Sequence polymorphisms and relationships of dimeric a-amylase inhibitor genes in the B genomes of Triticum and S genomes of Aegilops Ji-Rui Wang a,1 , Li Zhang a,1 , Yu-Ming Wei a , Ze-Hong Yan a , Bernard R. Baum b , Eviatar Nevo c , You-Liang Zheng a,d, * a Triticeae Research Institute, Sichuan Agricultural University, Yaan, Sichuan 625014, China b Agriculture & Agri-Food Canada, Eastern Cereal and Oilseed Research Centre, Ottawa, Ontario, Canada K1A 0C6 c Institute of Evolution, University of Haifa, Mt. Carmel, Haifa 31905, Israel d Key Laboratory of Crop Genetic Resources and Improvement in Southwest China, Ministry of Education, Sichuan Agricultural University, Yaan, Sichuan 625014, China Received 8 January 2007; received in revised form 8 March 2007; accepted 10 March 2007 Available online 23 March 2007 Abstract One hundred and fifty-nine sequences encoding dimeric a-amylase inhibitors were characterized from Triticum and Aegilops. These sequences had 375 nucleotides in length, among which there were 255 conserved sites, 50 singleton variable sites (the nucleotide polymorphism only observed in a single accession) and 70 polymorphic sites (the polymorphisms found in two or more accessions). The frequency of SNPs in the B (a.k.a. S) genomes coding a-amylase inhibitor genes was 1.7 out of 10 bases. A total of 59 haplotypes were defined, among which 4 main haplotypes occurring in more than 10 genes and 36 haplotypes with single gene were found, indicating that the dimeric a-amylase inhibitors might derive from a very limited number of ancestral genes. The phylogenetic median-joining network of the 59 haplotypes was highly star like with 5 haplotype groups, and at least 2 sub clusters emerged. Evolutionary distances of the 159 genes were calculated, and subjected to the construction of neighbour-joining trees that showed that the a-amylase inhibitor genes were divided into 5 groups, and each group had at least 2 subgroups. The neighbour-joining tree of the species indicated that the genes from common wheat and Triticum dicoccoides were closely related to those from Aegilops speltoides, and it was further supported by the median-joining networks analysis of the 59 haplotypes. These results revealed the important information on genome shaping events and processes occurring at dimeric a-amylase inhibitor genes loci and contributed to our understanding of functional aspects of the dimeric a-amylase inhibitor genes, as well as phylogenetic relationships between species. # 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Haplotype; Network; Recombination; Evolutionary analysis 1. Introduction The enzyme inhibitors act on the key insect gut digestive hydrolases, the a-amylases and proteinases [1]. It was known that the bulk of wheat albumins consisted of a few amylase isoinhibitor families very likely phylogenetically related and coded by a small number of parental genes [2]. a-Amylase inhibitors have been widely investigated for their possible use in strengthening plant defense against insect and microbial pests in a variety of plants. The wheat kernel contains a number of proteinaceous a- amylases inhibitors, which are by-products of starch and gluten manufacture [3]. In the absence of dissociating agents, wheat inhibitors active on exogenous a-amylases can be fractionated by gel filtration into three isoinhibitor families with apparent molecular weight close to 60, 24 and 12 kDa [4]. In cereal seeds, a-amylase inhibitors proteins with 120–130 amino acids, which include trypsin inhibitors as well as a-amylase inhibitors, were known as one great family on the basis of the homology of their amino acid sequence [5]. a-Amylase www.elsevier.com/locate/plantsci Plant Science 173 (2007) 1–11 * Corresponding author at: Triticeae Research Institute, Sichuan Agricultural University, Yaan, Sichuan 625014, China. Tel.: +86 835 2882620; fax: +86 835 2883153. E-mail address: ylzheng@sicau.edu.cn (Y.-L. Zheng). 1 These authors contributed equally. 0168-9452/$ – see front matter # 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2007.03.006