HK1138319A - Insect resistant cotton plants and methods for identifying same - Google Patents

Description

Insect-resistant cotton plants and methods for identifying same

Technical Field

The present invention relates to transgenic cotton plants, plant material and seeds characterized in that they comprise a specific transformation event, in particular the presence of a gene encoding a protein conferring insect resistance at a specific site in the cotton genome. The cotton plants of the invention combine an insect-resistant phenotype, an agronomic trait, genetic stability, and adaptability to different genetic backgrounds (equivalent to non-transformed cotton lines in the insect-free state). The invention further provides methods and kits for identifying the presence of plant material comprising in particular transformation event EF-GH5 in a biological sample.

Background

Phenotypic expression of transgenes in plants depends on: the structure of the gene itself, and its location in the plant genome. At the same time, the presence of transgenes (in the foreign DNA) at different locations in the genome will affect the overall phenotype of the plant in different ways. The successful agronomic or industrial introduction of a commercially interesting trait into a plant by genetic manipulation is a long-term process, depending on various factors. The actual transformation and regeneration of transgenic plants is only the first step in a series of selection steps, including extensive genetic characterization, breeding, field trial evaluation, and ultimately, selection of elite lines.

Cotton fiber is the most important textile raw material worldwide. Approximately 80,000,000 acres of cotton are harvested globally each year. Cotton is the fifth crop of the united states in terms of area yield, which was grown over 15,000,000 acres in 2000.

The most destructive insect species that live on cotton are: helicoverpa zea (corn ear bollworm or cotton bollworm), Helicoverpa armigera (corn earworm), Heliothis virescens (tobacco budworm) and Helicoverpa punctigera.

In view of the concerns for new foods and new feeds, the separation of GMO and non-GMO products, and the identification of proprietary materials, the clear identification of superior events is becoming increasingly important. Ideally, the identification method is fast and simple and does not require extensive laboratory equipment. Furthermore, the method should provide results that allow unambiguous determination of the superior event without expert interpretation, but if necessary, the results prove to be valid under expert scrutiny. Described herein are specific tools used to identify elite event EF-GH5 in biological samples.

In the development of insect-resistant cotton (Gossypium hirsutum), EE-GH5 was identified as an elite event from a population of transgenic cotton plants, comprising a gene encoding an insecticidal crystal protein from Bacillus thuringiensis (Bacillus thuringiensis). Transgenic cotton plants contain a chimeric gene encoding a Bt insecticidal crystal protein (as described in WO 03/093484) under the control of a plant-expressible promoter.

Cotton plants comprising insect-resistant genes have been disclosed in the art. However, no prior art disclosure teaches or suggests the present invention.

Summary of The Invention

The present invention relates to transgenic cotton plants, or seeds, cells or tissues thereof, comprising stably integrated in their genome an expression cassette comprising an insect-resistant gene comprising the coding sequence of the cry1Ab gene (as described in example 1.1 herein), which is insect-resistant, the transgenic plants and materials thereof having agronomic performance substantially equivalent to non-transgenic isogenic lines under insect-free pressure. Under insect stress, the transgenic plants will have an agronomic phenotype superior to that of non-transgenic plants.

According to the present invention, a cotton plant, or seed, cell or tissue thereof, comprises elite event EE-GH 5.

More precisely, the present invention relates to transgenic cotton plants, seeds, cells or tissues thereof, the genomic DNA of which is characterized in that a fragment specific for EE-GH5 is obtained when analyzed by the PCR identification method described herein using two primers directed to the 5 'or 3' flanking region of EE-GH5 and the foreign DNA, respectively. The primers can be directed to SEQ ID nos: 1 and the foreign DNA, for example, may comprise SEQ ID nos: 7 and SEQ ID No: 8, resulting in a DNA fragment of 100-700bp, preferably about 129 bp. The primers can also be directed to SEQ ID No: 2 and the foreign DNA, for example, may comprise SEQ ID nos: 3 and SEQ ID No: 6, to obtain a DNA fragment of 300-700bp, preferably about 369 bp.

Reference seeds containing elite events of the invention have been deposited at the ATCC under accession number PTA-8171. One embodiment of the present invention is seed comprising elite event EE-GH5, deposited under ATCC accession No. PTA-8171, which can be grown into cotton plants resistant to insects, particularly to helicoverpasp or Heliothis sp. The seed having ATCC deposit number PTA-8171 is a seed bank comprising at least about 95% of the transgenic seed grains homozygous for said transgene, comprising the elite event of the present invention, which can be grown into insect-resistant plants, and which can thus also be glufosinate-tolerant plants. Seeds can be sown and the growing plants can be treated with glufosinate as described herein to yield one hundred percent glufosinate tolerant plants containing the superior events of the invention. The invention further relates to cells, tissues and progeny and descendants from plants grown from the seed deposited with the ATCC under accession number PTA-8171 comprising the elite event of the invention. The present invention further relates to a plant obtainable by the propagation and/or breeding of a cotton plant comprising the elite event of the present invention grown from the seed deposited with the ATCC under accession No. PTA-8171. The present invention also relates to cotton plants comprising elite event EE-GH 5.

The invention further relates to a method for identifying a transgenic plant, or a cell or tissue thereof, comprising elite event EE-GH5, based on the identification of the presence of a characteristic DNA sequence, or an amino acid encoded by such a DNA sequence, in the transgenic plant, cell or tissue. According to a preferred embodiment of the invention, such characteristic DNA sequences are 15bp sequences, preferably 20bp, most preferably 30bp or more, which comprise the insertion site of the event, i.e.part of the foreign DNA and part of the cotton genome adjacent thereto (5 'or 3' flanking region), which allows the elite event to be identified in particular.

The invention further relates to a method for identifying elite event EE-GH5 in biological samples, based on primers or probes that can specifically recognize the 5 'and/or 3' flanking sequences of EE-GH 5.

More specifically, the present invention relates to a method comprising amplifying a nucleic acid sequence present in a biological sample. The method uses at least two primers for polymerase chain reaction, one recognizing the 5 'or 3' flanking region of EE-GH5 and the other recognizing the sequence in the foreign DNA, preferably to obtain a DNA fragment of 100-500 bp. The primers recognize the sequence within the 5 'flanking region of EE-GH5 (the sequence at positions 1-98 of SEQ ID No.1, or the sequence at positions 1-563 of SEQ ID No.16) or the 3' flanking region of EE-GH5 (the complementary sequence at positions 41-452 of SEQ ID No.2), and the sequence within the foreign DNA (the complementary sequence at positions 99-334 of SEQ ID No.1, or the sequence at positions 1-40 of SEQ ID No.2), respectively. The primer recognizing the 5' flanking region may comprise the nucleotide sequence of SEQ ID No.7 or SEQ ID No.17, and the primer recognizing the sequence within the foreign DNA may comprise the nucleotide sequence of SEQ ID No.8, SEQ ID No.18 or SEQ ID No.19 as described herein. The primer recognizing the 3' flanking region may comprise the nucleotide sequence of SEQ ID No.6 as described herein and the primer recognizing the sequence within the foreign DNA may comprise the nucleotide sequence of SEQ ID No.3 as described herein.

The invention more particularly relates to a method for identifying elite event EE-GH5 in biological samples, which method comprises amplifying a nucleic acid sequence in a biological sample by polymerase chain reaction using two primers having the nucleotide sequences of SEQ ID nos. 3 and 6, respectively, resulting in a DNA fragment of about 369 bp.

The current elite event comprises a foreign DNA sequence that is slightly rearranged compared to the DNA originally used in the transformation procedure. This rearrangement is unique to elite event EE-GH 5. Thus, the present invention also relates to a method for identifying elite event EE-GH5 in biological samples, which method comprises obtaining a DNA fragment of about 262bp by polymerase chain reaction, using primers flanking a unique rearrangement region of the foreign DNA in EE-GH5, such as primers comprising or having the nucleotide sequence of SEQ ID No.10 or SEQ ID No. 11.

The present invention further relates to the specific flanking sequences of EE-GH5 described herein, which can be used to develop specific identification methods for identifying EE-GH5 in biological samples. Such specific flanking sequences may also be used as reference control materials in identification assays. More particularly, the invention relates to 5 'and/or 3' flanking regions of EE-GH5 which may be used to develop specific primers and probes as further described herein. The invention also relates to nucleic acid molecules spanning a specific rearrangement within the foreign DNA of EE-GH5, e.g. a nucleic acid molecule comprising the nucleotide sequence from position 52 to 88 of SEQ ID No.12, or a nucleic acid molecule comprising the sequence of SEQ ID No. 12. Also suitable as reference material are nucleic acid molecules of preferably about 369bp, which comprise a sequence that can be amplified by primers having the nucleotide sequences of SEQ ID Nos. 3 and 6.

The invention further relates to methods for identifying the presence of EE-GH5 in a biological sample based on the use of these specific primers and probes. The primer may consist of a nucleotide sequence of 17 to about 200 contiguous nucleotides selected from the nucleotide sequence of nucleotides 1 to 98 of SEQ ID No.1, the nucleotide sequence of nucleotides 1 to 563 of SEQ ID No.16, or the complement of the nucleotide sequence of nucleotides 41 to 452 of SEQ ID No.2, in combination with a primer consisting of a nucleotide sequence of 17 to about 200 contiguous nucleotides selected from the complement of the nucleotide sequence of nucleotides 99 to 334 of SEQ ID No.1, and the nucleotide sequence of nucleotides 1 to 40 of SEQ ID No. 2. The primer may also comprise a nucleotide sequence located at the 3' end, further comprising an unrelated sequence, or a sequence derived from said nucleotide sequence but comprising a mismatch.

The invention further relates to a kit for identifying elite event EE-GH5 in biological samples, comprising at least one primer or probe that can specifically recognize the 5 'or 3' flanking region of EE-GH5 or can specifically recognize a specific rearrangement within a foreign DNA sequence in EE-GH 5.

In addition to primers that specifically recognize the 5 'or 3' flanking region of EE-GH5, the kits of the invention may comprise a second primer that specifically recognizes a sequence within the foreign DNA of EE-GH5 for use in a PCR identification protocol. The kit of the invention may comprise at least two specific primers, one recognizing a sequence within the 5' flanking region of EE-GH5 and the other recognizing a sequence within the foreign DNA. The primer identifying the 5' flanking region may comprise the nucleotide sequence of SEQ ID No.3 and the primer identifying the transgene may comprise the nucleotide sequence of SEQ ID No.6 or any other primer described herein.

The invention further relates to a kit for identifying elite event EE-GH5 in biological samples. The kit comprises PCR primers having the nucleotide sequences of SEQ ID Nos. 3 and 6 for use in the EE-GH5PCR identification protocol described herein. The invention further relates to a kit for identifying elite event EE-GH5 in biological samples. The kit comprises PCR primers having the nucleotide sequences of SEQ ID Nos. 10 and 11.

