Elite event EE-GM3 and methods and kits for identifying such event in biological samples

Patent metadata

Field	Value
Publication number	US-9631202-B2
Application number	US-201013511629-A
Country	US
Kind code	B2
Filing date	Nov 23, 2010
Priority date	Nov 23, 2009
Publication date	Apr 25, 2017
Grant date	Apr 25, 2017

Abstract

The invention provides specific transgenic soybean plants, plant material and seeds, characterized in that these products harbor a specific 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 method for identifying elite event EE-GM3 in biological samples, which method comprises detection of an EE-GM3 specific region with a specific primer pair or probe which specifically recognizes the 5′ or 3′ flanking region of foreign DNA comprising herbicide tolerance genes in EE-GM3 and the inserted foreign DNA contiguous therewith; reference seed comprising elite event EE-GM3 having been deposited at the NCIMB under deposit number NCIMB 41659.

2. The method of claim 1 , said method comprising amplifying a DNA fragment of between 50 and 1000 bp from a nucleic acid present in said biological samples using a polymerase chain reaction with at least two primers, one of said primers recognizing the 5′ flanking region of foreign DNA comprising herbicide tolerance genes in EE-GM3, said 5′ flanking region comprising the nucleotide sequence of SEQ ID No. 2 from nucleotide 1 to nucleotide 1451 or the 3′ flanking region of foreign DNA comprising herbicide tolerance genes in EE-GM3, said 3′ flanking region comprising the nucleotide sequence of the complement of SEQ ID No. 3 from nucleotide 241 to nucleotide 1408, the other primer of said primers recognizing a sequence within the foreign DNA comprising the nucleotide sequence of the complement of SEQ ID No. 2 from nucleotide 1452 to nucleotide 1843 or the nucleotide sequence of SEQ ID No. 3 from nucleotide 1 to nucleotide 240 or the other primer of said primers recognizing a sequence within the foreign DNA comprising the nucleotide sequence of SEQ ID No. 1 or its complement, or comprising the nucleotide sequence of SEQ ID No. 11 from nucleotide position 1452 to nucleotide position 16638 or its complement.

3. The method of claim 2 , wherein said primer recognizing the 5′ flanking region comprises a nucleotide sequence of 17 to 200 consecutive nucleotides selected from the nucleotide sequence of SEQ ID No. 2 from nucleotide 1 to nucleotide 1451 or said primer recognizing the 3′ flanking region of EE-GM3 comprises a nucleotide sequence of 17 to 200 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID No. 3 from nucleotide 241 to nucleotide 1408, and said primer recognizing a sequence within the foreign DNA comprises 17 to 200 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID No. 2 from nucleotide 1452 to nucleotide 1843 or the nucleotide sequence of SEQ ID No. 3 from nucleotide 1 to nucleotide 240, or the nucleotide sequence of SEQ ID No. 1 or its complement, or the nucleotide sequence of SEQ ID No. 11 from nucleotide position 1452 to nucleotide position 16638 or its complement.

4. The method of claim 2 , wherein said primer recognizing the 5′ flanking region comprises at its extreme 3′ end a nucleotide sequence of at least 17 consecutive nucleotides selected from the nucleotide sequence of SEQ ID No. 2 from nucleotide 1 to nucleotide 1451 or said primer recognizing the 3′ flanking region of EE-GM3 comprises at its extreme 3′ end a nucleotide sequence of at least 17 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID No. 3 from nucleotide 241 to nucleotide 1408, and said primer recognizing a sequence within the foreign DNA comprises at its 3′ end at least 17 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID No. 2 from nucleotide 1452 to nucleotide 1843 or the nucleotide sequence of SEQ ID No. 3 from nucleotide 1 to nucleotide 240 or the nucleotide sequence of SEQ ID No. 1 or its complement, or the nucleotide sequence of SEQ ID No. 11 from nucleotide position 1452 to nucleotide position 16638 or its complement.

5. The method of claim 4 , wherein said primers comprise the sequence of SEQ ID No. 5 and SEQ ID No. 4, respectively, or the sequence of SEQ ID No. 5 and SEQ ID No. 7 respectively.

6. The method of claim 5 , which method comprises amplifying a fragment of about 263 or 706 bp using the EE-GM3 PCR identification protocol.

