Brassica events LBFLFK and LBFDAU and methods for detection thereof

Patent metadata

Field	Value
Publication number	US-11033593-B2
Application number	US-201515526443-A
Country	US
Kind code	B2
Filing date	Nov 13, 2015
Priority date	Nov 14, 2014
Publication date	Jun 15, 2021
Grant date	Jun 15, 2021

Abstract

The present invention provides transgenic Brassica events LBFLFK and LBFDAU and progeny thereof, and cells, seeds, plants comprising DNA diagnostic for these events. The invention also provides artificial oligonucleotide primers and probes that are diagnostic for the LBFLFK and LBFDAU events and their progeny in a sample, and methods for detecting the presence of the LBFLFK and LBFDAU events and their progeny in a sample. The invention further provides oil and commodity products derived from the LBFLFK and LBFDAU events.

First claim

The invention claimed is:

1. A Brassica plant or seed comprising event LBFLFK, a sample of seed comprising transformation event LBFLFK having been deposited under ATCC Accession No. PTA-121703.

2. An F1 progeny of the Brassica plant of claim 1 , wherein the F1 progeny comprise polynucleotide having the sequence of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:13, or SEQ ID NO:14.

3. An artificial DNA molecule comprising a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:13.

4. A method of detecting the presence of DNA corresponding to the Brassica event LBFLFK in a sample comprising DNA, the method comprising the steps of:
(a) contacting the sample with an LBFLFK Locus 1 primer pair and an LBFLFK Locus 2 primer pair that, when used in a nucleic acid amplification reaction with genomic DNA from Brassica event LBFLFK, produces a Locus 1 amplicon and a Locus 2 amplicon that are diagnostic for Brassica event LBFLFK;
(b) performing a nucleic acid amplification reaction, thereby producing the Locus 1 and Locus 2 amplicons; and
(c) detecting the amplicons, wherein one amplicon comprises the LBFLFK Locus 1 junction region SEQ ID NO:4 or SEQ ID NO:5, or the complement thereof, and one amplicon comprises the LBFLFK Locus 2 junction region SEQ ID NO:13 or SEQ ID NO:14, or the complement thereof.

5. The method of claim 4 , wherein
(a) the LBFLFK Locus 1 primer pair comprises:
(i) a first primer selected from the group consisting of at least 11 consecutive nucleotides of SEQ ID NO:6, at least 11 consecutive nucleotides of the complement of SEQ ID NO:6, at least 11 consecutive nucleotides of SEQ ID NO:7, and at least 11 consecutive nucleotides of the complement of SEQ ID NO:7; and
(ii) a second primer selected from the group consisting of at least 11 consecutive nucleotides of SEQ ID NO:3 and at least 11 consecutive nucleotides of the complement of SEQ ID NO:3, and
(b) the LBFLFK Locus 2 primer pair comprises:
(i) a third primer selected from the group consisting of at least 11 consecutive nucleotides of SEQ ID NO:15 at least 11 consecutive nucleotides of the complement of SEQ ID NO:15, at least 11 consecutive nucleotides of SEQ ID NO:16, and at least 11 consecutive nucleotides of the complement of SEQ ID NO:16; and
(ii) a fourth primer selected from the group consisting of at least 11 consecutive nucleotides of SEQ ID NO:12 and at least 11 consecutive nucleotides of the complement of SEQ ID NO:12.

6. The method of claim 5 , wherein the first primer comprises SEQ ID NO:8, the second primer comprises SEQ ID NO:9, the third primer comprises SEQ ID NO:17, and the fourth primer comprises SEQ ID NO:18.

7. The method of claim 4 , further comprising the steps of:
d) contacting the sample with an LBFLFK Locus 1 wild type primer pair comprising at least 11 consecutive nucleotides of the Brassica genomic region of the LBFLFK Locus 1 transgene insertion and an LBFLFK Locus 2 wild type primer pair comprising at least 11 consecutive nucleotides of the Brassica genomic region of the LBFLFK Locus 2 transgene insertion;
e) performing a nucleic acid amplification reaction, thereby producing amplicons of the homologous wild type Brassica genomic regions corresponding to LBFLFK Locus 1 and LBFLFK Locus 2 insertions;
f) detecting the wild type Brassica amplicons; and
g) comparing the amplicons produced in step c) with the amplicons produced in step f), wherein the presence of both amplicons indicates that the sample is heterozygous for the LBFLFK Locus 1 and Locus 2 transgene insertions.

8. A meal derived from Brassica seed of claim 1 or progeny thereof, wherein the meal comprises a detectable amount of a nucleotide sequence diagnostic for Brassica event LBFLFK, wherein the sequence is selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:13.

9. A commodity product comprising the artificial DNA molecule of claim 3 .

10. A method for producing a Brassica plant, the method comprising the step of crossing a plant comprising Brassica event LBFLFK with a Brassica plant lacking event LBFLFK to obtain a plant comprising the Brassica event LBFLFK, wherein a sample of event LBFLFK has been deposited under ATCC Accession No. PTA-121703.

11. A Brassica plant comprising an artificial DNA molecule comprising a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:13, and SEQ ID NO:14, wherein the plant further comprises a polynucleotide comprising SEQ ID NO: 3 and/or a polynucleotide comprising SEQ ID NO: 12.

12. A Brassica seed comprising an artificial DNA molecule comprising a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:13, and SEQ ID NO:14, wherein the seed further comprises a polynucleotide comprising SEQ ID NO: 3 and/or a polynucleotide comprising SEQ ID NO: 13.

13. An oil derived from Brassica seed of claim 1 or progeny thereof, wherein the oil comprises a detectable amount of a nucleotide sequence diagnostic for Brassica event LBFLFK, wherein the sequence is selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:13.

Assignees

• Basf Plant Science Co Gmbh

Inventors

• Senger Toralf
• Marty Laurent
• Kunze Irene
• Rein Dietrich
• Bauer Joerg
• Andre Carl

Classifications

• C12N15/8247 Primary

  involving modified lipid metabolism, e.g. seed oil composition · CPC title

• A61K36/31 Primary

  Brassicaceae or Cruciferae (Mustard family), e.g. broccoli, cabbage or kohlrabi · CPC title

• C12Y114/19003

  Linoleoyl-CoA desaturase (1.14.19.3) · CPC title

• C12N9/93

  Ligases (6) · CPC title

• C12N9/0071

  acting on paired donors with incorporation of molecular oxygen (1.14) · CPC title