Soybean transgenic event mon 87708 and methods of use thereof

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TRANSGENIC SOYBEAN EVENT MON 87708 AND METHODS OF USE THEREOF

CROSS REFERENCE TO RELATED REQUESTS

This application claims the benefit of US Provisional Application No. 61/243,227 filed on September 17, 2009, which is incorporated herein by reference in its entirety.

INCORPORATION OF SEQUENCE LISTING

The sequence listing that is contained in the file with the name “55544-0001_seqlisting.txt”, which is 19.5 kilobytes (size as measured in Microsoft Windows®) and was created on August 13, 2010, is presented with this application by electronic filing and is incorporated by reference in this invention.

FIELD OF THE INVENTION

The invention relates to the transgenic event of Glycine max MON 87708. The event exhibits tolerance to the herbicide dicamba. The invention also relates to plants, plant parts, plant seeds, plant cells, agricultural products and methods related to the MON 87708 event and provides nucleotide molecules that are unique to the event and were created in connection with the insertion of DNA transgenic in the genome of a Glycine max plant.

BACKGROUND OF THE INVENTION

Soybean (Glycine max) is an important crop in many areas of the world, and biotechnology methods have been applied to this crop in order to produce soybeans with desirable characteristics. A desirable characteristic of this type is tolerance to a herbicide. Expression of a herbicide tolerance transgene in a plant can confer the desirable trait of herbicide tolerance in the plant, but expression of the transgene can be influenced

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by the chromosomal location and the genomic result of the transgene insertion. For example, it has been observed in plants that there is often variation in the level and pattern of transgene expression between individual events that differ in the site of chromosomal insertion of the transgene but are otherwise identical. There may also be undesirable and/or desirable phenotypic or agronomic differences between events. Because of this, it is often necessary to produce and analyze a large number of individual plant transformation events in order to select an event that has both the desired trait and the optimal phenotypic and agricultural characteristics necessary to make it suitable for commercial purposes. Such selection often requires greenhouse and field trials with many events over the course of several years, in multiple locations, and under a variety of conditions so that a significant amount of agronomic, phenotypic, and molecular data can be collected. The resulting data and observations must then be analyzed by groups of scientists and agronomists with the aim of selecting a commercially suitable event. That type of event, once selected, can then be used to introgress the desirable trait into other genetic backgrounds using plant breeding methods, and thus produce a number of different crop varieties containing the desirable trait. and are adequately adapted to specific local growing conditions.

SUMMARY OF THE INVENTION

The invention provides transgenic soybean plants designated event MON 87708, which exhibit commercially acceptable tolerance to applications of dicamba herbicide, the representative seed having been deposited with the American Type Culture Collection (ATCC, acronym corresponding to "American Type Culture Collection") with Accession No.

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PTA-9670. The invention further provides novel DNA molecules related to the soybean event MON 87708 and methods of using these molecules. The invention also provides seeds, progeny, plant parts, cells and staple products of soybean event MON 87708. The invention further provides methods for using soybean event MON 87708 and methods for producing dicamba-tolerant soybeans.

The invention provides recombinant DNA molecules related to the soybean event MON 87708. These recombinant DNA molecules may comprise nucleotide molecules having a nucleotide sequence representing a region of the genomic DNA flanking the transgene insertion, and/or a region of the transgene insertion, and/or a sequence contiguous to any of these regions. such as a junction region between the transgenic insert and the flanking genomic DNA of soybean event MON 87708. The invention further provides DNA molecules useful as primers and probes as diagnostic for soybean event MON 87708 and diagnostic amplicons for the presence of soybean event MON 87708. Soybean plants, plant cells, plant parts, goods are further described. of consumption, progeny and seeds that comprise these molecules.

The invention provides methods, compositions and kits useful for detecting the presence and/or absence of DNA derived from soybean event MON 87708 and therefore, the presence and/or absence of the event. The invention provides a method for the detection of MON 87708 by contacting a sample comprising DNA with a primer set that when used in a nucleic acid amplification reaction with genomic DNA from the soybean event MON 87708 produces an amplified DNA diagnostic of the MON 87708 event. soybean MON 87708, carrying out a nucleic acid amplification reaction thereby producing the amplified DNA, and detecting the presence and/or

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absence of amplified DNA. The invention further provides a method for the detection of MON 87708 by contacting a sample comprising DNA with a probe that when used in a hybridization reaction with DNA from soybean event MON 87708 hybridizes to a DNA molecule specific for the event. soybean MON 87708, carrying out a hybridization reaction, and detecting the hybridization of the probe to the DNA molecule. Also provided are kits comprising the methods and compositions of the invention useful for detecting the presence of DNA derived from soybean event MON 87708.

The invention provides a soybean plant, seed, plant cell, progeny plant, plant part, or commodity derived from a plant, plant cell or seed of soybean event MON 87708. The invention further provides a soybean plant, seed, plant cell, progeny plant, plant part or commodity comprising a recombinant DNA molecule having a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-8, and the complements and fragments thereof. The invention further provides a soybean plant, seed, plant cell, progeny plant, plant part or commodity derived from the plant or seed of soybean event MON 87708 and comprising a recombinant DNA molecule that produces an amplified DNA molecule. comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 7, and/or SEQ ID NO: 8 in a DNA amplification method.

The invention provides a method for controlling weeds in a field by planting soybean event MON 87708 and then applying an effective dose of dicamba herbicide capable of controlling weeds without injuring the soybean event MON 87708 plants. The invention further provides a method to control weeds in a field by applying an effective dose of dicamba herbicide to control weeds in a field and then planting soybean event MON 87708 in the field. The invention further provides a method for

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produce soybean seed essentially free of the seeds of toxic weed species by planting seed of a dicamba-tolerant soybean variety MON 87708 in a field, applying an effective post-emergence dose of dicamba herbicide sufficient to kill the toxic weed species to the field, and collecting seed from the field.

The invention provides methods for producing a soybean plant and/or seed that tolerates the application of dicamba herbicide by sexually crossing a soybean event plant MON 87708 comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 7 , and/or SEQ ID NO: 8 with a second soybean plant, thereby producing a seed, growing the seed to produce progeny plants, treating the progeny plants with dicamba, and selecting a progeny plant that is tolerant to dicamba. The methods may also include self-fertilizing the selected progeny plant to produce a plurality of second generation progeny plants and selecting among these a dicamba-tolerant plant. The methods may further include sexually crossing the selected progeny plant with another soybean plant to produce seed, growing the seed to produce a second generation of progeny plants, treating the second generation of progeny plants with dicamba, and selecting a progeny plant from second generation that is dicamba tolerant. The invention provides methods for producing a soybean plant and/or seed that tolerates the application of dicamba herbicide by self-fertilizing a dicamba-tolerant soybean event plant MON 87708 comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 7, and/or SEQ ID NO: 8, thereby producing a seed, cultivating the seed to produce progeny plants, treating the progeny plants with dicamba; and select a progeny plant that is tolerant to dicamba.

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The invention provides methods for determining the zygosity of a soybean event plant MON 87708 or seed comprising contacting a soybean DNA sample with a set of primers comprising SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14 and a probe set comprising SEQ ID NO: 15 and SEQ ID NO: 16; then carrying out a nucleic acid amplification reaction with the sample, primer set, and probe set; then detect in the nucleic acid amplification reaction a first fluorescent signal that is diagnostic for the MON 87708 event and a second fluorescent signal different from the first fluorescent signal and that is diagnostic for native soybean genomic DNA corresponding to the insertion location of the event transgene MON 87708; and analyze the presence and/or absence of the first fluorescent signal and the second fluorescent signal in the nucleic acid amplification reaction, where the presence of both fluorescent signals indicates that the sample is heterozygous for the MON 87708 event and the presence of only the first fluorescent signal indicates that the sample is homozygous for the MON 87708 event.

The invention further provides a soybean plant, seed, plant cell, or plant part comprising the soybean haplotype region at linkage group 9 at approximately map position 143.5 comprising a dicamba tolerance gene and further defined through the haplotype window 19743 and 19767, and methods of using it. The foregoing and other aspects of the invention will become more apparent from the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 illustrates the organization of the transgenic insert in the genome of soybean event MON 87708; [A] corresponds to the relative position of SEQ ID NO: 1, which is sixty nucleotides from the junction between the soybean genomic DNA and the

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DNA portion 5 of the transgenic DNA insert; [A'] corresponds to the relative position of SEQ ID NO: 7, which is one hundred nucleotides from the junction between the soybean genomic DNA and the 5' portion of the transgene insert DNA; [B] corresponds to the relative position of SEQ ID NO: 2, which is sixty nucleotides from the junction between the soybean genomic DNA and the 3' portion of the transgene insert DNA; [B'] corresponds to the relative position of SEQ ID NO: 8, which is one hundred nucleotides from the junction between the soybean genomic DNA and the 3' portion of the transgenic insert DNA; [C] corresponds to the relative position of SEQ ID NO: 3, which is the soybean genomic sequence flanking the arbitrarily assigned/designated 5' end of the expression cassette integrated into the genome in event MON 87708; [D] corresponds to the relative position of SEQ ID NO: 4, which is the soybean genomic sequence flanking the arbitrarily assigned/designated 3' end of the expression cassette integrated into the genome in event MON 87708; [E] represents the various elements that comprise SEQ ID NO: 5 and is the sequence of the expression cassette inserted into the genome of event MON 87708; and [F] represents the contiguous sequence (provided as SEQ ID NO: 6) comprising, as represented in the figure from left to right, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 4, in where SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 7, and SEQ ID NO: 8 are included, as these sequences are present in the genome at event MON 87708.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 is a sixty nucleotide sequence that represents the 5' junction between soybean genomic DNA and the integrated transgenic expression cassette. SEQ ID NO: 1 is positioned in SEQ ID NO: 6 at nucleotide position 1097-1156.

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SEQ ID NO: 2 is a sixty nucleotide sequence that represents the 3' junction between soybean genomic DNA and the integrated transgene expression cassette. SEQ ID NO: 2 is positioned in SEQ ID NO: 6 at nucleotide position 4100-4159.

SEQ ID NO: 3 is the 5' sequence flanking the inserted DNA from soybean event MON 87708 up to and including a transgenic DNA insertion region.

