Elite event EE-GM5 and methods and kits for identifying such events in biological samples

Abstract

The present invention provides specific transgenic soybean plants, plant materials, and seeds, which are characterized by carrying specific nematode resistance and herbicide tolerance transformation events at specific locations in the soybean genome. Tools are also provided that allow for the rapid and unambiguous identification of the events in biological samples. [Selection diagram] None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 437,874, filed December 22, 2016, and U.S. Provisional Patent Application No. 62 / 481,292, filed April 4, 2017, the contents of which are incorporated herein by reference in their entireties.

[0002] The present invention relates to novel nucleic acids and transgenic soybean plants, plant materials and seeds, characterized by the presence of specific transformation events at specific locations in the soybean genome, in particular by the presence of genes that confer nematode resistance and herbicide tolerance. The soybean plants of the present invention combine nematode resistance and herbicide tolerance phenotypes with agronomic performance, genetic stability and functionality in different genetic backgrounds equivalent to the corresponding non-transformed soybean genetic background in the absence of HPPD-inhibiting herbicides or nematode contamination. The present invention further provides methods and kits for identifying the presence of plant materials specifically containing the transformation event EE-GM5 in biological samples. [Background technology]

[0003] The phenotypic expression of a transgene in a plant is determined both by the structure of the gene or genes and by its or their location in the plant genome. At the same time, the presence of a transgene or "inserted T-DNA" at different locations in the genome affects the overall phenotype of the plant in different ways. The agronomically or industrially successful introduction of commercially interesting traits into plants by genetic engineering can be a redundant procedure that depends on different factors. The actual transformation and regeneration of genetically transformed plants is only the first in a series of selection steps that include extensive genetic characterization, introgression, and evaluation in field trials, ultimately leading to the selection of elite events.

[0004] Unequivocal identification of elite events is becoming increasingly important in light of the debate on novel foods / feeds, the separation of GMO and non-GMO products, and the identification of proprietary materials. Ideally, such an identification method would be rapid and simple, without the need for extensive laboratory settings. Furthermore, the method would allow unequivocal identification of elite events without expert interpretation, but provide results that would stand up under expert scrutiny if necessary.

[0005] Planting the nematode-resistant and herbicide-tolerant EE-GM5 soybean varieties offers growers new options for nematode and weed control using HPPD-inhibiting herbicides such as isoxaflutole (IFT), topramezone or mesotrione (MST) herbicides. HPPD-inhibiting herbicides offer an alternative weed control option for soybean growers, helping to manage problem weed species and slowing the spread of herbicide-resistant weeds as an alternative means of control tools.

[0006] The soybean cyst nematode (SCN) Heterodera glycines is a global challenge for soybean production and a continuing threat to growers. Since first detected in the United States in 1954 from a single county in North Carolina, SCN has spread to nearly every soybean-producing state in the United States and is estimated to cause yield losses of more than $1.2 billion annually in the United States, making it the most damaging soybean pathogen in the United States. SCN was first detected in Brazil in the early 1990s and has since spread throughout South America, where it is one of the most important pathogens in Brazil, causing losses in virtually all Brazilian growing areas. Similarly, SCN continues to spread in soybean-producing areas of China, where it has been detected in 15 provinces and is estimated to cause yield losses of more than $120 million. A multi-year study (2001-2015) in Iowa, USA, in which almost all SCN-resistant soybean varieties contain SCN resistance derived from PI 88788, found that the virulence of SCN populations increased over the years, and the PI 88788-source resistance resulted in increased SCN population density at the end of the season and reduced yields in SCN-resistant soybean varieties (Mitchum (2016), Phytopathology 106(12):1444-1450; Allen et al. (2017) Plant Health Progr. 18:19-27; Arias et al. (2017) www.researchgate.net / publication / 266907703_RESISTANCE_TO_SOYBEAN_CYST_NEMATODE_GENETICS_AND_BREEDING_IN_BRAZIL; McCarville et al. (2017) Plant Health Progress 18:146-155).

[0007] The root-lesion nematode, Pratylenchus brachyurus, has become an increasingly important pathogen of soybean. It has a wide host range and is widely distributed in tropical and subtropical regions, especially Brazil, Africa, and the southern United States. Pratylenchus brachyurus has become a concern among cotton and soybean growers in the Cerrado region of Brazil and is considered the major nematode pathogen of soybean in the region. In soybean, this nematode can reduce yields by 30-50%, with greater damage observed in sandy soils. The use of resistant soybean varieties would be the best way to control this nematode. However, to date no P. brachyurus-resistant soybean varieties have been identified.Although several soybean genotypes have been studied for Pratylenchus brachyurus resistance and several cultivars have been identified as being increasingly resistant, breeding of resistant cultivars against P. brachyurus is difficult due to the fact that this nematode is polyphagous and lacks close interaction with its host (Machado (2014) Current Agricultural Science and Technology 20:26-35; Antonio et al. (2012) Soil productivity losses in area infested by the nematoid of the root lesions in Vera, MT. In: Brazilian Congress of Soy, 6, 2012, Cuiaba. Abstracts. Londrina: Embrapa Soja, 4pp; Rios et al. (2016) Ciencia Rural 46:580-584; Lima et al. (2016 ... Soil productivity losses in area infested by the nematoid of the root lesions in Vera, MT. In: Brazilian Congress of Soy, 6, 2012, Cuiaba. Abstracts. Londrina: Embrapa Soja, 4pp; Rios et al. (2016) Soil productivity losses in area infested by the nematoid of the root lesions in Vera, MT al.,2017,Book Chapter 6:Soybean - The Basis of Yield,Biomass and Productivity;Edited by Minobu Kasai,ISBN 978-953-51-3118-2,Print ISBN 978-953-51-3117-5,InTech;Inomoto et al.(2011)Sucessao de culturas sob pivot central para controle de fitonematoides:variacao populacional,patogenicidade e estimativa de perdas.Tropical Plant Pathology 36:178-185).

[0008] It is known that protecting plants from nematodes such as SCN can help the plant better cope with other stresses such as soil composition / content, weather conditions, pathogen stress, herbicide application, etc. In particular, if such other stresses give a visible phenotype such as leaf chlorosis / yellowing, the effect of SCN control is more visible, but is otherwise often not "visible". For example, if soybean plants have sudden death syndrome (SDS) or iron deficiency chlorosis (IDC), protection from SCN will result in plants that are greener or have less severe SDS / IDC symptoms. Despite extensive research and varietal selection efforts, iron deficiency remains a challenge in the soybean producing region of the north-central United States. The expansion of soybean production in soils that are sensitive to iron deficiency and its possible interactions with changes in cropping systems has increased the importance of this issue. Iron deficiency occurs in soils with high pH and carbonates, but the expression of iron deficiency is highly variable in space due to interactions with spatially varying soil properties such as water content, salinity, iron availability, and other micronutrient and metal concentrations. In addition, iron deficiency expression interacts with biological factors such as nitrogen fixation, pests, and diseases, and with management-induced stresses such as herbicide application. Cultivar selection is the most important tool for managing iron deficiency, but cultivar selection is complicated by the large genotype by environment interactions associated with chlorosis resistance (Hansen et al. (2004) Soil Sci. Plant Nutr. 50(7):983-987).

[0009] Sudden soybean death syndrome (SDS) was first discovered in Arkansas in 1971 and has since been identified in most soybean-growing regions of the United States. SDS is a fungal disease that also occurs in disease complex with the soybean cyst nematode (SCN). SDS is one of the most devastating soil-borne diseases of soybean in the United States. When the disease occurs in the presence of SCN, symptoms develop earlier and are more severe. SDS is caused by a soil fungus within the Fusarium solani superspecies group. In North America, the pathogen is Fusarium virguliforme, formerly Fusarium solani var. glycines. In South America, F. brasiliense, F. cuneirostrum, F. tucumaniae, and F. virguliforme cause symptoms of SDS. Soybean varieties that are less susceptible to SDS have been developed, but there are no highly resistant varieties available. Although the fungus can infect the roots of soybean seedlings soon after planting, aboveground symptoms of SDS rarely appear until the soybean plant reaches the reproductive stage. The fungus produces a toxin in the roots that migrates to the leaves. The first noticeable symptoms of SDS are yellowing and defoliation of the upper leaves. If the disease manifests early, flowers and young pods do not develop. If the disease manifests later, the plant will have fewer seeds per pod or smaller seeds. The earlier and more severe the disease manifests, the lower the yield. SDS fungi can persist in the soil for long periods of time, so larger areas of a field will show disease symptoms each growing season until a large portion of the field is affected (Westphal et al. (2008). Sudden Death Syndrome of Soybean. The Plant Health Instructor. DOI: 10.1094 / PHI-I-2008-0102-01, www.apsnet.org / edcenter / intropp / lessons / fungi / ascomycetes / Pages / SuddenDeath.aspx).

[0010] Currently, soybean plants genetically engineered for nematode resistance have not been commercialized. Soybean plants that contain one or more herbicide resistance genes have been disclosed in the art. WO 2006 / 130436 describes a glyphosate-tolerant soybean event that contains the epsps gene, and WO 2011 / 034704 describes a dicamba-tolerant soybean event. WO 2012 / 082548 describes a soybean plant that contains both the hppd and pat genes. WO 2011 / 063411 describes a soybean event that has resistance to HPPD inhibitors and glyphosate, while WO 2011 / 063413 describes a soybean plant that has resistance to HPPD inhibitors, glufosinate and glyphosate. WO 2011 / 066384 describes soybean events with resistance to 2,4-D and glufosinate, while WO 2012 / 075426 describes soybean events with resistance to 2,4-D, glufosinate and glyphosate, and WO 2017 / 059795 describes soybean events with resistance to glyphosate. WO 2009 / 064652 describes soybean events with resistance to lepidopteran insects, and WO 2013 / 016527 describes soybean events with resistance to lepidopteran insects and glufosinate resistance.

[0011] HPPD genes and proteins that confer improved resistance to HPPD-inhibiting herbicides are disclosed, for example, in WO2015138394, WO2015135881, WO2014043435, and nematocidal activity of Cry proteins is described, for example, in WO2010027805, WO2010027809, WO2010027804, WO2010027799, WO2010027808 and WO2007147029.

[0012] None of the prior art disclosures teach or suggest elite events in soybeans containing nematode-active Cry genes, let alone elite events in soybeans containing nematode-active Cry genes in combination with genes that confer resistance to HPPD inhibitors.

[0013] It is known in the art that obtaining commercially elite transformation events in soybean plants with acceptable agronomic performance is far from straightforward. Summary of the Invention [Means for solving the problem]

[0014] The present invention provides nucleic acids encoding Cry14Ab-1 proteins, such as the cry14Ab-1.b coding sequence of SEQ ID NO: 7, or a sequence encoding a nematicidal Cry14Ab protein having at least 95, 96, 97, 98, or at least 99% sequence identity to SEQ ID NO: 7. Also provided herein are nucleic acids encoding HPPD-4 proteins, such as the hppdPf-4Pa coding sequence of SEQ ID NO: 9, or a sequence having at least 95, 96, 97, 98, or at least 99% sequence identity to SEQ ID NO: 9, which encode an HPPD protein that, when expressed in a plant, confers resistance to HPPD-inhibiting herbicides. Also applicable herein is a cry14Ab-1.b chimeric gene comprising the sequence of SEQ ID NO:11 from nucleotide position 131 to nucleotide position 5276, or its complement, or a cry14Ab-1.b chimeric gene comprising the sequence of SEQ ID NO:11 from nucleotide position 412 to nucleotide position 3969 operably linked to a plant expression promoter, or a sequence encoding a nematocidal Cry14Ab protein having at least 95, 96, 97, 98, or at least 99% sequence identity to the sequence of SEQ ID NO:11 from nucleotide position 131 to nucleotide position 5276, or its complement, or the sequence of SEQ ID NO:11 from nucleotide position 412 to nucleotide position 3969 (when operably linked to a plant expression promoter). Further provided herein is a hppdPf-4Pa chimeric gene comprising the sequence of SEQ ID NO:11 from nucleotide position 5382 to nucleotide position 7459, or a complement thereof, or the sequence of SEQ ID NO:11 from nucleotide position 5589 to nucleotide position 6665 operably linked to a plant expression promoter, or a sequence having at least 95, 96, 97, 98, or at least 99% sequence identity to the sequence of SEQ ID NO:11 from nucleotide position 5382 to nucleotide position 7459, or a complement thereof, or to the sequence of SEQ ID NO:11 from nucleotide position 5589 to nucleotide position 6665 (when operably linked to a plant expression promoter).The sequences encode HPPD proteins that, when expressed in plants, provide resistance to HPPD-inhibiting herbicides, as well as nucleic acids comprising the cry14Ab-1.b chimeric gene and the hppdPf-4Pa chimeric gene. These nucleic acids or genes are useful for transforming plants such as soybean, cotton, corn, rice, rapeseed, and wheat so that they control nematodes and / or have HPPD-inhibiting herbicide resistance.

[0015] Also provided herein is a chimeric DNA molecule comprising the nucleotide sequence of SEQ ID NO: 11 from nucleotide position 131 to nucleotide position 7941, or a nucleotide sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In one embodiment, the DNA molecule encodes a protein resistant to HPPD inhibitors and a protein that negatively affects plant disease nematodes, such as SCN, RKN, or Pratylenchus species nematodes. In one embodiment, the chimeric DNA molecule encodes a nematode control protein of SEQ ID NO: 8 or at least 99% identical thereto, and a HPPD inhibitor resistance protein of SEQ ID NO: 10 or at least 99% identical thereto. Also provided is the use of such DNA molecules to render plants, seeds, or cells, such as soybean plants, seeds, or cells, transformed to contain such DNA molecules, and plants or seeds, such as soybean plants or seeds, resistant to nematodes and tolerant to HPPD-inhibiting herbicides.

[0016] The present invention relates to a transgenic soybean plant, plant part, seed, cell or tissue thereof, comprising an expression cassette (both described in Example 1.1 herein and shown in SEQ ID NOs: 7 and 9, respectively) comprising a nematode resistance gene comprising a coding sequence of cry14Ab-1.b gene and a herbicide resistance gene comprising a coding sequence of hppdPf-4Pa gene stably integrated into its genome, providing resistance to plant parasitic nematodes such as Soybean Cyst Nematode, and resistance to HPPD-inhibiting herbicides such as Isoxaflutole, Topramezone or Mesotrione. In the absence of HPPD-inhibiting herbicides and nematode pressure, such soybean plants have substantially equivalent agronomic performance to non-transgenic isogenic lines. When encountering Soybean Cyst Nematode (SCN) pressure affecting plant performance in the field, the plants of the present invention will have superior agronomic phenotypes compared to non-transgenic plants. Also, following application of an HPPD-inhibiting herbicide to which resistance is conferred in the presence of weeds, plants of the invention will have a superior agronomic phenotype compared to plants not treated with the herbicide.

[0017] According to the present invention, the soybean plant or its seed, cell or tissue comprises elite event EE-GM5. In one embodiment, elite event EE-GM5 comprises any one of the sequences of SEQ ID NO: 1, 3, 5, or 24, or any one of the sequences of SEQ ID NO: 2, 4, 6, or 25, or any sequence essentially similar thereto. In one embodiment, EE-GM5 comprises any one of the sequences of SEQ ID NO: 1, 3, 5, or 24, and any one of the sequences of SEQ ID NO: 2, 4, 6, or 25, or any sequence essentially similar thereto, as well as the cry14Ab-1.b coding sequence of SEQ ID NO: 7 and the hppdPf-4Pa coding sequence of SEQ ID NO: 9, or any sequence essentially similar thereto. In one embodiment, elite event EE-GM5 is a T-DNA inserted at a specific location of the soybean genome contained in the reference seed deposited with ATCC under accession number PTA-123625. In one embodiment, such T-DNA in EE-GM5 comprises a gene encoding chimeric Cry14Ab-1 and a gene encoding HPPD-4. In another embodiment, the event is characterized by a 5' junction sequence of SEQ ID NO: 1 or 3, or a 3' junction sequence of SEQ ID NO: 2 or 4; or a 5' junction sequence of SEQ ID NO: 1 or 3 and a 3' junction sequence of SEQ ID NO: 2 or 4. In one embodiment, the EE-GM5-containing genomic DNA, when analyzed using polymerase chain reaction (herein "PCR") with two primers comprising the nucleotide sequences of SEQ ID NO: 12 and SEQ ID NO: 13, respectively, produces a DNA fragment of 85 bp. In one embodiment, the EE-GM5-containing genomic DNA, when analyzed using PCR with two primers comprising the nucleotide sequences of SEQ ID NO: 18 and SEQ ID NO: 19, respectively, produces a DNA fragment of 84 bp.

[0018] In one embodiment herein, there is provided a soybean plant, cell, plant part, seed or progeny thereof, each comprising an elite event EE-GM5 in its genome, and a reference seed comprising said event has been deposited with ATCC under accession number PTA-123625. In one embodiment, the plant or seed comprising EE-GM5 can be obtained by propagation of and / or breeding with a soybean plant grown from a seed deposited with ATCC under accession number PTA-123625.

[0019] More specifically, the present invention relates to transgenic soybean plants, plant parts, pollen, seeds, cells or tissues thereof, the genomic DNA of which, when analyzed by PCR as described herein, is characterized by the fact that a fragment specific for the event EE-GM5 is amplified using at least two primers directed to the region formed by part of the 5' or 3' T-DNA flanking region of EE-GM5 and part of the inserted T-DNA. The primers may be directed to the 3' T-DNA flanking region within SEQ ID NO:6 or SEQ ID NO:25, or the adjacent soybean plant genomic DNA downstream thereof, and the adjacent inserted T-DNA upstream thereof. The primers may also be directed to the 5' T-DNA flanking region within SEQ ID NO:5 or SEQ ID NO:24, or the adjacent soybean plant genomic DNA upstream thereof, and the adjacent inserted T-DNA downstream thereof. In one embodiment, such primers comprise or consist (essentially) of the nucleotide sequences of SEQ ID NO:12 and SEQ ID NO:13, or SEQ ID NO:18 and SEQ ID NO:19, or SEQ ID NO:26 and SEQ ID NO:28, or SEQ ID NO:27 and SEQ ID NO:29, respectively (e.g., a primer pair comprising a primer containing the nucleotide sequence of SEQ ID NO:12 at its 3' end and a primer containing the nucleotide sequence of SEQ ID NO:13 at its 3' end, or a primer pair comprising a primer containing the nucleotide sequence of SEQ ID NO:18 at its 3' end and a primer containing the nucleotide sequence of SEQ ID NO:19 at its 3' end, or a primer pair comprising a primer containing the nucleotide sequence of SEQ ID NO:26 at its 3' end and a primer containing the nucleotide sequence of SEQ ID NO:28 at its 3' end, or a primer pair comprising a primer containing the nucleotide sequence of SEQ ID NO:27 at its 3' end and a primer containing the nucleotide sequence of SEQ ID NO:29 at its 3' end), generating DNA fragments of 50 to 1000 bp, such as fragments of 85 bp or 84 bp.

[0020] A reference seed containing the elite event of the present invention has been deposited with the ATCC under accession number PTA-123625. One embodiment of the present invention is the elite event EE-GM5 contained in the seed deposited under accession number PTA-123625, which when introduced into soybean plants provides resistance to nematodes and tolerance to herbicides, in particular resistance to the soybean cyst nematode (Heterodera glycines, herein "SCN") and / or lesion nematodes (as used herein lesion nematodes refer to Pratylenchus species soybean disease nematodes, including but not limited to Pratylenchus brachyurus) and tolerance to HPPD inhibitors such as isoxaflutole, topramezone or mesotrione. Plants having EE-GM5 of the present invention also control root-knot nematodes (as used herein, root-knot nematodes refers to Meloidogyne species soybean disease nematodes, including but not limited to Meloidogyne incognita, Meloidogyne arenaria, Meloidogyne hapla, or Meloidogyne javanica, or any combination thereof), Rotylenchulus reniformis, and lance nematodes (Hoplolaimus species, such as H. columbus, H. galeatus, and H. magnistylus).A soybean event having HPPD inhibitor resistance and SCN nematode resistance, having a nucleotide sequence having at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity to the nucleotide sequence of EE-GM5 contained in the seed deposited with ATCC under accession number PTA-123625, or a sequence of 1-200, 1-150, 1-100, 1-75, 1-50, 1-30, 1-20, 1-10, or 1-5 nucleotides from the nucleotide sequence of EE-GM5 contained in the seed deposited with ATCC under accession number PTA-123625. The invention includes minor variants of this event, such as HPPD inhibitor-resistant and SCN nematode-resistant soybean events in which the nucleotide sequence differs from the nucleotide sequence formed by the following contiguous nucleotide sequences (5' to 3'): SEQ ID NO:5 or SEQ ID NO:24, SEQ ID NO:11 from nucleotide position 188 to nucleotide position 7101, and SEQ ID NO:6 or SEQ ID NO:25 by 1 to 200, 1 to 150, 1 to 100, 1 to 75, 1 to 50, 1 to 30, 1 to 20, 1 to 10, or 1 to 5 nucleotides. In one embodiment, EE-GM5 comprises a nucleotide sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity to the nucleotide sequence formed by the following contiguous nucleotide sequences (5' to 3'): SEQ ID NO:5 or SEQ ID NO:11 from nucleotide position 188 to nucleotide position 7101, and SEQ ID NO:6 or SEQ ID NO:25. Natural genetic variations, single DNA base differences, small insertions and deletions in homologous DNA sequences (eg, single nucleotide polymorphisms (SNPs)), are commonly found among plants of the same species (Zhu et al. (2003) Genetics 163:1123-1134).

[0021] The seed with ATCC deposit number PTA-123625 is a pure seed lot of transgenic seeds homozygous for the elite event EE-GM5 of the present invention, which grows into a nematode-resistant plant, whereby the plant is also resistant to HPPD inhibitors such as isoxaflutole, topramezone or mesotrione. The seed or progeny seed obtainable from the deposited seed (e.g., after crossing with other soybean plants having different genetic backgrounds) can be sown, and the growing plants can be treated with HPPD inhibitors such as isoxaflutole, topramezone or mesotrione as described herein, or can be tested for the presence of EE-GM5 as described herein to obtain plants containing the elite event of the present invention. The present invention further relates to cells, seeds, tissues, progeny, and descendants derived from plants containing the elite event of the present invention grown from the seed deposited with ATCC with accession number PTA-123625. The invention further relates to plants obtainable from (e.g., by propagation of and / or breeding with) soybean plants comprising the elite events of the invention (such as plants grown from seeds deposited with ATCC having accession number PTA-123625, or plants comprising the HPPD-4 coding sequence of SEQ ID NO:9 located between the sequence of SEQ ID NO:1, 3 or 5 and the sequence of SEQ ID NO:2, 4 or 6, and the cry14Ab-1.b coding sequence of SEQ ID NO:7, or plants comprising the hppdPf-4Pa coding sequence of SEQ ID NO:9 and the cry14Ab-1.b coding sequence of SEQ ID NO:7 located between any one of the sequences of SEQ ID NO:1, 3, 5, or 24 and any one of the sequences of SEQ ID NO:2, 4, 6, or 25). The invention also relates to progeny plants and seeds obtained from the above plants or seeds, which comprise the sequence of SEQ ID NO:1 and the sequence of SEQ ID NO:2, or the sequence of SEQ ID NO:3 and the sequence of SEQ ID NO:4, or the sequence of SEQ ID NO:5 and the sequence of SEQ ID NO:6, or the sequence of SEQ ID NO:24 and the sequence of SEQ ID NO:25.

[0022] The present invention further relates to a method for identifying a transgenic plant or its cells or tissues containing the elite event EE-GM5, the method being based on the identification of the presence of a characteristic DNA sequence or an amino acid encoded by such a DNA sequence in the transgenic plant, cell or tissue. According to a preferred embodiment of the present invention, such a characteristic DNA sequence is a sequence of 15 bp or at least 15 bp, preferably 20 bp or at least 20 bp, most preferably 30 bp or more, including the insertion site of the event, i.e. a sequence that contains both a part of the inserted T-DNA containing the HPPD inhibitor and nematode resistance transgene and a part of the 5' or 3' T-DNA flanking region adjacent thereto, extending into the soybean plant genome, thereby allowing specific identification of the elite event. The present invention also relates to plants, seeds and cells containing the event EE-GM5 identified herein.

[0023] The present invention further relates to a method for identifying elite event EE-GM5 in a biological sample, which method is based on a primer pair or a probe that specifically recognizes the 5' and / or 3' T-DNA flanking sequences in EE-GM5 and the inserted T-DNA sequence adjacent thereto. Other methods for identifying EE-GM5, e.g., for identifying its specific characteristic sequences, such as full or partial (directed) genome sequencing, are also included herein.

[0024] More specifically, the present invention relates to a method for identifying the elite event EE-GM5 in a biological sample, comprising the step of amplifying a sequence of a nucleic acid present in said biological sample using a polymerase chain reaction with at least two primers or a polymerase chain reaction with at least two primers and a probe to obtain a DNA fragment, preferably of a size between 50 and 1000 bp, where one of these primers recognizes the 5' or 3' T-DNA flanking region of EE-GM5 and the other primer recognizes a sequence within the inserted T-DNA containing herbicide resistance and nematode resistance genes adjacent to said 5' or 3' T-DNA flanking region. In one embodiment, to obtain a DNA fragment characteristic of the elite event EE-GM5, a first primer recognizes the 5'T-DNA flanking region of EE-GM5 and a second primer recognizes a sequence in the inserted T-DNA containing the herbicide resistance and nematode resistance genes adjacent downstream of the 5'T-DNA flanking region, or a first primer recognizes the 3'T-DNA flanking region of EE-GM5 and a second primer recognizes a sequence in the inserted T-DNA containing the herbicide resistance and nematode resistance genes adjacent upstream of the 3'T-DNA flanking region. In one embodiment, the polymerase chain reaction method further comprises the use of a probe that recognizes the DNA amplified by the primers, for example the junction DNA (either on the 5' or 3' side of the event, such as a probe comprising the nucleotide sequence of SEQ ID NO: 14 or 20 herein, or their complements, as the case may be) containing a portion of the inserted T-DNA in EE-GM5 and a portion of the DNA adjacent to the T-DNA, to detect the amplification product produced by the primers.The primers may recognize sequences within the 5' T-DNA flanking region of EE-GM5 (SEQ ID NO:5 from nucleotide position 1 to nucleotide position 166, or SEQ ID NO:24 from nucleotide position 1 to nucleotide position 1113), or within the 3' T-DNA flanking region of EE-GM5 (the complement of SEQ ID NO:6 from nucleotide position 359 to nucleotide position 691, or SEQ ID NO:25 from nucleotide position 359 to nucleotide position 1449), and within the inserted T-DNA (SEQ ID NO:5 from nucleotide position 167 to 353, or SEQ ID NO:6 from nucleotide position 1 to nucleotide position 358, or SEQ ID NO:23 from nucleotide position 1114 to 8572, or complements thereof), respectively. Primers recognizing 5' or 3' T-DNA flanking regions may comprise the nucleotide sequence of SEQ ID NO:13, SEQ ID NO:19, SEQ ID NO:26, or SEQ ID NO:27, and primers recognizing sequences within the inserted T-DNA containing the nematode resistance and herbicide tolerance genes may comprise the nucleotide sequence of SEQ ID NO:12, SEQ ID NO:18, SEQ ID NO:28, or SEQ ID NO:29 described herein. The present invention also relates to any event-specific primer pairs and specific DNA amplified using such primer pairs that can be obtained by one of skill in the art or that can be obtained from commercial sources from the EE-GM5 event sequences provided herein or contained in the seeds deposited with the ATCC under accession number PTA-123625.

[0025] The present invention more specifically relates to a method for identifying the elite event EE-GM5 in a biological sample, comprising amplifying a sequence of a nucleic acid present in the biological sample using the polymerase chain reaction with two primers comprising or consisting essentially of the nucleotide sequences SEQ ID NO: 12 and SEQ ID NO: 13, respectively, to obtain a DNA fragment of 85 bp, or with two primers comprising or consisting essentially of the nucleotide sequences SEQ ID NO: 18 and SEQ ID NO: 19, respectively, to obtain a DNA fragment of 84 bp. Plants containing the elite event EE-GM5 thus identified are also included in the present invention.

[0026] The present invention further relates to specific T-DNA flanking sequences of EE-GM5 described herein, which can be used to develop specific identification methods for EE-GM5 in biological samples. Such specific T-DNA flanking sequences can also be used as reference control materials in identification assays. More specifically, the present invention relates to 5' and / or 3' T-DNA flanking regions of EE-GM5, which can be used for the development of specific primers and probes, as further described herein. Also suitable as reference materials are nucleic acid molecules, preferably of 150-850 bp, comprising sequences that can be amplified by primers comprising or consisting (essentially) of the nucleotide sequences of SEQ ID NO: 12 and SEQ ID NO: 13, or SEQ ID NO: 18 and SEQ ID NO: 19.

[0027] The invention further relates to a method for the identification of the presence of EE-GM5 in a biological sample, based on the use of such specific primers or probes. The primers may comprise, consist or essentially consist of a nucleotide sequence of 17-200 consecutive nucleotides selected from the nucleotide sequence of SEQ ID NO:5 from nucleotide 1 to nucleotide 166, or the nucleotide sequence of SEQ ID NO:24 from nucleotide 1 to nucleotide 1113, or the complement of the nucleotide sequence of SEQ ID NO:6 from nucleotide 359 to nucleotide 691, or the complement of the nucleotide sequence of SEQ ID NO:25 from nucleotide 359 to nucleotide 1449, in combination with a primer comprising, consisting or essentially consisting of a nucleotide sequence of 17-200 consecutive nucleotides selected from the nucleotide sequence of SEQ ID NO:11 from nucleotide 1 to nucleotide 7459, or the nucleotide sequence of SEQ ID NO:23 from nucleotide position 1114 to nucleotide position 8572, such as a nucleotide sequence of 17 to about 200 consecutive nucleotides selected from the nucleotide sequence of SEQ ID NO:5 from nucleotide 167 to nucleotide 353, or the nucleotide sequence of SEQ ID NO:6 from nucleotide 1 to nucleotide 358, or the complement thereof. Primers may also contain these nucleotide sequences located at their 3' ends and may further contain unrelated sequences or sequences derived from the mentioned nucleotide sequences but containing mismatches. In one embodiment, the primers used herein may also be identical to the target DNA or its complement, where said target DNA is a hybrid containing nucleotide sequences of different origin and do not occur in such combination in nature.

[0028] The present invention relates to a kit for identifying the elite event EE-GM5 in a biological sample, said kit comprising at least one primer pair or probe specifically recognizing the 5' or 3' T-DNA flanking regions, the inserted T-DNA containing herbicide resistance and nematode resistance genes flanking it in EE-GM5.

[0029] In addition to the primer specifically recognizing the 5' or 3' T-DNA flanking region of EE-GM5, the kit of the present invention may contain a second primer specifically recognizing a sequence in the inserted T-DNA containing the HPPD-inhibiting herbicide resistance and nematode resistance genes of EE-GM5 for use in PCR identification protocols. The kit of the present invention may contain at least two specific primers, one of which recognizes a sequence in the 5' T-DNA flanking region of EE-GM5 or a sequence in the 3' T-DNA flanking region of EE-GM5, and the other of which recognizes a sequence in the inserted T-DNA containing the HPPD-inhibiting herbicide resistance and nematode resistance genes. The primer recognizing the 5' T-DNA flanking region may comprise the nucleotide sequence of SEQ ID NO: 19, and the primer recognizing the inserted T-DNA adjacent to said 5' T-DNA flanking region may comprise the nucleotide sequence of SEQ ID NO: 18, or the primer recognizing the 3' T-DNA flanking region may comprise the nucleotide sequence of SEQ ID NO: 13, and the primer recognizing the inserted T-DNA adjacent to said 3' flanking region may comprise the nucleotide sequence of SEQ ID NO: 12, or any other primer or combination of primers described herein or available from the description or seed deposit. The kit may further comprise a probe that recognizes a sequence located between the primer recognizing the 5' T-DNA flanking region and the primer recognizing a sequence within the inserted T-DNA, or that recognizes a sequence between the primer recognizing the 3' T-DNA flanking region and the primer recognizing a sequence within the inserted T-DNA, such as a probe comprising the sequence of SEQ ID NO: 14 or a probe comprising the sequence of SEQ ID NO: 20.

