Herbicide-resistant sunflower plants with multiple herbicide resistant alleles of ahasl1 and methods of use

Abstract

Herbicide resistant sunflower plants comprising two different herbicide-resistant alleles of the sunflower acetohydroxyacid synthase large subunit 1 (AHASL1) gene are described. Methods for making these sunflower plants and methods for controlling weeds or other undesired vegetation growing in the vicinity of these sunflower plants are disclosed. Such methods involve the use of acetohydroxyacid synthase-inhibiting herbicides. Methods for controlling parasitic weeds growing on sunflower plants are also described. Additionally provided are methods for determining the genotype of sunflower plants for AHASL1 gene.

1 - 38 . (canceled) 
     
     
         39 . A method for genotyping a sunflower acetohydroxyacid synthase large subunit 1 (AHASL1) gene, said method comprising:
 (a) obtaining DNA from a sunflower plant;   (b) using the DNA of (a) as a template for a first polymerase chain reaction (PCR) amplification comprising said DNA, polymerase, deoxyribonucleotide triphosphates, a forward AHASL1 primer, and a reverse wild-type AHASL1 primer comprising the nucleotide sequence of SEQ ID NO:4;   (c) using the DNA of (a) as a template for a second PCR amplification comprising said DNA, polymerase, deoxyribonucleotide triphosphates, a forward AHASL1 primer, and a reverse mutant AHASL1 primer comprising the nucleotide sequence of SEQ ID NO:5; and   (d) detecting products of said first PCR amplification and said second PCR amplification.   
     
     
         40 . The method of  claim 39 , wherein said forward AHASL1 primer comprises the nucleotide sequence of SEQ ID NO:3. 
     
     
         41 . The method of  claim 39 , wherein said first PCR amplification and said second PCR amplification are performed in separate reactions. 
     
     
         42 . The method of  claim 39 , wherein said first PCR amplification and said second PCR amplification are performed in the same reaction. 
     
     
         43 . A kit for genotyping a sunflower acetohydroxyacid synthase large subunit 1 (AHASL1) gene, said kit comprising
 (a) a forward AHASL1 primer,   (b) a reverse wild-type AHASL1 primer comprising the nucleotide sequence of SEQ ID NO:4, and   (c) a reverse mutant AHASL1 primer comprising the nucleotide sequence of SEQ ID NO:5.   
     
     
         44 . The kit of  claim 41 , further comprising deoxyribonucleotide triphosphates and a polymerase capable of catalyzing the PCR amplification of a fragment of a sunflower AHASL1 gene having an A122T allele; or a fragment of a wild-type sunflower AHASL1 gene. 
     
     
         45 . The kit of  claim 41 , wherein said forward AHASL1 primer comprises the nucleotide sequence of SEQ ID NO:3. 
     
     
         46 . A kit for genotyping a sunflower acetohydroxyacid synthase large subunit 1 (AHASL1) gene, said kit comprising:
 (a) a first forward AHASL1 primer comprising the nucleotide sequence of SEQ ID NO: 15, and a first reverse AHASL1 primer comprising the nucleotide sequence of SEQ ID NO:16; and   (b) a second forward AHASL1 primer comprising the nucleotide sequence of SEQ ID NO: 17, and a second reverse AHASL1 primer comprising the nucleotide sequence of SEQ ID NO: 18.   
     
     
         47 . The kit of  claim 46 , further comprising deoxyribonucleotide triphosphates and a polymerase capable of catalyzing the PCR amplification of a fragment of a sunflower AHASL1 gene having an A122T allele or a fragment of a wild type sunflower AHASL1 gene. 
     
     
         48 . A method for genotyping a sunflower acetohydroxyacid synthase large subunit 1 (AHASL1) gene, said method comprising:
 (a) obtaining DNA from a sunflower plant;   (b) using said DNA as a template for a first polymerase chain reaction (PCR) amplification comprising said DNA, polymerase, deoxyribonucleotide triphosphates, a first forward AHASL1 primer comprising the nucleotide sequence of SEQ ID NO: 15, and a first reverse AHASL1 primer comprising the nucleotide sequence of SEQ ID NO: 16;   (c) using said DNA as a template for a second PCR amplification comprising said DNA, polymerase, deoxyribonucleotide triphosphates, a second forward AHASL1 primer comprising the nucleotide sequence of SEQ ID NO: 17, and a second reverse AHASL1 primer comprising the nucleotide sequence of SEQ ID NO: 18; and   (d) detecting products of said first PCR amplification and said second PCR amplification.   
     
     
         49 . The method of  claim 48 , wherein said first PCR amplification and said second PCR amplification are performed in separate reactions. 
     