SEQ ID NO: 4 is the 3' sequence flanking the inserted DNA from soybean event MON 87708 up to and including a transgenic DNA insertion region.

SEQ ID NO: 5 is the sequence of the integrated transgene expression cassette.

SEQ ID NO: 6 is the nucleotide sequence that represents the contig of the 5' sequence flanking the inserted DNA of soybean event MON 87708 (SEQ ID NO: 3), the sequence of the inserted DNA (SEQ ID NO: 5) , and the 3' sequence flanking the inserted DNA from soybean event MON 87708 (SEQ ID NO: 4) and includes SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 7, and SEQ ID NO: 8.

SEQ ID NO: 7 is a sequence of one hundred nucleotides that represents the 5' junction between the soybean genomic DNA and the integrated transgene expression cassette.

SEQ ID NO: 8 is a sequence of one hundred nucleotides that represents the 3' junction between the soybean genomic DNA and the integrated transgene expression cassette.

SEQ ID NO: 9 is the sequence of a primer called Primer SQ13570 and used to identify the soybean event MON 87708. It is complementary to the expression cassette inserted in the region close to the insertion edge of the 3' transgene. A PCR amplicon produced from an assay

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TAQMAN® (PE Applied Biosystems, Foster City, CA) using the combination of primers SQ13570 and SQ13571 (SEQ ID NO: 10) is a positive result for the presence of the MON 87708 event.

SEQ ID NO: 10 is the sequence of a primer called Primer SQ13571 and used to identify the soybean event MON 87708. It is complementary to a 3' region that flanks the inserted expression cassette and close to the transgenic DNA insertion edge. A PCR amplicon produced from a TAQMAN® assay (PE Applied Biosystems, Foster City, CA) using the combination of primers SQ13570 (SEQ ID NO: 9) and SQ13571 is a positive result for the presence of the MON 87708 event.

SEQ ID NO: 11 is the sequence of a probe called Probe PB4655 and used to identify the soybean event MON 87708. It is complementary to a region that encompasses the 3' junction of the inserted expression cassette and the genomic DNA. This probe is a synthetic oligonucleotide labeled with 6-FAM™. The release of a fluorescent signal in an amplification reaction employing primers SQ13570 and SQ13571 (SEQ ID NO: 9-10) in combination with the 6-FAM™ labeled probe PB4655 is diagnostic of event MON 87708 in a TAQMAN® assay.

SEQ ID NO: 12 is the sequence of a primer called Primer SQ20632 and used to identify the zygosity of the MON 87708 event.

SEQ ID NO: 13 is the sequence of a primer called Primer SQ20636 and is used to identify the zygosity of the MON 87708 event and soybean wild type.

SEQ ID NO: 14 is the sequence of a primer called Primer SQ20637 and is used to identify the zygosity of wild type soybean.

SEQ ID NO: 15 is the sequence of a probe called Probe PB10130 and is used for a zygosity assay of the MON 87708 event.

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SEQ ID NO: 16 is the sequence of a probe called Probe PB10131 and is used for a soybean wild-type zygosity assay.

DETAILED DESCRIPTION

The following definitions and methods are provided to better define the invention and to guide those skilled in the art in the practice of the invention. Unless otherwise indicated, the terms should be understood in accordance with conventional usage by those skilled in the relevant art.

The invention provides a transgenic soybean event MON 87708 that exhibits commercially acceptable tolerance to dicamba herbicide applications. The event comprises a single insertion of transgenic DNA into the chromosome/genome of soybean germplasm. An “event” is produced by: (i) transformation of a plant cell with a nucleic acid construct that includes a transgene of interest, (ii) regeneration of a plant population that is the result of the insertion of the transgene into the genome of the plant, and (iii) selection of a particular plant characterized by insertion of the transgene at a particular location in the plant genome. The term “event” refers to the original transformant that includes the transgene inserted at the particular location in the plant genome. The term “event” further refers to the progeny of the transformant that includes the transgene inserted at the particular location in the plant genome. Said progeny can be produced through sexual exogamy between the transformant, or its progeny, and another plant. Said other plant may be a transgenic plant comprising the same transgene or a different transgene and/or a non-transgenic plant, such as one of a different variety. Even after repeated backcrossing to a recurrent parent, the inserted DNA and flanking DNA from the transformed parent are present in the progeny of the cross at the same genomic location.

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As used in this invention, the term "soybean" means Glycine max and includes all varieties of plants that can be grown with soybeans, including wild soybean species as well as those plants belonging to Glycine that allow interspecies cultivation.

The term "event" further refers to a DNA molecule of the original transformant comprising the inserted DNA and the flanking soybean genomic DNA immediately adjacent to either side of the inserted DNA. This DNA molecule is created through the act of inserting the transgenic DNA into the genome of the soybean plant, that is, through the act of transformation. Therefore, this DNA molecule comprises a nucleotide sequence that is both event-specific and unique to the genome of the soybean plant into which the transgenic DNA has been inserted, in the sense that this nucleotide sequence contains both the sequence of a particular region of soybean genomic DNA and of the transgenic DNA insert. The arrangement of the DNA inserted into soybean event MON 87708 in relation to the surrounding soybean plant genomic DNA is, therefore, specifically and unique to soybean event MON 87708. This DNA molecule is also an integral part of the chromosome soybean from event MON 87708 and, as such, is static in the plant and can be passed to the plant's progeny.

The MON 87708 event comprises a transgene that confers tolerance to dicamba herbicide applications to the soybean plant. “Dicamba” refers to 3,6-dichloro-2-methoxybenzoic acid. Dicamba is a synthetic auxin herbicide useful for controlling broadleaf weeds. Soybean plants were transformed with dicamba mono-oxygenase (DMO), an enzyme cloned from Stenotrophomonas maltophilia which is commonly found in soil rhizosphere. Dicamba mono-oxygenase is an enzyme that catalyzes the deactivation of dicamba through an O-demethylation reaction to the acidic non-herbicidal compound 3,5IF-2019-80266805-APN-ANP#INPI

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dichlorosalicylic. In some areas of the world, seeds from toxic weed species can contaminate harvested soybeans which can affect the health and nutrition of animals fed the contaminated soybean commodities. These plants can be eliminated from a soybean field by treatment with a dicamba herbicide. Members of this group of toxic weeds include Cardaria spp, Heliotropium spp, Centaurea spp., Senecio spp., Crotalaria spp., Solanum spp., Xanthium spp., Amsinckia spp., Cassia spp., Sesbania spp., Datura spp. , Ricinus spp., Argemone spp., Corchorus spp., Impomoea spp., and Echium spp.

As used in this invention, the term "recombinant" refers to a form of DNA and/or protein and/or an organism that would normally be found in nature and as such was created by human intervention. Such human intervention can produce a recombinant DNA molecule and/or a recombinant plant. As used in this invention, a “recombinant DNA molecule” is a DNA molecule that comprises a combination of DNA molecules that would naturally occur together and is the result of human intervention, e.g., a DNA molecule. DNA that is composed of a combination of at least two DNA molecules heterologous to each other, and/or a DNA molecule that is artificially synthesized and comprises a polynucleotide sequence that deviates from the polynucleotide sequence that would normally exist in nature, and/or a DNA molecule that comprises a transgene artificially incorporated into a host cell genomic DNA and the associated flanking DNA of the host cell genome. An example of a recombinant DNA molecule is a DNA molecule described in this invention that is the result of insertion of the transgene into soybean genomic DNA, which can ultimately result in the expression of an RNA molecule and/or recombinant protein in that organism.

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As used in this invention, a “recombinant plant” is a plant that would normally exist in nature, is the result of human intervention, and contains a transgene and/or heterologous DNA molecule incorporated into its genome. As a result of such genomic alteration, the recombinant plant is distinctively different from the related wild-type plant. An example of a recombinant plant is a soybean plant described in this invention as Event MON 87708.

As used in this invention, the term "transgene" refers to a nucleotide molecule artificially incorporated into a host cell genome. That type of transgene can be heterologous to the host cell. The term “transgenic plant” refers to a plant comprising that type of transgene.

As used in this invention, the term “heterologous” refers to a first molecule not normally found in combination with a second molecule in nature. For example, a molecule can be derived from a first species and inserted into the genome of a second species. The molecule would therefore be heterologous to the host and artificially incorporated into a genome of the host cell.

As used in this invention, the term "chimeric" refers to a single DNA molecule produced by the fusion of a first DNA molecule to a second DNA molecule, where neither the first nor the second DNA molecule would be normally found in that configuration, that is, fused to the other. Thus, the chimeric DNA molecule is a new DNA molecule not otherwise normally found in nature.

The invention provides DNA molecules and their corresponding nucleotide sequences. As used in this invention, the term “DNA”,

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"DNA molecule", "nucleotide molecule" refers to a DNA molecule of genomic or synthetic origin, that is, a polymer of deoxyribonucleotide bases or a polynucleotide molecule, read from the 5' end (upstream) to the 3' end. (downstream). As used in this invention, the term "DNA sequence", "nucleotide sequence" or "polynucleotide sequence" refers to the nucleotide sequence of a DNA molecule. The nomenclature used in this invention is that required by Title 37 of the United States Code of Federal Regulations § 1.822 and set forth in the tables in WIPO Standard ST.25 (1998), Appendix 2, Tables 1 and 3. By convention, the nucleotide sequences of the invention provided as SEQ ID NO: 1-8 and their fragments are described with reference to only one strand of the two strands of complementary nucleotide sequences. By implication, complementary sequences (i.e., complementary strand sequences), also referred to in the art as reverse complementary sequences, are within the scope of the invention and are expressly intended to be within the scope of the claimed subject matter.