[0030] The present invention relates to a kit for identifying elite event EE-GM5 in a biological sample, said kit comprising PCR primers comprising or consisting (essentially) of the nucleotide sequences of SEQ ID NO: 12 and SEQ ID NO: 13, or the nucleotide sequences of SEQ ID NO: 18 and SEQ ID NO: 19, for use in the EE-GM5 PCR protocol described herein. Said kit comprising primers comprising or consisting (essentially) of the nucleotide sequences of SEQ ID NO: 12 and SEQ ID NO: 13 may further comprise a probe comprising or consisting (essentially) of the nucleotide sequence of SEQ ID NO: 14, and said kit comprising primers comprising or consisting (essentially) of the nucleotide sequences of SEQ ID NO: 18 and SEQ ID NO: 19 may further comprise a probe comprising or consisting (essentially) of the nucleotide sequence of SEQ ID NO: 20. Said kit may further comprise reagents such as buffers and any or each of dNTPs, (Taq) DNA polymerase, MgCl2, stabilizers, and optionally dyes.

[0031] The present invention also relates to a kit for identifying elite event EE-GM5 in a biological sample, comprising a specific probe that comprises or (essentially) consists of a sequence that corresponds to (or is complementary to) a sequence having 80% to 100% sequence identity with a specific region of EE-GM5, such specific region comprising a part of the 5' or 3' T-DNA flanking region of EE-GM5 and a part of the inserted T-DNA adjacent thereto. In one embodiment, the sequence of the probe corresponds to a specific region comprising a part of the 5' or 3' T-DNA flanking region of EE-GM5 and a part of the inserted T-DNA adjacent thereto. Most preferably, the specific probe comprises or (essentially) consists of (or is complementary to) a sequence having 80% to 100% sequence identity with any one of the sequences of SEQ ID NO: 1, 3 or 5, or a sequence having 80% to 100% sequence identity with any one of the sequences of SEQ ID NO: 2, 4 or 6, or the specific probe comprises or (essentially) consists of (or is complementary to) a sequence having 80% to 100% sequence identity with a portion of at least 50 consecutive nucleotides of the sequence of SEQ ID NO: 5, or a sequence having 80% to 100% sequence identity with a portion of at least 50 consecutive nucleotides of the sequence of SEQ ID NO: 6, each of said portions of SEQ ID NO: 5 or 6 comprising sequences of inserted T-DNA and T-DNA flanking sequences of approximately equal length.

[0032] According to another aspect of the present invention, a DNA molecule is disclosed that comprises a polynucleotide of sufficient length of both the T-DNA flanking sequence and the inserted T-DNA of EE-GM5 to be useful as a primer or probe for the detection of EE-GM5 or to characterize a plant containing the event EE-GM5. Such a sequence may comprise any one of at least 9, at least 10, at least 15, at least 20, or at least 30 nucleotides on either side of the junction site, respectively, and either or both of the 5' and 3' junction sites of the EE-GM5 event, or may comprise any one of 9, 10, 15, 20 or 30 nucleotides of the T-DNA flanking sequence and the same number of nucleotides of the inserted T-DNA of EE-GM5. Most preferably, such a DNA molecule comprises any one of the sequences of SEQ ID NO: 1, 3, or 5, or any one of the sequences of SEQ ID NO: 2, 4, or 6. In one embodiment, such a DNA molecule comprises the sequence of SEQ ID NO: 23, 24, or 25. In one aspect of the present invention, soybean plants and seeds containing such specific DNA molecules are provided.

[0033] The methods and kits encompassed by the present invention can be used for different purposes, such as, but not limited to, to identify the presence or determine a (lower) threshold value of EE-GM5 in plants, plant material, or in products, such as, but not limited to, food or feed products (fresh or processed) that contain or are derived from plant material; additionally or alternatively, the methods and kits of the present invention can be used to identify transgenic plant material for the purposes of separating transgenic and non-transgenic material; additionally or alternatively, the methods and kits of the present invention can be used to determine the quality of plant material containing EE-GM5 (i.e., the percentage of pure material).

[0034] The present invention further relates to the 5' and / or 3' T-DNA flanking regions of EE-GM5, and specific primers and probes developed from the 5' and / or 3' T-DNA flanking sequences of EE-GM5.

[0035] The present invention also relates to genomic DNA obtained from plants containing the elite event EE-GM5, in particular genomic DNA containing EE-GM5 event specific sequences, such as one or both EE-GM5 junction sequences (containing a portion of the T-DNA flanking DNA and adjacent inserted DNA characteristic of EE-GM5), for example any one of the sequences of SEQ ID NO: 1, 3, 5, or 24, and / or any one of the sequences of SEQ ID NO: 2, 4, 6, or 25. Such genomic DNA can be used as reference control material in the identification assays described herein.

[0036] Also provided herein is a transgenic nematode-resistant and herbicide-tolerant soybean plant, or a cell, part, seed or progeny thereof, each comprising at least one elite event, said elite event being: i) a first chimeric gene comprising the cry14Ab-1.b gene from Bacillus thuringiensis encoding the Cry14Ab-1 protein under the control of a plant expression promoter, such as a chimeric gene comprising a plant expression promoter and the coding sequence of SEQ ID NO: 7; ii) A second chimeric gene comprising a modified hppdPf-4Pa gene from Pseudomonas encoding a more resistant HPPD enzyme under the control of a plant-expressible promoter, such as a chimeric gene comprising a plant-expressible promoter and the coding sequence of SEQ ID NO: 9. The inserted T-DNA comprises:

[0037] In one embodiment, the elite event comprises nucleotides 1 to 166 of SEQ ID NO:5 or 1 to 1113 of SEQ ID NO:24 immediately upstream and adjacent to the inserted T-DNA, and nucleotides 359 to 691 of SEQ ID NO:6 or nucleotides 359 to 1449 of SEQ ID NO:25 immediately downstream and adjacent to the inserted T-DNA.

[0038] In a further embodiment, said elite event can be obtained by breeding with a soybean plant grown from a reference seed containing said event deposited with the ATCC under accession number PTA-123625.

[0039] In another embodiment, the genomic DNA of said soybean plant, or a cell, part, seed or progeny thereof, when analyzed using PCR with two primers comprising the nucleotide sequences of SEQ ID NO:12 and SEQ ID NO:13, respectively, yields a 85 bp DNA fragment, or when analyzed using PCR with two primers comprising the nucleotide sequences of SEQ ID NO:18 and SEQ ID NO:19, respectively, yields a 84 bp DNA fragment.

[0040] Also provided herein is a method for identifying a transgenic soybean plant, or a cell, part, seed or progeny thereof, having nematode resistance, such as SCN and / or Pratylenchus and / or root-knot and / or Nematode resistance, and resistance to an HPPD inhibitor herbicide, such as isoxaflutole, topramezone or mesotrione, in a biological sample, the method comprising amplifying a 50-150 bp DNA fragment from a nucleic acid present in the biological sample using polymerase chain reaction with at least two primers, one of which recognizes the 5' T-DNA flanking region of elite event EE-GM5, the 5' T-DNA flanking region being sequenced as SEQ ID NO: 5 from nucleotide 1 to nucleotide 166, or SEQ ID NO: 6 from nucleotide 1 to nucleotide 166, the other of the primers recognizes a sequence within an inserted T-DNA comprising the nucleotide sequence of SEQ ID NO: 5 from nucleotide position 1 to nucleotide 353, or the nucleotide sequence of SEQ ID NO: 6 from nucleotide position 1 to nucleotide 358, or the inserted T-DNA comprises the nucleotide sequence of SEQ ID NO: 11 from nucleotide position 7459, or a complement thereof.

[0041] Also provided herein is a kit for identifying a transgenic soybean plant, or a cell, part, seed or progeny thereof, having nematode resistance and tolerance to an HPPD-inhibiting herbicide in a biological sample, the kit comprising one primer recognizing a 5' T-DNA flanking region of elite event EE-GM5, wherein the 5' T-DNA flanking region comprises the nucleotide sequence of SEQ ID NO:5 from nucleotide 1 to nucleotide 166, or the nucleotide sequence of SEQ ID NO:24 from nucleotide 1 to nucleotide 1113, or one primer recognizing a 3' T-DNA flanking region of the elite event, wherein the 3' T-DNA flanking region comprises the nucleotide sequence of SEQ ID NO:5 from nucleotide 1 to nucleotide 166, or the nucleotide sequence of SEQ ID NO:24 from nucleotide 1 to nucleotide 1113, a primer whose border region comprises the nucleotide sequence of the complement of SEQ ID NO:6 from nucleotide 359 to nucleotide 691, or the nucleotide sequence of the complement of SEQ ID NO:25 from nucleotide 359 to nucleotide 1449, and one primer that recognizes a sequence within the inserted T-DNA, wherein the inserted T-DNA comprises the nucleotide sequence of the complement of SEQ ID NO:5 from nucleotide 167 to nucleotide 353, or the nucleotide sequence of SEQ ID NO:6 from nucleotide 1 to nucleotide 358, or wherein the inserted T-DNA comprises the nucleotide sequence of SEQ ID NO:11 from nucleotide position 1 to nucleotide position 7459, or a complement thereof.

[0042] In one embodiment of the present invention, the inserted T-DNA of elite event EE-GM5 as used herein comprises the nucleotide sequence of SEQ ID NO:5 from nucleotide 167 to nucleotide 353, or a complement thereof, or the nucleotide sequence of SEQ ID NO:6 from nucleotide 359 to nucleotide 691, or a sequence having at least 95, 98, 99, 99.5, or 99.9% sequence identity to the nucleotide sequence of SEQ ID NO:11 from nucleotide position 7 to nucleotide position 7459, or a complement thereof.

[0043] Also provided herein are soybean plants, plant cells, tissues, or seeds comprising in their genome a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NO:1, 3, 5, or 24, or a nucleotide sequence having 80-100% sequence identity thereto, and / or SEQ ID NO:2, 4, 6, or 25, or a nucleotide sequence having 80-100% sequence identity thereto, and a nucleotide sequence having at least 80, 85, 90, 95, 97, 98, 99, 99.5, or at least 99.9% sequence identity to the nucleotide sequence of SEQ ID NO:11 from nucleotide position 188 to nucleotide position 7101, or its complement.

[0044] One embodiment of the present invention provides soybean plants, plant cells, tissues, or seeds that comprise in their genome a nucleic acid molecule that hybridizes under standard stringency conditions to the nucleotide sequence of any one of SEQ ID NOs: 1, 3, or 5, or its complement, or that hybridizes to the nucleotide sequence of any one of SEQ ID NOs: 2, 4, or 6, or its complement.

[0045] Also provided herein is an isolated nucleic acid molecule comprising a nucleotide sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 1, 3, 5, or 24, or its complement, or having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 2, 4, 6, or 25, or its complement, or an isolated nucleic acid molecule comprising a nucleic acid sequence that hybridizes under standard stringency conditions to the nucleotide sequence of any one of SEQ ID NOs: 1, 3, 5, or 24, or its complement, or to the nucleotide sequence of any one of SEQ ID NOs: 2, 4, 6, or 25, or its complement.

[0046] Also provided herein is an isolated nucleic acid molecule comprising a nucleotide sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the nucleotide sequence of SEQ ID NO: 7, or its complement, or an isolated nucleic acid molecule comprising a nucleic acid sequence that hybridizes to the nucleotide sequence of SEQ ID NO: 7, or its complement, under standard stringency conditions, wherein such a nucleic acid molecule encodes a nematocidal toxin active against cyst nematodes and / or disease nematodes and / or root-knot nematodes and / or pseudonematodes, such as Heterodera glycines and / or Pratylenchus brachyurus and / or Meloidogyne incognita and / or Rotylenchulus reniformis. In one embodiment, such a nucleic acid molecule is operably linked to a nucleic acid molecule comprising a (heterologous) plant-expressible promoter to form a chimeric gene. The use of said nucleic acid molecule in transformed plants or seeds for controlling plant pathogenic nematodes is also provided herein.Further provided herein is a method for controlling root-knot nematodes, such as Meloidogyne incognita, Meloidogyne arenaria, Meloidogyne hapla, or Meloidogyne javanica, in particular Meloidogyne incognita, comprising the step of using a Cry14Ab protein, or a DNA encoding a Cry14Ab protein, or a plant or seed containing said DNA, under the control of a plant expressible promoter, wherein said Cry14Ab protein is a protein comprising the amino acid sequence of SEQ ID NO: 8, or a protein having at least 96%, or at least 98%, or at least 99% sequence identity thereto, or a protein comprising the amino acid sequence of SEQ ID NO: 8 from amino acid position 1 to amino acid position 706, or a protein having at least 96%, or at least 98%, or at least 99% sequence identity thereto. Further provided herein is a method for controlling Rotylenchulus reniformis, comprising using a Cry14Ab protein, or DNA encoding a Cry14Ab protein, or a plant or seed containing said DNA, under the control of a plant-expressible promoter, wherein said Cry14Ab protein is a protein comprising the amino acid sequence of SEQ ID NO: 8, or a protein having at least 96%, or at least 98%, or at least 99% sequence identity thereto, or a protein comprising the amino acid sequence of SEQ ID NO: 8 from amino acid position 1 to amino acid position 706, or a protein having at least 96%, or at least 98%, or at least 99% sequence identity thereto.

[0047] Also provided herein is a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:11 from nucleotide position 131 to nucleotide position 7941, or a nucleotide sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein said nucleic acid molecule encodes a nematicidal Cry14Ab protein and an HPPD protein resistant to HPPD inhibitors. In one embodiment, the nucleic acid molecule encodes a protein of SEQ ID NO:8, or a protein at least 99% identical thereto, and a protein of SEQ ID NO:10, or a protein at least 99% identical thereto.

[0048] Also provided herein is a soybean plant cell comprising in its genome an elite event EE-GM5, a foreign DNA or inserted T-DNA at a defined locus, wherein the elite event EE-GM5 is contained in a reference seed deposited with the ATCC under accession number PTA-123625, said inserted T-DNA comprising a chimeric gene encoding Cry14Ab-1 and a chimeric gene encoding HPPD-4, said elite event being characterized by a 5' junction sequence of SEQ ID NO:1 or 3 and a 3' junction sequence of SEQ ID NO:2 or 4; or such a cell which is a seed cell, or such a cell, wherein the genomic DNA of said cell yields a 85 bp DNA fragment when analyzed using PCR with two primers comprising the nucleotide sequences of SEQ ID NO:12 and SEQ ID NO:13, respectively.

[0049] The present invention provides a nucleic acid molecule comprising the nucleotide sequence of elite event EE-GM5 contained in reference seed deposited with ATCC under accession number PTA-123625, said elite event comprising a gene encoding a chimeric Cry14Ab-1 and a gene encoding HPPD-4, and comprising the sequence of SEQ ID NO: 1 or 3 and the sequence of SEQ ID NO: 2 or 4.

[0050] The present invention also provides nucleic acid molecules comprising the following nucleotide sequences, in order, such as a nucleic acid molecule comprising a sequence b) that is at least 99.5% or at least 99.9% identical to the nucleotide sequence of SEQ ID NO:11 from nucleotide 188 to nucleotide 7101, or a sequence that is at least 99% identical thereto; b) the nucleotide sequence of SEQ ID NO:11 from nucleotide 188 to nucleotide 7101, or a sequence that is at least 99% identical thereto; and c) the nucleotide sequence of SEQ ID NO:6 from nucleotide 359 to nucleotide 691, or a sequence that is at least 99% identical thereto.

[0051] The present invention also provides nucleic acid molecules comprising the following nucleotide sequences in order, such as a nucleic acid molecule comprising a sequence b) that is at least 99.5% or at least 99.9% identical to the nucleotide sequence of SEQ ID NO:23: a) the nucleotide sequence of SEQ ID NO:24 from nucleotide 1 to 1113 or a sequence at least 99% identical thereto, b) the nucleotide sequence of SEQ ID NO:23 from nucleotide 1114 to nucleotide 8572 or a sequence at least 99% identical thereto, and c) the nucleotide sequence of SEQ ID NO:25 from nucleotide 359 to nucleotide 1449 or a sequence at least 99% identical thereto. According to the present invention, there is also provided a method for producing a soybean product, comprising obtaining a soybean seed comprising the above elite event EE-GM5, and producing the soybean product therefrom. In one embodiment, the soybean product in such a method is or comprises soybean meal, ground seeds, flour, or flakes, or soybean oil, soybean protein, lecithin, soy milk, tofu, margarine, biodiesel, biocomposite, adhesive, solvent, lubricant, detergent, foam, paint, ink, candle, or soybean oil or soybean protein containing food or feed products. In another embodiment, such soybean product comprises a nucleic acid specific for elite event EE-GM5. In one embodiment, said nucleic acid specific for elite event EE-GM5 comprises the sequence of SEQ ID NO: 1 or 3, or the sequence of SEQ ID NO: 2 or 4.

[0052] Also provided herein is a soybean product produced from a seed comprising the above-mentioned elite event EE-GM5, said soybean product being or comprising a soybean meal, ground seed, flour, or flake, and comprising a nucleic acid specific for elite event EE-GM5, said nucleic acid being detectable using the methods described herein. In one embodiment, said nucleic acid specific for elite event EE-GM5 comprises a sequence of SEQ ID NO: 1 or 3, or a sequence of SEQ ID NO: 2 or 4. In another embodiment, said nucleic acid specific for elite event EE-GM5 comprises a sequence of SEQ ID NO: 5 or 24, or a sequence of SEQ ID NO: 6 or 25. In one embodiment, said nucleic acid specific for elite event EE-GM5 comprises a sequence of SEQ ID NO: 5 or 24, and a sequence of SEQ ID NO: 6 or 25.

[0053] Also provided herein is the use of soybean seeds comprising the elite event EE-GM5 to obtain a soybean product, said elite event comprising any one of SEQ ID NOs: 1, 3, 5 or 24, and / or any one of SEQ ID NOs: 2, 4, 6 or 25. In one embodiment, in such use, the soybean product is any one of soybean meal, ground soybean seeds, soybean flour or soybean flakes.

[0054] Further provided herein is a method for producing a soybean plant or seed comprising the elite event EE-GM5 in combination with another SCN resistance locus / gene, such as by combining the elite event EE-GM5 with another SCN resistance locus / gene occurring in the same soybean plant / seed, and planting the seed comprising EE-GM5 and said other SCN resistance locus / gene. In one embodiment, the plant, cell or seed of the present invention contains one or more other SCN resistance loci / genes occurring in soybean, to obtain a combination of different SCN resistance sources in the soybean plant, cell or seed of the present invention. Several soybean SCN resistance loci or genes are known, including resistance sources PI 88788, PI 548402 (Peking), PI 548403 (Peking), PI 548404 (Peking), PI 548405 (Peking), PI 548406 (Peking), PI 548407 (Peking), PI 548409 ... One or more of them can be combined with EE-GM5 in the same plant, cell or seed, such as any one of the SCN resistance genes / locuses from 437654 (Hartwig or CystX®), or any combination thereof, or one or more of the natural SCN resistance loci / genes rhg1, rhg1-b, rhg2, rhg3, Rhg4, Rhg5, qSCN11, cqSCN-003, cqSCN-005, cqSCN-006, cqSCN-007, or any of the SCN resistance loci identified on any one of soybean chromosomes 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or any combination thereof (Kim et al., 2006). al.2016,Theor.Appl.Genet.129(12):2295-2311;Kim and Diers 2013,Crop Science 53:775-785;Kazi et al.2010,Theor.Appl.Gen.120(3):633-644;Glover et al.2004,Crop Science 44(3):936-941;www.soybase.org;Concibido et al.2004,Crop Science 44:1121-1131;Webb et al.1995,Theor.Appl.Genet.91:574-581).Also, in one embodiment, the plant or seed of the present invention may be selected from the group consisting of SCN resistance sources PI 548316, PI 567305, PI 437654, PI 90763, PI 404198B, PI 88788, PI 468916, PI 567516C, PI 209332, PI 438489B, PI 89772, Peking, PI 548402, PI 404198A, PI 561389B, PI 629013, PI 507471, PI 633736, PI 507354, PI 404166, PI 437655, PI 467312, PI 567328, PI 22897, or PI 494182. Table 3 herein provides a comprehensive list of soybean accessions reported as SCN tolerant, in which the SCN resistance gene / loci(s) can be combined with the EE-GM5 of the present invention in the same soybean plant, cell or seed.

[0055] [Table 1]

[0056] [Table 2]

[0057] Also provided herein is a method for protecting germinating soybean plants from weed competition, comprising treating a field sown with seeds containing the above elite event EE-GM5 with an HPPD-inhibiting herbicide, wherein the plants are tolerant to the HPPD-inhibiting herbicide.In one embodiment, in such a method, the HPPD-inhibiting herbicide is isoxaflutole, topramezone or mesotrione.

[0058] Also provided herein is a method for protecting germinating soybean plants from competition by weeds, comprising treating a field planted with soybean plants comprising the above elite event EE-GM5 with an HPPD-inhibiting herbicide prior to planting or sowing the soybean plants, and subsequently planting or sowing the soybean plants or seeds in the pre-treated field, wherein the plants are tolerant to the HPPD-inhibiting herbicide.

[0059] Also applicable herein is a method for controlling weeds in a field of soybean plants comprising the above-mentioned elite event EE-GM5, comprising treating said field with an effective amount of an HPPD-inhibiting herbicide, wherein the plants are tolerant to such herbicide.

[0060] Also provided herein is the use of a transgenic soybean plant, seed or progeny thereof for controlling weeds in a soybean field, wherein said plant, seed or progeny, respectively, comprises in its genome an elite event EE-GM5, which is a T-DNA at a defined locus contained in a reference seed deposited with ATCC under accession number PTA-123625, said T-DNA comprising a chimeric gene encoding Cry14Ab-1 and a chimeric gene encoding HPPD-4, said elite event being characterized by a 5' junction sequence of SEQ ID NO: 1 or 3 and a 3' junction sequence of SEQ ID NO: 2 or 4. In one embodiment, in such use, the transgenic soybean plant, seed or progeny thereof is resistant to nematodes and / or tolerant to HPPD-inhibiting herbicides. In one embodiment, the T-DNA comprises a chimeric gene encoding Cry14Ab-1 and a chimeric gene encoding HPPD-4, and the elite event is characterized by a 5' junction sequence of SEQ ID NO: 5 or 24 and a 3' junction sequence of SEQ ID NO: 6 or 25.

[0061] Also provided herein is the use of a soybean plant or seed comprising in its genome an elite event EE-GM5, said elite event EE-GM5 being an inserted T-DNA at a defined locus contained in a reference seed deposited with the ATCC under accession number PTA-123625, said inserted T-DNA comprising a chimeric gene encoding Cry14Ab-1 and a chimeric gene encoding HPPD-4, said elite event being characterized by a 5' junction sequence of SEQ ID NO: 1 or 3 and a 3' junction sequence of SEQ ID NO: 2 or 4. In one embodiment, in such use, the soybean plant or seed is resistant to SCN nematodes and / or tolerant to HPPD-inhibiting herbicides. In one embodiment, the T-DNA comprises a chimeric gene encoding Cry14Ab-1 and a chimeric gene encoding HPPD-4, and the elite event is characterized by a 5' junction sequence of SEQ ID NO: 5 or 24 and a 3' junction sequence of SEQ ID NO: 6 or 25.

[0062] Also provided herein is the use of a soybean seed comprising the elite event EE-GM5 to obtain a soybean product, wherein EE-GM5 is as described above.

[0063] Also provided herein is a method for producing a soybean plant or seed that contains elite event EE-GM5, comprising crossing a plant that contains EE-GM5 with another soybean plant, and planting the seed that contains EE-GM5 obtained from said cross. In one embodiment, such a method comprises applying an HPPD-inhibiting herbicide to said seed or plant.

[0064] According to the present invention, there is also provided a soybean seed comprising the above-mentioned elite event EE-GM5, and the use of an HPPD-inhibiting herbicide for controlling weeds in a soybean field, as well as the use of the soybean seed comprising the elite event EE-GM5 in a method for growing soybeans resistant to an HPPD-inhibiting herbicide, wherein the seed is as described above.

[0065] Further provided herein is the use of the above-mentioned elite event EE-GM5 to confer resistance to nematodes and / or tolerance to HPPD-inhibiting herbicides to soybean plants or seeds, or the use of soybean plants or seeds comprising the elite event EE-GM5 in combination with an HPPD-inhibiting herbicide to grow soybeans.

[0066] Also provided herein are primer pairs specific for EE-GM5, and kits or methods using such primer pairs, wherein at least one primer of the pair is labeled (e.g., with a detectable or screenable moiety) and the 5' end of at least one of the primers contains one or more mismatches or a nucleotide sequence that is unrelated to the 5' or 3' flanking sequence of EE-GM5 or unrelated to the T-DNA sequence of EE-GM5; or at least one of the primers contains a nucleotide sequence at its 3' end that spans the junction region between the T-DNA flanking sequence and the T-DNA sequence, said junction region being at nucleotides 166-167 of SEQ ID NO:5, nucleotides 1113-1114 of SEQ ID NO:24, or nucleotides 358-359 of SEQ ID NO:6 or 25, with the proviso that the 3' end are not derived exclusively from either the T-DNA or T-DNA flanking sequences of SEQ ID NO: 5 or 24, or 6 or 25; or at least one of the primers comprises a sequence that is 80-100% identical to a sequence within the 5' or 3' flanking region of EE-GM5 or within the inserted T-DNA of EE-GM5, respectively, and the primer sequence comprises at least one mismatch with the 5' or 3' flanking region or the T-DNA, provided that the at least one mismatch still allows specific identification of the elite event EE-GM5 by these primers under optimized detection conditions (e.g., optimized PCR conditions); or the nucleotide sequence of at least one of the primers comprises a nucleotide sequence of a nucleic acid fused to a nucleic acid from another source, or a complement thereof.

[0067] Other embodiments according to the present invention are summarized in the following paragraphs.

[0068] 1. A method for identifying elite event EE-GM5 in a biological sample, comprising detection of an EE-GM5 specific region using a specific primer pair or probe that specifically recognizes the 5' or 3' T-DNA adjacent region (at least a portion) in EE-GM5 and the adjacent inserted T-DNA (at least a portion).

[0069] 2. The method according to item 1, comprising the step of amplifying a 50-1000 bp DNA fragment from a nucleic acid present in the biological sample by polymerase chain reaction using at least two primers, one of which recognizes a 5'T-DNA flanking region of EE-GM5, the 5'T-DNA flanking region comprising the nucleotide sequence of SEQ ID NO: 5 from nucleotide 1 to nucleotide 166, or the nucleotide sequence of SEQ ID NO: 24 from nucleotide 1 to nucleotide 1113, or recognizes a 3'T-DNA flanking region of EE-GM5, the 3'T-DNA flanking region comprising the nucleotide sequence of SEQ ID NO: 359 to nucleotide 360. and wherein the other of the primers recognizes a sequence within the inserted T-DNA comprising the nucleotide sequence of SEQ ID NO:5, from nucleotide 167 to nucleotide 353, or a complement thereof, or the nucleotide sequence of SEQ ID NO:6, from nucleotide 1 to nucleotide 358, or a complement thereof, or the nucleotide sequence of SEQ ID NO:23, from nucleotide 1114 to nucleotide 8572, or a complement thereof.

[0070] 3. The method according to item 2, wherein the primer recognizing the 5' T-DNA flanking region comprises a nucleotide sequence of 17 to 200 consecutive nucleotides selected from the nucleotide sequence of SEQ ID NO: 5 from nucleotide 1 to nucleotide 166, or the nucleotide sequence of SEQ ID NO: 24 from nucleotide 1 to nucleotide 1113, or the primer recognizing the 3' T-DNA flanking region of EE-GM5 comprises a nucleotide sequence of 17 to 200 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID NO: 6 from nucleotide 359 to nucleotide 691, or the nucleotide sequence of the complement of SEQ ID NO: 25 from nucleotide 359 to nucleotide 1449. 5, from nucleotide 167 to nucleotide 353 or its complement, or the nucleotide sequence of SEQ ID NO:6, from nucleotide 1 to nucleotide 358 or its complement, or the nucleotide sequence of SEQ ID NO:11, from nucleotide 1 to nucleotide 7459 or its complement, or the nucleotide sequence of SEQ ID NO:23, from nucleotide 1114 to nucleotide 8572 or its complement.

[0071] 4. The method according to item 2, wherein the primer recognizing the 5' T-DNA flanking region comprises, at its 3' end, a nucleotide sequence of at least 17 consecutive nucleotides selected from the nucleotide sequence of SEQ ID NO:5 from nucleotide 1 to nucleotide 166, or the nucleotide sequence of SEQ ID NO:24 from nucleotide 1 to nucleotide 1113, or the primer recognizing the 3' T-DNA flanking region of EE-GM5 comprises, at its 3' end, a nucleotide sequence of the complement of SEQ ID NO:6 from nucleotide 359 to nucleotide 691, or a nucleotide sequence of the complement of SEQ ID NO:25 from nucleotide 359 to nucleotide 1449. and the primer recognizing a sequence within the inserted T-DNA comprises at its 3' end a nucleotide sequence of at least 17 contiguous nucleotides selected from the complement of the nucleotide sequence of SEQ ID NO:5 from nucleotide 167 to nucleotide 353, or the nucleotide sequence of SEQ ID NO:6 from nucleotide 1 to nucleotide 358, or the nucleotide sequence of SEQ ID NO:11 from nucleotide 1 to nucleotide 7459 or a complement thereof, or the nucleotide sequence of SEQ ID NO:23 from nucleotide 1114 to nucleotide 8572 or a complement thereof.

[0072] 5. The method of claim 4, wherein the primers comprise the sequences of SEQ ID NO: 12 and SEQ ID NO: 13, respectively, or the sequences of SEQ ID NO: 18 and SEQ ID NO: 19, respectively.

[0073] 6. The method according to item 5, comprising amplifying an 85 or 84 bp EE-GM5-specific fragment using PCR.

[0074] 7. The method according to any one of items 2 to 6, further comprising a step of hybridizing a probe specific to the DNA fragment amplified using the at least two primers.

[0075] 8. The method according to paragraph 7, wherein the probe recognizes a portion of the 5' T-DNA flanking region and a portion of the inserted T-DNA adjacent thereto, or the probe recognizes a portion of the 3' T-DNA flanking region and a portion of the inserted T-DNA adjacent thereto, or the probe recognizes a portion of the 5' T-DNA flanking region and a portion of the inserted T-DNA adjacent thereto, for example, the probe comprises the nucleotide sequence of SEQ ID NO: 1 or 3 or SEQ ID NO: 2 or 4.

[0076] 9. The method of claim 8, wherein the primers comprise the sequences of SEQ ID NO: 12 and SEQ ID NO: 13, respectively, and the probe comprises the sequence of SEQ ID NO: 14, or the primers comprise the sequences of SEQ ID NO: 18 and SEQ ID NO: 19, respectively, and the probe comprises the sequence of SEQ ID NO: 20.

[0077] 10. A kit comprising one primer recognizing a 5' T-DNA flanking region of EE-GM5, said 5' T-DNA flanking region comprising the nucleotide sequence of SEQ ID NO:5 from nucleotide 1 to nucleotide 166, or SEQ ID NO:24 from nucleotide 1 to nucleotide 1113, or one primer recognizing a 3' T-DNA flanking region of EE-GM5, said 3' T-DNA flanking region comprising the nucleotide sequence of the complement of SEQ ID NO:6 from nucleotide 359 to nucleotide 691, or the nucleotide sequence of the complement of SEQ ID NO:25 from nucleotide 359 to nucleotide 1449, and one primer recognizing a sequence within the inserted T-DNA, said inserted T-DNA comprising the complement of the nucleotide sequence of SEQ ID NO:5 from nucleotide 167 to nucleotide 353, or the nucleotide sequence of SEQ ID NO:6 from nucleotide 1 to nucleotide 358, or the nucleotide sequence of SEQ ID NO:11 from nucleotide 1 to nucleotide 7459 or its complement, or the nucleotide sequence of SEQ ID NO:23 from nucleotide 1114 to nucleotide 8572 or its complement.

[0078] 11. A kit according to item 10, wherein the primer recognizing the 5'T-DNA flanking region comprises a nucleotide sequence of 17 to 200 consecutive nucleotides selected from the nucleotide sequence of SEQ ID NO: 5 from nucleotide 1 to nucleotide 166, or the nucleotide sequence of SEQ ID NO: 24 from nucleotide 1 to nucleotide 1113, or the primer recognizing the 3'T-DNA flanking region of EE-GM5 comprises a nucleotide sequence of 17 to 200 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID NO: 6 from nucleotide 359 to nucleotide 691, or the nucleotide sequence of the complement of SEQ ID NO: 25 from nucleotide 359 to nucleotide 1449, and the primer recognizing a sequence within the inserted T-DNA comprises a nucleotide sequence of 17 to 200 consecutive nucleotides selected from the complement of the nucleotide sequence of SEQ ID NO: 5 from nucleotide 167 to nucleotide 353, or the nucleotide sequence of SEQ ID NO: 6 from nucleotide 1 to nucleotide 358, or the nucleotide sequence of SEQ ID NO: 11 from nucleotide 1 to nucleotide 7459 or its complement, or the nucleotide sequence of SEQ ID NO: 23 from nucleotide 1114 to nucleotide 8572 or its complement.

