BR122023026037A2 - PAIR OF INITIATORS, KIT AND METHODS FOR DETECTING THE PRESENCE OF THE EE-GM5 ELITE EVENT AND FOR DETERMINING THE STATE OF ZYGOSITY - Google Patents

Abstract

The invention provides specific transgenic plants, plant material and soybean seeds, characterized in that these products harbor a specific transformation event of nematode resistance and herbicide tolerance in a specific location of the soybean genome. Tools are also provided that allow rapid and unambiguous identification of the event in biological samples.

Description

CROSS REFERENCE WITH RELATED ORDERS

[0001] This application claims the benefit of U.S. Provisional Application Serial No. 62/437,874, filed on December 22, 2016, and U.S. Provisional Application Serial No. 62/481,292, filed on April 4, 2017, the contents of which are incorporated herein by reference in their entirety.

AREA OF INVENTION

[0002] This invention relates to novel nucleic acids and transgenic plants, plant material and soybean seeds, characterized by the fact that they harbor a specific transformation event, particularly by the presence of genes conferring resistance to nematodes and tolerance to herbicides, in a specific location in the soybean genome. The soybean plants of the invention combine the phenotype of nematode resistance and herbicide tolerance with agronomic performance, genetic stability and functionality in different genetic backgrounds equivalent to the corresponding untransformed soybean genetic background in the absence of inhibitory herbicide(s). of HPPD or nematode infestation. This invention further provides methods and kits for identifying the presence of plant material specifically comprising EE-GM5 transformation event in biological samples.

BACKGROUND OF THE INVENTION

[0003] The phenotypic expression of a transgene in a plant is determined both by the structure of the gene or genes themselves and by their location in the plant genome. At the same time, the presence of the transgene or “inserted T-DNA” in different locations in the genome will influence the overall phenotype of the plant in different ways. The agronomically or industrially successful introduction of a commercially interesting trait into a plant by genetic manipulation can be a time-consuming procedure dependent on different factors. The actual transformation and regeneration of genetically transformed plants is only the first in a series of selection steps, which include extensive genetic characterization, introgression, and evaluation in field trials, eventually leading to the selection of an elite event.

[0004] The unequivocal identification of an elite event is becoming increasingly important in view of the discussion on New Food/Feed, segregation of GMO and non-GMO products and the identification of protected material. Ideally, such an identification method is both rapid and simple, without the need for an extensive laboratory setting. Furthermore, the method must provide results that allow unambiguous determination of the elite event without interpretation by experts, but which remain after expert scrutiny if necessary.

[0005] Planting nematode-resistant and herbicide-tolerant EE-GM5 soybean varieties provides producers with new options for controlling nematodes and weeds using HPPD-inhibiting herbicides, such as isoxaflutole herbicide (IFT), topramezone or mesotrione (MST). HPPD inhibitor herbicides offer an alternative weed control option for the soybean producer to help manage problematic weed species and as an alternative mode of action to help slow the spread of herbicide-resistant weeds.

[0006] The soybean cyst nematode (SCN) Heterodera glycines (Ichinohe), a global problem for soybean production, is an ongoing threat to producers. Since its first detection in the US 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 more than $1.2 billion in annual losses. yield in the US, making it the most damaging soybean pathogen there. SCN was first detected in Brazil in the early 1990s and has since spread throughout South America and is one of the most important pathogens in Brazil causing losses in practically all crop regions in Brazil. Similarly, SCN continues to spread throughout China's soybean-producing regions with detections in 15 provinces and estimated yield losses of more than $120 million. A multi-year study in Iowa, USA (2001 to 2015) where almost all SCN-resistant soybean varieties contain SCN resistance to PI 88788, found that the virulence of SCN populations increased over the years, resulting in increased densities. of late-season SCN populations and reduced yields of SCN-resistant soybean varieties with resistance source PI88788 (Mitchum (2016), Phytopathology 106 (12): 1444-1450, Allen et al. (2017) Plant Health Progr .18:19-27, Arias et al.

[0007] The root lesion nematode Pratylenchus brachyurus has become an increasingly important pathogen of soybeans. It has a wide host range and is widely distributed in tropical and subtropical regions, especially in Brazil, Africa and the southern United States. Pratylenchus brachyurus has become a concern among cotton and soybean producers in the Cerrado region of Brazil and is considered the main pathogen of soybeans in the region. In soybeans, this nematode can reduce yields by 30 to 50%, with greater damage being observed in sandy soils. The use of resistant soybean varieties would be the best way to control this nematode, however, varieties resistant to P. brachyurus have not been identified to date. Although several soybean genotypes have been studied for resistance to Pratylenchus brachyurus, and some cultivars identified with increased tolerance, the cultivation of resistant cultivars against P.brachyurus is difficult due to the fact that this nematode is polyphagic and does not have a close interaction with its hosts. (Machado (2014) Current Agricultural Science and Technology 20: 26-35; Antonio et al. (2012) Soil productivity losses in area infested by the nematode of the root lesions in Vera, MT. In: Brazilian Congress of Soy, 6, 2012, Cuiabá. Londrina: Embrapa Soja, 4 pp; Rios et al. (2016) Ciência Rural 46: 580-584; Productivity; Edited by Minobu Kasai, ISBN 978-953-51-3118-2, Print ISBN 978-953-51-3117-5, InTech; population variation, pathogenicity and loss estimation. Tropical Plant Pathology 36: 178-185).

[0008] It is known that protecting plants against nematodes such as SCN can help plants better cope with other stresses such as soil composition/content, weather conditions, pathogen stress, herbicide applications, etc.. Particularly When such other stresses give a phenotype that is easily seen, such as chlorosis/yellowing of leaves, the effect of SCN control is easier to see while otherwise often not “visible”. For example, when soybean plants have Sudden Death Syndrome (SDS) or Iron Deficiency Chlorosis (IDC), SCN protection will result in plants that are greener or have less severe SDS/IDC symptoms. Despite extensive research and variety tracking efforts, iron deficiency remains a challenge in large areas of soybean production in the North Central U.S. The importance of this problem has increased due to expanded soybean production in soils susceptible to iron deficiency. and possible interactions with changes in the culture system. Iron deficiency occurs in soils with high pH and carbonates, but the expression of iron deficiency is highly spatially variable due to interactions with spatially variable soil properties such as moisture content, salinity, iron availability, and other micronutrient concentrations. and metals. Additionally, the expression of iron deficiency interacts with biotic factors such as nitrogen fixation, pests, diseases and management-induced stresses such as herbicide application. Variety selection is the most important means of managing iron deficiency, but variety selection is complicated by a large genotype by environmental interaction related to chlorosis tolerance (Hansen et al. (2004) Soil Sci. Plant Nutr. 50 (7): 983-987).

[0009] Soybean sudden death syndrome (SDS) was first discovered in 1971 in Arkansas and has since been confirmed throughout most US soybean growing areas. SDS is a fungal disease that also occurs in a disease complex with soybean cyst nematode (SCN). SDS is among the most devastating soil-borne diseases of soybeans in the United States. When this disease occurs in the presence of SCN, symptoms occur earlier and are more severe. SDS is caused by soil-borne fungi within a group of the Fusarium solani species complex. In North America, Fusarium virguliforme, formerly Fusarium solani f. sp. glycines, is the causative agent. In South America, F. brasiliense, F. cuneirostrum, F. tucumaniae, and F. virguliforme cause SDS symptoms. Although soybean cultivars that are less susceptible to SDS have been developed, no highly resistant cultivars are available. The fungus can infect soybean seedling roots immediately after planting, but above ground SDS symptoms rarely appear until soybean plants have reached reproductive stages. The fungus produces toxins in the roots that are translocated to the leaves. The first notable symptoms of SDS are yellowing and defoliation of upper leaves. If the disease develops early in the season, the flowers and young pods will be aborted. When the disease develops later, the plant will produce fewer seeds per pod or smaller seeds. The earlier serious illness develops, the more yield is reduced. Because the SDS fungus can persist in the soil for long periods, larger areas of a field will show symptoms of the disease each growing season until the majority 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, no soybean plants genetically manipulated for resistance to nematodes are commercialized. Soybean plants comprising one or more herbicide tolerance genes have been disclosed in the art. WO2006/130436 describes a glyphosate-tolerant soybean event comprising an epsps gene and WO2011/034704 describes a dicamba-tolerant soybean event. WO2012/082548 describes soybean plants comprising both an hppd and a pat gene. WO2011/063411 describes a soybean event with tolerance to HPPD inhibitors and glyphosate, while WO2011/063413 describes soybean plants with tolerance to HPPD inhibitors, glufosinate and glyphosate. WO2011/066384 describes a soybean event with tolerance to 2,4-D and glufosinate, while WO2012/075426 describes a soybean event with tolerance to 2,4-D, glufosinate and glyphosate and WO2017/059795 describes a soybean event with glyphosate tolerance. WO2009/064652 describes a soybean event with resistance to lepidopteran insects and WO2013/016527 describes a soybean event with resistance to lepidopteran insects and tolerance to glufosinate.

[0011] HPPD genes and proteins that confer improved tolerance to HPPD-inhibiting herbicides have been disclosed, e.g., in WO2015138394, WO2015135881, WO2014043435, and the nematicidal activity of Cry proteins has been described in, e.g., WO2010027805, WO2010027809, WO2010027804, WO2010027799, WO2010027808 and WO2007147029.

[0012] None of the prior art disclosures teach or suggest an elite event in soybean comprising a nematode-active Cry gene, and certainly not an elite event in soybean comprising a nematode-active Cry gene combined with a gene conferring tolerance to HPPD inhibitors.

[0013] It is known in the art that obtaining a commercial elite transformation event in soybean plants with acceptable agronomic performances is by no means straightforward.

SUMMARY OF PREFERRED EMBODIMENTS OF THE INVENTION

[0014] This invention provides a nucleic acid encoding a Cry14Ab-1 protein, such as the sequence encoding cry14Ab-1.b of SEQ ID NO: 7 or a sequence encoding a Cry14Ab nematicidal protein having at least 95, 96, 97, 98 or at least 99% sequence identity with SEQ ID NO:7. Also provided herein is a nucleic acid encoding an HPPD-4 protein, such as the sequence encoding hppdPf-4Pa of SEQ ID NO: 9 or a sequence having at least 95, 96, 97, 98 or at least 99% sequence identity. with SEQ ID NO: 9, wherein said sequence encodes an HPPD protein providing tolerance to HPPD-inhibiting herbicides when expressed in a plant. Also provided herein are a chimeric cry14Ab-1.b gene comprising the sequence of SEQ ID NO: 11 from nucleotide position 131 to nucleotide position 5276, or its complement, or a chimeric cry14Ab-1.b gene comprising the sequence of SEQ ID NO: 11 from nucleotide position 412 to nucleotide position 3969 operably linked to a plant-expressible promoter, or a sequence encoding a Cry14Ab nematicidal protein having at least 95, 96, 97, 98 or at least at least 99% sequence identity with the sequence of SEQ ID NO: 11 from nucleotide position 131 to nucleotide position 5276, or its complement, or with the sequence of SEQ ID NO: 11 from position nucleotide 412 to nucleotide position 3969 (operationally linked to a plant-expressible promoter). There is further provided a chimeric hppdPf-4Pa gene comprising the sequence of SEQ ID NO: 11 from nucleotide position 5382 to nucleotide position 7459, or its complement, or comprising the sequence of SEQ ID NO: 11 from nucleotide position 5589 to nucleotide position 6665 operably linked to a plant-expressible 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 its complement, or with the sequence of SEQ ID NO: 11 from nucleotide position 5589 to nucleotide position 6665 (when operably linked to a plant-expressible promoter ); wherein said sequence encodes an HPPD protein providing tolerance to HPPD-inhibiting herbicides when expressed in a plant, as well as a nucleic acid comprising said cry14Ab-1.bchimeric gene and said hppdPf-4Pachimeric gene. These nucleic acids or genes are useful for transforming plants such as soybeans, cotton, corn, rice, rapeseed and wheat such that they control nematodes and/or have tolerance to HPPD-inhibiting herbicides.

[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 with it. In one embodiment, this DNA molecule encodes a protein tolerant to an HPPD inhibitor and a protein negatively affecting plant pest nematodes, such as SCN, RKN or Pratylenchus spp. nematodes. In one embodiment, this chimeric DNA molecule encodes the protein of SEQ ID NO: 8 or a nematode control protein at least 99% identical to it and the protein of SEQ ID NO: 10 or an HPPD inhibitor tolerant protein at least 99% identical to it. Also provided are plants, seeds or cells, such as plants, seeds or soybean cells, transformed to contain such a DNA molecule, and the use of such a DNA molecule to make the plants or seeds, such as plants or seeds of soybeans, resistant to nematodes and tolerant to HPPD-inhibiting herbicides.

[0016] The present invention relates to a transgenic soybean plant, its plant part, seed, cell or tissue, comprising, stably integrated into its genome, an expression cassette comprising a nematode resistance gene comprising the coding sequence of the cry14Ab-1.b gene and a herbicide tolerance gene comprising the coding sequence of the hppdPf-4Pa gene (both as described in Example 1.1 and as represented in SEQ ID NO: 7 and 9, respectively), which provide resistance to parasitic nematodes of plants such as soybean cyst nematode and tolerance to an HPPD-inhibiting herbicide such as isoxaflutole, topramezone or mesotrione. In the absence of HPPD-inhibiting herbicide and nematode pressure, such a soybean plant has an agronomic performance that is substantially equivalent to the non-transgenic isogenic line. When encountering soybean cyst nematode (SCN) pressure affecting plant performance in the field, plants of the invention will have a superior agronomic phenotype compared to a non-transgenic plant. Likewise, in the presence of weeds, after application of an HPPD-inhibiting herbicide to which tolerance is provided, the plants of the invention will have a superior agronomic phenotype compared to plants that have not been treated with herbicides.

[0017] According to the present invention, the soybean plant or its seed, cells or tissues comprise the EE-GM5 elite event. In one embodiment, the elite event EE-GM5 comprises the sequence of any one of SEQ ID NO: 1, 3, 5 or 24 or the sequence of any one of SEQ ID NO: 2, 4, 6 or 25 or any sequences essentially similar to it. In one embodiment, EE-GM5 comprises the sequence of any one of SEQ ID NO: 1, 3, 5 or 24 and the sequence of any one of SEQ ID NO: 2, 4, 6 or 25, or any sequences essentially similar to it, and the sequence encoding cry14Ab-1.b of SEQ ID NO: 7 and the sequence encoding hppdPf-4Pa of SEQ ID NO: 9 or sequences essentially similar thereto. In one embodiment, the elite event EE-GM5 is a T-DNA inserted into a specific position in the soybean genome, as contained in the reference seed deposited with the ATCC under deposit 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, said event is characterized by the 5' junction sequence of SEQ ID NO: 1 or 3 or by the 3' junction sequence of SEQ ID NO: 2 or 4; or by the 5' splice sequence of SEQ ID NO: 1 or 3 and by the 3' splice sequence of SEQ ID NO: 2 or 4. In one embodiment, genomic DNA containing EE-GM5, when analyzed using a chain reaction of polymerase (“PCR” here) with two primers comprising the nucleotide sequence of SEQ ID NO: 12 and SEQ ID NO: 13, respectively, generating an 85 bp DNA fragment. In one embodiment, genomic DNA containing EE-GM5, when analyzed using PCR with two primers comprising the nucleotide sequence of SEQ ID NO: 18 and SEQ ID NO: 19, respectively, yields an 84 bp DNA fragment.

[0018] In one embodiment there is provided here a soybean plant, its cell, plant part, seed or progeny, each comprising the elite event EE-GM5 in its genome, with the reference seed comprising said event having been deposited at ATCC under filing number PTA-123625. In one embodiment, a plant or seed comprising EE-GM5 is obtainable by propagation of and/or crossing with a soybean plant grown from seed deposited with the ATCC under deposit number PTA-123625.

[0019] More specifically, the present invention relates to a transgenic soybean plant, its plant part, pollen, seed, cell or tissue, the genomic DNA of which is characterized by the fact that, when analyzed in PCR as described herein , using at least two primers directed against the region formed by a part of the 5' or 3' T-DNA flanking region of EE-GM5 and part of the inserted T-DNA, a fragment is amplified that is specific to the EE-GM5 event . The primers can be directed against the 3' T-DNA flanking region within SEQ ID NO: 6 or SEQ ID NO: 25 or soybean plant genomic DNA downstream thereof and contiguous thereto and the T-DNA inserted therein. upstream of it and contiguous with it. The primers can also be directed against the 5' T-DNA flanking region within SEQ ID NO: 5 or SEQ ID NO: 24 or soybean plant genomic DNA upstream thereof and contiguous thereto and the inserted T-DNA. downstream of it and contiguous with it. In one embodiment, such primers comprise or consist (essentially) of the nucleotide sequence of SEQ ID NO: 12 and SEQ ID NO: 13 or of SEQ ID NO: 18 and SEQ ID NO: 19 or of SEQ ID NO: 26 and SEQ ID NO: 28 or SEQ ID NO: 27 and SEQ ID NO: 29, respectively (e.g., a pair of primers comprising a primer containing at its extreme 3' end the nucleotide sequence of SEQ ID NO: 12 and a primer containing at its extreme 3' end the nucleotide sequence of SEQ ID NO: 13 or a pair of primers comprising a primer containing at its extreme 3' end the nucleotide sequence of SEQ ID NO: 18 and a primer containing at its extreme 3' end the nucleotide sequence of SEQ ID NO: 19 or a pair of primers comprising a primer containing at its extreme 3' end the nucleotide sequence of SEQ ID NO: 26 and a primer containing at its extreme 3' end the nucleotide sequence of SEQ ID NO: 28 or a pair of primers comprising a primer containing at its extreme 3' end the nucleotide sequence of SEQ ID NO: 27 and a primer containing at its extreme 3' end the nucleotide sequence of SEQ ID NO: 29) and give rise to a DNA fragment of between 50 and 1000 bp, such as an 85 bp or 84 bp fragment.

[0020] The reference seed comprising the elite event of the invention has been deposited with the ATCC under accession number PTA-123625. One embodiment of the invention is the elite event EE-GM5 as contained in the seed deposited under accession number PTA-123625, which when introduced into a soybean plant will provide nematode resistance and herbicide tolerance, particularly resistance to root cyst nematode. soybean (Heterodera glycines, “SCN” herein) and/or lesion-causing nematodes (lesion-causing nematode as used herein refers to soybean pest nematodes of Pratylenchus spp., including but not limited to Pratylenchus brachyurus) and tolerance to HPPD inhibitors such as isoxaflutole, topramezone or mesotrione. The EE-GM5 plants of this invention also control root knot nematode (root knot nematode as used herein refers to soybean pest nematodes of Meloidogyne spp., including but not limited to Meloidogyne incognita, Meloidogyne arenaria, Meloidogyne hapla or Meloidogyne javanica or any combination thereof), reniform nematode (Rotylenchulus reniformis) and lance nematode (Hoplolaimus spp. such as H. columbus, H. galeatus and H. magnistylus). Included in this invention are minimal variants of this event such as a soybean event with tolerance to HPPD inhibitors and resistance to SCN nematodes that has a nucleotide sequence of 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 as contained in the seed deposited with the ATCC under deposit number PTA-123625 or a soybean event with tolerance to HPPD inhibitors and resistance to nematodes SCN that has a nucleotide sequence differing 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 of the nucleotide sequence of EE-GM5 as contained in the deposited seed of ATCC deposit PTA-123625 or which has a nucleotide sequence differing 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 of the nucleotide sequence formed by the following consecutive 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. In one embodiment, EE-GM5 comprises a nucleotide sequence of at least 95%, at least at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% sequence identity with the sequence formed by the following consecutive nucleotide sequences (5' for 3'): SEQ ID NO: 5 or 24, SEQ ID NO: 11 from nucleotide position 188 to nucleotide position 7101 and SEQ ID NO: 6 or 25. Due to natural genetic variation, differences in DNA bases Unique and small insertions and deletions in homologous DNA sequences (e.g., single nucleotide polymorphisms (SNPs)) are commonly found in plants of the same species (Zhu et al. (2003) Genetics 163: 1123-1134).

[0021] The seed of ATCC filing number PTA-123625 is a pure seed lot of transgenic seeds homozygous for the EE-GM5 elite event of the invention, which will grow into nematode-resistant plants, whereby the plants are also tolerant to an HPPD inhibitor such as isoxaflutole, topramezone or mesotrione. Seed or progeny seed obtainable from the deposited seed (e.g., after crossing with other soybean plants of a different genetic background) can be sown and the cultured plants can be treated with an HPPD inhibitor such as isoxaflutole , topramezone or mesotrione as described herein or may be tested for the presence of EE-GM5 as described herein to obtain plants comprising the elite event of the invention. The invention further relates to cells, seeds, tissues, progeny and descendants of a plant comprising the elite event cultivated from the seed deposited with the ATCC having accession number PTA-123625. The invention relates to plants obtainable from (such as by propagation of and/or crossing with) a soybean plant comprising the elite event of the invention (such as a plant cultivated from seed deposited with the ATCC having the number of accession PTA-123625 or a plant comprising the sequence encoding HPPD-4 of SEQ ID NO: 9 and the sequence encoding cry14Ab-1.b of SEQ ID NO: 7 located between the sequence of SEQ ID NO: 1, 3 or 5 and the sequence of SEQ ID NO: 2, 4 or 6 or a plant 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 of sequences of SEQ ID NO: 1, 3, 5 or 24 and sequences of any one of SEQ ID NO: 2, 4, 6 or 25). The invention also relates to plants and progeny seeds obtained from the above plants or seed and 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 invention additionally relates to a method for identifying a transgenic plant, or its cells or tissues, comprising the EE-GM5 elite event, which method is based on the identification of the presence of characterizing DNA sequences or encoded amino acids by such DNA sequences in the transgenic plant, cells or tissues. According to a preferred embodiment of the invention, such characterizing DNA sequences are sequences of 15 bp or at least 15 bp, preferably 20 bp or at least 20 bp, most preferably 30 bp or more that comprise the event insertion site, i.e., a sequence containing both a part of the inserted T-DNA containing an HPPD inhibitor and nematode resistance transgene and a part of the 5' or 3' T-DNA flanking region contiguous therewith that extends into the plant genome of soybeans, allowing specific identification of the elite event. The invention also relates to plants, seeds and cells comprising the EE-GM5 event as identified herein.

[0023] The present invention additionally relates to methods for identifying the EE-GM5 elite event in biological samples, which methods are based on pairs of primers or probes that specifically recognize the 5' T-DNA flanking sequence and/or 3' and the inserted T-DNA sequence contiguous with it in EE-GM5. Any other methods to identify EE-GM5, e.g., to identify its specific characterizing sequences, are also included here, such as whole or partial (targeted) genome sequencing.

[0024] More specifically, the invention relates to a method for identifying the EE-GM5 elite event in biological samples comprising amplification of a nucleic acid sequence present in said biological samples, using a polymerase chain reaction with at least two primers or a polymerase chain reaction with at least two primers and a probe, wherein one of these primers recognizes the 5' or 3' T-DNA flanking region in EE-GM5, the other primer recognizes a sequence within the T -DNA comprising herbicide tolerance and nematode resistance genes that is contiguous with said 5' or 3' T-DNA flanking region, preferably to obtain a DNA fragment of 50 to 1000 bp in size. In one embodiment, a first primer recognizes the 5' T-DNA flanking region in EE-GM5 and a second primer recognizes a sequence within the T-DNA comprising herbicide tolerance and nematode resistance genes that is contiguous with and downstream of of said 5' T-DNA flanking region or a first primer recognizes the 3' T-DNA flanking region in EE-GM5 and a second primer recognizes a sequence within the T-DNA comprising the herbicide tolerance and herbicide resistance genes. nematodes that is contiguous with and upstream of said 3' T-DNA flanking region, to obtain a DNA fragment characteristic of the EE-GM5 elite event. In one embodiment, said polymerase chain reaction method further comprises the use of a probe that recognizes DNA amplified by said primers, e.g., junction DNA comprising part of the inserted T-DNA and part of the flanking DNA. said T-DNA in EE-GM5 (on the 5' or 3' side of the event, as applicable, such as a probe comprising the nucleotide sequence of SEQ ID NO: 14 or 20 herein or its complement), so as to detect the amplification product produced by said primers. The primers can recognize a sequence 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 (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 a sequence within the inserted T-DNA (SEQ ID NO: 5 from nucleotide position 167 to 353, or SEQ ID NO: 6 from from nucleotide position 1 to nucleotide position 358 or SEQ ID NO: 23 from nucleotide position 1114 to 8572 or its complement), respectively. The primer recognizing the 5' or 3' T-DNA flanking region may comprise the nucleotide sequence of SEQ ID NO: 13, SEQ ID NO: 19, SEQ ID NO: 26 or SEQ ID NO: 27 and the primer recognizing a sequence within the inserted T-DNA comprising 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. This invention also relates to any event-specific primer pair and the specific DNA amplified using such primer pair, as can be obtained by one of skill in the art or as can be obtained from commercial sources of the EE-GM5 event sequences. provided here or contained in the seed deposited with ATCC under accession number PTA-123625.