The invention also relates to a kit for identifying elite event EE-GH5 in biological samples. The kit comprises a specific probe having a sequence corresponding to (or complementary to) 80-100% sequence identity with a specific region of EE-GH 5. Preferably, the sequence of the probe corresponds to a specific region comprising part of the 5 'or 3' flanking region of the EE-GH5, or to a rearranged region of the foreign DNA specific for EE-GH 5. Most preferably, the specific probe has (or is complementary to) a sequence having 80-100% sequence identity with the nucleotide sequence at positions 78-119 of SEQ ID No.1, the nucleotide sequence at positions 20-61 of SEQ ID No.2 or the nucleotide sequence at positions 52-88 of SEQ ID No. 12.

In accordance with another aspect of the invention, the disclosed DNA sequences comprise the insertion site of the event, as well as a polynucleotide of genomic DNA from cotton and foreign DNA (transgene) long enough to be useful as primers or probes for detecting EE-GH 5. Such sequences, on each side of the insertion site, may comprise at least 9 nucleotides of cotton genomic DNA and a similar number of nucleotides of the exogenous DNA (transgene) of EE-GH5, respectively. Most preferably, such DNA sequences comprise at least 9 nucleotides of cotton genomic DNA and a similar number of nucleotides of foreign DNA (adjacent to the insertion site of SEQ ID No.1 or 2).

The methods and kits encompassed by the present invention may be used for various purposes, such as, but not limited to, the following: identifying the presence of EE-GH5 in a plant, plant material or product (such as, but not limited to: fresh or processed food or feed comprising or derived from plant material); additionally or alternatively, the methods and kits of the invention can be used to identify transgenic plant material with the aim of segregating transgenic or non-transgenic material; additionally or alternatively, the methods and kits of the present invention can be used to determine the quality of plant material comprising EE-GH5 (i.e. the percentage of pure material).

The invention further relates to the 5 'and/or 3' flanking regions of EE-GH5, and to specific primers and probes developed from the 5 'and/or 3' flanking sequences of EE-GH 5. The invention also relates to a special rearrangement region of EE-GH5, and a specific primer or probe designed according to the region.

The present invention also relates to genomic DNA obtained from a plant comprising elite event EE-GH 5. This genomic DNA can be used as a reference control material in performing the identification assays described herein.

Brief Description of Drawings

The following examples are not intended to limit the invention to the particular embodiments described, but may be understood in conjunction with the accompanying drawings (incorporated herein by reference). In these figures:

FIG. 1 shows by way of illustration the relationship between the referenced nucleotide sequences and the primers. Black bar shape: exogenous DNA; striped black bars: a rearrangement in EE-GH5 specific foreign DNA; light-colored bar shape: DNA of plant origin; striped, light-colored bars: deletion of the pre-insertion target site; arrow head: oligonucleotide primers, the numbers under the bars represent the nucleotide positions; (c) the complement of the indicated nucleotide sequence is represented.

FIG. 2 shows the results of a PCR identification protocol developed for EE-GH 5. Loading order of gel: lane 1: molecular weight markers (100bp ladder); lanes 2-3: a DNA sample from a cotton plant comprising transgenic event EE-GH 5; lanes 4-10: DNA samples from transgenic cotton plants that did not contain elite event EE-GH5 but contained a similar insect-resistant gene; lane 11: no template DNA control; lane 12: and (4) marking molecular weight.

FIG. 3 shows the results obtained for the zygosity scoring PCR protocol developed for EE-GH 5. Loading order of gel: lane 1: molecular weight markers (100bp ladder); lanes 2-3: a DNA sample from a cotton plant comprising transgenic event EE-GH5 in heterozygous form; lane 4: failure; lane 5: a control DNA sample from a wild-type cotton plant; lanes 6-7: a DNA sample from a cotton plant comprising homozygous form of transgenic event EE-GH 5; lane 8: no template DNA control; lane 9: and (4) marking molecular weight.

Detailed Description

Incorporation of recombinant DNA molecules into the genome of a plant is typically achieved by transformation of cells or tissues. The specific site of incorporation is usually determined by "random" integration.

DNA introduced into the genome of a plant by transforming a plant cell or tissue with recombinant DNA or "transforming DNA" and derived from the transforming DNA is hereinafter referred to as "exogenous DNA" and comprises one or more "transgenes". "plant DNA" in the context of the present invention refers to DNA derived from a transformed plant. Plant DNA is usually found at the same locus in corresponding wild-type plants. Exogenous DNA can be characterized by the location and structure of the incorporation site of the recombinant DNA molecule in the plant genome. The site of insertion of the recombinant DNA in the plant genome is also referred to as the "insertion site" or "target site". The insertion of recombinant DNA into a region of the plant genome called "pre-inserted plant DNA" is associated with a deletion of plant DNA, called "target site deletion". "flanking region" or "flanking sequence" as used herein refers to a DNA sequence of at least 20bp, preferably at least 50bp, up to 5000bp, different from the introduced DNA; preferably, the DNA is derived from the plant genome, either in and adjacent to the region immediately upstream of the exogenous DNA or in and adjacent to the region immediately downstream of the exogenous DNA. Transformation procedures that result in random integration of the exogenous DNA will result in transformants with different flanking regions that are characteristic and unique for each transformant. When recombinant DNA is introduced into a plant by traditional crossing, its insertion site in the plant genome, or its flanking regions, are generally not altered. The term "insertion region" as used herein refers to a region corresponding to at least 40bp, preferably at least 100bp, up to 10000bp, comprised in a sequence comprising upstream and/or downstream flanking regions of foreign DNA in the plant genome. In view of the slight differences due to intraspecies mutations, the insert region when crossed into a plant of the same species will retain at least 85%, preferably 90%, more preferably 95%, and most preferably 100% sequence identity to the sequences of the upstream and downstream flanking regions comprising the foreign DNA in the transformed plant.

The definition of an event is: (artificial) loci that, as a result of genetic engineering methods, carry a transgene comprising at least one copy of a gene of interest. A typical allelic state of an event is the presence or absence of exogenous DNA. The phenotypic characteristic of an event is the expression of a transgene. At the genetic level, an event is part of the genetic makeup of a plant. At the molecular level, the events are characterized by a restriction map (such as can be determined by Southern blotting), upstream and/or downstream flanking regions of the transgene, and the location and/or molecular structure of the molecular markers of the transgene. Generally, transformation of a plant with a transforming DNA comprising at least one gene of interest results in a population of transformants comprising a number of independent events, each of which is unique.

A good event, as used herein, is an event selected from a group of events. The event is obtained by transformation with the same transforming DNA or by backcrossing with the transformed plant, based on the expression and stability of the transgene and compatibility with the optimal agronomic traits of the plant comprising the elite event. Thus, the criteria for good event selection are one or more, preferably two or more, preferably all of the following criteria:

a) the presence of the exogenous DNA does not impair other desirable characteristics of the plant, such as those associated with agronomic performance or commercial value;

b) events are characterized by well-defined molecular structures that are stably inherited and for which appropriate tools for identifying controls can be developed;

c) under both heterozygous (or hemizygous) and homozygous conditions for the event, and at commercially acceptable levels within a range of environmental conditions (where the plant carrying the event may be used in a normal agricultural setting), the gene of interest exhibits correct, appropriate and stable spatio-temporal phenotypic expression.

The foreign DNA is preferably associated with a location in the plant genome which allows it to be easily introgressed into a desired commercial genetic background.

The event status as a elite event is confirmed by introgressing the elite event into different relevant genetic environments and observing its compliance with one, two or all of the above criteria (e.g. a, b, c).

Thus, a "elite event" refers to a locus comprising foreign DNA, which meets the above criteria. A plant, plant material or progeny thereof (e.g., a seed) contains one or more elite events in its genome.

The tools developed for identifying elite events, or plants, plant material comprising elite events, or products produced from plant material comprising elite events, are based on specific genomic characteristics of elite events, such as specific restriction maps of genomic regions comprising exogenous DNA, molecular markers, or flanking region sequences of exogenous DNA.

Once one or both flanking regions of the foreign DNA have been sequenced, primers and probes can be designed which specifically recognize this sequence(s) in the nucleic acid (DNA or RNA) of the sample by means of molecular biological techniques. For example, PCR methods can be developed to identify elite events in biological samples (e.g., plants, plant material, or products comprising plant material). This PCR method is based on at least two specific "primers", one recognizing sequences within the 5 'or 3' flanking region of the elite event and the other recognizing sequences within the foreign DNA. Preferably, the primer has a sequence of 15-35 nucleotides which, under optimized PCR conditions, "specifically recognizes" the sequence within the 5 'or 3' flanking region of the elite event and the foreign DNA of the elite event, respectively, such that a specific fragment ("integration fragment" or differential amplicon) can be amplified from a nucleic acid sample comprising the elite event. This means that under optimized PCR conditions only targeted integration fragments can be amplified, but not other sequences or foreign DNA within the plant genome.

Suitable PCR primers for the present invention may be the following:

-an oligonucleotide having a length of 17nt to about 100nt comprising at its 3 ' end a nucleotide sequence of at least 17 consecutive nucleotides, preferably 20 consecutive nucleotides, selected from a 5 ' flanking sequence (the nucleotide sequence of nucleotides 1 to 98 of SEQ ID No1, or the nucleotide sequence of nucleotides 1 to 563 of SEQ ID No 16) (a primer recognizing the 5 ' flanking sequence); or

-an oligonucleotide having a length of 17nt to about 200nt comprising at its 3 ' end a nucleotide sequence of at least 17 consecutive nucleotides, preferably 20 consecutive nucleotides, selected from the 3 ' flanking region (the complement of the nucleotide sequence of nucleotides 41-452 of seq id No 2) (a primer recognizing the 3 ' flanking sequence);

-an oligonucleotide having a length of 17nt to about 200nt, comprising at its 3' end a nucleotide sequence of at least 17 consecutive nucleotides, preferably 20 nucleotides, selected from the group consisting of the inserted DNA sequence (complementary sequence to the nucleotide sequence of nucleotides 99 to 334 of seq id No 1) (primer recognizing the foreign DNA);

-an oligonucleotide having a length of 17nt to about 40nt comprising a nucleotide sequence of at least 17 consecutive nucleotides, preferably 20 nucleotides, selected from the group consisting of an intervening DNA sequence (nucleotide sequence of nucleotides 1-40 of SEQ ID No 2).

The primer may of course be longer than the mentioned 17 consecutive nucleotides, and may for example be 20, 21, 30, 35, 50, 75, 100, 150, 200nt in length or longer. The primer may consist entirely of a nucleotide sequence selected from the mentioned flanking sequences and the foreign DNA sequence. However, the nucleotide sequence of the primer is less critical at the 5 'end (i.e., beyond the 17 consecutive nucleotides at the 3' end). Thus, the 5' sequence of the primer may consist of a nucleotide sequence selected from the flanking sequences or the foreign DNA, but may also suitably contain some (e.g.1, 2, 5, 10) mismatches. The 5' sequence of the primer may even consist entirely of a nucleotide sequence unrelated to the flanking sequence or the foreign DNA, for example, a nucleotide sequence representing a restriction enzyme recognition site. Preferably, such unrelated or flanking DNA sequences with mismatches should preferably be no longer than 100 nucleotides, more preferably no longer than 50 or even 25 nucleotides.

