Mitotic illegitimate recombination is a mechanism for novel changes in high-molecular-weight glutenin subunits in wheat-rye hybrids

Wide hybrids can have novel traits or changed expression of a quantitative trait that their parents do not have. These phenomena have long been noticed, yet the mechanisms are poorly understood. High-molecular-weight glutenin subunits (HMW-GS) are seed storage proteins encoded by Glu-1 genes that only express in endosperm in wheat and its related species. Novel HMW-GS compositions have been observed in their hybrids. This research elucidated the molecular mechanisms by investigating the causative factors of novel HMW-GS changes in wheat-rye hybrids. HMW-GS compositions in the endosperm and their coding sequences in the leaves of F(1) and F(2) hybrids between wheat landrace Shinchunaga and rye landrace Qinling were investigated. Missing and/or additional novel HMW-GSs were observed in the endosperm of 0.5% of the 2078 F(1) and 22% of 36 F(2) hybrid seeds. The wildtype Glu-1Ax null allele was found to have 42 types of short repeat sequences of 3-60 bp long that appeared 2 to 100 times. It also has an in-frame stop codon in the central repetitive region. Analyzing cloned allele sequences of HMW-GS coding gene Glu-1 revealed that deletions involving the in-frame stop codon had happened, resulting in novel ～1.8-kb Glu-1Ax alleles in some F(1) and F(2) plants. The cloned mutant Glu-1Ax alleles were expressed in Escherichia coli, and the HMW-GSs produced matched the novel HMW-GSs found in the hybrids. The differential changes between the endosperm and the plant of the same hybrids and the data of E. coli expression of the cloned deletion alleles both suggested that mitotic illegitimate recombination between two copies of a short repeat sequence had resulted in the deletions and thus the changed HMW-GS compositions. Our experiments have provided the first direct evidence to show that mitotic illegitimate recombination is a mechanism that produces novel phenotypes in wide hybrids.