[0079] 12. A kit according to Item 10, wherein the primer recognizing the 5' T-DNA flanking region comprises, at its 3' end, a nucleotide sequence of at least 17 consecutive nucleotides selected from the nucleotide sequence of SEQ ID NO:5 from nucleotide 1 to nucleotide 166, or the nucleotide sequence of SEQ ID NO:24 from nucleotide 1 to nucleotide 1113, or the primer recognizing the 3' T-DNA flanking region of EE-GM5 comprises, at its 3' end, a nucleotide sequence of the complement of SEQ ID NO:6 from nucleotide 359 to nucleotide 691, or a nucleotide sequence of the complement of SEQ ID NO:25 from nucleotide 359 to nucleotide 1449. and wherein the primer recognizing a sequence within the inserted T-DNA comprises at its 3' end a nucleotide sequence of at least 17 contiguous nucleotides selected from the complement of the nucleotide sequence of SEQ ID NO:5 from nucleotide 167 to nucleotide 353, or the nucleotide sequence of SEQ ID NO:6 from nucleotide 1 to nucleotide 358, or the nucleotide sequence of SEQ ID NO:11 from nucleotide 1 to nucleotide 7459 or a complement thereof, or the nucleotide sequence of SEQ ID NO:23 from nucleotide 1114 to nucleotide 8572 or a complement thereof.

[0080] 13. The kit according to item 10, comprising a primer comprising the sequence of SEQ ID NO: 12 and a primer comprising the sequence of SEQ ID NO: 13, or comprising a primer comprising the sequence of SEQ ID NO: 18 and a primer comprising the sequence of SEQ ID NO: 19.

[0081] 14. The kit described in item 10, further comprising a probe that recognizes a sequence between a primer that recognizes the 5' T-DNA flanking region and a primer that recognizes a sequence within the inserted T-DNA, or a probe that recognizes a sequence between a primer that recognizes the 3' T-DNA flanking region and a primer that recognizes a sequence within the inserted T-DNA.

[0082] 15. The kit described in item 14, wherein the probe recognizes a portion of the 5' T-DNA flanking region and a portion of the inserted T-DNA adjacent thereto, or the probe recognizes a portion of the 3' T-DNA flanking region and a portion of the inserted T-DNA adjacent thereto.

[0083] 16. The kit of item 15, wherein the primers comprise the sequences of SEQ ID NO:12 and SEQ ID NO:13 and the probe comprises the sequence of SEQ ID NO:14, or the primers comprise the sequences of SEQ ID NO:18 and SEQ ID NO:19 and the probe comprises the sequence of SEQ ID NO:20.

[0084] 17. A primer pair suitable for use in specific detection of EE-GM5, comprising a first primer comprising a sequence that specifically recognizes a sequence in the 5' or 3' T-DNA flanking region of the inserted T-DNA in EE-GM5 under optimized detection conditions, and a second primer comprising a sequence that specifically recognizes a sequence in the inserted T-DNA in EE-GM5 adjacent to said 5' or 3' flanking region under optimized detection conditions, wherein said 5' T-DNA flanking region comprises the nucleotide sequence of SEQ ID NO: 5 from nucleotide 1 to nucleotide 166, or SEQ ID NO: 24 from nucleotide 1 to nucleotide 1113, and said 3' T-DNA flanking region comprises the nucleotide sequence of SEQ ID NO: 25 from nucleotide 1 to nucleotide 1213, a primer pair comprising the nucleotide sequence of SEQ ID NO:6 from nucleotide 359 to nucleotide 691, or the nucleotide sequence of the complement of SEQ ID NO:25 from nucleotide 359 to nucleotide 1449, and said inserted T-DNA comprising the nucleotide sequence of SEQ ID NO:5 from nucleotide 167 to nucleotide 353, or the nucleotide sequence of SEQ ID NO:6 from nucleotide 1 to nucleotide 358, or the nucleotide sequence of SEQ ID NO:11 from nucleotide 1 to nucleotide 7459 or a complement thereof, or the nucleotide sequence of SEQ ID NO:23 from nucleotide 1114 to nucleotide 8572 or a complement thereof.

[0085] 18. A primer comprising at its 3' end the sequence of SEQ ID NO:12, or the sequence of SEQ ID NO:13, or the sequence of SEQ ID NO:18, or the sequence of SEQ ID NO:19.

[0086] 19. A primer pair comprising a first primer comprising the sequence of SEQ ID NO:12 at its 3' end and a second primer comprising the sequence of SEQ ID NO:13 at its 3' end, or comprising a first primer comprising the sequence of SEQ ID NO:18 at its 3' end and a second primer comprising the sequence of SEQ ID NO:19 at its 3' end.

[0087] 20. The method according to item 1, comprising a step of hybridizing nucleic acid of a biological sample with a specific probe for EE-GM5.

[0088] 21. The method according to item 20, wherein the sequence of the specific probe has at least 80% sequence identity with a sequence including the 5' T-DNA flanking sequence or the 3' T-DNA flanking sequence of EE-GM5 and a portion of the sequence of the inserted T-DNA adjacent thereto.

[0089] 22. The method of claim 21, wherein the sequence of the specific probe comprises any one of SEQ ID NOs: 1, 3, or 5, or any one of SEQ ID NOs: 2, 4, or 6, or a sequence having at least 80% sequence identity with the complement of said sequence.

[0090] 23. The method of claim 22, wherein the probe comprises any one of the sequences of SEQ ID NO:1 or 3, or any one of the sequences of SEQ ID NO:2 or 4.

[0091] 24. A kit for identifying elite event EE-GM5 in a biological sample, said kit comprising a specific probe capable of specifically hybridizing to a particular region of EE-GM5.

[0092] 25. The kit described in item 24, wherein the sequence of the specific probe has at least 80% sequence identity with a sequence including a portion of the 5' T-DNA flanking sequence or a portion of the 3' T-DNA flanking sequence of EE-GM5 and a portion of the sequence of the inserted T-DNA adjacent thereto.

[0093] 26. The kit of paragraph 25, wherein the sequence of the specific probe comprises a nucleotide sequence having at least 80% sequence identity with any one of SEQ ID NO: 1, 3, or 5, or any one of SEQ ID NO: 2, 4, or 6, or a complement of said sequences.

[0094] 27. Specific probe for identification of elite event EE-GM5 in biological samples

[0095] 28. The probe described in item 27, comprising a sequence including a portion of the 5' T-DNA flanking sequence or a portion of the 3' T-DNA flanking sequence of EE-GM5 and a portion of the sequence of the inserted T-DNA adjacent thereto, or a nucleotide sequence having at least 80% sequence identity to a complement thereof.

[0096] 29. The probe of paragraph 28, having at least 80% sequence identity with any one of SEQ ID NOs: 1, 3, or 5, or any one of SEQ ID NOs: 2, 4, or 6, or the complement of said sequences.

[0097] 30. A specific probe comprising a nucleotide sequence essentially similar to any one of SEQ ID NO: 1, 3, or 5, or any one of SEQ ID NO: 2, 4, or 6, or the complement of said sequences.

[0098] 31. A specific probe comprising the sequence of SEQ ID NO: 1 or 3 or the sequence of SEQ ID NO: 2 or 4.

[0099] 32. A method for confirming seed purity comprising detection of an EE-GM5 specific region using a specific primer pair or probe that specifically recognizes the 5' or 3' T-DNA flanking region in EE-GM5 and the adjacent inserted T-DNA in a seed sample.

[0100] 33. A step of amplifying a 50-1000 bp DNA fragment from a nucleic acid present in the biological sample using a polymerase chain reaction with at least two primers, one of which recognizes a 5'T-DNA flanking region of EE-GM5, the 5'T-DNA flanking region comprising the nucleotide sequence of SEQ ID NO: 5 from nucleotide 1 to nucleotide 166, or the nucleotide sequence of SEQ ID NO: 24 from nucleotide 1 to nucleotide 1113, or a 3'T-DNA flanking region of EE-GM5, the 3'T-DNA flanking region comprising the nucleotide sequence of the complement of SEQ ID NO: 6 from nucleotide 359 to nucleotide 691, or the nucleotide sequence of the complement of SEQ ID NO: 25 from nucleotide 359 to nucleotide 1449. 33. The method of claim 32, comprising the steps of: a) recognizing a sequence within an inserted T-DNA comprising the complement of the nucleotide sequence of SEQ ID NO:5 from nucleotide 167 to nucleotide 353, or the nucleotide sequence of SEQ ID NO:6 from nucleotide 1 to nucleotide 358, or within the nucleotide sequence of SEQ ID NO:11 from nucleotide 1 to nucleotide 7459 or its complement, or within the nucleotide sequence of SEQ ID NO:23 from nucleotide 1114 to nucleotide 8572 or its complement, and hybridizing a probe specific to the DNA fragment amplified using the at least two primers.

[0101] 34. The method of claim 33, comprising the steps of amplifying an 85 bp DNA fragment, wherein the primers comprise the sequences of SEQ ID NO: 12 and SEQ ID NO: 13, respectively, and the probe comprises the sequence of SEQ ID NO: 14, or amplifying an 84 bp DNA fragment, wherein the primers comprise the sequences of SEQ ID NO: 18 and SEQ ID NO: 19, respectively, and the probe comprises the sequence of SEQ ID NO: 20.

[0102] 35. A method for screening seeds for the presence of EE-GM5 comprising detection of an EE-GM5 specific region using a specific primer pair or probe that specifically recognizes the 5' or 3' T-DNA flanking regions in EE-GM5 and the inserted T-DNA adjacent thereto in a sample of a seed lot.

[0103] 36. Amplifying a 50-1000 bp DNA fragment from a nucleic acid present in the biological sample using a polymerase chain reaction with at least two primers, one of which recognizes a 5' T-DNA flanking region of an inserted T-DNA in EE-GM5, the 5' T-DNA flanking region comprising the nucleotide sequence of SEQ ID NO: 5 from nucleotide 1 to nucleotide 166, or SEQ ID NO: 24 from nucleotide 1 to nucleotide 1113, or a 3' T-DNA flanking region of an inserted T-DNA in EE-GM5, the 3' T-DNA flanking region comprising the nucleotide sequence of the complement of SEQ ID NO: 6 from nucleotide 359 to nucleotide 691, or the nucleotide sequence of the complement of SEQ ID NO: 25 from nucleotide 359 to nucleotide 1449. 36. The method of claim 35, wherein the other of the primers recognizes a sequence within the inserted T-DNA comprising the complement of the nucleotide sequence of SEQ ID NO:5 from nucleotide 167 to nucleotide 353, or the nucleotide sequence of SEQ ID NO:6 from nucleotide 1 to nucleotide 358, or the nucleotide sequence of SEQ ID NO:11 from nucleotide 1 to nucleotide 7459 or a complement thereof, or the nucleotide sequence of SEQ ID NO:23 from nucleotide 1114 to nucleotide 8572 or a complement thereof, and hybridizing a probe specific to the DNA fragment amplified with the at least two primers, such as a probe comprising the sequence of SEQ ID NO:1 or 3, or SEQ ID NO:2 or 4, or a complement thereof.

[0104] 37. The method of claim 36, comprising the step of amplifying an 85 bp DNA fragment, wherein the primers comprise the sequences of SEQ ID NO:12 and SEQ ID NO:13, respectively, and the probe comprises the sequence of SEQ ID NO:14.

[0105] 38. A method for determining the zygosity status of a plant, plant material or seed comprising elite event EE-GM5, the method comprising the step of amplifying a 50-1000 bp DNA fragment from nucleic acid present in the biological sample using polymerase chain reaction with at least three primers, two of which specifically recognize pre-insertion plant DNA, such as a primer comprising the nucleotide sequence of SEQ ID NO: 21 and a primer comprising the nucleotide sequence of SEQ ID NO: 19, and a third of which recognizes a sequence within the inserted T-DNA, such as the nucleotide sequence of SEQ ID NO: 18, wherein using the primers 84 and 72 bp DNA fragments are amplified.

[0106] 39. A method for detecting the presence of elite event EE-GM5 in a biological sample by hybridization with a substantially complementary labeled nucleic acid probe, wherein the probe:target nucleic acid ratio is amplified by recycling of the target nucleic acid sequence, said method comprising: a) hybridizing said target nucleic acid sequence to a first nucleic acid oligonucleotide comprising the nucleotide sequence of SEQ ID NO:5 from nucleotide position 167 to nucleotide position 184, or a complement thereof, or said first nucleic acid oligonucleotide comprising the nucleotide sequence of SEQ ID NO:6 from nucleotide position 341 to nucleotide position 358, or a complement thereof; b) hybridizing said target nucleic acid sequence to a second nucleic acid oligonucleotide comprising the nucleotide sequence of SEQ ID NO:5 from nucleotide 149 to nucleotide 166 or its complement, or said nucleic acid oligonucleotide comprising the nucleotide sequence of SEQ ID NO:6 from nucleotide 359 to nucleotide 376 or its complement, wherein said first and second oligonucleotides overlap by at least one nucleotide, and wherein either said first or second oligonucleotide is labeled to be said labeled nucleic acid probe; c) cleaving only the labeled probe within the probe:target nucleic acid sequence duplex with an enzyme that causes selective probe cleavage resulting in dissociation of the duplex, leaving the target sequence intact; d) reusing the target nucleic acid sequence by repeating steps (a)-(c); and e) detecting the cleaved labeled probe, thereby determining the presence of said target nucleic acid sequence and detecting the presence of elite event EE-GM5 in said biological sample. A method comprising:

[0107] 40. Transgenic soybean plants, or cells, parts, seeds or progeny thereof, each of which contains the elite event EE-GM5 in its genome, and reference seeds containing said event have been deposited with the ATCC under accession number PTA-123625.

[0108] 41. The transgenic soybean plant, seed, cell, part or progeny of paragraph 40, whose genomic DNA generates an 85 bp DNA fragment when analyzed using PCR for EE-GM5 with two primers containing the nucleotide sequences of SEQ ID NO:12 and SEQ ID NO:13, respectively.

[0109] 42. A seed containing elite event EE-GM5, an inserted T-DNA at a specific location in the soybean genome, contained in the seed deposited with ATCC under accession number PTA-123625 or a derivative thereof.

[0110] 43. A soybean plant, plant part, cell or tissue, or seed comprising the elite event EE-GM5, obtainable from the seed according to paragraph 42.

[0111] 44. A soybean plant, or a seed, cell or tissue thereof, each of which contains the elite event EE-GM5 in its genome and which can be obtained by propagation of and / or by breeding with a soybean plant grown from a seed deposited with ATCC under accession number PTA-123625.

[0112] 45. A soybean seed comprising the elite event EE-GM5, wherein a reference seed comprising said event has been deposited with the ATCC under accession number PTA-123625.

[0113] 46. ​​A transgenic soybean plant, cell or tissue comprising the elite event EE-GM5, obtainable from the seed according to paragraph 45.

[0114] 47. The soybean plant cell of any one of paragraphs 40, 41, 43, 44 and 46, which is a non-propagating plant cell.

[0115] 48. A method for producing a soybean plant or seed comprising the elite event EE-GM5, comprising crossing a plant according to any one of paragraphs 40, 41, 43, 44 and 46 with another soybean plant, and planting seeds obtained from said cross.

[0116] 49. Soybean genomic DNA containing elite event EE-GM5.

[0117] 50. A nucleic acid molecule comprising a nucleotide sequence essentially similar to any one of SEQ ID NOs: 1, 3, or 5, or any one of SEQ ID NOs: 2, 4, or 6, or the complement of said sequences, such as a nucleic acid that confers tolerance to HPPD-inhibiting herbicides and / or SCN resistance in soybean, such as a nucleic acid molecule comprising a nucleotide sequence having at least 99% or at least 99.5% sequence identity to the nucleotide sequence of SEQ ID NO: 5 or 24 or 6 or 25, or the complement thereof.

[0118] 51. The nucleic acid molecule of paragraph 50, comprising the nucleotide sequence of any one of SEQ ID NO: 1 or 3, or SEQ ID NO: 2 or 4, or a complement of said sequence, such as a nucleic acid molecule that also comprises the nucleotide sequence of SEQ ID NO: 7 and 9, or a nucleotide sequence having at least 98% sequence identity thereto, or a complement thereof, or a nucleic acid molecule that comprises the nucleotide sequence of SEQ ID NO: 11 from nucleotide position 188 to nucleotide position 7101, or a nucleotide sequence having at least 98% sequence identity thereto.

[0119] 52. A soybean plant, cell, plant part, seed or progeny thereof comprising a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, 3 or 5 or the nucleotide sequence of SEQ ID NO: 2, 4 or 6, such as a soybean plant also comprising a chimeric gene encoding Cry14Ab-1 and a chimeric gene encoding HPPD-4, in particular a chimeric gene comprising the nucleotide sequence of SEQ ID NO: 7 and 9, respectively; or a soybean plant, cell, plant part, seed or progeny thereof comprising a nucleotide sequence of SEQ ID NO: 3 and a nucleotide sequence of SEQ ID NO: 4 in its genome; or a soybean plant, cell, plant part, seed or progeny thereof comprising a nucleotide sequence of SEQ ID NO: 5 and a nucleotide sequence of SEQ ID NO: 6 in its genome; or a soybean plant, cell, plant part, seed or progeny thereof comprising a nucleic acid molecule according to any one of the preceding paragraphs, such as a soybean plant also comprising a chimeric gene encoding Cry14Ab-1 and a chimeric gene encoding HPPD-4, in particular a chimeric gene comprising the nucleotide sequence of SEQ ID NO: 7 and 9, respectively.

[0120] 53. A nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NO:1, 3, or 5, or SEQ ID NO:2, 4, or 6, such as a nucleic acid molecule comprising the nucleotide sequences of SEQ ID NO:5 and SEQ ID NO:6, or complements thereof, or a nucleic acid molecule comprising the nucleotide sequences of SEQ ID NO:24 and SEQ ID NO:25, or complements thereof.

[0121] 54. A transgenic soybean plant, plant cell, tissue, or seed, characterized by a nucleic acid molecule comprising a nucleotide sequence essentially similar to any one of SEQ ID NOs:1, 3, 5, or 24, or any one of SEQ ID NOs:2, 4, 6, or 25, or the complement of said sequences, wherein the soybean plant comprises event EE-GM5 in its genome, which also comprises a chimeric gene encoding Cry14Ab-1 and a chimeric gene encoding HPPD-4.

[0122] 55. A soybean plant, cell, tissue or seed that comprises EE-GM5 and that contains in the genome of its cells a nucleic acid sequence having at least 80%, 90%, 95% or 100% sequence identity to any one of SEQ ID NOs: 1, 3, or 5, or any one of SEQ ID NOs: 2, 4, or 6, or the complement of said sequences, such as a soybean plant that also contains a chimeric gene encoding Cry14Ab-1 and a chimeric gene encoding HPPD-4, or a soybean plant, cell, tissue or seed that contains in the genome of its cells a nucleic acid sequence having at least 80%, 90%, 95% or 100% sequence identity to the sequence of SEQ ID NO: 24 or SEQ ID NO: 25.

[0123] 56. A soybean plant, plant cell, tissue, or seed comprising in its genome a nucleic acid molecule comprising a nucleotide sequence having at least 99% sequence identity to the nucleotide sequence of SEQ ID NO:5 or 24 or SEQ ID NO:6 or 25, or a complement thereof, or a soybean plant, plant cell, tissue, or seed comprising in its genome a nucleic acid molecule comprising a nucleotide sequence having at least 99% sequence identity to the nucleotide sequence of SEQ ID NO:5 or 24 and SEQ ID NO:6 or 25, or a complement thereof.

[0124] 57. A soybean plant, plant cell, tissue, or seed, comprising in its genome a nucleic acid molecule that hybridizes under standard stringency conditions to the nucleotide sequence of SEQ ID NO: 5 or 6, or its complement.

[0125] 58. A nucleic acid molecule comprising a nucleotide sequence having at least 99% sequence identity with the nucleotide sequence of SEQ ID NO:5 or 24 or SEQ ID NO:6 or 25, or a complement thereof, such as a nucleic acid molecule comprising a nucleotide sequence having at least 99% sequence identity with the nucleotide sequence of SEQ ID NO:5 or 24 and SEQ ID NO:6 or 25, or a complement thereof.

[0126] 59. A nucleic acid molecule comprising a nucleic acid sequence that hybridizes under standard stringency conditions to the nucleotide sequence of SEQ ID NO: 5 or 6, or its complement.

[0127] 60. The nucleic acid molecule of any one of paragraphs 50, 51, 58, and 59, which also comprises the nucleotide sequences of SEQ ID NOs: 7 and 9.

[0128] 61. A chimeric DNA comprising a T-DNA 5' flanking region, an insert T-DNA, and a T-DNA 3' flanking region, wherein the sequence of the insert T-DNA comprises the sequence of SEQ ID NO: 11 from nucleotide 188 to nucleotide 7101, or a sequence at least 95, 96, 97, 98, 99, or at least 99.5% identical thereto, or the sequence of the insert T-DNA comprises the sequences of SEQ ID NOs: 7 and 9, and the T-DNA 5' flanking region is located immediately upstream of and adjacent to the insert T-DNA and comprises the sequence of SEQ ID NO: 5 from nucleotide 1 to nucleotide 166, or a sequence at least 95, 96, 97, 98, 99, or at least 99.5% identical thereto, or the sequence of SEQ ID NO: 24 from nucleotide 1 to nucleotide 1113, or a sequence at least 95, 96, 97, 98, 99, or at least 99.5% identical thereto, and the T-DNA a 3' flanking region is located immediately downstream of and adjacent to the inserted T-DNA and comprises the sequence of SEQ ID NO:6 from nucleotide 359 to nucleotide 691, or a sequence at least 95, 96, 97, 98, 99, or at least 99.5% identical thereto, or the nucleotide sequence of the complement of SEQ ID NO:25 from nucleotide 359 to nucleotide 1449, or a sequence at least 95, 96, 97, 98, 99, or at least 99.5% identical thereto, a chimeric DNA.

[0129] 62. A nucleic acid molecule comprising a nucleotide sequence having at least 98% sequence identity to the nucleotide sequence of SEQ ID NO:7 or its complement, such as the nucleotide sequence of SEQ ID NO:7, such as a DNA molecule comprising the nucleotide sequence of SEQ ID NO:11 from nucleotide 131 to 5276, or its complement, or a sequence encoding a nematocidal Cry14Ab protein having at least 95, 96, 97, 98, or at least 99% sequence identity to SEQ ID NO:7 or the sequence of SEQ ID NO:11 from nucleotide position 131 to nucleotide position 5276, or its complement.

[0130] 63. A nucleic acid molecule comprising a nucleotide sequence having at least 98% sequence identity to the nucleotide sequence of SEQ ID NO:9 or its complement, such as the nucleotide sequence of SEQ ID NO:9, such as a DNA sequence comprising the nucleotide sequence of SEQ ID NO:11 from nucleotide 5382 to 7459, or a complement thereof, or a sequence having at least 95, 96, 97, 98, or at least 99% sequence identity to SEQ ID NO:9 or the sequence of SEQ ID NO:11 from nucleotide position 5382 to nucleotide position 7459, or a complement thereof, wherein the sequence encodes an HPPD protein that, when expressed in a plant, provides tolerance to HPPD-inhibiting herbicides.

[0131] 64. A method for producing a soybean product comprising obtaining a soybean seed containing the above elite event EE-GM5, and producing the soybean product therefrom.

[0132] 65. The method of claim 64, wherein the soybean product is or comprises soybean meal, ground seeds, flour, or flakes.

[0133] 66. The method of claim 4 or 65, wherein the soybean product comprises a nucleic acid specific for elite event EE-GM5, such as the product comprising a nucleic acid that produces an amplicon diagnostic or specific for event EE-GM5, such as the sequence of SEQ ID NO: 1 or 3 or the sequence of SEQ ID NO: 2 or 4.

[0134] 67. A soybean product comprising the elite event EE-GM5 as above, such as a soybean product produced from a soybean plant, cell, part, seed, or progeny of any one of the preceding paragraphs.

[0135] 68. The soybean product of paragraph 67, wherein the soybean product is or comprises soybean meal, ground seeds, flour, or flakes.

[0136] 69. The soybean product of paragraph 67 or 68, wherein the soybean product comprises a nucleic acid specific for elite event EE-GM5, such as the product comprising a nucleic acid that produces an amplicon diagnostic or specific for event EE-GM5, such as the sequence of SEQ ID NO: 1 or 3, or the sequence of SEQ ID NO: 2 or 4, or a complement thereof.

[0137] 70. A method for protecting germinating soybean plants from competition by weeds comprising treating a field sown with seeds containing the elite event EE-GM5 described in any one of the preceding paragraphs with an HPPD-inhibiting herbicide, wherein the plants are tolerant to the HPPD-inhibiting herbicide.

[0138] 71. A method for protecting emerging soybean plants from competition by weeds comprising treating a field in which soybean plants containing the above elite event EE-GM5 are to be planted with an HPPD-inhibiting herbicide prior to planting or sowing the soybean plants, and subsequently planting or sowing the soybean plants or seeds in the pre-treated field, wherein the plants are tolerant to the HPPD-inhibiting herbicide.

[0139] 72. A method for controlling weeds in a field of soybean plants comprising the above elite event EE-GM5, comprising treating said field with an effective amount of an HPPD-inhibiting herbicide, wherein the plants are resistant to the HPPD-inhibiting herbicide.

[0140] 73. The method of any one of paragraphs 70 to 72, wherein the HPPD-inhibiting herbicide is isoxaflutole, topramezone, or mesotrione.

[0141] 74. Use of a transgenic soybean plant, seed or progeny thereof, comprising the above elite event EE-GM5, for producing soybean seeds or seeds.

[0142] 75. Use of a soybean plant or seed containing in its genome the above elite event EE-GM5 for producing a nematode-resistant and / or HPPD-inhibiting herbicide-tolerant plant.

[0143] 76. Use of soybean seeds containing elite event EE-GM5 to obtain a soybean product, such as the soybean product being or including ground soybean grains, soybean flour, soybean meal, or soybean flakes, wherein EE-GM5 is as described above.

[0144] 77. Use of soybean plants or seeds containing the elite event EE-GM5 as defined above in combination with an HPPD-inhibiting herbicide for growing a soybean field or for growing a soybean crop.

[0145] 78. A nucleic acid molecule obtainable from a seed deposited with ATCC under accession number PTA-123625, said nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOs:1, 3, or 5, and the nucleotide sequence of any one of SEQ ID NOs:2, 4, or 6.

[0146] 79. A soybean plant, cell, part, or seed, each containing in its genome an elite event EE-GM5, said elite event being a genetic locus comprising an inserted T-DNA containing a chimeric gene encoding an HPPD-4 protein and a chimeric gene encoding a Cry14Ab-1 protein, and 5' and 3' flanking sequences immediately surrounding said inserted T-DNA, as found in a reference seed deposited with ATCC under accession number PTA-123625.

[0147] 80. A progeny plant, cell, plant part or seed of the plant, cell, plant part or seed of paragraph 79, wherein the progeny plant, cell, plant part or seed comprises the nucleotide sequence of SEQ ID NO:3 and the nucleotide sequence of SEQ ID NO:4.

[0148] 81. The soybean plant, cell, part, seed or progeny of paragraph 79, whose genomic DNA generates an 84 bp DNA fragment when analyzed using PCR with two primers comprising the nucleotide sequences of SEQ ID NO:18 and SEQ ID NO:19, respectively.

[0149] 82. A plant according to any one of the preceding clauses, which is resistant to isoxaflutole and / or topramezone and / or mesotrione, such as a plant resistant to isoxaflutole, topramezone and mesotrione.

[0150] 83. A method for producing a soybean plant that is resistant to SCN and tolerant to HPPD-inhibiting herbicides, comprising the steps of: crossing a first soybean plant lacking a gene encoding Cry14Ab-1 and lacking a gene encoding HPPD-4 with a soybean plant described in any one of the preceding paragraphs, thereby introducing resistance to SCN and tolerance to HPPD-inhibiting herbicides into the genome of the soybean plant; and selecting progeny plants that are resistant to SCN and tolerant to HPPD-inhibiting herbicides.

[0151] 84. Use of a soybean plant or seed containing the elite event EE-GM5 as defined above to obtain a soybean crop, such as a soybean crop with a higher yield when contaminated with nematodes or sudden death syndrome.

[0152] 85. A method for producing a soybean crop having improved resistance to said nematodes or sudden death syndrome, comprising: (a) planting a field using the seeds described in any one of the above paragraphs, such as where the nematode is SCN or Pratylenchus species or Meloidogyne or Nematode spp.; and (b) harvesting the soybean seeds produced in the plants grown from said seeds, and optionally (c) applying to the field planted with said seeds or to said soybean plants, before or after germination of the seeds, one or more doses of an HPPD-inhibiting herbicide sufficient to control weeds but which are tolerated by said soybean seeds or plants.

[0153] 86. Use of soybean seeds as described in the above paragraphs for preparing a processed food or feed product, said processed food or feed product comprising a detectable amount of a nucleic acid comprising the nucleotide sequence of SEQ ID NO:1 and / or SEQ ID NO:2, or a complement thereof.

[0154] 87. The use of paragraph 86, wherein (i) the food or feed commodity comprises soybean meal, soybean flour, soybean flakes, or soybean oil; (ii) the nucleic acid comprises the nucleotide sequence of SEQ ID NO:3 and / or SEQ ID NO:4, or a complement thereof; or (iii) the nucleic acid further comprises the nucleotide sequence contained in SEQ ID NO:7 and SEQ ID NO:9.

[0155] 88. A soybean plant, seed or cell comprising an elite event EE-GM5 in its genome, wherein the elite event EE-GM5 comprises a nucleotide sequence that is at least 90% identical to the sequence set forth in SEQ ID NO:23, said elite event comprising a chimeric gene encoding HPPD-4 and a chimeric gene encoding Cry14Ab-1, and wherein said plant, seed or cell is tolerant to HPPD-inhibiting herbicides and has SCN resistance.

[0156] 89. The plant of paragraph 88, wherein elite event EE-GM5 comprises a nucleotide sequence that is at least 95% identical to the sequence set forth in SEQ ID NO:23.

[0157] 90. The plant of paragraph 88, wherein elite event EE-GM5 comprises a nucleotide sequence that is at least 99%, at least 99.5%, or at least 99.9% identical to the sequence set forth in SEQ ID NO:23.

[0158] 91. A nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:23 or a nucleotide sequence having at least 99% sequence identity to SEQ ID NO:23, which confers resistance to HPPD-inhibiting herbicides and / or nematode resistance, wherein the nematode is an SCN or Pratylenchus species or a root-knot nematode or a Nematode reniformis species.

[0159] 92. A nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:11 from nucleotide position 131 to nucleotide position 7941, or a nucleotide sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0160] 93. The nucleic acid molecule of paragraph 92, encoding an HPPD protein that is resistant to HPPD inhibitors and a protein that negatively affects plant disease nematodes, such as SCN, RKN or Pratylenchus species nematodes.

[0161] 94. The nucleic acid molecule of paragraph 93, encoding a protein of SEQ ID NO:8, or a protein at least 99% identical thereto, and a protein of SEQ ID NO:10, or a protein at least 99% identical thereto.

[0162] 95. A method for controlling weeds and / or nematodes in a field planted with soybean plants comprising the steps of 1) treating said field with an HPPD-inhibiting herbicide such as isoxaflutole, topramezone or mesotrione, and 2) planting or sowing in said treated field soybean plants or seeds containing the above-mentioned elite transformation event EE-GM5, wherein reference seeds containing said elite event have been deposited with the ATCC under deposit number PTA-123625.

[0163] 96. A method for weed control comprising the steps of 1) planting in a field soybean plants or seeds resistant to an HPPD-inhibiting herbicide, such as isoxaflutole, topramezone or mesotrione, and 2) applying an HPPD-inhibiting herbicide, such as isoxaflutole, topramezone or mesotrione, to the field before planting the plants or seeds, or to the soybean plants or seeds after planting (which may be before or after seed germination), wherein the plants or seeds contain the soybean elite transformation event EE-GM5 in their genome, and reference seeds containing the elite event have been deposited with the ATCC under accession number PTA-123625.