The nucleotide sequence corresponding to the complete nucleotide sequence of the inserted transgenic DNA and substantial segments of the soybean genomic DNA flanking either end of the inserted transgenic DNA is provided herein as SEQ ID NO: 6. A subsection of this is the Inserted transgenic DNA provided as SEQ ID NO: 5. The nucleotide sequence of the soybean genomic DNA physically linked by phosphodiester bond ligation to and therefore flanking the 5' end of the inserted transgenic DNA is set forth as shown in SEQ ID NO: 3. The nucleotide sequence of the soybean genomic DNA physically linked by

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The soybean event MON 87708 further comprises two regions, one spanning the 5' location and one spanning the 3' location where the transgenic DNA is inserted into the genomic DNA, referred to in this invention as the 5' and 3' junction, respectively. A “junction sequence” or “junction region” refers to the corresponding DNA sequence and/or DNA molecule that encompasses the inserted transgenic DNA and the adjacent flanking genomic DNA. The binding sequences can be arbitrarily represented by the two 10 nucleotide sequences provided as SEQ ID NO: 1 and SEQ ID NO: 2, each representing 30 nucleotides of flanking genomic DNA adjacent to, and contiguous with, 30 nucleotides of DNA inserted. Alternatively, the binding sequences may be arbitrarily represented by the two 100 nucleotide sequences provided as SEQ ID NO: 7 and SEQ ID NO: 8, each representing 50 nucleotides of flanking genomic DNA adjacent to, and contiguous with, 50 nucleotides. of inserted DNA. These nucleotides are connected by phosphodiester ligation and in soybean event MON 87708 they are present as part of the genome. In soybeans, the identification of one or more of SEQ ID NO: 1, SEQ ID NO. 2, SEQ ID NO: 7, and SEQ ID NO: 8 in a sample derived from a soybean plant, seed or plant part is determining that the DNA was obtained from soybean event MON 87708 and is diagnostic for the presence in a DNA sample from soybean event MON 87708. The invention therefore provides a DNA molecule containing at least the nucleotide sequence as set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 7, 25 and/or SEQ ID NO: 8 Any DNA segment derived from the transgenic soybean event MON 87708 that is sufficient to include SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 7, and/or SEQ ID NO: 8 is within the scope of the

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invention. Additionally, any polynucleotide comprising a sequence complementary to any of the sequences described within this paragraph is within the scope of the invention. Figure 1 illustrates the physical arrangement of SEQ ID NO: 1-5 and 7-8 in relation to SEQ ID NO. 6 arranged from 5' to 3'.

The invention provides exemplary DNA molecules that can be used either as primers or probes to diagnose the presence of DNA derived from soybean plant event MON 87708 in a sample. Such primers or probes are specific for a target nucleic acid sequence and as such are useful for the identification of the nucleic acid sequence of soybean event MON 87708 by the methods of the invention described in this invention.

A "primer" is typically an isolated, highly purified polynucleotide that is designated for use in specific annealing and hybridization methods involving thermal amplification. A pair of primers with template DNA, such as a soybean genomic DNA sample, can be used in a thermal amplification, such as polymerase chain reaction (PCR), to produce an amplicon, where the amplicon produced from said reaction would have a DNA sequence corresponding to the sequence of the template DNA located between the two sites where the primers hybridized to the template. As used in this invention, an "amplicon" is a piece or fragment of DNA that has been sintered using amplification techniques. An amplicon of the invention comprises at least SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 7, and/or SEQ ID NO: 8. A primer is typically designed to hybridize to a complementary target DNA strand to form a hybrid between the primer and the target DNA strand, and the presence of the primer is a point of recognition by a polymerase to begin primer extension (i.e. , polymerization of additional nucleotides into a molecule of

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elongation nucleotides) using the target DNA strand as a template. Primer pairs, as used in the invention, are intended to refer to the use of two primers that join opposite strands of a double-stranded nucleotide segment for the purpose of linearly amplifying the polynucleotide segment between the positions sought for the binding by the individual members of the primer pair, typically in a thermal amplification reaction or other conventional nucleic acid amplification methods. Exemplary DNA molecules useful as primers are provided as SEQ ID NO: 9-10. The pair of primers provided as SEQ ID NO: 9 and SEQ ID NO: 10 are useful as a first DNA molecule and a second DNA molecule that is different from the first DNA molecule, and both are, individually, of sufficient length of contiguous nucleotides of SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6 to function as DNA primers that, when used together in a thermal amplification reaction with template DNA derived from soybean event MON 87708, produce an amplicon comprising SEQ ID NO: 2.

A “probe” is an isolated nucleic acid that is complementary to a strand of a target nucleic acid. Probes according to the invention include not only deoxyribonucleic or ribonucleic acids but also polyamides and other probe materials that specifically bind to a target DNA sequence and the detection of such binding can be useful to diagnose, discriminate, determine or confirm the presence of that target DNA sequence in a particular sample. A probe may be linked to a conventional detectable reporter molecule or label, e.g., a radioactive isotope, ligand, chemiluminescent agents, or enzyme. An exemplary DNA molecule useful as a probe is provided as SEQ ID NO: 11.

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Probes and primers according to the invention may have complete sequence identity with the target sequence, although primers and probes that differ from the target sequence that retain the ability to preferentially hybridize to target sequences can be designed by conventional methods. For a nucleic acid molecule to serve as a primer or probe, it must only be sufficiently complementary in sequence to be able to form a stable double-stranded structure under the particular solvent and salt concentrations employed. Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of soybean event MON 87708 transgenic DNA in a sample. Probes and primers generally have at least about 11 nucleotides, at least about 18 nucleotides, at least about 24 nucleotides, or at least about 30 nucleotides or more in length. Such probes and primers hybridize specifically to a target DNA sequence under harsh hybridization conditions. Conventional severity conditions are described by Sambrook et al., 1989, and by Haymes et al., In: Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, DC (1985). As used in this invention, two nucleic acid molecules are capable of specifically hybridizing to each other if the two molecules are capable of forming a double-stranded, anti-parallel nucleic acid structure. A nucleic acid molecule is the “complement” of another nucleic acid molecule if they exhibit complete complementarity. As used in this invention, the molecules exhibit “complete complementarity” when each nucleotide of one of the molecules is complementary to a nucleotide of the other. Two molecules are “minimally complementary” if they can hybridize to each other with sufficient stability to allow them to remain paired to each other under at least

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fewer conventional “low severity” conditions. Similarly, molecules are “complementary” if they can hybridize with each other with sufficient stability to allow them to remain paired to each other under conventional “high stringency” conditions. Departures from complete complementarity are therefore permissible, as long as such departures do not completely exclude the ability of the molecules to form a double-stranded structure.

As used in this invention, the term "isolated" refers to at least partially separating a molecule from other molecules normally associated with it in its native or natural state. In one embodiment, the term "isolated" refers to a DNA molecule that is at least partially separated from the nucleic acids which normally flank the DNA molecule in its native or natural state. Therefore, DNA molecules fused to regulatory or coding sequences with which they are not normally associated, for example, as a result of recombinant techniques, are considered isolated in this invention. Such molecules are considered isolated even when they are integrated into the chromosome of a host cell or present in a nucleic acid solution with other DNA molecules.

Any number of methods well known to those skilled in the art may be employed to isolate and manipulate a DNA molecule, or fragment thereof, described in the invention. For example, PCR (polymerase chain reaction) technology may be employed to amplify a particular starting DNA molecule and/or to produce variants of the original molecule. DNA molecules or fragments thereof can also be obtained by other techniques such as directly synthesizing the fragment by chemical means, as is common practice using an automatic oligonucleotide synthesizer.

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The DNA molecules and corresponding nucleotide sequences provided in this invention are therefore useful for, among other things, identifying soybean event MON 87708, selecting plant varieties or hybrids comprising soybean event MON 87708, detecting the presence of DNA derived from the transgenic soybean event MON 87708 in a sample, and monitor samples to determine the presence and/or absence of soybean event MON 87708 or plant parts derived from soybean event MON 87708.

The invention provides soybean plants, progeny, seeds, plant cells, plant parts (such as pollen, ovule, pod, flower tissue, root tissue, stem tissue and leaf tissue), and commodities. These plants, progeny, seeds, plant cells, plant parts and commodities contain a detectable amount of a polynucleotide of the invention, that is, such as a polynucleotide having at least one of the sequences provided as SEQ ID NO: 1-8. The plants, progeny, seeds, plant cells, and plant parts of the invention may also contain one or more additional transgenes. That type of transgene can be any nucleotide sequence that encodes a protein or RNA molecule that confers a desirable characteristic including, but not limited to, increased insect resistance, increased water use efficiency, increased yield, increased drought resistance. , higher seed quality, improved nutritional quality, and/or greater tolerance to herbicides, wherein the desirable trait is measured relative to a soybean plant lacking said additional transgene.

The invention provides soybean plants, progeny, seeds, plant cells, and plant parts such as pollen, ovule, pod, flower, root or stem tissue, and leaves derived from a transgenic soybean plant event MON 87708. Representative seed sample from soybean event MON 87708 has been deposited in accordance with the Budapest Treaty for the purpose of making

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invention possible. The repository selected to receive the deposit is the American Type Culture Collection (ATCC), which has an address at 10801 University Boulevard, Manassas, Virginia USA, Zip Code 20110. ATCC repository has assigned accession number PTA-9670 to the seed with event MON 87708.

The invention provides a microorganism comprising a DNA molecule having SEQ ID NO: 1 and SEQ ID NO: 2 present in its genome. An example of that type of microorganism is a transgenic plant cell. Microorganisms, such as a plant cell of the invention, are useful in many industrial applications, including but not limited to: (i) use as a research tool for scientific pursuits or industrial research; (i) use in culture to produce endogenous or recombinant carbohydrate, lipid, nucleic or protein acid products or small molecules that can be used for subsequent scientific research or as industrial products; and (i¡) use with modern plant tissue culture techniques to produce transgenic plants or plant tissue cultures that can then be used for agricultural research or production. The production and use of microorganisms such as transgenic plant cells uses modern microbiological techniques and human intervention to produce a unique, man-made microorganism. In this process, recombinant DNA is inserted into a plant cell genome to create a transgenic plant cell that is separate and unique from natural plant cells. This transgenic plant cell can then be cultured much like bacteria and yeast cells using modern microbiology techniques and can exist in a single-cell, undifferentiated state. The new genetic composition of the plant cell and phenotype is a

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<img file="AR119652A2_D0021.tif" />

technical effect created by the integration of heterologous DNA into the cell's genome. Another aspect of the invention is a method of using a microorganism of the invention. Methods of using microorganisms of the invention, such as transgenic plant cells, include (i) methods of producing transgenic cells by integrating recombinant DNA into the genome of the cell and then employing this cell to derive additional cells possessing the same heterologous DNA. ; (i) methods of culturing cells containing recombinant DNA using modern microbiology techniques; (iii) methods for producing and purifying endogenous or recombinant carbohydrate, lipid, nucleic acid or protein products from cultured cells; and (iv) methods of using modern plant tissue culture techniques with transgenic plant cells to produce transgenic plants or transgenic plant tissue cultures.

