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

Summary A simple method based upon polymerase chain reaction (PCR) was developed to detect mutant alleles of the gene encoding α2‐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 (Leu136, Thr239) and resistant (Phe136, Ile239) α2‐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 α2‐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.