[0025] The present invention relates more specifically to a method for identifying the EE-GM5 elite event in biological samples, which method comprises amplification of a nucleic acid sequence present in a biological sample, using a chain reaction of polymerase with two primers comprising or consisting (essentially) of the nucleotide sequence of 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 of (essentially) ) in the nucleotide sequence of SEQ ID NO: 18 and SEQ ID NO: 19, respectively, to obtain an 84 bp DNA fragment. Also plants comprising the thus identified elite event EE-GM5 are included in this invention.

[0026] The present invention further relates to EE-GM5 specific T-DNA flanking sequences described herein, which can be used to develop EE-GM5 specific identification methods in biological samples. Such specific T-DNA flanking sequences can also be used as reference control material in identification assays. More particularly, the invention relates to the 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 material are nucleic acid molecules, preferably of about 150-850 bp, comprising sequence that can be amplified by primers comprising or consisting (essentially) of the nucleotide sequence of SEQ ID NO: 12 and SEQ ID NO: 13 or SEQ ID NO: 18 and SEQ ID NO: 19.

[0027] The invention additionally relates to methods of identifying the presence of EE-GM5 in biological samples based on the use of such specific primers or probes. The primers may comprise, consist of, or consist essentially of a nucleotide sequence of 17 to about 200 consecutive nucleotides selected from the nucleotide sequence of SEQ ID NO: 5 from nucleotide 1 to nucleotide 166 or SEQ ID NO: 24 from from nucleotide 1 to nucleotide 1113 or from the complement of the nucleotide sequence of SEQ ID NO: 25 from nucleotide 359 to nucleotide 691 or from the complement of the nucleotide sequence of SEQ ID NO: 25 from nucleotide 359 to nucleotide 1449, combined with primers, consisting or consisting essentially of a nucleotide sequence of 17 to about 200 consecutive nucleotides selected from the nucleotide sequence of SEQ ID NO: 11 from nucleotide 1 to nucleotide 7459 or its complement or nucleotide sequence of SEQ ID NO: 23 from nucleotide 1114 to nucleotide 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 its complement. Primers may also comprise these nucleotide sequences located at their extreme 3' end and further comprise unrelated sequences or sequences derived from the mentioned nucleotide sequences but comprising mismatches. In one embodiment, the primers, as used herein, may also be identical to the target DNA or its complement, wherein said target DNA is a hybrid containing nucleotide sequences of different origins, which do not occur in such a combination in nature.

[0028] The invention additionally relates to kits for identifying the EE-GM5 elite event in biological samples, said kits comprising at least one pair of primers or probe that specifically recognizes the 5' or 3' T-DNA flanking region and the inserted T-DNA comprising a herbicide tolerance and nematode resistance gene contiguous therewith in EE-GM5.

[0029] The kit of the invention may comprise, in addition to a primer that specifically recognizes the 5' or 3' T-DNA flanking region of EE-GM5, a second primer that specifically recognizes a sequence within the inserted T-DNA comprising a HPPD inhibitor herbicide tolerance and nematode resistance gene from EE-GM5, for use in a PCR identification protocol. Kits of the invention may comprise at least two specific primers, one of which recognizes a sequence within the 5' T-DNA flanking region of EE-GM5 or a sequence within the 3' T-DNA flanking region of EE-GM5 and the other which recognizes a sequence within the inserted T-DNA comprising a gene for tolerance to HPPD-inhibiting herbicides and nematode resistance. 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 contiguous with said 5' T-DNA flanking region may comprise the sequence of nucleotides 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 contiguous with said 3' flanking region may comprise the nucleotide sequence of SEQ ID NO: 12 or any other primer or combination of primers as described herein or obtainable from the description or seed deposit. The kit may further comprise a probe recognizing a sequence located between the primer recognizing the 5' T-DNA flanking region and the primer recognizing the sequence within the inserted T-DNA or recognizing a sequence between the primer recognizing the 3' DNA flanking region. ' and the primer recognizing the 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 invention additionally relates to a kit for identifying the EE-GM5 elite event in biological samples, said kit comprising PCR primers comprising or consisting (essentially) of the nucleotide sequence of SEQ ID NO: 12 and SEQ ID NO: 13 or nucleotide sequence of SEQ ID NO: 18 and SEQ ID NO: 19 for use in the EE-GM5 PCR protocol described here. Said kit comprising primers comprising or consisting (essentially) of the nucleotide sequence 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 sequence 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 additionally comprise buffer and reagents such as any or each of the following compounds: dNTPs, (Taq) DNA polymerase, MgCl2, stabilizers and, optionally, a dye.

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

[0032] According to another aspect of the invention, DNA molecules comprising sufficient length of polynucleotides from both the flanking T-DNA sequences and the inserted T-DNA of EE-GM5 are disclosed, so as to be useful as a primer or probe for the detection of EE-GM5 or to characterize plants comprising the EE-GM5 elite event. Such sequences may comprise any of at least 9, at least 10, at least 15, at least 20 or at least 30 nucleotides or may comprise any of 9, 10, 15, 20 or 30 nucleotides of the T-DNA flanking sequence. and a similar number of nucleotides of the inserted T-DNA of EE-GM5, on each side of the splice site, respectively, and this in either or both of the 5' and 3' splice sites of the EE-GM5 event. Most preferably, such DNA molecules comprise the sequence of any one of SEQ ID NO: 1, 3 or 5 or the sequence of any one of SEQ ID NO: 2, 4 or 6. In one embodiment, such DNA molecules comprise the sequence of SEQ ID NO: 23, 24 or 25. In one aspect of the invention soybean plants and seeds comprising 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, the following: to identify the presence or determine the (lower) threshold of EE-GM5 in plants, plant material or in products such as, but not limited to, food or feed products (fresh or processed) comprising or derived from plant material; additionally or alternatively, the methods and kits of the present invention can be used to identify transgenic plant material for the purposes of segregation between transgenic and non-transgenic material; Additionally or alternatively, the methods and kits of the present invention can be used to determine the quality (i.e., percentage of pure material) of plant material comprising EE-GM5.

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

[0035] The invention also relates to genomic DNA obtained from plants comprising the EE-GM5 elite event, particularly genomic DNA comprising sequences specific to the EE-GM5 event, such as one or both of the EE-GM5 junction sequences. GM5 (containing a portion of DNA flanking T-DNA and inserted T-DNA contiguous therewith, characteristic of EE-GM5), e.g., any of the sequences of SEQ ID NO: 1, 3, 5 or 24 and/or or any 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 cells, seeds or progeny thereof, each comprising at least one elite event, said elite event comprising an inserted T-DNA comprising : i) a first chimeric gene comprising a cry14Ab-1.b gene derived from Bacillus thuringiensis encoding a Cry14Ab-1 protein 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: 7 and ii) a second chimeric gene comprising a modified hppdPf-4Pa gene from Pseudomonas encoding a more tolerant 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.

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

[0038] In a further embodiment, said elite event is obtainable by crossing with a soybean plant grown from the reference seed comprising said event having been deposited with the ATCC under deposit number PTA-123625.

[0039] In another embodiment, the genomic DNA of said soybean plant, or its cells, parts, seeds or progeny, when analyzed using PCR with two primers comprising the nucleotide sequence of SEQ ID NO: 12 and SEQ ID NO: 13 , respectively, gives rise to an 85 bp DNA fragment or, when analyzed using PCR with two primers comprising the nucleotide sequence of SEQ ID NO: 18 and SEQ ID NO: 19, respectively, gives an 84 bp DNA fragment.

[0040] Also provided herein is a method for identifying a transgenic soybean plant, or its cells, parts, seed or progeny, with resistance to nematodes, such as resistance to SCN and/or Pratylenchus and/or root knot nematodes and /or reniforms, and tolerance to an HPPD inhibitor herbicide, such as isoxaflutole, topramezone or mesotrione, in biological samples, said method comprising amplification of a DNA fragment of between 50 and 150 bp from a nucleic acid present in samples biological processes using a polymerase chain reaction with at least two primers, one of said primers recognizing the 5' T-DNA flanking region of the elite event EE-GM5, said 5' T-DNA flanking region comprising the sequence of nucleotides of SEQ ID NO: 5 from nucleotide 1 to nucleotide 166 or of SEQ ID NO: 24 from nucleotide 1 to nucleotide 1113, or recognizing the T-DNA 3' flanking region of said event, comprising the said 3' T-DNA flanking region is 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, the other primer of said primers recognizing a sequence within the inserted T-DNA comprising the complement 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 wherein said inserted T-DNA comprises the nucleotide sequence of SEQ ID NO: 11 from nucleotide position 1 to nucleotide position 7459 or its complement.

[0041] Also provided here is a kit for identifying a transgenic soybean plant, or its cells, parts, seed or progeny, with resistance to nematodes and tolerance to an HPPD-inhibiting herbicide, in biological samples, said kit comprising a primer recognizing the 5' T-DNA flanking region of the elite event EE-GM5, said 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 primer recognizing the 3' T-DNA flanking region of said elite event, comprising said 3'a T-DNA flanking region complement nucleotide sequence of SEQ ID NO: 6 from nucleotide 359 to nucleotide 691 or the complement nucleotide sequence of SEQ ID NO: 25 from nucleotide 359 to nucleotide 1449 and a primer recognizing a sequence within the Inserted T-DNA, the inserted T-DNA comprising the complement 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 comprising to said inserted T-DNA the nucleotide sequence of SEQ ID NO: 11 from nucleotide position 1 to nucleotide position 7459 or its complement.

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

[0043] Also provided herein is a soybean plant, plant cell, tissue or seed, comprising in its 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 of 80 to 100% sequence identity thereto and/or SEQ ID NO: 2, 4, 6 or 25, or a nucleotide sequence of 80 to 100% sequence identity thereto, and a sequence of nucleotides with 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 the nucleotide position 188 to nucleotide position 7101 or its complement.

[0044] An embodiment of this invention provides a soybean plant, plant cell, tissue or seed, comprising in its genome a nucleic acid molecule hybridizing under standard stringency conditions with the nucleotide sequence of any one of SEQ ID NO: 1 , 3 or 5 or their complement or hybridizing to the nucleotide sequence of any of SEQ ID NO: 2, 4 or 6 or their complement.

[0045] Also provided herein is an isolated nucleic acid molecule comprising a nucleotide sequence with at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the nucleotide sequence of any of SEQ ID NO: 1, 3, 5 or 24, or their complement, or with at least 90%, at least 95%, at least 98% or at least 99% sequence identity with the nucleotide sequence of any one of SEQ ID NO: 2, 4, 6 or 25, or its complement, or an isolated nucleic acid molecule comprising a nucleotide sequence hybridizing under standard stringency conditions to the nucleotide sequence of any one of SEQ ID NO: 1, 3, 5 or 24, or their complement, or with the nucleotide sequence of any of SEQ ID NO: 2, 4, 6 or 25 or their complement.

[0046] Also provided herein is an isolated nucleic acid molecule comprising a nucleotide sequence with 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 nucleotide sequence hybridizing under standard stringency conditions to the nucleotide sequence of SEQ ID NO: 7 or its complement, wherein such nucleic acid molecule encodes a Nematicidal toxin active on cyst nematodes and/or lesion-forming nematodes and/or root nodule nematodes and/or reniform nematodes, 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 plant-expressible (heterologous) promoter so as to form a chimeric gene. Also provided herein is the use of said nucleic acid molecule in transformed plants or seeds to control plant pathogenic nematodes. Further provided herein is a method of controlling root knot nematodes such as Meloidogyne incognita, Meloidogyne arenaria, Meloidogyne hapla or Meloidogyne javanica, particularly Meloidogyne incognita, comprising use of a Cry14Ab protein or a DNA encoding a Cry14Ab protein or a plant or seed containing the said DNA under the control of a plant-expressible promoter, wherein said Cry14Ab protein is the protein comprising the amino acid sequence of SEQ ID NO: 8 or a protein with at least 96% or at least 98 or at least 99% of 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% of sequence identity with it. Further provided herein is a method of controlling kidney nematodes (Rotylenchulus reniformis), comprising use of 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, in that said Cry14Ab protein is the protein comprising the amino acid sequence of SEQ ID NO: 8 or a protein with at least 96% or at least 98% or at least 99% sequence identity thereto or a protein comprising the sequence of amino acids of SEQ ID NO: 8 from amino acid position 1 to amino acid position 706 or a protein with 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 Cry14Ab nematicidal protein and an HPPD inhibitor-tolerant HPPD protein. In one embodiment, this nucleic acid molecule encodes the protein of SEQ ID NO: 8 or a protein at least 99% identical to it and the protein of SEQ ID NO: 10 or a protein at least 99% identical to it.

[0048] Also provided here is a soybean plant cell comprising in its genome the elite event EE-GM5 which is a foreign DNA or a -DNA inserted into a defined locus, wherein the elite event EE-GM5 is as contained in the reference seed deposited with the ATCC under deposit number PTA-123625, wherein said gene encoding chimeric Cry14Ab-1 and a gene encoding chimeric HPPD-4 and wherein said elite event is characterized by the splice sequence 5 'of SEQ ID NO: 1 or 3 and by the junction sequence 3' of SEQ ID NO: 2 or 4; or such cell which is a seed cell, or such cell, wherein the genomic DNA of said cell, when analyzed using PCR with two primers comprising the nucleotide sequence of SEQ ID 12 and SEQ ID 13, respectively, gives rise to a fragment of 85 bp DNA.

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

[0050] The invention also provides a nucleic acid molecule comprising in order the following nucleotide sequences: a) the nucleotide sequence of SEQ ID NO: 5 from nucleotide 1 to 166 or a sequence at least 99% identical to it , b) the nucleotide sequence of SEQ ID NO: 11 from nucleotide 188 to nucleotide 7101 or a sequence at least 99% identical thereto and c) the nucleotide sequence of SEQ ID NO: 6 from nucleotide 359 to to nucleotide 691 or a sequence at least 99% identical thereto, such as such 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.

[0051] The invention also provides a nucleic acid molecule comprising in order the following nucleotide sequences: a) the nucleotide sequence of SEQ ID NO: 24 from nucleotide 1 to 1113 or a sequence at least 99% identical to it , 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 to nucleotide 1449 or a sequence at least 99% identical thereto, such as such 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. According to the invention there is also provided a method for producing a soybean product, comprising obtaining soybean seed comprising the elite event EE-GM5 as described above and producing the soybean product therefrom. In one embodiment, the soy product in such a method is or comprises soybean meal, crushed seeds, soy flour or flakes or oil, soy protein, lecithin, soy milk, tofu, margarine, biodiesel, biocomposite, adhesive, solvent, lubricant, cleaner, foam, paint, dye, candle, or a food or feed product containing soybean oil or soybean protein. In another embodiment, such soy product comprises a nucleic acid specific to the EE-GM5 elite event. In one embodiment, said EE-GM5 elite event-specific nucleic acid 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 the seed comprising the elite event EE-GM5 as described above, wherein said soybean product is or comprises soybean meal, crushed seeds, flour or flakes and comprises nucleic acids specific to the elite event EE-GM5, wherein said nucleic acids are detectable using the methods as described herein. In one embodiment, said EE-GM5 elite event-specific nucleic acid comprises the sequence of SEQ ID NO: 1 or 3 or the sequence of SEQ ID NO: 2 or 4. In another embodiment, said event-specific nucleic acid EE-GM5 elite event comprises the sequence of SEQ ID NO: 5 or 24 or the sequence of SEQ ID NO: 6 or 25. In one embodiment, said EE-GM5 elite event specific nucleic acid comprises the sequence of SEQ ID NO: 5 or 24 and the sequence of SEQ ID NO: 6 or 25.

[0053] Also provided here is the use of soybean seed comprising the elite event EE-GM5 to obtain a soybean product, wherein said elite event comprises the sequence of any one of SEQ ID NO: 1, 3 , 5 or 24 and/or the sequence of any of SEQ ID NO: 2, 4, 6 or 25. In one embodiment, in such use, the soy product is any of soybean meal, crushed soybean seeds, soy flour or soy flakes.

[0054] Additionally provided herein is a method for producing a soybean plant or seed comprising the EE-GM5 elite event combined with another SCN resistance locus/gene, such as by combining the EE-GM5 elite event with another SCN resistance locus/gene occurring in the same soybean plant/seed and planting of the seed comprising EE-GM5 and said other SCN resistance locus/gene. In one embodiment, the plants, cells or seeds of the invention contain one or more SCN resistance loci/genes that occur in soybeans, to obtain a combination of different sources of SCN resistance in the soybean plants, cells or seeds of the invention. . Several soybean SCN resistance loci or genes are known and one or more of these can be combined with EE-GM5 in the same plant, cell or seed, such as any of the SCN resistance genes/loci from PI 88788 resistance sources. , PI 548402 (Peking), PI 437654 (Hartwig or CystX®), or any combination thereof, or one or more of the native 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 of the soybean chromosomes 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 of any combination thereof (Kim et al. 2016, Theor. Appl. Genet. 129 (12): 2295-2311; Kim and Diers 2013, Crop Science 53: 775-785; org; Concibido et al. 2004, Crop Science 44: 574-581). Also, in one embodiment, the plants or seeds of the invention contain EE-GM5 when combined with one or more soybean SCN resistance loci obtained from any of the 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, 633736, PI 507354, PI 404166, PI 437655, PI 467312, PI 567328, PI 22897 or PI 494182. Table 3 attached herein provides a comprehensive list of soybean accessions reported to be resistant to SCN, of which the SCN resistance genes/loci (one or several) may be combined with EE-GM5 of the invention in the same soybean plant, cell or seed. TABLE 3

[0055] Also provided herein is a method for protecting emerging soybean plants from competition by weeds, comprising treating a field in which seeds containing the elite event EE-GM5 as described above have been sown, with an HPPD-inhibiting herbicide. , in which plants are tolerant to the HPPD-inhibiting herbicide. In one embodiment, in such a method, the HPPD-inhibiting herbicide is isoxaflutole, topramezone, or mesotrione.

[0056] Also provided herein is a method for protecting emerging soybean plants from weed competition, comprising treating a field to be planted with soybean plants comprising the EE-GM5 elite event as described above with an HPPD-inhibiting herbicide. , before the soybean plants are planted or the seeds are sown, followed by planting or sowing said soybean plants or seeds in said pretreated field, wherein the plants are tolerant to the HPPD-inhibiting herbicide.

[0057] Also provided herein is a method for controlling weeds in a field of soybean plants comprising the elite event EE-GM5 as described above, comprising treating said field with an effective amount of an HPPD-inhibiting herbicide, in that plants are tolerant to such herbicide.

[0058] Further provided herein is the use of a transgenic soybean plant, seed or progeny thereof, to control weeds in a soybean field, wherein each of said plant, seed or progeny comprises the elite event EE- GM5 in its genome, wherein EE-GM5 which is a T-DNA at a defined locus, as contained in the reference seed deposited with the ATCC under deposit number PTA-123625, wherein said T-DNA comprises a gene encoding chimeric Cry14Ab-1 and a gene encoding chimeric HPPD-4 and wherein said elite event is characterized by the 5' splice sequence of SEQ ID NO: 1 or 3 and the 3' splice sequence of SEQ ID NO: 2 or 4. In one embodiment, in such use, the transgenic soybean plant, its seed or progeny, is resistant to nematodes and/or tolerant to an HPPD-inhibiting herbicide. In one embodiment, said T-DNA comprises a gene encoding chimeric Cry14Ab-1 and a gene encoding chimeric HPPD-4 and said elite event is characterized by the 5' splice sequence of SEQ ID NO: 5 or 24 and the sequence 3' junction of SEQ ID NO: 6 or 25.

[0059] Also provided here is the use of a soybean plant or seed comprising the EE-GM5 elite event in its genome to cultivate a nematode-resistant and/or herbicide-tolerant plant, wherein said EE-GM5 elite event GM5 is a T-DNA inserted at a defined locus, as contained in the reference seed deposited with the ATCC under deposit number PTA-123625, wherein said inserted T-DNA comprises a gene encoding chimeric Cry14Ab-1 and a gene encoding Chimeric HPPD-4 and wherein said elite event is characterized by the 5' splice sequence of SEQ ID NO: 1 or 3 and the 3' splice 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 an HPPD-inhibiting herbicide. In one embodiment, said T-DNA comprises a gene encoding chimeric Cry14Ab-1 and a gene encoding chimeric HPPD-4 and said elite event is characterized by the 5' splice sequence of SEQ ID NO: 5 or 24 and the sequence junction 3'of SEQ ID NO: 6 or 25.

[0060] Also provided here 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.

[0061] Provided here is a method for producing a soybean plant or seed comprising the elite event EE-GM5, comprising crossing a plant comprising EE-GM5 with another soybean plant and planting the seed comprising EE-GM5 obtained from from the aforementioned intersection. In one embodiment, such a method includes the step of applying an HPPD-inhibiting herbicide to said seed or plant.

[0062] According to this invention there is also provided the use of a soybean seed comprising the elite event EE-GM5 as described above, and an HPPD inhibitor herbicide, to control weeds in a soybean field, and the use of a soybean seed comprising the elite event EE-GM5 in a method of growing soybeans tolerant to HPPD-inhibiting herbicides, wherein said seed is as described above.

[0063] Further provided is the use of the elite event EE-GM5 as described above to impart nematode resistance and/or tolerance to an HPPD-inhibiting herbicide to a soybean plant or seed, and the use of a soybean plant or seed soybean comprising the elite event EE-GM5, in combination with an HPPD-inhibiting herbicide, for soybean cultivation.

[0064] Also provided herein is an EE-GM5 specific primer pair, as well as kits or methods using such a primer pair, wherein at least one primer of said pair is labeled (such as with a detectable or traceable moiety) or wherein the 5' end of at least one of said primers comprises one or more mismatches or a nucleotide sequence unrelated to the 5' or 3' flanking sequences of EE-GM5 or unrelated to the T-DNA sequence of EE-GM5; or wherein at least one of said primers comprises a nucleotide sequence at its 3' end spanning the joining region between flanking T-DNA sequences and T-DNA sequences, said nucleotide joining region being 166- 167 in SEQ ID NO: 5, nucleotides 1113-1114 in SEQ ID NO: 24 or to nucleotides 358359 in SEQ ID NO: 6 or 25, provided that the 17 consecutive nucleotides at the 3' end are not derived exclusively from the T- DNA or T-DNA flanking sequences in SEQ ID Nos. 5 or 24 or 6 or 25; or wherein at least one of said primers comprises a sequence that is between 80 and 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 said primer sequence comprises at least one mismatch with said 5' or 3' flanking region or said T-DNA, provided that at least one mismatch further permits specific identification of the EE-GM5 elite event with said primers. under optimized detection conditions (e.g., optimized PCR conditions); or wherein the nucleotide sequence of at least one of said primers comprises the nucleotide sequence of a nucleic acid fused to a nucleic acid of another origin or its complement.

