Novel x-type high-molecular-weight glutenin genes from Aegilops tauschii and their implications on the wheat origin and evolution mechanism of Glu-D1-1 proteins.

Two new x-type high-molecular-weight glutenin subunits with similar size to 1Dx5, designated 1Dx5t and 1Dx5.1t in Aegilops tauschii, were identified by SDS-PAGE, RP-HPLC, and MALDI-TOF-MS. The coding sequences were isolated by AS-PCR and the complete ORFs were obtained. Allele 1Dx5t consists of 2481 bp encoding a mature protein of 827 residues with deduced Mr of 85,782 Da whereas 1Dx5.1t comprises 2526 bp encoding 842 residues with Mr of 87,663 Da. The deduced Mr's of both genes were consistent with those determined by MALDI-TOF-MS. Molecular structure analysis showed that the repeat motifs of 1Dx5t were correspondingly closer to the consensus compared to 1Dx5.1t and 1Dx5 subunits. A total of 11 SNPs (3 in 1Dx5t and 8 in 1Dx5.1t) and two indels in 1Dx5t were identified, among which 8 SNPs were due to C-T or A-G transitions (an average of 73%). Expression of the cloned ORFs and N-terminal sequencing confirmed the authenticities of the two genes. Interestingly, several hybrid clones of 1Dx5t expressed a slightly smaller protein relative to the authentic subunit present in seed proteins; this was confirmed to result from a deletion of 180 bp through illegitimate recombination as well as an in-frame stop codon. Network analysis demonstrated that 1Dx5t, 1Dx2t, 1Dx1.6t, and 1Dx2.2 represent a root within a network and correspond to the common ancestors of the other Glu-D-1-1 alleles in an associated star-like phylogeny, suggesting that there were at least four independent origins of hexaploid wheat. In addition to unequal homologous recombination, duplication and deletion of large fragments occurring in Glu-D-1-1 alleles were attributed to illegitimate recombination.