[0164] 97. A weed control process comprising the steps of: 1) treating a field for planting soybean plants or for sowing soybean seeds with an HPPD-inhibiting herbicide, such as isoxaflutole, topramezone or mesotrione, prior to planting soybean plants or sowing seeds, and 2) planting soybean plants comprising soybean elite transformation event EE-GM5 or sowing soybean seeds comprising soybean elite transformation event EE-GM5 in said pretreated field, wherein a reference seed comprising said soybean elite transformation event EE-GM5 has been deposited with the ATCC under deposit number PTA-123625.

[0165] 98. A method for reducing yield losses in a field planted with soybean plants, particularly a field containing or expected to contain nematodes such as SCN, RKN or Pratylenchus or Nematode reniformes or combinations thereof, comprising the steps of 1) obtaining a plant or seed containing the above-mentioned elite transformation event EE-GM5, and 2) planting or sowing the soybean plants or seeds, wherein a reference seed containing said elite event has been deposited with the ATCC under the accession number PTA-123625.

[0166] 99. A method for increasing the yield of soybean plants when planted in a field containing nematodes such as SCN, RKN or Pratylenchus or Nematode reniformes or combinations thereof, comprising the steps of 1) obtaining a plant or seed containing the above-mentioned elite transformation event EE-GM5, and 2) planting or sowing the soybean plant or seed, wherein a reference seed containing said elite event has been deposited with the ATCC under accession number PTA-123625.

[0167] 100. A method for the preparation of a soybean transformation event EE-GM5 as described above, comprising the steps of: introducing the elite soybean transformation event EE-GM5 as described above into the genome of a soybean plant or seed; and optionally treating said plant or seed with an HPPD-inhibiting herbicide, such as isoxaflutole, topramezone or mesotrione; or optionally treating a field in which said plant or seed will be planted with an HPPD-inhibiting herbicide, such as isoxaflutole, topramezone or mesotrione, and planting said plant or seed in the pre-treated field. A method for producing soybean plants or seeds that are resistant to HPPD-inhibiting herbicides such as mesotrione, or that are resistant to nematodes such as SCN, RKN or Pratylenchus or Nematode nematode, or that are resistant to HPPD-inhibiting herbicides such as isoxaflutole, topramezone or mesotrione, or that are resistant to nematodes such as SCN, RKN or Pratylenchus or Nematode nematode.

[0168] 101. A nucleic acid molecule specifically characterizing soybean elite transformation event EE-GM5, characterized in that it comprises the nucleotide sequence of any one of SEQ ID NO: 1, 3 or 5, which contains a portion of the inserted foreign DNA of EE-GM5 adjacent to and downstream of a portion of the inserted foreign DNA of EE-GM5, and / or that it comprises the nucleotide sequence of SEQ ID NO: 2, 4 or 6, which contains a portion of the inserted foreign DNA of EE-GM5 adjacent to and downstream of a portion of the inserted foreign DNA of EE-GM5.

[0169] 102. A plant or seed comprising EE-GM5 as described above and also comprising resistance or tolerance to SCN, RKN or Pratylenchus or Nematode reniformes, or combinations thereof, provided by soybean resistance loci / genes.

[0170] 103. The plant or seed contains EE-GM5 and PI 548316, PI 567305, PI 437654, PI 90763, PI 404198B, PI 88788, PI 468916, PI 567516C, PI 209332, PI 438489B, PI 89772, Peking, PI 548402, PI 404198A, PI 561389B, PI 629013, PI 507471, PI 633736, PI 507354, PI 404166, PI 437655, PI 467312, PI 567328, PI 22897, or PI 103. The plant or seed of paragraph 102, comprising any one or combination of SCN resistance alleles / locuses of 494182.

[0171] 104. A plant or seed comprising EE-GM5 as above and also comprising tolerance to other herbicides provided by herbicide tolerance genes (natural or mutant soybean genes or transgenes), such as tolerance to glyphosate, glufosinate, sulfonylurea, imidazolinone, HPPD inhibitor, dicamba, 2,4-D, or PPO inhibitor based herbicides, or any combination thereof.

[0172] 105. The plant or seed of paragraph 103, wherein the plant or seed comprises EE-GM5 as described above and one or more of the following soybean transformation events conferring herbicide tolerance: MST-FGφ72-3, SYN-φφφH2-5, DAS-68416-4, DAS-444φ6-6, MON-877φ8-9, MON89788, MON-φ4φ32-6, ACS-GMφφ5-3, BPS-CV127-9, ACS-GMφφ6-4, MON-877φ5-6, or event DP-3φ5423-1.

[0173] 106. A method for reducing the severity of the effects of sudden death syndrome or iron deficiency chlorosis on soybean plants in the presence of SCN contamination or for increasing the yield of soybean plants in an SCN-containing field contaminated with sudden death syndrome or in an SCN-containing field that causes iron deficiency chlorosis in soybeans, said method comprising planting a soybean plant or sowing a soybean seed comprising an elite event EE-GM5, and a reference seed comprising said elite event has been deposited with the ATCC under accession number PTA-123625.

[0174] The following examples are not intended to limit the invention to the specific embodiments described, but may be understood in conjunction with the accompanying figures, which are incorporated herein by reference. [Brief description of the drawings]

[0175] [Figure 1]Schematic representation of the relationship between the cited nucleotide sequences and primers. Black bar: insert T-DNA; striped bar: DNA flanking the T-DNA; checkered arrow (a): cry14Ab-1.b chimeric gene (see Table 1 for the composition of the chimeric gene); striped arrow (b): hppdPf-4Pa chimeric gene (see Table 1 for the composition of the chimeric gene); black arrow: oligonucleotide primer; (c) indicates the complement of the indicated nucleotide sequence; black line: oligonucleotide probe (numbers below are representative SEQ ID NOs). The numbers below the bars representing SEQ ID NOs. 5 and 6 are the nucleotide positions of the different elements in said sequences. Note: the scheme is not drawn to scale. [Diagram 2] Endpoint method for EE-GM5 identity analysis. Figure 2 shows an example of the results of the method described in Example 2.1 for a series of soybean samples containing EE-GM5 and conventional soybean samples. For each sample, the S / B ratios are displayed for both the EE-GM5 specific reaction and the endogenous reaction. In this figure, samples within the line marked "a" are soybean samples that do not contain EE-GM5, samples within the line marked "b" are soybean samples that contain EE-GM5, and samples within the box formed by the line marked "c" are indeterminate samples. [Diagram 3] EE-GM5 An end-point method for GM5 identity and zygosity analysis. Figure 3 shows an example of the results of the method described in Example 2.2 for a series of soybean samples containing EE-GM5 in homozygous state, soybean samples containing EE-GM5 in hemizygous state and conventional soybean samples. In this figure, the samples within the line marked "a" are soybean samples containing EE-GM5 in homozygous state, the samples within the line marked "b" are soybean samples containing EE-GM5 in hemizygous state, the samples within the line marked "c" are soybean samples not containing EE-GM5, and the samples within the box formed by the line marked "d" are indeterminate samples. [Figure 4]Real-time PCR method for EE-GM5 low-level abundance analysis. Figure 4 shows an example of the results of the RT-PCR method described in Example 2.4 for low-level abundance analysis performed on calibration samples. "a", "b", "c", "d", and "e" indicate the Ct values ​​of calibration samples "A", "B", "C", "D", and "E", respectively. Calibration samples "A", "B", "C", "D", and "E" have decreasing amounts of EE-GM5 DNA. [Diagram 5] Average maximum phytotoxicity results of herbicide treatments. Figure 5 shows the average of maximum phytotoxicity data recorded for herbicide treatments in several field trials over a two-year period for soybean plants containing event EE-GM5 compared to non-transformed / conventional soybean plants (Thorne). The numbers in parentheses under the treatments indicate the number of trials included in the bar, and the numbers above each bar indicate the average maximum phytotoxicity value for that treatment. The treatments applied were as follows: IFT = isoxaflutole, MST = mesotrione, PE = pre-emergence, PO = post-emergence (at V2-V3 stage, in conjunction with adjuvant crop oil concentrate and ammonium sulfate to enhance herbicide activity). The rates shown are grams active ingredient / hectare (4x dose pre-emergence, 2x dose post-emergence). [Figure 6] Grain yield of EE-GM5 in Thorne in SCN-infested fields. EE-GM5 in the original transformant background (Thorne) was tested in SCN-infested fields (ranging from low to high SCN contamination) in nine different locations throughout Iowa, Illinois, Indiana, Missouri, and Tennessee in 2015 and 2016. Points are the estimated yield (as percent difference relative to the null) of homozygous events in each test, and horizontal lines represent the 95% confidence limits of the contrast between homozygous events and the null segregant (if the line does not overlap the vertical line at 100 percent yield of the null segregant, the event was significantly different from the null segregant). "Across Locs" is the estimated yield of the combined analysis across all nine locations. [Figure 7]Grain yield of EE-GM5 in an SCN-susceptible elite background in an SCN-infested field. EE-GM5 was introgressed (BC2F3) into an SCN-susceptible elite MG I (maturity group I) line and tested in 2016 at one location in Minnesota and one location in North Dakota (each with high SCN contamination levels). The points are the estimated yields (as percent difference relative to the null) of homozygous events in each test, and the horizontal lines near the points represent the 95% confidence limits of the contrast between the homozygous events and the null segregants (if the lines do not overlap the vertical lines at 100 percent yield of the null segregants, the events were significantly different from the null segregants). "Across Locs" is the estimated yield of the combined analysis across both locations. [Figure 8] Grain yield of EE-GM5 in an elite SCN-resistant background in an SCN-infested field. EE-GM5 was crossed to an elite MG III (maturity group III) line that is resistant to SCN (due to the rhg1 locus from PI88788), and EE-GM5 was tested across low to high SCN contamination levels at three locations in 2016 (trials beginning with "16", e.g., 16-IN1) and seven locations in 2017 (trials beginning with "17", e.g., 17-IN1) (see arrows, locations with low SCN pressure are at the bottom of the diagram (e.g., 17-IL2) and locations with high SCN pressure are at the top (e.g., 17-IN1)). SCN pressure was assigned by considering several factors, including known field history, SCN populations in the soil, relative yield of resistant and susceptible control varieties, soil properties (pH and % sand), and visual assessment of root contamination at the susceptible entry. The points are the average yield difference (tons per hectare) of homozygous events in each trial compared to the null segregant, and the horizontal lines near the points represent the 95% confidence limits of the contrast between the homozygous events and the null segregant (if the line does not overlap the vertical line at 0 difference from the null segregant, the yield of the event was significantly different from the null segregant). "Avg" is the average yield across all locations for each year. [Figure 9]US Pratylenchus Resistance Greenhouse Assay. Elite soybean plants with EE-GM5 control pineapple root lesion nematode (Pratylenchus brachyurus) in a US greenhouse assay. Plants with EE-GM5 ("EE-GM5") were compared to other elite soybean lines: one SCN susceptible maturity group (MG) 3 line ("THORNE"), one MG3 SCN susceptible line, one MG6.2 SCN susceptible line, and one MG9 SCN susceptible line ("Susc WT" represents the average of these three lines), one MG3 SCN resistant line (with rhg1 resistance allele from PI88788, "SCN Res(PI88788)"), and one MG6.2 SCN resistant line with rhg1 and Rhg4 SCN resistance from Peking ("SCN Res(Peking)"). Plotted is the average number of Pratylenchus in the roots 30 days after contamination (5 plants per entry), also showing the variation observed among varieties (as is typically seen in greenhouse assays). Results show approximately 90% control of Pratylenchus across the EE-GM5 lines. Soybean lines with natural SCN resistance (from Peking or PI88788) do not control pineapple Pratylenchus brachyurus. [Figure 10]Greenhouse assay for Pratylenchus resistance in Brazil. Soybean plants homozygous for EE-GM5 ("EE-GM5 HH") significantly reduced Pratylenchus brachyurus in soybean roots. Pratylenchus brachyurus was isolated from a local field in Brazil. EE-GM5 plants (two different US elite lines, both maturity group 6.2, one SCN susceptible and one Peking SCN resistant ("EE-GM5")) and five Brazilian soybean lines with limited Pratylenchus control ("Brazilian lines"), one Brazilian line labeled as low Rf (reproductive factor) for Pratylenchus ("BRS 7380 (Low Rf)"), one US elite line (maturity group 6.2) that is SCN susceptible ("SCN Susc") and one US elite line (MG 6.2) with Peking SCN resistance ("SCN Res(Peking)) were evaluated for Pratylenchus control in greenhouse assays in Brazil. Plotted are the averages of those entries, and also show the variation observed among varieties (as is typical in greenhouse assays). One Brazilian soybean line (BRS 7380) showed about 89% reduction in Pratylenchus. The EE-GM5 line controlled Pratylenchus by about 99%. Soybean lines carrying Peking natural resistance to SCN do not control Pratylenchus brachyurus. [Figure 11]Iron deficiency chlorosis (IDC) scores of EE-GM5 plants compared to null. Figure 11 shows the IDC scores of soybean plants with EE-GM5 at one location (with high SCN contamination). The study was a split-plot design (four plots per entry) examining the effect of the event in three different backgrounds (two susceptible soybean lines and one with SCN resistance derived from PI88788). The average IDC scores of plants with the event EE-GM5 ("EE-GM5") and the corresponding null segregants ("Null", lacking EE-GM5) across three genetic backgrounds (one SCN resistant, one SCN susceptible, and one SCN susceptible Thorne background) are shown. One bar represents 12 total plots. Vertical lines indicate standard error ("SEM" is standard error of the mean). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0176] In the present invention, in the development of nematode-resistant soybean (Glycine max) containing a gene encoding 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor resistance combined with a gene conferring resistance to nematodes (each under the control of a plant-expressible promoter), EE-GM5 has been identified as an elite event from a population of transgenic soybean plants. Specific tools for use in identifying the elite event EE-GM5 in biological samples are described herein.

[0177] Integration of recombinant DNA molecules into the plant genome typically results from cell or tissue transformation, with specific integration sites usually being by random integration.

[0178] DNA introduced into a plant genome as a result of transformation of a plant cell or tissue with recombinant DNA or "transforming DNA" and derived from such transforming DNA is hereinafter referred to as "inserted T-DNA" containing one or more "transgenes". The transgenes of EE-GM5 are nematode resistance and HPPD-inhibiting herbicide tolerance genes. "Plant DNA" in the context of the present invention refers to DNA derived from a transformed plant. The plant DNA is usually found at the same locus in the corresponding wild-type plant. The inserted T-DNA can be characterized by the position and configuration at the site of integration of the recombinant DNA molecule in the plant genome. The site in the plant genome where the recombinant DNA is inserted is also referred to as the "insertion site" or "target site". The insertion of recombinant DNA into a region of the plant genome referred to as the "pre-insertion plant DNA" (or "pre-insertion locus") can be associated with a deletion of the plant DNA referred to as a "target site deletion". As used herein, "flanking region" or "flanking sequence" refers to a sequence of DNA from the plant genome that is different from the introduced T-DNA by at least 10 bp, at least 20 bp, at least 50 bp, and up to 5000 bp, preferably located immediately upstream or immediately downstream of the inserted T-DNA. Transformation procedures that result in random integration of the inserted T-DNA will result in transformants with different flanking regions that are characteristic and unique for each transformant. When recombinant DNA is introduced into a plant by traditional crossing, its insertion site in the plant genome, or its flanking regions, will generally not change.

[0179] As used herein, an "isolated nucleic acid (sequence / molecule)" or "isolated DNA (sequence / molecule)" refers to a nucleic acid or DNA (sequence / molecule) that is no longer present in the natural environment from which it was isolated, for example, a nucleic acid sequence in another (bacterial) host or plant genome, or a nucleic acid or DNA (sequence / molecule) fused to a DNA or nucleic acid (sequence / molecule) of another origin, such as when contained in a chimeric gene under the control of a (heterologous) plant expression promoter. Any nucleic acid or DNA of the present invention, including any primer, may also be non-naturally occurring, such as a nucleic acid or DNA having a sequence identical to a naturally occurring sequence but with a label (lacking from the naturally occurring counterpart), or having a sequence with at least one nucleotide addition or substitution or at least one internal nucleotide deletion compared to the naturally occurring nucleotides, or having a sequence with less than 100% (non-identical) identity to a naturally occurring nucleic acid or DNA or a fragment thereof, or a nucleic acid or DNA having a sequence consisting of nucleotide sequences of different origins that do not occur together in nature (chimeric or hybrid DNA), or an artificial synthetic nucleic acid or DNA having a sequence different from a naturally occurring nucleic acid or DNA or a fragment thereof.

[0180] An event is defined as a (man-made) genetic locus carrying an inserted T-DNA or transgene containing at least one copy of a gene of interest as a result of genetic manipulation. The typical allelic state of an event is the presence or absence of an inserted T-DNA. An event is phenotypically characterized by the expression of one or more transgenes. At the genetic level, an event is part of the genetic makeup of the plant. At the molecular level, an event can be characterized by flanking sequences upstream and / or downstream of the transgene, the position of molecular markers and / or a restriction map by the molecular location of the transgene (as determined, for example, by Southern blotting). Usually, transformation of a plant with a transforming DNA containing at least one gene of interest results in a population of transformants containing a large number of separate events, each of which is unique. An event is characterized by an inserted T-DNA and at least one flanking sequence.

[0181] As used herein, an elite event is an event selected from a group of events obtained by transformation with the same transforming DNA, based on the effectiveness and superior expression of optimal traits, the stability of the transgene and its compatibility with optimal agronomic properties of the plant containing it. Thus, the criteria for elite event selection are one or more, preferably two or more, advantageously all of the following: a) Trait validity; b) the presence of the inserted T-DNA does not impair other desirable characteristics of the plant, such as those related to agronomic performance or commercial value; c) the event is characterized by a clearly defined molecular structure that is stably inherited and allows the development of appropriate tools for identity control; d) The gene of interest exhibits precise, appropriate and stable spatial and temporal phenotypic expression at commercially acceptable levels over the range of environmental conditions to which plants carrying the event are likely to be exposed in normal agronomic use.

[0182] The inserted T-DNA is preferably associated with a location in the plant genome that allows for easy introgression into the desired commercial genetic background.

[0183] An event's status as an elite event is confirmed by introgression of the elite event in a different related genetic background and observing compliance with one, two, three or all criteria, e.g., a), b), c) and d) above.

[0184] Thus, an "elite event" refers to a locus that contains an inserted T-DNA, which meets the above criteria. A plant, plant material, or progeny such as a seed, may contain one or more different elite events in its genome.

[0185] Tools developed to identify elite events, or plants or plant material containing elite events, or products containing plant material containing elite events, are based on specific genomic characteristics of the elite events, such as the inserted T-DNA, molecular markers or specific restriction maps of genomic regions containing sequences of the flanking regions of the inserted T-DNA.

[0186] Once one or both of the flanking regions of the inserted T-DNA have been sequenced, it is possible by molecular biology techniques to develop primers and / or probes that specifically recognize this (these) sequence in the nucleic acid (DNA or RNA) of the sample. For example, a PCR method can be developed to identify elite events in a biological sample (such as a sample of plants, plant material or products containing plant material). Such a PCR is based on at least two specific "primers", one recognizing a sequence in the 5' or 3' T-DNA flanking region of the elite event and the other recognizing a sequence in the inserted T-DNA. The primers preferably have a sequence of 15 to 35 nucleotides that "specifically recognize" a sequence in the 5' or 3' T-DNA flanking region of the elite event and in the inserted T-DNA of the elite event, respectively, under optimized PCR conditions, such that a specific fragment (the "integration fragment" or discriminating amplicon) is amplified from the nucleic acid sample containing the elite event. This means that only the targeted integration fragment is amplified under optimized PCR conditions, and no other sequences in the plant genome or the inserted T-DNA are amplified.

[0187] PCR primers suitable for the present invention may be: - oligonucleotides ranging from 17 nt to about 200 nt in length (primers recognizing 5' T-DNA flanking sequences) containing at their 3' end a nucleotide sequence of at least 17 consecutive nucleotides, preferably at least 20 consecutive nucleotides, selected from the 5' T-DNA flanking sequences (SEQ ID NO: 5 from nucleotide 1 to nucleotide 166, or SEQ ID NO: 24 from nucleotide 1 to nucleotide 1113, or a plant genomic sequence adjacent thereto upstream); or - oligonucleotides ranging from 17 nt to about 200 nt in length (primers recognizing 3' T-DNA flanking sequences) containing at their 3' end a nucleotide sequence of at least 17 consecutive nucleotides, preferably at least 20 consecutive nucleotides, selected from the 3' T-DNA flanking sequences (the nucleotide sequence of the complement of SEQ ID NO: 6 from nucleotide 359 to nucleotide 691, or the nucleotide sequence of the complement of SEQ ID NO: 25 from nucleotide 359 to nucleotide 1449, or a downstream adjacent plant genomic sequence); or - oligonucleotides (primers recognizing the inserted T-DNA) ranging in length from 17 nt to about 200 nt, comprising at their 3' end a nucleotide sequence of at least 17 consecutive nucleotides, preferably at least 20 consecutive nucleotides, selected from the inserted T-DNA sequence (the complement of SEQ ID NO: 5 from nucleotide 167 to nucleotide 353, or the sequence of SEQ ID NO: 6 from nucleotide 1 to nucleotide 358, or the sequence of SEQ ID NO: 11 from nucleotide 1 to nucleotide 7459, or the sequence of SEQ ID NO: 23 from nucleotide 1114 to nucleotide 8572, or their complements).

[0188] It will be appreciated that a primer recognizing a 5' T-DNA flanking sequence can be used in a PCR reaction with a primer recognizing an inserted T-DNA selected from the complement of SEQ ID NO:5 from nucleotide 167 to nucleotide 353, or the downstream adjacent T-DNA sequence thereof, while a primer recognizing a 3' T-DNA flanking sequence can be used in a PCR reaction with a primer recognizing an inserted T-DNA selected from the sequence of SEQ ID NO:6 from nucleotide 1 to nucleotide 358, or the upstream adjacent T-DNA thereof. A primer recognizing an inserted T-DNA can also be selected from the sequence of SEQ ID NO:11 from nucleotide 1 to nucleotide 7459, or the sequence of SEQ ID NO:23 from nucleotide 1114 to nucleotide 8572, or the complements thereof.

[0189] The primers may, of course, be longer than the mentioned 17 consecutive nucleotides, for example, 20, 21, 30, 35, 50, 75, 100, 150, 200 nt long or even longer. The primers may be entirely composed of nucleotide sequences selected from the mentioned nucleotide sequences of the flanking sequence and the inserted T-DNA sequence. However, the nucleotide sequences of the primers at their 5' ends (i.e., outside the 17 consecutive nucleotides at the 3' end) are not critical. Thus, the 5' sequence of the primers may optionally comprise or consist of a nucleotide sequence selected from the flanking sequence or the inserted T-DNA, but may contain some (for example, 1, 2, 5, or 10) mismatches compared to the T-DNA or the T-DNA flanking DNA. The 5' sequence of the primer may be a nucleotide sequence that is completely unrelated to the adjacent sequence or the inserted T-DNA, such as, for example, a nucleotide sequence that represents one or more restriction enzyme recognition sites, or a nucleotide sequence to which other oligonucleotides, such as a labeled oligonucleotide such as a FRET cassette, can be bound (see LGC genomics; Semagn et al., 2014, Mol Breeding 33:1-14, and US Pat. No. 7,615,620). Such an unrelated sequence or an adjacent DNA sequence with a mismatch should preferably be 100 or less, more preferably 50 or less, or even 25 nucleotides. The primer may also be modified with a label, such as a fluorescent label.

[0190] Furthermore, suitable primers may comprise or (essentially) consist of a nucleotide sequence at their 3' end spanning the junction region between sequences from the 5' or 3' T-DNA flanking regions and the inserted T-DNA sequence (located at nucleotides 166 and 167 of SEQ ID NO:5 and nucleotides 358 and 359 of SEQ ID NO:6, or nucleotides 1113 and 1114 of SEQ ID NO:24 and nucleotides 358 and 359 of SEQ ID NO:25), with the proviso that said 17 contiguous nucleotides located at 3' are not derived exclusively from either the inserted T-DNA or the T-DNA flanking sequences of SEQ ID NO:5 or 6, or 24 or 25.

[0191] It will also be readily apparent to one of skill in the art that appropriately selected PCR primer pairs should not contain sequences that are complementary to each other.

[0192] For purposes of this invention, a "complement of the nucleotide sequence represented in SEQ ID NO:X" is a nucleotide sequence that can be derived from the represented nucleotide sequence by replacing nucleotides with their complementary nucleotides according to Chargaff's rules (A⇔T; G⇔C) and reading the sequence in the 5' to 3' direction, i.e., in the opposite direction to the represented nucleotide sequence.

[0193] Examples of suitable primers are the oligonucleotide sequences of SEQ ID NO:13 or SEQ ID NO:19 or SEQ ID NO:26 or 27 (3' or 5' T-DNA flanking sequence recognition primers), or SEQ ID NO:12 or SEQ ID NO:18 or SEQ ID NO:28 or 29 (insertion T-DNA recognition primers for use with the 3' or 5' T-DNA flanking sequence recognition primers).

[0194] Preferably, the amplified fragment has a length of 50-500 nucleotides, such as 50-150 nucleotides. The specific primers may have sequences 80-100% identical to sequences within the 5' or 3' T-DNA flanking regions of the elite event and within the insert T-DNA of the elite event, respectively, provided that mismatches still allow specific identification of the elite event with these primers under optimized PCR conditions. However, the range of acceptable mismatches can be easily determined experimentally and is known to the skilled person.

[0195] Detection of the integrated fragments can occur in various ways, for example, by size estimation after gel analysis. The integrated fragments can also be sequenced directly. Other sequence-specific methods for detecting amplified DNA fragments are also known in the art. The amplified DNA fragments can also be detected using detection of a labeled sequence and label. For example, a labeled probe can be included in the reaction mixture that specifically binds to the amplified fragment. In one embodiment, the labeled probe (FRET hybridization probe) can include a fluorescent label and a quencher, such that the FRET cassette is no longer quenched and fluoresces when bound to the PCR product. Alternatively, a labeled FRET cassette, i.e., an oligonucleotide labeled with a fluorescent label and a quencher, that specifically binds to one of the primers in the reaction mixture, such as a FRET cassette directed to the 5' extension of the primer used in the reaction mixture, can be included in the reaction mixture (see, for example, Semagn et al., 2014, Mol Breeding 33:1-14, and US Pat. No. 7,615,620). Fluorescence can be measured using methods known in the art. Fluorescence can be measured in real time, i.e. during each cycle of the PCR reaction. Fluorescence can also be measured at the end of the PCR reaction.

[0196] Since the sequences of the primers and their relative positions in the genome are specific to the elite event, amplification of the integration fragment will only occur in biological samples containing (the nucleic acid of) the elite event. Preferably, when performing PCR to identify the presence of EE-GM5 in an unknown sample, a control is included for a set of primers capable of amplifying a fragment in a "housekeeping gene" of the plant species of the event. A housekeeping gene is a gene that is expressed in most cell types and is involved in basic metabolic activities common to all cells. Preferably, the fragment amplified from the housekeeping gene is a fragment larger than the amplified integration fragment. Other controls can be included depending on the sample to be analyzed.

[0197] Standard PCR protocols are described in the "PCR Application Manual" (Roche Molecular Biochemicals, 2nd edition, 1999, or 3rd edition, 2006) and other references. Optimal conditions for PCR, including the sequences of specific primers, are specified in the "PCR (or polymerase chain reaction) identification protocol" for each elite event. However, it is understood that some parameters in the PCR identification protocol may need to be adjusted to specific laboratory conditions and may be slightly modified to obtain similar results. For example, the use of different methods for the preparation of DNA may require adjustments, for example, of the amount of primers used, the polymerase and the annealing conditions. Similarly, the selection of other primers may determine other optimal conditions for the PCR identification protocol. However, these adjustments will be clear to the skilled artisan and are further detailed in current PCR application manuals such as those cited above.

[0198] Alternatively, specific primers can be used to amplify the integrated fragment, which can be used as a "specific probe" to identify EE-GM5 in biological samples. Contacting the nucleic acid of the biological sample with the probe under conditions that allow hybridization of the probe with its corresponding fragment in the nucleic acid results in the formation of a nucleic acid / probe hybrid. The formation of this hybrid can be detected (e.g., via the label of the nucleic acid or the probe), thereby indicating the presence of EE-GM5. Such identification methods based on hybridization with a specific probe (on a solid support or in solution) have been described in the art. The specific probe is preferably a sequence that specifically hybridizes under optimized conditions to a region that includes a part of the 5' or 3' T-DNA flanking region of the elite event and a part of the inserted T-DNA adjacent thereto (hereinafter referred to as the "specific region"). Preferably, the specific probe comprises a sequence of 50-500 bp, or 100-350 bp that is at least 80%, or 80-85%, or 85-90%, or 90-95%, or 95%-100% identical (or complementary) to the nucleotide sequence of the specific region of EE-GM5. Preferably, the specific probe will comprise a sequence of about 15 to about 100 contiguous nucleotides that is identical (or complementary) to the specific region of the elite event.

[0199] Oligonucleotides suitable as PCR primers for detecting the elite event EE-GM5 can also be used to develop PCR-based protocols for determining the zygosity status of plants containing the elite event. For this purpose, two primers that recognize the wild-type locus before integration are designed so that they are directed towards each other and the insertion site is located between the primers. These primers may contain primers that specifically recognize the 5' and / or 3' T-DNA flanking sequences of EE-GM5. This set of primers that recognize the wild-type locus before integration, together with a third primer complementary to the transformed DNA sequence (inserted T-DNA), allows for simultaneous diagnostic PCR amplification of the EE-GM5 specific locus as well as the wild-type locus. If the plant is homozygous for the transgenic locus or the corresponding wild-type locus, the diagnostic PCR will result in a single PCR product, preferably typical in length, typical of either the transgenic or wild-type locus. If the plant is hemizygous for the transgenic locus, two locus-specific PCR products will appear, reflecting the amplification of both the transgenic and wild-type locus.

[0200] Alternatively, to determine the zygosity status of plants containing elite events, two primers recognizing the wild-type locus before integration are designed such that they are directed to each other and one primer specifically recognizes the 5' or 3' T-DNA flanking sequence contained in SEQ ID NO: 5 or 6, or SEQ ID NO: 24 or 25, and one primer specifically recognizes the 3' or 5' T-DNA flanking sequence contained within SEQ ID NO: 6 or 5, or SEQ ID NO: 24 or 25, or specifically recognizes the pre-insertion locus. For the present invention, a suitable primer pair recognizing the wild-type locus before integration is a primer pair containing one primer comprising or consisting (essentially) of the nucleotide sequence of SEQ ID NO: 21, and one primer comprising or consisting (essentially) of the nucleotide sequence of SEQ ID NO: 19. This set of primers allows for simultaneous diagnostic PCR amplification of the EE-GM5 specific locus and the wild-type locus in the direction towards a primer that specifically recognizes the 5' or 3' T-DNA flanking sequence (such as a primer that comprises or consists (essentially) of the nucleotide sequence of SEQ ID NO: 18 in the direction towards a primer that comprises or consists (essentially) of the nucleotide sequence of SEQ ID NO: 19) together with a third primer that is complementary to the transformed DNA sequence (inserted T-DNA) or to the transformed DNA sequence and the 5' or 3' T-DNA flanking sequence adjacent to it. If the plant is homozygous for the transgenic locus or the corresponding wild-type locus, the diagnostic PCR will result in a single PCR product typical of either the transgenic or wild-type locus. If the plant is hemizygous for the transgenic locus, two locus-specific PCR products will appear, reflecting the amplification of both the transgenic and the wild-type locus.

[0201] The detection of PCR products typical of wild-type and transgenic loci can be based on the determination of the length of PCR products that can be typical of wild-type and transgenic loci. Alternatively, the detection of PCR products typical of wild-type and transgenic loci can be carried out by modifying the primer specific to the pre-insertion locus and by modifying the primer specific to the insertion T-DNA and detecting the incorporation of the modified primer into the PCR product. For example, the primer specific to the pre-insertion locus and the primer specific to the insertion T-DNA can be labeled with a fluorescent label, where the label is different for the two primers. Fluorescence can be detected when the primer is incorporated into the PCR product. When the plant is homozygous for the transgenic locus or the corresponding wild-type locus, the fluorescence of only the label of the primer specific to the insertion T-DNA or only the label of the primer specific to the pre-insertion locus can be detected. If the plant is hemizygous for the transgenic locus, the fluorescence of both the label of the primer specific for the inserted T-DNA and the label of the primer specific for the pre-insertion locus can be detected, reflecting amplification of both the transgenic and wild-type loci.

