| Wild hybridization and allopolyploidization is an important way for speciation as well as one of the major forces in plant evolution. Previous studies indicated that the process of allopolyploidization is associated with rapid and extensive genetic and epigenetic changes. However, the underlying mechanisms for these alterations are poorly understood.High-molecular-weight glutenin subunit (HMW-GS) is one of the important quality agronomic traits for major determinants of the processing and bread-making quality of flour of wheat. In this study, we aim to observe the HMW-GS variations in a large number of hybrids between common wheat Shinchunaga (Triticum aestivum L.,2n=42, AABBDD) and Qinling rye (Secale cereale L. cv. Qinling,2n=14, RR), and to explore the molecular mechanisms for HMW-GSs variation. The results were as follows:1. It was found that most of the 28 chromosomes in Shinchunaga-rye F1 hybrids showed univalent pairing configuration. In situ hybridization (GISH) analysis on F3 and F5 plants found some plants contained 1-3 rye chromosomes or fragment. Some plants had 42 chromosomes or close to this number. They might be formed by the union of gametes with most or all wheat chromosomes by unreduced wheat chromosomes and with few rye chromosomes by reduced rye chromosomes. This may be related to asynchronous of chromosome segregation between wheat and rye.2. Wide hybrids can generate novel traits, yet the mechanism is poorly understood. This study tried to understand the mechanism at molecular level by simultaneously investigating changes of HMW-GSs in endosperm and their coding sequences in leaves of F1 and F2 hybrids between wheat landrace Shinchunaga and rye landrace Qinling. Novel HMW-GS patterns with missing subunits and/or added novel HMW-GSs were observed in the endosperm of 0.5%of the 2078 Fl and 22% of 36 F2 hybrid seeds. Analyzing allele sequences of HMW-GS coding gene Glu-1 revealed that DNA deletion mutation led to novel-1.8kb Glu-1 Ax alleles in some F1 and F2 plants. The wild type Glu-1 Ax gene was found to have 42 types of short repeat sequences of 3-60 bp long that appeared 2-100 times, respectively, in the central repetitive region, which was involved in the deletions. Different changes between the endosperm and the plant of the same hybrids and data of Escherichia coli expression of the cloned deletion alleles suggested that mitotic illegitimate recombination between two copies of these short direct repeat sequences in the hybrids resulted in the deletions and thus the changes of HMW-GS patterns. Our experiments provided evidence to show that mitotic illegitimate recombination is at least one of the mechanisms that produce novel phenotypes in wide hybrids, and thus heterosis.3. A total of 826 seeds in F2～F5 generations from ten families were analyzed by SDS-PAGE. The results indicated that a large number of variations occurred in different families and different generations, including subunit disappearance and novel subunit appearance. The disappearance ratio of subunits from high to low was Ry (75.06%), Dx2.2 (37.77%), By8 (15.50%), Rx (15.25%), Bx7 (7.02%), Dy12 (0%). This meant that Ry was normally disappearing, while Dy12 was always expression. A total of 151 seeds (18.28%) appeared 1-3 new bands. There were 205 (24.82%) seeds with subunit combination Rx-, Bx7+By8,Dy12 and 168 (20.34%) seeds with Dx2.2+Dyl2,Rx,Bx7+By8. The varied frequency in F4 generation was higher than in F3. According to HMW-GS variations from F2 to F5 generations in 10 families, we discussed genetic diversity during wide hybridization as well as the potential values for quality breeding.
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