Molecular characterisation of the inactive allele of the gene Glu-A1 and the development of a set of AS-PCR markers for HMW glutenins of wheat

The present work reports new PCR markers that amplify the complete coding s equence of the specific alleles of the high molecular weight (HMW) glutenin genes. A set of AS-PCR molecular markers was designed which use primers fr om nucleotide sequences of the Glu-A1 and Glu-D1 genes, making use of the m inor diffeences between the sequences of the x1, x2* of Glu-A1, and the x5 and y10 of Glu-D1. These primers were able to distinguish between x2* and t he x1 or xNull of Glu-A1. Also x5 was distinguishable from x2, and y10 from y12. The primers amplified the complete coding regions and corresponded to the upstream and downstream flanking positions of Glu-A1 and Glu-D1. Prime rs designed to amplify the Glu-A1 gene amplified a single product when used with genomic DNA of common wheats and the xNull allele of this gene. This work also describes the cloning and characterisation of the nucleotide sequ ence of this allele. It possesses the same general structure as x2* and x1 (previously determined) and differs from these alleles in the extension of the coding sequence for a presumptive mature protein with only 384 residues . This is due to the presence of a stop codon (TAA) 1215-bp downstream from the start codon. A further stop codon (TAG), 2280-bp downstream from the s tarting codon is also found. The open reading frame of xNull and x1 alleles has the same size in bp. Both are larger than x2* which shows two small de letions. The reduced size of the presumptive mature protein encoded by xNul l could explain the negative effect of this allele on grain quality.