Furthermore, if the 17 contiguous nucleotides at the 3 'end are not derived solely from the foreign DNA or plant-derived sequence of SEQ ID No1 or 2, a suitable primer may comprise or consist of a nucleotide sequence spanning the junction region between the plant DNA-derived sequence and the foreign DNA sequence (located between nucleotides 98-99 of SEQ ID No1 and nucleotides 40-41 of SEQ ID No 2) at its 3' end.

It will be immediately apparent to the skilled artisan that appropriately selected pairs of PCR primers should not comprise sequences that are complementary to each other.

For the purposes of the present invention, "SEQ ID No: the complementary sequence of the nucleotide sequence represented by X "is a nucleotide sequence in which a nucleotide is replaced with a complementary nucleotide according to the Chargaff's ruleAnd a nucleotide sequence derived from the represented nucleotide sequence and read in the 5 '-3' direction, which is the reverse direction of the represented nucleotide sequence.

Examples of suitable primers are: the oligonucleotide sequences of SEQ ID No 7 or SEQ ID No 17 (primers recognizing the 5 'flanking sequence), the oligonucleotide sequences of SEQ ID No 8, SEQ ID No 18 or SEQ ID No 19 (primers recognizing the foreign DNA, used together with the primers recognizing the 5' flanking sequence), the oligonucleotide sequences of SEQ ID No 3 (primers recognizing the foreign DNA, used together with the primers recognizing the 3 'flanking sequence), the oligonucleotide sequences of SEQ ID No 4, SEQ ID No 5, SEQ ID No6 (primers recognizing the 3' flanking sequence), and the oligonucleotide sequences of SEQ ID Nos 10 and 11 (primers recognizing the rearrangement of the specific foreign DNA in EE-GH 5).

Other examples of suitable oligonucleotide primers include or consist of the following sequences at their 3' end:

a. primers recognizing the 5' flanking sequence:

nucleotide sequence of nucleotides 58 to 77 of SEQ ID No1

Nucleotide sequence of nucleotides 85 to 104 of SEQ ID No 16

Nucleotide sequence of nucleotides 150-166 of SEQ ID No 16

Nucleotide sequence of nucleotides 150 and 171 of SEQ ID No 16

Nucleotide sequence of nucleotides 154-171 of SEQ ID No 16

Nucleotide sequence of nucleotides 87 to 104 of SEQ ID No 16

Nucleotide sequence of nucleotides 189 and 206 of SEQ ID No 16

b. Primers recognizing the foreign DNA sequence used together with primers recognizing the 5' flanking sequence:

the complement of the nucleotide sequence at nucleotide 149-170 of SEQ ID No1

The complement of the nucleotide sequence at nucleotide 169-186 of SEQ ID No1

The complement of the nucleotide sequence of nucleotide 193-212 of SEQ ID No1

The complement of the nucleotide sequence at nucleotide 161-178 of SEQ ID No1

The complement of the nucleotide sequence of nucleotide 194 and 211 of SEQ ID No1

The complement of the nucleotide sequence of nucleotide 193-211 of SEQ ID No1

The complement of the nucleotide sequence at nucleotide number 163-185 of SEQ ID No1

The complement of the nucleotide sequence of nucleotide number 194 and 210 of SEQ ID No1

The complement of the nucleotide sequence of nucleotide No. 193-210 of SEQ ID No1

The complement of the nucleotide sequence of nucleotide No. 193-209 of SEQ ID No1

c. Primers recognizing the 3' flanking sequence:

the complement of the nucleotide sequence of nucleotide 149-168 of SEQ ID No 2

The complement of the nucleotide sequence of nucleotides 154 and 173 of SEQ ID No 2

The complement of the nucleotide sequence of nucleotide No. 140-159 of SEQ ID No 2

The complement of the nucleotide sequence of nucleotide 141-160 of SEQ ID No 2

The complement of the nucleotide sequence at nucleotide 147-166 of SEQ ID No 2

The complement of the nucleotide sequence of nucleotides 148 and 167 of SEQ ID No 2

The complement of the nucleotide sequence at nucleotide 149-167 of SEQ ID No 2

The complement of the nucleotide sequence of nucleotide 153-172 of SEQ ID No 2

The complement of the nucleotide sequence of nucleotides 154-174 of SEQ ID No 2

The complement of the nucleotide sequence of nucleotide No. 160-177 of SEQ ID No 2

The complement of the nucleotide sequence of nucleotide No. 232-251 of SEQ ID No 2

The complement of the nucleotide sequence of nucleotide 140-160 of SEQ ID No 2

The complement of the nucleotide sequence of nucleotides 141-161 of SEQ ID No 2

Complementary sequence of the nucleotide sequence at nucleotide 147-165 of SEQ ID No 2

The complement of the nucleotide sequence at nucleotide number 149-166 of SEQ ID No 2

The complement of the nucleotide sequence of nucleotides 148 and 166 of SEQ ID No 2

The complement of the nucleotide sequence of nucleotide 147-167 of SEQ ID No 2

The complement of the nucleotide sequence of nucleotide 148-168 of SEQ ID No 2

The complement of the nucleotide sequence of nucleotide 153-173 of SEQ ID No 2

The complement of the nucleotide sequence of nucleotides 154-175 of SEQ ID No 2

The complement of the nucleotide sequence at nucleotide number 156 and 175 of SEQ ID No 2

The complement of the nucleotide sequence of nucleotides 232-250 of SEQ ID No 2

The complement of the nucleotide sequence of nucleotide 140-157 of SEQ ID No 2

The complement of the nucleotide sequence of nucleotide 140-161 of SEQ ID No 2

The complement of the nucleotide sequence of nucleotides 141-162 of SEQ ID No 2

The complement of the nucleotide sequence of nucleotide 148-165 of SEQ ID No 2

The complement of the nucleotide sequence of nucleotide 149-165 of SEQ ID No 2

The complement of the nucleotide sequence at nucleotide 147-168 of SEQ ID No 2

The complement of the nucleotide sequence of nucleotide 148-169 of SEQ ID No 2

The complement of the nucleotide sequence at nucleotide 156-174 of SEQ ID No 2

The complement of the nucleotide sequence of nucleotide 153-174 of SEQ ID No 2

The complement of the nucleotide sequence of nucleotide 232-249 of SEQ ID No 2

The complement of the nucleotide sequence of nucleotide No. 234 and 251 of SEQ ID No 2

The complement of the nucleotide sequence of nucleotide 141-163 of SEQ ID No 2

The complement of the nucleotide sequence of nucleotides 148 and 164 of SEQ ID No 2

The complement of the nucleotide sequence of nucleotide 147-169 of SEQ ID No 2

The complement of the nucleotide sequence of nucleotide 153-175 of SEQ ID No 2

The complement of the nucleotide sequence at nucleotide number 156-177 of SEQ ID No 2

Complementary sequence of the nucleotide sequence of nucleotides 181-198 of SEQ ID No 2

The complement of the nucleotide sequence of nucleotide 181-202 of SEQ ID No 2

The complement of the nucleotide sequence of nucleotide No. 234 and 250 of SEQ ID No 2

The complement of the nucleotide sequence at nucleotide 140-162 of SEQ ID No 2

The complement of the nucleotide sequence of nucleotide 181-203 of SEQ ID No 2

d. Primers recognizing the foreign DNA sequence used together with primers recognizing the 3' flanking sequence:

-nucleotide sequence of nucleotides 30 to 49 of SEQ ID No 2

-nucleotide sequence of nucleotides 30 to 48 of SEQ ID No 2

-nucleotide sequence of nucleotides 32 to 48 of SEQ ID No 2

-nucleotide sequence of nucleotides 32 to 49 of SEQ ID No 2

-nucleotide sequence of nucleotides 31 to 49 of SEQ ID No 2

-nucleotide sequence of nucleotides 31 to 48 of SEQ ID No 2

Nucleotide sequence of nucleotides 30 to 46 of SEQ ID No 2

As used herein, "the nucleotide sequence of nucleotides at positions X-Y of SEQ ID No. Z" means a nucleotide sequence including both nucleotide end points.

Preferably, the amplified fragment has a length of 50-500 nucleotides, preferably 100-350 nucleotides. Specific primers may have sequences that are 80-100% identical to the 5 'or 3' flanking region of the elite event and sequences within its foreign DNA, respectively, provided that mismatches may still allow specific identification of the elite event using these primers under optimized PCR conditions. However, the range of permissible mismatches can be readily determined experimentally and is known to those skilled in the art.

The following table lists the expected DNA amplicon (or integrated fragment) sizes obtained using the selected PCR primer pairs.

Primer 1	Starting position	Primer 2	End position	Amplicon length
					HVH024	58	DPA312	186	128
GHI057	1	MAE115	175	175
					GHI057	1	HVH022	251	251
GHI057	1	GHI065	369	369

Detection of the integrated fragment can be performed in various ways, such as size estimation after gel analysis. The integrated fragment can also be directly sequenced. Other sequence-specific methods for detecting amplified DNA fragments are also well known in the art.

Since the sequence of the primers and their relative positions in the genome are unique for the elite event, amplification of the integrated fragment can only occur in a biological sample comprising (the nucleic acid of) the elite event. Preferably, when PCR is performed to identify the presence of EE-GH5 in an unknown sample, control primers should be included in a set of primers with which sequences within the "housekeeping gene" of the plant variety containing the event can be amplified. Housekeeping genes are genes that are expressed in most cell types and are involved in the fundamental metabolic activity common to all cells. Preferably, the fragment amplified from the housekeeping gene is a larger fragment than the amplified integrated fragment. Other controls may also be included depending on the sample being analyzed.

Standard PCR protocols are described in the art, for example "PCR application Manual" (Roche molecular Biochemicals, 2nd edition, 1999) and other references. The conditions for optimization of PCR, including the sequence of specific primers, are specified in the "PCR identification protocol" for each elite event. However, it will be appreciated that many of the parameters of a PCR identification protocol may need to be adjusted to specific laboratory conditions and modified somewhat to achieve similar results. For example, using different methods for preparing DNA may require adjusting parameters such as the amount of primers used, the polymerase, and the annealing conditions used. Similarly, the selection of other primers may dictate other optimization conditions for the PCR identification protocol. However, these adjustments are obvious to the person skilled in the art and are furthermore described in detail in the existing PCR application manual (e.g.one of the above cited).

