 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277 , 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302,303,304,305 m 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330 , 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355 , 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377,378,379,380,381,382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421,

i 28/32 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, or 500, 750, 1000, 1250, 1500 , 1750, 2000, or more nucleotide base pairs (plus or minus any of the increments listed above). Alternatively, the primer pair can be derived from the flanking sequence on both sides of the inserted DNA in order to produce an amplicon that includes the nucleotide sequence of the total insert. A member of a primer pair derived from the plant genomic sequence may be located a distance from the inserted DNA sequence. This distance can vary from a base pair of nucleotides to about twenty thousand base pairs of nucleotides. The use of the term "amplicon" specifically excludes dimer primers that can be formed in the DNA thermal amplification reaction.

Nucleic acid amplification can be achieved by any of the various methods of nucleic acid amplification known in the art, including the polymerase chain reaction (PCR). A variety of amplification methods are known in the art and are described, among others, in United States Patent No. 4,683,195 and United States Patent No. 4,683,202. The PCR amplification methods were developed to amplify up to 22 kb of genomic DNA. These methods, as well as other methods known in the DNA amplification technique, can be used in the practice of the present invention. The sequence of the heterologous transgene DNA insert, or genomic flanking sequence of a soybean e-wind, can be verified (and corrected if necessary) by amplifying such sequences from the event using primers derived from the sequences provided herein , followed by standard DNA sequencing of the cloned DNA PCR amplicon.

The amplicon produced by these methods can be detected by a variety of techniques. Agarose gel electrophoresis and etidium bromide staining is a well-known common method of detecting DNA amplicons.

Another such method is Genetic Bit Analysis where a DNA oligonucleotide is designated, which overlaps both the adjacent genomic flanking DNA sequence and the inserted DNA sequence.

The oligonucleotide is immobilized in wells of a micro-well plate.

Following PCR of the region of interest (using a primer in the inserted sequence and an adjacent flanking genomic sequence), a single stranded PCR product can be hybridized to the immobilized oligonucleotide and serves as a template for a reaction of simple base extension using a specific DNA polymerase and ddNTPs labeled "10 specifics for the next expected base.

The reading can be fluorescent or ELISA-based.

A signal indicates the presence of the flanking insert / sequence due to successful amplification, hybridization, and simple base extension.

All patents, patent applications, provisional applications, and publications referred to, or cited here, are incorporated by reference in their entirety to the extent that they are not inconsistent with the explicit teachings of this specification.

The following examples are included to illustrate procedures for practicing the invention and to demonstrate certain preferred embodiments of the invention.

These examples should not be construed as limiting.

It should be appreciated by those skilled in the art that the techniques revealed in the following examples represent specific approaches used to illustrate preferred models for their practice.

However, those skilled in the art must, in the light of the present disclosure, appreciate that any changes can be made in these specific embodiments, while similar results are still obtained without departing from the spirit and scope of the invention.

Unless otherwise indicated, all percentages are by weight, and all solvent mixture ratios are by volume, unless otherwise noted.

The following abbreviations are used, unless otherwise indicated.

: 30/32 AAD-12 aryloxyalkanoate dioxigenase-1 bp base ºC degrees Celsius DNA deoxyribonucleic acid DIG digoxigenin EDTA ethylenedioaminatetraacetic acid Kb kilobase ug microgram. upL microliter À 10 mL milliliter à M molar mass OLP overlap probe PCR polymerase chain reaction PTU plant transcription unit SDS sodium dodecyl sulfate SOP standard operating procedure SSC a buffer solution containing a mixture of sodium chloride and sodium citrate, pH 8.3 V volts

EXAMPLES Example 1. Event Specific Tagman Assay A specific TAQMAN ASSAY has been developed to detect the presence of DAS-68416-44 soy event and to determine plant zygism status in population generation. To develop a specific event assay, specific Tagman primers and probes were designed according to the DNA sequences located at the 5 'insert-to-plant junction. For specific detection of DAS-68416-4 soybean event, a 128-bp DNA fragment that reinforces this 5 'integration junction was amplified using two specific primers. The amplification of this PCR product was measured by a specific target MGB probe synthesized by Applied Biosystems containing the FAM reporter in its 5th terminal. The specificity of this Taqgman detection method for DAS-soy event

. 31/32 68416-4 was tested against 15 different soy events aad-12 and non-transgenic soy variety (Maverick) in duplex format with the specific endogenous soybean reference gene, lectin.

Example 1.1. GDNA isolation GDNA samples from 15 different AAD-12 soybean events and non-GM soy varieties were tested in this study.