[0202] Alternatively, the primer specific to the pre-insertion locus and the primer specific to the insertion T-DNA can have a 5' extension that specifically binds to the labeled FRET cassette, i.e., an oligonucleotide labeled with a fluorescent label and a quencher, and the 5' extension and the corresponding FRET cassette are different for the two primers (see, for example, Semagn et al., 2014, Mol Breeding 33:1-14, and US Pat. No. 7,615,620). When the primer is incorporated into the PCR product and then the FRET cassette is incorporated into the PCR product, the fluorescence can be detected. When the plant is homozygous for the transgenic locus or the corresponding wild-type locus, the fluorescence of only the FRET cassette that specifically binds to the primer specific to the insertion T-DNA or only the FRET cassette that specifically binds to the primer specific to the pre-insertion locus can be detected. If the plant is hemizygous for the transgenic locus, the fluorescence of both the FRET cassette that binds specifically to the primer specific for the inserted T-DNA and the FRET cassette that binds specifically to the primer specific for the pre-insertion locus can be detected, reflecting amplification of both the transgenic and wild-type loci.

[0203] If the plant is homozygous for the transgenic locus or the corresponding wild-type locus, the diagnostic PCR will yield a single PCR product, preferably typical in length, for either the transgenic or wild-type locus If the plant is hemizygous for the transgenic locus, two locus-specific PCR products will appear, reflecting amplification of both the transgenic and wild-type loci.

[0204] Alternatively, to determine the zygosity status of plants containing elite events, the presence of the event can be determined in a PCR in a quantitative manner as described in the examples. For this purpose, two primers recognizing the event EE-GM5 are designed so that they are directed to each other, one primer specifically recognizing the 5' or 3' T-DNA flanking sequence contained in SEQ ID NO: 5 or 6, or in SEQ ID NO: 24 or 25, and one primer specifically recognizing the inserted T-DNA in SEQ ID NO: 5 or 6, or in SEQ ID NO: 24 or 25, or in SEQ ID NO: 11 or 23. This set of primers allows PCR amplification of the EE-GM5 specific locus. The amplified DNA fragments can be quantitatively detected using a labeled probe contained in the reaction mixture that specifically binds to the amplified fragments. The labeled probe may contain a fluorescent label and a quencher so that the label is no longer quenched and fluoresces when bound to the PCR product. Using methods known in the art, the fluorescence can be measured in real time, i.e. during each cycle of the PCR reaction. The PCR cycle at which fluorescence exceeds a certain threshold level is an indication of the amount of EE-GM5 specific locus in the biological sample analyzed, and the zygosity status can be calculated based on reference homozygous and heterozygous samples.

[0205] Alternatively, the zygosity status of plants containing EE-GM5 can be determined based on copy number analysis using Taqman chemistry and real-time PCR principles. Alternative methods would typically include an EE-GM5-specific reaction to quantify EE-GM5 copy number, and an endogenous gene-specific reaction for normalization of EE-GM5 copy number. Samples containing EE-GM5 events in homozygous state would have a relative copy number two times higher than hemizygous samples. Unpaired samples would not amplify EE-GM5 sequences in such a way.

[0206] In addition, detection methods specific to elite event EE-GM5 that are different from PCR-based amplification methods can also be developed using elite event specific sequence information provided herein.Such alternative detection methods include linear signal amplification detection methods based on invasive cleavage of specific nucleic acid structures, also known as Invader™ technology (for example, as described in U.S. Patent No. 5,985,557 "Invasive Cleavage of Nucleic Acids" and U.S. Patent No. 6,001,567 "Detection of Nucleic Acid sequences by Invader Directed Cleavage", which are incorporated herein by reference). For this purpose, the target sequence is hybridized by a labeled first nucleic acid oligonucleotide comprising the nucleotide sequence of SEQ ID NO:5 from nucleotide position 167 to nucleotide position 184 or its complement, or comprising the nucleotide sequence of SEQ ID NO:6 from nucleotide position 341 to nucleotide position 358 or its complement, and is further hybridized by a second nucleic acid oligonucleotide comprising the nucleotide sequence of SEQ ID NO:5 from nucleotide 149 to nucleotide 166 or its complement, or said nucleic acid oligonucleotide comprising the nucleotide sequence of SEQ ID NO:6 from nucleotide 359 to nucleotide 376 or its complement, wherein said first and second oligonucleotides overlap by at least one nucleotide.

[0207] The double- or triple-stranded structure produced by this hybridization allows for selective probe cleavage by an enzyme (Cleavase®) that leaves the target sequence intact. The cleaved labeled probe is then detected, potentially through an intermediate step that results in further signal amplification.

[0208] In one embodiment, a method for detecting the presence of elite event EE-GM5 in a biological sample by hybridization with a substantially complementary labeled nucleic acid probe, wherein the probe:target nucleic acid ratio is amplified by recycling of the target nucleic acid sequence, said method comprising: a) hybridizing said target nucleic acid sequence to a first nucleic acid oligonucleotide comprising the nucleotide sequence of SEQ ID NO:5 from nucleotide position 167 to nucleotide position 184, or a complement thereof, or said first nucleic acid oligonucleotide comprising the nucleotide sequence of SEQ ID NO:6 from nucleotide position 341 to nucleotide position 358, or a complement thereof; b) hybridizing said target nucleic acid sequence to a second nucleic acid oligonucleotide comprising the nucleotide sequence of SEQ ID NO:5 from nucleotide 149 to nucleotide 166 or its complement, or said second nucleic acid oligonucleotide comprising the nucleotide sequence of SEQ ID NO:6 from nucleotide 359 to nucleotide 376 or its complement, wherein said first and second oligonucleotides overlap by at least one nucleotide, and wherein either said first or second oligonucleotide is labeled to be said labeled nucleic acid probe; c) cleaving only the labeled probe within the probe:target nucleic acid sequence duplex with an enzyme that causes selective probe cleavage resulting in dissociation of the duplex, leaving the target sequence intact; d) reusing the target nucleic acid sequence by repeating steps (a)-(c); and e) detecting the cleaved labeled probe, thereby determining the presence of said target nucleic acid sequence and detecting the presence of elite event EE-GM5 in said biological sample. A method is provided, comprising:

[0209] As used herein, two nucleic acids are "substantially complementary" if they are not complete complements of each other (as defined herein), such as if their sequences are at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% complementary to each other.

[0210] "Kit" as used herein refers to a set of reagents for carrying out the method of the invention, more specifically for identifying elite event EE-GM5 in a biological sample or for determining the zygosity status of EE-GM5-containing plant material. More specifically, a preferred embodiment of the kit of the invention comprises at least one or two specific primers as described above for identifying elite events, or three specific primers, or two specific primers and one specific probe as described above for determining the zygosity status. Optionally, the kit may further comprise any other reagents as described herein in the PCR identification protocol, or any other protocol as described herein for EE-GM5 detection. Alternatively, according to another embodiment of the invention, the kit may comprise a specific probe, as described above, that specifically hybridizes with the nucleic acid of the biological sample to identify the presence of EE-GM5 therein. Optionally, the kit may further comprise any other reagents (hybridization buffer, label, etc.) for identifying EE-GM5 in a biological sample using the specific probe.

[0211] The kits of the invention can be used for purposes of quality control (e.g. seed lot purity), detection of the presence or absence of elite events in plant material or materials containing or derived from plant material (such as but not limited to food or feed or industrial products), and their components can be specifically tailored.

[0212] As used herein, "sequence identity" with respect to nucleotide sequences (DNA or RNA) refers to the number of positions with identical nucleotides divided by the number of nucleotides of the shorter of the two sequences.The alignment of two nucleotide sequences is carried out by the Wilbur and Lipmann algorithm (Wilbur and Lipmann, 1983, Proc. Nat. Acad. Sci. USA 80:726) using a window size of 20 nucleotides, a word length of 4 nucleotides, and a gap penalty of 4.Computer-assisted analysis and interpretation of sequence data, including sequence alignment as described above, can be conveniently carried out using, for example, the sequence analysis software package of the Genetics Computer Group (GCG, University of Wisconsin Biotechnology Center). A sequence is designated as "essentially similar" if such sequences have at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%, or at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity. When an RNA sequence is said to be essentially similar or have a degree of sequence identity with a DNA sequence, it is clear that thymidine (T) in the DNA sequence is considered equivalent to uracil (U) in the RNA sequence.It is also clear that small differences or mutations may appear in DNA sequences over time and that some mismatches may be tolerated for the event-specific primers or probes of the invention, and any DNA sequence shown herein in any embodiment of the invention for any 3' or 5' T-DNA flanking DNA, or any insert or inserted T-DNA, or any primer or probe of the invention, also includes sequences essentially similar to the sequences provided herein, such as sequences that hybridize to or have at least 90%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with the given sequence for any 3' or 5' T-DNA flanking DNA, any primer or probe, or any insert or inserted T-DNA of the invention, such as nucleotide sequences that differ from any given sequence by 1 to 200, 1 to 150, 1 to 100, 1 to 75, 1 to 50, 1 to 30, 1 to 20, 1 to 10, 1 to 5, or 1 to 3 nucleotides.

[0213] The term "primer" as used herein encompasses any nucleic acid capable of initiating the synthesis of a nascent nucleic acid in a template-dependent process such as PCR. Typically, primers are oligonucleotides of 10-30 nucleotides, although longer sequences can be used. Although the single-stranded form is preferred, primers may be provided in double-stranded form. Probes can be used as primers, but are designed to bind to target DNA or RNA and need not be used in the amplification process.

[0214] The term "recognition" as used herein when referring to a particular primer or probe refers to the fact that the particular primer or probe specifically hybridizes to a nucleic acid sequence in an elite event under conditions described in the method (such as the conditions of a PCR identification protocol), whereby specificity is determined by the presence of positive and negative controls.

[0215] The term "hybridize" as used herein when referring to a specific probe refers to the fact that the probe binds to a specific region in the nucleic acid sequence of the elite event under standard stringency conditions. Standard stringency conditions as used herein refer to the conditions of hybridization described herein or those described by Sambrook et al., 1989 (Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, NY), which may include, for example, the following steps: 1) immobilizing plant genomic DNA fragments on a filter; 2) prehybridizing the filter in 50% formamide, 5xSSPE, 2xDenhardt's reagent, and 0.1% SDS at 42°C for 1-2 hours, or in 6xSSC, 2xDenhardt's reagent, and 0.1% SDS at 68°C for 1-2 hours; 3) adding a labeled hybridization probe; 4) incubating for 16-24 hours; 5) washing the filter in 1xSSC, 0.1% SDS at room temperature for 20 minutes; 6) washing the filter three times for 20 minutes each in 0.2xSSC, 0.1% SDS at 68°C; and 7) exposing the filter to X-ray film at -70°C for 24-48 hours with intensifying screens.

[0216] As used herein, a biological sample is a sample of a plant, plant material, or product containing plant material. The term "plant" is intended to encompass any cell, tissue, or organ taken from or derived from any such plant, including, but not limited to, soybean (Glycine max) plant tissue at any stage of maturity, and any seed, leaf, stem, flower, root, single cell, gamete, cell culture, tissue culture, protoplast. As used herein, "plant material" refers to material obtained from or derived from a plant. A product containing plant material relates to food, feed, or other products that are produced using or may be contaminated by plant material. In the context of the present invention, it is understood that such biological samples are tested for the presence of nucleic acid specific to EE-GM5, which indicates the presence of nucleic acid in the sample. Thus, the method referred to herein for identifying elite event EE-GM5 in a biological sample relates to the identification in the biological sample of nucleic acid containing elite event.

[0217] As used herein, "comprising" should be interpreted as specifying the presence of the recited described features, integers, steps, reagents or components, but does not exclude the presence or addition of one or more features, integers, steps or components, or groups thereof. Thus, for example, a nucleic acid or protein comprising a sequence of nucleotides or amino acids may contain more nucleotides or amino acids than those actually recited, i.e., embedded in a larger nucleic acid or protein. A chimeric gene comprising a functionally or structurally defined DNA sequence may contain additional DNA sequences, such as promoters, leaders, trailers, and / or transcription termination sequences (possibly also including DNA encoding a targeting or transport peptide).

[0218] The present invention also relates to the development of the elite event EE-GM5 in soybean plants containing this event, progeny plants and seeds containing the elite event EE-GM5 obtained from these plants, and plant materials derived from plants containing this event. As described in the Examples, plants containing the elite event EE-GM5 can be obtained. The present invention also relates to seeds containing the elite event EE-GM5 deposited with the ATCC under the accession number PTA-123625, or derivatives thereof containing the elite event EE-GM5. As used herein, "(seed) derivatives" refers to plants that can be grown from such seeds, progeny resulting from selfing, crossing or backcrossing, as well as plant cells, organs, parts, tissues, cell cultures, protoplasts, and plant materials thereof.

[0219] Soybean plants or plant materials containing EE-GM5 can be identified according to any of the identification protocols for EE-GM5 described in the Examples, including the end-point method for EE-GM5 identity analysis in Example 2.1, the end-point method for EE-GM5 identity and zygosity analysis described in Example 2.2, the real-time PCR method for EE-GM5 low-level abundance analysis described in Example 2.3, or the real-time PCR method for EE-GM5 low-level abundance analysis described in Example 2.4. Briefly, soybean genomic DNA present in a biological sample is amplified by PCR using a primer that specifically recognizes a sequence within the 5' or 3' T-DNA flanking sequence of EE-GM5, such as a primer having the sequence of SEQ ID NO: 13 or SEQ ID NO: 19, and a primer that recognizes a sequence in the inserted T-DNA, such as a primer having the sequence of SEQ ID NO: 12 or SEQ ID NO: 18, or a primer that recognizes the 5' or 3' T-DNA flanking sequence of EE-GM5 and the adjacent inserted T-DNA. DNA primers that amplify a portion of the endogenous soybean sequence are used as a positive control for PCR amplification. If, upon PCR amplification, the material produces fragments of the expected size or causes fluorescence of the expected fluorescent label, then the material contains plant material from a soybean plant harboring the elite event EE-GM5.

[0220] Plants carrying EE-GM5 are characterized by their nematode resistance, in particular SCN, disease nematode and / or root-knot nematode ("RKN") and / or Nematode nematode, as well as by their resistance to HPPD inhibitors such as isoxaflutole, topramezone or mesotrione. Different commercial varieties of soybean plants carrying EE-GM5 are also characterized by having agronomic properties comparable to the corresponding non-transgenic isogenic commercial varieties in the absence of HPPD-inhibiting herbicide application and SCN contamination. It has been observed that the presence of the inserted T-DNA in the insertion region of the soybean plant genome described herein confers particularly interesting phenotypic and molecular properties to the plants containing this event.

[0221] Also provided herein is a method for producing a soybean plant that is resistant to SCN and tolerant to HPPD-inhibiting herbicides, comprising: crossing a first soybean plant that lacks the gene encoding Cry14Ab-1 and lacks the gene encoding HPPD-4 with an EE-GM5-containing soybean plant to introduce resistance to SCN and tolerance to HPPD-inhibiting herbicides into the genome of the soybean plant; and selecting progeny plants that are resistant to SCN and tolerant to HPPD-inhibiting herbicides.Resistance to SCN can be measured using standard SCN greenhouse assays, such as those described at www.plantpath.iastate.edu / tylkalab / greenhouse-resistance-screening and www.plantmanagementnetwork.org / pub / php / review / 2009 / sce08 / .

[0222] One embodiment of the present invention provides an elite event in soybean plants that can be obtained by inserting two transgenes at specific locations in the soybean genome, which confers resistance to nematodes and tolerance to HPPD-inhibiting herbicides such as isoxaflutole, topramezone or mesotrione to such soybean plants, and such elite event has essentially similar agronomic performance to an isogenic line (as used herein, an "isogenic line" or "near isogenic line" is a soybean line with the same genetic background, such as a plant with the same genetic background as the plant used for transformation, but lacking the transgene, or a segregating sister line ("null") that has lost the transgene). In particular, the present invention provides an elite event in soybean plants, the insertion or presence of said elite event in the genome of such soybean plants does not appear to cause increased susceptibility to diseases, does not cause a reduction in yield, or does not cause increased lodging compared to isogenic lines or commercial soybean varieties. Thus, the present invention provides an elite event in soybean plants, designated EE-GM5, which has improved resistance to nematodes compared to isogenic lines, without negatively affecting the yield of said soybean plants, and which can withstand the application of HPPD-inhibiting herbicides, such as isoxaflutole, topramezone or mesotrione, and which are not statistically significantly different from isogenic soybean plants or commercial soybean varieties in their disease susceptibility or lodging. These properties make the elite event of the present invention a valuable tool in nematode control and weed resistance management programs. In one embodiment, event EE-GM5 is selected from the group consisting of event EE-GM3 (also known as FG-072, MST-FGφ72-3, described in WO 2011063411 and USDA-APHIS Petition 09-328-01p), event SYHT0H2 (also known as 0H2, SYN-φφφH2-5, described in WO 2012 / 082548 and 12-215-01p), event DAS-68416-4 (also known as EnlistSoybean, described in WO 2011 / 066384 and WO 2011 / 066360, and USDA-APHIS Petition 09-349-01p), Event DAS-44406-6 (also known as Enlist E3, DAS-444φ6-6, described in WO 2012 / 075426 and USDA-APHIS Petition 11-234-01p), Event MON87708 (Dicamba Tolerance Event in Roundup Ready 2 Xtend Soybeans, described in WO 2011 / 034704 and USDA-APHIS Petition 10-188-01p, MON-877φ8-9), Event MON89788 (also known as Genuity Roundup Ready 2 Yield, as described in WO 2006 / 130436 and USDA-APHIS Petition 06-178-01p), Event 40-3-2 (also known as Roundup Ready, GTS 40-3-2, MON-φ4φ32-6, as described in USDA-APHIS Petition 93-258-01), Event A2704-12 (also known as LL27, ACS-GMφ5-3, as described in WO 2006108674 and USDA-APHIS Petition 96-068-01p), Event 127 (also known as BPS-CV127-9, as described in WO 2010 / 080829), Event A5547-127 (also known as LL55, ACS-GMφφ6-4, as described in WO 2006108675 and USDA-APHIS Petition 96-068-01p), Petition 96-068-01p), event MON87705 (MON-877φ5-6, Vistive Gold, International Publication No. 2010 / 037016, USDA-APHIS Petition 09-201-01p), or event DP305423 (also known as DP-3φ5423-1, International Publication No. 2008 / 054747, USDA-APHIS Petition 09-201-01p).or EE-GM5 is combined with one or more soybean GM events that provide resistance to any one or combination of glyphosate, glufosinate, HPPD inhibitor, sulfonylurea or imidazolinone, AHAS or ALS inhibitor and / or auxin (e.g., dicamba, 2,4-D) herbicides, such as Petition 06-354-01p, ... MON 89788 (aka Intacta RR2 Pro Soybean, MON-877φ1-2 x MON-89788-1), DAS-81419-2 x DAS-44406-6 (aka Conkest™ Enlist E3™ Soybean, DAS-81419-2 x DAS-444φ6-6), Event DAS-68416-4 x Event MON 89788 (aka Enlist™ RoundUp Ready™ 2 Soybean, DAS-68416-4 x MON-89788-1), Event MON-87769-7 x Event MON-89788-1 (aka Omega-3 x Genuity Roundup Ready 2 Yield Soybeans), Event MON 87705 x Event MON 89788 (aka Vistive Gold, MON-877φ5-6 x MON-89788-1), or Event MON87769 x Event MON89788 (aka Omega-3 x Genuity Roundup Ready 2 Yield Soybeans, MON-87769-7 x MON-89788-1).

[0223] Also provided herein is a soybean plant or part thereof comprising event EE-GM5, and a representative soybean seed comprising event EE-GM5 has been deposited under ATCC Accession No. PTA-123625. Further provided herein are seeds of such plants comprising such events, and soybean products produced from such seeds, said soybean products comprising event EE-GM5. Such soybean products may be or may comprise soybean meal, ground soybean grains, soybean flakes, or products comprising any of these processed soybean products. In particular, such soybean products comprise nucleic acids producing diagnostic or specific amplicons for event EE-GM5, such amplicons comprising any one of the sequences of SEQ ID NO: 1 or 3, or SEQ ID NO: 2 or 4. Also provided herein is a method of producing a soybean product, comprising obtaining a soybean seed or grain comprising event EE-GM5, and producing such a soybean product therefrom. Also provided herein is a method for obtaining processed food, feed, or industrial products from soybean seeds, such as soybean oil, soybean protein, lecithin, soy milk, tofu, margarine, biodiesel, biocomposite, adhesive, solvent, lubricant, detergent, foam, paint, ink, candle, soybean oil or soybean protein-containing food or (animal) feed products, said method comprising obtaining seeds containing EE-GM5, and producing said processed food, feed, or industrial products from said seeds. In an embodiment, this process may also comprise obtaining soybean seeds or plants containing event EE-GM5, growing said seeds or plants in a field, and harvesting soybean seeds. Optionally, this method comprises applying an HPPD-inhibiting herbicide, such as IFT, topramezone, or mesotrione, before planting, before emergence, after emergence, or on top of the plants containing EE-GM5. In one embodiment, the invention includes the above soybean-derived processed food, feed or industrial products, such as processed products that produce an EE-GM5 event-specific amplicon using the methods described herein or that contain the nucleotide sequence of any one of SEQ ID NO: 1, 3 or 5, or SEQ ID NO: 2, 4 or 6.

[0224] Also provided herein is a soybean plant that is a descendant of any of the above soybean plants and comprises event EE-GM5, such as a progeny plant or seed of any one of the above soybean plants comprising the sequence of SEQ ID NO:1 or 3, or the sequence of SEQ ID NO:2 or 4, or a progeny plant or seed of any one of the above soybean plants comprising the sequence of SEQ ID NO:1 or 3 and the sequence of SEQ ID NO:2 or 4, or a progeny plant or seed of any one of the above soybean plants comprising the sequence of SEQ ID NO:5 or SEQ ID NO:24, or the sequence of SEQ ID NO:6 or SEQ ID NO:25, or a progeny plant or seed of any one of the above soybean plants comprising the sequence of SEQ ID NO:5 or SEQ ID NO:24 and the sequence of SEQ ID NO:6 or SEQ ID NO:25.

[0225] In particular, further provided herein is a method for producing a soybean plant that is resistant to nematodes and tolerant to isoxaflutole and / or topramezone and / or mesotrione herbicides, comprising the steps of introducing the event EE-GM5 into the genome of a first soybean plant lacking the event EE-GM5 by crossing such a plant with a soybean plant containing EE-GM5, and selecting progeny plants that are resistant to nematodes and tolerant to isoxaflutole and / or topramezone and / or mesotrione herbicides.

[0226] Also provided herein is a soybean plant with acceptable agronomic properties, resistant to nematodes and tolerant to isoxaflutole, topramezone or mesotrione herbicides, which contains a gene encoding Cry14Ab-1 and a gene encoding HPPD-4 and can produce a diagnostic amplicon for event EE-GM5.Also provided herein are such specific isolated amplicons (DNA sequence fragments) that can be obtained using the specific detection tools described herein, in particular amplicons that contain in their sequence DNA fragments derived from 5' or 3' T-DNA flanking DNA and DNA inserted into the plant genome by transformation as defined herein.

[0227] Further provided herein is a method for controlling weeds in a field of soybean plants containing event EE-GM5, or in a field in which such soybean plants are planted (said plants are planted in said field after treatment), comprising treating the field with an effective amount of an HPPD-inhibiting herbicide, such as an isoxaflutole or topramezone or mesotrione herbicide, wherein such plants are resistant to such herbicide.

[0228] Further provided herein is DNA comprising the sequence of SEQ ID NO: 5 or 6 or a sequence essentially similar thereto, and any plant, cell, tissue or seed, particularly soybean, comprising such a DNA sequence, such as a plant, cell, tissue or seed comprising EE-GM5. Also included herein is any soybean plant, cell, tissue or seed comprising the DNA sequence of SEQ ID NO: 5 or 6 (heterologous or foreign to a conventional soybean plant, seed, tissue or cell) or comprising a DNA sequence having at least 99% or 99.5% sequence identity to the sequence of SEQ ID NO: 5 or 24 or SEQ ID NO: 6 or 25.

[0229] Also described is a chimeric DNA comprising an inserted T-DNA, the sequence of said inserted T-DNA having at least 97, 98, 99, 99.5 or at least 99.9% sequence identity thereto, and wherein the 5' and 3' T-DNA flanking regions comprise adjacent sequences, wherein the 5' T-DNA flanking region immediately upstream of said inserted T-DNA is characterized by a sequence comprising the sequence of SEQ ID NO:5, from nucleotide 1 to nucleotide 166, or the sequence of SEQ ID NO:24, from nucleotide 1 to nucleotide 1113, and the 3' T-DNA flanking region immediately downstream of said inserted T-DNA is characterized by a sequence comprising the nucleotide sequence of SEQ ID NO:6, from nucleotide 359 to nucleotide 691, or the complement of SEQ ID NO:25, from nucleotide 359 to nucleotide 1449. In one embodiment, the sequence of the inserted T-DNA is SEQ ID NO:11, from nucleotide 1 to nucleotide 7459, or SEQ ID NO:23, from nucleotide 1114 to nucleotide 8572, or has at least 97, 98, 99, 99.5 or at least 99.9% sequence identity thereto, and a portion of the 5' and 3' T-DNA flanking regions comprises adjacent sequences, and a portion of the 5' T-DNA flanking region immediately adjacent upstream of the inserted T-DNA comprises SEQ ID NO:5, from nucleotide 1 to nucleotide 166, or SEQ ID NO:13, from nucleotide 1 to nucleotide 1113. 24, or a sequence having at least 97, 98, 99, 99.5 or at least 99.9% sequence identity thereto, and a portion of the 3' T-DNA flanking region immediately downstream adjacent to said inserted T-DNA is characterized by a sequence consisting of the nucleotide sequence of SEQ ID NO:6 from nucleotide 359 to nucleotide 691, or the nucleotide sequence of the complement of SEQ ID NO:25 from nucleotide 359 to nucleotide 1449, or a sequence having at least 97, 98, 99, 99.5 or at least 99.9% sequence identity thereto.

[0230] Chimeric DNA refers to a DNA sequence that contains regulatory and coding sequences that are not found together in nature.Thus, chimeric DNA can contain adjacent DNA regions that are derived from different sources or are arranged in a different way from that found in nature.An example of chimeric DNA is the sequence of SEQ ID NO:5 or 6.

[0231] Also provided herein are transgenic soybean plants, plant cells, tissues, or seeds that comprise in their genome an event EE-GM5 characterized by a nucleic acid molecule comprising a nucleotide sequence essentially similar to SEQ ID NO:1 or 3, and a nucleic acid molecule comprising a nucleotide sequence essentially similar to SEQ ID NO:2 or 4, or the complement of said sequences, as well as soybean plants, plant cells, tissues, or seeds that comprise in their genome an event EE-GM5 characterized by a nucleic acid molecule comprising a nucleotide sequence essentially similar to SEQ ID NO:5 or 24 and SEQ ID NO:6 or 25, or the complement of said sequences.

[0232] Further provided herein is a soybean plant, cell, tissue or seed comprising EE-GM5, characterized by comprising in the genome of the cell a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of SEQ ID NOs: 1, 3, 5 or 24, and a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of SEQ ID NOs: 2, 4, 6 or 25, or a complement of said sequence.

[0233] The term "isoxaflutole," as used herein, refers to the herbicide isoxaflutole [i.e., (5-cyclopropyl-4-isoxazolyl)[2-(methylsulfonyl)-4-(trifluoromethyl)phenyl]methanone], its active metabolite, the diketonitrile, and any mixture or solution containing said compound. HPPD-inhibiting herbicides useful for application in the present invention include diketonitriles such as 2-cyano-3-cyclopropyl-1-(2-methylsulfonyl-4-trifluoromethylphenyl)-propane-1,3-dione and 2-cyano-1-[4-(methylsulfonyl)-2-trifluoromethylphenyl]-3-(1-methylcyclopropyl)propane-1,3-phione; other isoxazoles; and pyrazolinates such as topramezone [i.e., [3-(4,5-dihydro-3-isoxazolyl)-2-methyl-4-(methylsulfonyl)phenyl](5-hydroxy-1-methyl-1H-pyrazol-4-yl)methanone], and pyrasulfotole [(5-hydroxy-1,3-di or mesotrione [2-[4-(methylsulfonyl)-2-nitrobenzoyl]cyclohexane-1,3-dione]; or 2-chloro-3-(methylsulfanyl)-N-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide]; or 2-methyl-N-(5-methyl-1,3,4-oxadiazol-2-yl)-3-(methylsulfonyl)-4-(trifluoromethyl)benzamide; or pyrazofen [2-[4-(2,4-dichlorobenzoyl)-1,3-dimethylpyrazol-5-yloxy]acetophenone].

[0234] In one embodiment of the present invention, the field in which the soybean plants containing the EE-GM5 event are planted can be treated with an HPPD-inhibiting herbicide such as isoxaflutole ("IFT"), topramezone or mesotrione, or with both an HPPD-inhibiting herbicide and glyphosate before the soybeans are sown, which removes the weeds in the field that are killed by the HPPD-inhibitor and / or glyphosate, and allows the soybeans to be subsequently planted or sown in the same pre-treated field in a no-till method (burn-off application with an HPPD-inhibiting herbicide). The residual activity of IFT will also protect the emerging and developing soybean plants from competition by weeds at an early growth stage. Once the soybean plants have reached a certain size and are prone to weed recurrence, an HPPD inhibitor or a mixture of an HPPD inhibitor and a selective (conventional) soybean herbicide, or a mixture of an HPPD inhibitor and an herbicide that is non-selective in soybeans (e.g., glyphosate or glufosinate), to which the plant is tolerant because the plant contains a resistance gene / locus, can be applied over the plants as a postemergence herbicide.

[0235] In another embodiment of the invention, a field sown with seeds containing the EE-GM5 event can be treated with an HPPD-inhibiting herbicide such as IFT, topramezone or mesotrione before the soybean plants emerge but after the seeds are sown (the field can be made weed-free before sowing using tillage or other methods including traditional tillage methods such as chisel tillage or seedbed preparation), where the soybean plants that emerge and grow will not have competition from weeds because residual activity keeps the field free of weeds killed by the herbicide (pre-emergence application of the HPPD-inhibiting herbicide). Once the soybean plants have reached a certain size and are prone to weed recurrence, an HPPD inhibitor or an HPPD inhibitor-soybean selective (conventional) herbicide mixture, or a mixture of an HPPD inhibitor and an herbicide that is non-selective in soybeans (e.g., glyphosate or glufosinate), to which the plant is tolerant because the plant contains a resistance gene / locus, can be applied as a post-emergence herbicide over the plants. In one embodiment of the present invention, a weed control process is provided, comprising sowing EE-GM5-containing soybean seeds in a field, and treating the field with an HPPD-inhibiting herbicide before plants germinate from the seeds but after the seeds are sown.

[0236] In another embodiment of the invention, plants containing the EE-GM5 event can be treated from the soybean plants germinated from the sown seeds with an HPPD-inhibiting herbicide such as IFT, topramezone or mesotrione to remove field weeds that are killed by the HPPD inhibitor, and the application can be carried out together with (e.g. in a spray tank mixture), before or after an over-the-plant treatment with a selective soybean post-emergence herbicide, or a herbicide that is non-selective in soybeans (e.g. glyphosate or glufosinate) to which the plant is tolerant because the plant contains a resistance gene / locus (post-emergence application of an HPPD-inhibiting herbicide with or without said soybean selective or non-selective herbicide).

[0237] Also in accordance with the present invention, soybean plants carrying EE-GM5 (which may also contain other herbicide-tolerant soybean events / traits as described herein) may be treated with a soybean insecticide, herbicide or fungicide, or soybean seeds carrying EE-GM5 may be coated with a soybean insecticide, herbicide or fungicide.

[0238] The following examples describe the development and identification of the elite event EE-GM5, the development of different soybean lines containing this event, and the development of tools for the specific identification of the elite event EE-GM5 in biological samples.

[0239] Unless otherwise stated in the examples, all recombinant techniques are carried out according to standard protocols as described in Sambrook J and Russell DW (eds.) (2001) Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press, New York and Ausubel FA, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA and Struhl K (eds.) (2006) Current Protocols in Molecular Biology. John Wiley & Sons, New York.