7. A kit comprising one primer recognizing the 5′ flanking region of foreign DNA comprising herbicide tolerance genes in EE-GM3, said 5′ flanking region comprising the nucleotide sequence of SEQ ID No. 2 from nucleotide 1 to nucleotide 1451 or one primer recognizing the 3′ flanking region of foreign DNA comprising herbicide tolerance genes in EE-GM3, said 3′ flanking region comprising the nucleotide sequence of the complement of SEQ ID No. 3 from nucleotide 241 to nucleotide 1408, and one primer recognizing a sequence within the foreign DNA, said foreign DNA comprising the nucleotide sequence of the complement of SEQ ID No. 2 from nucleotide 1452 to nucleotide 1843 or the nucleotide sequence of SEQ ID No. 3 from nucleotide 1 to nucleotide 240 or the nucleotide sequence of SEQ ID No. 1 or its complement, or the nucleotide sequence of SEQ ID No. 11 from nucleotide position 1452 to nucleotide position 16638 or its complement, wherein
at least one of said primers is labeled;
the 5′ end of at least one of said primers comprises one or more mismatches or a nucleotide sequence unrelated to the 5′ or 3′ flanking sequence of foreign DNA sequence;
at least one of said primers comprises a nucleotide sequence at their 3′ end spanning the joining region between the plant DNA derived sequences and the foreign DNA sequences, said joining region being at nucleotides 1451-1452 in SEQ ID No 2 or nucleotides 240-241 in SEQ ID No 3, provided that the 17 consecutive nucleotides at the 3′ end are not derived exclusively from either the foreign DNA or plant-derived sequences in SEQ ID Nos. 2 or 3; or
at least one of said primers comprises a sequence which is between 80 and 100% identical to a sequence within the 5′ or 3′ flanking region of the elite event or the foreign DNA of the elite event, respectively, and said primer sequence comprises at least one mismatch with said 5′ or 3′ flanking region or said foreign DNA, provided the at least one mismatch still allows specific identification of the elite event with these primers under optimized PCR conditions.

8. The kit of claim 7 , wherein said primer recognizing the 5′ flanking region comprises a nucleotide sequence of 17 to 200 consecutive nucleotides selected from the nucleotide sequence of SEQ ID No. 2 from nucleotide 1 to nucleotide 1451 or said primer recognizing the 3′ flanking region of EE-GM3 comprises a nucleotide sequence of 17 to 200 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID No. 3 from nucleotide 241 to nucleotide 1408, and said primer recognizing a sequence within the foreign DNA comprises 17 to 200 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID No. 2 from nucleotide 1452 to nucleotide 1843 or the nucleotide sequence of SEQ ID No. 3 from nucleotide 1 to nucleotide 240, or the nucleotide sequence of SEQ ID No. 1 or its complement, or the nucleotide sequence of SEQ ID No. 11 from nucleotide position 1452 to nucleotide position 16638 or its complement.

9. The kit of claim 7 , wherein said primer recognizing the 5′ flanking region comprises at its extreme 3′ end a nucleotide sequence of at least 17 consecutive nucleotides selected from the nucleotide sequence of SEQ ID No. 2 from nucleotide 1 to nucleotide 1451 or said primer recognizing the 3′ flanking region of EE-GM3 comprises at its extreme 3′ end a nucleotide sequence of at least 17 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID No. 3 from nucleotide 241 to nucleotide 1408, and said primer recognizing a sequence within the foreign DNA comprises at its 3′ end at least 17 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID No. 2 from nucleotide 1452 to nucleotide 18

Assignees

• Mason Justin Thomas
• Lettow Leslie James
• Eby Mark Alan
• Eby William H
• Welz Günter
• Verhaeghe Steven
• De Beuckeleer Marc
• Habex Veerle
• Ferrulo Jean-Marc
• Ms Technologies Llc
• Bayer Cropscience Nv

Inventors

• Mason Justin Thomas
• Lettow Leslie James
• Eby Mark Alan
• Eby William H
• Welz Günter
• Verhaeghe Steven
• De Beuckeleer Marc
• Habex Veerle
• Ferrulo Jean-Marc

Classifications

• C12N15/8275

  Glyphosate · CPC title

• C12N15/8274 Primary

  for herbicide resistance · CPC title

• C12Q2600/13

  Plant traits · CPC title

• C12Q1/6895 Primary

  for plants, fungi or algae · CPC title

• C12Q2600/156

  Polymorphic or mutational markers · CPC title