Use of Pyrosequencing Technology to Genotype Imidazolinone-Tolerant Wheat

Cultivars of several cereal crops have been developed with acetohydroxyacid synthase (AHAS) insensitivity to imidazolinone herbicides and are now an important tool for weed management. Options for screening for imidazolinone resistant lines include direct herbicide application, biochemical assays for AHAS activity and DNA-based methods. Herbicide and biochemical assays for AHAS activity provide limited information as to mutation copy number and provide no information as to the genome on which the mutation is located without extensive test crossing. Wheat (Triticum aestivum L.) can have between one and six copies of the resistant acetohydroxyacid synthase on any of three genomes. A novel DNA-based screening protocol is described here in which pyrosequencing is used to screen for the S653N imidazolinone tolerant mutation in wheat. One assay is shown to successfully detect zero to four copies of the S653N mutation, while additional assays can detect the presence of S653N in individual wheat genomes. All of these assays are based on a single 298-bp PCR fragment and can be easily scaled up or down depending on the number lines that need to be screened. Potential applications include detection of mutant copy number in segregating populations, and the selection of parental lines with genome specific mutant composition