[0065] Other modalities according to the invention are summarized in the following paragraphs: 1. Method for identifying the EE-GM5 elite event in biological samples, which method comprises specific detection of EE-GM5 with a specific pair of primers or probe which specifically recognize(s) the (at least a part of) the region containing part of the 5' or 3' T-DNA flanking region and the (at least a part of) the T-DNA contiguous therewith in EE-GM5; 2. The method of paragraph 1, said method comprising amplification of a DNA fragment of between 50 and 1000 bp from a nucleic acid present in said biological samples using a polymerase chain reaction with at least two primers, recognizing one of said primers the 5' T-DNA flanking region in EE-GM5, said 5' T-DNA flanking region comprising the nucleotide sequence of SEQ ID NO: 5 from nucleotide 1 to nucleotide 166 or of SEQ ID NO: 24 from nucleotide 1 to nucleotide 1113 or recognizing the 3' T-DNA flanking region in 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 complement nucleotide sequence of SEQ ID NO: 25 from nucleotide 359 to nucleotide 1449, the other primer of said primers recognizing a sequence within the inserted T-DNA comprising the nucleotide sequence of SEQ ID NO: 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 sequence of nucleotides of SEQ ID NO: 23 from nucleotide 1114 to nucleotide 8572 or its complement; 3. The method of paragraph 2, wherein said 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 starting from nucleotide 1 to nucleotide 166 or from SEQ ID NO: 24 from nucleotide 1 to nucleotide 1113 or said primer recognizing the 3' T-DNA flanking region of EE-GM5 comprises a selected nucleotide sequence of 17 to 200 consecutive nucleotides 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 and said primer recognizing a sequence within the Inserted T-DNA comprises 17 to 200 consecutive nucleotides selected from the nucleotide sequence of SEQ ID NO: 5 from nucleotide 167 to nucleotide 353 or its complement or from the nucleotide sequence of SEQ ID NO: 6 from nucleotide 1 up 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 starting from nucleotide 1114 up to nucleotide 8572 or its complement; 4. The method of paragraph 2, wherein said primer recognizing the 5' T-DNA flanking region comprises at its extreme 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 from SEQ ID NO: 24 from nucleotide 1 to nucleotide 1113 or said primer recognizing the 3' T-DNA flanking region of EE-GM5 comprises at its 3' end extreme a nucleotide sequence of at least 17 consecutive nucleotides selected from the nucleotide sequence of SEQ ID NO: 6 from nucleotide 359 to nucleotide 691 or the complement nucleotide sequence of SEQ ID NO: 25 from nucleotide 359 to to nucleotide 1449 and said primer recognizing a sequence within the inserted T-DNA comprises at its extreme 3' end at least 17 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 sequence of nucleotides of SEQ ID NO: 23 from nucleotide 1114 to nucleotide 8572 or its complement; 5. The method of paragraph 4, wherein said primers comprise the sequence of SEQ ID NO: 12 and SEQ ID NO: 13, respectively, or the sequence of SEQ ID NO: 18 and SEQ ID NO: 19, respectively; 6. The method of paragraph 5, which method comprises amplification of a specific fragment of EE-GM5 of 85 or 84 bp using PCR; 7. The method of any one of paragraphs 2 to 6, further comprising the step of hybridizing a probe specific to the amplified DNA fragment with said at least two primers; 8. The method of paragraph 7, wherein said probe recognizes part of said 5' T-DNA flanking region and part of the inserted T-DNA contiguous therewith or wherein said probe recognizes part of said T-DNA flanking region. 3' DNA and part of the inserted T-DNA contiguous thereto or recognizes part of said 5' T-DNA flanking region and part of the inserted T-DNA contiguous thereto, such as wherein said probe comprises the nucleotide sequence of SEQ ID NO: 1 or 3 or SEQ ID No 2 or 4; 9. The method of paragraph 8, wherein said primers comprise the sequence of SEQ ID NO: 12 and SEQ ID NO: 13, respectively, and wherein said probe comprises the sequence of SEQ ID NO: 14 or wherein the said primers comprise the sequence of SEQ ID NO: 18 and SEQ ID NO: 19, respectively, and wherein said probe comprises the sequence of SEQ ID NO: 20; 10. A kit comprising a primer recognizing the 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 of SEQ ID NO: 24 from nucleotide 1 to nucleotide 1113 or a primer recognizing the 3' T-DNA flanking region of EE-GM5, said 3' T-DNA flanking region comprising the sequence of complement nucleotides of SEQ ID NO: 6 from nucleotide 359 to nucleotide 691 or the complement nucleotide sequence of SEQ ID NO: 25 from nucleotide 359 to nucleotide 1449 and a primer recognizing a sequence within the T- Inserted DNA, said inserted T-DNA comprising the complement of the nucleotide sequence of SEQ ID NO: 5 from nucleotide 167 to nucleotide 353 or of 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 her complement; 11. The kit of paragraph 10, wherein said 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 starting from nucleotide 1 to nucleotide 166 or from SEQ ID NO: 24 from nucleotide 1 to nucleotide 1113 or said primer recognizing the 3' T-DNA flanking region of EE-GM5 comprises a selected nucleotide sequence of 17 to 200 consecutive nucleotides 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 and said primer recognizing a sequence within the Inserted T-DNA comprises 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 from 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; 12. The kit of paragraph 10, wherein said primer recognizing the 5' T-DNA flanking region comprises at its extreme 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 from SEQ ID NO: 24 from nucleotide 1 to nucleotide 1113 or said primer recognizing the 3' T-DNA flanking region of EE-GM5 comprises at its 3' end a nucleotide sequence of at least 17 consecutive nucleotides selected from the nucleotide sequence of SEQ ID NO: 6 from nucleotide 359 to nucleotide 691 or the complement nucleotide sequence of SEQ ID NO: 25 from nucleotide 359 to nucleotide 1449 and said primer recognizing a sequence within the inserted T-DNA comprises at its extreme 3' end at least 17 consecutive nucleotides selected from the complement of the nucleotide sequence of SEQ ID NO: 5 from nucleotide 167 to nucleotide 353 or from the nucleotide sequence of SEQ ID NO: 6 from nucleotide 1 to nucleotide 358 or from 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; 13. The kit of paragraph 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; 14. The kit of paragraph 10, further comprising a probe recognizing a sequence between the primer recognizing the 5' T-DNA flanking region and the primer recognizing the sequence within the inserted T-DNA or recognizing a sequence between the primer recognizing the region 3' flanking DNA and the primer recognizing the sequence within the inserted T-DNA; 15. The kit of paragraph 14, wherein said probe recognizes part of said 5' T-DNA flanking region and part of the inserted T-DNA contiguous therewith or wherein said probe recognizes part of said T-DNA flanking region. DNA 3'' and part of the inserted T-DNA contiguous with it; 16. The kit of paragraph 15, wherein said primers comprise the sequence of SEQ ID NO: 12 and SEQ ID NO: 13 and wherein said probe comprises the sequence of SEQ ID NO: 14 or wherein said primers comprise the sequence of SEQ ID NO: 18 and SEQ ID NO: 19 and wherein said probe comprises the sequence of SEQ ID NO: 20; 17. Primer pair suitable for use in a specific detection of EE-GM5, comprising a first primer comprising a sequence that, under optimized detection conditions, specifically recognizes a sequence within the 5' or 3' T-DNA flanking region of the T-DNA inserted into EE-GM5 and a second primer comprising a sequence that, under optimized sequence conditions, specifically recognizes a sequence within the T-DNA inserted into EE-GM5 contiguous with said 5' or 3' flanking region, comprising said flanking region of T-DNA the nucleotide sequence of SEQ ID NO: 5 from nucleotide 1 to nucleotide 166 or of SEQ ID NO: 24 from nucleotide 1 to nucleotide 1113, said flanking region of T-DNA 3' 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, comprising said T-DNA inserted 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; 18. A primer comprising at its extreme 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; 19. A pair of primers comprising a first primer comprising at its extreme 3' end the sequence of SEQ ID NO: 12 and a second primer comprising at its extreme 3' end the sequence of SEQ ID NO: 13 or comprising a first primer comprising at its extreme 3' end the sequence of SEQ ID NO: 18 and a second primer comprising at its extreme 3' end the sequence of SEQ ID NO: 19; 20. The method of paragraph 1, which method comprises hybridization of a nucleic acid from biological samples with an EE-GM5 specific probe; 21. The method of paragraph 20, wherein the sequence of said specific probe has at least 80% sequence identity with a sequence comprising part of the 5' T-DNA flanking sequence or the 3' T-DNA flanking sequence of EE-GM5 and the inserted T-DNA sequence contiguous with it; 22. The method of paragraph 21, wherein the sequence of said specific probe comprises a sequence with at least 80% sequence identity to the sequence of any of SEQ ID NO: 1, 3 or 5 or the sequence of any of SEQ ID NO: 2, 4 or 6 or the complement of said sequences; 23. The method of paragraph 22, wherein said probe comprises the sequence of any one of SEQ ID NO: 1 or 3 or the sequence of any one of SEQ ID NO: 2 or 4; 24. A kit for identifying the EE-GM5 elite event in biological samples, said kit comprising a specific probe, capable of specifically hybridizing with a specific region of EE-GM5; 25. The kit of paragraph 24, wherein the sequence of said specific probe has at least 80% sequence identity with a sequence comprising part of the 5' T-DNA flanking sequence or part of the 3' T-DNA flanking sequence of EE-GM5 and part of the inserted T-DNA sequence contiguous with it; 26. The kit of paragraph 25, wherein the sequence of said 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 the complement of said sequences; 27. A specific probe for the identification of the EE-GM5 elite event in biological samples; 28. The probe of paragraph 27, which comprises a nucleotide sequence having at least 80% sequence identity with a sequence comprising part of the 5' T-DNA flanking sequence or part of the 3' T-DNA flanking sequence of EE -GM5 and part of the T-DNA sequence inserted contiguous with it or its complement; 29. The probe of paragraph 28, which has at least 80% sequence identity to the sequence of any one of SEQ ID NO: 1, 3 or 5 or the sequence of any one of SEQ ID NO: 2, 4 or 6 or the complement of said sequences; 30. A specific probe comprising a nucleotide sequence being essentially 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; 31. A specific probe comprising the sequence of SEQ ID NO: 1 or 3 or the sequence of SEQ ID NO: 2 or 4; 32. Method for confirming seed purity, which method comprises specific detection of EE-GM5 with a pair of specific primers or probe that specifically recognize(s) the 5' or 3' T-DNA flanking region and the T- DNA contiguous with it in EE-GM5, in seed samples; 33. The method of paragraph 32, comprising amplification of a DNA fragment of between 50 and 1000 bp from a nucleic acid present in said biological samples using a polymerase chain reaction with at least two primers, recognizing one of said primers the 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 of SEQ ID NO: 24 from nucleotide 1 to nucleotide 1113 or the 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 from nucleotide 359 to nucleotide 691 or the nucleotide sequence of the complement of SEQ ID NO: 25 from nucleotide 359 to nucleotide 1449, the other primer of said primers recognizing a sequence within the inserted T-DNA comprising the complement of the sequence of nucleotides of SEQ ID NO: 5 from nucleotide 167 to nucleotide 353 or of the nucleotide sequence of SEQ ID NO: 6 from nucleotide 1 to nucleotide 358 or within the nucleotide sequence of SEQ ID NO: 11 to 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 hybridization of a probe specific to the amplified DNA fragment with the said at least two initiators; 34. The method of paragraph 33, comprising amplification of an 85 bp DNA fragment and wherein said primers comprise the sequence of SEQ ID NO: 12 and SEQ ID NO: 13, respectively, and wherein said probe comprises the sequence of SEQ ID NO: 14 or amplification of an 84 bp DNA fragment and wherein said primers comprise the sequence of SEQ ID NO: 18 and SEQ ID NO: 19, respectively, and wherein said probe comprises the sequence of SEQ ID NO: 20; 35. Method for screening seeds for the presence of EE-GM5, which method comprises specific detection of EE-GM5 with a specific primer pair or probe that specifically recognizes the 5' or 3' T-DNA flanking region ' and the T-DNA contiguous with it in EE-GM5, in seed lot samples; 36. The method of paragraph 35, comprising amplification of a DNA fragment of between 50 and 1000 bp from a nucleic acid present in said biological samples using a polymerase chain reaction with at least two primers, recognizing one of said primers the 5' T-DNA flanking region of the T-DNA inserted into EE-GM5, said 5' T-DNA flanking region comprising the nucleotide sequence of SEQ ID NO: 5 from nucleotide 1 to nucleotide 166 or of SEQ ID NO: 24 from nucleotide 1 to nucleotide 1113 or the 3' T-DNA flanking region of the T-DNA inserted into 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, the other primer of said primers recognizing a sequence within of the inserted T-DNA comprising the complement of the nucleotide sequence of SEQ ID NO: 5 from nucleotide 167 to nucleotide 353 or of the nucleotide sequence of SEQ ID NO: 6 from nucleotide 1 to nucleotide 358 or comprising the nucleotide sequence of SEQ ID NO: 11 from nucleotide 1 to nucleotide 7459 or its complement or comprising the nucleotide sequence of SEQ ID NO: 23 from nucleotide 1114 to nucleotide 8572 or its complement and hybridization of a probe specific to the DNA fragment amplified with said 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 their complement; 37. The method of paragraph 36, comprising amplification of an 85 bp DNA fragment and wherein said primers comprise the sequence of SEQ ID NO: 12 and SEQ ID NO: 13, respectively, and wherein said probe comprises the sequence of SEQ ID NO: 14; 38. A method for determining the zygosity status of a plant, plant material or seed comprising the elite event EE-GM5, said method comprising amplification of DNA fragments of between 50 and 1000 bp from a nucleic acid present in said biological samples using a polymerase chain reaction with at least three primers, two of said primers recognizing specifically pre-insert 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, the third of said primers recognizing a sequence within the inserted T-DNA, such as the nucleotide sequence of SEQ ID NO: 18, such as using said method said primers in which the DNA fragments of 84 and 72 bp are amplified; 39. A method of detecting the presence of the EE-GM5 elite event in biological samples through hybridization with a substantially complementary labeled nucleic acid probe in which the probe:target nucleic acid ratio is amplified by recycling the target nucleic acid sequence , said method comprising: a) hybridizing said target nucleic acid sequence with a 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 said first nucleic acid oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 6 from nucleotide position 341 to nucleotide position 358 or its complement; b) hybridizing said target nucleic acid sequence with 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 sequence of nucleotides 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 in which any of said first or second oligonucleotides is labeled for said nucleic acid probe being labeled; c) cleavage of only the labeled probe within the probe duplex: target nucleic acid sequence with an enzyme that causes selective cleavage of the probe resulting in dissociation of the duplex, leaving the target sequence intact; d) recycling the target nucleic acid sequence by repeating steps (a) to (c); and e) detecting the cleaved labeled probe, thereby determining the presence of said target nucleic acid sequence, and detecting the EE-GM5 elite event in said biological sample; 40. A transgenic soybean plant, or its cells, parts, seed or progeny, each comprising the elite event EE-GM5 in its genome, the reference seed comprising said event having been deposited with the ATCC under deposit number PTA-123625; 41. The transgenic soybean plant, seed, cells, parts or progeny of paragraph 40, the genomic DNA of which, when analyzed using PCR for EE-GM5 with two primers comprising the nucleotide sequence of SEQ ID 12 and SEQ ID 13 respectively, originates a DNA fragment of 85 bp; 42. Seed comprising the elite event EE-GM5, which is a T-DNA inserted at a specific position in the soybean genome, as contained in the seed deposited with the ATCC under deposit number PTA-123625 or in derivatives thereof ; 43. A soybean plant, plant part, cell or tissue or seed comprising elite event EE-GM5 obtainable from the seed of paragraph 42; 44. A soybean plant, or its seed, cells or tissues, each comprising the elite event EE-GM5 in its genome, obtainable by propagation of and/or crossing with a soybean plant grown from the seed deposited at ATCC under filing number PTA-123625; 45. A soybean seed comprising the elite event EE-GM5, having been the reference seed comprising said event deposited with the ATCC under deposit number PTA-123625; 46. A transgenic soybean plant, cell or tissue, comprising the elite event EE-GM5 obtainable from the seed of paragraph 45; 47. The soybean plant cell according to any of paragraphs 40, 41, 43, 44 and 46, which is a non-self-propagating plant cell; 48. A method for producing a soybean plant or seed comprising 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 the seed obtained from said crossing; 49. Soybean genomic DNA comprising the elite event EE-GM5; 50. A nucleic acid molecule comprising a nucleotide sequence essentially similar to the sequence of any one of SEQ ID NO: 1, 3 or 5 or to the sequence of any one of SEQ ID NO: 2, 4 or 6, or to the complement of said sequences, such as a nucleic acid molecule comprising a nucleotide sequence having at least 99% or at least 99.5% sequence identity with the nucleotide sequence of SEQ ID NO: 5 or 24 or 6 or 25, or its complement, such as said nucleic acid that confers tolerance to an HPPD-inhibiting herbicide and/or resistance to SCN in soybean; 51. The nucleic acid molecule of paragraph 50 comprising the nucleotide sequence of either SEQ ID NO: 1 or 3 or SEQ ID NO: 2 or 4, or the complement of said sequences, such as such 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 its complement, or such a nucleic acid molecule 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% sequence identity thereto. 52. A soybean plant, its cell, plant part, seed or progeny comprising a nucleic acid molecule of any of these paragraphs, such as a soybean plant, its cell, plant part, seed or progeny comprising a nucleic acid molecule nucleic acid comprising the nucleotide sequence of SEQ ID NO: 1, 3 or 5 or the nucleotide sequence of SEQ ID NO: 2, 4 or 6 or a soybean plant, its cell, plant part, seed or progeny, comprising in its genome the nucleotide sequence of SEQ ID NO: 3 and the nucleotide sequence of SEQ ID NO: 4 or a soybean plant, its cell, plant part, seed or progeny, comprising in its genome the nucleotide sequence of SEQ ID NO: 5 and the nucleotide sequence of SEQ ID NO: 6 or a soybean plant, its cell, plant part, seed or progeny, comprising in its genome the nucleotide sequence of SEQ ID NO: 24 and the sequence of nucleotides of SEQ ID NO: 25, such as such a soybean plant further comprising a chimeric gene encoding Cry14Ab-1 and a chimeric gene encoding HPPD-4, particularly such chimeric genes comprising the nucleotide sequence of SEQ ID NO: 7 and 9 , respectively; 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 sequence of SEQ ID NO: 5 and SEQ ID NO: 6, or their complement, or such as a nucleic acid molecule, which comprises the nucleotide sequence of SEQ ID NO: 24 and SEQ ID NO: 25 or their complement; 54. A transgenic soybean plant, plant cell, tissue or seed, comprising in its genome the EE-GM5 event characterized by a nucleic acid molecule comprising a nucleotide sequence essentially similar to the sequence of any of SEQ ID NO: 1 , 3, 5 or 24 or to the sequence of any one of SEQ ID NO: 2, 4, 6 or 25, or to the complement of said sequences, wherein said soybean plant also comprises a chimeric gene encoding Cry14Ab-1 and a chimeric encoding HPPD-4; 55. A soybean plant, cell, tissue or seed, comprising EE-GM5 and comprising in the genome of its cells a nucleic acid sequence with at least 80%, 90%, 95% or 100% sequence identity with a sequence of any one of SEQ ID NO: 1, 3 or 5 or a sequence of any one of SEQ ID NO: 2, 4 or 6, or the complement of said sequences, such as a soybean plant also comprising a chimeric gene encoding Cry14Ab -1 and a chimeric encoding HPPD-4 or such soybean plant, cell, tissue or seed, comprising in the genome of its cells a nucleic acid sequence with at least 80%, 90%, 95% or 100% sequence identity with the sequence SEQ ID NO: 24 or SEQ ID NO: 25; 56. A plant, plant cell, tissue or soybean seed, comprising in its genome a nucleic acid molecule comprising a nucleotide sequence with at least 99% sequence identity to the nucleotide sequence of SEQ ID NO: 5 or 24 or SEQ ID NO: 6 or 25, or its complement, or such plant, plant cell, tissue or soybean seed, comprising in its genome a nucleic acid molecule comprising a nucleotide sequence with at least 99% identity of sequences with the nucleotide sequence of SEQ ID NO: 5 or 24 and SEQ ID NO: 6 or 25 or their complement; 57. A plant, plant cell, tissue or soybean seed, comprising in its genome a nucleic acid molecule hybridizing under standard stringency conditions with the nucleotide sequence of SEQ ID NO: 5 or 6 or its complement; 58. A nucleic acid molecule comprising a nucleotide sequence with at least 99% sequence identity to the nucleotide sequence of SEQ ID NO: 5 or 24 or SEQ ID NO: 6 or 25, or its complement, such as a nucleic acid molecule comprising a nucleotide sequence with at least 99% sequence identity to the nucleotide sequence of SEQ ID NO: 5 or 24 and SEQ ID NO: 6 or 25 or their complement; 59. Nucleic acid molecule comprising a nucleotide sequence hybridizing under standard stringency conditions to the nucleotide sequence of SEQ ID NO: 5 or 6 or its complement; 60. The nucleic acid molecule of any one of paragraphs 50, 51, 58 and 59, which also comprises the nucleotide sequence of SEQ ID NO: 7 or 9; 61. A chimeric DNA comprising a 5' flanking region of T-DNA, an inserted T-DNA and a 3' flanking region of T-DNA, wherein the sequence of said 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 wherein the sequence of said inserted T-DNA comprises the sequence of SEQ ID NO: 7 and 9 and wherein said 5' flanking region of T-DNA is located immediately upstream of and contiguous with said inserted 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 from 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 wherein said 3' flanking region of T-DNA is located immediately downstream of and contiguous with said 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; 62. A nucleic acid molecule comprising a nucleotide sequence with 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 nematicidal Cry14Ab protein having at least 95, 96, 97, 98 or at least 99 % identity of sequences with SEQ ID NO: 7 or with the sequence of SEQ ID NO: 11 from nucleotide position 131 to nucleotide position 5276 or its complement; 63. A nucleic acid molecule comprising a nucleotide sequence with 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 molecule comprising the nucleotide sequence of SEQ ID NO: 11 from nucleotide 5382 to 7459 or its complement or a sequence having at least 95, 96, 97, 98 or at least 99% sequence identity with SEQ ID NO: 9 or with the sequence of SEQ ID NO: 11 from nucleotide position 5382 to nucleotide position 7459 or its complement, wherein said sequence encodes an HPPD protein providing tolerance to HPPD-inhibiting herbicides when expressed in a plant; 64. A method of producing a soybean product, comprising obtaining soybean seed comprising the EE-GM5 elite event as described above and producing the soybean product therefrom; 65. The method of paragraph 64, wherein the soy product is or comprises soybean bran, crushed seeds, flour or flakes; 66. The method of paragraph 4 or 65, wherein such soybean product comprises an EE-GM5 elite event specific nucleic acid, such product comprising a nucleic acid that produces an EE-GM5 elite event specific or diagnostic amplicon. GM5, such as the sequence of SEQ ID NO: 1 or 3 or the sequence of SEQ ID NO: 2 or 4; 67. A soybean product, comprising elite event EE-GM5 as described above, such as a soybean product produced from the soybean plant, cell, part, seed or progeny of any of these paragraphs; 68. The soy product of paragraph 67, wherein the soy product is or comprises soybean bran, crushed seeds, flour or flakes; 69. The soybean product of paragraph 67 or 68, wherein said soybean product comprises an EE-GM5 elite event specific nucleic acid, such as such product comprising a nucleic acid that produces an amplicon or specific 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 its complement; 70. A method for protecting emerging soybean plants from competition by weeds, comprising treating a field in which seeds containing the elite event EE-GM5 as described in any of these paragraphs have been sown, with an HPPD-inhibiting herbicide, wherein the plants are tolerant to the HPPD-inhibiting herbicide; 71. A method for protecting emerging soybean plants from competition by weeds, comprising treating a field to be planted with soybean plants comprising the elite event EE-GM5 as described above with an HPPD-inhibiting herbicide, prior to soybean plants are planted or seeds are sown, followed by planting or sowing said soybean plants or seeds in said pre-treated field, wherein the plants are tolerant to the HPPD-inhibiting herbicide; 72. A method for controlling weeds in a field of soybean plants comprising the elite event EE-GM5 as described above, comprising treating said field with an effective amount of an HPPD-inhibiting herbicide, wherein the plants are tolerant to HPPD inhibitor herbicide; 73. The method of any one of paragraphs 70 to 72, wherein the HPPD-inhibiting herbicide is isoxaflutole, topramezone or mesotrione; 74. Use of a transgenic soybean plant, its seed or progeny, comprising the EE-GM5 elite event as described above to produce soybean grain or seed; 75. Use of a soybean plant or seed comprising the elite event EE-GM5 as described above in its genome to cultivate a plant resistant to nematodes and/or tolerant to HPPD-inhibiting herbicides; 76. Use of a soybean seed comprising the elite event EE-GM5 to obtain a soybean product, wherein EE-GM5 is as described above, such as wherein such soybean product is or comprises ground soybeans, soy flour, soy bran or soy flakes; 77. Use of a soybean plant or seed comprising the elite event EE-GM5 as defined above, in combination with an HPPD-inhibiting herbicide, for cultivation of a soybean field or for cultivation of a soybean crop; 78. A nucleic acid molecule obtainable from the seed deposited with the ATCC under accession number PTA-123625, wherein said nucleic acid molecule comprises the nucleotide sequence of any one of SEQ ID NO: 1, 3 or 5 and the nucleotide sequence of any one of SEQ ID NO: 2, 4 or 6; 79. A soybean plant, cell, plant part or seed, each comprising in its genome the elite event EE-GM5, wherein said elite event is the genetic locus comprising an inserted T-DNA containing a gene encoding the chimeric HPPD-4 protein and a gene encoding the chimeric Cry14Ab-1 protein and 5' and 3' flanking sequences immediately surrounding said inserted T-DNA, as found in the reference seed deposited with the ATCC under deposit number PTA-123625 ; 80. A progeny plant, cell, plant part or seed of the plant, cell, plant part or seed of paragraph 79, wherein said 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; 81. The soybean plant, cell, part, seed or progeny of paragraph 79, the genomic DNA of which, when analyzed using PCR with two primers comprising the nucleotide sequence of SEQ ID NO: 18 and SEQ ID NO: 19 respectively, originates an 84 bp DNA fragment; 82. The plant of any of the above paragraphs that is tolerant to isoxaflutole and/or topramezone and/or mesotrione, such as such a plant tolerant to isoxaflutole, topramezone and mesotrione; 83. A method for producing a soybean plant resistant to SCN and tolerant to HPPD-inhibiting herbicides, comprising introducing resistance to SCN and tolerance to HPPD-inhibiting herbicides into the genome of a soybean plant by crossing a first soybean plant not having a gene encoding Cry14Ab-1 and not having a gene encoding HPPD-4 with the soybean plant of any of the above paragraphs and selecting a progeny plant resistant to SCN and tolerant to HPPD-inhibiting herbicides; 84. Use of a soybean plant or seed comprising the elite event EE-GM5 as defined above to obtain a soybean crop, such as a soybean crop producing better when infested by nematodes or Sudden Death Syndrome; 85. A method of producing a soybean crop with improved resistance to nematodes or Sudden Death Syndrome, comprising the steps of (a) planting a field using seed as described in any of the above paragraphs; and (b) collecting soybean seed produced from plants grown from said seed and, optionally, (c) applying to the field planted with said seeds before or after emergence of the seeds or to said soybean plants of one or more doses of an HPPD-inhibiting herbicide sufficient to kill weeds but which is tolerated by said soybean seeds or plants, such as wherein said nematodes are SCN species nematodes or Pratylenchus species or root knot nematodes or reniform nematodes; 86. Use of the soybean seed described in the above paragraphs to prepare a food or containing processed feed event, wherein said processed food or feed comprises a detectable amount of a nucleic acid comprising the nucleotide sequence of SEQ ID NO : 1 and/or SEQ ID NO: 2 or its complement; 87. The use of paragraph 86, in which (i) said food or said type of feed comprises soybean meal, soybean flour, soybean flakes or soybean oil; (ii) said nucleic acid comprises the nucleotide sequence of SEQ ID NO: 3 and/or SEQ ID NO: 4 or its complement; or (iii) said nucleic acid further comprises the nucleotide sequence contained in SEQ ID NO: 7 and SEQ ID NO: 9; 88. A soybean plant, seed or cell comprising in its genome the elite event EE-GM5, wherein the elite event EE-GM5 comprises a nucleotide sequence that is at least 90% identical to the sequence presented in SEQ ID NO : 23, wherein said elite event comprises a gene encoding chimeric HPPD-4 and a gene encoding chimeric Cry14Ab-1, wherein said plant, seed or cell is tolerant to an HPPD-inhibiting herbicide and has resistance to SCN; 89. The blueprint of paragraph 88, wherein the elite event EE-GM5 comprises a nucleotide sequence that is at least 95% identical to the sequence set forth in SEQ ID NO: 23; 90. The blueprint of paragraph 88, wherein the 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; 91. A nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 23 or a nucleotide sequence with at least 99% sequence identity to SEQ ID NO: 23, which confers tolerance to an HPPD-inhibiting herbicide and/or or resistance to nematodes, such as wherein said nematode is a SCN species nematode or Pratylenchus species or root knot nematode or reniform nematode; 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; 93. The nucleic acid molecule of paragraph 92, which encodes an HPPD protein tolerant to an HPPD inhibitor and a protein negatively affecting plant pest nematodes, such as SCN, RKN or Pratylenchus spp. nematodes. ; 94. Nucleic acid molecule of paragraph 93, which encodes the protein of SEQ ID NO: 8 or a protein at least 99% identical to it and the protein of SEQ ID NO: 10 or a protein at least 99% identical to it; 95. A method for controlling weeds and/or nematodes in a field to be 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 of soybean plants or seeds comprising elite transformation event EE-GM5 as described above in said treated field, wherein the reference seed comprising said elite event is deposited with the ATCC under deposit number PTA- 123625; 96. A method of weed control, characterized by the fact that it comprises the steps of: 1) planting soybean plants or seeds tolerant to an HPPD-inhibiting herbicide such as isoxaflutole, topramezone or mesotrione, in a field and 2) application of an HPPD-inhibiting herbicide, such as isoxaflutole, topramezone or mesotrione, to said field before planting said plants or seeds or to said soybean plants or seeds after planting (may be before or after seed germination), in that said plants or seeds comprise the EE-GM5 soybean elite transformation event in their genome, with the reference seed comprising said elite event being deposited with the ATCC under deposit number PTA-123625; 97. A process for controlling weeds, characterized by the fact that it comprises the steps of: 1) treating a field to be planted with soybean plants or a field to be sown with soybean seeds with an HPPD-inhibiting herbicide, such as isoxaflutole, topramezone or mesotrione, before soybean plants are planted or seeds are sown, and 2) planting of soybean plants comprising the EE-GM5 soybean elite transformation event or sowing of soybean seeds comprising the event of elite transformation of EE-GM5 soybeans in said pre-treated field, wherein the reference seed comprising said elite transformation event of EE-GM5 soybeans is deposited with the ATCC under deposit number PTA-123625; 98. A method for reducing yield loss in a field to be planted with soybean plants, particularly a field that contains or is expected to contain nematodes such as SCN, RKN or Pratylenchus or reniform nematodes or a combination thereof, comprising the step of 1) obtaining plants or seed comprising elite transformation event EE-GM5 as described above and 2) planting or sowing soybean plants or seeds, wherein the reference seed comprising said elite event is deposited with the ATCC under the filing number PTA-123625; 99. A method for increasing the yield of soybean plants when planted in a field containing nematodes such as SCN, RKN or Pratylenchus or reniform nematodes or a combination thereof, comprising the step of 1) obtaining plants or seed comprising an elite EE transformation event -GM5 as described above and 2) planting or sowing of soybean plants or seeds, wherein the reference seed comprising said elite event is deposited with the ATCC under deposit number PTA-123625; 100. A method for producing a soybean plant or seed tolerant to an HPPD-inhibiting herbicide, such as isoxaflutole, topramezone or mesotrione, or for producing a soybean plant or seed tolerant to nematodes, such as SCN, RKN or Pratylenchus or reniform nematodes, or for production of a soybean plant or seed tolerant to an HPPD-inhibiting herbicide, such as isoxaflutole, topramezone or mesotrione, and tolerant to nematodes, such as SCN, RKN or Pratylenchus or reniform nematodes, characterized by the step of introducing into the genome of a soybean plant or seed the soybean elite transformation event EE-GM5 as described above and, optionally, treating said plant or seed with an HPPD-inhibiting herbicide, such as isoxaflutole, topramezone or mesotrione or, optionally, treating the field in which said plant or seed is to be planted with an HPPD-inhibiting herbicide, such as isoxaflutole, topramezone or mesotrione, and planting said plant or seed in said pre-treated field; 101. A nucleic acid molecule that specifically characterizes the elite transformation event of soybean EE-GM5, characterized in that it comprises the nucleotide sequence of any one of SEQ ID NO: 1, 3 or 5, which contains a part of soybean plant genomic DNA and a portion of foreign DNA inserted from EE-GM5 downstream thereof and contiguous therewith and/or characterized by the fact that it comprises the nucleotide sequence of any of SEQ ID NO: 2, 4 or 6, which contains a portion of foreign DNA inserted from EE-GM5 and a portion of soybean plant genomic DNA downstream thereof and contiguous therewith; 102. A plant or seed comprising EE-GM5 as described above and further comprising tolerance or resistance to SCN, RKN or Pratylenchus or reniform nematodes, or a combination thereof, as provided by soybean resistance loci/genes; 103. The plant or seed of paragraph 102, wherein said plant or seed comprises EE-GM5 and any one or a combination of the SCN resistance alleles/loci of 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; 104. A plant or seed comprising EE-GM5 as described above, also comprising tolerance to other herbicides, as provided by herbicide tolerance genes (native or mutated soybean genes or transgenes), such as tolerance to glyphosate-based herbicides, glufosinate, sulfonylurea, imidazolinone, HPPD inhibitors, dicamba, 2,4-D or PPO inhibitors or any combination thereof; 105. The plant or seed of paragraph 103 wherein said plant or seed comprises EE-GM5 as described above and one or more of the following soybean transformation events conferring herbicide tolerance: MST-FG072-3, SYN-000H2-5 , DAS-68416-4, DAS-44406-6, MON-87708-9, MON89788, MON-04032-6, ACS-GM005-3, BPS-CV127-9, ACS-GM006-4, MON-87705-6 or event DP-305423-1; and 106. A method for reducing the severity of the effects of Sudden Death Syndrome or Chlorine Iron Deficiency on soybean plants in the presence of SCN infestation or for increasing the yield of soybean plants in fields containing SCN infested with Death Syndrome Sudden or in fields containing SCN causing Iron Deficiency Chlorosis in soybeans, which method comprises planting soybean plants or sowing soybean seeds comprising the EE-GM5 elite event, in which the reference seed comprising said event of elite is deposited with the ATCC under deposit number PTA-123625.

