Soybean event 3560.4.3.5 and compositions and methods for the identification and detection thereof

Abstract

Compositions and methods related to transgenic glyphosate/ALS inhibitor-tolerant soybean plants are provided. Specifically, soybean plants having a 3560.4.3.5 event which imparts tolerance to glyphosate and at least one ALS-inhibiting herbicide are provided. The soybean plant harboring the 3560.4.3.5 event at the recited chromosomal location comprises genomic/transgene junctions having at least the polynucleotide sequence of SEQ ID NO:10 and/or 11. The characterization of the genomic insertion site of the 3560.4.3.5 event provides for an enhanced breeding efficiency and enables the use of molecular markers to track the transgene insert in the breeding populations and progeny thereof. Various methods and compositions for the identification, detection, and use of the soybean 3560.4.3.5 events are provided.

1 . An isolated polynucleotide comprising SEQ ID NO: 10 or 11. 
     
     
         2 . The isolated polynucleotide of  claim 1 , wherein said polynucleotide is selected from the group consisting of:
 (a) a nucleotide sequence set forth in SEQ ID NO:6, 12, 13, 14, 15, 27, 28, 41, or 42; and,   (b) a nucleotide sequence comprising a fragment of SEQ ID NO: 6, 12, 13, 14, 15, 27, 28, 41 or 42.   
     
     
         3 . A kit for identifying event 3560.4.3.5 in a biological sample, said kit comprising a first and a second primer, wherein said first and said second primer amplify a polynucleotide comprising a 3560.4.3.5 specific region. 
     
     
         4 . The kit of  claim 3 , wherein said kit further comprises a polynucleotide for the detection of the 3560.4.3.5 specific region. 
     
     
         5 . The kit of  claim 3 , wherein said first primer comprises a first fragment of SEQ ID NO: 6 and the second primer comprises a second fragment of SEQ ID NO:6, wherein said first and said second primer flank said 3560.4.3.5 specific region and share sufficient sequence homology or complementarity to said polynucleotide to amplify said 3560.4.3.5 specific region. 
     
     
         6 . The kit of  claim 5 , wherein
 a) said first primer comprises a fragment of SEQ ID NO:3 and said second primer comprises a fragment of SEQ ID NO: 5;   b) said first primer comprises a fragment of SEQ ID NO:3 and said second primer comprises a fragment of SEQ ID NO:4;   c) said first primer comprises a fragment of SEQ ID NO: 4 and said second primer comprises a fragment of SEQ ID NO:5.   
     
     
         7 . The kit of  claim 5 , wherein said first and said second primer comprises at least 8 consecutive polynucleotides of SEQ ID NO: 6. 
     
     
         8 . The kit of  claim 6 , wherein said first or said second primer comprises at least 8 consecutive polynucleotides of SEQ ID NO:3, 4, or 5. 
     
     
         9 . The kit of  claim 3 , wherein said first or said second primer comprise SEQ ID NO:7, 8, 9, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 37, 38, 39, 40, 44, 45, 46, 51, 52, 53, 54, 55. 
     
     
         10 . A DNA detection kit comprising at least one polynucleotide that can specifically detect a 3560.4.3.5 specific region, wherein said polynucleotide comprises at least one DNA molecule of a sufficient length of contiguous nucleotides identical or complementary to SEQ ID NO: 6. 
     
     
         11 . The DNA detection kit of  claim 10 , wherein said polynucleotide that can specifically detect a 3560.4.3.5 specific region comprises a polynucleotide having SEQ ID NO:10 or 11. 
     
     
         12 . The DNA detection kit of  claim 10 , wherein said polynucleotide comprises a sequence which hybridizes under stringent conditions with sequences selected from the group consisting of:
 (a) the sequences of SEQ ID NO:5 and SEQ ID NO:3; and,   (b) the sequences of SEQ ID NO:4 and SEQ ID NO: 3.   
     
     
         13 . A method for identifying event 3560.4.3.5 in a biological sample, comprising
 (a) contacting said sample with a first and a second primer; and,   (b) amplifying a polynucleotide comprising a 3560.4.3.5 specific region.   
     
     
         14 . The method of  claim 13 , wherein the polynucleotide comprising the 3560.4.3.5 specific region comprises SEQ ID NO:11. 
     
     
         15 . The method of  claim 13 , wherein the polynucleotide comprising the 3560.4.3.5 specific region comprises SEQ ID NO:10. 
     
     
         16 . The method of  claim 13 , further comprising detecting the 3560.4.3.5 specific region. 
     
     
         17 . The method of  claim 13 , wherein said first primer comprises a first fragment of SEQ ID NO: 6 and the second primer comprises a second fragment of SEQ ID NO:6, wherein said first and said second primer flank said 3560.4.3.5 specific region and share sufficient sequence homology or complementarity to said polynucleotide to amplify said 3560.4.3.5 specific region. 
     
     
         18 . The method of  claim 17  , wherein
 a) said first primer comprises a fragment of SEQ ID NO:3 and said second primer comprises a fragment of SEQ ID NO:5;   b) said first primer comprises a fragment of SEQ ID NO:3 and said second primer comprises a fragment of SEQ ID NO:4;   c) said first primer comprises a fragment of SEQ ID NO: 5 and said second primer comprises a fragment of SEQ ID NO:4.   
     
     
         19 . The method of  claim 17 , wherein said first and said second primer comprise at least 8 consecutive polynucleotides of SEQ ID NO: 6. 
     
     
         20 . The method of  claim 18 , wherein said first and said second primer comprise at least 8 consecutive polynucleotides of SEQ ID NO:5, 4 or 3. 
     
     
         21 . The method of  claim 13  , wherein said first and said second primer comprise
 SEQ ID NO:7, 8, 9, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 37, 38, 39 40, 44, 45, 46, 51, 52, 53, 54, or 55.   
     
