The Molecular Mechanisms Of High-molecular-weight Glutenin Subunit Variations In Derivatives Of Wheat-rye Hybrids

As an important way to the speciation, wide hybridization, which can simulate the evolutionary process of species, is a significant mean for biological evolution research. Previous studies indicated that there are wide ranges of genetic and epigenetic variations during the wide hybridization. Underlying molecular mechanisms for novel phenotypes, however, are still poorly understood.Common wheat (Triticum aestivum L.,2n=6x=42, AABBDD) is widely cultivated as one of important cereal crops in the world. High-molecular-weight glutenin subunit (HMW-GS) is one of the major storage proteins in wheat endosperm, which affects the processing and bread-making quality of flour of wheat. Rye (Secale cereale L.,2n=2x=14, RR), a relative species of wheat, has been widely used in wheat genetics and breeding improvement since it has many desirable traits and rich genetic diversity. Previous studies found novel HMW-GS variations in some plants derived from hybrids between common wheat Shinchunaga and rye cv. Qinling. In the present study, we investigated the mechanism for HMW-GS variations by using SDS-PAGE, cytological observation, genomic in situ hybridization, gene sequencing, and prokaryotic expression. The main results are as follows:1. The HMW-GS compositions of F4seeds from two lines were analyzed by SDS-PAGE. All the50F4seeds, derived from the F3plant C-4-1, were absent for subunits1Dx2.2and Ry. All the50F4seeds, derived from the F3plant C-4-2, were absent for subunits Rx, Ry and1By8. We investigated their F3and F5seeds, and found same HMW-GS composition as in F4, which indicated their stable inheritance. Further studied found that there were all six HMW-GS in the F1seed as wild-type has. However, Rx subunit was absent in the F2seeds.2. In agronomic traits, the F5plants of the two lines were more closed to its maternal donor, common wheat Shinchunaga. Consistently agronomic traits and normal seed set among plants in the two lines indicated their stability.3. The chromosome number of root-tip cells in F5seeds was42. Genomic in situ hybridization (GISH) analysis indicated that there was no rye chromosome in F5seeds. Most of their chromosomes were paired into21bivalents at meiotic metaphase I, thus indicating that their cytology were stable.4. The genomic DNA from F3(C-4-1and C-4-2), F1(C) and F2(C-4) was amplified by using a pair of common primers. Sequences analysis of HMW-GS genes revealed that DNA mutation led to a nove1-2.6kb Glu-1Dx2.2allele which named Glu-1Dx2.2v (GenBank number:KC881262) in C-4-1. It was originated by the illegitimate recombination between two copies of direct repeat sequence (DR). The Glu-1Dx2.2v was further confirmed by its successful expression in Escherichia coli, and the Glu-1Dx2.2v and Rx subunit were similar in mobility. We also investigated the mutant genes Glu-1By8null (GenBank number:KC881264) and Glu-1Bx7v (GenBank number:KC881263) in C-4-2. The Glu-1By8null was induced by a nonsense mutation (Câ†'T) of the wild type gene Glu-1By8, which led to a termination codon. The Glu-1BxT was caused by the DNA deletion of a18bp tandem repeat (TR). In the F2plant, we detected the wild-type and mutated HMW-GS genes at the same time.In F1,however, we only found the wild-type genes. Therefore, these variations might originate in F1generation.5. Cytological observations did not find rye chromosome from F3to F5 generations. In addition, Glu-1Dx2.2v and Rx subunit were similar in mobility. Using specific molecular markers for Glu-1Dx2.2and Glu-Rx, we only amplified1Dx2.2v. These results proved the absence of Glu-Rx.