Molecular Identification Of High-molecular-weight Glutenin Subunit Variation In Wheat-Rye Hybrid

The study of variation induced by wide hybridization is one of the research hotspots. But previous studies almost focused on whole genome level, little is known about the molecular mechanism of gene variation. It’s difficult to build specific links between changed genes and novel phenotypes due to orthologous genes impact. High-molecular-weight glutenin subunits (HMW-GSs) genes, which can address this issue, provide us a useful model for the study of genetic variation induced by wide hybridization.In the present study, we study the molecular mechanism of HMW-GS variations in hybrids and derivates of common wheat Shinchunaga (Triticum aestivum L.,2n=42, AABBDD) with Qinling rye (Secale cereale L. cv. Qinling,2n=14, RR) by SDS-PAGE analysis, cytological identification, gene clone and prokaryotic expression analysis. The results are as follows:The HMW-GS compositions of D family seeds were analyzed by SDS-PAGE. The F4 seeds D-1-7-1 was wide-type. But the subunits 1Dx2.2 and Ry were absent in F5 seeds D-1-7-1-3.The chromosome number of root-tip cells in D-1-7-1-3 seeds was 42. Genomic in situ hybridization (GISH) and fluorescence in situ hybridization (FISH) analysis indicated that there were 41 wheat chromosomes and a single rye chromosome. The singe rye chromosome was 1R. The results indicated that D-1-7-1-3 was monosomic substitution.The molecular identification of Glu-Dx2.2 in F5 plant D-1-7-1-3 indicated that a deletion mutation of Glu-Dx2.2 by illegitimated recombination led to a novel allele Glu-1Dx2.2v, which have intact open reading frame (ORF). The prokaryotic expression indicated that the 1Dx2.2v protein band has the same electrophoretic mobility with the Rx. This result explained the reason for 1Dx2.2 absence in D-1-7-1-3.4.The molecular identification of Glu-Rx and Glu-Ry coding region from DNA and cDNA were carried out by molecular methods. The results indicated that the sequences of Glu-Ry coding region were not mutated in DNA and cDNA. It has intact ORF and could be transcribed into messenger RNA (mRNA). Although the sequences of Glu-Rx coding region were also unchanged in DNA, it could not be transcribed into mRNA. A further prokaryotic expression of Glu-Rx revealed that the electrophoretic mobility for expression protein was above 1Bx7. The position of expression protein just corresponded with the absent subunit in D-1-7-1-3. This result indicated that the absent subunit above 1Bx7 was Rx.In order to find out the reason of Rx absence, a further molecular identification of Rx promoter region was carried out. The sequences were no difference as compared with controls. The results indicated that Rx absence was not caused by sequence variation of promoter region. This result suggested there may be an alternative molecular mechanism for Rx absence in D-1-7-1-3.