A single polymerase chain reaction-based assay for simultaneous detection of two mutations conferring resistance to tubulin-binding herbicides in Setaria viridis

A simple method based upon polymerase chain reaction (PCR) was developed to detect mutant alleles of the gene encoding alpha 2-tubulin, which confers recessive resistance to tubulin-binding herbicides in Setaria viridis. Multiplex, bidirectional allele-specific PCR (Mbi-PASA) was shown to specifically and reliably detect the presence of all sensitive (Leu(136), Thr(239)) and resistant (Phe(136), Ile(239)) alpha 2-tubulin alleles in a single reaction. Double-blind analysis of 2000 S. viridis seedlings using seed bioassay and Mbi-PASA confirmed that the presence of two mutant alpha 2-tubulin alleles in a seedling was always associated with cross-resistance to dinitroaniline and benzoic acid herbicides, sensitivity to a benzamide herbicide, and hypersensitivity to carbamate herbicides. No other resistance mechanism was detected in the S. viridis populations screened. Successful Mbi-PASA genotyping was achieved with fresh and dried plant fragments from the field. Compared with bioassays, Mbi-PASA is faster and more robust, removing the need for live plant material. It is the only way of detecting recessive resistance before resistant plants occur in a field. Mbi-PASA can be performed with basic molecular biology laboratory equipment, and is suitable for high-throughput genotyping adaptation. It is the tool of choice for resistance diagnosis in such cases where only a few recessive, target-derived, genes control resistance to herbicides.