BRIEF DESCRIPTION OF THE DRAWINGS

[0066] The following Examples, not intended to limit the invention to the specific embodiments described, may be understood in conjunction with the accompanying Figures, incorporated herein by reference, in which: Figure 1. SCHEMATIC REPRESENTATION OF THE RELATIONSHIP BETWEEN NUCLEOTIDE SEQUENCES AND INITIATORS CITED

[0067] Black bar: inserted T-DNA; dotted bar: DNA flanking T-DNA; checkered arrow (a): chimeric cry14Ab-1.b gene (see Table 1 for chimeric gene composition); dotted arrow (b): chimeric hppdPf- 4Pa gene (see Table 1 for chimeric gene composition); black arrows: oligonucleotide primers; (c) refers to the complement of the indicated nucleotide sequence; black line: oligonucleotide probes (number below is representative SEQ ID NO:). The numbers below the bars representing SEQ ID NO: 5 and 6 are the nucleotide positions of the different elements in said sequences. Note: the diagram is not drawn to scale. Figure 2. END POINT METHOD FOR DEEE-GM5 IDENTITY ANALYSIS

[0068] Figure 2 shows an example of the result 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 for both the EE-GM5 specific reaction and the endogenous reaction are displayed. In this figure, the samples within the lines marked “a” are soybean samples not containing EE-GM5, the samples within the lines marked “b” are soybean samples containing EE-GM5, and the samples within the box formed by the lines marked with “c” are inconclusive samples. Figure 3. ENDPOINT METHOD FOR ANALYSIS OF DEEE-GM5 IDENTITY AND ZYGOSITY

[0069] Figure 3 shows an example of the result of the method described in Example 2.2 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. In this figure, the samples within the lines marked “a” are soybean samples containing EE-GM5 in a homozygous state, the samples within the lines marked “b” are soybean samples containing EE-GM5 in a hemizygous state, samples within the lines marked “c” are soybean samples not containing EE-GM5 and samples within the box formed by the lines marked “d” are inconclusive samples. Figure 4. REAL-TIME PCR METHOD FOR ANALYSIS OF THE LOW-LEVEL PRESENCE OFEE-GM5

[0070] Figure 4 shows an example of the results of the RT-PCR method described in Example 2.4 for low-level presence analysis as performed on calibration samples. “a”, “b”, “c”, “d”, “e” indicate the Ct values for calibration samples “A”, “B”, “C”, “D”, “E”, respectively . Calibration samples “A”, “B”, “C”, “D”, “E” have decreasing amounts of EE-GM5 DNA Figure 5. AVERAGE PHYTO MAX RESULTS FOR HERBICIDES TREATMENTS.

[0071] Figure 5 shows the average maximum plant phytotoxicity data recorded for herbicide treatment in various field trials over 2 years, for soybean plants containing event EE-GM5 compared to untransformed soybean plants /conventional (Thorne). The numbers in ( ) below a treatment give the number of trials included in the bar, the number at the top of each bar gives the average maximum phytotoxicity value for that treatment. The treatments applied were: IFT = isoxaflutole, MST = mesotrione, PE = pre-emergence, PO = post-emergence (in stage V2-V3, with adjuvants culture oil concentrate and ammonium sulfate added to increase herbicide activity) . Rates shown are in grams of active ingredient/hectare (4x pre-emergence dose, 2x post-emergence dose). Figure 6. EE-GM5 GRAIN YIELD IN THORNE IN SCN INFESTED FIELDS

[0072] EE-GM5 on original transform background (Thorne) was tested in 9 different locations throughout Iowa, Illinois, Indiana, Missouri and Tennessee in 2015 and 2016, in SCN-infested fields (ranging from low to low SCN infestation). high). The point is the estimated yield of the homozygous event for each assay (as percentage difference from the null), the horizontal lines represent the 95% confidence limits of the contrast between the homozygous event and the null segregate (if the line does not match). overlap with the vertical line at the 100 percent null segregated yield, then the event was significantly different from the null segregated one). “Across Locs” is the estimated yield from a combined analysis across all 9 locations. Figure 7. DEEE-GM5 GRAIN YIELD IN ELITE BACKGROUND SUSCEPTIBLE TO SCN IN FIELDS INFESTED BY SCN

[0073] EE-GM5 was introgressed (BC2F3) into an elite MG I (maturity group I) line that is susceptible to SCN and was tested at a location in Minnesota and a location in North Dakota in 2016 (each one with high levels of SCN infestation). The point is the estimated yield of the homozygous event for each assay (as percentage difference from the null), the horizontal line around the point represents the 95% confidence limits of the contrast between the homozygous event and the null segregant (if line did not overlap with the vertical line at 100 percent null segregant yield, then the event was significantly different from the null segregant). “Across Locs” is the estimated yield from a combined analysis across both locations. Figure 8. EE-GM5 GRAIN YIELD ON ELITE SCN-RESISTANT BACKGROUND IN SCN-INFESTED FIELDS

[0074] EE-GM5 was crossed into an elite MG III (maturity group III) line that is resistant to SCN (due to the rhg1 locus of PI88788) and was tested in 3 locations in 2016 (trials starting with “16 ” such as 16-IN1) and at 7 locations in 2017 (trials starting with “17” such as 17-IN1), ranging from low to high SCN infestation levels (see arrows, locations with low SCN pressure are at the bottom of the figure (e.g., 17- IL2) and the locations with high SCN pressure at the top (e.g., 17-IN1)). SCN pressure was assigned by consideration of several known factors including known field history, SCN populations in the soil, relative yields of resistant and susceptible control varieties, soil characteristics (pH and % sand), and a visual assessment of infestation. of roots in susceptible inputs. The point is the average difference in yield (in tons per hectare) of the homozygous event in each trial relative to the null segregant, the horizontal line around the point represents the 95% confidence limits of the contrast between the homozygous event and the segregant null (if the line does not overlap with the vertical line at the difference of 0 with the null segregant, then the yield for the event was significantly different from the null segregant). “Average” is the average yield across all locations in each year. Figure 9. GREENHOUSE TEST OF RESISTANCE TO PRATYLENCHUS IN THE USA

[0075] Elite soybean plants with EE-GM5 control Pratylenchus brachyurus in US greenhouse trials. Plants with EE-GM5 (“EE-GM5”) were compared with other elite soybean lines: a Maturity Group (MG)3 line susceptible to SCN (“THORNE”), a line susceptible to SCN MG3, a line susceptible to SCN MG 6.2 and a line susceptible to SCN MG9 (“Susc WT” shows the average for these 3 lines), a line resistant to SCN MG3 (with the rhg1 resistance allele of PI88788, “SCN Res (PI88788)” ) and a SCN-resistant line MG 6.2 with the SCN resistance of rhg1 and Rhg4 from Peking (“SCN Res (Peking)”). Average numbers of Pratylenchus on roots 30 days after infestation (5 plants per entry) are graphed, also showing the variation observed across varieties (as typically seen in greenhouse trials). Results show ~90% control of Pratylenchus along EE-GM5 lines. Soybean lines with resistance to native SCN (from Peking or PI88788) do not control Pratylenchus brachyurus. Figure 10. GREENHOUSE TEST OF RESISTANCE TO PRATYLENCHUS IN BRAZIL

[0076] Soybean plants homozygous for EE-GM5 (“EE-GM5 HH”) significantly reduce Pratylenchus brachyurus in soybean roots. Pratylenchus brachyurus were isolated from local fields in Brazil. EE-GM5 plants (in two different US elite lines (both maturity group 6.2, one susceptible to SCN and one resistant to SCN Peking (“EE-GM5”)) and five Brazilian soybean lines, with limited control from Pratylenchus (“Brazil lines”), a marked line labeled as low Rf (breeding factor) to Pratylenchus (“BRS 7380 (low Rf)”), an elite US line (maturity group 6.2) that is susceptible to SCN (“SCN Susc”) and an elite US line (MG 6.2) with resistance to SCN Peking (“SCN Res (Peking)”) were evaluated for control of Pratylenchus in a greenhouse trial in Brazil. the averages of these inputs, also showing the variation observed across varieties (as typically seen in greenhouse trials). A Brazilian soybean line (BRS 7380) showed ~89% reduction of Pratylenchus. % control of Pratylenchus. Soybean lines carrying native Peking resistance to SCN do not control Pratylenchus brachyurus. Figure 11. IRON DEFICIENCY CHHLORSIS (IDC) SCORES FOR EE-GM5 PLANTS COMPARED TO NULLS

[0077] Figure 11 shows the IDC scores of soybean plants with EE-GM5 in one location (with high SCN infestation). The trial was a split-batch design (4 lots per entry) looking at the effect of the event on 3 different backgrounds (2 susceptible soybean lines and 1 with resistance to PI88788 SCN). Shown are the average IDC scores for plants with event EE-GM5 (“EE-GM5”) and the corresponding null segregant (“Null”, having no EE-GM5) across three genetic backgrounds (1 SCN resistant, 1 susceptible to SCN and the Thorne fund susceptible to SCN). One bar represents 12 total lots. The vertical lines indicate the standard error (“SEM” is the Standard Error of the Mean).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

[0078] In this invention, EE-GM5 was identified as an elite event from a population of transgenic soybean plants in the development of nematode-resistant soybean (Glycine max) comprising a gene encoding tolerance to the inhibitor 4-hydroxy phenylpyruvate dioxygenase (HPPD) combined with a gene conferring resistance to nematodes, each under the control of a plant-expressible promoter. Specific tools for use in identifying the EE-GM5 elite event in biological samples are described here.

[0079] The incorporation of a recombinant DNA molecule into the plant genome typically results from the transformation of a cell or tissue. The particular embedding location is usually due to random integration.

[0080] DNA introduced into the plant genome as a result of transformation of a plant cell or tissue with a recombinant DNA or “transforming DNA” and originating from such transforming DNA is hereinafter referred to as “inserted T-DNA” comprising one or more "transgenes”. The EE-GM5 transgenes are a nematode resistance gene and a tolerance gene for HPPD inhibitor herbicides. “Plant DNA” in the context of the present invention will refer to DNA originating from the plant that is transformed. The plant DNA will usually be found at the same genetic locus in the corresponding wild-type plant. The inserted T-DNA can be characterized by the location and configuration at the site of incorporation of the recombinant DNA molecule into the plant genome. plant where a recombinant DNA has been inserted is also referred to as the “insertion site” or “target site”. The insertion of the recombinant DNA into the region of the plant genome is referred to as the “pre-insertion plant DNA” (or “pre-locus”. insertion”) may be associated with a deletion of plant DNA, referred to as a “target site deletion”. A “flanking region” or “flanking sequence” as used herein refers to a sequence of at least 10 bp, at least 20 bp, at least 50 bp and up to 5000 bp of DNA other than the introduced T-DNA, preferably genome DNA plant that is located immediately upstream of and contiguous with or immediately downstream of and contiguous with the inserted T-DNA. Transformation procedures leading to random integration of the inserted T-DNA will result in transformants with different flanking regions, which are characteristic and unique to each transformant. When recombinant DNA is introduced into a plant through traditional breeding, its insertion site in the plant genome, or its flanking regions, will generally not be changed.

[0081] An “isolated nucleic acid (sequence/molecule)” or “isolated DNA (sequence/molecule)”, as used herein, refers to a nucleic acid or DNA (sequence/molecule) that is no longer in the natural environment at from which it was isolated, e.g., the nucleic acid sequence in another host (bacterial) or in a plant genome, or a nucleic acid or DNA (sequence/molecule) fused to DNA or nucleic acid (sequence/molecule) ) of another origin, such as when contained in a chimeric gene under the control of a plant-expressible (heterologous) promoter. Any nucleic acid or DNA of this invention, including any primer, may also be non-naturally occurring, such as a nucleic acid or DNA with a sequence identical to a naturally occurring sequence but having a tag (absent the naturally occurring counterpart) or with a sequence having at least one nucleotide addition or substitution or at least one internal nucleotide deletion compared to a naturally existing nucleotide or to a sequence having less than 100% sequence identity (not identical) to a naturally existing nucleic acid or DNA or a fragment thereof or a nucleic acid or DNA with a sequence consisting of nucleotide sequences of different origins that do not occur together in nature (a chimeric or hybrid DNA) or a man-made synthetic nucleic acid or DNA with a sequence different from the nucleic acid or natural DNA or a fragment thereof.

[0082] An event is defined as an (artificial) genetic locus that, as a result of genetic manipulation, carries an inserted T-DNA or transgene comprising at least one copy of a gene or genes of interest. Typical allelic states of an event are the presence or absence of the inserted T-DNA. An event is characterized phenotypically by the expression of the transgene or transgenes. At the genetic level, an event is part of the genetic makeup of a plant. At the molecular level, an event can be characterized by the restriction map (e.g., as determined by Western blotting), the flanking sequences upstream and/or downstream of the transgene, the location of molecular markers, and/or the configuration molecular structure of the transgene. Usually, transformation of a plant with a transforming DNA comprising at least one gene of interest leads to a population of transformants comprising a multitude of separate events, each of which is unique. An event is characterized by the inserted T-DNA and at least one of the flanking sequences.

[0083] An elite event, as used herein, is an event that is selected from a group of events, obtained by transformation with the same transforming DNA, based on optimal trait efficacy and superior expression, stability of the transgene(s) and their compatibility with ideal agronomic characteristics of the plant comprising the same(s). Thus, the criteria for selection of elite events are one or more of, preferably two or more of, advantageously all of the following: a) trait effectiveness; b) that the presence of the inserted T-DNA does not compromise other desired characteristics of the plant, such as those relating to agronomic performance or commercial value; c) that the event is characterized by a well-defined molecular configuration that is stably inherited and for which appropriate tools for identity control can be developed; d) that the gene(s) of interest show correct, appropriate and stable spatial and temporal phenotypic expression at a commercially acceptable level over a range of environmental conditions in which plants carrying the event are likely to are exposed in normal agronomic use.

[0084] It is preferred that the inserted T-DNA is associated with a position in the plant genome that allows easy introgression into desired commercial genetic backgrounds.

[0085] The status of an event as an elite event is confirmed by introgressing the elite event into different relevant genetic backgrounds and observing compliance with one of, two of, three of, or all of the criteria, e.g., the ), b), c) and d) above.

[0086] An “elite event” thus refers to a genetic locus comprising an inserted T-DNA, which meets the criteria described above. A plant, plant material or progeny such as seeds may comprise one or more elite events in its genome.

[0087] Tools developed to identify an elite event or the plant or plant material comprising an elite event, or products comprising plant material comprising the elite event, are based on specific genomic characteristics of the elite event, such as a specific restriction map of the genomic region comprising the inserted T-DNA, molecular markers or the sequence of the region(s) flanking the inserted T-DNA.

[0088] Once one or both flanking regions of the inserted T-DNA have been sequenced, primers and/or probes can be developed that specifically recognize this sequence(s) in the nucleic acid (DNA). or RNA) from a sample using a molecular biological technique. For example, a PCR method can be developed to identify the elite event in biological samples (such as plant samples, plant material or products comprising plant material). Such a PCR is based on at least two specific “primers”, one recognizing a sequence within the 5' or 3' T-DNA flanking region of the elite event and the other recognizing a sequence within the inserted T-DNA. The primers preferably have a sequence of between 15 and 35 nucleotides that under optimized PCR conditions “specifically recognize” a sequence within the 5' or 3' T-DNA flanking region of the elite event and the inserted T-DNA of the elite event. elite, respectively, such that a specific fragment (“integration fragment” or discrimination amplicon) is amplified from a nucleic acid sample comprising the elite event. This means that only the targeted integration fragment, and no other sequences in the plant genome or inserted T-DNA, are amplified under optimized PCR conditions.

[0089] Suitable PCR primers for the invention may be the following: - oligonucleotides varying in length from 17 nt to about 200 nt, comprising a nucleotide sequence of at least 17 consecutive nucleotides, preferably 20 consecutive nucleotides, selected of the 5' T-DNA flanking sequence (SEQ ID NO: 5 from nucleotide 1 to nucleotide 166 or SEQ ID NO: 24 from nucleotide 1 to nucleotide 1113 or plant genomic sequences upstream thereof and contiguous with it) at its 3' end (primers recognizing 5' T-DNA flanking sequences); or - oligonucleotides ranging in length from 17 nt to about 200 nt, comprising a nucleotide sequence of at least 17 consecutive nucleotides, preferably 20 consecutive nucleotides, selected from the 3' T-DNA flanking sequence (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 plant genomic sequences downstream thereof and contiguous thereto) at its end 3' (primers recognizing 3' T-DNA flanking sequences); or - oligonucleotides ranging in length from 17 nt to about 200 nt, comprising a nucleotide sequence of at least 17 consecutive nucleotides, preferably 20 consecutive nucleotides, selected from the inserted T-DNA sequences (complement of SEQ ID NO: 5 from nucleotide 167 to nucleotide 353 or 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 its complement) at its 3' end (primers recognizing inserted T-DNA).