Alternatively, specific primers can be used to amplify the integrated fragment that serves as a "specific probe" for identifying EE-GH5 in a biological sample. Contacting nucleic acids of the biological sample with the probe under conditions that allow hybridization of the probe to its corresponding fragment of the nucleic acid, resulting in the formation of a nucleic acid/probe hybrid. The formation of a hybrid can be detected (e.g., by labeling the nucleic acid or probe), wherein the formation of a hybrid indicates the presence of EE-GH 5. Such identification methods based on hybridization (on a solid support or in solution) with specific probes have been described in the art. Preferably, the specific probe is a sequence that specifically hybridizes under optimized conditions to a region located within the 5 'or 3' flanking region of the elite event and, preferably, also to a region comprising part of the foreign DNA adjacent thereto (hereinafter referred to as the "specific region"). Preferably, the specific probe comprises a sequence of 50-500bp, preferably 100-350bp, which is at least 80%, preferably 80-85%, more preferably 85-90%, particularly preferably 90-95%, most preferably 95-100% identical (or complementary) to the nucleotide sequence of the specific region. Preferably, the specific probe will comprise a sequence of about 15-100 contiguous nucleotides that are identical (or complementary) to the specific region of the elite event.

Oligonucleotides suitable as PCR primers for detection of elite event EE-GH5 can also be used to develop PCR-based protocols to determine the zygosity status of elite events. To this end, two primers recognizing the wild type (wt) locus were designed: they point towards each other with an insertion site in between. These primers may be primers which specifically recognize the 5 'and 3' flanking sequences contained within SEQ ID Nos 1 or 2, respectively. These primers may also be those which specifically recognize the 5 'or 3' flanking sequence (for example those having the nucleotide sequence of SEQ ID No 7 or SEQ ID Nos 4-6), as well as those which recognize the region deleted upon insertion of EE-GH5 in the pre-inserted plant DNA. This set of primers, together with a third primer complementary to the transforming DNA sequence (e.g. a primer having the nucleotide sequence of SEQ ID No 3) allows a diagnostic PCR method to amplify the EE-GH5 specific locus and the wt locus. If the transgenic locus or the corresponding wt locus of the plant is homozygous the diagnostic PCR will produce a single PCR product of typical, preferably typical length for the transgenic locus or wt locus. If the plant is hemizygous for the transgenic locus, two locus specific PCR products will appear, reflecting that both the transgenic locus and the wt locus were amplified.

In addition, using the elite event specific sequence information provided herein, detection methods specific for elite event EE-GH5, different from PCR-based amplification methods, can also be developed. These alternative Detection methods include linear signal amplification Detection methods based on invasive Cleavage of specific Nucleic acid structures, also known as the Invader (TM) technique (e.g., as described in U.S. Pat. Nos. 5,985,557, "invasive Cleavage of Nucleic Acids", and 6,001,567, "Detection of Nucleic acid sequences by Invader Directed Cleavage", both of which are incorporated herein by reference). To this end, the target sequence may be a labeled first oligonucleotide comprising the nucleotide sequence of nucleotides 99 to 116 of SEQ ID No1 or a complementary sequence thereof or a labeled first oligonucleotide comprising the nucleotides 23 to 40 of SEQ ID No 2The labelled nucleic acid probe comprising the nucleotide sequence of nucleotides or the complement thereof of SEQ ID No1 may be hybridised or may be further hybridised to a second oligonucleotide comprising the nucleotide sequence of nucleotides 81 to 98 or the complement thereof of SEQ ID No 2 or the labelled nucleic acid probe comprising the nucleotide sequence of nucleotides 41 to 58 or the complement thereof of SEQ ID No 2, wherein the first and second oligonucleotides overlap by at least one nucleotide. The duplex or triplex structure obtained by hybridization may be enzymatically usedSelective probe cleavage was performed to leave the target sequence intact. The cleaved labeled probe is then detected and may produce further signal enhancement through intermediate steps.

As used herein, "kit" refers to a set of reagents for performing the methods of the invention, and more particularly, for performing the identification of elite event EE-GH5 in biological samples or determining the zygosity status of plant material comprising EE-GH 5. More particularly, a preferred embodiment of the kit of the invention comprises at least one or two specific primers for identifying elite events as described above, or three specific primers for determining the zygosity status. Alternatively, the kit further comprises any other reagents mentioned in the PCR identification protocol. Alternatively, according to another embodiment of the invention, the kit may comprise the above-mentioned specific probe, which can specifically hybridize to a nucleic acid of a biological sample to identify the presence of EE-GH 5. Alternatively, the kit may further comprise any other reagents (such as, but not limited to: hybridization buffer, label) used for the identification of EE-GH5 in a biological sample with specific probes.

The kits of the invention can be used and their ingredients specifically adjusted for quality control (e.g., purity of seed lots), for detecting the presence of elite events in plant material or materials comprising or derived from plant material (e.g., but not limited to: food products or feed products).

"sequence identity" as used herein with respect to a nucleotide sequence (DNA or RNA) refers to the number of positions having the same nucleotide divided by the number of nucleotides in the shorter of the two sequences. The alignment of the two nucleotide sequences was performed according to the Wilbur-Lipmann algorithm (Wilbur and Lipmann, 1983, Proc. Nat. Acad. Sci. USA 80: 726) using a window size of 20 nucleotides, a word length of 4 nucleotides and a gap penalty of 4. Computer-assisted analysis and interpretation of sequence data, including the sequence alignments described above, may be conveniently performed using, for example, the sequence analysis software package of the Genetics Computer Group (GCG, University of Wisconsin Biotechnology center). The sequences analyzed were considered "substantially similar" based on: these sequences have at least about 75% sequence identity, particularly at least about 80%, more particularly at least about 85%, very particularly about 90%, particularly about 95%, more particularly about 100% identity. It is clear that when an RNA sequence is expressed as being substantially similar to or having a certain sequence identity to a DNA sequence, thymine (T) of the DNA sequence is considered to be identical to uracil (U) in the RNA sequence.

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

The term "recognition", as used herein when referring to specific primers, refers to the fact that: specific primers specifically hybridize to the nucleic acid sequence in the elite event under the conditions given in the method (e.g., conditions of a PCR identification protocol), where specificity is determined by the presence of positive and negative controls.

The term "hybridization" as used herein when referring to a specific probe refers to the fact that: under standard stringency conditions, probes bind to specific regions in the nucleic acid sequence of the elite event. Standard stringency conditions as described herein refer to hybridization conditions as described herein, or conventional hybridization conditions (see Sambrook et al, 1989, Molecular Cloning: A Laboratory Manual, second edition, Cold Spring harbor Laboratory Press, NY). For example, the hybridization may comprise the steps of: 1) immobilizing plant genomic DNA fragments on filter paper, 2) prehybridizing the filter paper in 50% formamide, 5X SSPE, 2X Denhardt's reagent and 0.1% SDS at 42 ℃ for 1-2h, or prehybridizing the filter paper in 6X SSC, 2X Denhardt's reagent and 0.1% SDS at 68 ℃ for 1-2h, 3) adding labeled hybridization probes, 4) incubating for 16-24h, 5) washing the filter paper in 1X SSC, 0.1% SDS at room temperature for 20min, 6) washing the filter paper in 0.2X SSC, 0.1% SDS at 68 ℃ three times, 20min each, 7) exposing the filter paper to X-ray film at-70 ℃ for 24-48h using a sensitizing screen.

As used herein, a "biological sample" is a sample of a plant, plant material, or product comprising plant material. The term "plant" encompasses cotton upland (Gossypium hirsutum) plant tissue at any stage of maturity, and also encompasses any cell, tissue or organ harvested or derived from such plants, including but not limited to: seed, leaf, stem, flower, root, single cell, gamete, cell culture, tissue culture or protoplast. As used herein, "plant material" refers to material obtained from a plant. Products comprising plant material relate to food, feed, or other products made from or which may be contaminated with plant material. It will be appreciated that in the context of the present invention, these biological samples are tested for the presence of a nucleic acid specific for EE-GH5, indicating the presence of the nucleic acid in the sample. Thus, the methods of identifying elite event EE-GH5 in biological samples referred to herein relate to the identification of nucleic acids comprising said elite event in biological samples.

As used herein, "comprising" may be interpreted as: the presence of stated features, integers, steps, reagents or components are specified but does not preclude the presence or addition of one or more features, integers, steps, 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 actually cited, i.e. be comprised in a larger nucleic acid or protein. Chimeric genes comprising functionally or structurally defined DNA sequences may comprise additional DNA sequences and the like.

The invention also relates to the development of elite event EE-GH5 in cotton into plants comprising the event, progeny derived from these plants, plant cells, or plant material derived from the event. Plants comprising elite event EE-GH5 can be obtained as described in example 1.

Cotton plants or plant material comprising EE-GH5 can be determined according to the PCR identification protocol described in example 2 for EE-GH 5. Briefly, cotton genomic DNA present in a biological sample is amplified by PCR using primers that are: primers which specifically recognize sequences in the 5 'or 3' flanking sequence of EE-GH5 (e.g.primers having the sequence SEQ ID No: 6), and primers which recognize sequences in the foreign DNA (e.g.primers having the sequence SEQ ID No: 3). DNA primers that amplify a portion of the endogenous cotton sequence can be used as a positive control for PCR amplification. If the material produced fragments of the expected size when subjected to PCR amplification, the material comprised plant material from a cotton plant containing elite event EE-GH 5.

Plants containing EE-GH5 are characterized by their insect resistance, and by their glufosinate tolerance. Plants containing EE-GH5 are also characterized by their agronomic traits (comparable to those of the american commercially available variety under no insect stress). It has been observed that the presence of exogenous DNA in the insertion region of the cotton plant genome as described herein can confer a phenotypic and molecular signature of particular interest to plants comprising this event.

The following example describes the identification of elite event EE-GH5 and the development of a tool for the specific identification of elite event EE-GH5 in biological samples.

Unless otherwise indicated in the examples, all recombinant techniques were performed according to the Molecular Cloning described in "SambrookJ and Russell DW (eds.) (2001): a Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press, New York "and the standard procedures of" Ausubel FA, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JAand Struhl K (eds.) (2006) Current Protocols in Molecular biology, John Wiley & Sons, New York ".

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 "Brown TA, (1998) Molecular biology LabFax, 2nd Edition, Academic Press, Diego". Standard materials and methods for Polymerase Chain Reaction (PCR) are described in "McPherson MJ andSG (2000) PCR (the bases), BIOS Scientific Publishers Ltd., Oxford "and" PCR applications Manual, 3rd Edition (2006), Roche Diagnostics GmbH, Mannheim orwww.roche-applied-science.com ".