     
         22 . A method for identifying event 3560.4.3.5 in a biological sample, comprising
 (a) contacting said sample with a first and a second primer;   (b) performing a DNA amplification reaction, thereby producing a DNA amplicon molecule; and   (c) detecting said DNA amplicon molecule, wherein the detection of said DNA amplicon molecule in said DNA amplification reaction indicates the presence of soybean event 3560.4.3.5.   
     
     
         23 . The method of  claim 22 , wherein said first primer comprises a first fragment of SEQ ID NO: 6 and the second primer comprises a second fragment of SEQ ID NO:6, wherein said first and said second primer flank said 3560.4.3.5 specific region and share sufficient sequence homology or complementarity to said polynucleotide to amplify said 3560.4.3.5 specific region. 
     
     
         24 . The method of  claim 22 , wherein said first and second primer are selected from the group consisting of:
 (a) the sequences comprising SEQ ID NO:16 and SEQ ID NO:17;   (b) the sequences comprising SEQ ID NO:7 and SEQ ID NO:8;   (c) the sequences comprising SEQ ID NO:23 and SEQ ID NO:20;   (d) the sequences comprising SEQ ID NO:23 and SEQ ID NO:19;   (e) the sequences comprising SEQ ID NO:18 and SEQ ID NO:22;   (f) the sequences comprising SEQ ID NO:21 and SEQ ID NO:22;   (g) the sequences comprising SEQ ID NO:7 and SEQ ID NO:9;   (h) the sequences comprising SEQ ID NO:8 and SEQ ID NO:9; and,   (i) the sequences comprising SEQ ID NO:44 and SEQ ID NO:45.   
     
     
         25 . A method of detecting the presence of DNA corresponding to a 3560.4.3.5 event in a sample, the method comprising:
 (a) contacting the sample with a polynucleotide probe that hybridizes under stringent hybridization conditions with DNA from soybean event 3560.4.3.5 and specifically detects the 3560.4.3.5 event;   (b) subjecting the sample and probe to stringent hybridization conditions; and   (c) detecting hybridization of the probe to the DNA, wherein detection of hybridization indicates the presence of the 3560.4.3.5 event.   
     
     
         26 . The method of  claim 25 , wherein said sample comprises soybean tissue. 
     
     
         27 . A polynucleotide comprising a sequence selected from the group consisting of SEQ ID NO: 7, 8, 9, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 37, 38, 39, 40, 43, 44, 45, 46, 51, 52, 53, 54, 55, or its complement. 
     
     
         28 . A pair of DNA molecules comprising a first DNA molecule and a second DNA molecule, wherein said first DNA molecule comprising a first fragment of SEQ ID NO: 6 and the second DNA molecule comprising a second fragment of SEQ ID NO:6, wherein said first and said second DNA molecule flank said 3560.4.3.5 specific region of SEQ ID NO:6 and share sufficient sequence homology or complementarity to said polynucleotide to amplify said 3560.4.3.5 specific region. 
     
     
         29 . A method for confirming seed purity or a method for screening seeds in a seed lot for a 3560.4.3.5 event comprising
 (a) contacting said sample with a first and a second primer;   (b) performing a DNA amplification reaction, thereby producing a DNA amplicon molecule; and   (c) detecting said DNA amplicon molecule, wherein the detection of said DNA amplicon molecule in said DNA amplification reaction indicates the presence of soybean event 3560.4.3.5.   
     
     
         30 . The method of  claim 29 , wherein said first primer comprises a first fragment of SEQ ID NO: 6 and the second primer comprises a second fragment of SEQ ID NO:6, wherein said first and said second primer flank said 3560.4.3.5 specific region and share sufficient sequence homology or complementarity to said polynucleotide to amplify said 3560.4.3.5 specific region. 
     
     
         31 . The method of  claim 30 , wherein
 a) said first primer comprises a fragment of SEQ ID NO:3 and said second primer comprises a fragment of SEQ ID NO:5;   b) said first primer comprises a fragment of SEQ ID NO:3 and said second primer comprises a fragment of SEQ ID NO:4;   c) said first primer comprises a fragment of SEQ ID NO: 4 and said second primer comprises a fragment of SEQ ID NO:5.   
     
     
         32 . The method of  claim 30 , wherein said first and said second primer comprises at least 8 consecutive polynucleotides of SEQ ID NO: 6. 
     
     
         33 . The method of  claim 31 , wherein said second primer and said first primer comprise at least 8 consecutive polynucleotides of SEQ ID NO:3, 4, or 5. 
     
     
         34 . The method of  claim 29 , wherein said first or said second primer comprise SEQ ID NO: 7, 8, 9, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 37, 38, 39, 40, 44, 45, 46, 51, 52, 53, 54, or 55. 
     
     
         35 . A method for confirming seed purity or for screening for the presence of a 3560.4.3.5 event in a seed lot comprising
 (a) contacting a sample comprising soybean DNA with a polynucleotide probe that hybridizes under stringent hybridization conditions with DNA from soybean event 3560.4.3.5 and specifically detects the 3560.4.3.5 event;   (b) subjecting the sample and probe to stringent hybridization conditions; and   (c) detecting hybridization of the probe to the DNA, wherein detection of hybridization indicates the presence of the 3560.4.3.5 event.   
     
     
         36 . The method of  claim 35 , wherein said polynucleotide is selected from the group consisting of:
 (a) a nucleotide sequence set forth in SEQ ID NO:6, 10, 11, 12, 13, 14, 15, 27, 28, 41, 42, or 43; or   (b) a nucleotide sequence comprising a fragment of SEQ ID NO: 6, 10, 11, 12, 13, 14, 15, 27, 28, 41, 42, or 43.