Development of chromosome 6D-specific markers for α-gliadin genes and their use in assessing dynamic changes at the <i>Gli</i>-<i>2</i> loci

To develop chromosome 6D-specific point mutation (PM) markers for α-gliadin genes, 79 α-gliadin sequences cloned from Aegilops tauschii and another 40 α-gliadin genes with known chromosome locations were used in multi-sequence alignment and phylogenic analysis. Additional multiple alignment adjustments were performed manually to facilitate discovery of putative chromosome 6D-specific point mutations. A total of 85 PM primers were designed to detect 68 candidate chromosome 6D-specific point mutations. Experimental tests revealed 11 chromosome 6D-specific PM markers by using genomic DNA from homoeologous group 6 nullisomic–tetrasomic lines of Chinese Spring and putative diploid and tetraploid ancestors of hexaploid wheat as PCR templates. Detection of PM markers in one synthetic hexaploid wheat and its parental lines indicated that some α-gliadin genes were lost from Gli - 2 loci during the formation of hexaploid wheat by amphidiploidization of the genomes of Triticum turgidum and Ae. tauschii . Detection of these PM markers in Ae. tauschii , T. aestivum and its four subspecies indicated that at least two genetically distinct sources of Ae. tauschii contributed germplasm to the D genome of T. aestivum .