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US 2004/0171027 A1Published application
Assay for imidazolinone resistance mutations in Brassica species
AssigneeAdvanta Canada, Inc.
InventorsStephen Barnes, Sigrid Vanstraelen
Life of this patent
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Abstract
The invention provides methods and oligonucleotide primers for assaying Brassica napus plants for the presence or absence of mutations that confer resistance to imidazolinone herbicides. Specifically, the methods and primers of the invention are useful for detecting the PM1 mutation of the B. napus AHAS1 gene and the PM2 mutation of the B. napus AHAS3 gene.
Claims25
A method of assaying a Brassica plant for imidazolinone herbicide tolerance conferred by the PM1 mutation of the B. napus AHAS1 gene, the method comprising the steps of:
- a) isolating genomic DNA from the plant;
- b) selectively amplifying an AHAS1 gene from the genomic DNA using an AHAS1 forward primer and an AHAS1 reverse primer in a first amplification step, thereby producing an AHAS1 reaction mixture;
- c) removing the AHAS1 primers from the AHAS1 reaction mixture to produce a purified AHAS1 reaction mixture;
- d) in a second amplification step, further amplifying a portion of the amplified AHAS1 gene containing the site of the PM1 mutation, by combining the purified AHAS1 reaction mixture with a PM1 forward primer and a PM1 reverse primer, wherein the PM1 forward primer and the PM1 reverse primer are nested within the AHAS1 forward and reverse primers;
- e) denaturing the product of the second amplification step to produce single stranded polynucleotides that are allowed to adopt unique conformations by intramolecular interactions;
- and
- f) detecting the presence or absence of the PM1 mutation on the basis of the mobility of said single stranded polynucleotide conformers in a substrate.
4 more elements
A method for assaying a Brassica plant for imidazolinone herbicide tolerance conferred by the PM2 mutation of the B. napus AHAS3 gene, the method comprising the steps of:
- a) isolating genomic DNA from the plant;
- b) selectively amplifying the AHAS3 gene from the genomic DNA using an AHAS3 forward primer and an AHAS3 reverse primer in a first amplification step to produce an AHAS3 reaction mixture;
- c) removing the AHAS3 primers from the AHAS3 reaction mixture to produce a purified AHAS3 reaction mixture;
- d) in a second amplification step, further amplifying the amplified AHAS3 gene, by combining a first aliquot of the purified AHAS3 reaction mixture with a PM2 region forward primer, a PM2 region reverse primer, and a primer selective for a wild type allele of the PM2 region at position 1712 of the AHAS3 gene as depicted in SEQ ID NOs:5 and 8;
- e) in a third amplification step further amplifying the amplified AHAS3 gene, by combining a second aliquot of the purified AHAS3 reaction mixture with a PM2 region forward primer, a PM2 region reverse primer, and a primer selective for the PM2 mutation;
- and
- f) analyzing the amplified first and second aliquots for the presence or absence of the PM2 mutation.
4 more elements
An amplification primer selected from the group consisting of an oligonucleotide having a sequence as set forth in SEQ ID NO:9;
- an oligonucleotide having a sequence as set forth in SEQ ID NO:10;
- an oligonucleotide having a sequence as set forth in SEQ ID NO:11;
- an oligonucleotide having a sequence as set forth in SEQ ID NO:12;
- an oligonucleotide having a sequence as set forth in SEQ ID NO:13;
- an oligonucleotide having a sequence as set forth in SEQ ID NO:14;
- an oligonucleotide having a sequence as set forth in SEQ ID NO:15;
- an oligonucleotide having a sequence as set forth in SEQ ID NO:16;
- an oligonucleotide having a sequence as set forth in SEQ ID NO:17;
- and an oligonucleotide having a sequence as set forth in SEQ ID NO:18.
