Rapid diagnosis of sulfonylurea‐resistant S choenoplectus juncoides [ R oxb.] Palla using polymerase chain reaction–restriction fragment length polymorphism and isogene‐specific direct sequencing

Rapid diagnostic methods to detect known mutations in acetolactate synthase ( ALS ) genes that confer sulfonylurea ( SU ) resistance to S choenoplectus juncoides were developed in this study. By using 11 SU ‐resistant accessions (nine accessions with a P ro 197 substitution in ALS 1 or ALS 2 , one accession with an A sp 376 G lu substitution in ALS 2 and one accession with a T rp 574 L eu substitution in ALS 2 ), polymerase chain reaction–restriction fragment length polymorphism ( PCR–RFLP ) analysis for DNA fragments that were amplified simultaneously from genomic ALS 1 and ALS 2 and PCR–RFLP analysis for DNA fragments that were amplified from either of the genomic ALS 1 or ALS 2 were carried out. In each of the two PCR–RFLP analyses, a common PCR product was digested separately with the restriction enzymes, Bsp LI , Mbo I and Mun I , in order to detect Pro 197 substitutions, an A sp 376 G lu substitution and a T rp 574 L eu substitution, respectively. In each of the lanes where the detection of SU ‐resistant substitutions was aimed, a specific band to suggest the existence of the said substitutions was observed in theoretically assumable ways. Separately, a direct sequencing method also was established, which was able to selectively sequence ALS 1 or ALS 2 from common templates containing both ALS 1 and ALS 2 by the isogene‐selective primers that were designed to anneal either of the ALS genes. It is expected that these methods could be used for the genetic analysis of SU ‐resistant S . juncoides by providing rapid and accurate diagnosis.