In the detailed description and examples, reference is made to the following sequences:

SEQ ID No. 1: nucleotide sequence comprising the 5' flanking region of EE-GH5

SEQ ID No. 2: nucleotide sequence comprising the 3' flanking region of EE-GH5

SEQ ID No. 3: primer GHI057

SEQ ID No. 4: primer MAE115

SEQ ID No. 5: primer HVH022

SEQ ID No. 6: primer GHI065

SEQ ID No. 7: primer HVH024

SEQ ID No. 8: primer DPA312

SEQ ID No. 9: primer GHI066

SEQ ID No. 10: primer GHI047

SEQ ID No. 11: primer GHI048

SEQ ID No. 12: from plants containing EE-GH5 using primers GHI047 and GHI048

Nucleotide sequence of amplified fragment in cotton DNA

SEQ ID No. 13: primer 1 for amplifying control fragment

SEQ ID No. 14: primer 2 for amplifying control fragment

SEQ ID No. 15: nucleotide sequence of plant genome target sequence before insertion of EE-GH5

Column(s) of

SEQ ID No. 16: nucleotide sequence (Long) comprising the 5' flanking region of EE-GH5

SEQ ID No. 17: primer HVH036

SEQ ID No. 18: primer SHA028

SEQ ID No. 19: primer MAE067

Examples

1. Identification of elite event EE-GH5

Insect-resistant cotton obtained by transforming cotton with a vector comprising the coding sequence of the modified cry1ab gene under the control of the S7 promoter of subterranean clover stunt virus.

Elite event EE-GH5 was screened by a broad selection procedure based on good expression and stability of insect-resistant genes, and its compatibility with optimal agronomic traits (e.g., plant height, node height, boll retention, stand-up capacity, vigor, fiber length, fiber strength, and lint yield) was also evaluated.

The screened events were introduced into different commercial genetic backgrounds and the results of field trials in different regions were compared. The plants are attacked by the pests by different treatments. The plants showed good insect control.

In addition, the events have normal leaf, flower and boll morphology, excellent fertility, and exhibit no disease or abnormal insect susceptibility in a variety of genetic backgrounds. During the process of introgressing into various genetic backgrounds, no abnormal problems or abnormal conditions were found.

2. Identification of flanking regions of elite event EE-GH5

The sequence of the region flanking the foreign DNA in elite event EE-GH5 was determined using the thermal asymmetric interlacing (TAIL-) PCR method described by Liu et al (1995, Plant J.8 (3): 457- & 463). The method uses three nested primers and a shorter arbitrary degenerate primer in a sequential reaction so that the relative amplification efficiencies of specific and non-specific products can be thermally controlled. Specific primers are selected for annealing reactions at the boundaries of the foreign DNA and based on their annealing conditions. Small amounts (5. mu.l) of unpurified secondary and tertiary PCR products were analyzed on a 1% agarose gel. The tertiary PCR product was purified and sequenced.

2.1 Right (5') flank region

Fragments identified as containing the 5' flanking region were sequenced (SEQ ID No. 1). The sequence of nucleotides 1 to 98 corresponds to plant DNA, while the sequence of nucleotides 99 to 334 corresponds to foreign DNA. A longer fragment containing the 5' flanking region was also sequenced (SEQ ID No. 16). The sequence between nucleotides 1-563 corresponds to plant DNA, while the sequence between 564-799 corresponds to foreign DNA.

2.2 left (3') flanking region

The fragment identified as comprising the 3' flanking region obtained by the TAIL-PCR method was sequenced (SEQ ID No. 2). The sequence between nucleotides 1 to 40 corresponds to the foreign DNA, while the sequence between nucleotides 41 to 452 corresponds to the plant DNA.

2.3 regions comprising an EE-GH 5-specific rearrangement in foreign DNA

A nucleotide sequence comprising a specifically rearranged region of EE-GH5 within the foreign DNA was identified (SEQ ID No. 12). The region between positions 52 and 88 can be identified as rearranged when compared to the foreign DNA used to obtain the transformants from which elite event EE-GH5 was selected.

Development of a polymerase chain reaction identification protocol for EE-GH5

3.1. Primer and method for producing the same

Specific primers were designed which recognize sequences within elite events.

More particularly, two primers were developed which recognize sequences within the foreign DNA which flank a particular rearrangement of EE-GH 5. The primers span a sequence of approximately 262 bp. The following primers were found to give particularly clear and reproducible results in a PCR reaction of EE-GH5 DNA:

GHI047：5’-TgC.CTC.TTg.AAC.TgT.AgC-3’ (SEQ ID No.：10)

GHI048：5’-ACT.TgC.AgT.TgC.TgA.TgA.Tg-3’(SEQ ID No.：11)

alternatively, the designed primer may recognize a sequence within the 5' flanking region of EE-GH 5. A second primer is then selected within the sequence of the exogenous DNA such that the primers span a sequence of about 369 nucleotides. The following primers were demonstrated to give particularly clear and reproducible results in a PCR reaction of EE-GH5 DNA:

GHI065：5’-ggg.CCg.gAT.AAA.ATT.AgC.CT-3’(SEQ ID No.：6)

(target sequence: plant DNA)

GHI057：5’-ATA.gCg.CgC.AAA.CTA.ggA-3’(SEQ ID No.：3)

(target sequence: insert DNA)

Primers targeting endogenous sequences are preferably included in the PCR mix. These primers serve as internal controls in unknown samples and in DNA positive controls. A positive result was obtained with the endogenous primer pair, indicating that sufficient DNA of the appropriate quality was contained in the genomic DNA preparation used to generate the PCR product. Endogenous primers were selected to recognize housekeeping genes in cotton.

GHI001：5’-AAC.CTA.ggC.TgC.TgA.Agg.AgC-3’(SEQ ID No.：13)

GHI002：5′-CAA.CTC.CTC.CAg.TCA.TCT.CCg-3′(SEQ ID No.：14)

3.2 amplified fragments

The expected amplified fragments in the PCR reaction were:

for primer pair GHI001-GHI 002: 445bp (endogenous control)

For primer pair GHI047-GHI 048: 262bp (EE-GH5 good event)

For primer pair GHI057-GHI 065: 369bp (EE-GH5 good event)

3.3 template DNA

Template DNA was prepared by leaf punch method according to Edwards et al (Nucleic Acid Research, 19, p1349, 1991). When using DNA prepared by other methods, tests using different amounts of template should be performed. Usually 50ng of genomic template DNA gives the best results.

3.4 Positive and negative controls specified

To avoid false positives or negatives, the following positive and negative controls should be included in the PCR program:

master mix control (DNA negative control). The PCR reaction was carried out without any DNA added. When the expected result, no PCR product, was observed, this indicates that the PCR reaction mixture was not contaminated with the target DNA.

DNA positive control (genomic DNA sample known to contain transgene sequences). Successful amplification of the positive control indicates that the PCR reaction was performed under conditions that allow amplification of the target sequence.

Wild type DNA control. When a PCR reaction is performed, the template DNA provided is genomic DNA prepared from a non-transgenic plant. When the expected result, no amplification of the transgenic PCR product but amplification of the endogenous PCR product, was observed, this indicates that there was no detectable background amplification of the transgene in the genomic DNA sample.

3.5PCR conditions

The best results were obtained under the following conditions:

the PCR mix for the 25 μ Ι reaction contains:

2.5. mu.l template DNA

2.5. mu.l 10x amplification buffer (supplied by the manufacturer of Taq polymerase)

0.5μl10mM dNTPs

0.4μl GHI001(10pmoles/μl)

0.4μl GHI002(10pmoles/μl)

0.7. mu.l GHI047(10 pmoles/. mu.l) or GHI057(PCR program 2)

0.7. mu.l GHI048(10 pmoles/. mu.l) or GHI065(PCR program 2)

0.1. mu.l Taq DNA polymerase (5 units/. mu.l)

Adding water to 25 μ l

The thermal cycling procedure followed with the best results obtained is:

at 95 ℃ for 4min.

Then: at 95 ℃ for 1min.

At 57 deg.C for 1min

At 72 deg.C for 2min

5 cycles

Then: 30sec at 92 DEG C

30sec at 57 DEG C

At 72 deg.C for 1min

25 cycles

Then: at 72 deg.C for 10min

3.6 agarose gel analysis

In order to best observe the PCR results, 10-20. mu.l of the PCR sample should be spotted on a 1.5% agarose gel (Tris-borate buffer) and labeled with an appropriate molecular weight marker (e.g., 100bp ladder PHARMACIA).

3.7 validation of results

Data obtained from transgenic plant DNA samples by a single PCR procedure and a single PCR mix should not be accepted unless: 1) the DNA positive control showed the expected PCR product (transgene and endogenous fragment); 2) PCR amplification of the DNA negative control was negative (no fragment); 3) the wild type DNA control showed the expected results (amplification of the endogenous fragment).

When following the PCR identification protocol for EE-GH5 described above, the lanes show visible amounts of transgene and endogenous PCR products of the expected sizes, indicating that the corresponding plants used to prepare the genomic template DNA inherit the EE-GH5 elite event. Lanes show no visible amount of transgenic PCR product and visible amount of endogenous PCR product, indicating that the corresponding plants used to prepare the genomic template DNA do not contain elite events. Lanes show that there were no visible amounts of endogenous and transgenic PCR products, indicating that the quality and/or quantity of genomic DNA did not allow for the production of PCR products. These plants could not be scored. The preparation of genomic DNA should be repeated and a new PCR procedure performed using appropriate controls.

3.8 identification of EE-GH5 Using differential PCR protocols

Test procedures using all appropriate controls must be performed before attempting to screen for unknown substances. Development protocols may need to be optimized for different components (template DNA preparations, Taq DNA polymerase, primer quality, dNTPs, thermocyclers, etc.) between laboratories.

Amplification of endogenous sequences plays a key role in the protocol. The experimenter must determine the conditions for PCR and thermocycling. Using these conditions, equimolar amounts of amplification of endogenous and transgenic sequences of known transgenic genomic DNA templates can be performed. As long as the target endogenous fragment is not amplified, or the target sequence is not amplified to the same intensity of ethidium bromide staining (as judged by agarose gel electrophoresis), the PCR conditions need to be optimized.

Leaf material from a number of cotton plants, some of which contained EE-GH5, was tested according to the protocol described above. Samples derived from elite event EE-GH5 and cotton wild type were used as positive and negative controls, respectively.

FIG. 2 shows the results of Excellent event PCR identification protocol 1 according to EE-GH5 on a number of cotton plant samples. The samples of lanes 2 and 3 were found to contain a 305bp band, confirming that it contained a elite event; whereas the samples of lanes 4 to 10 do not contain EE-GH 5. Lanes 4 and 10 contain other cotton elite events (including plants containing different insect-resistant chimeric genes). Lane 11 represents the negative control (water) and lanes 1 and 12 represent molecular weight markers (100bp ladder).