Genomic DNA was extracted using the Qiagen DNeasy 96 Plant Kit. Fresh soybean leaf discs, eight per sample, were used for gDNA extraction using a modified Qiagen DNeasy 96 Plant Kit protocol. GD- í 10 NA was quantified with the Pico Green method according to the instructions. of the seller (Molecular probes, Eugene, OR). The samples were diluted with DNase-free water resulting in a concentration of 10 ng / yuE for the purpose of this study.

Example 1.2. Tagman Assay and Results Specific Tagman initiators and probes were designed for a DAS tag-specific assay DAS-68416-4. These reagents can be used with the conditions listed below to detect aad-12 within the DAS-68416-4 soy event. Table 1 lists the initiator and probe sequences that were developed specifically for the detection of DAS-68416-4 event. Table 1. PCR initiators and probes Advance EEE emo Reverse ACT O AA AATAAATI Advance

. 32/32 | 002 TTCTCTGCTGCCACGGGACTCGA-BHQ1 The multiplex PCR conditions for amplification are as follows: IX PCR buffer, 0.5 - 2.5 MM MgCl7, 0.2 mM dNTP, 0.2 µM Soybean Initiator16-F, 0.2 uM of Soy Starter 16-R, 0.2 uM of ZNOO7 Starter, 0.2 UM of ZNOO8 Starter, 0.08 µM of Soy416-Probe, 0.08 µM of ZNLTOO2, 40 U / mL of HotStart Tag, 30 ng gONA in a total reaction of 25 ul.

The cocktail was amplified using the following conditions: i) 95ºC per. 15 min ,, ii) 95ºC for 20 sec, iii) 60ºC for 60 sec, iv) repeat step ii-lii for - 35 cycles, v) 4ºC maintain.

Real-time PCR was performed on BIO-RAD ICYCLER'Y and ABI Gene Amp PCR System 9700 thermocyclists. Data analysis was based on measuring the cycle limit (CT), which is the number of PCR cycles when the fluorescence measurement reaches an adjusted value.

The CT value was calculated automatically by iCycler software.

The Tagman detection method for the DAS-68416-4 soybean event was tested against 16 different soybean events aad-12 and non-transgenic soybean varieties in duplex format with lectin-specific soy endogen as a reference gene.

This assay specifically detects the DAS-68416-4 soy event and does not produce or amplify any false-positive results from the controls (ie, the 15 different soy events aad-12 and non-GM soy varieties). Specific event initiators and probes can be used for the detection of the DAS-68416-4 soy event and these conditions and reagents are applicable for zygosity assays.

Claims (22)