     
         50 . The method of  claim 48 , wherein said first PCR amplification and said second PCR amplification are performed in the same reaction. 
     
     
         51 . A herbicide-resistant sunflower plant identifiable by the method of  claim 39 , wherein said sunflower plant comprises in its genome a first allele of an acetohydroxyacid synthase large subunit (AHASL1) gene and a second allele of said AHASL1 gene, wherein said first allele encodes an AHASL1 protein comprising the A122T amino acid substitution and said second allele encodes an AHASL1 protein comprising an amino acid substitution selected from the group consisting of the A205V amino acid substitution and the P197L amino acid substitution. 
     
     
         52 . The sunflower plant of  claim 51 , wherein said herbicide-resistant sunflower plant is resistant to at least one AHAS-inhibiting herbicide. 
     
     
         53 . The sunflower plant of  claim 51 , wherein said AHAS-inhibiting herbicide is selected from the group comprising: imidazolinone herbicides, sulfonylurea herbicides, triazolopyrimidine herbicides, pyrimidinyloxybenzoate herbicides, and sulfonylamino-carbonyltriazolinone herbicides. 
     
     
         54 . The sunflower plant of  claim 51 , wherein said sunflower plant is a seed. 
     
     
         55 . A method of controlling weeds in the vicinity of the sunflower plant of  claim 51 , said method comprising applying an effective amount of an AHAS-inhibiting herbicide to the weeds and to the sunflower plant. 
     
     
         56 . The method of  claim 55 , wherein said AHAS-inhibiting herbicide comprises one or more of an imidazolinone herbicide, a sulfonylurea herbicide, a triazolopyrimidine herbicide, a pyrimidinyloxybenzoate herbicide, and a sulfonylamino-carbonyltriazolinone herbicide. 
     
     
         57 . The method of  claim 56 , wherein said imidazolinone herbicide comprises one or more of 2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-nicotinic acid, 2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-3-quinolinecarboxylic acid, 5-ethyl-2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-nicotinic acid, 2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-5-(methoxymethyl)-nicotinic acid, 2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-5-methylnicotinic acid, and a mixture of methyl 6-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-m-toluate and methyl 2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-p-toluate. 
     
     
         58 . The method of  claim 56 , wherein said sulfonylurea herbicide comprises one or more of chlorsulfuron, metsulfuron methyl, sulfometuron methyl, chlorimuron ethyl, thifensulfuron methyl, tribenuron methyl, bensulfuron methyl, nicosulfuron, ethametsulfuron methyl, rimsulfuron, triflusulfuron methyl, triasulfuron, primisulfuron methyl, cinosulfuron, amidosulfuron, flazasulfuron, imazosulfuron, pyrazosulfuron ethyl, and halosulfuron. 
     
     
         59 . A method for combating undesired vegetation comprising contacting the sunflower seed of  claim 54  before sowing and/or after pregermination with an effective amount of AHAS-inhibiting herbicide. 
     
     
         60 . The method of  claim 59 , wherein said AHAS-inhibiting herbicide is selected from the group comprising one or more of an imidazolinone herbicide, a sulfonylurea herbicide, a triazolopyrimidine herbicide, a pyrimidinyloxybenzoate herbicide, and a sulfonylamino-carbonyltriazolinone herbicide. 
     
     
         61 . The seed of  claim 54 , wherein the seed comprises an AHAS-inhibiting herbicide seed treatment. 
     
     
         62 . The seed of  claim 61 , wherein said AHAS-inhibiting herbicide comprises one or more of an imidazolinone herbicide, a sulfonylurea herbicide, a triazolopyrimidine herbicide, a pyrimidinyloxybenzoate herbicide, and a sulfonylamino-carbonyltriazolinone herbicide. 
     
     
         63 . The seed of  claim 62 , wherein said imidazolinone herbicide comprises one or more of 2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-nicotinic acid, 2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-3-quinolinecarboxylic acid, 5-ethyl-2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-nicotinic acid, 2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-5-(methoxymethyl)-nicotinic acid, 2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-5-methylnicotinic acid, and a mixture of methyl 6-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-m-toluate and methyl 2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-p-toluate. 
     
     
         64 . The seed of  claim 62 , wherein said sulfonylurea herbicide comprises one or more of chlorsulfuron, metsulfuron methyl, sulfometuron methyl, chlorimuron ethyl, thifensulfuron methyl, tribenuron methyl, bensulfuron methyl, nicosulfuron, ethametsulfuron methyl, rimsulfuron, triflusulfuron methyl, triasulfuron, primisulfuron methyl, cinosulfuron, amidosulfuron, flazasulfuron, imazosulfuron, pyrazosulfuron ethyl, and halosulfuron.