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

Satoshi Iwakami,Masaki Endo,Yoshiko Shimono,Yohei Manabe,Tohru Tominaga,Hiroyuki Shibaike,Akira Uchino

doi:10.3389/fpls.2017.00254

Abstract

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.

Highlights

Herbicides that inhibit acetolactate synthase (ALS) cause depletion of branched chain amino acids such as valine, leucine and isoleucine, leading to plant death (Duggleby et al, 2008; Yu and Powles, 2014)
Thifensulfuron-methyl resistant A. aequalis accessions were found to carry a mutation at the Pro197 residue in either the ALS1 or ALS2 gene
It is unlikely that the higher-copy number accessions are tetraploid, ploidy level variation within a single species has been reported in Alopecurus spp. including A. aequalis (Sieber and Murray, 1980; Koul and Gohil, 1990)

Summary

Introduction

Herbicides that inhibit acetolactate synthase (ALS) cause depletion of branched chain amino acids such as valine, leucine and isoleucine, leading to plant death (Duggleby et al, 2008; Yu and Powles, 2014). Resistance to ALS inhibitors has been reported in 159 weed species. ALS inhibitors are categorized into five groups based on their chemical structure: sulfonylurea (SU), triazolopyrimidine (TP), pyrimidinylthiobenzoate (PTB), imidazolinone (IMI), and sulfonylaminocarbonyltriazolinone (SCT). In Japan, SUs are predominantly used due to their excellent crop safety, broad spectrum of weed control and low toxicity to animals. Most cases of resistance to ALSinhibiting herbicides reported in Japan, such as Monochoria vaginalis (Ohsako and Tominaga, 2007), Schoenoplectus juncoides (Uchino et al, 2007), and Sagittaria trifolia (Iwakami et al, 2014b), have evolved under SU selection (Uchino et al, 2016)

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Conclusion