The plants of the invention can pass the event DNA, including the transgene, to progeny. As used in this invention, "progeny" includes any plant, seed, plant cell, and/or regenerable plant part that comprises the DNA of the event derived from an ancestor plant and/or a polynucleotide that has at least one of the sequences provided as SEQ ID NO: 1 and SEQ ID NO: 2. Plants, progeny, and seeds may be homozygous or heterozygous for the transgene. The progeny can be grown from seeds produced by a plant with the soybean event MON 87708 and/or from seeds produced by a plant fertilized with pollen from a plant with the soybean event MON 87708.

Progeny plants can be self-pollinated (also known as “selfing”) to generate a true plant breeding line, that is, plants homozygous for the transgene. The

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<img file="AR119652A2_D0022.tif" />

Selfing of appropriate progeny can produce plants that are homozygous for both added exogenous genes.

Alternatively, progeny plants can be crossed by outcrossing, e.g., crossed with another unrelated plant, to produce a varietal or hybrid seed or plant. The other unrelated plant may be transgenic or non-transgenic. A varietal or hybrid seed or plant of the invention can therefore be derived by crossing a first parent lacking the specific and unique DNA of soybean event MON 87708 with a second parent comprising soybean event MON 87708, giving resulting in a hybrid that comprises the specific and unique DNA of soybean event MON 87708. Each parent can be a hybrid or an inbred/varietal, as long as the crossing or reproduction results in a plant or seed of the invention, that is, a seed that has at least one allele that contains the unique and specific DNA. from soybean event MON 87708 and/or SEQ ID NO: 1 and SEQ ID NO: 2. Therefore, two different transgenic plants can be mated to produce hybrid offspring containing two independently segregating, aggregated, exogenous genes. For example, the dicamba-tolerant soybean MON 87708 can be crossed with another transgenic soybean plant to produce a plant that has the characteristics of both transgenic parents. An example of this would be a cross of the dicamba-tolerant soybean MON 87708 with a plant that has one or more additional traits such as herbicide tolerance (e.g., soybean event 40-3-2 or soybean event MON89788 (Publication US Patent Application Publication No. 20060282915), insect control (e.g., soybean event MON87701 (US Patent Application Publication No. 20090130071)), and/or other desirable characteristics (e.g., higher oil composition such as soybean event MON87769 (PCT Patent Publication

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<img file="AR119652A2_D0023.tif" />

W02009102873)), resulting in a progeny plant or seed that is tolerant to dicamba and has one or more other characteristics. Herbicides for which tolerance of transgenic plants has been demonstrated and for which the methods of the invention can be applied include, but are not limited to: glyphosate, glufosinate, sulfonylureas, imidazolinones, bromoxynil, delapon, cyclohexandione, protoporphyrinogen oxidase inhibitors. , and isoxasflutole herbicides. Nucleotide molecules encoding proteins involved in herbicide tolerance are known in the art and include, but are not limited to, a nucleotide molecule encoding: glyphosate-tolerant 5-enolpyruvylshikimate-3phosphate synthase (EPSPS) (see, for example, example, US Patents 5,627,061; glyphosate oxidoreductase (GOX) (see, for example, US Patent No. 5,776,760); glyphosate-n-acetyltransferase (GAT); a herbicide-tolerant acetolactate synthase (ALS, also known as acetohydroxyacid synthase (AHAS)) for tolerance to sulfonylureas, imidazolinones, triazolopyrimidines, pyrimidinyloxybenzoates, sulfonylamino carbonyl triazolinones, and/or heteroaryl ethers; an acetyl coenzyme A carboxylase (ACCase) tolerant to a herbicide or R-2,4-dichlorophenoxypropionate dioxygenase (rdpA) for tolerance to an aryloxyphenoxypropionate (AOPP) (such as haloxyfop, quizalofop, dichlorofop, and diclofop); a detoxification protein such as a 2,4-D dioxygenase (tfdA), R-2,4-dichlorophenoxypropionate dioxygenase (rdpA), AryloxyAlkanoate Dioxygenase (AAD), and/or S-2,4-dichlorophenoxypropionate dioxygenase (sdpA) to tolerance to synthetic auxin herbicides; a bromoxynyl nitrilase (Bxn) for tolerance to Bromoxynil (see, for example, US Patent No. 4,810,648); a phytoene desaturase (crtl) for tolerance to norflurazon; resistance to bialafos resistance (bar) or phosphinothricin acetyltransferase (PAT) protein (see, for example, US Patent Nos. 5,646,024 and 5,276,268) for tolerance to glufosinate

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<img file="AR119652A2_D0024.tif" />

and bialafos; and a protein for tolerance to the herbicide triketone (mezotrione, tembotrione, topromezone, isoxazole) such as tolerant to 4-HydroxyPhenylPyruvate Dioxygenase (HPPD), a detoxifying cytochrome P450, or a bypass of the HPPD pathway such as Artbrobacter globiformis HPP oxidase (HPPO ) and Pseudomonas acidovorans 4-HPA 1-hydroxylase (HPAH) and NADH oxidoreductase (HPAC).

Backcrossing to a parent plant and outcrossing with a non-transgenic plant are also contemplated, as is vegetative propagation. Descriptions of other breeding methods that are commonly employed for different traits and crops can be found in one of several references, e.g., Fehr, in Breeding Methods for Cultivar Development, Wilcox J. ed., American Society of Agronomy, Madison Wl (1987).

The invention provides a plant part that is derived from soybean event MON 87708. As used in this invention, a "plant part" refers to any part of a plant which is composed of material derived from a plant with the soybean event MON 87708. Plant parts include, but are not limited to, pollen, ovule, pod, flower, root or stem tissue, fibers, and leaves. Plant parts may be viable, non-viable, regenerable, and/or non-regenerable.

The invention provides a commodity that is derived from the soybean event MON 87708. As used in this invention, a “commodity product” refers to any composition or product that is composed of material derived from a plant with the soybean event MON 87708, seed, plant cells or plant part. Commodities can be sold to consumers and can be viable or non-viable. Non-viable commodities include, but are not limited to: non-viable seeds and grains; processed seeds, seed parts, and plant parts; dehydrated plant tissue,

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<img file="AR119652A2_D0025.tif" />

frozen plant tissue and processed plant tissue; seeds and plant parts processed for animal feed for consumption by terrestrial and/or aquatic animals, oil, ground grain, flour, flakes, bran, fiber, milk, cheese, paper, cream, wine and any other food for human consumption; and biomasses and fuel products. Viable commodities include, but are not limited to, seeds and plant cells. Soybean event MON 87708 can, therefore, be used to manufacture any commodity typically purchased from soybeans. Any such commodity that is derived from soybean event MON 87708 may contain at least a detectable amount of the specific and unique DNA corresponding to soybean event MON 87708, and may specifically contain a detectable amount of a polynucleotide containing at least 15 contiguous nucleotides of SEQ ID NO: 1 or SEO ID NO: 2. Any conventional detection method for nucleotide molecules can be used, including detection methods described in this invention. A commodity is within the scope of the invention if there is any detectable amount of SEQ ID NO: 1 or SEQ ID NO: 2 in the commodity.

The plants, progeny, seeds, plant cells, plant parts (such as pollen, ovule, pod, flower, root or stem tissue and leaves), and basic products of the invention are, therefore, useful for, among other things, other things, cultivating plants for the purpose of producing seed and/or plant parts from soybean event MON 87708 for agricultural purposes, producing progeny from soybean event MON 87708 for plant breeding and research purposes, use with microbiological techniques for industrial and research applications and sale to consumers.

The invention provides methods for controlling weeds and methods for producing plants using dicamba herbicide and soybean event MON 87708. A

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<img file="AR119652A2_D0026.tif" />

Method for controlling weeds in a field is proportionate and consists of planting varietal or hybrid plants with soybean event MON 87708 in a field and applying a herbicidally effective dose of dicamba to the field for the purpose of controlling weeds in the field without injuring the plants. plants with MON 87708. That type of dicamba herbicide application can be pre-emergence, that is, at any time after the MON 87708 seed is planted and before MON 87708 plants emerge, or post-emergence, that is, at any time after MON plants emerge. 87708. Another method of controlling weeds in a field is also provided and is to apply an effective dose of dicamba herbicide to control weeds in a field and then plant soybean event MON 87708 in the field. That type of dicamba herbicide application would be preplant, that is, before the MON 87708 seed is planted, and could be done at any time prior to planting including, but not limited to, approximately 14 days preplant to approximately 1 day prior to planting. . The invention further provides a method of producing a soybean seed essentially free of the seeds of toxic weed species by planting seeds of a dicamba-tolerant soybean variety MON 87708 in a field, applying an effective post-emergence dose of dicamba herbicide sufficient to kill the species of toxic weed to the field, and harvesting the seed from the field. An effective dicamba herbicide rate for use in the field should range from approximately 0.0056 kilograms per hectare (0.005 pounds per acre) to approximately 8.96 kilograms per hectare (8 pounds dicamba per acre) during a growing season. growth. Multiple applications of dicamba may be used during a growing season, for example, two applications (such as a pre-plant application and a post-emergence application or a pre-emergence application and a post-emergence application).

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<img file="AR119652A2_D0027.tif" />

or three applications (such as a preplant application, a preemergence application, and a postemergence application).

Methods are provided for producing a herbicide-tolerant soybean plant comprising DNA sequences specific and unique to the MON 87708 transgenic event of the invention. The transgenic plants used in these methods can be homozygous or heterozygous for the transgene. Progeny plants produced by these methods can be varietal or hybrid plants; They can be grown from seeds produced by a plant with soybean event MON 87708 and/or from seeds produced by a plant fertilized with pollen from a plant with soybean event MON 87708; and they can be homozygous or heterozygous for the transgene. The progeny plants may subsequently be self-pollinated to generate a true plant breeding line, i.e. plants homozygous for the transgene, or alternatively they may be outcrossed, e.g., bred with another unrelated plant, to produce a plant variety. or seed or a hybrid.

