Evolutionary origin of the segmental duplication encompassing the wheat GLU-B1 locus encoding the overexpressed Bx7 (Bx7OE) high molecular weight glutenin subunit

Sequencing of a BAC clone encompassing the Glu-B1 locus in Glenlea, revealed a 10.3 Kb segmental duplication including the Bx7 gene and flanking an LTR retroelement. To better understand the evolution of this locus, two collections of wheat were surveyed. The first consisted of 96 diploid and tetraploid species accessions while the second consisted of 316 Triticum aestivum cultivars and landraces from 41 countries. The genotypes were first characterized by SDS-PAGE and a total of 40 of the 316 T. aestivum accessions were found to display the overexpressed Bx7 phenotype (Bx7(OE)). Three lines from the 96 diploid/tetraploid collection also displayed the stronger intensity staining characteristic of the Bx7(OE) subunit. The relative amounts of the Bx7 subunit to total HMW-GS were quantified by RP-HPLC for all Bx7(OE) accessions and a number of checks. The entire collection was assessed for the presence of four DNA markers namely an 18 bp indel of the coding region of Bx7 variant alleles, a 43 bp indel of the 5'-region and the left and right junctions of the LTR retrotransposon borders and the duplicated segment. All 43 accessions found to have the Bx7(OE) subunit by SDS-PAGE and RP-HPLC produced the four diagnostic PCR amplicons. None of the lines without the Bx7(OE) had the LTR retroelement/duplication genomic structure. However, the 18 and 43 bp indel were found in accessions other than Bx7(OE). These results indicate that the overexpression of the Bx7 HMW-GS is likely the result of a single event, i.e., a gene duplication at the Glu-B1 locus mediated by the insertion of a retroelement. Also, the 18 and 43 bp indels pre-date the duplication event. Allelic variants Bx7*, Bx7 with and without 43 bp insert and Bx7(OE) were found in both tetraploid and hexaploid collections and shared the same genomic organization. Though the possibility of introgression from T. aestivum to T. turgidum cannot be ruled out, the three structural genomic changes of the B-genome taken together support the hypothesis of multiple polyploidization events involving different tetraploid progenitors.