4. Use of a specific integration fragment as a probe for the detection of material containing EE-GH5

A specific integrated fragment of EE-GH5 was obtained by PCR amplification using specific primers GHI047(SEQ ID No.10) and GHI048(SEQ ID No.11), or by chemical synthesis, and labeled. This integrated fragment was used as a specific probe for the detection of EE-GH5 in biological samples. Nucleic acid was extracted from the sample according to standard procedures. The nucleic acid is then contacted with a specific probe under hybridization conditions optimized to allow formation of hybrids. The formation of hybrids is then detected to indicate the presence of EE-GH5 nucleic acid in the sample. Optionally, the nucleic acid in the sample is amplified using specific primers prior to contacting with the specific probes. Alternatively, the nucleic acid is labeled prior to contact with the specific probe (rather than the integrated fragment). Optionally, the specific probes are attached to a solid support (such as, but not limited to, a filter paper, test strip or bead) prior to contacting the sample.

5. PCR-based protocol for determining the zygosity status of EE-GH5 cotton plant material

5.1 primers

Two primers recognizing the nucleotide sequence of the wild-type locus before insertion of the elite event were designed: they point towards each other with an insertion site in between. This set of primers, together with a third primer complementary to the foreign DNA sequence and directed against the flanking DNA, performs PCR amplification of the EE-GH5 locus and the wt locus.

The following primers were found to give particularly clear and reproducible results in a zygosity scoring PCR reaction on EE-GH5 DNA:

GHI0665’-AgA.TAA.AAT.CgT.CAg.TgC.Tg-3’(SEQ ID No.：9)

(target sequence: plant DNA upstream of 5' flanking sequence)

GHI0575’-ATA.gCg.CgC.AAA.CTA.ggA-3’(SEQ ID N0.：3)

(target sequence: insert DNA)

GHI0655’-ggg.CCg.gAT.AAA.ATT.AgC.CT-3’(SEQ ID No.：6)

(target sequence: plant DNA of 3' flanking sequence)

5.2 amplifying fragments

The expected amplified fragments in the PCR reaction were:

for primer pair GHI065-GHI 066: 517bp (wt locus)

For primer pair GHI057-GHI 065: 369bp (EE-GH5 locus)

5.3 template DNA

According to Edwards et al (Nucleic Acid Research, 19, p1349, 1991), template DNA is prepared by leaf punching. When using DNA prepared by other methods, different amounts of template should be used for the test procedure. Typically 50ng of genomic template DNA will give the best results.

5.4 Positive and negative controls specified

To avoid false positive or negative results, the following positive and negative controls should be included in the PCR program:

master mix control (DNA negative control). The PCR reaction was carried out without any DNA added. When the expected result, no PCR product, was observed, this indicated that the PCR mixture was not contaminated with the target DNA.

DNA positive control (genomic DNA sample known to contain transgene sequences). Successful amplification of the positive control indicates that the PCR was performed under conditions that allow amplification of the target sequence.

Wild type DNA control. When a PCR reaction is performed, the template DNA provided is genomic DNA prepared from a non-transgenic plant. When the expected result, no amplification of the transgenic PCR product but amplification of the endogenous PCR product, was observed, this indicates that there was no detectable background amplification of the transgene in the genomic DNA sample.

5.5PCR conditions

The best results are obtained under the following conditions:

the PCR mix for the 25 μ Ι reaction contains:

x μ l template DNA (150ng)

2.5 μ l10 Xamplification buffer (supplied by the manufacturer with Taq polymerase)

0.5μl 10mM dNTPs

1.5μl GHI053(10pmoles/μl)

1.0μl GHI054(10pmoles/μl)

0.5μl GHI041(10pmoles/μl)

0.1. mu.l Taq DNA polymerase (5 units/. mu.l)

Adding water to 25 μ l

The thermal cycling program followed for optimal results is:

at 95 ℃ for 4min.

Then: at 95 ℃ for 1min.

At 57 deg.C for 1min

At 72 deg.C for 2min

5 cycles

Then: 30sec at 92 DEG C

30sec at 57 DEG C

At 72 deg.C for 1min

25 cycles

Then: at 72 deg.C for 10min

5.6 agarose gel analysis

In order to best observe the PCR results, 10-20. mu.l of the PCR sample should be spotted on a 1.5% agarose gel (Tris-borate buffer) and labeled with an appropriate molecular weight marker (e.g., 100bp ladder PHARMACIA).

5.7 validation of the results

Data obtained from transgenic plant DNA samples by a single PCR program and a single PCR master mix should not be accepted unless:

positive control shows the expected PCR product (transgene target sequence amplification)

Wild-type positive DNA control shows the expected result (wild-type target sequence amplification)

Negative control PCR amplification was negative (no fragment).

Lanes with visible amounts of the expected size of the transgenic PCR product and no visible amounts of the wild-type PCR product indicate that the corresponding plants used to prepare the genomic DNA template are homozygous for the transgenic gene cassette.

The presence of visible amounts of the expected size of the transgenic and wild-type PCR products lanes indicates that the corresponding plants used to prepare the genomic template DNA are hemizygous for the transgenic gene cassette. Lanes with visible amounts of wild-type PCR product and no visible amounts of transgenic PCR product indicate that the corresponding plants used to prepare the genomic template DNA do not inherit the transgene sequence to be detected and are homozygous for the wild-type locus.

Lanes where no visible amount of transgene and wild type PCR product was present indicate that the quality and/or quantity of genomic DNA does not allow for the generation of PCR products. These plants could not be scored. The preparation of genomic DNA should be repeated and a new PCR procedure must be performed using appropriate controls.

5.8 identification of zygosity status in plants comprising EE-GH5 Using a zygosity scoring protocol

FIG. 3 shows the results of EE-GH5 zygosity score PCR on a number of cotton plant samples. The samples of lanes 2-3 and 6-7 were found to contain a PCR fragment (369bp) characteristic of elite event EE-GH5, whereas the samples of lanes 2, 3 and 5 contained a fragment characteristic of the presence of the wt locus. Thus, lanes 6 and 7 contain the homozygous form of EE-GH5, lanes 2 and 3 contain the hemizygous form of EE-GH5, and lane 5 contains the homozygous form of the wt locus (i.e. unpaired for EE-GH 5). Lane 8 represents the negative control (water) sample, and lanes 1 and 9 represent molecular weight markers (100bp ladder).

Introgression of EE-GH5 into preferred cultivars

Through repeated backcrossing, elite event EE-GH5 was introduced into commercial cotton cultivars, such as, but not limited to: FM5013, FM5015, FM5017, FM989, FM832, FM966 and FM958, FM989, FM958, FM832, FM991, FM819, FM800, FM960, FM966, FM981, FM5035, FM5044, FM5045, FM5013, FM5015, FM5017 or FM 5024.

The observation was that introgression of elite event genes into these cultivars did not significantly affect any of their desired phenotypic or agronomic characteristics (no linkage drag), while transgene expression (as determined by glufosinate tolerance) reached commercially acceptable levels. This confirms the state of event EE-GH5 as a superior event.

Elite lines may be advantageously combined with other elite events available on the market, in particular other elite event pest-resistance genes for pest-resistance management purposes, such as, but not limited to: events 3006-210-23(USDA achis evaluation 03-036-02 p); event 281-24-236(USDAaphis petition 03-036-01 p); event MON158985(USDA aphis petition00-342-01 p); event MON531(USDA aphis rejection 94-308-01p), or event COT102(═ Syngenta vip3A, USDA aphis rejection 03-155-01 p). Elite event EE-GH5 can also be combined with herbicide-tolerant elite events, such as event MON1445(USDAaphis petition 95-045-01p), or event MON88913(USDA aphis petition 04-086-01 p).

Unless specifically stated otherwise, the term "plant" as used in the claims below is intended to encompass plant tissue at any stage of maturity, as well as any cell, tissue or organ taken from or derived from any such plant, including but not limited to: any seed, leaf, stem, flower, root, single cell, gamete, cell culture, tissue culture, or protoplast.

Reference seed comprising elite event EE-GH5 was deposited at ATCC (10801University blvd, Manassas, VA 20110-: PTA-8171. The alternate name for EE-GH5 is T304-40.

Unless specifically stated otherwise, the term "plant" as used in the claims below is intended to encompass plant tissue at any stage of maturity, as well as any cell, tissue or organ taken from or derived from any such plant, including but not limited to: any seed, leaf, stem, flower, root, single cell, gamete, cell culture, tissue culture, or protoplast.

The foregoing description of the invention is intended to be illustrative and not limiting.

Various changes or modifications to the described embodiments will occur to those skilled in the art. Such changes or modifications can be made without departing from the spirit or scope of the present invention.

Sequence listing

<110> Bayer biosciences

Trolinder，Linda

Moens，Sofie

Paelinck，Dimitri

Habex，Veerle

Van Herck，Hans

<120> insect-resistant cotton plant and method for identifying the same

<130>BCS06-2011

<150>EP07075263.9

<151>2007-04-05

<150>EP07075299.3

<151>2007-04-23

<150>US60/923142

<151>2007-04-12

<160>19

<170> PatentIn version 3.3

<210>1

<211>334

<212>DNA

<213>Artificial

<220>

<223> nucleotide sequence comprising 5' flanking sequence of EE-GH5

<220>

<221>misc_feature

<222>(1)..(98)

<223>plant DNA

<220>

<221>misc_feature

<222>(99)..(334)

<223> insert DNA

<400>1

cttttatcat ataagtgcct atataaataa agtttagtca gtcaaattag agagattgtc 60

attgtaggga gtttgtccaa ttattggtgt ttttaatccc agtactcggc cgtcgaccgc 120

ggtaccccgg aattgaaaac agatttgagg tttcaaagtg gtgttgtggc tacagttcaa 180

gaggcattaa aagctgattt ggccggatct tttgaagctt tcaaacacaa ggaaatcatc 240

catcaacctc caatcgaatg gctttttgct tggcacaata attcccctac tttcgacttg 300

aggacaagtc gattttccgg gccggatgta ttga 334

<210>2

<211>452

<212>DNA

<213> Artificial

<220>

<223> nucleotide sequence comprising 3' flanking sequence of EE-GH5

<220>

<221>misc_feature

<222>(1)..(40)

<223>insert DNA

<220>

<221>misc_feature

<222>(41)..(452)