A soybean plant that tolerates the application of dicamba herbicide can be produced by sexual crossing of a plant with the event MON 87708 comprising a nucleotide molecule comprising the sequence of SEQ ID NO: 1 and SEQ ID NO: 2 with another soybean plant. soybeans and thereby produce the seed, which is then grown into progeny plants. These progeny plants can then be treated with dicamba herbicide to select progeny plants that are tolerant to the dicamba herbicide. Alternatively, these progeny plants can be analyzed using diagnostic methods to select progeny plants that contain the DNA of the MON 87708 event. The other plant used in the cross may or may not be tolerant to the dicamba herbicide and may or may not be transgenic. The progeny plant and/or seed produced may be a variety of

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<img file="AR119652A2_D0028.tif" />

seed or hybrid seed. In the practice of this method, the step of sexually crossing a plant with another plant, that is, cross-pollination, can be achieved or facilitated by human intervention, for example: by human hands collecting pollen from a plant and putting it into contact of this pollen with the style or stigma of a second plant; by human hands and/or actions that remove, destroy or cover the stamen or anthers of a plant (e.g., by plucking or by application of a chemical gametocide) so as to prevent natural self-pollination and cross-pollination would have to be carried out. out in order for fertilization to occur; by human placement of pollinating insects in a position for “directed pollination” (e.g., placing hives in orchards or fields or caging plants with pollinating insects); by human opening or removal of parts of the flower to allow the placement or contact of foreign pollen on the style or stamen (e.g., in soybeans that naturally have flowers that prevent or prevent cross-pollination, making them naturally obligate self-pollinators without human intervention ); through selective plant placement (e.g., intentionally planting plants in pollination proximity); and/or by applying chemical products to precipitate flowering or to promote receptivity (of the stigma for pollen).

A soybean plant that tolerates the application of dicamba herbicide can be produced by self-fertilizing a plant with the event MON 87708 comprising a nucleotide molecule comprising the sequence of SEQ ID NO: 1 and SEQ ID NO: 2 and thereby produce the seed, which is then grown into progeny plants. These progeny plants can then be treated with dicamba herbicide to select progeny plants that are tolerant to dicamba herbicide. Alternatively, these progeny plants can be analyzed using diagnostic methods to select progeny plants.

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<img file="AR119652A2_D0029.tif" />

containing the DNA of the MON 87708 event. By putting this method into practice, the step of sexually crossing a plant with itself, that is, self-pollination or self-fertilization, can be achieved or facilitated by human intervention, for example: by human hands collecting pollen from the plant and bringing it into contact to this pollen with the style or stigma of the same plant and then optionally avoiding additional fertilization of the plant; by human hands and/or actions that remove, destroy or cover the stamen or anthers of other nearby plants (e.g., by plucking or by application of a chemical gametocide) so that cross-pollination is prevented and self-pollination would have to occur in order for fertilization to occur; by human placement of pollinating insects in a position for “directed pollination” (e.g., caging a plant alone with pollinating insects); by human manipulation of the flower or its parts to allow self-pollination; through selective plant placement (e.g., intentionally planting plants beyond pollination proximity); and/or by application of chemical products to precipitate flowering or to promote receptivity (of the stigma for pollen).

The progeny soybean plants or seeds covered by these methods and produced using these methods will be different from other soybean plants, for example because the progeny soybean plants and seeds: are recombinant and as such created by human intervention; They are tolerant to the herbicide dicamba; contain at least one allele consisting of the transgenic DNA of the invention; and/or contain a detectable amount of a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2. A seed may be selected from an individual progeny plant, provided that the seed comprises SEQ ID NO: 1 and SEQ ID NO: 2, will be within the scope of the invention.

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<img file="AR119652A2_D0030.tif" />

In practicing the invention, two different transgenic plants can be crossed to produce hybrid offspring containing two independently segregating heterologous genes. Selfing of appropriate progeny can produce plants that are homozygous for both genes. Backcrossing to a parent plant and outcrossing with a non-transgenic plant are also contemplated, as is vegetative propagation. Descriptions of other methods that are commonly employed for different traits and crops can be found in one of several references, e.g., Fehr, in Breeding Methods for Cultivar Development, Wilcox J. ed., American Society of Agronomy, Madison Wl (1987) .

The plants and seeds used in the methods described in this invention may also contain one or more additional transgenes. Said transgene can be any nucleotide sequence that encodes a protein or RNA molecule that confers a desirable characteristic including, but not limited to, greater resistance to insects, greater efficiency in water use, greater yield, greater resistance to drought, greater quality. of seed, improved nutritional quality, and/or greater tolerance to herbicide, wherein the desirable trait is measured relative to a soybean plant lacking said additional transgene.

The methods of the invention are therefore useful for, among other things, controlling weeds in a field while growing plants for the purpose of producing seed and/or plant parts of soybean event MON 87708 for agricultural or research purposes, select progeny from soybean event MON 87708 for plant breeding or research purposes, and produce progeny plants and seeds from soybean event MON 87708.

Plants, progeny, seeds, plant cells, plant parts (such as pollen, ovule, pod, flower, root or stem tissue, and leaves), and commodities

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<img file="AR119652A2_D0031.tif" />

of the invention can be evaluated to determine DNA composition, gene expression, and/or protein expression. That type of evaluation can be performed using any conventional method such as PCR, northern blotting, southern analysis, western blotting, immunoprecipitation, and ELISA or using the detection methods and/or detection kits provided in this invention.

Methods of detecting the presence of DNA derived from a soybean cell, tissue, seed, or event plant of soybean MON 87708 in a sample are provided. One method consists of (i) extracting a DNA sample from at least one soybean cell, tissue, seed or plant, (i) contacting the DNA sample with a pair of primers that is capable of producing an amplicon of the DNA of event MON 87708 under conditions appropriate for DNA amplification, (iii) carry out a DNA amplification reaction, and then (iv) detecting the amplicon molecule and/or confirming that the nucleotide sequence of the amplicon comprises a nucleotide sequence specific to the MON 87708 event, such as one selected from the group consisting of SEQ ID NO: 1-8. The amplicon should be one that is specific to the MON 87708 event, such as an amplicon comprising SEQ ID NO: 1 or SEQ ID NO: 2. The detection of a specific nucleotide sequence for the MON 87708 event in the amplicon is decisive and/or diagnoses the presence of the specific DNA for the MON 87708 soybean event in the sample. An example of a primer pair that is capable of producing an amplicon from the DNA of event MON 87708 under conditions appropriate for DNA amplification is provided as SEQ ID NO: ΙΟΙ 1. Other primer pairs can be easily designed by one skilled in the art and would comprise at least one fragment of SEQ ID NO: 6. Another method for detecting the presence of DNA derived from a soybean tissue, seed or plant cell from the event of soybean MON 87708 in a sample consists of (i)

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extract a DNA sample from at least one soybean cell, tissue, seed or plant, (i) contact the DNA sample with a DNA probe specific for the DNA of event MON 87708, (iii) allow The probe and the DNA sample hybridize under severe hybridization conditions, and then (iv) detect the hybridization between the probe and the target DNA sample. An example of a DNA probe sequence that is specific for the DNA of event MON 87708 is provided as SEQ ID NO: 11. Other probes can be easily designed by one skilled in the art and would comprise at least one fragment of SEQ ID NO: 6. Detection of hybridization of the probe to the DNA sample diagnoses the presence of the specific DNA for soybean event MON 87708 in the sample. The absence of hybridization alternatively diagnoses the absence of DNA specific for the soybean event MON 87708 in the sample.

DNA detection kits are provided that are useful for the identification of soybean event MON 87708 DNA in a sample and can also be applied to methods for the genetic improvement of soybean plants containing the appropriate event DNA. Said kits contain DNA primers and/or probes comprising fragments of SEQ ID NO: 1-8. An example of such a kit comprises at least one DNA molecule of sufficient length of contiguous nucleotides of SEQ ID NO: 6 to function as a DNA probe useful for detecting the presence and/or absence of DNA derived from the soybean event. transgenic MON 87708 in a sample. DNA derived from transgenic soybean event MON 87708 would comprise SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 7, and/or SEQ ID NO: 8. A DNA molecule sufficient for use as a DNA probe that is useful for determining, detecting, or diagnosing the presence and/or absence of soybean event DNA MON 87708 in a sample is provided as SEQ ID NO: 11. Other probes They may be

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<img file="AR119652A2_D0033.tif" />

easily designed by one skilled in the art and should comprise at least 15 contiguous nucleotides of SEQ ID NO: 6 and should be sufficiently unique to soybean event DNA MON 87708 in order to identify DNA derived from the event. Another type of kit comprises a pair of primers useful for producing an amplicon useful for detecting the presence and/or absence of DNA derived from transgenic soybean event MON 87708 in a sample. That type of kit would employ a method comprising contacting a target DNA sample with a pair of primers as described in this invention, then carrying out a nucleic acid amplification reaction sufficient to produce an amplicon comprising SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 7, and/or SEQ ID NO: 8, and then detect the presence and/or absence of the amplicon. That type of method may also include sequencing the amplicon or a fragment thereof, which would be determinant of, that is, diagnostic for, the presence of the DNA specific for the soybean event MON 87708 in the target DNA sample. Other primer pairs can be easily designed by one skilled in the art and should comprise at least 15 contiguous nucleotides of SEQ ID NO: 6 and should be sufficiently unique to the soybean event DNA MON 87708 DNA in order to identify derived DNA. of the event.

Nucleic acid amplification can be achieved by any of the various nucleic acid amplification methods known in the art, including thermal amplification methods. Many techniques are known to detect, quantify and/or sequence the amplicon produced by these methods. An exemplary technique useful for practicing this invention is TAQMAN® (PE Applied Biosystems, Foster City, CA).

The detection kits and methods of the invention are useful for, among other things, identifying soybean event MON 87708, selecting varieties or hybrids of plants that comprise soybean event MON 87708, detecting the presence

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<img file="AR119652A2_D0034.tif" />

of DNA derived from the transgenic soybean event MON 87708 in a sample, and monitor the samples to determine the presence and/or absence of the soybean event MON 87708 or plant parts derived from the soybean event MON 87708.

