Pesticide Biochemistry and Physiology 88 (2007) 143–148 www.elsevier.com/locate/ypest 0048-3575/$ - see front matter  2006 Elsevier Inc. All rights reserved. doi:10.1016/j.pestbp.2006.10.006 Discovery of single-nucleotide mutations in acetolactate synthase genes by Ecotilling Guang-Xi Wang a,¤ , Mui-Keng Tan b , Sujay Rakshit c , Hiromasa Saitoh c , Ryohei Terauchi c , Toshiyuki Imaizumi a , Takanori Ohsako d , Tohru Tominaga a a Laboratory of Weed Science, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan b NSW Department of Primary Industries, Elizabeth Macarthur Agricultural Institute, PMB 8, Menangle, NSW 2568, Australia c Iwate Biotechnology Research Center, Kitakami, Iwate 024-0003, Japan d Laboratory of Agroecology, Graduate School of Agriculture, Kyoto Prefectural University, Kyoto 619-0244, Japan Received 25 July 2006; accepted 18 October 2006 Available online 26 October 2006 Abstract TILLING (Targeting Induced Local Lesions IN Genomes) is a powerful reverse genetic technique that employs a mismatch-speciWc endonuclease to discover induced point mutations in genes of interest. The use of the TILLING technique to survey natural variation in genes is called Ecotilling. We report an adaptation of Ecotilling for rapid detection of single-nucleotide mutations in the acetolactate syn- thase (ALS) genes of sulfonylurea (SU)-resistant (R) Monochoria vaginalis (Pontederiaceae), a paddy weed, in Japan. Genomic DNA of a SU-R plant (target DNA) was mixed with the DNA of a SU-susceptible (S) plant (reference DNA). Ecotilling detected two nucleotide mutations in the ALS gene of SU-R M. vaginalis. These 2 mutations were conWrmed by DNA sequencing. A single nucleotide mutation (C to A), in the codon CCT to CAT and another mutation (C to T), in the codon CCT to TCT were identiWed by sequencing. Both muta- tions result in the disruption of a Pro codon in the conserved Domain A region with the consequent substitution of a His residue in the Wrst mutation and a Ser residue in the second. Substitution of the Pro residue in Domain A of the ALS gene has been reported to result in insensitivity to SUs in many weed biotypes. This study demonstrates that Ecotilling is a fast, reliable, economical method for detecting single-nucleotide mutations in genes arising from herbicide selection.  2006 Elsevier Inc. All rights reserved. Keywords: Acetolactate synthase gene; Ecotilling; Herbicide resistance; Mismatch cleavage; Single-nucleotide mutation defection 1. Introduction Acetolactate synthase (ALS, EC 4.1.3.18; also called acetohydroxyacid synthase) 1 is the Wrst common enzyme in the biosynthesis of the branched chain amino acids valine, leucine, and isoleucine. Several classes of herbicides are known to inhibit ALS, such as the sulfonylureas, imidazoli- nones, triazolopyrimidine sulfonanilides, and pyrimidinyl oxybenzoates, by binding to a relic quinone-binding site [1]. These highly selective ALS-inhibiting herbicides are very valuable for weed management in a wide range of crops worldwide. However, biotypes resistant to the ALS-inhibit- ing herbicides have been reported in 93 plant species [2]. Resistant biotypes in many cases have modiWed ALS genes with one or more point mutations causing reduced sensitiv- ity to the ALS-inhibiting herbicides [1,3–7]. Substitution in any of the Wve conserved amino acids (Ala 122 , Pro 197 , Ala 205 , Trp 574 , or Ser 653 : numbered based on the ALS precursor in Arabidopsis thaliana) is known to result in ALS inhibitor resistance. The Wrst molecular biological investigation of SU-R biotype of Monochoria vaginalis is that of Wang et al. * Corresponding author. Fax: +81 75 753 6062. E-mail address: WANG@weed.mbox.media.kyoto-u.ac.jp (G.-X. Wang). 1 Abbreviations used: ALS, acetolactate synthase; ASPCR, allele-speciWc PCR; DGGE, denaturing gradient gel electrophoresis; EMS, ethylme- thane sulfonate; PASA, PCR ampliWcation of speciWc alleles; PCR, poly- merase chain reaction; R, resistant; S, susceptible; SNP, single nucleotide polymorphisms; SSCP, single strand conformation polymorphism; SU, sulfonylurea.