Molecular characterization of dimeric alpha-amylase inhibitor genes in wheat and development of genome allele-specific primers for the genes located on chromosome 3BS and 3DS

Alpha-amylase inhibitors are attractive candidates for the control of seed weevils as these insects are highly dependent on starch as an energy source. For weevil control, alpha-amylase inhibitors and their genes could be used to genetically engineer weevil resistant seeds. Thirty genes encoding dimeric alpha-amylase inhibitors were isolated from Triticum aestivum L. 'Chinese Spring' and characterized by nucleotide and amino acid sequence analysis. Eleven representative alpha-amylase inhibitor genes were identified, and the deduced amino acid sequences of these genes were of high coherence (95.1%). These inhibitors and others obtained from the wheat EST database were clustered into three groups, the genes from 'Chinese Spring' were present in each group. Specific primer sets were designed for each group, based on the SNPs of these genes, and the chromosome locations of each group of inhibitor genes investigated by amplification of the 'Chinese Spring' ditelosomic lines. There were two and one groups of inhibitor genes on chromosomes 3BS and 3DS, respectively, whereas no group of inhibitor genes was found on chromosome 3AS. Thus, the primer set for each group of inhibitor genes was genome allele-specific. The two known inhibitors, 0.53 and 0.19, were located on chromosomes 3BS and 3DS, respectively. The validity of the three genome allele-specific primer sets was confirmed by amplifications in 15 accessions of Triticum urartu, Triticum monococcum, Aegilops tauschii and Triticum dicoccoides. These results gave further support at the molecular level, that the 24 kDa dimeric alpha-amylase inhibitors in cultivated wheat are encoded by a multigene family.