'18 CLAIMS 1. “Method to determine zygiosity event of a soybean plant comprising an AAD-12 soybean event pDAB4468-0416, said event comprising a transgene construct comprising an AAD-12 gene, referred to as the transgene construct being flanked by a 5 'flanking of genomic DNA from soy and a 3' flanking of genomic DNA from soy, said method comprising: obtaining a DNA sample of genomic DNA from said soy plant; produce a contacted sample that contacts said DNA sample with q 10 a. a first initiator event and a second initiator event, wherein said first initiator event specifically binds to the transgene construct, said second initiator event specifically binds to 5 'genomic soy flanking DNA or referred to 3 'soybean genomic flanking DNA, and in which said first primer event and said second primer event produce an amplicon event when subjected to TAQMAN PCR b conditions. a reference advance primer and a reverse reference primer that produce a reference amplicon from an endogenous soy reference gene when subjected to conditions of TAQMAN c. a flourishing event probe that hybridizes to said amplicon event d. a flourishing reference probe that hybridizes to said reference amplicon; submit said contacted sample to TAQMAN PCR end point conditions based on flowering; quantifying said blooming e-wind probe that hybridizes to said amplicon event; quantifying said flourishing reference probe which hybridizes to said reference amplicon; compare amounts of hybridized blooming event probe to hybridized blooming reference probe; and determine zygosity of pDAB4468-0416 by comparing flourishing proportions of hybridized flowering event probe and probe . 2/3 hybridized flourishing reference. A method according to claim 1, wherein said amplicons consist of 50-150 residues. A method according to claim 1, wherein said 5 flanking DNA comprises residues 1-2730 of SEQ ID NO: 1, and said 3 'flanking DNA comprises 9122-10,212 of SEQ ID NO: L. A method according to claim 1, wherein said transgene construct consists of residues 2731-9121 of SEQ ID NO: 1. 5. The method of claim 1, wherein said reference gene is an endogenous soy lectin gene. A method according to claim 1, wherein said second initiator event binds residues 2530-2730 of SEQ ID NO: 1, or the complements thereof. Method according to claim 1, wherein said second initiator event binds residues 9122-9322 of SEQ ID NO: |. 8. Method, according to claim 1, in which the said method is used to generate introgression of the event in another soybean line. Method according to claim 8, wherein said other soy line lacks said event. 10. The method of claim 1, wherein said amplicons consist of 100-200 base pairs. 11. The method of claim 1, wherein said reference gene comprises or hybridizes to a sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7. 12. The method of claim 1, wherein said reference primers comprise SEQ ID NO: 5 and SEQ ID NO: 6, and the reference probe comprising SEQ ID NO: 7. 13. The method of claim 1, wherein said probes are labeled with a fluorescent dye and an extinguisher. '33 A method according to claim 13, wherein said probe event comprises FAM as said fluorescent dye at the 5 'end of said probe event and an MGB extinguisher at the 3' end of said probe event . A method according to claim 13, wherein said reference probe is labeled with HEX at the 5 'end of said reference probe and a Black Hole Quencher 1 (BHQ!) At the 3' end of said reference probe . 16. The method of claim 1, wherein said probe event comprises SEQ ID NO: 4. Í 17. Method according to claim 1, wherein said initiating events are selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 3. 18. Method according to claim 1, in which the results of said method are read directly on a plate reader. 19. The method of claim 1, wherein said DNA sample is obtained from a soybean plant in a field. 20. Kit for carrying out the method as defined in claim 1, said kit comprising said first initiator event, said second initiator event, said reference advance initiator, said reverse reference initiator, said event probe, and said reference probe. 21. The kit of claim 20, wherein said initiator events consist of SEQ ID NO: 2 and SEQ ID NO: 3, said reference primers consist of SEQ ID NO: 5 and SEQ ID NO: 6, said probe event consists of SEQ ID NO: 4, and said reference probe consists of SEQ ID NO: 7. 22. Isolated polynucleotide comprising a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQIDNO4, SEQID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7. Pvull 2 CSVMV - ORF1 | | MARÇS AUDIO ORF23 PAT EEN SSEsSass Y Genome Soy Genome | DNA Digested with Pvull and IcoRV | EcoRV v Pvull CSVNV EcoR) Open AUTO pan17 ORI R AAD12 iM AIR oR PAT MAR ARO 2s sc - [SS | | Connect to Genome Walker Adapter + y Du p — D SDS QuE ES) EE caea: THESE IS - -> e = +> = ++ AP ES LENdo3 AP1 ES LENdO3 ES PATENdO3 API PCR Primary PCR Primary PCR Primary PCR Secondary PCR Secondary PCR Secondary AP2 ES LEnd04 AP2 ES LENdO4 ES PATENdo04 AP2> <<> +> = << mea | A o PA —— fo A End limit 5th End limit 5th End limit 3rd EcoRV Genomic DNA from the soy event DAS-68416-4 was digested with EcoRV, or Pvu II, and used to generate corresponding GENOMEWALKERTY libraries, which were used as templates to amplify the target DNA sequences. - 2/4 AAD-12 - ORF 23 - AtUbi1O and CsVMV Promoter RB7 MAR ORF1 Promoter Flanking Region 5 'PAT Flanking Region 3' | First | Primer Primer Primer - Primer | Primer 416-5-1] / 4468-1R [4488-1 4450-2R | 4468-2 - 4468-3R | 4468-9 416-3-1R Amplicon 1 i Í Í Amplicon 4 Amplicon 2 Amplicon 3 The schematic diagram represents the locations of the primer and cloning strategy for sequencing the full length of the DAS-68416-4 soy event from the 5 'to limits 3 'FIG. two . 3/4 y AAD-12 ORF 23 q AtUbi1O Promoter CSVMV Promoter PAT RB7 MAR ORF 1 Soy 416F Soy 416R Flanking Region 3 'Flanking Region 5 N | 16PATGO3 16LENdGO04 16PATGO4 16LENdGO3 ANLENdOS PATENdO6S 16LENdGO1 ANIILENdO6 ISPATOO2 16PATGO1 16LENdG02 The schematic diagram represents the locations of the primer to confirm the total length sequence of the DAS-68416-4 soybean event from the limits of 5-to-5 '. 3 . . 4/4 -. Flanking Region 5 'T-DNA Insertion Site Flanking Region 3' 192: | Insertion BP 9 463HR EEE SEE a in ESSA AAA A HOUSES T - AAD-12 aryloxyalkanoate dioxigenase-1 The schematic diagram represents the locations of the primer to confirm the insertion site sequence of the AAD-12 soy event DAS-68416-4.