Elite event EE-GM4 and methods and kits for identifying such event in biological samples

Patent metadata

Field	Value
Publication number	US-11242539-B2
Application number	US-201716471752-A
Country	US
Kind code	B2
Filing date	Dec 22, 2017
Priority date	Dec 22, 2016
Publication date	Feb 8, 2022
Grant date	Feb 8, 2022

Abstract

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

First claim

The invention claimed is:

1. A nucleic acid molecule comprising
(a) a nucleotide sequence having at least 95% sequence identity to the nucleotide sequence of SEQ ID No. 3, 4, 5, or 6;
(b) a nucleotide sequence having at least 99% sequence identity to the nucleotide sequence of SEQ ID No. 3, 4, 5, 6, 24 or 25;
(c) the nucleotide sequence of SEQ ID No. 1, 2, 3, or 4, or the complement of said sequences; or
(d) the nucleotide sequence of SEQ ID No. 1 or 3 and SEQ ID No. 2 or 4, or the complement thereof,
wherein said nucleic acid optionally further comprises
(e) the nucleotide sequence of SEQ ID No. 7 and 9, or the complement thereof; or
(f) the nucleotide sequence of SEQ ID No. 11 from nucleotide position 188 to nucleotide position 7368 or a nucleotide sequence having at least 98%, or least 99%, or at least 99.5% or at least 99.9% sequence identity thereto.

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

3. The plant, cell, plant part, seed or progeny of claim 2 , further comprising the nucleotide sequence of SEQ ID No. 7 and 9, or the nucleotide sequence of SEQ ID No. 23 from nucleotide position 1059 to nucleotide position 8663.

4. A soybean plant, cell, part, or seed comprising in its genome elite event EE-GM4, wherein said elite event is the genetic locus comprising an inserted T-DNA containing a chimeric HPPD-4 protein-encoding gene and a chimeric Cry14Ab-1 protein-encoding gene, and 5′ and 3′ flanking sequences immediately surrounding said inserted T-DNA, as found in reference seed deposited at the ATCC under deposit number PTA-123624.

5. A progeny plant, cell, plant part or seed of the plant of claim 4 , wherein said progeny plant, cell, plant part or seed comprises the nucleotide sequence of SEQ ID No. 3 and the nucleotide sequence of SEQ ID No. 4, optionally wherein the genomic DNA of which, when analyzed using PCR with two primers comprising the nucleotide sequence of SEQ ID No, 12 and SEQ ID No. 13 respectively, yields a DNA fragment of 126 bp.

6. A soybean product produced from the soybean plant, cell, part, seed or progeny of claim 2 , wherein said soybean product comprises a nucleic acid that produces an amplicon diagnostic of or specific for event EE-GM4, wherein said event is as found in reference seed deposited at the ATCC under deposit number PTA-123624.

7. A method for protecting emerging soybean plants of claim 2 from competition by weeds, the method comprising treating a field to be planted with said soybean plants with an HPPD inhibitor herbicide, before the soybean plants are planted or the seeds are sown, followed by planting or sowing of said soybean plants or seeds in said pre-treated optionally wherein said HPPD inhibitor herbicide is selected from isoxaflutole, topramezone, or mesotrione.

8. A method for producing a soybean plant or seed comprising elite event EE-GM4 comprising crossing a plant according to claim 2 with another soybean plant, and planting the seed comprising EE-GM4 obtained from said cross, wherein said event is as found in reference seed deposited at the ATCC under deposit number PTA-123624.

9. A method for identifying elite event EE-GM4 in biological samples, the method comprising detecting an EE-GM4 specific region comprising SEQ ID NO: 1 and/or SEQ ID NO: 2 with a specific primer pair or a specific probe which specifically recognizes the region in said samples.

