Rapid diagnosis of sulfonylurea-resistant Schoenoplectus juncoides [Roxb.] 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 Schoenoplectus juncoides were developed in this study. By using 11 SU-resistant accessions (nine accessions with a Pro197 substitution in ALS1 or ALS2, one accession with an Asp376Glu substitution in ALS2 and one accession with a Trp574Leu substitution in ALS2), polymerase chain reactionrestriction fragment length polymorphism (PCRRFLP) analysis for DNA fragments that were amplified simultaneously from genomic ALS1 and ALS2 and PCRRFLP analysis for DNA fragments that were amplified from either of the genomic ALS1 or ALS2 were carried out. In each of the two PCRRFLP analyses, a common PCR product was digested separately with the restriction enzymes, BspLI, MboI and MunI, in order to detect Pro197 substitutions, an Asp376Glu substitution and a Trp574Leu 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 ALS1 or ALS2 from common templates containing both ALS1 and ALS2 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.