[0090] It will be understood that primers recognizing the 5' T-DNA flanking sequences can be used in a PCR reaction in conjunction with primers recognizing the inserted T-DNA that are selected from the complement of SEQ ID NO: 5 starting at nucleotide 167 up to nucleotide 353 or T-DNA sequences downstream thereof and contiguous therewith, whereas primers recognizing the 3' flanking T-DNA sequences can be used in a PCR reaction in conjunction with primers recognizing the inserted T-DNA which are selected from the sequence of SEQ ID NO: 6 from nucleotide 1 to nucleotide 358 or T-DNA downstream thereof and contiguous therewith. Primers recognizing inserted T-DNA can also be selected from the sequence of SEQ ID NO: 11 from nucleotide 1 to nucleotide 7459 or from the sequence of SEQ ID NO: 23 from nucleotide 1114 to nucleotide 8572 or its complement .

[0091] The primers can obviously be longer than the mentioned 17 consecutive nucleotides and can, for example, be 20, 21, 30, 35, 50, 75, 100, 150, 200 nt in length or even longer . The primers may consist entirely of nucleotide sequence selected from the aforementioned nucleotide sequences of flanking sequences and inserted T-DNA sequences. However, the nucleotide sequence of the primers at their 5' end (i.e., outside the 17 consecutive nucleotides at the 3' end) is less critical. Thus, the 5' sequence of the primers may comprise or consist of a nucleotide sequence selected from the flanking sequences or inserted T-DNA, as appropriate, but may retain several (e.g., 1, 2, 5, or 10) mismatches. compared to T-DNA or DNA flanking T-DNA. The 5' sequence of the primers may even be entirely a nucleotide sequence unrelated to the flanking sequences or inserted T-DNA, such as, e.g., a nucleotide sequence representing one or more restriction enzyme recognition sites or such as nucleotide sequences capable of binding to other oligonucleotides, such as labeled oligonucleotides, such as FRET cassettes (LGC genomics; see Semagn et al., 2014, Mol Breeding 33: 1-14, and US 7615620). Such unrelated sequences or flanking DNA sequences with mismatches should preferably be no longer than 100, more preferably no longer than 50 or even 25 nucleotides. Primers can also be modified with a tag, such as a fluorescent tag.

[0092] Furthermore, suitable primers may comprise or consist (essentially) of a nucleotide sequence at their 3' end spanning the joining region between the sequences derived from the 5' or 3' T-DNA flanking region and the inserted T-DNA sequences (located at nucleotides 166 and 167 in SEQ ID NO: 5 and nucleotides 358 and 359 in SEQ ID NO: 6 or nucleotides 1113 and 1114 in SEQ ID NO: 24 and nucleotides 358 and 359 in SEQ ID NO : 25) provided that the 17 consecutive 3'-located nucleotides mentioned are not derived exclusively from the inserted T-DNA or flanking T-DNA sequences in SEQ ID NO: 5 or 6 or SEQ ID NO: 24 or 25.

[0093] It will also be immediately clear to the skilled artisan that appropriately selected PCR primer pairs should not also comprise sequences complementary to each other.

[0094] For the purpose of the invention, the “complement of a nucleotide sequence represented in SEQ ID No: according to Chargaff's rules (AOT; GoC) and reading the sequence in the 5' to 3' direction, i.e., in the opposite direction of the represented nucleotide sequence.

[0095] 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 (primer recognizing 3' or 5' T-DNA flanking sequences) or SEQ ID NO: 12 or SEQ ID NO: 18 or SEQ ID NO: 28 or 29 (primer recognizing T-DNA inserted for use with primers recognizing 3' or 5' T-DNA flanking sequences).

[0096] Preferably, the amplified fragment has a length of between 50 and 500 nucleotides, such as a length of between 50 and 150 nucleotides. Specific primers may have a sequence that is between 80 and 100% identical to a sequence within the 5' or 3' T-DNA flanking region of the elite event and the inserted T-DNA of the elite event, respectively, as long as mismatches still allow specific identification of the elite event with these primers under optimized PCR conditions. The range of permissible mismatches, however, can be easily determined experimentally and is known to one skilled in the art.

[0097] Detection of integration fragments can occur in several ways, e.g., through size estimation after gel analysis. Integration fragments can also be directly sequenced. Other sequence-specific methods for detecting amplified DNA fragments are also known in the art. Amplified DNA fragments can also be detected using tagged sequences and tag detection. 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) may comprise a fluorescent label and a quencher, such that the FRET cassette is not already quenched and emits fluorescence when bound to the PCR product. Alternatively, a labeled FRET cassette, i.e., an oligonucleotide labeled with a fluorescent label and a quencher, may be included in the reaction mixture that specifically binds to one of the primers in the reaction mixture, such as a FRET cassette directed to a 5' extension of the primer used in the reaction mixture ( see, e.g., Semagn et al., 2014, Mol Breeding 33: 114, and US 7615620). 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.

[0098] As the sequence of the primers and their relative location in the genome are unique to the elite event, amplification of the integration fragment will only occur in biological samples comprising (the nucleic acid of) the elite event. Preferably when performing a PCR to identify the presence of EE-GM5 in unknown samples, a control is included from a set of primers with which a fragment within a “housekeeping gene” of the event plant species can be amplified. Housekeeping genes are genes that are expressed in most cell types and that concern basic metabolic activities common to all cells. Preferably, the fragment amplified from the housekeeping gene is a fragment that is larger than the amplified integration fragment. Depending on the samples to be analyzed, other controls may be included.

[0099] Standard PCR protocols are described in the art, such as in “PCR Applications Manual” (Roche Molecular Biochemicals, 2nd Edition, 1999, or 3rd Edition, 2006) and other references. The optimal conditions for PCR, including the sequence of specific primers, are specified in a “PCR (or Polymerase Chain Reaction) Identification Protocol” for each elite event. It is however understood that a number of parameters in the PCR Identification Protocols may need to be adjusted to specific laboratory conditions and may be modified slightly to obtain similar results. For example, using a different method for DNA preparation may require adjustment, for example, of the amount of primers, polymerase and annealing conditions used. Similarly, the selection of other primers may dictate other optimal conditions for the PCR Identification Protocol. These adjustments will however be apparent to one skilled in the art and are further detailed in current PCR application manuals such as the one cited above.

[0100] Alternatively, specific primers can be used to amplify an integration fragment that can be used as a “specific probe” for identifying EE-GM5 in biological samples. Contact of nucleic acid from a 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., by nucleic acid or probe labeling), whereby the formation of this hybrid indicates the presence of EE-GM5. Such identification methods based on hybridization with a specific probe (on a solid phase carrier or in solution) have been described in the art. The specific probe is preferably a sequence that, under optimized conditions, specifically hybridizes to a region comprising part of the 5' or 3' T-DNA flanking region of the inserted T-DNA elite event contiguous therewith (hereinafter referred to as the “region specific"). Preferably, the specific probe comprises a sequence of between 50 and 500 bp or of 100 to 350 bp that is at least 80% or between 80 and 85% or between 85 and 90% or between 90 and 95% or between 95% and 100 % identical (or complementary) or is identical (or complementary) to the nucleotide sequence of a specific region of EE-GM5. Preferably, the specific probe will comprise a sequence of about 15 to about 100 contiguous nucleotides identical (or complementary) with a specific region of the elite event.

[0101] Oligonucleotides suitable as PCR primers for detecting the EE-GM5 elite event can also be used to develop a PCR-based protocol to determine the zygosity status of plants containing the elite event. For this purpose, two primers recognizing the wild-type locus before integration are designed in such a way that they are directed towards each other and have the insertion site located in the middle of the primers. These primers may contain primers specifically recognizing the 5' and/or 3' T-DNA flanking sequences of EE-GM5. This set of primers recognizing the wild-type locus prior to integration, in conjunction with a third primer complementary to the transforming DNA sequences (inserted T-DNA), allows simultaneous diagnostic PCR amplification of the EE-GM5 specific locus as well as of the wild-type locus. If the plant is homozygous for the transgenic locus or the corresponding wild-type locus, the diagnostic PCR will give rise to a single typical PCR product, preferably typical in length, for 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 the transgenic and wild-type locus.

[0102] Alternatively, to determine the zygosity state of plants containing the elite event, two primers recognizing the wild-type locus prior to integration are designed in such a way that they are directed toward each other and that one primer recognizes specifically the 5' or 3' T-DNA flanking sequences contained in SEQ ID NO: 5 or 6 or in SEQ ID NO: 24 or 25 and that a primer specifically recognizes the 3' or 5' T-DNA flanking sequences contained within SEQ ID NO: 6 or 5 or SEQ ID NO: 24 or 25 or specifically recognizes the pre-insertion locus. For the current invention, a suitable primer pair recognizing the wild-type locus prior to integration is a primer pair containing a primer comprising or consisting (essentially) of the nucleotide sequence of SEQ ID NO: 21 and a primer comprising the or consisting (essentially) of the nucleotide sequence of SEQ ID NO: 19. This set of primers, together with a third primer complementary with transforming DNA sequences (inserted T-DNA) or complementary with transforming DNA sequences and the flanking sequences of 5' or 3' T-DNA contiguous therewith and in a direction toward the primer that specifically recognizes the 5' or 3' T-DNA flanking sequences (such as a primer comprising or consisting (essentially) of the nucleotide sequence of SEQ ID NO: 18, which is in a direction in the direction of the primer comprising or consisting (essentially) of the nucleotide sequence of SEQ ID NO: 19) allows simultaneous diagnostic PCR amplification of the specific EE-GM5 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 give rise to a single PCR product typical for 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 the transgenic and wild-type locus.

[0103] Detection of PCR products typical for the wild-type and transgenic locus can be based on determining the length of PCR products that may be typical for the wild-type and transgenic locus. Alternatively, detection of typical PCR products for the wild-type and transgenic locus can be accomplished by modifying the pre-insertion locus-specific primer and by modifying the inserted T-DNA-specific primer and detecting incorporation into a PCR product. of modified primers. For example, the pre-insertion locus-specific primer and the inserted T-DNA-specific primer can be labeled using a fluorescent label, where the labels are different for the two primers. Fluorescence can be detected when the primer is incorporated into a PCR product. If the plant is homozygous for the transgenic locus or the corresponding wild-type locus, fluorescence can be detected from the label of the inserted T-DNA-specific primer only or the pre-insertion locus-specific primer only. If the plant is hemizygous for the transgenic locus, fluorescence can be detected from both the inserted T-DNA-specific primer label and the pre-insertion locus-specific primer, reflecting amplification of the transgenic and wild-type locus.

[0104] Alternatively, the pre-insertion locus specific primer and the inserted T-DNA specific primer may have a 5' extension that specifically binds to a labeled FRET cassette, i.e., an oligonucleotide labeled with a fluorescent tag and a quencher, where the 5' extension and corresponding FRET cassettes are different for the two primers (see, e.g., Semagn et al., 2014, Mol Breeding 33: 1-14, and US 7615620). Fluorescence can be detected when the primer is incorporated into a PCR product and subsequently the FRET cassette is incorporated into the PCR product. If the plant is homozygous for the transgenic locus or the corresponding wild-type locus, fluorescence can be detected from the FRET cassette binding specifically to the inserted T-DNA-specific primer only or from the FRET cassette specifically binding to the T-DNA-specific primer. pre-insertion locus only. If the plant is hemizygous for the transgenic locus, fluorescence can be detected from both the FRET cassette specifically binding to the inserted T-DNA-specific primer and the FRET cassette specifically binding to the pre-insertion locus-specific primer, reflecting amplification of the transgenic and wild-type locus.

[0105] If the plant is homozygous for the transgenic locus or the corresponding wild-type locus, the diagnostic PCR will give rise to a single typical PCR product, preferably typical in length, for 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 the transgenic and wild-type locus.

[0106] Alternatively, to determine the zygosity state of plants containing the elite event, the presence of the event can be determined in a PCR reaction in a quantitative manner as described in the Examples. For this purpose, two primers recognizing the EE-GM5 event are designed in such a way that they are directed toward each other and in which one primer specifically recognizes the 5' or 3' T-DNA flanking sequence contained within SEQ ID NO: 5 or 6 or within SEQ ID NO: 24 or 25 and wherein a primer specifically recognizes the T-DNA inserted within SEQ ID NO: 5 or 6 or within SEQ ID NO: 24 or 25 or within SEQ ID NO: 11 or 23. This primer set allows PCR amplification of the specific EE-GM5 locus. The amplified DNA fragment can be quantitatively detected using a labeled probe that is included in the reaction mixture that specifically binds to the amplified fragment. The labeled probe may comprise a fluorescent label and a quencher, such that the label is not already quenched and emits fluorescence when bound to the PCR product. Fluorescence may be measured in real time, i.e., during each cycle of the PCR reaction, using methods known in the art. The PCR cycle at which fluorescence exceeds a certain threshold level is a measure for the amount of specific EE-GM5 locus in the biological sample that is analyzed, and the zygosity status can be calculated based on homozygous and heterozygous reference samples. .

[0107] Alternatively, the zygosity state of plants comprising EE-GM5 can also be determined based on copy number analysis, using Taqman chemistry and Real-Time PCR principles. The alternative method will 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 the EE-GM5 event in a homozygous state will have a relative copy number that is twice as high as hemizygous samples. Azygous samples will not amplify the EE-GM5 sequence in such a method.

[0108] Furthermore, EE-GM5 elite event-specific detection methods that differ from PCR-based amplification methods can also be developed using the elite event-specific sequence information provided here. Such alternative detection methods include linear signal amplification detection methods based on invasive cleavage of particular nucleic acid structures, also known as InvaderTM technology (as described, e.g., U.S. Patent 5,985,557 “Invasive Cleavage of Nucleic Acids ”, 6,001,567 “Detection of Nucleic Acid sequences by Invader Directed Cleavage”, incorporated herein by reference). For this purpose, the target sequence is hybridized to a first labeled 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 with a second nucleic acid oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 5 from nucleotide 149 to 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 a nucleotide.

[0109] The duplex or triplex structure that is produced by this hybridization allows specific probe cleavage with an enzyme (Cleavase®) leaving the target sequence intact. The cleaved labeled probe is subsequently detected, potentially via an intermediate step resulting in additional signal amplification.

[0110] In one embodiment, a method of detecting the presence of the elite event EE-GM5 in biological samples is provided through hybridization with a substantially complementary labeled nucleic acid probe in which the probe:target nucleic acid ratio is amplified through recycling of the target nucleic acid sequence, said method comprising: a) hybridizing said target nucleic acid sequence with a first nucleic acid oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 5 from nucleotide position 167 to position of nucleotides 184 or its complement or said first nucleic acid oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 6 from nucleotide position 341 to nucleotide position 358 or its complement; b) hybridizing said target nucleic acid sequence with 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 in which any of said first or second oligonucleotides is labeled to be said labeled nucleic acid probe; c) cleavage of only the labeled probe within the probe duplex: target nucleic acid sequence with an enzyme that causes selective cleavage of the probe resulting in dissociation of the duplex, leaving the target sequence intact; d) recycling the target nucleic acid sequence by repeating steps (a) to (c); and e) detecting the cleaved labeled probe, thereby determining the presence of said target nucleic acid sequence, and detecting the elite event EE-GM5 in said biological samples.

[0111] Two nucleic acids are “substantially complementary,” as used herein, when they are not the full complement of each other (as defined herein), such as when their sequences are at least 80%, at least 85%, at least 90% , at least 95% or at least 99% complementary to each other.

[0112] A “kit” as used herein refers to a set of reagents for the purpose of carrying out the method of the invention, more particularly, the identification of the EE-GM5 elite event in biological samples or the determination of the zygosity status of plant material containing EE-GM5. More particularly, a preferred embodiment of the kit of the invention comprises at least one or two specific primers, as described above for identifying the elite event, or three specific primers, or two specific primers and one specific probe, as described above for determining the state of zygosity. Optionally, the kit may additionally comprise any other reagent described herein in the PCR Identification Protocol or any of the other protocols as described herein for detection of EE-GM5. Alternatively, according to another embodiment of this invention, the kit may comprise a specific probe, as described above, that specifically hybridizes with nucleic acid from biological samples to identify the presence of EE-GM5 therein. Optionally, the kit may additionally comprise any other reagent (such as but not limited to hybridization buffer, label) for identifying EE-GM5 in biological samples using the specific probe.

[0113] The kit of the invention can be used, and its components can be specifically adjusted, for quality control purposes (e.g., purity of seed lots), detection of the presence or absence of the elite event in seed material. plant or material comprising or derived from plant material, such as but not limited to food or feed or industrial products.

[0114] 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 in the shorter of the two sequences. Alignment of the two nucleotide sequences is performed 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-aided analysis and interpretation of sequence data, including sequence alignment as described above, can, for example, be conveniently performed using the Genetics sequence analysis software package Computer Group (GCG, University of Wisconsin Biotechnology Center). Sequences are indicated as “essentially similar” when such sequences have a sequence identity of 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 minus 99.9%. Of course, when RNA sequences are said to be essentially similar or have a certain degree of sequence identity with DNA sequences, thymidine (T) in the DNA sequence is considered to be the same as 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 permitted for the event-specific primers or probes of this invention, so any DNA sequence indicated herein in any embodiment of this invention for any 3' or 5' T-DNA flanking DNA or to any insert or inserted T-DNA or any primer or probe of this invention also includes sequences essentially similar to the sequences provided herein, such as sequences hybridizing to or 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, for any primer or probe, or for any insertion or T- Inserted DNA of this invention, such as a nucleotide sequence differing 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 of any given sequence.

[0115] The term “primer” as used herein encompasses any nucleic acid that is capable of initiating the synthesis of a nascent nucleic acid in a template-dependent process, such as PCR. Typically, primers are oligonucleotides of 10 to 30 nucleotides, but longer sequences may be employed. The primers may be provided in double-stranded form, although the single-stranded form is preferred. Probes can be used as primers, but they are designed to bind to target DNA or RNA and do not need to be used in an amplification process.

[0116] The term “recognizing” as used herein when referring to specific primers or probes refers to the fact that the specific primers or probes specifically hybridize to a nucleic acid sequence in the elite event under the conditions set forth in the method (such as the conditions of the PCR Identification Protocol), whereby specificity is determined by the presence of positive and negative controls.

[0117] The term “hybridization” as used here when referring to specific probes 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 for hybridization described herein or to conventional hybridization conditions as described by Sambrook et al., 1989 (Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, NY) which for example may comprise the following steps: 1) immobilization of plant genomic DNA fragments on a filter, 2) pre-hybridization of the filter for 1 to 2 hours at 42°C in 50% formamide, 5X SSPE, 2X Denhardt's reagent and 0.1% SDS or for 1 to 2 hours at 68°C in 6X SSC, 2X Denhardt's reagent and 0.1% SDS, 3) addition of the hybridization probe that has been labeled, 4 ) incubation for 16 to 24 hours, 5) washing the filter for 20 min. at room temperature in 1X SSC, 0.1% SDS, 6) wash the filter three times for 20 min. each at 68°C in 0.2X SSC, 0.1% SDS and 7) exposure of the filter for 24 to 48 hours to x-ray film at -70°C with an intensifying screen.

[0118] As used herein, a biological sample is a sample of a plant, plant material or products comprising plant material. The term “plant” is intended to encompass tissues of the soybean plant (Glycine max), at any stage of maturity, as well as any cells, tissues or organs taken from or derived from any such plant, including without limitation any seeds, leaves, stems, flowers, roots, single cells, gametes, cell cultures, tissue cultures, or protoplasts. “Plant material,” as used herein, refers to material that is obtained or derived from a plant. Products comprising plant material relate to food, feed or other products that are produced using plant material or may be contaminated by plant material. It is understood that, in the context of the present invention, such biological samples are tested for the presence of EE-GM5 specific nucleic acids implying the presence of nucleic acids in the samples. Thus, the methods referred to here for identifying the EE-GM5 elite event in biological samples relate to the identification in biological samples of nucleic acids that comprise the elite event.

[0119] As used herein, “comprising” is to be interpreted as specifying the presence of the features, integers, steps, reagents or components set forth as referred to, 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 comprise more nucleotides or amino acids than are actually mentioned, i.e., be embedded in a larger nucleic acid or protein. A chimeric gene comprising a DNA sequence that is functionally or structurally defined may comprise additional DNA sequences, such as promoter, leader, trailer and/or transcript termination sequences (possibly also including a DNA encoding a targeting or transit peptide). ).

[0120] The present invention also relates to the development of an EE-GM5 elite event in soybean plants comprising this event, progeny plants and seeds comprising the EE-GM5 elite event obtained from these plants and with cells of plant or plant material derived from plants comprising this event. Plants comprising the EE-GM5 elite event can be obtained as described in the Examples. This invention also relates to seed comprising the elite event EE-GM5 deposited with the ATCC under deposit number PTA-123625 or derivatives thereof comprising the elite event EE-GM5. “(Seed) derivatives”, as used herein, refer to plants that can be grown from such seed, offspring resulting from self-fertilization, crossing or backcrossing, as well as plant cells, organs, parts, tissues, cell cultures , protoplasts and plant material thereof.

[0121] Soybean plants or plant material comprising EE-GM5 can be identified according to any of the identification protocols for EE-GM5 as described in the Examples, including the Endpoint method for analyzing the identity of EE-GM5 in Example 2.1, the Endpoint method for analyzing the identity and zygosity of EE-GM5 as described in Example 2.2, the Real-Time PCR method for analyzing the Low Level Presence of EE-GM5 as described in Example 2.3, or the Real-Time PCR to analyze the low-level presence of EE-GM5 as described in Example 2.4. Briefly, soybean genomic DNA present in the 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 the primer with 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 the primer with the sequence of SEQ ID NO: 12 or SEQ ID NO: 18 or with a primer that recognizes the flanking sequence of T-DNA 5' or 3' of EE-GM5 and the inserted T-DNA contiguous therewith. DNA primers that amplify part of an endogenous soybean sequence are used as a positive control for PCR amplification. If, after PCR amplification, the material gives rise to a fragment of the expected size or gives rise to the expected fluorescent tag fluorescence, the material contains plant material from a soybean plant harboring the elite event EE-GM5.

[0122] Plants harboring EE-GM5 are characterized by their resistance to nematodes, particularly SCN, resistance to lesion-forming nematodes and/or root knot nematodes (“RKN”) and/or reniform nematodes, as well as their tolerance to HPPD inhibitors such as isoxaflutole, topramezone or mesotrione. Soybean plants in different commercially available varieties harboring EE-GM5 are also characterized by having agronomic characteristics that are comparable to the corresponding commercially available isogenic non-transgenic varieties in the absence of application of HPPD-inhibiting herbicides and SCN infestation. It was observed that the presence of a T-DNA inserted in the insertion region of the soybean plant genome described here confers particularly interesting phenotypic and molecular characteristics to the plants comprising this event.

[0123] Also provided herein is a method for producing a soybean plant resistant to SCN and tolerant to HPPD-inhibiting herbicides, comprising introducing resistance to SCN and tolerance to HPPD-inhibiting herbicides into the genome of a soybean plant by crossing a first soybean plant not having a gene encoding Cry14Ab-1 and not having a gene encoding HPPD-4 with a soybean plant containing EE-GM5 and selection of a progeny plant resistant to SCN and tolerant to HPPD inhibitor herbicides. SCN resistance can be measured using a standard SCN greenhouse assay, e.g., www.plantpath.iastate.edu/tylkalab/greenhouse-resistance-screening and www.plantmanagementnetwork.org/pub/php/review/2009 /sce08/.

