Inbred transgenic canola line ns-b50027-4 and seeds thereof

Abstract

The present embodiments relate to inbred transgenic canola line NS-B50027-4; seeds and oils obtained from NS-B50027-4; and progeny derived from NS-B50027-4. In particular, NS-B50027-4 is a true-breeding canola line capable of producing at least 5% DHA in its seed oil.

We claim: 
     
         1 . A method of detecting the presence of an elite event NS-B50027-4, representative seed of an inbred NS-B50027-4 canola line having been deposited under ATCC Accession Number PTA-123186, in a sample comprising a plant DNA, said method comprises the steps of:
 (a) contacting said sample with:
 (i) a first nucleic acid primer that binds to a flanking junction region of  Brassica  genome of NS-B50027-4, and 
 (ii) a second nucleic acid primer that binds to a transgene of NS-B50027-4; 
   (b) subjecting said sample to a polymerase chain reaction assay; and   (c) assaying the amplicons generated between said first and second nucleic acid primers, wherein the presence of said generated amplicons is diagnostic of elite event NS-B50027-4 in said sample, and   wherein said inbred canola line NS-B50027-4 contains within its genome:   a first transgenic locus, located on a first chromosome, comprising one copy of each of a M  pusilla Δ 6-desaturase, a  P. cordata Δ 5-elongase, a  P. salina Δ 5-desaturase, and a  P. pastoris Δ 15/ω3-desaturase gene; and   a second transgenic locus, located on a second chromosome, comprising two copies of each of a  Micromonas pusilla Δ 6-desaturase, a  Pyramimonas cordata Δ 5-elongase, a  Pavlova salina  Δ5-desaturase, a  Pichia pastoris Δ 15/ω3-desaturase, a  Pavlova salina  Δ4-desaturase, a Lachancea  kluyveri Δ 12-desaturase, and a  P. cordata Δ 6-elongase gene.   
     
     
         2 . The method of  claim 2 , wherein said first and second nucleic acid primers are the primers as shown in SEQ. ID NO:1 to SEQ ID NO:90. 
     
     
         3 . A kit comprising components to carry out the method of  claim 1  or  claim 2 . 
     
     
         4 . A method of detecting the presence of at least one transgenic locus in a sample, wherein said method comprises the steps of:
 (a) contacting said sample that comprises a plant DNA with:
 (i) at least one first nucleic acid primer that binds to a first transgene-flanking junction region of a first transgenic locus, said at least one first nucleic acid primer selected from the group of primers consisting of SEQ ID NO:17-SEQ ID NO:31 and SEQ ID NO:83-SEQ ID NO:86; or 
 (ii) at least one second nucleic acid primer that binds to a second transgene-flanking junction region of said first transgenic locus, said at least one second nucleic acid primer selected from the group of primers consisting of SEQ ID NO:32-SEQ ID NO:41; or 
 (iii) at least one third nucleic acid primer that binds to a first transgene-flanking junction region of a second transgenic locus, said at least one third nucleic acid primer selected from the group of primers consisting of SEQ ID NO:42-SEQ ID NO:62; or 
 (iv) at least one fourth nucleic acid primer that binds to a second transgene-flanking junction region of said second transgenic locus, said at least one fourth nucleic acid primer selected from the group of primers consisting of SEQ ID NO:63-SEQ ID NO:90; or 
 (v) at least ten contiguous nucleotides of any preceding nucleic acid primers of (i), (ii), (iii) and (iv) or complements thereof; 
   (b) subjecting said sample to polymerase chain reaction assay; and   (c) assaying for amplicons generated between said primers, wherein the presence of said amplicons is diagnostic of the presence of at least one transgenic locus from a segregant of an inbred canola line NS-B50027-4,   wherein said inbred canola line NS-B50027-4, representative seed of an inbred NS-B50027-4 canola line having been deposited under ATCC Accession Number PTA-123186.   
     
     
         5 . The method of  claim 4 , wherein said polymerase chain reaction assay is a KASP™ genotyping assay. 
     
     
         6 . The method of  claim 4 , further comprising the step of contacting said sample with a nucleic acid primer that binds to a transgene sequence, said nucleic acid primer is selected from the group of primers consisting of those depicted in SEQ ID NO:1-SEQ ID NO:8, at least ten contiguous nucleotides thereof, or complements thereof. 
     
     
         7 . The method of  claim 4 , further comprising the step of contacting said sample with a nucleic acid primer that binds to a transgene sequence, said nucleic acid primer is selected from the group consisting of those depicted in SEQ ID NO:1-SEQ ID NO:16, at least ten contiguous nucleotides thereof, or complements thereof. 
     
     
         8 . A kit comprising components to carry out the method of  claim 4 . 
     
     
         9 . A recombinant nucleic acid molecule extracted from an inbred canola line NS-B50027-4, or a progeny or segregant thereof, wherein a representative seed of said inbred canola line NS-B50027-4 has been deposited under ATCC Accession Number PTA-123186, wherein said NS-B50027-4 contains within its genome:
 a first transgenic locus comprising one copy of each of a  M. pusilla Δ 6-desaturase, a  P. cordata Δ 5-elongase, a  P. salina Δ 5-desaturase, and a  P. pastoris Δ 15/ω3-desaturase gene,   a second transgenic locus comprising two copies of each of a  Micromonas pusilla Δ 6-desaturase, a  Pyramimonas cordata Δ 5-elongase, a  Pavlova salina  Δ5-desaturase, a  Pichia pastoris Δ 15/ω3-desaturase, a  Pavlova salina  Δ4-desaturase, a  Lachancea kluyveri Δ 12-desaturase, and a  P. cordata Δ 6-elongase gene;   wherein said first transgenic locus is located on a first chromosome, and said second transgenic locus is located on a second chromosome;   wherein said inbred canola line NS-B50027-4, or a progeny or segregant thereof, comprises either said first transgenic locus, said second transgenic locus, or both said transgenic loci.   
     
     
         10 . The recombinant nucleic acid molecule of  claim 9 , wherein said recombinant nucleic acid molecule has the nucleic acid sequence of (a) nucleotides 1160 to 47773 of SEQ ID NO:41 or a complement thereof; (b) nucleotides 2090 to 14201 of SEQ ID NO:40 or a complement thereof; or (c) both (a) and (b). 
     
     
         11 . The recombinant nucleic acid molecule of  claim 10 , wherein said nucleic acid molecule of (a) is at least 80%, 95%, 97%, 98%, 99%, or 99.5% identical to the nucleotide sequence of SEQ ID NO:41 from nucleotide position 1268 to nucleotide position 47662 or a complement thereof. 
     
     
         12 . The recombinant nucleic acid molecule of  claim 10 , wherein said nucleic acid molecule of (b) is at least 80%, 95%, 97%, 98%, 99%, or 99.5% identical to the nucleotide sequence of SEQ ID NO:40 from nucleotide position 2090 to nucleotide position 14201 or a complement thereof.