Characterisation of the acetolactate synthase (ALS) gene of Raphanus raphanistrum L. and the molecular assay of mutations associated with herbicide resistance

Abstract

The coding sequence of the entire acetolactate synthase (ALS) gene of Raphanus raphanistrum L., wild radish was determined to be 1758 bp. Twelve ALS coding sequences were generated from representative populations collected from different geographical regions in Australia and consisted of populations that were either resistant or susceptible to ALS-inhibiting herbicides. Comparative analysis of the ALS gene sequences identified 81 base polymorphisms, 74 of which were neutral resulting in no amino acid change. There were seven loci where base changes resulted in amino acid substitutions in the gene. Five of these occurred randomly amongst the susceptible and resistant R. raphanistrum populations. Two of these five base polymorphisms were adjacent and accounted for three possible amino acids at the loci, indicating there are at least three alleles of the ALS gene in R. raphanistrum. The remaining two base mutations were observed only in the herbicide resistant populations. Three of the four resistant populations examined had a mutation, which disrupted the Pro codon in Domain A, a conserved region widely reported as conferring resistance to sulfonylurea herbicides in many weeds. The fourth herbicide resistant population had a mutation that caused a Trp→Leu substitution in Domain B. Studies have shown that this single point mutation endows a broad resistance to all four classes of ALS-inhibiting herbicides. An important aspect of our work was the development of molecular diagnostic assays for rapid screening of the pivotal mutations in Domains A and B in natural populations. Not every plant in the resistant, natural populations had the associated mutations, and some plants were heterozygotes for the resistant ALS gene allele characterised.