[0124] An embodiment of this invention provides an elite event in soybean plants, obtainable by insertion of 2 transgenes in a specific location in the soybean genome, an elite event that confers resistance to nematodes and tolerance to an HPPD inhibitor herbicide such as such as isoxaflutole, topramezone or mesotrione in such soybean plants and wherein such elite event has an agronomic performance essentially similar to isogenic lines (as used herein, “isogenic lines” or “near-isogenic lines” are soybean lines of the same genetic background but not having the transgenes, such as plants of the same genetic background as the plant used for transformation or sister segregating (“null”) lines having lost the transgenes). Particularly, the current invention provides an elite event in soybean plants, wherein the insertion or presence of said elite event in the genome of such soybean plants does not appear to cause an increased susceptibility to disease, does not cause a yield penalty or does not causes increased lodging compared to isogenic lines or commercial soybean cultivars. Accordingly, the current invention provides an elite event in soybean plants, designated as EE-GM5, which results in soybean plants that have improved resistance to nematodes and can tolerate the application of an HPPD-inhibiting herbicide such as isoxaflutole, topramezone or mesotrione without negatively affecting the yield of said soybean plants compared to isogenic lines, which soybean plants are not statistically significantly different in their susceptibility to disease, or lodging, from isogenic soybean plants or commercial soybean cultivars. These characteristics make the current elite event a valuable tool in a nematode control and weed resistance management program. In one embodiment, event EE-GM5 is combined with one or more soybean events providing tolerance to any one or a combination of glyphosate-based, glufosinate-based, HPPD inhibitor-based, sulfonylurea-based or imidazolinones, AHAS or ALS and/or auxin-type inhibitors (e.g., dicamba, 2,4-D), such as Event EE-GM3 (also known as FG-072, MST-FG072-3, described in WO2011063411 , USDA-APHIS Petition 09-328-01p), Event SYHT0H2 (aka 0H2, SYN-000H2-5, described in WO2012/082548 and 12-215-01p), Event DAS-68416-4 (aka Enlist Soybean, described in WO2011/066384 and WO2011/066360, USDA-APHIS Petition 09-349-01p), Event DAS-444066 (also known as Enlist E3, DAS-44406-6, described in WO2012/075426 and USDA- APHIS 11-234-01p), Event MON87708 (dicamba tolerant event of Roundup Ready 2 known as Genuity Roundup Ready 2 Yield, described in WO2006/130436 and USDA-APHIS Petition 06-178-01p), Event 40-3-2 (also known as Roundup Ready, GTS 40-3-2, MON-04032- 6, described in USDA-APHIS Petition 93-258-01), Event A2704-12 (also known as LL27, ACS-GM005-3, described in WO2006108674 and USDA-APHIS Petition 96-068-01p), Event 127 (also known as BPS-CV127-9, described in WO2010/080829), Event A5547-127 (also known as LL55, ACS-GM006-4, described in WO2006108675 and in USDA-APHIS Petition 96-068-01p), event MON87705 (MON-87705-6, Vistive Gold, published PCT patent application WO2010/037016, USDA-APHIS Petition 09-201-01p) or event DP305423 (also known as DP-305423-1, published PCT patent application WO2008/054747, USDA-APHIS Petition 06-354-01p) or EE-GM5 is combined with a combination of the following events: Event MON98788 x MON87708 (also known as Roundup Ready 2 Xtend Soybeans, MON-87708-9 x MON- 89788-1), Event HOS x Event 40-3-2 (also known as Plenish High Oleic Soybeans x Roundup Ready Soybeans), Event EE-GM3 x EE-GM2 (also known as FG-072xLL55, described in WO2011063413), Event MON 87701 x MON 89788 (aka Intacta RR2 Pro Soybean, MON-87701-2 x MON-89788-1), DAS-81419-2 x DAS-44406-6 (aka Conkesta™ Enlist E3™ Soybean, DAS -81419-2 x DAS-44406-6), 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 (aka Omega-3 1) or Event MON87769 x Event MON89788 (also known as Omega-3 x Genuity Roundup Ready 2 Yield Soybeans, MON-87769-7 x MON-89788-1).

[0125] Also provided here is a soybean plant or part thereof comprising event EE-GM5, which representative soybean seed comprising event EE-GM5 has been deposited under ATCC accession number PTA-123625. Further provided herein are seeds of such plants comprising such an event, as well as a soybean product produced from such seeds, wherein said soybean product comprises the EE-GM5 event. Such soy product may be or may comprise soybean meal, crushed soybean grain, soybean flakes or a product comprising any of these processed soybean products. Particularly, such soy product comprises a nucleic acid that produces a diagnostic or specific amplicon of the EE-GM5 event, such amplicon comprising the sequence of either SEQ ID NO: 1 or 3 or SEQ ID NO: 2 or 4. Also provided is a method for 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 of obtaining food, feed or processed industrial products derived from soybeans, such as soybean oil, soybean protein, lecithin, soybean milk, tofu, margarine, biodiesel, biocomposites, adhesives, solvents, lubricants, cleaners, foam, paint, dye, candles, food or feed (animal) products containing soybean oil or soy protein, said method comprising obtaining grain comprising EE-GM5 and producing said food, feed or industrial product processed from said grain. In one embodiment, this process may also include the step of obtaining a soybean seed or plant comprising event EE-GM5, cultivating said seed or plant in a field, and collecting soybean grain. Optionally, this method includes application of an HPPD-inhibiting herbicide such as IFT, topramezone or mesotrione before planting, before emergence, after emergence or on top of plants comprising EE-GM5. In one embodiment, processed food, feed or industrial products derived from soybeans are included in this invention, such as such processed products that produce an EE-GM5 event specific amplicon using the methods described herein or that comprise the nucleotide sequence of either SEQ ID NO: 1, 3 or 5 or SEQ ID NO: 2, 4 or 6.

[0126] Also provided herein is a soybean plant, which is a progeny of any of the above soybean plants, and which comprises event EE-GM5, such as a progeny plant or seed of any of the above soybean plants that comprises 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 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 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 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.

[0127] Additionally provided here is a method for producing a soybean plant resistant to nematodes and tolerant to the herbicide isoxaflutole and/or topramezone and/or mesotrione, comprising introducing into the genome of such a plant the event EE-GM5, particularly by crossing a first soybean plant not having the EE-GM5 event with a soybean plant comprising EE-GM5 and selection of a nematode-resistant progeny plant tolerant to the herbicide isoxaflutole and/or topramezone and/or mesotrione.

[0128] Also provided here is a nematode-resistant soybean plant tolerant to the herbicide isoxaflutole, topramezone or mesotrione with acceptable agronomic characteristics, comprising a gene encoding Cry14Ab-1 and a gene encoding HPPD-4 and capable of producing an amplicon diagnostic for the EE-GM5 event. Also provided herein are specific isolated amplicons (fragments of DNA sequences) as such, which can be obtained using the specific detection tools described herein, particularly amplicons including in their sequence a DNA fragment originating from T-DNA flanking DNA. 5' or 3' and the DNA inserted into the plant genome by transformation, as defined here.

[0129] Further provided herein is a method for controlling weeds in a field of soybean plants comprising event EE-GM5 or a field to be planted with such soybean plants (wherein 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-based or topramezone-based or mesotrione-based herbicide, wherein such plants are tolerant to such herbicide.

[0130] Further provided herein is a 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 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 (heterologous or foreign to a conventional soybean plant, seed, tissue or cell) of SEQ ID NO: 5 or 6 or comprising a sequence of DNA with 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.

[0131] Also described is a chimeric DNA comprising an inserted T-DNA, wherein the sequence of said inserted T-DNA comprises the sequence of SEQ ID NO: 11 from nucleotide 1 to nucleotide 7459 or SEQ ID NO: 23 from nucleotide 1114 to nucleotide 8572 or a sequence with at least 97, 98, 99, 99.5 or at least 99.9% sequence identity thereto, flanked by a 5' T-DNA flanking region or 3', wherein the 5' T-DNA flanking region immediately upstream of and contiguous with said inserted T-DNA is characterized by a sequence comprising the sequence of SEQ ID NO: 5 from nucleotide 1 to nucleotide 166 or of SEQ ID NO: 24 from nucleotide 1 to nucleotide 1113 wherein the 3' T-DNA flanking region immediately downstream of and contiguous with said inserted T-DNA is characterized by a sequence comprising the 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. In one embodiment, the sequence of said inserted T-DNA consists of sequence of SEQ ID NO: 11 from nucleotide 1 to nucleotide 7459 or SEQ ID NO: 23 from nucleotide 1114 to nucleotide 8572 or a sequence of at least 97, 98, 99, 99.5 or at least 99 .9% sequence identity thereto, flanked by part of a 5' or 3' T-DNA flanking region, wherein the portion of said 5' T-DNA flanking region immediately upstream of and contiguous with said Inserted T-DNA is characterized by a sequence consisting of the sequence of SEQ ID NO: 5 from nucleotide 1 to nucleotide 166 or of SEQ ID NO: 24 from nucleotide 1 to nucleotide 1113 or a sequence of at least 97 , 98, 99, 99.5 or at least 99.9% sequence identity thereto and wherein the portion of the 3' T-DNA flanking region immediately downstream of and contiguous with said inserted T-DNA is characterized by a sequence consisting of the sequence of SEQ ID NO: 6 from nucleotide 359 to nucleotide 691 or the complement nucleotide sequence of SEQ ID NO: 25 from nucleotide 359 to nucleotide 1449 or a sequence of at least 97 , 98, 99, 99.5 or at least 99.9% sequence identity with it.

[0132] Chimeric DNA refers to DNA sequences, including regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric DNA may comprise regions of DNA adjacent to each other that are derived from different sources or that are arranged in a manner different from that found in nature. Examples of a chimeric DNA are the sequences of SEQ ID NO: 5 or 6.

[0133] Also provided here is a transgenic soybean plant, plant cell, tissue or seed, comprising in its genome the EE-GM5 event 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 a plant, plant cell, tissue or soybean seed, comprising in its genome the 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.

[0134] Further provided here is a plant, plant cell, tissue or soybean seed, comprising EE-GM5, characterized by comprising in the genome of its cells a nucleic acid sequence with 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 with any of SEQ ID NO: 1, 3, 5 or 24 and a nucleic acid sequence with 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% of sequence identity with any of SEQ ID NO: 2, 4, 6 or 25 or the complement of said sequences.

[0135] The term “isoxaflutol”, as used herein, refers to the herbicide isoxaflutole [i.e., (5-cyclopropyl-4-isoxazolyl)[2-(methylsulfonyl)-4-(trifluoromethyl)phenyl]methanone], its metabolite active, diketonitrile, and any mixtures or solutions comprising said compound. HPPD inhibitor herbicides useful for application in the embodiment of this invention are the diketonitriles, e.g., 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 the pyrazolinates, e.g., topramezone [i.e., [3-(4,5-dihydro-3-isoxazolyl)-2-methyl-4-(methylsulfonyl)phenyl](5-hydroxy-1-methyl- 1H-pyrazol-4-yl)methanone] and pyrasulfotol [(5-hydroxy-1,3-dimethylpyrazol-4-yl(2-mesyl-4-trifluoromethylphenyl)methanone]; 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 pyrazophene [2-[4-(2, 4-dichlorobenzoyl)-1,3-dimethylpyrazol-5-yloxy]acetophenone].

[0136] In one embodiment of this invention, a field to be planted with soybean plants containing event EE-GM5 can be treated with an HPPD-inhibiting herbicide, such as isoxaflutole ("IFT"), topramezone or mesotrione or with either HPPD inhibitor herbicide such as glyphosate, before the soybeans are seeded, which clears the field of weeds that are killed by the HPPD inhibitor and/or glyphosate, allowing no-till practices, followed by planting or seeding the soybeans in that same field pretreated later (burn application using an HPPD-inhibiting herbicide). size, and weeds tend to reappear, an HPPD inhibitor or a mixture of an HPPD inhibitor with a soybean-selective (conventional) herbicide or a mixture of an HPPD inhibitor with a herbicide that is not soybean-selective (e.g., glyphosate or glufosinate) but for which the plants contain a tolerance gene/locus such that said plants are tolerant to said herbicide can be applied as post-emergence herbicides to the tops of the plants.

[0137] In another embodiment of this invention, a field in which seeds containing the EE-GM5 event have been sown 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 weed-free before sowing using other means, including conventional tillage practices such as plowing, chiseling or seed bed preparation), where residual activity will keep the field weed-free weeds killed by the herbicide such that emerging and cultivated soybean plants do not have weed competition (pre-emergence application of an HPPD-inhibiting herbicide). Once soybean plants reach a certain size, and weeds tend to reappear, an HPPD inhibitor or a HPPD inhibitor-selective (conventional) soybean herbicide mixture or a mixture of HPPD inhibitor with a herbicide that is not selective in soybeans (e.g., glyphosate or glufosinate) but for which the plants contain a tolerance gene/locus such that said plants are tolerant to said herbicide can be applied as post-emergent herbicides on the tops of the plants. In one embodiment of the invention there is provided a process for controlling weeds comprising sowing a field of soybean seeds containing EE-GM5 and treating said field with an HPPD-inhibiting herbicide before plants emerge from said seed, but after the seeds are sown.

[0138] In another embodiment of this invention, plants containing event EE-GM5 can be treated with an HPPD-inhibiting herbicide, such as IFT, topramezone or mesotrione, on the tops of soybean plants that emerged from seeds that were seeded, which clears the field of weeds killed by the HPPD inhibitor, the application of which may be in conjunction with (e.g., in a spray tank mix) followed by or preceded by a treatment with a post-emergent herbicide selective soybean or a herbicide that is not selective on soybeans (e.g., glyphosate or glufosinate) for which the plants contain a tolerance gene/locus such that said plants are tolerant to said herbicide, on the top of the plants (post-emergence application of an HPPD-inhibiting herbicide (with or without said selective or non-selective soybean herbicide)).

[0139] Likewise, according to the current invention, soybean plants harboring EE-GM5 (which may also contain another soybean event/trait of herbicide tolerance as described herein) can be treated with, or soybean seeds harboring EE -GM5 can be coated with any soybean insecticide, herbicide or fungicide.

[0140] The following examples describe the development and identification of the EE-GM5 elite event, the development of different soybean lines comprising this event, and the development of tools for the specific identification of the EE-GM5 elite event in biological samples.

[0141] Unless otherwise shown 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 in “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.”

[0142] Standard materials and references are described in “Croy RDD (ed.) (1993) Plant Molecular Biology LabFax, BIOS Scientific Publishers Ltd., Oxford and Blackwell Scientific Publications, Oxford” and in “Brown TA, (1998) Molecular Biology LabFax, 2nd Edition, Academic Press, San Diego.” Standard materials and references for polymerase chain reactions (PCR) can be found in “McPherson MJ and M0ller SG (2000) PCR (The Basics), BIOS Scientific Publishers Ltd., Oxford” and in “PCR Applications Manual, 3rd Edition ( 2006), Roche Diagnostics GmbH, Mannheim or www.roche-applied-science.com”.

[0143] It should be understood that a number of parameters in any laboratory protocol such as the PCR protocols in the Examples below may need to be adjusted to specific laboratory conditions and may be modified slightly to obtain similar results. For example, using a different method for DNA preparation or selecting other primers in a PCR method may dictate other optimal conditions for the PCR protocol. These adjustments will however be apparent to one skilled in the art and are further detailed in current PCR application manuals.

[0144] In the description and examples, reference is made to the following sequences in the attached Sequence Listing: SEQ ID NO: 1: 5' EE-GM5 junction SEQ ID NO: 2: 3' EE-GM5 junction SEQ ID NO: 3: junction 5' of EE-GM5 SEQ ID NO: 4: 3' junction of EE-GM5 SEQ ID NO: 5: 5' region of EE-GM5 SEQ ID NO: 6: 3' region of EE-GM5 SEQ ID NO: 7 : sequence encoding cry14Ab-1.b SEQ ID NO: 8: amino acid sequence of the Cry14Ab-1 protein SEQ ID NO: 9: sequence encoding hppdPf-4Pa SEQ ID NO: 10: amino acid sequence of the HPPD-4 protein SEQ ID NO : 11: transformation plasmid pSZ8832 - T-DNA cross-border sequence SEQ ID NO: 12: PRIM1038 primer SEQ ID NO: 13: PRIM1039 primer SEQ ID NO: 14: TM1788 probe SEQ ID NO: 15: KVM164 primer SEQ ID NO : 16: KVM165 initiator SEQ ID NO: 17: TM1242 probe SEQ ID NO: 18: PRIM1041 initiator SEQ ID NO: 19: PRIM1040 initiator SEQ ID NO: 20: TM1789 probe SEQ ID NO: 21: PRIM1629 initiator SEQ ID NO: 22 : TM2083 probe SEQ ID NO: 23: EE-GM5 soybean event SEQ ID NO: 24: 5' junction sequence of EE-GM5 SEQ ID NO: 25: 3' junction sequence of EE-GM5 SEQ ID NO: 26 : GLPA210 primer SEQ ID NO: 27: GLPA212 primer SEQ ID NO: 28: GLPB167 primer SEQ ID NO: 29: GLPB170 primer SEQ ID NO: 30: PRIM2123 primer SEQ ID NO: 31: PRIM2122 primer SEQ ID NO: 32: probe TM2327 SEQ ID NO: 33: pre-insertion locus sequence

EXAMPLES 1. TRANSFORMATION OF GLYCINE MAX WITH A NEMATODE RESISTANCE GENE AND A HERBICIDES TOLERANCE GENE 1.1. DESCRIPTION OF THE INSERTED T-DNA COMPRISING THE CHIMERIC GENES CRY14AB-1.B AND HPPDPF-4PA

[0145] Soybean EE-GM5 was developed through Agrobacterium-mediated transformation using the pSZ8832 vector containing hppdPf-4Pa and cry14Ab-1.b expression cassettes: (i) The mutant hppdPf-4P gene encoding the HPPD-4 protein (the amino acid sequence of which is shown in SEQ ID NO: 10). The sequence encoding hppdPf-4Pa was developed by introducing point mutations at position 335 (substitution of Glu for Pro), at position 336 (substitution of Gly for Trp), at position 339 (substitution of Lys for Ala) and at position 340 ( replacement of Ala by Gln) in a DNA encoding the HPPD protein derived from the Pseudomonas fluorescens A32 strain. Expression of the HPPD-4 protein confers tolerance 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.

[0146] Plasmid pSZ8832 is a plant transformation vector that contains a cry14Ab-1.bchimeric gene and a chimeric hppdPf-4Pa gene located between the right border (RB) of T-DNA and the left border (LB) of T -DNA. A description of the genetic elements comprised in T-DNA between the right and left boundary of T-DNA is given in Table 1 below. Confirmatory sequencing of the T-DNA (between the T-DNA boundaries) of this plasmid resulted in the sequence of SEQ ID NO: 11. The nucleotide sequence of the sequences encoding cry14Ab-1.b and hppdPf-4Pa (showing the coding strand) is represented in SEQ ID NO: 7 and 9, respectively.

[0147] Table 1: Description of genetic elements between the boundaries of the T-DNA in pSZ8832 and nucleotide positions in SEQ ID NO: 11.

1.2. EE-GM5 EVENT

[0148] The T-DNA vector pSZ8832 was introduced into Agrobacterium tumefaciens and transformed soybean plants (var. Thorne) were selected using tolerance to HPPD inhibitors according to methods known in the art. Surviving plants were then self-pollinated to generate T1 seed. Subsequent generations were produced through self-pollination or through crossing into other soybean germplasm.

1.2.1 IDENTIFICATION OF THE EE-GM5 ELITE EVENT

[0149] The elite event EE-GM5 was selected based on an extensive selection procedure (based on parameters including but not limited to trait efficacy in the greenhouse and field, molecular characteristics and agronomic characteristics) from a wide range of different transformation events obtained using the same chimeric genes. It was discovered that soybean plants containing EE-GM5 had an insertion of the transgenes at a unique locus in the soybean plant genome, had similar agronomy to the parent plants used for transformation, did not cause a yield penalty for the insertion of the transforming DNA (in comparison with a corresponding isogenic line without the event, such as a “null” plant line obtained from a transformed plant in which the transgenes were not segregated), resulted in a significant reduction in adult females infesting the roots in a greenhouse trial of standard SCN and had improved yield under SCN nematode pressure in the field compared to the isogenic null line not containing EE-GM5. Additionally, tolerance to the application of HPPD-inhibiting herbicides was measured in field trials, but herbicide tolerance was not a selection criterion for elite event selection.

1.2.1.1 MOLECULAR ANALYSIS OF THE EVENT

[0150] Western blot results showed that EE-GM5 contain a single transgenic locus that contains a single copy of the cry14Ab-1.b chimeric gene and a single copy of the hppdPf-4Pa chimeric gene. EE-GM5 lacks a part of the 35S promoter of the hppdPf-4Pa chimeric gene (indicating that not all of the entire T-DNA of SEQ ID NO: 11 was inserted into the soybean genome during transformation). No PCR fragments were obtained after PCR analysis using primers targeting vector backbone sequences that are flanking the left and right border of the T-DNA as well as the aadA sequence. Likewise, the presence of identical EE-GM5 integration fragments in multiple generations of EE-GM5 demonstrates the structural stability of the event.

1.2.1.2 EVENT INHERITANCE

[0151] The inheritance of inserted T-DNA insertion in subsequent generations by testing the genotype of hppdPf-4Pa and cry14Ab-1.b genes by PCR analysis shows that the hppdPf-4Pa and cry14Ab-1.b genes contained within the EE-GM5 insertions are inherited in a predictable manner and as expected for a single insertion. These data are consistent with Mendelian principles and support the conclusion that the EE-Gm5 event consists of a single integrated insertion at a unique chromosomal locus within the soybean nuclear genome.

[0152] Likewise, analysis of EE-GM5 segregation patterns in subsequent generations after EE-GM5 introgression into 5 elite soybean lines confirmed normal Mendelian segregation. Table 2 shows the observed segregation of EE-GM5 in different segregating populations. TABLE 2. EE-GM5 SEGREGATION ANALYSIS

[0153] In Table 2, “HH” represents homozygous plants, “Hemi” hemizygous plants and “null” null segregants having lost EE-GM5 and “ns” means not statistically significant (as for any variation from normal/expected segregation ). In these assays, Progenitor 1 was an MG VI line with resistance to native SCN of Rhg1 and Rhg4, Progenitor 2 was an MG VI line susceptible to SCN, Progenitor 3 was a MG IX line susceptible to SCN, Progenitor 4 was an MG III line with resistance to native Rhg1 SCN and Progenitor 5 was an MG I line susceptible to SCN.

1.2.1.3 STABILITY OF PROTEIN EXPRESSION

[0154] Protein expression levels of HPPD-4 and Cry14Ab-1 proteins in greenhouse-grown plants were determined by enzyme-linked immunosorbent assay (ELISA) on leaf, root and seed samples collected from different generations (p .e.g., T4, T6 and BC2F3) of EE-GM5 soybeans. HPPD-4 and Cry14Ab-1 exhibit similar average expression levels in leaf, root and seed across all generations tested. Any observed differences in Cry14Ab-1 and HPPD-4 concentrations were attributed to natural plant-to-plant variability.

1.2.1.4 AGRONOMIC PERFORMANCE AND TOLERANCE TO HPPD INHIBITOR HERBICIDES

[0155] In agronomic equivalence assays, plants comprising EE-GM5 in the original transformation background (Thorne) were compared with segregating nulls (not having EE-GM5) and with wild-type Thorne plants when grown in the absence of SCN. Plots were not treated with HPPD herbicides but were maintained weed-free through the use of conventional herbicides and manual weed removal where necessary. No differences impacting agronomic performance in a biologically significant manner were observed between plants containing the event and segregating nulls (not having EE-GM5) when grown in comparable trials at different locations when verifying qualitative plant characteristics such as flower color. , pod color, seed color and pubescence and quantitative characteristics such as yield, height, lodging, support and days to maturity. Consequently, plants comprising EE-GM5 showed normal agronomic characteristics comparable to the corresponding non-transgenic plants.

[0156] Additional trials with EE-GM5 in the original Thorne transformation background were conducted in 2017. Preliminary trials in which EE-GM5 was in elite MG1 and MG3 genetic backgrounds were also established in a limited number of locations in 2017. When qualitative characteristics of plants such as flower color, pod color, seed color and pubescence and quantitative characteristics such as yield, height, lodging, support and days to maturity were verified, no consistent and significant differences were detected between the EE event -GM5 and the segregating nulls (not having EE-GM5) in any of the three genetic backgrounds, confirming that plants comprising EE-GM5 showed normal agronomic characteristics.

[0157] The tolerance of plants comprising EE-GM5 to HPPD-inhibiting herbicides was tested at different locations in the field over 2 years. In these trials it was found that plants with EE-GM5 had commercially relevant tolerance to isoxaflutole (IFT) when applied pre-emergence as well as when applied post-emergence, but crop damage was somewhat higher for pre-emergence applications. of IFT. These trials also showed that plants containing the EE-GM5 event had commercially relevant tolerance to mesotrione (MST) when applied pre-emergence or when applied post-emergence. All post-emergence treatments were in the V2-V3 stage, with adjuvants concentrated in culture oil and additional ammonium sulfate to increase herbicide activity.

[0158] Fig. 5 shows the average of the maximum phytotoxicity (plant damage) data recorded for herbicide treatment in various field trials over 2 years, for soybean plants containing event EE-GM5 compared to plants unprocessed/conventional soybeans. Control untransformed Thorne plants showed average maximum phytotoxicity values of about 80 to 90% in these same trials, showing that these HPPD-inhibiting herbicides are not tolerated by (non-GM) soybeans. “Maximum phytotoxicity” as used here is the maximum phytotoxicity rating recorded in any observation during the duration of a trial (with 3 to 4 observations per trial). In existing weed control applications, a normal dose (1x) for isoxaflutole (IFT) in pre- or post-emergence application and for MST in post-emergence application is 105 gr/ha and a normal dose (1x) for mesotrione in pre-emergence application it is 210 gr/ha. Consequently, in these trials reported in Fig. 5, the applications used pre-emergence in Fig. 5 (420 gr/ha for IFT, 840 gr/ha for mesotrione) were at 4 times the normal dose, and in post-emergence (210 gr/ha for each of IFT and mesotrione), were at 2 times the normal dose.

[0159] In the 3rd year, plants with EE-GM5 (on Thorne background) when treated with isoxaflutole (IFT, at 410 g/ha) pre-emergence in a field trial location had 9% maximum phytotoxicity and, when treated with isoxaflutole (IFT) post-emergence (stage V2-V3, at 210 h/ha) in 4 locations, they had an average of 10.9% maximum phytotoxicity, confirming the tolerance observed before.

