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Severe infestations of  Alopecurus aequalis  (shortawn foxtail), a noxious weed in wheat and barley cropping systems in Japan, can occur even after application of thifensulfuron-methyl, a sulfonylurea (SU) herbicide. In the present study, nine accessions of  A. aequalis  growing in a single wheat field were tested for sensitivity to thifensulfuron-methyl. Seven of the nine accessions survived application of standard field rates of thifensulfuron-methyl, indicating that severe infestations likely result from herbicide resistance. Acetolactate synthase (ALS) is the target enzyme of SU herbicides. Full-length genes encoding ALS were therefore isolated to determine the mechanism of SU resistance. As a result, differences in  ALS  gene copy numbers among accessions were revealed. Two copies,  ALS1  and  ALS2 , were conserved in all accessions, while some carried two additional copies,  ALS3  and  ALS4 . A single-base deletion in  ALS3  and  ALS4  further indicated that they represent pseudogenes. No differences in ploidy level were observed between accessions with two or four copies of the  ALS  gene, suggesting that copy number varies. Resistant plants were found to carry a mutation in either the  ALS1  or  ALS2  gene, with all mutations causing an amino acid substitution at the Pro197 residue, which is known to confer SU resistance. Transcription of each  ALS  gene copy was confirmed by reverse transcription PCR, supporting involvement of these mutations in SU resistance. The information on the copy number and full-length sequences of  ALS  genes in  A. aequalis  will aid future analysis of the mechanism of resistance.

Copy Number Variation in Acetolactate Synthase Genes of Thifensulfuron-Methyl Resistant Alopecurus aequalis (Shortawn Foxtail) Accessions in Japan