[0240] Standard materials and references are found in Croy RDD (ed.) (1993) Plant Molecular Biology LabFax, BIOS Scientific Publishers Ltd., Oxford and Blackwell Scientific Publications, Oxford and Brown TA, (1998) Molecular Biology LabFax, 2nd Edition, Academic Press, San Diego. Standard materials and methods for polymerase chain reaction (PCR) can be found in McPherson MJ and Møller SG (2000) PCR (The Basics), BIOS Scientific Publishers Ltd., Oxford and PCR Applications Manual, 3rd Edition (2006), Roche Diagnostics GmbH, Mannheim or www.roche-applied-science.com.

[0241] It should be understood that some parameters in any experimental protocol, such as the PCR protocol in the following examples, may need to be adjusted to specific laboratory conditions and may be slightly modified to obtain similar results. For example, the use of different methods for DNA preparation or selection of other primers in the PCR method may determine other optimal conditions for the PCR protocol. However, these adjustments are clear to those skilled in the art and are further detailed in the current PCR application manual.

[0242] In the description and examples, reference is made to the following sequences from the accompanying sequence listing: SEQ ID NO:1: 5' junction EE-GM5 SEQ ID NO:2: 3' junction EE-GM5 SEQ ID NO: 3: EE-GM5 5' junction SEQ ID NO: 4: EE-GM5 3' junction SEQ ID NO: 5: EE-GM5 5' region SEQ ID NO: 6: EE-GM5 3' region SEQ ID NO: 7: cry14Ab-1.b coding sequence SEQ ID NO: 8: Cry14Ab-1 protein amino acid sequence SEQ ID NO: 9: hppdPf-4Pa coding sequence SEQ ID NO: 10: HPPD-4 protein amino acid sequence SEQ ID NO: 11: Transformation plasmid pSZ8832-T-DNA border sequence SEQ ID NO: 12: Primer PRIM1038 SEQ ID NO: 13: Primer PRIM1039 SEQ ID NO: 14: Probe TM1788 SEQ ID NO: 15: primer KVM164 SEQ ID NO: 16: primer KVM165 SEQ ID NO: 17: Probe TM1242 SEQ ID NO: 18: Primer PRIM1041 SEQ ID NO: 19: Primer PRIM1040 SEQ ID NO: 20: Probe TM1789 SEQ ID NO: 21: Primer PRIM1629 SEQ ID NO: 22: Probe TM2083 SEQ ID NO: 23: Soybean event EE-GM5 SEQ ID NO: 24: EE-GM5 5' junction sequence SEQ ID NO: 25: EE-GM5 3' junction sequence SEQ ID NO: 26: primer GLPA210 SEQ ID NO: 27: primer GLPA212 SEQ ID NO: 28: primer GLPB167 SEQ ID NO: 29: primer GLPB170 SEQ ID NO: 30: primer PRIM2123 SEQ ID NO: 31: primer PRIM2122 SEQ ID NO: 32: Probe TM2327 SEQ ID NO: 33: Pre-insertion locus sequence EXAMPLES

[0243] 1. Transformation of soybean (Glycine max) with nematode resistance and herbicide tolerance genes 1.1. Description of the inserted T-DNA containing the cry14Ab-1.b and hppdPf-4Pa chimeric genes EE-GM5 soybean was developed by Agrobacterium-mediated transformation with vector pSZ8832, which contains the following hppdPf-4Pa and cry14Ab-1.b expression cassettes. (i) A mutant hppdPf-4Pa gene encoding the HPPD-4 protein (the amino acid sequence of which is shown in SEQ ID NO: 10). The hppdPf-4Pa coding sequence was expressed by introducing point mutations at position 335 (substitution of Glu by Pro), position 336 (substitution of Gly by Trp), position 339 (substitution of Lys by Ala) and position 340 (substitution of Ala by Gln) into DNA encoding the HPPD protein from Pseudomonas fluorescens strain A32. Expression of the HPPD-4 protein confers resistance to HPPD-inhibiting herbicides such as isoxaflutole, topramezone or mesotrione. (ii) the cry14Ab-1.b gene encodes the Cry14Ab-1 protein, the amino acid sequence of which is shown in SEQ ID NO: 8. Expression of the Cry14Ab-1 protein confers resistance to nematodes, such as the soybean cyst nematode Heterodera glycines.

[0244] Plasmid pSZ8832 is a plant transformation vector containing the cry14Ab-1.b and hppdPf-4Pa chimeric genes located between the right T-DNA border (RB) and the left T-DNA border (LB). A description of the genetic elements contained in the T-DNA between the right and left T-DNA borders is provided in Table 1 below. Confirmatory sequencing of the T-DNA (between the T-DNA borders) of this plasmid yielded the sequence of SEQ ID NO: 11. The nucleotide sequences of the cry14Ab-1.b and hppdPf-4Pa coding sequences (coding strands shown) are presented in SEQ ID NOs: 7 and 9, respectively.

[0245] [Table 3]

[0246] [Table 4]

[0247] 1.2. Event EE-GM5 The T-DNA vector pSZ8832 was introduced into Agrobacterium tumefaciens and transformed soybean plants (variety Thorne) were selected for HPPD inhibitor resistance according to methods known in the art. Surviving plants were self-pollinated to generate T1 seeds. Subsequent generations were produced by self-pollination or by crossing to other soybean germplasm.

[0248] 1.2.1 Identification of the elite event EE-GM5 The elite event EE-GM5 was selected from a wide range of different transformation events obtained with the same chimeric gene based on an extensive selection procedure (based on parameters including but not limited to trait efficacy, molecular characteristics, and agronomic characteristics in the greenhouse and field). Soybean plants containing EE-GM5 were found to have an insertion of the transgene at a single locus in the soybean plant genome, have similar overall agronomy as the parent plants used for transformation, cause no yield penalty due to the insertion of the transformation DNA (compared to the corresponding isogenic lines without the event, such as "null" plant lines obtained from transformed plants where the transgene segregated), result in a significant reduction in female adults contaminating the roots in standard SCN greenhouse assays, and have improved yield under SCN nematode pressure in the field compared to isogenic null lines not containing EE-GM5. Furthermore, although resistance to the application of HPPD-inhibiting herbicides was measured in field trials, herbicide resistance was not a selection criterion for the elite event selection.

[0249] 1.2.1.1 Molecular analysis of events Southern blot results showed that EE-GM5 contained a single transgenic locus, containing a single copy of the cry14Ab-1.b chimeric gene and a single copy of the hppdPf-4Pa chimeric gene. EE-GM5 lacks a portion of the 35S promoter of the hppdPf-4Pa chimeric gene, indicating that the entire T-DNA of SEQ ID NO:11 was not inserted into the soybean genome during transformation. PCR analysis using primers targeting the left and right borders of the T-DNA and vector backbone sequences flanking the aadA sequence did not yield any PCR fragments. The presence of identical EE-GM5 integration fragments in multiple generations of EE-GM5 also demonstrates the structural stability of the event.

[0250] 1.2.1.2 Event inheritance Inheritance of the inserted T-DNA insert in successive generations by testing the genotypes of the hppdPf-4Pa and cry14Ab-1.b genes by PCR analysis indicates that the hppdPf-4Pa and cry14Ab-1.b genes contained within the EE-GM5 insert are inherited in a predictable manner and as expected for a single insertion. These data are consistent with Mendel's principles and support the conclusion that the EE-GM5 event consists of a single insertion integrated into a single chromosomal locus within the soybean nuclear genome.

[0251] Furthermore, analysis of the segregation pattern of EE-GM5 in the successive generations following introgression of EE-GM5 into five elite soybean lines confirmed normal Mendelian segregation. Table 2 shows the segregation of EE-GM5 observed in different segregating populations.

[0252] [Table 5]

[0253] In Table 2, "HH" stands for homozygous plants, "Hemi" stands for hemizygous plants, "null" stands for null segregants that lost EE-GM5, and "ns" means not statistically significant (for any variation from normal / expected segregation). In these tests, parent 1 was a MG VI line with Rhg1 and Rhg4 natural SCN resistance, parent 2 was a MG VI line susceptible to SCN, parent 3 was a MG IX line susceptible to SCN, parent 4 was a MG III line with Rhg1 natural SCN resistance, and parent 5 was a MG I line susceptible to SCN.

[0254] 1.2.1.3 Stability of protein expression Protein expression levels of HPPD-4 and Cry14Ab-1 proteins in greenhouse-grown plants were determined by sandwich enzyme-linked immunosorbent assay (ELISA) in leaf, root and seed samples collected from different generations (e.g., T4, T6 and BC2F3) of EE-GM5 soybean. HPPD-4 and Cry14Ab-1 show similar average expression levels in leaves, roots and seeds across all generations tested. Any differences observed in Cry14Ab-1 and HPPD-4 concentrations were due to natural plant-to-plant variability.

[0255] 1.2.1.4 Agronomic performance and tolerance to HPPD-inhibiting herbicides In an agronomic equivalence test, plants containing EE-GM5 in the original transformation background (Thorne) were compared to segregating null (lacking EE-GM5) and wild-type Thorne plants when grown in the absence of SCN. The plots were not treated with HPPD herbicide, but were kept weed-free by the use of conventional herbicides and by manual weeding when necessary. When grown in comparative tests at different locations, no differences were observed between event-containing plants and segregating null (lacking EE-GM5) that affected agronomic performance in a biologically significant way when examined for qualitative plant characteristics such as flower color, pod color, seed color and pubescence, and for quantitative characteristics such as yield, total height, lodging, planting, days to maturity. Thus, plants containing EE-GM5 showed normal agronomic characteristics comparable to the corresponding non-transgenic plants.

[0256] Further trials with EE-GM5 in the original Thorne transgenic background were conducted in 2017. Preliminary trials with EE-GM5 in elite MG1 and MG3 genetic backgrounds were also constructed in 2017 at a limited number of locations. When examined for qualitative plant traits such as flower color, pod color, seed color and pubescence, and for quantitative traits such as yield, total height, lodging, planting, test weight and days to maturity, no consistent meaningful differences were detected between the EE-GM5 event and segregating nulls (lacking EE-GM5) in any of the three genetic backgrounds, thus confirming that plants containing EE-GM5 exhibit normal agronomic traits.

[0257] The tolerance of plants containing EE-GM5 to HPPD-inhibiting herbicides was tested at different locations in the field over a two-year period. In these tests, it was found that plants with EE-GM5 had commercially relevant tolerance to isoxaflutole (IFT) when applied pre-emergence and post-emergence, but crop damage was slightly higher for IFT pre-emergence applications. These tests also showed that plants containing event EE-GM5 had commercially relevant tolerance to mesotrione (MST) when applied pre-emergence or post-emergence. All post-emergence treatments were performed at the V2-V3 stage, with adjuvant crop oil concentrate and ammonium sulfate to enhance herbicidal activity.

[0258] Figure 5 shows the average maximum phytotoxicity data (plant damage) recorded for herbicide treatments in several field trials over a two-year period for soybean plants containing event EE-GM5 compared to non-transformed / conventional soybean plants. The control non-transformed Thorne plants showed average maximum phytotoxicity values ​​of approximately 80-90% in these same trials, indicating that these HPPD-inhibiting herbicides are not tolerated by (non-GM) soybeans. As used herein, "maximum phytotoxicity" is the highest phytotoxicity rating recorded in any observation (3-4 observations per trial) during the test period. For existing weed control applications, the normal (1x) dose of isoxaflutole (IFT) in pre- or post-emergence applications and MST in post-emergence applications is 105gr / ha, and the normal (1x) dose of mesotrione in pre-emergence applications is 210gr / ha. Thus, in these trials reported in Figure 5, the applications used pre-emergence in Figure 5 (420gr / ha for IFT and 840gr / ha for mesotrione) were four times the normal doses, and post-emergence (210gr / ha for each of IFT and mesotrione) were twice the normal doses.

[0259] In the third year, plants carrying EE-GM5 (Thorne background) had a maximum phytotoxicity of 9% when treated pre-emergence with isoxaflutole (IFT, 410 g / ha) at one field test location and an average maximum phytotoxicity of 10.9% when treated post-emergence with isoxaflutole (IFT) (V2-V3 stage, 210 h / ha) at four locations, thus confirming the resistance previously observed.

[0260] Also, in several field trials over a two-year period, soybean plants with event EE-GM5 had good tolerance (average maximum phytotoxicity values ​​for each treatment were less than 20%) to the HPPD inhibitor test compound 2-methyl-N-(5-methyl-1,3,4-oxadiazol-2-yl)-3-(methylsulfonyl)-4-(trifluoromethyl)benzamide (US Pat. No. 9,101,141) when applied pre-emergence at 400 gr ai / ha or post-emergence at 200 gr ai / ha, respectively. In these trials, soybean plants with event EE-GM5 also showed good tolerance (average maximum phytotoxicity of 20%) to the HPPD inhibitor test compound 2-chloro-3-(methylsulfanyl)-N-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide (US Pat. No. 8,481,749) when applied post-emergence at 100-150 gr ai / ha. All post-emergence treatments were performed at the V2-V3 stage with adjuvant crop oil concentrate and ammonium sulfate to enhance herbicidal activity. In year 3, plants carrying EE-GM5 (Thorne background) had an average maximum phytotoxicity of 13.3% when treated with 150 g / ha of 2-chloro-3-(methylsulfanyl)-N-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide post-emergence at three field test locations.

[0261] Adding data from the third season of herbicide tolerance field trials, the same or very similar mean maximum phytotoxicity ratings were obtained for IFT as those shown in Figure 5 when EE-GM5 was pre- or post-applied with the same dose of isoxaflutole herbicide, but in a different geographic location.

[0262] Additionally, plants bearing EE-GM5 when treated postemergence (V2-V3) with topramezone (+COC and AMS) at 36 g ai / ha in two field trials in the United States conferred an average maximum phytotoxicity of 11%, indicating that EE-GM5 also confers good resistance to this HPPD inhibitor.

[0263] 1.2.1.5 Nematode resistance Standard SCN assays measuring female index in greenhouses showed a significant reduction in SCN cysts in the roots of plants containing EE-GM5 when compared to Thorne wild-type soybean plants. Furthermore, standard SCN assays measuring female index in greenhouses also showed that soybean plants containing event EE-GM5 and natural SCN resistance showed a significant reduction in SCN cysts in the roots compared to SCN-resistant elite soybean lines without EE-GM5. When EE-GM5 was introgressed into elite soybean lines with PI88788 soybean resistance (maturity group 3) or Peking soybean resistance (maturity group 6.2), there was a consistent reduction in the number of SCN cysts in the roots compared to roots with only natural resistance.

[0264] In field trials over two years at several locations, soybean plants containing EE-GM5 provided significant yield increases compared to isogenic null segregants in SCN-infested fields. Figure 6 shows grain yield of EE-GM5 in the original transformant background (Thorne) tested in SCN-infested fields (ranging from low to high SCN contamination) at nine different locations throughout Iowa, Illinois, Indiana, Missouri, and Tennessee in 2015 and 2016. Additional trials with EE-GM5 in the original transformant background (Thorne) were conducted in 2017 at a total of 12 locations with varying SCN pressures. Across all 12 trials, plants containing EE-GM5 produced an average of 10% higher yield than null segregants lacking EE-GM5 (p=0.003). Figure 7 shows the grain yield of EE-GM5 when introgressed (BC2F3) into an elite MG I (maturity group I) line susceptible to SCN and tested in 2016 at one location in Minnesota and one location in North Dakota (each with high SCN contamination levels). The same MG I line was tested in 2017 at the same two locations (again each with high SCN contamination) and an additional facility in Wisconsin (the latter with moderate SCN pressure), and grain yield of plants containing EE-GM5 was consistently higher than the corresponding null segregants lacking EE-GM5. Finally, a pilot study across three locations in Brazil with moderate to high SCN pressure in trials planted late in 2017 showed a significant average increase of 31% in elite susceptible lines of plants with EE-GM5 when compared to the segregant null (lacking EE-GM5) (p=0.01). Although overall yields in these Brazilian pilot trials tended to be lower due to late planting and the variation within a trial was relatively large, which may have influenced the magnitude of yield increases, a clearly significant and visually observable yield increase was found for plants with EE-GM5. Thus, event EE-GM5 confers a significant yield increase to soybean plants in SCN-infected fields.

[0265] In a study to evaluate the effect on yield of event EE-GM5 when combined with natural SCN resistance, a series of F3 populations were developed from single crosses of EE-GM5 with a conventional elite MG III line carrying the rhg1 resistance gene from PI88788. In the F3 populations, one "stacked" population homozygous for both event EE-GM5 and the rhg1 allele was compared to a population homozygous only for the rhg1 allele (lacking EE-GM5). Yield trials were constructed with these populations at three locations with medium to high levels of SCN contamination in 2016 and at seven locations spanning low to high SCN pressure in 2017. The results are shown in Figure 8. All 2017 trials included three different seed treatments. No significant yield differences or interactions were observed for any of these seed treatments alone, so data were pooled across seed treatments to provide the best statistical estimate of yield differences between homozygous (HH) events and null segregants. As shown in Figure 8, across all three locations in 2016, the "stacked" population (plants homozygous for the EE-GM5 event and the rhg1 allele) produced 8% higher yields than the population carrying only the rhg1 allele (p=0.08), and in the 2017 trials provided an average yield increase of 11% for plants homozygous for the EE-GM5 event and the rhg1 allele compared to the population carrying only the rhg1 allele (p=1.24-11). For reference, the average yield increase for lines containing EE-GM5 using only basal seed treatments (Evergol® Energy + Allegiance® fungicide + Poncho® insecticide) across the 2017 trials was 0.27 T / ha (10.2% yield increase, p=0.0002). The basal seed treatment used in all trials in 2016 was Evergol® Energy + Allegiance® fungicide. As shown in Figure 8, a close relationship was seen between yield response and SCN pressure across all 10 trials in both years, with higher yields observed in locations with higher SCN pressure (towards the top of Figure 8).These results indicate that the addition of the EE-GM5 event to a conventional SCN-resistant soybean cultivar can result in significant yield increases in SCN-infested fields.

[0266] Conducting yield trials under moderate to high SCN contamination is difficult due to many factors that affect the results. Because SCN population density within a field can be variable in nature, the overall impact of SCN on yield can also vary from one plot to the next (see, for example, www.plantmanagementnetwork.org / pub / php / review / 2009 / sce08 / ). Favorable soil type, good fertility, and sufficient rainfall can mitigate the effects of SCN contamination on soybean plants, minimizing the impact on yield even under high SCN populations. Many fields with very high SCN populations tend to have poor soils and, as a result, lower yield potential, making it difficult to discern statistically significant effects on yield. Thus, yield data from SCN field trials can be highly variable, and significant improvements in yield would not be expected in all trials with high SCN populations. The overall trend between trials is the most relevant criterion for judging the performance of an event.

[0267] SCN field trials conducted with plants containing EE-GM5 were constructed in fields with natural SCN contamination. Experimental units consisted of field plots containing 2–4 rows ranging from 3.8–9.1 m with 0.76 m spacing. The number of rows per plot and the length of the plot varied from site to site based on field size and equipment configuration. Plots were sown at 26 seeds per meter, so each experimental unit contained 200–960 seeds. Plots were randomized within the field using a split-plot or split-split-plot design. Split-plot designs are well suited to help minimize the effects of soil type or the high variability of SCN populations prevalent in SCN-contaminated fields. In SCN field trials, plants containing EE-GM5 were planted in subplots next to or very close to an accompanying subplot containing a separate null plant (without EE-GM5). The proximity of the two plots helps to minimize the effect of field variation on estimates of the difference between plants with or without event EE-GM5 (SCN). Most trials were repeated four times, a few were repeated three times, and a few were repeated five or six times.

[0268] Moderate to heavy contamination with Sudden Death Syndrome was observed in two locations (Indiana and Iowa) in 2016. Plots at these two locations were assessed for incidence and severity of SDS symptoms, and SDS disease index (DX) was calculated using the "SIUC method of SDS scoring" (www.scnresearch.info / 462.pdf). DX scores of plants homozygous for EE-GM5 were 61% lower in Indiana and 55% lower in Iowa than susceptible null segregants (lacking EE-GM5), indicating that the event provided protection against SDS infection. SDS and SCN are often closely associated in the field and will show some interactions in plants (see, for example, www.soybeanresearchinfo.com / pdf_docs / sdsupdate.pdf, and www.apsnet.org / edcenter / intropp / lessons / fungi / ascomycetes / pages / suddendeath.aspx).

[0269] In 2017, iron deficiency chlorosis (IDC) scores were collected for plants carrying EE-GM5 (and their null segregants) at one test location in the United States (with high SCN contamination) where IDC symptoms were observed. The test was a split-plot design examining the effect of the event in three different backgrounds. IDC evaluation was performed as described by Cianzio et al. (1979) Crop Science 19:644-646. Figure 11 shows the average of the IDC scores of plants carrying the event EE-GM5 and the corresponding null segregants (lacking EE-GM5) across three genetic backgrounds (one SCN resistant (PI88788 resistant), one SCN susceptible, and one SCN susceptible Thorne background). Significantly lower IDC scores were found for plants containing EE-GM5 compared to their null segregants. Thus, in a field study in which soybean plants were exposed to both SCN and IDC, EE-GM5 significantly reduced IDC leaf severity, and this reduction occurred across three soybean accessions, one of which contained PI 88788-type natural SCN resistance.

[0270] Non-transformed Thorne and EE-GM5 seeds were also germinated and planted in the greenhouse to test for control of the disease nematode Pratylenchus brachyurus. When the plants were 2 weeks old, Pratylenchus brachyurus (1500 nematodes / plant, at different growth stages) were applied to the plants. Thirty days after application, Pratylenchus were extracted from the roots and counted. The average number of nematodes found on the roots of plants containing EE-GM5 was compared to the average number of Pratylenchus found on the roots of wild-type Thorne plants. An average of approximately 80-90% less Pratylenchus was found in the roots of plants containing EE-GM5 compared to the Thorne control roots, indicating significant control of the disease nematode by soybean event EE-GM5.

[0271] Figure 9 shows the results of a Pratylenchus brachyurus greenhouse assay in the United States comparing elite lines with EE-GM5 in five elite soybean lines with SCN susceptible and SCN resistant US soybean lines (one SCN susceptible (MG 1), one SCN resistant (PI88788, MG 3), one SCN susceptible (MG 6.2), one SCN resistant (Peking, MG 6.2), and one SCN susceptible (MG 9)). Soybean plants were grown in small conical pots and kept in a greenhouse at temperatures varying between 25-32 °C. Pratylenchus brachyurus obtained from a field in South Carolina and grown in the greenhouse were used to inoculate plants at the V2-V3 growth stage. Approximately 1500 eggs + adults were inoculated per plant, with each entry having 5 plants. After 30 days of contamination, nematodes and eggs were extracted from the roots and counted. Each entry was performed in two independent experiments. SCN-susceptible and SCN-resistant US soybean lines showed no control of Pratylenchus, while plants carrying EE-GM5 showed approximately 90% control of Pratylenchus.

[0272] Figure 10 shows the results of a Pratylenchus brachyurus greenhouse assay in Brazil, comparing soybean plants with EE-GM5 to a Brazilian soybean line with no resistance and one low Rf line, as well as SCN-susceptible and SCN-resistant plants. The soybean lines were grown in small conical pots and kept in a greenhouse at temperatures varying between 25 and 32 °C. Pratylenchus brachyurus obtained from a field in Brazil and grown in a greenhouse were used to inoculate plants at the V2-V3 growth stage. Approximately 1000 eggs + adults were inoculated per plant, with each entry having five plants. After 30 days of contamination, nematodes and eggs were extracted from the roots and counted. The results shown are from a single experiment. One Brazilian soybean line (BRS 7380) classified as having a low reproductive factor for Pratylenchus showed approximately 89% reduction in Pratylenchus. Plants with EE-GM5 controlled Pratylenchus by approximately 99%. Soybean lines carrying natural resistance to SCN (rhg1+rhg4) do not control Pratylenchus brachyurus.

[0273] Plants containing EE-GM5 can also be used to control root-knot nematodes (RKN), such as Meloidogyne incognita. Although Meloidogyne incognita populations contaminate Thorne wild-type soybeans less frequently, Thorne plants with EE-GM5 show a further reduction in RKN egg counts / root mass on average compared to untransformed Thorne plants.

[0274] 1.2.2 Identification of the flanking regions and inserted T-DNA of elite event EE-GM5 The sequences of the inserted T-DNA and the adjacent T-DNA flanking regions contained in the EE-GM5 elite event are shown in the attached sequence listing.

[0275] 1.2.2.1 5'T-DNA flanking region The fragment identified as containing the 5' T-DNA flanking region of EE-GM5 was sequenced and its nucleotide sequence is represented in SEQ ID NO:5, nucleotides 1 to 166. The 5'-T-DNA flanking region is comprised of soybean genomic sequence corresponding to the pre-insertion locus sequence (SEQ ID NO:5, nucleotides 1 to 166). The 5' junction region, including a portion of the inserted T-DNA sequence and a portion of the adjacent T-DNA 5' flanking sequence, is represented in SEQ ID NOs:1 and 3.

[0276] 1.2.2.2 3'T-DNA flanking region The fragment identified as containing the 3' T-DNA flanking region of EE-GM5 was sequenced and its nucleotide sequence is represented in SEQ ID NO:6, nucleotides 359-691. The 3' T-DNA flanking region is composed of a 39 nucleotide filler DNA sequence (position 359 to position 397 of SEQ ID NO:6) followed by maize genomic sequence corresponding to the pre-insertion locus sequence (position 398 to position 691 of SEQ ID NO:6). The 3' junction region, including a portion of the inserted T-DNA sequence and a portion of the adjacent T-DNA 3' flanking sequence, is represented in SEQ ID NOs:2 and 4.

[0277] 1.2.2.3 Insertion of T-DNA in EE-GM5 The inserted T-DNA adjacent to the 5'-T-DNA flanking sequences described above was sequenced and its nucleotide sequence is represented in SEQ ID NO:5, nucleotides 167 to 353. The inserted T-DNA adjacent to the 3'-T-DNA flanking sequences described above was sequenced and its nucleotide sequence is represented in SEQ ID NO:6, nucleotides 1 to 358. During transformation, 63 bp of genomic DNA were deleted in the pre-insertion locus sequences and replaced by the inserted DNA (composed of the T-DNA and a small amount of filler DNA).

[0278] Sequencing of the T-DNA region (the portion between the T-DNA borders) in the transformation plasmid pSZ8832 gave the sequence reported in SEQ ID NO: 11. The chimeric cry14Ab-1.b gene sequence (including the Ubi10 promoter and the 35S 3' untranslated region) is represented in SEQ ID NO: 11 from nucleotides 131 to 5276 (counterclockwise). The inserted T-DNA sequence in the 5' flanking region of SEQ ID NO: 5 (nucleotides 167 to 353) is identical to the nucleotide sequence of SEQ ID NO: 11 from nucleotide 1 to nucleotide 187, and the inserted T-DNA sequence in the 3' flanking region of SEQ ID NO: 6 (nucleotides 1 to 358) is identical to the nucleotide sequence of SEQ ID NO: 11 from nucleotide 7102 to nucleotide 7459. Thus, the 5' end of the T-DNA inserted into EE-GM5 corresponds to nucleotide 1 in the transformation plasmid sequence of SEQ ID NO: 11, and therefore the 3' end of the T-DNA inserted into EE-GM5 corresponds to nucleotide 7459 in the transformation plasmid sequence of SEQ ID NO: 11. The T-DNA inserted into EE-GM5 between the sequence of SEQ ID NO: 5 and the sequence of SEQ ID NO: 6, contained in the seed deposited with the ATCC under accession number PTA-123625, has a sequence essentially similar or identical to the sequence of SEQ ID NO: 11 from nucleotide 188 to nucleotide 7101.

[0279] The insertion locus of event EE-GM5 can be determined from the wild type soybean variety Thorne based on the 5' and 3' T-DNA flanking sequences provided herein (SEQ ID NO:5 from nt 1 to nt 166 and SEQ ID NO:6 from nt 359 to nt 691) by methods known in the art. The pre-insertion locus sequence in the soybean genome corresponds in order to the following sequences: nucleotide position 1 to nucleotide position 166 of SEQ ID NO:5, the 63 nt deletion, and nucleotide position 398 to nucleotide position 691 of SEQ ID NO:6. The complete pre-insertion locus sequence is given in SEQ ID NO:33, where nt 1 to 1000 is the 5' flanking genomic sequence, nt 1001 to 1063 is the target site deletion, and 1064 to 2063 is the 3' flanking genomic sequence.

[0280] 1.2.3 Confirmation of flanking regions and inserted T-DNA of elite event EE-GM5 PCR amplification using primers targeting plant DNA upstream and downstream of the inserted T-DNA as well as the inserted T-DNA in EE-GM5 confirmed and extended the 5' and 3' flanking sequences of EE-GM5.

[0281] 1.2.3.1. 5' junction sequence EE-GM5 specific reaction Two primers, GLPA210 and GLPB167, were designed to amplify an approximately 5118 bp amplicon spanning the junction region of the 5' T-DNA flanking sequence with the T-DNA insert of event EE-GM5. The sequence of primer GLPA210 was derived from the soybean reference sequence of Glycine max Williams 82.a2.v1.

[0282] EE-GM5 Forward primer targeting the T-DNA 5' flanking sequence: GLPA210 5'-CTCTCACCCAgATTTCAC-3' (SEQ ID NO: 26)

[0283] Reverse primer targeting the EE-GM5 insertion T-DNA sequence: GLPB167 5'-TACAACgTgCTCgCTATTCC-3' (SEQ ID NO: 28)

[0284] Composition of reaction mixture for 5' junction sequencing reaction: 5μl Expand™ buffer (Roche) 1 μl dNTP (10 mM) 2μl forward primer (10pmol / μl) 2μl reverse primer (10pmol / μl) 0.75 μl Expand™ High Fidelity Enzyme Mix (3.5 U / μL; Roche) 50ng template DNA Water up to 50μl

[0285] Thermocycling conditions for 5' Junction Sequence Reaction:

[0286] [Table 6]

[0287] The sequence of the resulting extended T-DNA 5' flanking sequence, which is adjacent upstream of the portion of the inserted T-DNA shown in SEQ ID NO:5, is shown in SEQ ID NO:24.

[0288] 1.2.3.2. 3' junction sequence EE-GM5 specific reaction Two primers, GLPB170 and GLPA212, were designed to amplify an approximately 4982 bp amplicon spanning the junction region of the T-DNA insert of event EE-GM5 with the 3' T-DNA flanking sequence. The sequence of primer GLPA212 was derived from the reference sequence of soybean (Glycine max) Williams 82.a2.v1.

[0289] Forward primer targeting the EE-GM5 inserted T-DNA sequence: GLPB170 5'-TCTCggTATCAgCgTTCTTg-3' (SEQ ID NO: 29)

[0290] Reverse primer targeting the EE-GM5 T-DNA 3' flanking sequence: GLPA212 5'-CCCATgCggTATTATgTg-3' (SEQ ID NO: 27)

[0291] Composition of reaction mixture for 3' junction sequencing reaction: 5μl Expand™ buffer (Roche) 1 μl dNTP (10 mM) 2μl forward primer (10pmol / μl) 2μl reverse primer (10pmol / μl) 0.75 μl Expand™ High Fidelity Enzyme Mix (3.5 U / μL; Roche) 50ng template DNA Water up to 50μl

[0292] Thermocycling conditions for 3' Junction Sequence Reaction:

[0293] [Table 7]

[0294] The sequence of the resulting extended T-DNA 3' flanking sequence, which is adjacent downstream to the portion of the inserted T-DNA shown in SEQ ID NO:6, is shown in SEQ ID NO:25.

[0295] Since the amplicons obtained in the two reactions overlapped, this allowed the reconstruction of the sequence of the EE-GM5 insertion T-DNA and the extended 5' and 3' flanking sequences shown in SEQ ID NO: 23. The 5' T-DNA flanking sequence of SEQ ID NO: 23 is from nucleotide position 1 to nucleotide position 1113 (corresponding to the pre-insertion locus genomic sequence), the insertion T-DNA sequence is from nucleotide position 1114 to nucleotide position 8572, and the 3' T-DNA flanking sequence of SEQ ID NO: 23 is from nucleotide position 8573 to nucleotide position 9663 (corresponding to the 39 nt filler DNA (nt 8573 to 8611 in SEQ ID NO: 23) and the pre-insertion locus genomic sequence (nt 8612 to 9663 in SEQ ID NO: 23)).

[0296] 2. Development of EE-GM5 Identification Protocol 2.1. Endpoint method for EE-GM5 identity analysis This method describes a polymerase chain reaction detection method for analyzing the presence of event EE-GM5-specific DNA sequences in DNA samples obtained from biological samples such as plant material (e.g., leaves or seeds) using standard DNA extraction procedures.