[0160] Likewise, in several field trials over 2 years, soybean plants with event EE-GM5 had good tolerance towards the experimental HPPD inhibitor compound 2-methyl-N-(5-methyl-1,3 ,4-oxadiazol-2-yl)-3-(methylsulfonyl)-4-(trifluoromethyl)benzamide (US patent 9101141) when applied pre-emergence at 400 g of ai/ha or post-emergence at 200 g of ai/ ha, respectively (the average maximum phytotoxicity value for each treatment was below 20%). In these trials, soybean plants with event EE-GM5 also showed good tolerance (average maximum phytotoxicity of 20%) to the experimental HPPD inhibitor compound 2-chloro-3-(methylsulfanyl)-N-(1-methyl-1H- tetrazol-5-yl)-4-(trifluoromethyl)benzamide (US patent 8481749) when applied post-emergence at 100-150 g ai/ha. All post-emergence treatments were in the V2-V3 stage, with adjuvants concentrated in culture oil and additional ammonium sulfate to increase herbicide activity. In the 3rd year, plants with EE-GM5 (on Thorne background) when treated with 2-chloro-3-(methylsulfanyl)-N-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl) benzamide at 150 g/ha post-emergence in 3 field trial locations had an average maximum phytotoxicity of 13.3%.

[0161] Average maximum phytotoxicity ratings equal to or very similar to those described in Fig. 5 were obtained for IFT when data obtained from a 3rd station of herbicide tolerance field trials applying isoxaflutole herbicide at the same dosages were added. pre or post EE-GM5 when in another geographic location.

[0162] Likewise, plants with EE-GM5 when treated post-emergent (V2-V3) with topramezone at 36g ai/ha (+ COC and AMS) in 2 field trials in the USA gave an average maximum phytotoxicity of 11% , showing that EE-GM5 also confers good tolerance to this HPPD inhibitor.

1.2.1.5 RESISTANCE TO NEMATODES

[0163] Standard SCN assays measuring the female index in the greenhouse showed a significant reduction of SCN cysts in roots of plants containing EE-GM5 when compared to wild-type Thorne soybean plants. Additionally, standard SCN assays measuring the female index in the greenhouse also showed that soybean plants containing the EE-GM5 event and resistance to native SCN showed a significant reduction in SCN cysts in roots compared to elite soybean lines resistant to SCN without EE-GM5. When EE-GM5 was introgressed into an elite soybean line with resistance to soybean PI88788 (maturity group 3) or an elite soybean line with resistance to Peking soybean (maturity group 6.2), consistently a reduced number of cysts of SCN was seen in roots compared to roots with native resistance alone.

[0164] In field trials over 2 years in various locations, soybean plants containing EE-GM5 gave a significant yield increase compared to isogenic null segregants in SCN-infested fields. Figure 6 shows the grain yield of EE-GM5 in the original transform background (Thorne) as tested at 9 different locations throughout Iowa, Illinois, Indiana, Missouri and Tennessee in 2015 and 2016, in SCN-infested fields (ranging from low to high SCN infestation). Additional trials with EE-GM5 in the original transform background (Thorne) were conducted in 2017 at a total of 12 locations with varying SCN pressure. Across all 12 trials, plants containing EE-GM5 produced an average of 10% higher yields than null segregants lacking EE-GM5 (p = 0.003). Fig. 7 shows the grain yield of EE-GM5 when introgressed (BC2F3) into an elite MG I (maturity group I) line that is susceptible to SCN and was tested at a location in Minnesota and a location in the Dakotas. of the north in 2016 (each with a high level of SCN infestation). The same MG I line was tested at the same two locations (each again with high SCN infestation) and an additional location in Wisconsin in 2017 (the latter having moderate SCN pressure), and the grain yield of plants containing EE- GM5 was consistently higher than corresponding null segregants lacking EE-GM5. Finally, preliminary studies across three locations with moderate to high SCN pressure in Brazil in late-planted trials in 2017 showed a significant average increase of 31% (p = 0.01) in an elite susceptible line for plants with EE- GM5 when compared with the null segregant (not having EE-GM5). Due to the later planting date, glocal yields in these preliminary trials in Brazil tended to be low and variability within a trial was somewhat high, which may have influenced the magnitude of the yield increase, but a clearly significant increase was discovered. and visually observable yield for plants with EE-GM5. Consequently, the EE-GM5 event confers a significant increase in yield in soybean plants in SCN-infested fields.

[0165] In a study to evaluate the effect of the EE-GM5 event on yield when combined with resistance to native SCN, a series of F3 populations were developed from the single cross of EE-GM5 with an elite conventional MG III line carrying the rhg1 resistance gene from PI88788. In F3 populations, a “stacked” population that is homozygous for both the EE-GM5 event and the rhg1 allele was compared with a population homozygous for the rhg1 allele only (having no EE-GM5). Field trials were established with these populations in 2016 in three locations with moderate to high SCN infestation and in 2017 in seven locations ranging from low to high SCN pressure. The results are shown in Figure 8. All 2017 trials included three different seed treatments. No significant differences or interactions in yield were observed for any of these seed treatments alone, so data were pooled across treatments to provide the best statistical estimates of the difference in yield between the homozygous event (HH) and the null segregant. As shown in Fig. 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. rhg1 (p = 0.08) and the 2017 trials provided an average yield increase of 11% for plants homozygous for the EE-GM5 event and the rhg1 event (p = 1.24-11), compared to population carrying only the rhg1 allele. For reference, the average yield increase for lines containing EE-GM5 over the 2017 trials with the seed-based treatment alone (Evergol® Energy + Allegiance® fungicide + Poncho® insecticide) was 0.27 T/ha (increase in yield of 10.2%; p = 0.0002). The base seed treatment used in all 2016 trials was Evergol® Energy + Allegiance® fungicide. As shown in Figure 8 a close relationship was found across all 10 trials in both years between yield response and SCN pressure with greater yield gains being observed at locations with high SCN pressure (towards the top in Fig .8). These results show that the addition of the EE-GM5 event to soybean varieties with resistance to conventional SCN can provide a significant yield increase in SCN-infested fields.

[0166] The condition of field trials under moderate to high SCN infestation is challenging due to many factors that have an impact on the results. Densities of SCN populations within fields can vary substantially and therefore the overall impact of SCN on yield can also vary from one plot to the next (see, e.g., www.plantmanagementnetwork.org/pub/php/review/ 2009/sce08/). Favorable soil types, good fertility and adequate rainfall can mitigate the impact of SCN infestation on the soybean plant and can minimize yield impacts even under high SCN populations. Many fields with very high SCN populations tend to have weak soils and thus lower yield potential, making it difficult to distinguish significant impacts on yield. Therefore, yield data from SCN field trials can be quite variable and one would not expect to see significant improvements in yield in all trials with high SCN populations. Global trends throughout trials are the most relevant criteria for evaluating an event's performance.

[0167] SCN field trials that were carried out with plants containing EE-GM5 were established in a field with natural SCN infestation. The experimental units consisted of a field plot containing 2 to 4 lines spaced 0.76 m apart and ranging from 3.8 to 9.1 m in length. The number of lines per lot and length of the lots would vary from location to location based on field size and equipment settings. The lots were sown at 26 seeds per meter and therefore each experimental unit contained between 200 and 960 seeds. Plots were randomized in the field using a split-plot and sub-split-plot design. Split-plot designs are well adjusted to help minimize the effect of high variability in soil type or SCN populations that is common in SCN-infested fields. In SCN field trials, plants comprising EE-GM5 were planted in a subplot close to, or very close to, a companion subplot containing segregating null plants (without EE-GM5). The close proximity of the two plots helps to minimize the effect of field variability (SCN) in estimating the difference between plants with and without the EE-GM5 event. Most trials were replicated four times, but some were replicated three times and some were replicated five or six times.

[0168] Moderate to severe infestations of Sudden Death Syndrome were observed in two locations (Indiana and Iowa) in 2016. Batches in these two locations were classified for the incidence and severity of SDS symptoms and the Disease Index (DX) of SDS was calculated using the “SIUC SDS Scoring Method” (www.scnresearch.info/462.pdf). DX scores in plants homozygous for EE-GM5 were 61% lower in Indiana and 55% lower in Iowa than in the susceptible null segregant (having no EE-GM5), indicating that the event was providing protection against infection by SDS. SDS and SCN are often closely associated in the field and will show some interactions in the plant (see, e.g., www.soybeanresearchinfo.com/pdf_docs/sdsupdate.pdf and www.apsnet.org/edcenter/intropp/lessons/fungi/ascomycetes /page s/suddendeath.aspx).

[0169] In 2017, Iron Deficient Chlorosis (IDC) scores were gathered on plants with EE-GM5 (and its null segregants) at a US location (with high SCN infestation) where IDC symptoms were observed. The trial was a split-plot design looking at the effect of the event on three different backgrounds. IDC classifications were taken as described by Cianzio et al. (1979) Crop Science 19: 644-646. Fig. 11 shows the average IDC scores for plants with event EE-GM5 and those for the corresponding null segregants (not having EE-GM5) across three genetic backgrounds (1 resistant to SCN (PI88788 resistance), 1 susceptible the SCN and the Thorne fund susceptible to SCN). Significantly lower IDC scores were found for plants containing EE-GM5 compared to its null segregants. Consequently, EE-GM5 significantly reduced foliar IDC severity in a field trial where soybean plants were challenged by SCN and IDC. This reduction occurred across three soybean lines, one of which included resistance to native SCN type PI 88788.

[0170] Likewise, untransformed Thorne and EE-GM5 seeds were germinated and planted in the greenhouse to verify control of the lesion-forming nematode, Pratylenchus brachyurus. Pratylenchus brachyurus nematodes (#1500/plant, different developmental stages) were applied to plants when 2 weeks old. 30 days after application, Pratylenchus nematodes were extracted from the roots and counted. The average number of nematodes found in the roots of plants containing EE-GM5 was compared with the average number of Pratylenchus nematodes found in roots of wild-type Thorne plants. On average, approximately 80 to 90% fewer Pratylenchus nematodes were found in roots of plants containing EE-GM5 when compared to Thorne control roots, indicating significant control of lesion-forming nematodes by the EE-GM5 soybean event.

[0171] Figure 9 shows results from a greenhouse trial of Pratylenchus brachyurus in the USA, comparing elite lines with EE-GM5 in 5 elite soybean lines (one susceptible to (MG 1), one resistant to SCN (PI88788 , MG 3), one SCN-susceptible (MG 6.2), one SCN-resistant (Peking, MG 6.2), and one SCN-susceptible (MG 9)) with SCN-susceptible and SCN-resistant US soybean lines. Soybean plants were grown in small conical pots and maintained in greenhouses with temperatures varying between 25-32 °C. Pratylenchus brachyurus nematodes, obtained from South Carolina and raised in the greenhouse, were used to inoculate plants at the V2-V3 developmental stage. Approximately 1500 eggs + adults were inoculated per plant and each entry had 5 plants. 30 days after infestation, nematodes and eggs were extracted from the roots and counted. Each input was operated in two independent experiments. While SCN-susceptible and SCN-resistant U.S. soybean lines showed no control of Pratylenchus, plants with EE-GM5 showed about 90% control of Pratylenchus.

[0172] Figure 10 shows results from a greenhouse trial of Pratylenchus brachyurus in Brazil, comparing soybean plants with EE-GM5 with soybean lines from Brazil without resistance and 1 line with low Rf and plants susceptible and resistant to SCN. The soybean lines were grown in small conical pots and maintained in greenhouses with temperatures varying between 25-32 °C. Pratylenchus brachyurus nematodes, obtained from fields in Brazil and raised in the greenhouse, were used to inoculate plants at the V2-V3 developmental stage. Approximately 1000 eggs + adults were inoculated per plant and each entry had 5 plants. 30 days after infestation, nematodes and eggs were extracted from the roots and counted. The results shown are from a single experiment. A Brazilian soybean line (BRS 7380), labeled as having a low reproductive factor for Pratylenchus, shows about 89% reduction of Pratylenchus. Plants with EE-GM5 gave ~99% control of Pratylenchus. Soybean lines carrying native resistance to SCN (rhg1 + rhg4) do not control Pratylenchus brachyurus.

[0173] Likewise, plants containing EE-GM5 can be used to control root knot nematodes (RKN) such as Meloidogyne incognita. Although the Meloidogyne incognita population does not infest wild-type Thorne soybean very well, Thorne plants with EE-GM5 show an additional reduction in RKN egg number/root mass on average compared to untransformed Thorne plants.

1.2.2 IDENTIFICATION OF FLANKING REGIONS AND INSERTED T-DNA FROM THE ELITEEE-GM5 EVENT

[0174] The sequence of the regions flanking the inserted T-DNA and the T-DNA contiguous therewith as contained in the elite event EE-GM5 are shown in the attached Sequence Listing.

1.2.2.1 T-DNA 5' FLANKING REGION

[0175] A fragment identified as comprising the 5' T-DNA flanking region of EE-GM5 was sequenced and its nucleotide sequence is represented in SEQ ID NO: 5, nucleotides 1-166. This 5' T-DNA flanking region is comprised of soybean genomic sequences corresponding to the pre-insertion locus sequence (SEQ ID NO: 5, nucleotides 1-166). The 5' junction region comprising part of the inserted T-DNA sequence and part of the 5' flanking T-DNA sequence contiguous therewith is represented in SEQ ID NO: 1 and 3.

1.2.2.2 T-DNA 3' FLANKING REGION

[0176] A fragment identified as comprising 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. This 3' T-DNA flanking region consists of a 39-nucleotide filler DNA sequence (from position 359 to position 397 in SEQ ID NO: 6), followed by soybean genomic sequences corresponding to the locus sequence pre-insertion (from position 398 to position 691 in SEQ ID NO: 6). The 3' junction region comprising part of the inserted T-DNA sequence and part of the 3' flanking T-DNA sequence contiguous therewith is represented in SEQ ID NO: 2 and 4.

1.2.2.3 DEEE-GM5 INSERTED T-DNA

[0177] The inserted T-DNA contiguous with the 5' T-DNA flanking region above was sequenced and its nucleotide sequence is represented in SEQ ID NO: 5, nucleotides 167-353. Likewise, the inserted T-DNA contiguous with the above 3' T-DNA flanking region was sequenced and its nucleotide sequence is represented in SEQ ID NO: 6, nucleotides 1-358. During transformation, 63 bp of genomic DNA were deleted in the pre-insertion locus sequence, and these were replicated by the inserted DNA (consisting of T-DNA and a small part of filler DNA).

[0178] Sequencing of the T-DNA region in the transformation plasmid pSZ8832 (the part between the T-DNA borders) resulted in the sequence reported in SEQ ID NO: 11. The chimeric cry14Ab-1.b gene sequence (comprising the Ubi10 promoter and the 3' untranslated region of 35S) is represented in SEQ ID NO: 11 from nucleotides 131-5276 (counterclockwise). The T-DNA sequence inserted in the 5' flanking region in SEQ ID NO: 5 (nucleotides 167-353) is identical to the nucleotide sequence in SEQ ID NO: 11 from nucleotide 1 to nucleotide 187 and the sequence of T -DNA inserted in the 3' flanking region in SEQ ID NO: 6 (nucleotides 1358) is identical to the nucleotide sequence in SEQ ID NO: 11 from nucleotide 7102 to nucleotide 7459. Consequently, 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 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 is contained in the seed deposited with the ATCC under deposit number PTA-123625 and has an essentially similar sequence or identical to the sequence of SEQ ID NO: 11 from nucleotide 188 to nucleotide 7101.

[0179] The insertion locus for the EE-GM5 event can be determined from wild-type soybean var. Thorne based on the 5' and 3' T-DNA flanking sequences provided here (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 to the following sequences in order: nucleotide position 1 to nucleotide position 166 in SEQ ID NO: 5, a 63 nt deletion and nucleotide position 398 to nucleotide position 691 in SEQ ID NO: 6. The complete pre-insertion locus sequence is given in SEQ ID NO: 33, where nt 1-1000 are 5' flanking genomic sequences, nt 1001-1063 are the target site deletion and 1064-2063 are 3' flanking genomic sequences.

1.2.3 CONFIRMATION OF FLANKING REGIONS AND INSERTED T-DNA FROM THE ELITEEE-GM5 EVENT

[0180] PCR amplification using primers targeting plant DNA upstream and downstream of the inserted T-DNA and the inserted T-DNA in EE-GM5 confirmed and extended the 5' and 3' flanking sequences of EE-GM5.

1.2.3.1. SPECIFIC REACTION OF EE-GM5 WITH 5' JUNCTION SEQUENCE

[0181] Two primers, GLPA210 and GLPB167, were designed to amplify an amplicon of approximately 5118 bp spanning the junction region of the 5' T-DNA flanking sequence with the T-DNA insertion fragment for the EE-GM5 event. The GLPA210 primer sequence originates from the Glycine max Williams soybean reference sequence 82.a2.v1.

[0182] Forward primer targeting the 5' flanking sequence of T-DNA of EE-GM5: GLPA210 5'- CTCTCACCCAgATTTCAC -3' (SEQ ID NO: 26) Reverse primer targeting the inserted T-DNA sequence of EE-GM5: GLPB167 5'- TACAACgTgCTCgCTATTCC -3' (SEQ ID NO: 28) Composition of the reaction mixture for the reaction with 5' junction sequence:

[0183] The sequence of the extended T-DNA 5' flanking sequence that was obtained and which is contiguous with and upstream of part of the inserted T-DNA as shown in SEQ ID NO: 5 is shown in SEQ ID NO: 24.

1.2.3.2. SPECIFIC REACTION OF EE-GM5 WITH 3' JUNCTION SEQUENCE

[0184] Two primers, GLPB170 and GLPA212, were designed to amplify an amplicon of approximately 4982 bp spanning the region of the T-DNA insertion fragment for the EE-GM5 event with the 3' T-DNA flanking sequence. The GLPA212 primer sequence originates from the Glycine max Williams reference sequence 82.a2.v1.

[0185] Forward primer targeting the inserted T-DNA sequence of EE-GM5: GLPB170 5'- TCTCggTATCAgCgTTCTTg -3' (SEQ ID NO: 29) Reverse primer targeting the 3' flanking sequence of EE-GM5 T-DNA: GLPA212 5'- CCCATgCggTATTATgTg -3' (SEQ ID NO: 27) Composition of the reaction mixture for the reaction with 3' junction sequence:

[0186] The sequence of the extended T-DNA 3' flanking sequence that was obtained and is contiguous with and downstream of part of the inserted T-DNA as shown in SEQ ID NO: 6 is shown in SEQ ID NO: 25.

[0187] Since the resulting amplicons in the 2 reactions above overlapped, this allowed a reconstruction of the sequence of the inserted T-DNA of EE-GM5 and the extended 5' and 3' flanking sequences, which is shown in SEQ ID NO: 23. The 5' T-DNA flanking sequence in SEQ ID NO: 23 is from nucleotide position 1 to nucleotide position 1113 (corresponding to pre-insertion locus genomic sequences), the inserted T-DNA sequence is from nucleotide position 1114 to nucleotide position 8572 and the 3' T-DNA flanking sequence in SEQ ID NO: 23 is from nucleotide position 8573 to nucleotide position 9663 (corresponding to filler DNA with 39 nt (nt 8573-8611 in SEQ ID NO: 23) and pre-insertion locus genomic sequences (nt 8612-9663 in SEQ ID NO: 23)).

2. DEVELOPMENT OF IDENTIFICATION PROTOCOLS FOR EE-GM5 2.1 END POINT METHOD FOR EE-GM5 IDENTITY ANALYSIS

[0188] This method describes a polymerase chain reaction detection method for analyzing the presence of DNA sequences specific to the EE-GM5 event in DNA samples obtained from biological samples, such as plant materials (e.g. ., leaf or seed) using standard DNA extraction procedures.

[0189] The method description outlines the method design, including the oligonucleotide primer and probe sequences, the composition of the reaction mixture, the thermocycling conditions required to perform the reaction, and the fluorescent reader scenarios found appropriate for amplicon detection. . It also provides general recommendations on the nature and use of control samples. Additionally, guidance for data analysis and interpretation is provided, including an example of a method result taking into account recommendations on the use of control materials and guidance for data analysis.

2.1.1. METHOD DESIGN

[0190] The method uses Taqman chemistry to amplify and detect two target sequences: an EE-GM5 specific reaction determines the presence of the event, a taxon specific reaction validates negative results for the event specific reaction.

2.1.1.1. DEEE-GM5 SPECIFIC REACTION

[0191] 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 insertion fragment for the EE-GM5 event.

[0192] A probe, TM1788, using FAM as a fluorescent label and BHQ1 as a quencher, was designed to detect the amplified sequence.

[0193] Forward primer targeting the EE-GM5 T-DNA sequence: PRIM1038 5'- gAgCCACCTTCCTTTTCCACTA -3' (SEQ ID NO: 12) Reverse primer targeting the 3' flanking sequence of EE-GM5 T-DNA: PRIM1039 5'- ATAgggTTACTgCTTCgTAAAATAAgCA -3' (SEQ ID NO: 13) Probe targeting the T-DNA junction of EE-GM5 and its 3' flanking sequence: TM1788 FAM 5'- CgCgTCCATgATgCTgCgACTATg -3' BHQ1 (SEQ ID NO: 14) 2.1.1.2. Taxon-specific reaction

[0194] Two primers, KVM164 and KVM165, were designed to amplify a 102 bp amplicon from the endogenous soybean lectin1 gene sequence.

[0195] A probe, TM1242, using JOE as the fluorescent label and BHQ1 as, was designed to detect the amplified sequence.

[0196] Forward primer targeting the endogenous Lectin 1 gene sequence: KVM164 5'- CTTTCTCgCACCAATTgACA -3' (SEQ ID NO: 15) Reverse primer targeting the endogenous Lectin 1 gene sequence: KVM165 5'- TCAAACTCAACAgCgACgAC -3' (SEQ ID NO: 16) Probe targeting the endogenous Lectin 1 gene sequence: TM1242 JOE 5'- CCACAAACACATgCAggTTATCTTgg -3' BHQ1 (SEQ ID NO: 17) 2.1.2. COMPOSITION OF THE REACTIONAL MIXTURE Notes: • The 2x PerfeCta qPCR FastMix II, ROX was provided by Quanta Bioscience. Other enzyme buffers can be used but performance must be verified. • Primers and labeled probes were ordered from Integrated DNA Technologies. • * The amount of template DNA per reaction may vary but must be checked. 2.1.3. THERMOCYCLING CONDITIONS • Thermocycling conditions have been validated for use in a BIORAD C1000 thermal cycler. Other equipment can be used but performance must be checked. 2.1.4. Wavelength and bandwidth scenarios Notes: • Wavelength and bandwidth scenarios have been validated for use on a Tecan M1000 plate reader. Other equipment and scenarios can be used but performance must be verified. 2.1.5. CONTROL SAMPLES • The following control samples must be included in the experiment to validate test sample results: • Positive control: a DNA sample containing the target and endogenous sequences; • Negative control: a DNA sample containing only the endogenous sequence; • Control without mold: a water sample (without DNA) 2.1.6. DATA ANALYSIS; • For all samples, the ratio of Fluorescent Signal to Background (S/B) is calculated for both target and endogenous reactions; • Control samples must give the expected result, ie: o The positive control must be scored “detected”; o The negative control must be scored “not detected”; o The no-template control should only show background fluorescent levels; • A sample is scored as follows: o Detected: target S/B and endogenous S/B exceed an acceptable threshold ratio, e.g., 2; o Not detected: the target S/B is below an acceptable threshold ratio, e.g., 1, and additionally, the endogenous S/B exceeds an acceptable threshold ratio, e.g., 2; o Inconclusive: target and endogenous S/B are below an acceptable threshold, e.g. 1.

[0197] Figure 2 shows an example of the result 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 reaction and the endogenous reaction are displayed.

2.2 FINAL POINT METHOD FOR ANALYSIS OF DEEE-GM5 IDENTITY AND ZYGOSITY

[0198] This method describes a polymerase chain reaction detection method for analyzing the presence and zygosity status of DNA sequences specific to the EE-GM5 event in DNA samples obtained from biological samples, such as biomaterials. plant (e.g., leaf or seed) using standard DNA extraction procedures.

[0199] The method description outlines the reaction reagents, the oligonucleotide primer and probe sequences, the thermocycling conditions required to perform the reaction, and the fluorescent reader scenarios found appropriate for amplicon detection. It also provides general recommendations on the nature and use of control samples. Additionally, guidance for data analysis and interpretation is provided, including an example of a method result taking into account recommendations on the use of control materials and guidance for data analysis.

[0200] It is noted that the performance of the method for analyzing zygosity (from option 1 below) may be variety dependent due to the nature of the pre-insertion locus sequence. Therefore, performance verification is required for each variety into which the event is introgressed. For cases of inadequate performance, an alternative endpoint method design for zygosity can be used (as in option 2 below) or an alternative Real-Time PCR method based on copy number analysis can be used to determine zygosity. zygosity, such as that described below in section 2.3.