The sequence of the heterologous DNA insert, junction sequences, or flanking sequences from soybean event MON 87708 (with representative seed samples deposited as ATCC PTA-9670) can be verified (and corrected if necessary) by amplifying those types of sequences from the event using primers derived from the sequences provided in this invention followed by conventional DNA sequencing of the amplicon or cloned DNA.

As used in this invention, the term “comprising” means “including but not limited to.”

The following examples are included to demonstrate examples of certain preferred embodiments of the invention. Those skilled in the art should appreciate that the techniques described in the examples below represent approaches that the inventors have found to work well in the practice of the invention, and therefore may be considered to constitute examples of preferred embodiments for its practice. . However, those skilled in the art should appreciate, in light of the present description, that many changes can be made to the specific embodiments that are described and still obtain a similar or similar result without departing from the spirit and scope of the invention.

EXAMPLES

Example 1: Transformation of Soybean A3525 and selection of event MON 87708

The soybean plant MON 87708 was produced by soybean Agrobacterium-mediated transformation. The soybean cells were transformed and

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<img file="AR119652A2_D0035.tif" />

regenerated into intact soybean plants and individual plants were selected from the plant population that showed plant expression cassette integrity and dicamba resistance. From this population, the soybean plant event MON 87708 was selected and characterized.

The transgenic dicamba-tolerant soybean plant MON 87708 was developed through Agrobacterium-mediated transformation of soybean meristem tissue using the transformation vector PV-GMHT4355. The method was described in US Patent No. 6,384,301 (herein incorporated by reference), which allows the generation of transformed plants without the use of callus. Briefly, meristem tissues were excised from embryos of germinated A3525 soybean seed (Asgrow, St Louis, MO). After coculture with Agrobacterium carrying the vector, meristems were placed in selection medium containing glyphosate (Monsanto, St Louis, MO), carbenicillin disodium salt, cefotaxime sodium salt, and ticarcillin disodium salt/potassium clavulanate mixture. to inhibit the growth of non-transformed plant cells and excess Agrobacterium. The meristems were then placed in media conducive to the development of shoots and roots. Rooted plants with normal phenotypic characteristics were selected and transferred to soil for growth evaluation and further evaluation.

The R0 plants generated through the aforementioned transformation were transferred to soil for growth and then self-fertilized to produce the R1 seed. During subsequent selfing of R0 plants to produce the R1 generation, unligated insertions of T-DNA I (dmo expression cassette) and T-DNA II (cp4 epsps expression cassette) were segregated. A non-lethal dose of glyphosate was applied to R1 plants. Plants with minor lesions were selected for further analysis, while plants

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<img file="AR119652A2_D0036.tif" />

that do not show any lesion, that is, that contain T-DNA II (cp4 epsps expression cassette) were eliminated from further development. Subsequently, R0 plants containing only a single T-DNA insert (i.e., dmo gene cassette) were identified. The T-DNA I expression cassette comprised the Peanut Chlorotic Streak Virus (PCISV) promoter with a duplicated enhancer region (P-PCISV.FLt-enh) operatively linked to a DNA guide derived from the RNA transcript of the Tobacco Etch Virus (L-TEV); operably linked to a DNA molecule encoding an N-terminal chloroplast transit peptide of the small subunit of ribulose 1,5-bisphosphate carboxylase (SSU) of Pisum sativum (TS-RbcS-3C); operatively linked to part of the mature ribulose 1,5-bisphosphate carboxylase small subunit (SSU) protein from Pisum sativum (CR-RbcS-3C); operatively linked to a DNA molecule encoding a dicamba monooxygenase (DMO) from Stenotrophomonas maltophilia (Pseudomonas maltophilia was the original name for the source of the DMO gene. This source organism was subsequently reclassified first as Xanthomonas maltophilia and then as Stenotrophomonas maltophilia); operably linked to a 3' UTR DNA molecule derived from the ribulose 1,5-bisphosphate carboxylase small subunit gene of Pisum sativum (TPs.RbcS2-E9). Plants were selected using a combination of analytical techniques, including TaqMan, PCR analysis, and herbicide spraying. The MON 87708 event was selected from approximately 2,400 individual transgenic events based on its superior phenotypic characteristics, a comprehensive molecular profile analysis, and its desirable haplotype association. Then, event MON 87708 was crossed with event MON 89788 (glyphosate tolerant). The progeny of this cross were treated with dicamba (Clarity®, BASF,

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Research Triangle Park, NC), glyphosate (Roundup WeatherMAX®, Monsanto Co., St Louis, MO), or a combination of dicamba and glyphosate. Treatments were carried out in the preplant stage, postplant in the vegetative growth stage 3 (V3), and postplant in the reproduction stage 1 (R1). The treated plants were classified to determine the percentage of growth inhibition at 14 days after treatment (DAT) for the preplant treatment with the herbicide, 3 DAT for the post-emergence treatment in the VE stage, and 3 DDT post-emergence treatment in the stage R1. The herbicide(s) were applied at various rates per acre as shown in Table 10 1. Percent inhibition measurements represent an average of replicates.

Table 1: Dicamba and/or Roundup WeatherMAX® tolerance test with

MON89788 x MON87708

<td>Herbicide (ea Proportion gm/ha (lb/a))</td><td>% of inhibition to 14 DDT PRE</td><td>% inhibition at 3 DDT POST (V3)</td><td>% inhibition at 3 DDT POST (R1)</td>

<td>Untreated/No herbicide</td><td> 0,0</td><td> 0,0</td><td> 0,0</td>

<td>Roundup WeatherMAX® (3364 (3.0))</td><td> 0,0</td><td> 0,0</td><td> 0,0</td>

<td>Clarity® (2244 (2.0))</td><td> 0,0</td><td> 10,0</td><td> 20,0</td>

<td>Clarity®561 (0.5) and Roundup WeatherMAX® (841 (0.75))</td><td> 0,0</td><td> 5,0</td><td> 10,0</td>

<td>Clarity® (1120 (1.0)) and Roundup WeatherMAX® (1682 (1.5))</td><td> 0,0</td><td> 7,5</td><td> 12,5</td>

<td>Clarity® (2244 (2.0)) and Roundup WeatherMAX® (3364 (3.0))</td><td> 0,0</td><td> 22,5</td><td> 25,0</td>

Dicamba tolerance transgene mapped in soybean event

MON 87708 to ligation group 9 at approximately map position 143.5. The associated haplotype window 19743 and 19767 has no effect on yield, maturity, height or lodging. It is provided

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Table 2: Association of haplotype LG9, Pos 143.5

<td>Event</td><td>Haplotype Window</td><td>ID of the Haplotype</td><td>Performance</td><td>Maturity</td><td>Height</td><td>Acame</td><td>Sequence of Haplotype</td><td>Linkage group</td>

<td>GM_A92205</td><td> 19743</td><td> 1573355</td><td> 0,00</td><td> -0,03</td><td> 0,06</td><td> 0,04</td><td>CGCTG</td><td> 9</td>

<td>GM_A92205</td><td> 19743</td><td> 1573357</td><td> 0,00</td><td> 0,07</td><td> -0,03</td><td> -0,04</td><td>CGCTA</td><td> 9</td>

<td>GM_A92205</td><td> 19743</td><td> 1573371</td><td> 0,00</td><td> -0,09</td><td> -0,41</td><td> -0,09</td><td>CCCTG</td><td> 9</td>

<td>GM_A92205</td><td> 19743</td><td> 1573373</td><td> 0,00</td><td> -0,20</td><td> -0,01</td><td> -0,03</td><td>TG*GG</td><td> 9</td>

<td>GM_A92205</td><td> 19743</td><td> 1573374</td><td> 0,00</td><td> -0,08</td><td> -0,07</td><td> 0,05</td><td>TG*GA</td><td> 9</td>

<td>GM_A92205</td><td> 19743</td><td> 1573375</td><td> 0,00</td><td> -0,15</td><td> 0,05</td><td> 0,04</td><td>CCCTA</td><td> 9</td>

<td>GM_A92205</td><td> 19743</td><td> 1573376</td><td> 0,00</td><td> -0,45</td><td> -0,14</td><td> 0,00</td><td>TC*GG</td><td> 9</td>

<td>GM_A92205</td><td> 19767</td><td> 1573486</td><td> 0,00</td><td> 0,00</td><td> -0,03</td><td> 0,00</td><td>TACGGTC</td><td> 9</td>

<td>GM_A92205</td><td> 19767</td><td> 1573493</td><td> 0,00</td><td> 0,00</td><td> 0,22</td><td> 0,00</td><td>AACAATT</td><td> 9</td>

<td>GM_A92205</td><td> 19767</td><td> 1573494</td><td> 0,00</td><td> 0,00</td><td> 0,03</td><td> 0,00</td><td>TACAATC</td><td> 9</td>

<td>GM_A92205</td><td> 19767</td><td> 1573495</td><td> 0,00</td><td> 0,00</td><td> 0,07</td><td> 0,00</td><td>TGAAACC</td><td> 9</td>

<td>GM_A92205</td><td> 19767</td><td> 1573497</td><td> 0,00</td><td> 0,00</td><td> 0,41</td><td> 0,00</td><td>TACGGTT</td><td> 9</td>

<td>GM_A92205</td><td> 19767</td><td> 1573499</td><td> 0,00</td><td> 0,00</td><td> -0,01</td><td> 0,00</td><td>TGAAACT</td><td> 9</td>

<td>GM_A92205</td><td> 19767</td><td> 1573500</td><td> 0,00</td><td> 0,00</td><td> 0,06</td><td> 0,00</td><td>TGAGACC</td><td> 9</td>

<td>GM_A92205</td><td> 19767</td><td> 1573502</td><td> 0,00</td><td> 0,00</td><td> -0,07</td><td> 0,00</td><td>AACAATC</td><td> 9</td>

<td>GM_A92205</td><td> 19767</td><td> 1573503</td><td> 0,00</td><td> 0,00</td><td> 0,08</td><td> 0,00</td><td>AACGATC</td><td> 9</td>

<td>GM_A92205</td><td> 19767</td><td> 1573504</td><td> 0,00</td><td> 0,00</td><td> 0,07</td><td> 0,00</td><td>TACAGTC</td><td> 9</td>

<td>GM_A92205</td><td> 19767</td><td> 1573506</td><td> 0,00</td><td> 0,00</td><td> -0,03</td><td> 0,00</td><td>AACGATT</td><td> 9</td>

<td>GM_A92205</td><td> 19767</td><td> 1573507</td><td> 0,00</td><td> 0,00</td><td> 0,20</td><td> 0,00</td><td>TGAAATT</td><td> 9</td>

Example 2: Characterization of DNA Sequences of MON 87708

The DNA inserted into the genome of soybean plant MON 87708 and the flanking sequence was characterized by detailed molecular analyses. These analyzes included: the insertion sequence, the number of insertions (number of integration sites within the soybean genome), the copy number (number of copies of the transgenic DNA within a locus), the

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integrity of the inserted gene cassette, flanking sequences, and association of the insertion with haplotype regions of the soybean genome.