<223> plant DNA

<400>2

atagcgcgca aactaggata aattatcgcg cgcggtgtca tctatctcct ttttcttttc 60

aagttatccc aagatctagg ggtattttgc atcattaaga gaaaactttg tttaattaga 120

cttccaatca ccagtcttag gtataaggga aggctgatgc cgataacatt gccgaacaaa 180

ccatcaaggc aactagagat gccttgatgc aaaagagctc ttttttagac cctcaaatgc 240

caagcagtgg atagaaagaa gctcaatgtt aagtgcaaac aaaattcgaa aggctaaaag 300

tttatcgaga ttcgaatctc taaatcaatg tcattaacaa ctatataaca ggctaatttt 360

atccggccca ataaataaaa caaaataaaa ataataaatc aaggtcaatt tacaaaatag 420

attggcccaa acaaaaaaga gcccaataac cc 452

<210>3

<211>18

<212>DNA

<213>Artificial

<220>

<223> oligonucleotide primer GHI057

<400>3

atagcgcgca aactagga 18

<210>4

<211>20

<212>DNA

<213> Artificial

<220>

<223> oligonucleotide primer MAE115

<400>4

tcggcaatgt tatcggcatc 20

<210>5

<211>19

<212>DNA

<213> Artificial

<220>

<223> oligonucleotide primer HVH022

<400>5

ccactgcttg gcatttgag 19

<210>6

<211>20

<212>DNA

<213> Artificial

<220>

<223> oligonucleotide primer GHI065

<400>6

gggccggata aaattagcct 20

<210>7

<211>20

<212>DNA

<213> Artificial

<220>

<223> oligonucleotide primer HVH024

<400>7

gtcattgtag ggagtttgtc 20

<210>8

<211>18

<212>DNA

<213> Artificial

<220>

<223> oligonucleotide primer DPA312

<400>8

tgcctcttga actgtagc 18

<210>9

<211>20

<212>DNA

<213> Artificial

<220>

<223> oligonucleotide primer GHI066

<400>9

agataaaatc gtcagtgctg 20

<210>10

<211>18

<212>DNA

<213> Artificial

<220>

<223> oligonucleotide primer GHI047

<400>10

tgcctcttga actgtagc 18

<210>11

<211>20

<212>DNA

<213> Artificial

<220>

<223> oligonucleotide primer GHI048

<400>11

acttgcagtt gctgatgatg 20

<210>12

<211>262

<212>DNA

<213> Artificial

<220>

<223> nucleotide sequence of fragment amplified from cotton DNA comprising EE-GH5 using GHI047 and GHI048

<220>

<221>misc_feature

<222>(1)..(51)

<223> sequence from 3' mel

<220>

<221>misc_feature

<222>(86)..(88)

<223> sequence derived from right border of T-DNA

<220>

<221>misc_feature

<222>(89)..(262)

<223> sequence from 3' mel

<400>12

tgcctcttga actgtagcca caacaccact ttgaaacctc aaatctgttt tcaattccgg 60

ggtaccgcgg tcgacggccg agtactggcc aaatttagtt tttggataaa gtaccagatt 120

tagggcaata aaactataaa cggccagctt ttagctacat acaatgagca ttcctgataa 180

acaaagaacc caaccggtat ttgtaagtga ataaacaaaa gtagtattaa gctttaagtt 240

tccatcatca gcaactgcaa gt 262

<210>13

<211>21

<212>DNA

<213> Artificial

<220>

<223> oligonucleotide primer GHI001

<400>13

aacctaggct gctgaaggag c 21

<210>14

<211>21

<212>DNA

<213> Artificial

<220>

<223> oligonucleotide primer GHI002

<400>14

caactcctcc agtcatctcc g 21

<210>15

<211>558

<212>DNA

<213> Artificial

<220>

<223> nucleotide sequence of plant genome target sequence before insertion of EE-GH5

<220>

<221>misc_feature

<222>(1)..(113)

<223> sequences corresponding to 5' flanking sequences

<220>

<221>misc_feature

<222>(114)..(145)

<223> Pre-indel deletion in Excellent event EE-GH5

<220>

<221>misc_feature

<222>(146)..(558)

<223> sequences corresponding to 3' flanking sequences

<220>

<221>misc_feature

<222>(457)..(475)

<223> sequence corresponding to the complementary sequence of oligonucleotide GHI065

<400>15

agtttatatt tcttactttt atcatataag tgcctatata aataaagttt agtcagtcaa 60

attagagaga ttgtcattgt agggagtttg tccaattatt ggtgttttta atcacaagtt 120

gtttaaggat tccaaaccat acattatcta tctccttttt cttttcaagt tatcccaaga 180

tctaggggta ttttgcatca ttaagagaaa actttgttta attagacttc caatcaccag 240

tcttaggtat aagggaaggc tgatgccgat aacattgccg aacaaaccat caaggcaact 300

agagatgcct tgatgcaaaa gagctctttt ttagaccctc aaatgccaag cagtggatag 360

aaagaagctc aatgttaagt gcaaacaaaa ttcgaaaggc taaaagttta tcgagattcg 420

aatctctaaa tcaatgtcat taacaactat ataacaggct aattttatcc ggcccaataa 480

ataaaacaaa ataaaaataa taaatcaagg tcaatttaca aaatagattg gcccaaacaa 540

aaaagagccc aataaccc 558

<210>16

<211>799

<212>DNA

<213> Artificial

<220>

<223> nucleotide sequence (long) comprising 5' flanking sequence of EE-GH5

<220>

<221>misc_feature

<222>(1)..(563)

<223>plant DNA

<220>

<221>misc_feature

<222>(564)..(799)

<223> insert DNA

<400>16

aacaaataag ccaattccaa agagaggata gctgatcttt tcactaatca taacttgaat 60

ttttttagtg gtgcttatct gtttcaggtg ttaggagcac aatcatacca ttccctgcat 120

aattttcaga ctctaggatg aatccttttc gaagaggagg tgaatgatac gaactcagaa 180

agggtaaatt catttgagtt caagtttgga acactgacca acttgcaatg aatttttggg 240

ttaaattatt ttctttatgg atcatgaaat gatatattat ttaaaatttt aagatgttta 300

tttattcatt taattatagt ttgattatat acatttgtaa aattgaatat ttatttggtt 360

ttaaaagtta ttaatttctt gtttattcat taaataaagt tcaattatag ataaaatcgt 420

cagtgctgaa ttttatatta ttttattaga agtttatatt tcttactttt atcatataag 480

tgcctatata aataaagttt agtcagtcaa attagagaga ttgtcattgt agggagtttg 540

tccaattatt ggtgttttta atcccagtac tcggccgtcg accgcggtac cccggaattg 600

aaaacagatt tgaggtttca aagtggtgtt gtggctacag ttcaagaggc attaaaagct 660

gatttggccg gatcttttga agctttcaaa cacaaggaaa tcatccatca acctccaatc 720

gaatggcttt ttgcttggca caataattcc cctactttcg acttgaggac aagtcgattt 780

tccgggccgg atgtattga 799

<210>17

<211>20

<212>DNA

<213> Artificial

<220>

<223> oligonucleotide primer HVH036

<400>17

caggtgttag gagcacaatc 20

<210>18

<211>24

<212>DNA

<213> Artificial

<220>

<223> oligonucleotide primer SHA028

<400>18

cacaacacca ctttgaaacc tcaa 24

<210>19

<211>19

<212>DNA

<213> Artificial

<220>

<223> oligonucleotide primer MAE067

<400>19

ccattcgatt ggaggttga 19

Claims (45)

1. A transgenic cotton plant, or a cell, part, seed or progeny thereof, each comprising elite event EE-GH5 in its genome, reference seed comprising said event being deposited with the ATCC under deposit number PTA-8171.

2. The transgenic cotton plant, seed, cell, part or progeny of claim 1, having genomic DNA which, when analyzed with the elite event identification scheme of EE-GH5 with two primers comprising the nucleotide sequences of SEQ ID No 10 and SEQ ID No 11, respectively, produces a DNA fragment of about 262bp, or, when analyzed with the elite event identification scheme of EE-GH5 with two primers comprising the nucleotide sequences of SEQ ID No 3 and SEQ ID No6, respectively, produces a DNA fragment of about 369 bp.

3. Seed comprising elite event EE-GH5 deposited at the ATCC under accession number PTA-8171, or a derivative thereof.

4. A cotton plant or part thereof, or seed thereof, obtained from the seed of claim 3.

5. A cotton plant, or seed, cell or tissue thereof, each comprising elite event EE-GH5 in its genome, produced by propagation and/or breeding of a cotton plant grown from the seed deposited with the ATCC under accession No. PTA-8171.

6. Cotton seed comprising elite event EE-GH5, reference seed comprising said event having been deposited at the ATCC under accession number PTA-8171.

7. A transgenic cotton plant, cell or tissue comprising elite event EE-GH5 produced from the seed of claim 6.

8. A method of producing a cotton plant or seed comprising elite event EE-GH5, comprising crossing the plant of any one of claims 1-7 with another cotton plant and planting seed resulting from said crossing.

9. Cotton genomic DNA comprising elite event EE-GH 5.

10. Genomic DNA produced from a cotton plant, plant cell or seed according to any one of claims 1-7.

11. A method for identifying elite event EE-GH5 in biological samples, which method comprises detection of an EE-GH5 specific region with a specific primer or probe that specifically recognizes the 5 'or 3' flanking region of EE-GH 5.

12. The method of claim 11, which comprises amplifying a DNA fragment of 100 to 150bp from a nucleic acid present in said biological sample using a polymerase chain reaction with at least two primers, one of which recognizes the 5 ' flanking region of EE-GH5 or the 3 ' flanking region of EE-GH5, said 5 ' flanking region having the nucleotide sequence of nucleotides 1 to 98 of SEQ ID No1 or the nucleotide sequence of nucleotides 1 to 563 of SEQ ID No 16; the 3' flanking region has the nucleotide sequence of the complementary sequence of nucleotides 41-452 of SEQ ID No 2, and the other primer of the primers recognizes the sequence in the exogenous DNA, which has the nucleotide sequence of the complementary sequence of nucleotides 99-334 of SEQ ID No1 or the nucleotide sequence of nucleotides 1-40 of SEQ ID No 2.

13. The method of claim 12, wherein said primer that recognizes the 5 'flanking region consists of a nucleotide sequence of 17 to 200 consecutive nucleotides, said 5' flanking region being selected from the nucleotide sequence of nucleotides 1 to 98 of SEQ ID No1 or the nucleotide sequence of nucleotides 1 to 563 of SEQ ID No 16, or said primer that recognizes the 3 'flanking region of EE-GH5 consists of a nucleotide sequence of 17 to 200 consecutive nucleotides, said 3' flanking region being selected from the nucleotide sequence of the complement of nucleotides 41 to 452 of SEQ ID No 2; the primer for identifying the sequence in the exogenous DNA consists of 17-200 continuous nucleotides, and the exogenous DNA is selected from a nucleotide sequence of a complementary sequence of 99-334 nucleotides of SEQ ID No1 or a nucleotide sequence of 1-40 nucleotides of SEQ ID No 2.

14. The method of claim 2, wherein said primer identifying the 5 'flanking region comprises at its 3' end a nucleotide sequence of at least 17 consecutive nucleotides, said 5 'flanking region being selected from the nucleotide sequence of nucleotides 1 to 98 of SEQ ID No1 or the nucleotide sequence of nucleotides 1 to 563 of SEQ ID No 16, or said primer identifying the 3' flanking region of EE-GH5 comprises at its 3 'end a nucleotide sequence of at least 17 consecutive nucleotides, said 3' flanking region being selected from the nucleotide sequence of the complement of nucleotides 41 to 452 of SEQ ID No 2; the primer recognizing a sequence in the foreign DNA comprising at least 17 continuous nucleotides at its 3' end is selected from the nucleotide sequence of the complementary sequence of nucleotides 99 to 334 of SEQ ID No1 or the nucleotide sequence of nucleotides 1 to 40 of SEQ ID No 2.