2.2.1. METHOD DESIGN

[0201] The method uses Taqman chemistry to amplify and detect two target sequences: an EE-GM5 specific reaction determines the presence of the event, a Pre-Insertion Locus specific reaction determines the presence of the event pre-insertion locus.

[0202] Detection of only the specific EE-GM5 sequence indicates the presence of the EE-GM5 event in a state of homozygous zygosity.

[0203] Detection of only the EE-GM5-specific and Pre-Insertion Locus-specific sequence indicates the presence of the EE-GM5 event in a state of hemozygous zygosity.

[0204] Detection of only the specific Pre-Insertion Locus sequence indicates the absence of the EE-GM5 event.

2.2.1.1. DEEE-GM5 SPECIFIC REACTION A.OPTION 1

[0205] Two primers, PRIM1040 and PRIM1041, were designed to amplify an 84 bp amplicon spanning the junction region of the 5' flanking T-DNA sequence with the T-DNA insertion fragment for the EE-GM5 event.

[0206] A probe, TM1789, using FAM as a fluorescent label and BHQ1 as a quencher, was designed to detect the amplified sequence.

[0207] Forward primer targeting the EE-GM5 T-DNA sequence: PRIM1041 5'- CATTgTgCTgAATAggTTTATAgCTATgAT -3' (SEQ ID NO: 18) Reverse primer targeting the 5' flanking sequence of EE-GM5 T-DNA: PRIM1040 5'- TCAAATCAACATgggTgACTAgAAA -3' (SEQ ID NO: 19) Probe targeting the junction of EE-GM5 T-DNA and its 5' flanking sequence: TM1789 FAM 5'- CAgTACTgggCCCTTgTggCgCT -3' BHQ-1 (SEQ ID NO: 20)

B.OPTION 2

[0208] Two primers, PRIM2123 and PRIM1041, were designed to amplify a 134 bp amplicon spanning the junction region of the 5' flanking T-DNA sequence with the T-DNA insertion fragment for the EE-GM5 event.

[0209] A probe, TM1789, using FAM as a fluorescent label and BHQ1 as a quencher, was designed to detect the amplified sequence.

[0210] Forward primer targeting the EE-GM5 T-DNA sequence: PRIM1041 5'- CATTgTgCTgAATAggTTTATAgCTATgAT -3' (SEQ ID NO: 18) Reverse primer targeting the 5' flanking sequence of EE-GM5 T-DNA: PRIM2123 5'- gCACTgTTTAACTTTAAATAACTCATTTgAg - 3'(SEQ ID NO:30) Probe targeting the T-DNA junction of EE-GM5 and its 5' flanking sequence: TM1789 FAM 5'- CAgTACTgggCCCTTgTggCgCT -3' BHQ-1 (SEQ ID NO: 20)

2.2.1.1. PRE-INSERTION LOCUS-SPECIFIC REACTION A.OPTION 1

[0211] Two primers, PRIM1629 and PRIM1040, were designed to amplify a 72 bp amplicon spanning the junction of the pre-insertion locus and the 5' flanking sequence of the EE-GM5 pre-insertion locus.

[0212] An MGB probe, TM2083, using VIC as a fluorescent label and MGB-NFQ as a quencher, is designed to detect the amplified sequence.

[0213] Forward primer targeting the EE-GM5 pre-insertion locus sequence: PRIM1629 5'- TTggTgAAAAACAATTTggTgTACA -3' (SEQ ID NO: 21) Reverse primer targeting the 5' flanking sequence of EE-GM5 T-DNA: PRIM1040 5'- TCAAATCAACATgggTgACTAgAAA -3' (SEQ ID NO: 19) Wild-type probe targeting pre-insertion locus function and 5' flanking sequence: TM2083 VIC 5'- AATCAAATCgACATCAATgT -3' MGB-NFQ (SEQ ID NO: 22)

B.OPTION2

[0214] Two primers, PRIM2122 and PRIM2123, were designed to amplify a 193 bp amplicon spanning the junction of the pre-insertion locus and the 5' flanking sequence of the EE-GM5 pre-insertion locus.

[0215] An MGB probe, TM2327, using VIC as a fluorescent label and MGB-NFQ as a quencher, is designed to detect the amplified sequence.

[0216] Forward primer targeting the EE-GM5 pre-insertion locus sequence: PRIM2122 5' CAAgCAAAATAAgCAACTAgATCTATTgg -3' (SEQ ID NO: 31) Reverse primer targeting the 5' flanking sequence of EE-GM5 T-DNA: PRIM2123 5'- gCACTgTTTAACTTTAAATAACTCATTTgAg -3' (SEQ ID NO: 30) Wild-type probe targeting pre-insertion locus function and 5' flanking sequence: TM2327 VIC 5'- TTTggTgAAAAACAATTTggTgT -3' MGB-NFQ (SEQ ID NO: 32 ) 2.2.2. COMPOSITION OF THE REACTIONAL MIXTURE A.OPTION 1 • Primers and labeled probes were ordered from Integrated DNA Technologies • * The amount of template DNA per reaction may vary but must be verified 2.2.3. THERMOCYCLING CONDITIONS (THE SAME FOR OPTIONS 1 AND 2) Notes: • Thermocycling conditions have been validated for use in a BIORAD C1000 thermal cycler. Other equipment can be used but performance must be checked. 2.2.4. WAVELENGTH AND BANDWIDTH SCENARIOS (THE SAME FOR OPTIONS 1 AND 2) • Wavelength and bandwidth scenarios have been validated for use on a Tecan M1000 plate reader. Other equipment and scenarios can be used but performance must be verified. 2.2.5. CONTROL SAMPLES (SAME FOR OPTIONS 1 AND 2) • The following control samples must be included in the experiment to validate test sample results: • Homozygous control: a DNA sample containing the target sequence in a homozygous state • Hemozygous control: a DNA sample containing the target sequence in a hemozygous state • Wild-type control: a DNA sample not containing the target sequence • No template control: a water sample 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 target and pre-insertion locus reactions. • Control samples must give the expected result, ie: o The homozygous control must be scored “homozygous” o The hemozygous control must be scored “hemozygous” o The wild-type control must be scored “wild type” o The control without template should only show background levels of fluorescence • A sample is scored as follows: the homozygote: the target S/B exceeds an acceptable threshold ratio, e.g., 2 and the S/B of the pre-insertion locus is below an acceptable threshold ratio, e.g., 1 o hemizygote: both the target and pre-insertion locus S/B exceed an acceptable threshold ratio, e.g., 2 o wild type: the target S/B is below an acceptable threshold ratio, e.g., 1 and the S/B of the pre-insertion locus exceeds an acceptable threshold ratio, e.g., 2 o Inconclusive: the target and pre-insertion locus S/B are below an acceptable threshold, e.g. 1

[0217] Figure 3 shows an example of the result of the method (from 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 soybean samples conventional.

2.3. REAL-TIME PCR METHOD FOR ANALYSIS OF EE-GM5 IDENTITY AND ZYGOSITY

[0218] The method describes a Real-Time quantitative polymerase chain reaction detection method for analyzing the zygosity status of DNA sequences from the EE-GM5 event in DNA samples obtained from biological samples, such as materials from plant (e.g., leaf or seed) using standard DNA extraction procedures.

[0219] The method description outlines the reaction reagents, the oligonucleotide primer and probe sequences, the thermocycling conditions required to perform the reaction, including the fluorescent reader scenarios found appropriate for amplicon detection. It also provides general recommendations on the nature and use of control samples. Additionally, guidance is provided for data analysis and interpretation.

[0220] This method is not variety dependent and can be used as an alternative method of analyzing zygosity to the method described in section 2.2 above.

2.3.1. METHOD DESIGN

[0221] The method uses Taqman chemistry and Real-Time PCR principles to quantify the relative copy number of a specific EE-GM5 sequence.

[0222] The method includes an EE-GM5 specific reaction to quantify EE-GM5 copy number and a taxon specific reaction for normalization of EE-GM5 copy number.

[0223] Samples containing the EE-GM5 insertion sequence in a homozygous state have a relative copy number that is twice as high as hemizygous samples. Azygous samples do not amplify the EE-GM5 sequence.

2.3.2. DEEE-GM5 SPECIFIC REACTION

[0224] 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 insertion fragment for the EE-GM5 event.

[0225] A probe, TM1788, using FAM as a fluorescent label and BHQ1 as a quencher, was designed to quantify the amplified sequence.

[0226] Forward primer targeting the T-DNA sequence of EE-GM5: PRIM1038 5'- gAgCCACCTTCCTTTTCCACTA -3' (SEQ ID NO: 12) Reverse primer targeting the 3' flanking sequence of EE-GM5: PRIM1039 5'- ATAgggTTACTgCTTCgTAAAATAAgCA -3' (SEQ ID NO: 12) Probe targeting the junction of EE-GM5 T-DNA and its 3' flanking sequence: TM1788 FAM 5'- CgCgTCCATgATgCTgCgACTATg -3' BHQ-1 (SEQ ID NO: 14)

2.3.3. TAXON-SPECIFIC REACTION

[0227] Two primers, KVM164 and KVM165, were designed to amplify a 102 bp amplicon from the endogenous soybean lectin1 gene sequence.

[0228] A probe, TM1242, using JOE as a fluorescent label and BHQ1 as a quencher, was designed to quantify the amplified sequence.

[0229] Forward primer targeting the endogenous Lectin 1 gene sequence: KVM164 5'- CTTTCTCgCACCAATTgACA -3' (SEQ ID NO: 15) Reverse primer targeting the endogenous Lectin 1 gene sequence: KVM165 5'- TCAAACTCAACAgCgACgAC -3' (SEQ ID NO: 16) Probe targeting the endogenous Lectin 1 gene sequence: TM1242 JOE 5'- CCACAAACACATgCAggTTATCTTgg -3' BHQ1 (SEQ ID NO: 17) 2.3.4. COMPOSITION OF THE REACTIONAL MIXTURE Notes: • The 2x PerfeCta qPCR FastMix II, LOW ROX was provided by Quanta Bioscience. Other enzyme buffers can be used but performance must be verified. • The primer and labeled probes were ordered from Integrated DNA Technologies • * The amount of template DNA per reaction may vary but must be verified 2.3.5. THERMOCYCLING CONDITIONS 10°C Notes: • ** Fluorescent reading is performed in each cycle, after completion of the primer elongation step at 60°C • Thermocycling conditions have been validated for use on a ViiA7 and Quantstudio 7 Real-Time PCR device Other equipment can be used but performance must be checked.

2.3.6. CONTROL SAMPLES

[0230] The following control samples must be included in the experiment to validate test sample results • Homozygous control: a DNA sample containing the target sequence in a homozygous state • Hemozygous control: a DNA sample containing the target sequence in a a hemozygous state • Wild-type control: a DNA sample not containing the target sequence • No template control: a water sample 2.3.7. DATA ANALYSIS • Data analysis is performed using the ddCt method. In this method, the copy number is calculated for all samples relative to a chosen reference sample. It is recommended to use the hemizygous control as the reference sample. • Control samples must give the expected result, i.e.: o The homozygous control must be scored “homozygous” o The hemozygous control must be scored “hemozygous” o The wild-type control must be scored “wild type” o The control without mold should only show background levels of fluorescence • A sample is scored as follows: o homozygote: relative copy number is 2 +/- an acceptable threshold, e.g., 0.5 o hemozygote: relative copy number is 1 +/- an acceptable threshold, e.g., 0.25 o Wild type: the relative copy number is 0 + an acceptable threshold, e.g., 0.1 o Inconclusive: the relative copy number is outside the acceptable ranges for homozygous, hemizygous, and wild-type samples.

2.3.8. REAL-TIME PCR METHOD FOR ANALYSIS OF LOW-LEVEL PRESENCE OFEE-GM5

[0231] This method describes a detection method for analyzing the Low Level Presence of event EE-GM5 DN sequences obtained from bulk plant materials (e.g., leaf or seed) or processed materials (e.g., .e.g., food or feed containing processed soybeans) using standard DNA extraction procedures.

[0232] The method description outlines the reaction reagents, the oligonucleotide primer and probe sequences, and the thermocycling conditions required to carry out the reaction. It also provides general recommendations on the simultaneous use of a taxon-specific method to support data analysis and interpretation of results. Additionally, recommendations on the nature and use of control samples are provided.

[0233] It is noted that alternative methods may be available for the intended purpose, including but not limited to digital droplet PCR methods. Digital droplet PCR methods use Endpoint methods for event identity analysis, as described in section 1.1, in combination with subsampling principles on the extracted DNA sample. In this method, the low-level presence of the event is determined based on the ratio of DNA subsamples considered positive and negative for the event sequence.

2.4.1 METHOD DESIGN

[0234] The method uses Taqman chemistry and Real-Time PCR principles to detect or quantify low levels of EE-GM5 in a DNA sample.

[0235] Two primers, PRIM1040 and PRIM1041, are designed to amplify an 84 bp amplicon spanning the junction region of the 5' flanking sequence with the T-DNA insertion fragment for the EE-GM5 event.

[0236] A probe, TM1789, using FAM as a fluorescent label and BHQ1 as a quencher, is designed to quantify the amplified sequence.

[0237] Forward primer targeting the EE-GM5 T-DNA sequence: PRIM1041 5'- CATTgTgCTgAATAggTTTATAgCTATgAT - 3' (SEQ ID NO: 18) Reverse primer targeting the 5' flanking sequence of EE-GM5 T-DNA: PRIM1040 5'- TCAAATCAACATgggTgACTAgAAA -3' (SEQ ID NO: 19) Probe targeting the junction of EE-GM5 T-DNA and its 5' flanking sequence: TM1789 FAM 5'- CAgTACTgggCCCTTgTggCgCT -3' BHQ-1 (SEQ ID NO: 20) 2.4.2 COMPOSITION OF THE REACTIONAL MIXTURE Water up to 20 μL Notes: • The 2x PerfeCta qPCR FastMix II, LOW ROX was provided by Quanta Bioscience. Other enzyme buffers can be used but performance must be verified. • The primer and labeled probes were ordered from Integrated DNA Technologies. • * The amount of template DNA per reaction may vary but must be checked. 2.4.3 THERMOCYCLING CONDITIONS Notes: • ** Fluorescent reading is performed in each cycle, after completion of the primer stretching step at 60 °C. • Thermocycling conditions have been validated for use in a ViiA7 and Quantstudio 7 Real-Time PCR device. Other equipment can be used but performance must be verified.

2.4.4 TAXON-SPECIFIC METHOD

[0238] A Real-Time PCR detection method targeting an endogenous sequence must be performed simultaneously on an identical amount of template DNA as used in the target-specific Real-Time PCR method. The output 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. 2.4.5 TEST SAMPLES, CALIBRATION SAMPLES AND CONTROL SAMPLES • It is recommended that all test samples be analyzed in duplicate. • A set of calibration samples is included in the experiment to generate standard curves for both target and taxon specific methods. • Additionally, the following control samples are included: o Positive control: a DNA sample containing the target sequence at the Detection Limit level, o Negative control: a DNA sample containing only the endogenous sequence o No template control: a sample of water 2.4.6 DATA ANALYSIS • For all samples, threshold cycle values (i.e., Ct values) are determined for both target and taxon specific methods. A threshold cycle is defined as the cycling number at which the amplification plot for a given sample reaches a defined signal threshold (see Figure 4) • Standard curve formulas are calculated for both target and taxon specific methods using the Ct values and the number of genome copies of the calibration samples • Standard curve parameters must meet the acceptance criteria for slope and linearity (R2), e.g. o -3.2 < slope < -3, 6 o R2 > 0.98 • For all samples, the number of copies in the genome for the target and endogenous method is calculated using linear regression analysis. • The amount of low-level presence relative to the total amount of taxon-specific DNA is determined by calculating the % ratio of copy numbers in the genome for the target and taxon-specific method. • Control samples must give the expected result, i.e.: o The positive control must be scored “detected” o The negative control must be scored “not detected” o The no template control must only show background fluorescent levels • A sample is scored as follows: o Detected: low-level presence is above the detection limit for all replicates, taking into account method measurement uncertainty o Not detected: low-level presence is below the detection limit for all replicates, taking into account the measurement uncertainty of the method o Inconclusive: replicate samples are inconsistent scores

[0239] Figure 4 shows an example of the result of the method performed on calibration samples.

3. INTROGRESSION OF EE-GM5 IN PREFERRED CULTIVARS

[0240] The elite event EE-GM5 was introduced by repeated backcrossing into six different elite soybean lines. The lines were selected to represent a range of maturities: two lines of MG I, one line of MG III, two lines of MG VI and one line of MG IX. One of the MG I lines and the MG III line carried the Rhg1 native resistance allele from PI88788 and one of the MG VI lines carried the Rhg1 and Rhg4 native resistance alleles from PI437654. The other three lines were susceptible to SCN.

[0241] Likewise, in initial testing, in several experiments, no biologically significant differences were observed for Cry14Ab-1 from HPPD-4 protein expression levels measured in leaves of greenhouse-grown plants (as measured with ELISA or Western blot (only normal assay variation was seen)) and no significant differences were seen in the standard greenhouse SCN assay results (measuring % reduction in SCN cysts vs. the Thorne control) when event EE-GM5 was introgressed from the Thorne background into other soybean germplasm backgrounds (of different maturity, at different stages of introgression), compared to what was found for EE-GM5 in the Thorne background.

[0242] The introgression of the EE-GM5 elite event into other soybean cultivars does not significantly influence any of the desirable phenotypic or agronomic characteristics of these cultivars (no linkage drag) while expression of the transgenes meets commercially acceptable levels. This confirms the status of the EE-GM5 event as an elite event.

[0243] Furthermore, the EE-GM5 elite event is advantageously combined with other elite soybean transformation events. Particularly useful plants according to the invention are plants containing EE-GM5 combined with another soybean transformation event, or a combination of more than one other soybean transformation event, such as those listed in the databases of various regulatory agencies. national or regional events, including but not limited to Event MON87751 (described in WO2014201235 and USDA-APHIS Petition 13-337-01p), Event pDAB8264.42.32.1 (described in WO2013010094), Event DAS-81419-2 (also known as as ConkestaTM Soybean, described in WO2013016527 and USDA-APHIS Petition 12-272-01p), Event EE-GM3 (also known as FG-072, MST-FG072-3, described in WO2011063411, USDA-APHIS Petition 09-328 -01p), Event SYHT0H2 (also known as 0H2, SYN- 000H2-5, described in WO2012/082548 and 12-215-01p), Event DAS-68416-4 (also known as Enlist Soybean, described in WO2011/066384 and WO2011/066360, USDA-APHIS Petition 09-349-01p), Event DAS-81615-9 (described in WO2014004458), Event DAS-44406-6 (also known as Enlist E3, DAS-44406-6, described in WO2012 /075426 and USDA-APHIS 11-234-01p), Event MON87708 (Xtend Soybeans, described in WO2011/034704 and USDA-APHIS Petition 10-188-01p), Event MON89788 (also known as Genuity Roundup Ready 2 Yield, described in WO2006/130436 and USDA-APHIS Petition 06-178-01p), Event DAS-14536-7 (described in WO2012/075429), Event 40-3-2 (also known as RoundUp Ready, MON-04032-6, described in USDA-APHIS Petition 93-258-01), Event A2704-12 (also known as LL27, ACS-GM005-3, described in WO2006108674 and USDA-APHIS Petition 96-068-01p), Event 127 (also known as BPS-CV127-9, described in WO2010/080829), Event A5547-127 (also known as LL55, ACS-GM006-4, described in WO2006108675 and in USDA-APHIS Petition 96-068-01p), Event MON87754 (also known as Vistive III, MON-87754-1, described in WO2010/024976), Event HOS (also known as DP-305423-1, Plenish High Oleic Soybean, described in WO2008054747), Event MON87701 (also known as MON- 87701-2, described in WO2009064652 and USDA-APHIS Petition 09-082-01p), Event MON 87705 (also known as MON-87705-6, described in WO2010/037016 and USDA-APHIS Petition 09-201-01p) , Event MON87712 (also known as MON-87712-4, described in WO2012/051199), Event pDAB4472-1606 (also known as Event 1606, described in WO2012/033794), Event 3560.4.3.5 (also known as DP-356043- 5, described in WO2008/002872), Event MON87769 (also known as MON-87769-7, described in WO2009102873 and USDA-APHIS Petition 09-183-01p) or any combination of EE-GM5 with several of these other events GM soybeans, such as a combination of EE-GM5 with any of the following combinations: Event MON98788 x MON87708 (also known as Roundup Ready 2 Xtend Soybeans, MON-87708-9 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 WO2011063413), Event MON 87701 x MON 89788 (aka Intacta RR2 Pro Soybean, MON-87701-2 x MON-89788-1), DAS-81419-2 x DAS-44406-6 (also known as Conkesta™ Enlist E3™ Soybean, DAS-81419-2 x DAS-44406-6 ), Event DAS- 81419-2 x Event DAS-68416-4 (described in WO2013016516), Event DAS- 68416-4 x MON Event 89788 (also known as Enlist™ RoundUp Ready® 2 Soybean, DAS-68416-4 -89788-1), Event MON-87769-7 x Event MON-89788-1 (aka Omega-3 -6 x MON-89788-1), MON 87769 x MON 89788 (also known as Omega-3 x Genuity Roundup Ready 2 Yield Soybeans, MON-87769-7 x MON-89788-1).

[0244] As used in the claims below, unless otherwise clearly indicated, the term “plant” is intended to encompass plant tissues, at any stage of maturity, as well as any cells, tissues or organs taken from of or derived from any such plant, including without limitation any seeds, leaves, stems, flowers, roots, single cells, gametes, cell cultures, tissue cultures or protoplasts.

[0245] The reference seed comprising the elite event EE-GM5 was deposited with the ATCC (10801 University Blvd., Manassas, VA 201102209) on November 9, 2016, under the ATCC accession number PTA-123625, and the its viability has been confirmed. Alternative names for EE-GM5 are event GMB151 or BCS-GM151-6.

[0246] The above description of the invention is intended to be illustrative and not limiting.

[0247] Various changes or modifications to the described modalities may occur to those skilled in the art. These can be done without departing from the spirit or scope of the invention.

Claims (5)

1. PAIR OF PRIMER, suitable for use in a specific detection of EE-GM5, characterized in that it comprises a first primer comprising a sequence that, under optimized detection conditions, specifically recognizes a sequence within the T-DNA 5' flanking region or 3' of the EE-GM5-inserted T-DNA and a second primer comprising a sequence that, under optimized sequence conditions, specifically recognizes a sequence within the EE-GM5-inserted T-DNA contiguous with said 5' or 3' region flanking region, said T-DNA flanking region comprising the nucleotide sequence of SEQ ID NO: 5 from nucleotide 1 to nucleotide 166 or of SEQ ID NO: 24 from nucleotide 1 to nucleotide 1113, said flanking region comprising 3' T-DNA flanking region the complement nucleotide sequence of SEQ ID NO: 6 from nucleotide 359 to nucleotide 691 or the complement nucleotide sequence of SEQ ID NO: 25 from nucleotide 359 to nucleotide 1449, said 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 position 1 to nucleotide position 7459 or its complement or the nucleotide sequence of SEQ ID NO: 23 from nucleotide 1114 to nucleotide 8572 or the its complement. 2. KIT, suitable for use in a specific detection of EE-GM5, characterized in that it comprises the pair of primers, as defined in claim 1. 3. METHOD OF DETECTING THE PRESENCE OF THE EE-GM5 ELITE EVENT, in biological samples by means of hybridization with a substantially complementary labeled nucleic acid probe in which the probe:target nucleic acid ratio is amplified by recycling the acid sequence target nucleic acid, the method being characterized by comprising: a) hybridizing said target nucleic acid sequence with a 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 said first nucleic acid oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 6 from nucleotide position 341 to nucleotide position 358 or its complement; b) hybridizing said target nucleic acid sequence with 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 sequence of nucleotides 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 of said first or second oligonucleotides is labeled for said nucleic acid probe being labeled; c) cleaving only the labeled probe within the probe duplex: target nucleic acid sequence with an enzyme that causes selective cleavage of the probe resulting in dissociation of the duplex, leaving the target sequence intact; d) recycling the target nucleic acid sequence by repeating steps (a) to (c); and e) detecting cleaved labeled probe, thereby determining the presence of said target nucleic acid sequence. 4. METHOD FOR DETECTING THE PRESENCE OF THE EE-GM5 ELITE EVENT in biological samples, or for determining the state of zygosity of a plant, plant material or seed, characterized by comprising EE-GM5, wherein said method is according to any one of Examples 2.1, 2.2, 2.3 or 2.4 5. METHOD FOR DETERMINING THE ZYGOSITY STATE of a plant, plant material or seed comprising the elite event EE-GM5, characterized by comprising the amplification of DNA fragments of between 50 and 1000 bp from a nucleic acid present in said biological samples using a polymerase chain reaction with at least three primers, two of said primers specifically recognizing pre-insertion plant DNA, the third of said primers recognizing a sequence within the inserted T-DNA.