[0297] The method description outlines the method design, including oligonucleotide primer and probe sequences, the composition of the reaction mixture, the thermocycling conditions required to carry out the reaction, and the fluorescence reader settings that were found to be suitable for amplicon detection. It also provides general recommendations regarding the nature and use of control samples. In addition, guidelines for data analysis and interpretation are provided, including examples of method results that take into account recommendations for the use of control substances and guidelines for data analysis.

[0298] 2.1.1. Method design This method uses Taqman chemistry to amplify and detect two target sequences: an EE-GM5-specific reaction determines the presence of the event, and a taxon-specific reaction verifies a negative result for the event-specific reaction.

[0299] 2.1.1.1.EE-GM5 specific reaction Two primers, PRIM1038 and PRIM1039, were designed to amplify an 85-bp amplicon spanning the junction region of the 3′ flanking sequence with the T-DNA insert of event EE-GM5.

[0300] A probe, TM1788, using FAM as the fluorescent label and BHQ1 as the quencher, was designed to detect the amplified sequence.

[0301] Forward primer targeting the EE-GM5 T-DNA sequence: PRIM1038 5'-gAgCCACCTTCCTTTTCCACTA-3' (SEQ ID NO: 12)

[0302] Reverse primer targeting the EE-GM5 T-DNA 3' flanking sequence: PRIM1039 5'-ATAgggTTACTgCTTCgTAAAATAAgCA-3' (SEQ ID NO: 13)

[0303] EE-GM5 Probe targeted to the junction of the T-DNA and its 3' flanking sequence: TM1788 FAM 5'-CgCgTCCATgATgCTgCgACTATg-3'BHQ1 (SEQ ID NO: 14)

[0304] 2.1.1.2. Taxon-specific reactions Two primers, KVM164 and KVM165, were designed to amplify a 102 bp amplicon of the soybean endogenous lectin 1 gene sequence.

[0305] A probe, TM1242, using JOE and BHQ1 as the fluorescent label was designed to detect the amplified sequence.

[0306] Forward primer targeting the endogenous lectin 1 gene sequence: KVM164 5'-CTTTCTCgCACCAATTgACA-3' (SEQ ID NO: 15)

[0307] Reverse primer targeting the endogenous lectin 1 gene sequence: KVM165 5'-TCAAACTCAACAgCgACgAC-3' (SEQ ID NO: 16)

[0308] Probes targeting the endogenous lectin 1 gene sequence: TM1242 JOE 5'-CCACAAACACATgCAggTTATCTTgg-3'BHQ1 (SEQ ID NO: 17)

[0309] 2.1.2. Composition of the reaction mixture 5.0μl 2x PerfeCta qPCR FastMix II,ROX 0.2μl PRIM1038[10pmol / μl] 0.2μl PRIM1039[10pmol / μl] 0.05μl KVM164[10pmol / μl] 0.05μl KVM165[10pmol / μl] 0.1μl TM1788[10pmol / μl] 0.1μl TM1242[10pmol / μl] ×μl Template DNA (20ng*) Water up to 10μl Note: 2x PerfeCta qPCR FastMix II, ROX was provided by Quanta Bioscience. Other enzyme buffers may be used, but performance should be verified. Primers and labeled probes were ordered from Integrated DNA Technologies. *The amount of template DNA per reaction may vary but should be confirmed.

[0310] Thermocycling Conditions

[0311] [Table 8]

[0312] 2.1.4. Wavelength and Bandwidth Settings

[0313] [Table 9]

[0314] 2.1.5. Control Samples To verify the results of the test samples, the following control samples should be included in the experiment. Positive control: DNA sample containing the target and endogenous sequences. Negative control: DNA sample containing only endogenous sequences ● No template control: Water sample (no DNA)

[0315] Data Analysis • For all samples, the ratio of fluorescent signal to background (S / B) is calculated for both target and endogenous responses. The control sample should give the expected result, i.e. ○ Positive controls should be rated as "detection" ○ Negative controls should be scored as "no detection." ○ No template controls should show only background levels of fluorescence ● The samples are evaluated as follows: ○ Detection: Target S / B and endogenous S / B exceed an acceptable threshold ratio (e.g., 2) o Not detected: target S / B is below an acceptable threshold ratio (eg, 1) and endogenous S / B is above an acceptable threshold ratio (eg, 2). Indeterminate: Target and intrinsic S / B are below an acceptable threshold (e.g., 1)

[0316] Figure 2 shows an example of the results of the method for a series of soybean samples containing EE-GM5 and conventional soybean samples. For each sample, the S / B ratios for both the EE-GM5 specific and endogenous responses are displayed.

[0317] 2.2. Endpoint Methods for EE-GM5 Identity and Zygosity Analysis This method describes a polymerase chain reaction detection method for analyzing the presence and zygosity status of event EE-GM5-specific DNA sequences in DNA samples obtained from biological samples such as plant material (e.g., leaves or seeds) using standard DNA extraction procedures.

[0318] The method description outlines the reaction reagents, oligonucleotide primer and probe sequences, the thermocycling conditions required to carry out the reaction, and the fluorescence reader settings that were found to be suitable for amplicon detection. It also provides general recommendations regarding the nature and use of control samples. In addition, guidelines for data analysis and interpretation are provided, including examples of method results that take into account recommendations regarding the use of control substances and guidelines for data analysis.

[0319] Note that method performance for zygosity analysis (option 1 below) may vary due to the nature of the pre-insertion locus sequence. Therefore, validation of performance is required for each variety into which the event is introgressed. If performance is insufficient, alternative endpoint method designs for zygosity can be used (as in option 2 below), or alternative real-time PCR methods based on copy number analysis, such as those described below in section 2.3, can be used for zygosity determination.

[0320] 2.2.1. Method design This method uses Taqman chemistry to amplify and detect two target sequences: an EE-GM5 specific reaction determines the presence of the event, and a pre-insertion locus specific reaction determines the presence of the pre-insertion locus of the event.

[0321] Detection of only the EE-GM5 specific sequence indicates the presence of the event EE-GM5 in the homozygous state.

[0322] Detection of EE-GM5-specific and pre-insertion locus-specific sequences indicates the presence of the event EE-GM5 in the hemizygous state.

[0323] Detection of only the pre-insertion locus-specific sequence indicates the absence of event EE-GM5.

[0324] 2.2.1.1. EE-GM5 specific reaction A. Option 1 Two primers, PRIM1040 and PRIM1041, were designed to amplify an 84-bp amplicon spanning the junction region of the T-DNA 5′ flanking sequence with the T-DNA insert of event EE-GM5.

[0325] A probe, TM1789, using FAM as the fluorescent label and BHQ1 as the quencher, was designed to detect the amplified sequence.

[0326] Forward primer targeting the EE-GM5 T-DNA sequence: PRIM1041 5'-CATTgTgCTgAATAggTTTATAgCTATgAT-3' (SEQ ID NO: 18)

[0327] Reverse primer targeting the EE-GM5 T-DNA 5' flanking sequence: PRIM1040 5'-TCAAATCAACATgggTgACTAgAAA-3' (SEQ ID NO: 19)

[0328] EE-GM5 Probe targeted to the junction of the T-DNA and its 5' flanking sequence: TM1789 FAM 5'-CAgTACTgggCCCTTgTggCgCT-3'BHQ-1 (SEQ ID NO: 20)

[0329] B. Option 2 Two primers, PRIM2123 and PRIM1041, were designed to amplify a 134-bp amplicon spanning the junction region of the T-DNA 5′-flanking sequence with the T-DNA insert of event EE-GM5.

[0330] A probe, TM1789, using FAM as the fluorescent label and BHQ1 as the quencher, was designed to detect the amplified sequence.

[0331] Forward primer targeting the EE-GM5 T-DNA sequence: PRIM1041 5'-CATTgTgCTgAATAggTTTATAgCTATgAT-3' (SEQ ID NO: 18)

[0332] Reverse primer targeting the EE-GM5 T-DNA 5' flanking sequence: PRIM2123 5'-gCACTgTTTAACTTTAAATAACTCATTTgAg-3' (SEQ ID NO: 30)

[0333] EE-GM5 Probe targeted to the junction of the T-DNA and its 5' flanking sequence: TM1789 FAM 5'-CAgTACTgggCCCTTgTggCgCT-3'BHQ-1 (SEQ ID NO: 20)

[0334] 2.2.1.1. Pre-insertion locus-specific responses A. Option 1 Two primers, PRIM1629 and PRIM1040, are designed to amplify a 72 bp amplicon spanning the junction between the pre-insertion locus and the 5′ flanking sequence of the EE-GM5 pre-insertion locus.

[0335] An MGB probe, TM2083, using VIC as the fluorescent label and MGB-NFQ as the quencher, is designed to detect the amplified sequence.

[0336] Forward primer targeting the EE-GM5 pre-insertion locus sequence: PRIM1629 5'-TTggTgAAAAACAATTTggTgTACA-3' (SEQ ID NO: 21)

[0337] Reverse primer targeting the EE-GM5 T-DNA 5' flanking sequence: PRIM1040 5'-TCAAATCAACATgggTgACTAgAAA-3' (SEQ ID NO: 19)

[0338] Wild-type probe targeting the junction of the pre-insertion locus and the 5' flanking sequence: TM2083 VIC 5'-AATCAAATCgACATCAATgT-3'MGB-NFQ (SEQ ID NO: 22)

[0339] B. Option 2 Two primers, PRIM2122 and PRIM2123, are designed to amplify a 193 bp amplicon spanning the junction between the pre-insertion locus and the 5' flanking sequence of the EE-GM5 pre-insertion locus.

[0340] An MGB probe, TM2327, using VIC as the fluorescent label and MGB-NFQ as the quencher, is designed to detect the amplified sequence.

[0341] Forward primer targeting the EE-GM5 pre-insertion locus sequence: PRIM2122 5'CAAgCAAAATAAgCAACTAgATCTATTgg-3' (SEQ ID NO: 31)

[0342] Reverse primer targeting the EE-GM5 T-DNA 5' flanking sequence: PRIM2123 5'-gCACTgTTTAACTTTAAATAACTCATTTgAg-3' (SEQ ID NO: 30)

[0343] Wild-type probe targeting the junction of the pre-insertion locus and the 5' flanking sequence: TM2327 VIC 5'-TTTggTgAAAAACAATTTggTgT-3'MGB-NFQ (SEQ ID NO: 32)

[0344] 2.2.2. Composition of the reaction mixture A. Option 1 5.0μl 2x PerfeCta qPCR FastMix II,ROX 0.4μl PRIM1040[10pmol / μl] 0.2μl PRIM1041[10pmol / μl] 0.2μl PRIM1629[10pmol / μl] 0.1μl TM1789[10pmol / μl] 0.1μl TM2083[10pmol / μl] ×μl Template DNA (20ng*) Water up to 10μl

[0345] B. Option 2 5.0μl 2x PerfeCta qPCR FastMix II,ROX 0.2μl PRIM1041[10pmol / μl] 0.2μl PRIM2122[10pmol / μl] 0.2μl PRIM2123[10pmol / μl] 0.1μl TM1789[10pmol / μl] 0.1μl TM2327[10pmol / μl] ×μl Template DNA (20ng*) Water up to 10μl Note: 2x PerfeCta qPCR FastMix II, ROX was provided by Quanta Bioscience. Other enzyme buffers may be used, but performance should be verified. Primers and labeled probes were ordered from Integrated DNA Technologies. *The amount of template DNA per reaction may vary but should be confirmed.

[0346] 2.2.3. Thermocycling conditions (same for options 1 and 2)

[0347] [Table 10]

[0348] 2.2.4. Wavelength and Bandwidth Settings (same for options 1 and 2)

[0349] [Table 11]

[0350] 2.2.5. Control Samples (same for options 1 and 2) To verify the results of the test samples, the following control samples should be included in the experiment. Homozygous control: A DNA sample containing the target sequence in the homozygous state. Hemizygous control: A DNA sample containing the target sequence in a hemizygous state. Wild-type control: A DNA sample that does not contain the target sequence. ● No template control: water sample

[0351] 2.2.6. Data Analysis (same for options 1 and 2) • For all samples, the ratio of fluorescent signal to background (S / B) is calculated for both the targeted and pre-insertion locus reactions. The control sample should give the expected result, i.e. Homozygous controls should be scored as "homozygous." ○ Hemizygous controls should be scored as "hemizygous" ○ Wild-type controls should be rated as "wild-type" ○ No template controls should show only background levels of fluorescence ● The samples are evaluated as follows: o Homozygous: Target S / B exceeds an accepted threshold ratio (e.g., 2) and pre-insertion locus S / B is below an accepted threshold ratio (e.g., 1). Hemizygous: both the target and pre-insertion locus S / B exceed an acceptable threshold ratio (e.g., 2) Wild type: target S / B is below an acceptable threshold ratio (e.g., 1) and pre-insertion locus S / B is above an acceptable threshold ratio (e.g., 2). Indeterminate: the target and pre-insertion locus S / B are below an acceptable threshold (e.g., 1)

[0352] FIG. 3 shows examples of the results of the method (option 1 above) for a series of soybean samples containing EE-GM5 in a homozygous state, soybean samples containing EE-GM5 in a hemizygous state and conventional soybean samples.

[0353] 2.3. Real-time PCR method for EE-GM5 identity and zygosity analysis This method describes a quantitative real-time polymerase chain reaction detection method for analyzing the zygosity status of event EE-GM5 DNA sequences in DNA samples obtained from biological samples such as plant material (e.g., leaves or seeds) using standard DNA extraction procedures.

[0354] The method description outlines the reaction reagents, oligonucleotide primer and probe sequences, the thermocycling conditions required to perform the reaction, and includes fluorescence reader settings that were found to be suitable for amplicon detection. It also provides general recommendations regarding the nature and use of control samples. In addition, guidelines for data analysis and interpretation are provided.

[0355] This method is species independent and can be used as an alternative method of zygosity analysis to the method described in section 2.2 above.

[0356] 2.3.1. Method design This method uses Taqman chemistry and real-time PCR principles to quantify the relative copy number of EE-GM5 specific sequences.

[0357] The method includes an EE-GM5-specific reaction for quantification of EE-GM5 copy number, and a taxon-specific reaction for normalization of EE-GM5 copy number.

[0358] Samples containing the EE-GM5 insert in a homozygous state have a relative copy number two-fold higher than hemizygous samples. Unpaired samples do not amplify the EE-GM5 sequence.

[0359] EE-GM5 specific reaction Two primers, PRIM1038 and PRIM1039, were designed to amplify an 85-bp amplicon spanning the junction region of the 3′ flanking sequence with the T-DNA insert of event EE-GM5.

[0360] A probe, TM1788, using FAM as the fluorescent label and BHQ1 as the quencher, was designed to quantify the amplified sequence.

[0361] Forward primer targeting the EE-GM5 T-DNA sequence: PRIM1038 5'-gAgCCACCTTCCTTTTCCACTA-3' (SEQ ID NO: 12)

[0362] Reverse primer targeting EE-GM5 3' flanking sequence: PRIM1039 5'-ATAgggTTACTgCTTCgTAAAATAAgCA-3' (SEQ ID NO: 12)

[0363] EE-GM5 Probe targeted to the junction of the T-DNA and its 3' flanking sequence: TM1788 FAM 5'-CgCgTCCATgATgCTgCgACTATg-3'BHQ-1 (SEQ ID NO: 14)

[0364] 2.3.3. Taxon-specific reactions Two primers, KVM164 and KVM165, were designed to amplify a 102 bp amplicon of the soybean endogenous lectin 1 gene sequence.

[0365] A probe, TM1242, using JOE as the fluorescent label and BHQ1 as the quencher, was designed to quantify the amplified sequence.

[0366] Forward primer targeting the endogenous lectin 1 gene sequence: KVM164 5'-CTTTCTCgCACCAATTgACA-3' (SEQ ID NO: 15)

[0367] Reverse primer targeting the endogenous lectin 1 gene sequence: KVM165 5'-TCAAACTCAACAgCgACgAC-3' (SEQ ID NO: 16)

[0368] Probes targeting the endogenous lectin 1 gene sequence: TM1242 JOE 5'-CCACAAACACATgCAggTTATCTTgg-3'BHQ1 (SEQ ID NO: 17)

[0369] 2.3.4. Composition of the reaction mixture 5.0μl 2x PerfeCta qPCR FastMix II,Low ROX 0.2μl PRIM1038[10pmol / μl] 0.2μl PRIM1039[10pmol / μl] 0.2μl KVM164[10pmol / μl] 0.2μl KVM165[10pmol / μl] 0.05μl TM1788[10pmol / μl] 0.05μl TM1242[10pmol / μl] × μl template DNA (20 ng*) Water up to 10μl Note: ● 2×Perfecta qPCR FastMix II, LOW ROX was provided by Quanta Bioscience. Other enzyme buffers may be used, but performance should be verified. Primers and labeled probes were ordered from Integrated DNA Technologies. *The amount of template DNA per reaction may vary but should be confirmed.

[0370] 2.3.5. Thermocycling Conditions

[0371] [Table 12]

[0372] 2.3.6. Control Samples To verify the results of the test samples, the following control samples should be included in the experiment. Homozygous control: A DNA sample containing the target sequence in the homozygous state. Hemizygous control: A DNA sample containing the target sequence in a hemizygous state. Wild-type control: A DNA sample that does not contain the target sequence. ● No template control: water sample

[0373] Data Analysis Data analysis is performed using the ddCt method, where copy numbers are calculated for all samples relative to a chosen reference sample. It is recommended to use a hemizygous control as a reference sample. The control sample should give the expected result, i.e. Homozygous controls should be scored as "homozygous." ○ Hemizygous controls should be scored as "hemizygous" ○ Wild-type controls should be rated as "wild-type" ○ No template controls should show only background levels of fluorescence ● The samples are evaluated as follows: Homozygous: Relative copy number is 2+ / - the accepted threshold (e.g., 0.5). Hemizygous: Relative copy number is 1+ / - the accepted threshold (e.g., 0.25). Wild-type: Relative copy number is 0 + an accepted threshold (e.g., 0.1) Indeterminate: Relative copy number is outside the acceptable range for homozygous, hemizygous and wild-type samples

[0374] 2.4. Real-time PCR method for analysis of low levels of EE-GM5 This method describes a detection method to analyze the low-level presence of event EE-GM5 DNA sequences obtained from bulk plant material (e.g., leaves or seeds) or processed material (e.g., food or feed containing processed soybean seeds) using standard DNA extraction procedures.

[0375] The method description outlines the reaction reagents, oligonucleotide primer and probe sequences, and the thermocycling conditions required to perform the reaction. It also provides general recommendations for the concurrent use of taxon-specific methods to support data analysis and result interpretation. In addition, recommendations regarding the nature and use of control samples are also provided.

[0376] It should be noted that alternative methods, including but not limited to digital droplet PCR, may be available for the intended purpose. Digital droplet PCR uses an end-point method for event identity analysis, as described in section 1.1, in combination with the principle of subsampling on the extracted DNA sample. In this method, the low-level presence of an event is determined based on the ratio of DNA subsamples found to be positive and negative for the event sequence.

[0377] 2.4.1 Method design This method uses Taqman chemistry and real-time PCR principles to detect or quantitate low levels of EE-GM5 in DNA samples.

[0378] Two primers, PRIM1040 and PRIM1041, were designed to amplify an 84-bp amplicon spanning the junction region of the 5′ flanking sequence with the T-DNA insert of event EE-GM5.

[0379] A probe, TM1789, using FAM as the fluorescent label and BHQ1 as the quencher, is designed to quantitate the amplified sequence.

[0380] Forward primer targeting the EE-GM5 T-DNA sequence: PRIM1041 5'-CATTgTgCTgAATAggTTTATAgCTATgAT-3' (SEQ ID NO: 18)

[0381] Reverse primer targeting the EE-GM5 T-DNA 5' flanking sequence: PRIM1040 5'-TCAAATCAACATgggTgACTAgAAA-3' (SEQ ID NO: 19)

[0382] EE-GM5 Probe targeted to the junction of the T-DNA and its 5' flanking sequence: TM1789 FAM 5'-CAgTACTgggCCCTTgTggCgCT-3'BHQ-1 (SEQ ID NO: 20)

[0383] 2.4.2 Composition of the reaction mixture 10.0μl 2x PerfeCta qPCR Fastmix II, Low ROX 0.5μl PRIM1040[10pmol / μl] 0.5μl PRIM1041[10pmol / μl] 0.5μl TM1789[10pmol / μl] ×μl Template DNA (200ng*) Water up to 20μl Note: ● 2×PerfeCta qPCR FastMix II, LOW ROX was provided by Quanta Bioscience. Other enzyme buffers may be used, but performance should be verified. Primers and labeled probes were ordered from Integrated DNA Technologies. *The amount of template DNA per reaction may vary but should be confirmed.

[0384] 2.4.3 Thermocycling conditions

[0385] [Table 13]

[0386] 2.4.4 Taxon-specific methods Real-time PCR detection methods targeting endogenous sequences should be performed simultaneously with the same amount of template DNA as used in the target-specific real-time PCR method. The results of the taxon-specific method should be used to support data analysis and interpretation, i.e., to normalize the amount of input DNA and to validate any negative results for the target-specific reaction.

[0387] 2.4.5 Test Samples, Calibrators and Control Samples • It is recommended that all test samples be analyzed in duplicate. • A set of calibration samples that generate standard curves for both the target and taxon specific methods are included in the experiment. In addition, the following control samples are included: Positive control: A DNA sample containing the target sequence at the limit of detection. Negative control: DNA sample containing only endogenous sequences ○ No template control: water sample

[0388] 2.4.6 Data Analysis For all samples, the threshold cycle value (i.e., Ct value) is determined for both target and taxon specific methods. The threshold cycle is defined as the cycle number at which the amplification plot of a given sample reaches a defined signal threshold (see Figure 4). • Standard curve equations are calculated for both target and taxon specific methods using the Ct values ​​and genome copy amounts of the calibration samples. Standard curve parameters are slope and linearity (R 2 ) acceptance criteria, e.g., the following should be met: ○ -3.2<gradient<-3.6 ○ R 2 >0.98 For all samples, the genome copy numbers of the target and endogenous methods are calculated using linear regression analysis. • The abundance of low levels relative to the total amount of taxon-specific DNA is determined by calculating the % ratio of genome copy numbers for the target and taxon-specific methods. The control sample should give the expected result, i.e. ○ Positive controls should be rated as "detection" ○ Negative controls should be scored as "no detection." ○ No template controls should show only background levels of fluorescence ● The samples are evaluated as follows: Detection: Low levels present are above the detection limit of all replicates, taking into account the measurement uncertainty of the method. No detection: Low levels present are below the detection limit of all replicate measurements, taking into account the measurement uncertainty of the method. Inconclusive: Duplicate samples give inconsistent scores

[0389] FIG. 4 shows an example of the results of the method carried out on a calibration sample.

[0390] 3. Introgression of EE-GM5 into preferred cultivars The elite event EE-GM5 was introduced by repeated backcrossing to six different elite soybean lines. Lines were selected to represent a range of maturity: two from MG I, one from MG III, two from MG VI, and one from MG IX. One of the MG I and MG III lines contained the Rhg1 natural resistance allele from PI88788, and one of the MG VI lines carried the Rhg1 and Rhg4 natural resistance alleles from PI437654. The other three lines were susceptible to SCN.

[0391] Also, in initial testing, in several experiments, no biologically significant differences were observed for Cry14Ab-1 in HPPD-4 protein expression levels measured in leaves of greenhouse grown plants when measured by ELISA or Western blot (only routine assay changes were seen) and when event EE-GM5 was introgressed from the Thorne background into other soybean germplasm backgrounds (at different maturities, different introgression stages), no significant differences were seen in the results of standard greenhouse SCN assays (measured as % reduction in SCN cysts relative to the Thorne control) compared to those found for EE-GM5 in the Thorne background.

[0392] Introgression of the elite event EE-GM5 into other soybean varieties does not significantly affect any of the desirable phenotypic or agronomic traits of these varieties (no linkage drag), while expression of the transgene meets commercially acceptable levels, confirming the status of event EE-GM5 as an elite event.

[0393] Furthermore, the elite event EE-GM5 is advantageously combined with other soybean elite transformation events. Particularly useful plants according to the invention include event MON87751 (described in WO 2014201235 and USDA-APHIS Petition 13-337-01p), event pDAB8264.42.32.1 (described in WO 2013010094), event DAS-81419-2 (also known as Conkesta™ Soybean, described in WO 2013016527 and USDA-APHIS Petition 12-272-01p), event EE-GM3 (also known as FG-072, MST-FG072-3, described in WO 2011063411 and USDA-APHIS Petition 12-272-01p), event EE-GM4 (also known as FG-072, MST-FG42-4, described in WO 2011063411 and USDA-APHIS Petition 12-272-01p), event EE-GM5 (also known as FG-072, MST-FG42-5, described in WO 2011063411 and USDA-APHIS Petition 12-272-01p), event EE-GM6 (also known as FG-072, MST-FG42-6, described in WO 2011063411 and USDA-APHIS Petition 12-272-01p), event EE-GM7 (also known as FG-072, MST-FG42-7, described in WO 2011063411 and USDA-APHIS Petition 12-272-01p), event EE-GM8 (also known as FG-072 Event SYHT0H2 (also known as 0H2, SYN-φφφH2-5, as described in WO 2012 / 082548 and WO 2012-215-01p), Event DAS-68416-4 (also known as Enlist Soybean, as described in WO 2011 / 066384 and WO 2011 / 066360, as described in USDA-APHIS Petition 09-349-01p), Event DAS-81615-9 (also known as WO 2014004458), Event DAS-44406-6 (also known as Enlist E3, DAS-444φ6-6, as described in WO 2012 / 075426 and USDA-APHIS Event MON87708 (Xtend Soybeans, described in WO 2011 / 034704 and USDA-APHIS Petition 10-188-01p), Event MON89788 (aka Genuity Roundup Ready 2 Yield, described in WO 2006 / 130436 and USDA-APHIS Petition 06-178-01p), Event DAS-14536-7 (described in WO 2012 / 075429), Event 40-3-2 (aka RoundUp Ready, MON-φ4φ32-6, USDA-APHISEvent A2704-12 (also known as LL27, ACS-GMφφ5-3, described in WO 2006108674 and USDA-APHIS Petition 96-068-01p), Event 127 (also known as BPS-CV127-9, described in WO 2010 / 080829), Event A5547-127 (also known as LL55, ACS-GMφφ6-4, described in WO 2006108675 and USDA-APHIS Petition 96-068-01p), Event MON87754 (also known as Vistive III, MON-87754-1, described in WO 2010 / 024976), Event HOS (also known as DP-3φ5423-1, Plenish High Oleic Soybean, as described in WO 2008054747; Event MON 87701 (also known as MON-877φ1-2, as described in WO 2009064652 and USDA-APHIS Petition 09-082-01p); Event MON 87705 (also known as MON-877φ5-6, as described in WO 2010 / 037016 and USDA-APHIS Petition 09-082-01p); EE-GM5 in combination with another soybean transformation event, such as those listed in various national or regional regulatory agency databases, including, but not limited to, event MON87712 (also known as MON-87712-4, described in WO 2012 / 051199), event pDAB4472-1606 (also known as event 1606, described in WO 2012 / 033794), event 3560.4.3.5 (also known as DP-356043-5, described in WO 2008 / 002872), event MON87769 (also known as MON-87769-7, described in WO 2009102873 and USDA-APHIS Petition 09-183-01p), or a combination of two or more other soybean transformation events, or a combination of the following: event MON98788 x MON87708 (aka Roundup Ready 2 XtendSoybeans, MON-877φ8-9 x MON-89788-1), Event HOS x Event 40-3-2 (aka Plenish High Oleic Soybeans x Roundup Ready Soybeans), Event EE-GM3 x EE-GM2 (aka FG-072xLL55, described in the brochure of International Publication No. 2011063413), Event MON 87701 x MON 89788 (aka Intacta RR2 Pro Soybean, MON-877φ1-2 x MON-89788-1), DAS-81419-2 x DAS-44406-6 (aka Conkest(TM) Enlist E3(TM) Soybean, DAS-81419-2 x DAS-444φ6-6), Event DAS-81419-2 x Event DAS-68416-4 (described in WO 2013016516), Event DAS-68416-4 x Event MON 89788 (also known as Enlist™ RoundUp Ready™ 2 Soybean, DAS-68416-4 x MON-89788-1), Event MON-87769-7 x Event MON-89788-1 (also known as Omega-3 x Genuity Roundup Ready 2 Yield Soybeans), MON 87705 x MON 89788 (also known as Vistive Gold, MON-877φ5-6 x MON-89788-1), or MON87769 x MON89788 (also known as Omega-3 x Genuity Roundup Ready 2 Yield Soybeans, MON-87769-7 x The transgenic soybean events may be transgenic plants containing any combination of EE-GM5 with some of these other transgenic soybean events, such as combinations of EE-GM5 with any one of the other transgenic soybean events (MON-89788-1).

[0394] As used in the claims which follow, unless expressly indicated otherwise, the term "plant" is intended to encompass plant tissue, at any stage of maturity, and any cell, tissue, or organ taken from or derived from any such plant, including, but not limited to, any seed, leaf, stem, flower, root, single cell, gamete, cell culture, tissue culture, protoplast.

[0395] Reference seeds containing elite event EE-GM5 were deposited with ATCC (10801 University Blvd., Manassas, VA 20110-2209) under ATCC accession number PTA-123625 on November 9, 2016, and their germination ability was confirmed. Alternative designations for EE-GM5 are event GMB151 or BCS-GM151-6.

[0396] The above description of the invention is intended to be illustrative and not limiting.

