Acetolactate Synthase Target-Site Mutations and Single Nucleotide Polymorphism Genotyping in ALS-Resistant Kochia (Kochia scoparia)

Abstract

The molecular basis for acetolactate synthase (ALS)–inhibitor resistance was determined for 6 susceptible (HS) and 24 resistant (HR) kochia populations from western Canada. The latter included 3 HR populations from Alberta (AB), 3 from Manitoba (MB), and 18 from Saskatchewan (SK). HR plants survived application of the ALS-inhibitor herbicide thifensulfuron–tribenuron mixture in the greenhouse. Most of the HR populations were heterogeneous and contained both HR and HS individuals. The molecular basis for resistance was determined in 273 HR individuals by sequencing the ALS gene (2,270 base pair [bp]) or by conducting a TaqMan genotyping assay developed in this study using real-time polymerase chain reaction (PCR) for single nucleotide polymorphism (SNP) 1709, where a G to T substitution resulted in a Leu for Trp substitution at amino acid position 574 (Trp574Leu mutation). A total of 16 SNPs were identified in the ALS gene sequences (0.7% polymorphism), 5 of which resulted in amino acid changes that confer resistance to ALS-inhibiting herbicides. The SNPs correspond to three target-site mutations: Pro197 (SNPs 565 and 566), Asp376 (SNP 1116), and Trp574 (SNPs 1708 and 1709). The Trp574Leu mutation was predominant (189 HR plants). The next most common mutation was the highly variable residue Pro197 (44 HR plants) with substitution by one of nine amino acids. The least-frequent were Asp376Glu (9 plants) and Trp574Arg (3 plants) substitutions. The presence of two ALS target-site mutations was found in 30 individual kochia plants, the first report from field-selected weed populations. These include combinations Pro197 + Trp574 (23 plants) and Pro197 + Asp376 (7 plants). The detection of Pro197, Asp376, and Trp574 mutations, as well as both combinations, from geographically separate regions suggests multiple origins of these mutations.