Molecular DNA probes were used that included the intact coding region and its respective regulatory elements, the promoters, introns and polyadenylation sequences of the plant expression cassettes. Analysis showed that MON 87708 contains a single transgenic DNA insertion with one copy of the expression cassette. DNA sequence analyzes and inverse PCR that were carried out to determine the 5' and 3' links of the insert-to-plant genome confirm the organization of the elements within the insert (Figure 1), and determine the sequence of Complete DNA of the insert in the soybean plant MON 87708 (provided in this invention as SEQ ID NO: 5). A soybean plant that comprises in its genome the linked transgenic genetic elements shown in Figure 1 and is resistant to dicamba is one aspect of the invention.

The sequences flanking the transgenic DNA insertion in MON 87708 were determined using inverse PCR as described in Ochman et al., 1990 (PCR Protocols: A guide to Methods and Applications, Academic Press, Inc.) and/or Genome Walker techniques. . Plant genomic DNA from A3525 and transgenic soybean lines was isolated from tissue grown under conventional greenhouse conditions. Approximately 1 gram of young leaf tissue was combined with liquid nitrogen and ground to a fine powder using a mortar and pestle. DNA was extracted using a Nucleon™ PhytoPure™ Genomic DNA Extraction Kit (RPN8511, Amersham, Piscataway, NJ) according to the manufacturer's protocol. After the final precipitation step, the DNA was resuspended in 0.5 ml of TE (10mM Tris-HCI pH 8.0, 1mM EDTA). This method can be modified by one skilled in the art to extract DNA from any soybean tissue, including, but not limited to,

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the seed tissue. An aliquot of DNA was digested with restriction endonucleases selected based on restriction analysis of the transgenic DNA. After self-ligation of restriction fragments, PCR was carried out using primers designed from the transgenic DNA sequence that would amplify sequences extending from the 5' and 3' ends of the transgenic DNA. PCR products were separated by agarose gel electrophoresis and purified using a QIAGEN gel purification kit (Qiagen, Valencia, CA). Subsequent DNA products were directly sequenced using standard DNA sequencing protocols. The 5' flanking sequence extending into the right border sequence of the transgenic DNA of the expression cassette is presented as SEQ ID NO: 3 ([C], see Figure 1). The 3' flanking sequence extending into the left border sequence of the transgenic DNA of the expression cassette is presented as SEQ ID NO: 4 ([D], see Figure 1). The portion of the expression cassette DNA that was fully integrated into the A3525 genomic DNA is presented as SEQ ID NO: 5 ([E], see Figure 1).

The sequences of the isolated DNA molecule were compared with the sequence of the transgenic DNA to identify the flanking sequence and the co-isolated transgenic DNA fragment. Confirmation of the presence of the expression cassette was achieved by PCR with primers designed based on the deduced flanking sequence data and the known transgenic DNA sequence. The wild type sequence corresponding to the same region into which the transgenic DNA was integrated into the transformed line was isolated using primers designed from the flanking sequences in MON 87708. PCR reactions were carried out using the amplification system Elongase® (Invitrogen, Carlsbad, CA). The flanking DNA sequences in MON 87708 and the wild type A3525 sequence were

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<img file="AR119652A2_D0039.tif" />

analyzed against multi-nucleotide and protein databases. This information was used to examine the relationship of the transgene to the plant genome and to look for the integrity of the insertion site. The flanking sequence and wild-type sequences were used to design primers for TAQMAN® endpoint assays used to identify events. Zygosity assays were developed using this information.

Example 3: TAQMAN® Event-Specific Endpoint Assays

This example describes an event-specific endpoint TAQMAN® thermal amplification method developed to identify the MON 87708 event in a sample. Examples of conditions useful with the TAQMAN® Specific Endpoint method for event MON 87708 are as follows: Step 1: 18 megahoms of water adjusted for a final volume of 10 pl. Step 2: 5.0 μΙ of 2X Universal Master Mix (dNTPs, enzyme, buffer) to a final 1X concentration. Step 3: 0.5 μΙ Event Primer 1 (SQ13570) and Event Primer 2 (SQ13571) Mix (resuspended in 18 megahoms of water to a concentration of 20 uM for each primer) to 1.0 μΜ final concentration (for For example, in a microcentrifuge tube, the following must be added to achieve 500 μΙ at a final concentration of 20 uM: 100 μΙ of Primer SQ13570 (SEQ ID NO: 9) at a concentration of 100 μΜ; NO: 10) at a concentration of 100 μΜ; 300 μΙ of 18 megahoms of water). Step 4: 0.2 μΙ Event Probe 6-FAM™ MGB PB4655 (resuspended in 18 megahoms of water to a concentration of 10 μΜ (SEQ ID NO: 11) to a final concentration of 0.2 μΜ. Step 5: 0.5 μΙ Mixture of Internal Control Primer 1 and Internal Control Primer 2 (resuspended in 18 megahoms of

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<img file="AR119652A2_D0040.tif" />

water up to a concentration of 20_μΜ for each primer) up to 1.0 μΜ final concentration. Step 6: 0.2 pl of VIC™ Internal Control Probe to a final concentration of 0.2 μΜ (resuspended in 18 megahoms of water to a concentration of 10_μΜ) Step 7: 3.0 μΙ of Extracted DNA (template) for each sample with one each of the following comprising 1. Leaf Samples to be analyzed; 2. Negative control (non-transgenic DNA); 3. Negative water control (no mold); 4. Positive control DNA MON 87708. Step 8: The thermal cycler conditions are as follows: One Cycle at 50°C for 2 minutes; One Cycle at 95°C for 10 minutes; Ten Cycles of 95°C for 15 seconds then 64°C for 1 minute with -1°C/cycle; Thirty Cycles of 95°C for 15 seconds then 54°C for 1 minute; final cycle of 10°C.

The DNA primers used in the endpoint assay are primers SQ13570 (SEQ ID NO: 9), SQ13571 (SEQ ID NO: 10), and the 6-FAM™ labeled probe PB4655 (SEQ ID NO: 11). . 6-FAM™ is a fluorescent dye product from Applied Biosystems (Foster City, CA) linked to the DNA probe. For TAQMAN® MGB™ probes, the 5' exonuclease activity of Taq DNA polymerase dissociates the probe from the 5' end, between the fluorophore and the annealer. When hybridized to the target DNA strand, the annealer and fluorophore are separated sufficiently to produce a fluorescent signal, thereby releasing fluorescence. SQ13570 (SEQ ID NO: 9) and SQ13571 (SEQ ID NO: 10) when used with these reaction methods with PB4655 (SEQ ID NO: 11) produce a DNA amplicon that is diagnostic for the MON 87708 event DNA. Controls for this analysis should include a positive soybean control containing DNA from the MON 87708 event, a negative control of non-transgenic soybean, and a negative control containing no template DNA. In addition, a control for the PCR reaction includes Internal Control Primers and an Internal Control Probe, specific for a

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<img file="AR119652A2_D0041.tif" />

single copy gene in the Glycine genome. One skilled in the art will know how to design primers specific for a single copy gene in the Glycine genome. These assays are optimized for use with either an Applied Biosystems GeneAmp® POR System 9700 (operated at full speed) or the MJ Research DNA Engine PTC-225 thermal cycler. Other methods and apparatus known to those skilled in the art that produce amplicons that identify DNA from event MON 87708 are within the knowledge of the art.

R0 plants demonstrating the presence of the expression cassette were allowed to develop into fully mature plants. Probes designed based on the cassette sequences of the dicamba tolerance transgene were used to probe Southern blots for ligation. R0 plants were also evaluated for expression cassette copy number using a combination of Southern analysis and TAQMAN® endpoint analysis.

A zygosity assay is useful to determine whether a plant comprising an event is homozygous for the event DNA; That is, it comprises exogenous DNA in the same location on each chromosome of a chromosomal pair; or heterozygous for an event DNA, i.e. comprising exogenous DNA on only one chromosome of a chromosome pair; or is null for the event DNA, i.e., wild type. The endpoint TAQMAN® thermal amplification method was also employed to develop zygosity assays for event MON 87708. This example describes an event-specific endpoint TAQMAN® thermal amplification method developed to determine event zygosity. MON 87708 in a sample. For this assay, a three-primer assay was employed where primer SQ20632 (SEQ ID NO: 12) hybridizes and extends

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<img file="AR119652A2_D0042.tif" />

specifically from the 3' junction of the inserted exogenous DNA and genomic DNA, primer SQ20636 (SEQ ID NO: 13) specifically hybridizes and extends from the DNA flanking the 3' side of the inserted exogenous DNA, and primer SQ20637 (SEQ ID NO: 14) specifically hybridizes and extends from the genomic DNA into which the inserted exogenous DNA was integrated. All three primers are diagnostic for the event. In this example, primer SQ20636 (SEQ ID NO: 13) and primer SQ20632 (SEQ ID NO: 12) and 6-FAM™ labeled oligonucleotide probe PB10130 (SEQ ID NO: 15) are diagnostic when one copy exists. of the inserted exogenous DNA. In this example, SQ20636 (SEQ ID NO: 13) and primer SQ20637 (SEQ ID NO: 14) and VIC™ labeled oligonucleotide probe PB10131 (SEQ ID NO: 16) are diagnostic when there is no copy of the exogenous DNA inserted present in the genomic DNA, that is, the wild type. When the three primers and two probes are mixed together in a PCR reaction with DNA extracted from a plant homozygous for the MON 87708 event, there is a fluorescent signal only from the oligonucleotide probe labeled with 6-FAM™ PB10130 (SEQ ID NO : 15) which is indicative of, and diagnostic of, a plant homozygous for the MON 87708 event. When the three primers and the two probes are mixed together in a PCR reaction with DNA extracted from a plant heterozygous for the MON 87708 event, there is a fluorescent signal from both the 6-FAM™ labeled oligonucleotide probe PB10130 (SEQ ID NO: 15) as well as the VIC™ labeled oligonucleotide probe PB10131 (SEQ ID NO: 16) which is indicative of, and diagnostic of, a plant heterozygous for the MON 87708 event. When the three primers and the two probes are mixed together in a PCR reaction with DNA extracted from a plant that is null for the MON 87708 event (i.e., wild type), there is a fluorescent signal only from the oligonucleotide probe labeled with VIC™ PB10131