6 more elements
A nucleic acid selected from the group consisting of a nucleic acid having a sequence as set forth from nucleotide 96 to nucleotide 2330 of SEQ ID NO:19;
- a nucleic acid having a sequence as set forth from nucleotide 1817 to nucleotide 2063 of SEQ ID NO:1;
- a nucleic acid having a sequence as set forth from nucleotide 1735 to nucleotide 1980 of SEQ ID NO:2;
- a nucleic acid having a sequence as set forth from nucleotide 1809 to nucleotide 2054 of SEQ ID NO:3;
- a nucleic acid having a sequence as set forth from nucleotide 1720 to nucleotide 1966 of SEQ ID NO:4;
- a nucleic acid having a sequence as set forth from nucleotide 64 to nucleotide 2310 of SEQ ID NO:20;
- a nucleic acid having a sequence as set forth from nucleotide 1383 to nucleotide 1770 of SEQ ID NO:5;
- a nucleic acid having a sequence as set forth from nucleotide 1518 to nucleotide 1905 of SEQ ID NO:6;
- a nucleic acid having a sequence as set forth from nucleotide 1352 to nucleotide 1739 of SEQ ID NO:7;
- a nucleic acid having a sequence as set forth from nucleotide 1308 to nucleotide 1695 of SEQ ID NO:8;
- a nucleic acid having a sequence as set forth from nucleotide 1560 to nucleotide 1770 of SEQ ID NO:5;
- a nucleic acid having a sequence as set forth from nucleotide 1695 to nucleotide 1905 of SEQ ID NO:6;
- a nucleic acid having a sequence as set forth from nucleotide 1529 to nucleotide 1739 of SEQ ID NO:7;
- and a nucleic acid having a sequence as set forth from nucleotide 1485 to nucleotide 1695 of SEQ ID NO:8.
10 more elements
A method of marker assisted breeding of plants of Brassica species using a PM1 mutation of the B. napus AHAS1 gene as a marker, the method comprising the steps of:
- a) isolating genomic DNA from a Brassica plant;
- b) selectively amplifying an AHAS1 gene from the genomic DNA using an AHAS1 forward primer and an AHAS1 reverse primer in a first amplification step, thereby producing an AHAS1 reaction mixture;
- c) removing the AHAS1 primers from the AHAS1 reaction mixture to produce a purified AHAS1 reaction mixture;
- d) in a second amplification step, further amplifying a portion of the amplified AHAS1 gene containing the site of the PM1 mutation, by combining the purified AHAS1 reaction mixture with a PM1 forward primer and a PM1 reverse primer, wherein the PM1 forward primer and the PM1 reverse primer are nested within the AHAS1 forward and reverse primers;
- e) denaturing the product of the second amplification step to produce single stranded polynucleotides that are allowed to adopt unique conformations by intramolecular interactions;
- f) detecting the presence or absence of the PM1 mutation on the basis of the mobility of said single stranded polynucleotide conformers in a substrate:
- and
- g) selecting said plant as a parent for further breeding if the PM1 mutation is present.
5 more elements
A method of marker assisted breeding of plants of Brassica species using a PM2 mutation of the B. napus AHAS3 gene as a marker, the method comprising the steps of:
- a) isolating genomic DNA from the plant;
- b) selectively amplifying the AHAS3 gene from the genomic DNA using an AHAS3 forward primer and an AHAS3 reverse primer in a first amplification step to produce an AHAS3 reaction mixture;
- c) removing the AHAS3 primers from the AHAS3 reaction mixture to produce a purified AHAS3 reaction mixture;
- d) in a second amplification step, further amplifying the amplified AHAS3 gene, by combining a first aliquot of the purified AHAS3 reaction mixture with a PM2 region forward primer, a PM2 region reverse primer, and a primer selective for a wild type allele of the PM2 region at position 1712 of the AHAS3 gene as depicted in SEQ ID NOs:5 and 8;
- e) in a third amplification step further amplifying the amplified AHAS3 gene, by combining a second aliquot of the purified AHAS3 reaction mixture with a PM2 region forward primer, a PM2 region reverse primer, and a primer selective for the PM2 mutation;
- f) analyzing the amplified first and second aliquots for the presence or absence of the PM2 mutation, and
- g) selecting said plant as a parent for further breeding if the PM2 mutation is present.
4 more elements
Dependencies are inferred from the claim text.