[0397] Various changes or modifications in the described embodiments will occur to those skilled in the art, and can be made without departing from the spirit or scope of the invention. For example, the present invention encompasses the following embodiments: [Embodiment 1] A nucleic acid molecule comprising a nucleotide sequence essentially similar to any one of SEQ ID NOs: 1, 3, or 5, or any one of SEQ ID NOs: 2, 4, or 6, or the complement of said sequences. [Embodiment 2] A nucleic acid molecule comprising a nucleotide sequence having at least 99% sequence identity to the nucleotide sequence of SEQ ID NO: 3, 4, 5, 6, 24 or 25 or its complement. [Embodiment 3] A nucleic acid molecule according to embodiment 1 or 2, comprising the nucleotide sequence of any one of SEQ ID NO: 1 or 3 or SEQ ID NO: 2 or 4, or a complement of said sequence, or comprising the nucleotide sequences of SEQ ID NO: 1 or 3 and SEQ ID NO: 2 or 4. [Embodiment 4] A nucleic acid molecule described in any one of embodiments 1 to 3, which also comprises the nucleotide sequences of SEQ ID NOs: 7 and 9, or their complements. [Embodiment 5] A nucleic acid molecule described in any one of embodiments 1 to 4, comprising the nucleotide sequence of SEQ ID NO: 11 from nucleotide position 188 to nucleotide position 7101, or a nucleotide sequence having at least 98%, or at least 99%, or at least 99.5%, or at least 99.9% sequence identity thereto. [Embodiment 6] A nucleic acid molecule described in embodiment 5, comprising the nucleotide sequence of SEQ ID NO: 5 or 24, and SEQ ID NO: 6 or 25, or a complement thereof. [Embodiment 7] A nucleic acid molecule obtainable from a seed deposited with ATCC under accession number PTA-123625, the nucleic acid molecule comprising any one of the nucleotide sequences set forth in SEQ ID NO:1, 3, or 5, and any one of the nucleotide sequences set forth in SEQ ID NO:2, 4, or 6. [Embodiment 8] Soybean genomic DNA comprising the nucleic acid molecule described in any one of embodiments 1 to 7. [Embodiment 9] A soybean plant, cell, plant part, seed, or progeny thereof, comprising the nucleic acid molecule according to any one of embodiments 1 to 7. [Embodiment 10] A soybean plant, cell, plant part, seed or progeny thereof comprising the nucleotide sequence of SEQ ID NO:3 and the nucleotide sequence of SEQ ID NO:4 in its genome. [Embodiment 11] A soybean plant, cell, plant part, seed or progeny thereof comprising in its genome the nucleotide sequence of SEQ ID NO:5 and the nucleotide sequence of SEQ ID NO:6, or the nucleotide sequence of SEQ ID NO:24 and the nucleotide sequence of SEQ ID NO:25. [Embodiment 12] A plant, cell, plant part, seed or progeny according to embodiment 10 or 11, which also comprises the nucleotide sequence of SEQ ID NO: 7 and 9, or the nucleotide sequence of SEQ ID NO: 23 from nucleotide position 1114 to nucleotide position 8572. [Embodiment 13] A soybean plant, cell, part, or seed, each of which contains an elite event EE-GM5 in its genome, wherein the elite event is a genetic locus comprising an inserted T-DNA containing a chimeric gene encoding an HPPD-4 protein and a chimeric gene encoding a Cry14Ab-1 protein, and 5' and 3' flanking sequences immediately surrounding the inserted T-DNA, as found in a reference seed deposited with the ATCC under accession number PTA-123625. [Embodiment 14] A progeny plant, cell, plant part or seed of the plant, cell, plant part or seed of embodiment 13, wherein the progeny plant, cell, plant part or seed comprises the nucleotide sequence of SEQ ID NO:3 and the nucleotide sequence of SEQ ID NO:4. [Embodiment 15] A soybean plant, cell, part, or seed described in embodiment 13 or 14, whose genomic DNA generates an 84 bp DNA fragment when analyzed using PCR with two primers containing the nucleotide sequences of SEQ ID NO: 18 and SEQ ID NO: 19, respectively. [Embodiment 16] A plant according to any one of embodiments 9 to 15, which is resistant to isoxaflutole, topramezone or mesotrione. [Embodiment 17] A soybean product produced from the soybean plant, cell, part, or seed according to any one of embodiments 9 to 16. [Embodiment 18] The soybean product of embodiment 17, which is or comprises soybean meal, ground seeds, soybean flour or soybean flakes. [Embodiment 19] A soybean product according to embodiment 17 or 18, wherein the soybean product comprises a nucleic acid that produces an amplicon diagnostic or specific for event EE-GM5. [Embodiment 20] A method for producing a soybean product, comprising the steps of obtaining a soybean plant, cell, part, seed or progeny described in any one of embodiments 9 to 16, and producing a soybean product therefrom. [Embodiment 21] The method of embodiment 20, wherein the soybean product is or comprises soybean meal, ground seeds, soybean flour or soybean flakes. [Embodiment 22] The method described in embodiment 20 or 21, wherein the soybean product comprises a nucleic acid that produces an amplicon diagnostic or specific for event EE-GM5. [Embodiment 23] A weed control process comprising the step of treating a field sown with soybean seeds according to any one of embodiments 9 to 16 with an HPPD-inhibiting herbicide before the soybean plants germinate but after the seeds are sown. [Embodiment 24] A weed control process comprising treating a soybean plant according to any one of embodiments 9 to 16 with an HPPD inhibitor herbicide from above the plant. [Embodiment 25] A method for protecting germinating soybean plants according to any one of embodiments 9 to 16 from competition by weeds, comprising treating a field in which the soybean plants are to be planted with an HPPD-inhibiting herbicide before planting or sowing the soybean plants, and then planting or sowing the soybean plants or seeds in the pre-treated field. [Embodiment 26] The process of any one of embodiments 23 to 25, wherein the HPPD inhibitor herbicide is isoxaflutole, topramezone, or mesotrione. [Embodiment 27] A method for producing a soybean plant that is resistant to SCN and / or tolerant to HPPD-inhibiting herbicides, comprising the steps of: crossing a first soybean plant that lacks a gene encoding Cry14Ab-1 and a gene encoding HPPD-4 with a soybean plant described in any one of embodiments 9 to 16, thereby introducing resistance to SCN and / or tolerance to HPPD-inhibiting herbicides into the genome of the soybean plant; and selecting progeny plants that are resistant to SCN and / or tolerant to HPPD-inhibiting herbicides. [Embodiment 28] The plant, seed, part, cell or progeny thereof or the use described in any of embodiments 9 to 16 for producing soybean seeds. [Embodiment 29] Use of a soybean plant or seed according to any one of embodiments 9 to 16 for growing a nematode-resistant and / or HPPD-inhibiting herbicide-tolerant plant. [Embodiment 30] Use of a soybean seed according to any one of embodiments 9 to 16 for obtaining a soybean product, wherein the soybean product is or comprises ground soybean grains, soybean flour, soybean meal, or soybean flakes. [Embodiment 31] A method for producing a soybean plant or seed containing the elite event EE-GM5, comprising the steps of crossing a plant described in any one of embodiments 9 to 16 with another soybean plant, and planting a seed containing EE-GM5 obtained from the cross. [Embodiment 32] A nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:11 from nucleotide position 131 to nucleotide position 7941, or a nucleotide sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the nucleic acid molecule encodes a nematocidal Cry14Ab protein and an HPPD protein resistant to HPPD inhibitors. [Embodiment 33] A nucleic acid molecule described in embodiment 32, encoding a protein of sequence number 8 or a protein at least 99% identical thereto, and a protein of sequence number 10 or a protein at least 99% identical thereto. [Embodiment 34] A method for identifying elite event EE-GM5 in a biological sample, comprising detection of an EE-GM5-specific region using a specific primer pair or a specific probe that specifically recognizes a region containing a portion of the 5' or 3' T-DNA adjacent region in EE-GM5 and a portion of the inserted T-DNA adjacent thereto. [Embodiment 35] A primer pair suitable for use in specific detection of EE-GM5, comprising a first primer comprising a sequence that specifically recognizes a sequence in the 5' or 3' T-DNA flanking region of the inserted T-DNA in EE-GM5 under optimized detection conditions, and a second primer comprising a sequence that specifically recognizes a sequence in the inserted T-DNA in EE-GM5 adjacent to the 5' or 3' flanking region under optimized detection conditions, wherein the 5' T-DNA flanking region comprises the nucleotide sequence of SEQ ID NO: 5 from nucleotide 1 to nucleotide 166, or SEQ ID NO: 24 from nucleotide 1 to nucleotide 1113, and the 3' T-DNA flanking region comprises the nucleotide sequence of SEQ ID NO: 25 from nucleotide 1 to nucleotide 1113. a nucleotide sequence of the complement of SEQ ID NO:6 from nucleotide position 359 to nucleotide 691, or a nucleotide sequence of the complement of SEQ ID NO:25 from nucleotide position 359 to nucleotide 1449, and said inserted T-DNA comprises the complement of the nucleotide sequence of SEQ ID NO:5 from nucleotide position 167 to nucleotide 353, or the nucleotide sequence of SEQ ID NO:6 from nucleotide position 1 to nucleotide 358, or the nucleotide sequence of SEQ ID NO:11 from nucleotide position 1 to nucleotide position 7459 or a complement thereof, or the nucleotide sequence of SEQ ID NO:23 from nucleotide position 1114 to nucleotide position 8572 or a complement thereof. [Embodiment 36] The primer pair according to embodiment 35, wherein the first primer comprises a nucleotide sequence of 17 to 200 consecutive nucleotides selected from the nucleotide sequence of SEQ ID NO:5 from nucleotide 1 to nucleotide 166, or the nucleotide sequence of SEQ ID NO:24 from nucleotide 1 to nucleotide 1113, or a complement thereof, or the nucleotide sequence of SEQ ID NO:6 from nucleotide 358 to nucleotide 691, or the nucleotide sequence of SEQ ID NO:25 from nucleotide 359 to nucleotide 1449, or a complement thereof, and the second primer comprises a nucleotide sequence of 17 to 200 consecutive nucleotides selected from the nucleotide sequence of SEQ ID NO:5 from nucleotide 167 to nucleotide 353 or a complement thereof, or the nucleotide sequence of SEQ ID NO:6 from nucleotide 1 to nucleotide 358 or a complement thereof, or the nucleotide sequence of SEQ ID NO:11 from nucleotide position 1 to nucleotide position 7459 or a complement thereof, or the nucleotide sequence of SEQ ID NO:23 from nucleotide 1114 to nucleotide 8572 or a complement thereof. [Embodiment 37] The primer pair according to embodiment 35, wherein the first primer comprises at its 3' end a nucleotide sequence of at least 17 consecutive nucleotides selected from SEQ ID NO:5 from nucleotide 1 to nucleotide 166, or SEQ ID NO:24 from nucleotide 1 to nucleotide 1113, or a complement thereof, or comprises at its 3' end a nucleotide sequence of at least 17 consecutive nucleotides selected from SEQ ID NO:6 from nucleotide 358 to nucleotide 691, or SEQ ID NO:25 from nucleotide 359 to nucleotide 1449, or a complement thereof, and the second primer comprises at its 3' end a nucleotide sequence of at least 17 consecutive nucleotides selected from SEQ ID NO:5 from nucleotide 167 to nucleotide 353, or a complement thereof, or SEQ ID NO:6 from nucleotide 1 to nucleotide 358, or a complement thereof, or SEQ ID NO:11 from nucleotide position 1 to nucleotide position 7459, or a complement thereof, or SEQ ID NO:23 from nucleotide 1114 to nucleotide 8572, or a complement thereof. [Embodiment 38] A primer pair as described in embodiment 35, wherein the first primer comprises the nucleotide sequence of SEQ ID NO: 19 or SEQ ID NO: 13, or the second primer comprises the nucleotide sequence of SEQ ID NO: 18 or SEQ ID NO: 12, respectively. [Embodiment 39] A primer pair as described in embodiment 35, comprising a first primer comprising the nucleotide sequence of SEQ ID NO: 19 or the nucleotide sequence of SEQ ID NO: 13 at its 3' end, or a second primer comprising the nucleotide sequence of SEQ ID NO: 18 or the nucleotide sequence of SEQ ID NO: 12 at its 3' end. [Embodiment 40] A primer pair described in embodiment 38 or 39, for amplifying an 85 or 84 bp EE-GM5-specific fragment using PCR. [Embodiment 41] A primer pair comprising a first primer comprising the sequence of SEQ ID NO: 13 at its 3' end and a second primer comprising the sequence of SEQ ID NO: 12 at its 3' end, or a first primer comprising the sequence of SEQ ID NO: 18 at its 3' end and a second primer comprising the sequence of SEQ ID NO: 19 at its 3' end. [Embodiment 42] A specific probe for identifying elite event EE-GM5 in a biological sample, the probe specifically recognizing a sequence including a part of the 5' T-DNA flanking sequence of EE-GM5 and a part of the inserted T-DNA adjacent to it downstream, or its complement, or a sequence including a part of the 3' T-DNA flanking sequence of EE-GM5 and a part of the inserted T-DNA adjacent to it upstream, or its complement. [Embodiment 43] A specific probe for identifying elite event EE-GM5 in a biological sample, comprising a nucleotide sequence having at least 80% sequence identity to a sequence including a portion of the 5' T-DNA flanking sequence and a portion of the inserted T-DNA adjacent to it downstream, or its complement, or a sequence including a portion of the 3' T-DNA flanking sequence in EE-GM5 and a portion of the inserted T-DNA adjacent to it upstream, or its complement. [Embodiment 44] A probe described in embodiment 43, having at least 80% sequence identity with the sequence of SEQ ID NO: 1 or 3, or the sequence of SEQ ID NO: 2 or 4, or its complement. [Embodiment 45] The probe described in embodiment 44, comprising any one of the sequences of SEQ ID NO: 1, 3, or 5, or any one of the sequences of SEQ ID NO: 2, 4, or 6, or a complement thereof. [Embodiment 46] A probe described in any one of embodiments 42 to 45, comprising the sequence of SEQ ID NO: 3 or the sequence of SEQ ID NO: 4. [Embodiment 47] The method described in embodiment 34, comprising amplifying a 50 to 1000 bp DNA fragment from nucleic acid present in the biological sample using a polymerase chain reaction with at least two primers, the at least two primers being primers described in any one of embodiments 35 to 41. [Embodiment 48] The method described in embodiment 47, further comprising a step of hybridizing a probe specific to the DNA fragment amplified using the at least two primers. [Embodiment 49] The method described in embodiment 48, wherein the probe recognizes a portion of the 5' T-DNA flanking region and a portion of the adjacent inserted T-DNA, or the probe recognizes a portion of the 3' T-DNA flanking region and a portion of the adjacent inserted T-DNA. [Embodiment 50] The method described in embodiment 47, wherein the primers comprise the sequences of SEQ ID NO: 12 and SEQ ID NO: 13, respectively, and the probe comprises the sequence of SEQ ID NO: 14, or the primers comprise the sequences of SEQ ID NO: 18 and SEQ ID NO: 19, respectively, and the probe comprises the sequence of SEQ ID NO: 20. [Embodiment 51] A kit suitable for use in specific detection of EE-GM5, comprising the primer pair described in any one of embodiments 35 to 41. [Embodiment 52] The kit described in embodiment 51, further comprising a probe specific to the DNA fragment amplified by the primer pair. [Embodiment 53] The kit described in embodiment 52, wherein the probe recognizes a portion of the 5' T-DNA flanking region and a portion of the inserted T-DNA adjacent thereto, or the probe recognizes a portion of the 3' T-DNA flanking region and a portion of the inserted T-DNA adjacent thereto. [Embodiment 54] The kit described in embodiment 52, wherein the primers comprise the sequences of SEQ ID NO: 12 and SEQ ID NO: 13 and the probe comprises the sequence of SEQ ID NO: 14, or the primers comprise the sequences of SEQ ID NO: 18 and SEQ ID NO: 19 and the probe comprises the sequence of SEQ ID NO: 20. [Embodiment 55] A kit suitable for use in specific detection of EE-GM5, comprising a probe according to any one of embodiments 42 to 46. [Embodiment 56] The method described in embodiment 34, comprising a step of hybridizing the nucleic acid of the biological sample with a probe described in any one of embodiments 42 to 46. [Embodiment 57] A method for verifying seed purity or for screening seeds for the presence of elite event EE-GM5, the method comprising detection of an EE-GM5 specific region in a sample of the seeds using a primer pair described in any one of embodiments 35 to 41 or a probe described in any one of embodiments 42 to 46. [Embodiment 58] The method described in embodiment 57, comprising a step of amplifying an 85 bp DNA fragment, wherein the primers comprise the sequences of SEQ ID NO: 12 and SEQ ID NO: 13, respectively, and the probe comprises the sequence of SEQ ID NO: 14, or a step of amplifying an 84 bp DNA fragment, wherein the primers comprise the sequences of SEQ ID NO: 18 and SEQ ID NO: 19, respectively, and the probe comprises the sequence of SEQ ID NO: 20.

[0081] [Embodiment 59] A method for detecting the presence of elite event EE-GM5 in a biological sample by hybridization with a substantially complementary labeled nucleic acid probe, wherein the probe:target nucleic acid ratio is amplified by recycling of the target nucleic acid sequence, said method comprising: a) hybridizing said target nucleic acid sequence to a first nucleic acid oligonucleotide comprising the nucleotide sequence of SEQ ID NO:5 from nucleotide position 167 to nucleotide position 184, or a complement thereof, or said first nucleic acid oligonucleotide comprising the nucleotide sequence of SEQ ID NO:6 from nucleotide position 341 to nucleotide position 358, or a complement thereof; b) hybridizing said target nucleic acid sequence to a second nucleic acid oligonucleotide comprising the nucleotide sequence of SEQ ID NO:5 from nucleotide 149 to nucleotide 166 or its complement, or said nucleic acid oligonucleotide comprising the nucleotide sequence of SEQ ID NO:6 from nucleotide 359 to nucleotide 376 or its complement, wherein said first and second oligonucleotides overlap by at least one nucleotide, and wherein either said first or second oligonucleotide is labeled to be said labeled nucleic acid probe; c) cleaving only the labeled probe within the probe:target nucleic acid sequence duplex with an enzyme that causes selective probe cleavage resulting in dissociation of the duplex, leaving the target sequence intact; d) reusing the target nucleic acid sequence by repeating steps (a)-(c); and e) detecting the cleaved labeled probe, thereby determining the presence of said target nucleic acid sequence. A method comprising: [Embodiment 60] A method for determining the zygosity status of a plant, plant material or seed comprising elite event EE-GM5, the method comprising the step of amplifying a 50-1000 bp DNA fragment from nucleic acid present in the biological sample using polymerase chain reaction with at least three primers, two of which specifically recognize pre-insertion plant DNA and a third of which recognizes a sequence within the inserted T-DNA. [Embodiment 61] The method described in embodiment 60, wherein the two primers that specifically recognize the pre-insertion plant DNA are a primer comprising the nucleotide sequence of SEQ ID NO: 19 and a primer comprising the nucleotide sequence of SEQ ID NO: 21, and the primer that recognizes a sequence within the inserted T-DNA is a primer comprising the nucleotide sequence of SEQ ID NO: 18. [Embodiment 62] A method for detecting the presence of elite event EE-GM5 in a biological sample, or a method for determining the zygosity status of a plant, plant material or seed containing EE-GM5, wherein the method is described in any one of Examples 2.1, 2.2, 2.3, or 2.4. [Embodiment 63] A method for reducing yield loss in a field in which soybean plants are planted, particularly in a field that contains, has contained, or is expected to contain nematodes such as SCN, RKN, or Pratylenchus, or nematode eggs, or Nematode reniforme or eggs thereof, comprising 1) obtaining a plant or seed containing the elite transformation event EE-GM5, and 2) planting or sowing the soybean plants or seeds, wherein a reference seed containing the elite event has been deposited with the ATCC under the accession number PTA-123625. [Embodiment 64] A method for increasing the yield of soybean plants when planted in a field containing nematodes such as SCN, RKN or Pratylenchus or nematode eggs or Nematode reniforme or its eggs, comprising: 1) obtaining a plant or seed containing the elite transformation event EE-GM5; and 2) planting or sowing the soybean plant or seed, wherein a reference seed containing the elite event has been deposited with the ATCC under the accession number PTA-123625. [Embodiment 65] A method for the preparation of a soybean plant or seed, comprising the steps of: introducing an elite soybean transformation event EE-GM5 into the genome of a soybean plant or seed; and optionally treating said plant or seed with an HPPD-inhibiting herbicide, such as isoxaflutole, topramezone or mesotrione; or optionally treating a field in which said plant or seed will be planted with an HPPD-inhibiting herbicide, such as isoxaflutole, topramezone or mesotrione, and planting said plant or seed in the pre-treated field. or a method for producing soybean plants or seeds that are resistant to HPPD-inhibiting herbicides such as mesotrione, or a method for producing soybean plants or seeds that are resistant to nematodes such as SCN, RKN or Pratylenchus or Nematode nematode, or a method for producing soybean plants or seeds that are resistant to HPPD-inhibiting herbicides such as isoxaflutole, topramezone or mesotrione, or that are resistant to nematodes such as SCN, RKN or Pratylenchus or Nematode nematode. [Embodiment 66] A nucleic acid molecule specifically characterizing soybean elite transformation event EE-GM5, characterized in that it comprises any one of the nucleotide sequences of SEQ ID NO: 1, 3 or 5, which contains a portion of soybean plant genomic DNA and a portion of the inserted foreign DNA of EE-GM5 adjacent to its downstream, and / or that it comprises the nucleotide sequence of SEQ ID NO: 2, 4 or 6, which contains a portion of soybean plant genomic DNA and a portion of the inserted foreign DNA of EE-GM5 adjacent to its downstream. [Embodiment 67] The method according to embodiment 27 or 31, comprising applying an HPPD inhibitor such as isoxaflutole, topramezone or mesotrione to a soybean seed growing on the plant, or to a field in which the seed is planted, or onto the plant. [Embodiment 68] A method for increasing the yield of soybean plants in a field containing SCN contaminated with sudden death syndrome or in a field containing SCN causing iron deficiency chlorosis in soybeans, the method comprising planting or sowing a plant containing an elite event EE-GM5, and a reference seed containing the elite event has been deposited with the ATCC under the accession number PTA-123624.

Claims

1. In the 5' to 3' direction, A nucleotide sequence of SEQ ID NO: 5 or 24, or a nucleotide sequence having at least 99% sequence identity thereto; The nucleotide sequence of SEQ ID NO:11 from nucleotide position 188 to nucleotide position 7101, or a nucleotide sequence having at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.5%, or at least 99.9% sequence identity thereto; and The nucleotide sequence of SEQ ID NO: 6 or 25, or a nucleotide sequence having at least 99% sequence identity thereto. or a complement thereof.

2. 2. The nucleic acid molecule of claim 1, comprising the nucleotide sequence of SEQ ID NO: 5 or 24, and SEQ ID NO: 6 or 25, or a complement thereof.

3. A nucleic acid molecule obtainable from the seed deposited with the ATCC under accession number PTA-123625, said nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:5 and the nucleotide sequence of SEQ ID NO:

6.

4. 2. Soybean genomic DNA comprising the nucleic acid molecule of claim 1.

5. 10. A soybean plant, cell, plant part, seed or progeny thereof, comprising the nucleic acid molecule of claim 1.

6. 13. A soybean plant, cell, plant part, seed or progeny thereof, comprising in its genome the nucleic acid molecule of claim 1.

7. 7. The plant, cell, plant part, seed or progeny thereof according to claim 5 or 6, which is resistant to isoxaflutole, topramezone or mesotrione.

8. 1. A soybean plant, cell, part, or seed, each comprising an elite event EE-GM5 in its genome, said elite event EE-GM5 being a genetic locus comprising an inserted T-DNA containing a chimeric gene encoding a HPPD-4 protein and a chimeric gene encoding a Cry14Ab-1 protein, and 5' and 3' flanking sequences immediately surrounding said inserted T-DNA, as contained in a reference seed deposited with the ATCC under accession number PTA-123625.

9. 9. The soybean plant, cell, part or seed of claim 8, whose genomic DNA generates an 84 bp DNA fragment when analyzed using PCR with two primers comprising the nucleotide sequences of SEQ ID NO:18 and SEQ ID NO:19, respectively.

10. a) treating a field sown with soybean seeds according to any one of claims 5 to 8 with an HPPD-inhibiting herbicide before the soybean plants have germinated but after the seeds have been sown; or b) treating the soybean plant according to any one of claims 5 to 8 with an HPPD-inhibiting herbicide from above the plant. wherein the plant is tolerant to the herbicide.

11. 9. A method for protecting germinating soybean plants according to any one of claims 5 to 8 from competition by weeds, comprising the steps of treating a field in which to plant soybean plants with an HPPD-inhibiting herbicide prior to planting or sowing soybean plants, and subsequently planting or sowing said soybean plants or seeds in said pre-treated field.

12. 12. The method of claim 10 or 11, wherein the HPPD-inhibiting herbicide is isoxaflutole, topramezone or mesotrione.

13. A method for identifying elite event EE-GM5 in a biological sample, comprising detection of an elite event EE-GM5 specific region using a specific primer pair or specific probe that specifically recognizes a region containing a portion of the 5' or 3' T-DNA flanking region in elite event EE-GM5 and a portion of the inserted T-DNA adjacent thereto, wherein said elite event EE-GM5 is a genetic locus comprising an inserted T-DNA containing a chimeric gene encoding a HPPD-4 protein and a chimeric gene encoding a Cry14Ab-1 protein, and 5' and 3' flanking sequences immediately surrounding said inserted T-DNA, contained in a reference seed deposited with the ATCC under accession number PTA-123625.

14. 1. A kit suitable for use in the specific detection of elite event EE-GM5, said elite event EE-GM5 being a genetic locus comprising an inserted T-DNA containing a chimeric gene encoding a HPPD-4 protein and a chimeric gene encoding a Cry14Ab-1 protein, and 5' and 3' flanking sequences immediately surrounding said inserted T-DNA, contained in a reference seed deposited with the ATCC under accession number PTA-123625, comprising the following primer pair: a) a primer pair comprising a first primer comprising a sequence that specifically recognizes a sequence in the 5' or 3' T-DNA flanking region of the inserted T-DNA in the elite event EE-GM5 under optimized detection conditions, and a second primer comprising a sequence that specifically recognizes a sequence in the inserted T-DNA in the elite event EE-GM5 adjacent to said 5' or 3' flanking region under optimized detection conditions, wherein said 5' T-DNA flanking region comprises the nucleotide sequence of SEQ ID NO: 5 from nucleotide 1 to nucleotide 166, or SEQ ID NO: 24 from nucleotide 1 to nucleotide 1113. a primer pair comprising: said 3' T-DNA flanking region comprising a nucleotide sequence of the complement of SEQ ID NO:6 from nucleotide 359 to nucleotide 691, or a nucleotide sequence of the complement of SEQ ID NO:25 from nucleotide 359 to nucleotide 1449, and said inserted T-DNA comprising a complement of the nucleotide sequence of SEQ ID NO:5 from nucleotide 167 to nucleotide 353, or a nucleotide sequence of SEQ ID NO:6 from nucleotide 1 to nucleotide 358, or a nucleotide sequence of SEQ ID NO:11 from nucleotide position 1 to nucleotide position 7459, or a complement thereof; b) a primer pair comprising a first primer comprising a nucleotide sequence of 17-200 contiguous nucleotides selected from SEQ ID NO:5 from nucleotide 1 to nucleotide 166, or the nucleotide sequence of SEQ ID NO:24 from nucleotide 1 to nucleotide 1113, or a complement thereof, or comprising a nucleotide sequence of 17-200 contiguous nucleotides selected from the nucleotide sequence of SEQ ID NO:6 from nucleotide 358 to nucleotide 691, or the nucleotide sequence of SEQ ID NO:25 from nucleotide 359 to nucleotide 1449, or a complement thereof, and a second primer comprising a nucleotide sequence of 17-200 contiguous nucleotides selected from the nucleotide sequence of SEQ ID NO:5 from nucleotide 167 to nucleotide 353, or a complement thereof, or the nucleotide sequence of SEQ ID NO:6 from nucleotide 1 to nucleotide 358, or a complement thereof, or the nucleotide sequence of SEQ ID NO:11 from nucleotide position 1 to nucleotide position 7459, or a complement thereof; c) a primer pair comprising a first primer comprising at its 3' end a nucleotide sequence of at least 17 contiguous nucleotides selected from SEQ ID NO:5 from nucleotide 1 to nucleotide 166, or the nucleotide sequence of SEQ ID NO:24 from nucleotide 1 to nucleotide 1113, or a complement thereof, or comprising at its 3' end a nucleotide sequence of at least 17 contiguous nucleotides selected from the nucleotide sequence of SEQ ID NO:6 from nucleotide 358 to nucleotide 691, or the nucleotide sequence of SEQ ID NO:25 from nucleotide 359 to nucleotide 1449, or a complement thereof, and a second primer comprising at its 3' end a nucleotide sequence of at least 17 contiguous nucleotides selected from the nucleotide sequence of SEQ ID NO:5 from nucleotide 167 to nucleotide 353, or a complement thereof, or the nucleotide sequence of SEQ ID NO:6 from nucleotide 1 to nucleotide 358, or a complement thereof, or the nucleotide sequence of SEQ ID NO:11 from nucleotide position 1 to nucleotide position 7459, or a complement thereof; d) a primer pair comprising a first primer comprising the nucleotide sequence of SEQ ID NO:19 or SEQ ID NO:13, or a second primer comprising the nucleotide sequence of SEQ ID NO:18 or SEQ ID NO:12, respectively; or e) A primer pair comprising a first primer comprising the nucleotide sequence of SEQ ID NO:19 or the nucleotide sequence of SEQ ID NO:13 at its 3' end, or a second primer comprising the nucleotide sequence of SEQ ID NO:18 or the nucleotide sequence of SEQ ID NO:12 at its 3' end.

15. The kit of claim 14, wherein the primer pair amplifies an 82 bp or 84 bp elite event EE-GM5 specific fragment using PCR.

16. A kit for the identification of elite event EE-GM5 in a biological sample, said elite event EE-GM5 being a genetic locus comprising an inserted T-DNA containing a chimeric gene encoding a HPPD-4 protein and a chimeric gene encoding a Cry14Ab-1 protein, and 5' and 3' flanking sequences immediately surrounding said inserted T-DNA, contained in a reference seed deposited with the ATCC under accession number PTA-123625, said kit comprising a specific probe for: a) a probe that specifically recognizes a sequence comprising a part of the 5' T-DNA flanking sequence of elite event EE-GM5 and a part of the inserted T-DNA adjacent thereto downstream, or a complement thereof, or a sequence comprising a part of the 3' T-DNA flanking sequence of elite event EE-GM5 and a part of the inserted T-DNA adjacent thereto upstream, or a complement thereof; b) a probe comprising a nucleotide sequence having at least 90% sequence identity with a sequence comprising a portion of the 5' T-DNA flanking sequence of elite event EE-GM5 and a portion of the inserted T-DNA adjacent thereto downstream, or a complement thereof, or a sequence comprising a portion of the 3' T-DNA flanking sequence of elite event EE-GM5 and a portion of the inserted T-DNA adjacent thereto upstream, or a complement thereof; or c) A probe comprising the nucleotide sequence of SEQ ID NO:5 or SEQ ID NO:6 or their complements.

17. 16. The method according to claim 13, comprising amplifying a 50-1000 bp DNA fragment from nucleic acids present in the biological sample using polymerase chain reaction with at least two primers, the at least two primers being primers contained in the kit according to claim 14 or 15.

18. 18. The method of claim 17, further comprising the step of hybridizing a probe specific to the DNA fragment amplified using the at least two primers.

19. The method according to claim 18, wherein the probe recognizes a part of the 5' T-DNA flanking region and a part of the inserted T-DNA adjacent thereto, or the probe recognizes a part of the 3' T-DNA flanking region and a part of the inserted T-DNA adjacent thereto.

20. 19. The method of claim 18, wherein the primers comprise the sequences of SEQ ID NO:12 and SEQ ID NO:13, respectively, and the probe comprises the sequence of SEQ ID NO:14, or the primers comprise the sequences of SEQ ID NO:18 and SEQ ID NO:19, respectively, and the probe comprises the sequence of SEQ ID NO:

20.

21. 16. A kit suitable for use in the specific detection of elite event EE-GM5, comprising the primer pair contained in the kit of claim 14 or 15, wherein said elite event EE-GM5 is a genetic locus comprising an inserted T-DNA containing a chimeric gene encoding a HPPD-4 protein and a chimeric gene encoding a Cry14Ab-1 protein, and 5' and 3' flanking sequences immediately surrounding said inserted T-DNA, contained in a reference seed deposited with the ATCC under accession number PTA-123625.

22. 22. The kit of claim 21, further comprising a probe specific to the DNA fragment amplified by the primer pair.

23. 17. The method of claim 13, comprising the step of hybridizing nucleic acid of the biological sample with a probe contained in the kit of claim 16.

24. 17. A method for verifying seed purity or for screening seeds for the presence of elite event EE-GM5, said method comprising detection of an elite event EE-GM5 specific region in a sample of said seeds with a primer pair comprised in the kit of claim 14 or 15 or a probe comprised in the kit of claim 16, said elite event EE-GM5 being a genetic locus comprising an inserted T-DNA containing a chimeric gene encoding HPPD-4 protein and a chimeric gene encoding Cry14Ab-1 protein, and 5' and 3' flanking sequences immediately surrounding said inserted T-DNA, as contained in a reference seed deposited with the ATCC under accession number PTA-123625.

25. The method of claim 24, comprising the steps of: amplifying an 82 bp DNA fragment, wherein the primers comprise the sequences of SEQ ID NO:12 and SEQ ID NO:13, respectively, and the probe comprises the sequence of SEQ ID NO:14; or amplifying an 84 bp DNA fragment, wherein the primers comprise the sequences of SEQ ID NO:18 and SEQ ID NO:19, respectively, and the probe comprises the sequence of SEQ ID NO:

20.

26. 1. A method for determining the zygosity status of a plant, plant material or seed comprising elite event EE-GM5, said method comprising the steps of amplifying a 50-1000 bp DNA fragment from nucleic acid present in a biological sample using polymerase chain reaction with at least three primers, two of which specifically recognize pre-insertion plant DNA and a third of which recognizes a sequence within an inserted T-DNA, said elite event EE-GM5 being a genetic locus comprising an inserted T-DNA containing a chimeric gene encoding a HPPD-4 protein and a chimeric gene encoding a Cry14Ab-1 protein, and 5' and 3' flanking sequences immediately surrounding said inserted T-DNA, contained in a reference seed deposited with the ATCC under accession number PTA-123625.

27. The method according to claim 26, wherein the two primers that specifically recognize the pre-insertion plant DNA are a primer comprising the nucleotide sequence of SEQ ID NO: 19 and a primer comprising the nucleotide sequence of SEQ ID NO: 21, and the primer that recognizes a sequence within the inserted T-DNA is a primer comprising the nucleotide sequence of SEQ ID NO:

18.

28. 10. A method for reducing yield loss or increasing yield of soybean plants in a field containing nematodes or nematode eggs, comprising the steps of: 1) obtaining a soybean plant or seed comprising the elite event EE-GM5 of claim 8; and 2) planting or sowing said soybean plant or seed.

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