Development and application of allele-specific PCR assays for imazethapyr resistance in rice (Oryza sativa)

Outcrossing or cross hybridization is a potential concern in herbicide-resistant crop management strategies such as in the Clearfield[trade mark sign] rice system. Recent studies have shown that the mutated acetolactate synthase (ALS) gene that confers resistance to imazethapyr (Newpath) herbicide can be transferred from Clearfield rice cultivars via cross pollination under field conditions to weedy red rice. Resistance of commercial Clearfield rice cultivars to imazethapyr is due to the presence of two point mutations in the ALS gene that result in amino acid substitutions from serine to asparagine (S to D) and glycine to glutamic acid (G to E). We report here development of a DNA-based method that involves application of allele-specific PCR assays to distinguish herbicide-susceptible and resistant ALS alleles in either homozygous or heterozygous genotypes produced from natural outcrosses between Clearfield varieties CL121, CL141, CL161 and weedy red rice. PCR assays that can distinguish between the homozygous and heterozygous imazethapyr-resistant S₆₅₃D and G₆₅₄E SNP alleles of the rice ALS gene were developed and evaluated. A total of 483 individual red rice plants were successfully screened for the presence of S₆₅₃D SNP and another 145 F₂ individuals from natural red rice x CL121 hybridizations were screened for the presence of the G₆₅₄E SNP. The PCR-based assays produced during this study are simple, rapid, inexpensive, reproducible and require only standard PCR and electrophoretic instruments that can be applied toward outcrossing evaluation and effective weed management strategies for the Clearfield crop system.