15. The method of claim 4, wherein said primers comprise the sequences of SEQ ID Nos 3 and 6, respectively.

16. The method of claim 5, which comprises amplifying a fragment of about 369bp using the EE-GH5 identification protocol.

17. A kit for identifying elite event EE-GH5 in biological samples, said kit comprising a primer recognizing the 5 ' flanking region of EE-GH5, or a primer recognizing the 3 ' flanking region of EE-GH5, and a primer recognizing a sequence within the foreign DNA, said 5 ' flanking region having the nucleotide sequence of nucleotides 1 to 98 of SEQ ID No1 or the nucleotide sequence of nucleotides 1 to 563 of SEQ ID No 16; the 3' flanking region has a nucleotide sequence of a complementary sequence of nucleotides 41 to 452 of SEQ ID No 2; the exogenous DNA has a nucleotide sequence of a complementary sequence of 99-334 nucleotides of SEQ ID No1 or a nucleotide sequence of 1-40 nucleotides of SEQ ID No 2.

18. The kit of claim 17, wherein said primer recognizing the 5 'flanking region consists of a nucleotide sequence of 17 to 200 consecutive nucleotides, said 5' flanking region being selected from the nucleotide sequence of nucleotides 1 to 98 of SEQ ID No1 or the nucleotide sequence of nucleotides 1 to 563 of SEQ ID No 16, or said primer recognizing the 3 'flanking region of EE-GH5 consists of a nucleotide sequence of 17 to 200 consecutive nucleotides, said 3' flanking region being selected from the nucleotide sequence of the complement of nucleotides 41 to 452 of SEQ ID No 2; the primer for identifying the sequence in the exogenous DNA consists of 17-200 continuous nucleotides, and the exogenous DNA is selected from a nucleotide sequence of a complementary sequence of 99-334 nucleotides of SEQ ID No1 or a nucleotide sequence of 1-40 nucleotides of SEQ ID No 2.

19. The kit of claim 17, wherein said primer recognizing the 5 'flanking region comprises at its 3' end a nucleotide sequence of at least 17 consecutive nucleotides selected from the nucleotide sequence of nucleotides 1 to 98 of SEQ ID No1 or the nucleotide sequence of nucleotides 1 to 563 of SEQ ID No 16, or said primer recognizing the 3 'flanking region of EE-GH5 comprises at its 3' end a nucleotide sequence of at least 17 consecutive nucleotides selected from the nucleotide sequence of the complement of nucleotides 41 to 452 of SEQ ID No 2; the primer recognizing a sequence in the foreign DNA comprises at least 17 continuous nucleotides at its 3' end, selected from the nucleotide sequence of the complementary sequence of nucleotides 99 to 334 of SEQ ID No1 or the nucleotide sequence of nucleotides 1 to 40 of SEQ ID No 2.

20. The kit of claim 17, comprising a primer consisting of the sequence of SEQ ID No 3 and a primer consisting of the sequence of SEQ ID No 6.

21. A primer suitable for use in EE-GH5 specific detection, the sequence of which specifically recognizes under optimized detection conditions a sequence within the 5 ' or 3 ' flanking region of EE-GH5, said 5 ' flanking region having the nucleotide sequence of nucleotides 1 to 98 of SEQ ID No1 or the nucleotide sequence of nucleotides 1 to 563 of SEQ ID No 16; the 3' flanking region has the nucleotide sequence of the complementary sequence of nucleotides 41-452 of SEQ ID No 2.

22. The primer of claim 21, wherein said primer consists of a nucleotide sequence of 17-200 contiguous nucleotides selected from the nucleotide sequence of nucleotides 1-98 of SEQ ID No1 or the nucleotide sequence of nucleotides 1-563 of SEQ ID No 16, or the nucleotide sequence of 17-200 contiguous nucleotides selected from the nucleotide sequence of the complement of nucleotides 41-452 of SEQ ID No 2.

23. The primer of claim 21, wherein said primer comprises at its 3 'end a nucleotide sequence of at least 17 contiguous nucleotides selected from the nucleotide sequence of nucleotides 1 to 98 of SEQ ID No1 or the nucleotide sequence of nucleotides 1 to 563 of SEQ ID No 16 or at its 3' end a nucleotide sequence of at least 17 contiguous nucleotides selected from the nucleotide sequence of the complement of nucleotides 41 to 452 of SEQ ID No 2.

24. A primer comprising at its 3' end the sequence of SEQ ID No 3.

25. A primer comprising at its 3' end the sequence of SEQ ID No 6.

26. The method of claim 11, which comprises hybridizing nucleic acids of the biological sample with a specific probe for EE-GH 5.

27. The method of claim 26, wherein the sequence of said specific probe has at least 80% sequence identity with a sequence comprising the 5 'or 3' flanking sequence of EE-GH5 and the portion of the foreign DNA sequence adjacent thereto.

28. The method of claim 27, wherein the sequence of said specific probe has at least 80% sequence identity with the nucleotide sequence at positions 78-119 of SEQ ID No1 or the nucleotide sequence at positions 20-61 of SEQ ID No 2 or the complement of said sequences.

29. A kit for identifying elite event EE-GH5 in biological samples, comprising a specific probe capable of specifically hybridizing to a specific region of EE-GH 5.

30. The kit of claim 29, wherein the sequence of said specific probe has at least 80% sequence identity with a sequence comprising part of the 5 'flanking sequence or the 3' flanking sequence of EE-GH5 and the sequence of the foreign DNA contiguous therewith.

31. The kit of claim 30, wherein the sequence of said specific probe has at least 80% sequence identity with the nucleotide sequence at positions 78-119 of SEQ ID No1 or the nucleotide sequence at positions 20-61 of SEQ ID No 2 or the complement of said sequences.

32. A specific probe for the identification of elite event EE-GH5 in biological samples.

33. The probe of claim 32, which has at least 80% sequence identity to a sequence comprising part of the 5 'flanking sequence or the 3' flanking sequence of EE-GH5 and the sequence of the foreign DNA contiguous therewith, or the complement thereof.

34. The probe of claim 33, which has at least 80% sequence identity with nucleotides 78 to 119 of SEQ ID No1 or nucleotides 20 to 61 of SEQ ID No 2 or the complement of said sequences.

35. A specific probe for the identification of elite event EE-GH5 in biological samples, the sequence of which is substantially similar to the nucleotide sequence at positions 78-119 of SEQ ID No1 or the nucleotide sequence at positions 20-61 of SEQ ID No 2 or the complement of said sequences.

36. A method for confirming seed purity, which method comprises detecting an EE-GH5 specific region in a seed sample with a specific primer or probe that specifically recognizes the 5 'or 3' flanking region of EE-GH 5.

37. A method of screening seeds for the presence of EE-GH5, said method comprising detecting an EE-GH5 specific region in samples of seed lots with a specific primer or probe that specifically recognizes the 5 'or 3' flanking region of EE-GH 5.

38. A method for determining the zygosity status of a plant, plant material or seed comprising elite event EE-GH5, said method comprising amplifying a DNA fragment of 100-500bp from a nucleic acid present in said biological sample by polymerase chain reaction using at least three primers, two of which specifically recognize a pre-inserted plant DNA, such as the 5 'flanking region of EE-GH5, said 5' flanking region having the nucleotide sequence of nucleotides 1 to 98 of SEQ ID No1 or the nucleotide sequence of nucleotides 1 to 563 of SEQ ID No 16, or the 3 'flanking region of EE-GH5, said 3' flanking region having the nucleotide sequence of the complement of nucleotides 41 to 452 of SEQ ID No 2 or said pre-inserted DNA having the nucleotide sequence of SEQ ID No 15 or the complement thereof, the third of which recognizes a sequence within the foreign DNA, the exogenous DNA has a complementary sequence of the nucleotide sequence of nucleotides 99-334 of SEQ ID No1 or the nucleotide sequence of nucleotides 1-40 of SEQ ID No 2.

39. The method according to claim 38, wherein said first primer comprises the nucleotide sequence of SEQ ID No6, said second primer comprises the sequence of SEQ ID No 9, and said third primer comprises the sequence of SEQ ID No 3.

40. A method for detecting the presence of elite event EE-GH5 in biological samples by hybridization with a substantially complementary labeled nucleic acid probe, wherein the ratio of probe to target nucleic acid is increased by recovery of the target nucleic acid sequence, the method comprising:

a) hybridizing said target nucleic acid sequence to a first nucleic acid oligonucleotide comprising the nucleotide sequence of nucleotides 99 to 116 of SEQ ID No1 or the complement thereof, or to said first nucleic acid oligonucleotide comprising the nucleotide sequence of nucleotides 23 to 40 of SEQ ID No 2 or the complement thereof;

b) hybridizing said target nucleic acid sequence to a second nucleic acid oligonucleotide comprising the nucleotide sequence of nucleotides 81-98 of SEQ ID No1 or the complement thereof, or to said labeled nucleic acid probe comprising the nucleotide sequence of nucleotides 41-58 of SEQ ID No 2 or the complement thereof; wherein said first and second oligonucleotides overlap by at least one nucleotide, and wherein said first or second oligonucleotide is labeled as said labeled nucleic acid probe;

c) cleavage of the probe with enzyme alone: a labeled probe within the duplex of the target nucleic acid sequence, the enzyme causing selective probe cleavage resulting in duplex dissociation leaving the target sequence intact;

d) recovering the target nucleic acid sequence by repeating steps (a) to (c);

e) detecting the cleaved labeled probe, thereby determining the presence of the target nucleic acid sequence.

41. An isolated nucleic acid molecule comprising a nucleotide sequence substantially similar to the nucleotide sequence of nucleotides 88 to 109 of SEQ ID No1 or the nucleotide sequence of nucleotides 30 to 50 of SEQ ID No 2, or the complement of said sequence.

42. The isolated nucleic acid molecule of claim 41, comprising a nucleotide sequence substantially similar to the nucleotide sequence of nucleotides 78 to 119 of SEQ ID No1 or the nucleotide sequence of nucleotides 20 to 60 of SEQ ID No 2, or the complement of said sequence.

43. An isolated nucleic acid fragment comprising the nucleotide sequence of SEQ ID No 12.

44. The method of claim 11, comprising amplifying a DNA fragment of approximately 262bp using primers consisting essentially of the nucleotide sequences of SEQ ID Nos 10 and 11, respectively.

45. A kit comprising two oligonucleotide primers consisting essentially of the nucleotide sequences of SEQ ID nos 10 and 11, respectively.