EMS induced SNP changes led to mutation of Wx protein in common wheat

Three EMS mutant wheat lines with varied mobility of Wx-A1 and Wx-B1 were identified. In the mutant lines M2-97-8 and M2-104-2, Wx-B1 showed slightly faster mobility compared to those of wild line, and Wx-A1 in the mutant line M2-101-1 has a greatly faster mobility, which co-migrates with Wx-D1. The results of gene sequencing indicated that SNP mutations appeared within exon of Wx-1 and further resulted in the mutation of amino acid residues in the mutant lines. The prediction analysis showed that the isoelectric point of the mutants M2-97-8 and M2-104-2 was more basic than that of the wild type while the isoelectric point of M2-101-1 was not changed; and the Waxy protein of the three mutations were all around 66 kDa. The SDS-PAGE results revealed that the mobility variation of Wx-B1 of M2-97-8 and M2-104-2 is due to the change of isoelectric point, while M2-101-1 may be caused by variation or modification of protein structure but not by the change of isoelectric point. The prokaryotic expression results further indicated that these mutated Wx-A1 and Wx-B1 genes migrated faster than those of wild Wx-A1 and Wx-B1 and were consistent with the results of molecular weight prediction. Although, the increased or decreased contents of total starch and amylose were observed in Wx-A1 or Wx-B1mutant, the correlation between the contents of total starch and amylose and the mutant Wx-A1 and Wx-B1 subunits cannot be determined. These mutants can not only increase the polymorphism of Waxy gene to investigate the function of these alleles, but also be valuable for wheat quality breeding.