10. A primer pair suitable for use in a EE-GM4 specific detection, comprising:
(a) a first primer comprising a sequence which specifically recognizes a sequence within the 5′ or 3′ T-DNA flanking region of the inserted T-DNA in ER-GM4, and a second primer comprising a sequence which specifically recognizes a sequence within the inserted T-DNA in EE-GM4 contiguous with said flanking 5′ or 3′ region, said 5′ T-DNA flanking region comprising the nucleotide sequence of SEQ ID No. 5 from nucleotide 1 to nucleotide 227 or of SEQ 1D No. 24 from nucleotide 1 to nucleotide 1058, said 3′ T-DNA flanking region comprising the nucleotide sequence of the complement of SEQ ID No. 6 from nucleotide 254 to nucleotide 501 or the nucleotide sequence of the complement of SEQ ID No, 25 from nucleotide 254 to nucleotide 1339, said inserted T-DNA comprising the complement of the nucleotide sequence of SEQ ID No. 5 from nucleotide 228 to nucleotide 398, or the nucleotide sequence of SEQ ID No. 6 from nucleotide 1 to nucleotide 253, or the nucleotide sequence of SEQ ID No. 11 from nucleotide position 17 to nucleotide position 7621, or the complement thereof, or the nucleotide sequence of SEQ ID No. 23 from nucleotide position 1059 to nucleotide position 8663, or the complement thereof;
(b) a first primer comprising a nucleotide sequence of 17 to 200 consecutive nucleotides selected from the nucleotide sequence of SEQ ID No. 5 from nucleotide 1 to nucleotide 227 or from SEQ ID No. 24 from nucleotide 1 to nucleotide 1058, or the complement thereof, or comprises a nucleotide sequence of 17 to 200 consecutive nucleotides selected from the nucleotide sequence of SEQ ID No. 6 from nucleotide 254 to nucleotide 501 or from the nucleotide sequence of SEQ ID No. 25 from nucleotide 254 to nucleotide 1339, or the complement thereof, and a second primer comprising a nucleotide sequence of 17 to 200 consecutive nucleotides selected from the nucleotide sequence of SEQ ID No. 5 from nucleotide 228 to nucleotide 398 or the complement thereof, or the nucleotide sequence of SEQ ID No. 6 from nucleotide 1 to nucleotide 253 or the complement thereof, or the nucleotide sequence of SEQ ID No. 11 from nucleotide position 17 to nucleotide position 7621 or the complement thereof, or the nucleotide sequence of SEQ ID No. 23 from nucleotide position 1059 to nucleotide position 8663, or the complement thereof;
(c) a first primer comprising at its extreme 3′ end a nucleotide sequence of at least 17 consecutive nucleotides selected from the nucleotide sequence of SEQ ID No. 5 from nucleotide, 1to nucleotide 227 or from SEQ ID No. 24 from nucleotide 1 to nucleotide 1058, or the complement thereof, or comprising at its extreme 3′ end a nucleotide sequence of at least 1.7 consecutive nucleotides selected from the nucleotide sequence of SEQ ID No. 6 from nucleotide 254 to nucleotide 501 or from the nucleotide sequence of S 1 Q ID No. 25 from nucleotide 254 to nucleotide 1339, or the complement thereof, and a second primer comprising at its extreme 3′ end at least 17 consecutive nucleotides selected from the nucleotide sequence of SEQ ID No. 5 from nucleotide 228 to nucleotide 398 or the complement thereof, or the nucleotide sequence of SEQ ID No. 6 from nucleotide 1 to nucleotide 253 or the complement thereof, or the nucleotide sequence of SEQ U) No. 11 from nucleotide position 17 to nucleotide, position 7621or the complement thereof, or the nucleotide sequence of SEQ ID No. 23 from nucleotide position 1059 to nucleotide position 8663, or the complement thereof;
(d) a first primer comprising the nucleotide sequence of SEQ ID No. 13 or SEQ ID No. 21, and a second primer comprising the nucleotide sequence of SEQ ID No. 12 or SEQ ID No. 20, respectively;
(e) a first primer comprising at its extreme 3′ end the nucleotide sequence of SEQ ID No. 13, or the nucleotide sequence of SEQ 1D No. 21, and a second primer comprising at its extreme 3′ end the nucleotide sequence of SEQ ID No. 1.2, or the nucleotide sequence of SEQ ID No. 20; or
(f) a first primer comprising at its extreme 3′ end the sequence of SEQ ID No. 13 and a second primer comprising at its extreme 3′ end the sequence of SEQ ID NO. 12, or comprising a first primer comprising at its extreme 3′ end the sequence of SEQ ID No. 21 and a second primer comprising at it

Assignees

• BASF Agricultural Solutions Seed US LLC

Inventors

• Moser Hal
• Buyse Maxim
• Slabbinck Filip
• Beilinson Vadim
• Kleven Thomas W
• Daum Julia
• Habex Veerle
• Aartsen Wendy
• Mccarville Michael

Classifications

• C12Q2600/13

  Plant traits · CPC title

• C12N15/8279

  for biotic stress resistance, pathogen resistance, disease resistance · CPC title

• C12N15/8285 Primary

  for nematode resistance · CPC title

• C12N15/8274 Primary

  for herbicide resistance · CPC title

• C12Q1/6895

  for plants, fungi or algae · CPC title