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<img file="AR119652A2_D0043.tif" />

(SEQ ID NO: 16) which is indicative of, and diagnostic of, a null plant for the MON 87708 event, that is, wild type. Examples of conditions useful with this method are as follows. Step 1: 18 megahoms of water adjusted for a final volume of 10 μΙ. Step 2: 5.0 μΙ of 2X Universal Master Mix (Applied Biosystems cat # 4304437; dNTPs, enzyme, buffer) to a final concentration of 1X. Step 3: 0.5 μΙ of Zygosity Primers SQ20632, SQ20636, SQ20637 (resuspended in 18 megahoms of water to a concentration of 20 μΜ for each primer) to a final concentration of 1.0 μΜ. Step 4: 0.2 μΙ of Probe 6-FAM™ PB10130 (SEQ ID NO: 15) (resuspended in 18 megahoms of water to a concentration of 10 μΜ) to 0.2 μΜ final concentration. Step 5: 0.2 μΙ VIC™ Probe PB10131 (SEQ ID NO: 16) (resuspended in 18 megahoms of water to a concentration of 10 μΜ) to a final concentration of 0.2 μΜ. Step 6: 3.0 μΙ of Extracted DNA (template) for each sample with one each of the following comprising 1. Leaf samples to be analyzed (4-80 ng of genomic DNA diluted in water); 2. Negative control (non-transgenic soy DNA; 4ng diluted in water); 3. Negative water control (no template; solution in which DNA was resuspended); 4. MON 87708 genomic DNA positive control of known heterozygous event (4 ng diluted in water); 5. MON 87708 genomic DNA positive control of the known homozygous event (4 ng diluted in water). Step 7: Mix gently. Step 8: Thermocycler conditions when using the Applied Biosystems GeneAmp® 9700 PCR System (operated at full speed) or MJ Research DNA Engine PTC-225 thermal cycler are as follows. One Cycle at 50°C for 2 minutes; a cycle at 95°C for 10 minutes; Ten Cycles of (95°C for 15 seconds, then 64°C for 1 minute (-1°C/cycle); Thirty Cycles of (95°C for 15 seconds, then 54°C for 1 minute); 10 to 20 optional additional cycles (95°C for 15 seconds, then 64°C for 1 minute

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<img file="AR119652A2_D0044.tif" />

(-1 C/cycle) may provide more distinct population separation during EndPoint TaqMan® Endpoint analysis; One cycle at 10°C maintain.

Example 4: Identification of the MON 87708 event in any MON 87708 breeding activity

The following example describes how event MON 87708 can be identified within the progeny of any breeding activity using soybean event MON 87708.

DNA event primer pairs are used to produce a diagnostic amplicon for soybean event MON 87708. A diagnostic amplicon for MON 87708 comprises at least one junction sequence, provided as SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 7 or SEQ ID NO: 8. The event primer pairs that will produce a diagnostic amplicon for MON 87708 include primer pairs based on the flanking sequences and the inserted expression cassette. To acquire a diagnostic amplicon in which SEQ ID NO: 1 is found, one would design a forward primer molecule based on SEQ ID NO: 3 from bases 1 to 1126 and a reverse primer molecule based on the DNA sequence of the inserted expression cassette (SEQ ID NO: 5 from positions 1 to 3003) wherein the primer molecules are of sufficient length of contiguous nucleotides to hybridize specifically to SEQ ID NO: 3 and SEQ ID NO: 5. To acquire a diagnostic amplicon in which SEQ ID NO: 2 is found, one would design a forward primer molecule based on the DNA sequence of the inserted expression cassette (SEQ ID NO: 5 from positions 1 to 3003) and a reverse primer molecule based on the 3 'flanking sequence (SEQ ID NO: 4 of bases 131 to 1947), wherein the primer molecules are of sufficient length of contiguous nucleotides to specifically hybridize to

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<img file="AR119652A2_D0045.tif" />

SEQ ID NO: 4 and SEQ ID NO: 5. For practical purposes, one should design primers which produce amplicons of a limited size range, for example, between 100 and 1000 bases. Amplicons with smaller size (shorter polynucleotide length) are generally produced more reliably in PCR reactions, allow shorter cycle times, and can be easily separated and visualized on agarose gels or adapted for use in assays. TAQMAN® endpoint. Smaller amplicons can be produced and detected by methods known in the art of DNA amplicon detection. In addition, the amplicons produced using the primer pairs can be cloned into vectors, propagated, isolated and sequenced or can be sequenced directly with methods well established in the art. Any primer pair derived from the combination of SEQ ID NO: 3 and SEQ ID NO: 5 or the combination of SEQ ID NO: 4 and SEQ ID NO: 5 that is useful in a DNA amplification method to produce a diagnostic amplicon for MON 87708 or its progeny is one aspect of the invention. Any single isolated DNA polynucleotide primer molecule comprising at least 11 contiguous nucleotides of SEQ ID NO: 3, or its complement that is useful in a DNA amplification method to produce a diagnostic amplicon for MON 87708 or its progeny is an aspect of the invention. Any single isolated DNA polynucleotide primer molecule comprising at least 11 contiguous nucleotides of SEQ ID NO: 4, or its complement that is useful in a DNA amplification method to produce a diagnostic amplicon for MON 87708 or its progeny is a aspect of the invention. Any single isolated DNA polynucleotide primer molecule comprising at least 11 contiguous nucleotides of SEQ ID NO: 5, or its complement that is useful in a DNA amplification method to produce a

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<img file="AR119652A2_D0046.tif" />

Diagnostic amplicon for MON 87708 or its progeny is one aspect of the invention.

An example of the amplification conditions for this analysis is illustrated in Example 3. However, any modification of these methods or the use of DNA primers homologous or complementary to SEQ ID NO: 3 or SEQ ID NO: 4 or sequences of DNA of the genetic elements contained in the transgene insert (SEQ ID NO: 5) of MON 87708 that produce a diagnostic amplicon for MON 87708 is within the art. A diagnostic amplicon comprises a DNA molecule homologous or complementary to at least one genomic/transgenic junction DNA (SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 7 or SEQ ID NO: 8), or its substantial portion.

An analysis for the plant tissue sample from event MON 87708 should include a positive tissue control from event MON 87708, a negative control from a soybean plant other than event MON 87708 (for example, but not limited to A3525), and a negative control containing no soybean genomic DNA. A primer pair that will amplify an endogenous soybean DNA molecule will serve as an internal control for the DNA amplification conditions. Additional primer sequences may be selected from SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5 by those skilled in the art of DNA amplification methods, and the conditions selected for the production of an amplicon. by the methods shown in Example 3 may differ, but result in a diagnostic amplicon for the MON 87708 event DNA. The use of these DNA primer sequences with modifications to the methods of Example 3 is within the scope of the invention. The amplicon produced by at least one DNA primer sequence derived from SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5 that is diagnostic for MON 87708 is one aspect of the invention.

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<img file="AR119652A2_D0047.tif" />

DNA detection kits contain at least one DNA primer of sufficient length of contiguous nucleotides derived from SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, which when used in an amplification method of DNA produces a diagnostic amplicon for MON 87708 or its progeny is one aspect of the invention. A MON 87708 soybean plant, plant part, plant cell, seed or commodity that will produce a diagnostic amplicon for MON 87708 when tested in a DNA amplification method is one aspect of the invention. The assay for the MON 87708 amplicon can be carried out using an Applied Biosystems GeneAmp® 9700 PCR System (operated at full speed) or an MJ Research DNA Engine PTC-225 thermal cycler or any other amplification system that can be used to produce a diagnostic amplicon of MON 87708 as shown in Example 3.

A deposit of a representative sample of soybean seed from event MON 87708 described above and mentioned in the claims has been made pursuant to the Treaty of Budapest with the American Standard Crop Collection (ATCC), 10801 University Boulevard, Manassas, VA. 20110. The ATCC accession number for this repository is PTA-9670. The deposit will be held by the depositary for a period of 30 years, or 5 years after the last application, or for the effective life of the patent whichever is longer, and will be replaced as necessary during that period.

Having illustrated and described the principles of the invention, it should be apparent to those skilled in the art that the invention can be modified in arrangement and detail without departing from said principles. We claim all modifications that are within the spirit and scope of the attached claims.

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Argentine Republic - National Executive Branch 2019 - Year of Export

Additional Signature Sheet Graphic report

Number: IF-2019-80266805-APN-ANP#INPI

BUENOS AIRES CITY

Thursday, September 5, 2019

Reference: 20190101909

The document was imported by the GEDO system with a total of 50 page/s.

Digitally signed by GESTION DOCUMENTAL ELECTRONICA - GDE

DN: cn=ELECTRONIC DOCUMENT MANAGEMENT - GDE, c=AR, o=GOVERNMENT SECRETARY OF MODERNIZATION, ou=ADMINISTRATIVE MODERNIZATION SECRETARY, serialNumber=CUIT 30715117564

Date: 2019.09.05 04:38:21 -03'00'

Darío Julio Martin Mayares

Administrative Advisor

National Patent Administration

National Institute of Industrial Property

Digitally signed by GESTION DOCUMENTAL ELECTRONICA GDE

DN: cn=GEST10N ELECTRONIC DOCUMENTARY - GDE, c=AR, o=GOVERNMENT SECRETARY OF MODERNIZATION, ou=ADMINISTRATIVE MODERNIZATION SECRETARY, serialNumber=CUIT 30715117564

Date: 2019.09.05 04:38:23 -03'00'