Genomic Variation Of Somatic Hybrid Introgression Lines Between Triticum Aestivum And Agropyron Elongatum

Asymmetric somatic hybridization between protoplast of common wheat and UV-irradiated protoplast of Agropyron elongatum generated fertile introgression hybrid lines and these lines have inherited to F₁₀ generation. Some of these introgression lines showed stable superior agronomic traits, including dwarf habit, salt and/or drought tolerance, disease resistance, high yield and good processing quality etc. SDS-PAGE analysis also indicated that novel HMW-GS presented in some of these hybrid lines. The presence of phenotypic diversity in these introgression lines indicates that there may be some changes in hybrid genomes. Studies on the manner and mechanism of genetic variation of these introgression lines are the basis for understanding these variations and will be helpful for wheat improvement using these hybrid lines. However, investigation on genomic variation of hybrid lines is very limited. In this paper, we investigated the genetic variation of these hybrid lines in the level of whole genome and that of HMW-GS gene family. Different results are involved in this work: the variation and mechanism of repeat sequences; epigenetic changes; silencing or activation of functional genes; alteration of gene structure and origination of novel genes; comparison of natural evolution with rapid evolution induced by somatic hybridization of HMW-GS genes. This work makes a molecular foundation for wheat genetic breeding through asymmetric somatic hybridization. The main results of this research are listed as follows:1. Analysis of SSR loci in hybrid genomeAnalyzes of SSR loci will be useful for exploring the microsatellite polymorphism and understanding mechanisms of the changes of the hybrid genomes. We screened 102 SSR loci of three hybrid lines with different phenotypes and both parents, along with the calli mixture of parent wheat Jinanl77 that was used as control to distinguish somaclonal variation. Only a few SSR sequences of hybrid lines have been found directly derive from A. elongatum. About 30% of detected loci were changed in the three hybrid lines when compared with parent wheat. The varied loci in hybrid lines whose band patterns coincident with those of the calli mixture account for only about 6.2% of all the checked loci, which indicates that somaclonal variation is subordinate for the variation of SSR loci in hybrid lines. Whereas the higher variation frequency of 26.5% SSR loci is deduced to arose from hybridization and the introgression of alien genetic material to the wheat genome. On the other hand, the variation of the 32 loci changed in the mixed calli and that of the other 70 loci unchanged showed aberrance with frequencies of 20.7/32 and 12.3/70 in the hybrid lines, respectively. This means that the former are prone to alter in both somaclonal variation and somatic hybridization. We speculate that they are likely hypervariation regions (or hot spots) in the wheat genome. Sequencing and aligning of some SSR sequences varied in hybrids indicates that the variation of the SSR loci is mainly due to the change of the number of repeat motifs.2. AFLP and MSAP analysis of hybrid genomeAFLP and MSAP have been shown to be the efficient way for detecting genomic polymorphism and epigenetic changes. We surveyed three hybrid lines and parents by screening 453 loci in all using AFLP fingerprinting. Among these loci, the total eliminated fragments are 43 while the novel fragments are 19 in the three hybrid lines. The varied loci of hybrid lines account for about 4.6% of all the scanned loci while 1.8% variance is brought by somaclonal variation. The frequency of the lost sequences is about 3.2%, while that of the novel fragments is only 1.4% among all the 453 loci. It is noted that the presence of novel fragments mainly result from somaclonal variation (11/19); the remainder (8/19) directly come from A. elongatum or biparental recombination, even due to alterations in DNA methylation patterns, which in agreement with the changes of methylation of the hybrid lines. We investigated the extent of cytosine methylation of 122 loci of hybrid lines and parent wheat using MSAP analysis. The total loci whose methylation patterns changed in hybrids account for about 18.6% of all the detected loci; out of them, 12% loci showed hypermethylation while only 6.6% loci demethylation. Changes of only 6.3% loci were caused by somaclonal variation. 3. SSCP analysis of hybrid introgression linesSSCP has the ability of exploring expression changes of homoeoalleles of given genes in hybrid introgression lines. Under the help of SSCP platform, we have analyzed the change of gene expression between hybrid lines and Jinanl77, the silencing or activation of homoeologous alleles. The expression of about 12.3% (7/57) and 8.8% (5/57) of the 57 EST loci we examined changed in leaf and root respectively, which means that one or two members of a gene set (homoeoalleles) silenced or activated in hybrids. Among the seven genes whose expression patterns changed in the hybrids, three genes encode proteins with unknown functions, while proteins encoded by the remainder four genes are phosphatidylinositol 3- and 4-kinase family protein, peptidylprolyl isomerase, monodehydroascorbate reductase and 1, 3-beta-glucan synthase, respectively.4. The origination of novel HMW-GS in hybrid linesComparison of novel HMW-GS genes of hybrid lines with published HMW-GS genes of common wheat is beneficial for investigating the structural variation of hybrid novel HMW-GS genes. We cloned five and four HMW-GS genes from hybrid lines and parent wheat Jinan 177 using PCR amplification respectively. We also successfully cloned 15 HMW-GS genes from another parent A. elongatum, eleven of them are novel genes. Through alignment of novel HMW-GS genes of hybrids with those genes from both parents, we found four possible mechanisms through which did the novel HMW-GS genes of hybrids come from. According to the results: (1) both H11-3-3 and H11-4-3 of the hybrid 11-4-6 directly inherited from A. elongatum; (2) H1Dx5 of the hybrid II-12 comes from point mutation of parent wheat 1Bx2.1; (3) H1By8 and H1By16 of the hybrid 11-4-6 likely result from slippage of parent wheat 1By9.1; (4) HlDy12 of II-12 maybe come from recombination of biparental genes 1Dy12.1 and Aey2. Therefore, we explained for the first time the mechanisms of generation of some novel HMW-GS genes of hybrid lines in molecular level, which is similar to the mechanism of natural evolution of these genes supposed before. We also described gene shuffling as a new mechanism of novel HMW-GS gene formation in hybrids. Therefore, we demonstrate that through asymmetric somatic hybridization, novel HMW-GS genes can be rapidly generated in a short time when compared with the time-consuming natural evolution. The results suggest that asymmetric somatic hybridization is an important approach for widening HMW-GS genebank of wheat quality improvement.5. Evolutionary status of three genes from A. elongatumPhylogenetic analysis of HMW-GS genes of A. elongatum will make it easy for understanding the natural evolution of this gene family and helpful for further explaining the origination of the other novel HMW-GS genes in the wheat somatic introgression lines. In the dendrogram of phylogenetic tree of the fifteen HMW-GS genes cloned from A. elongatum, we found that C-terminal regions of three y-type genes Aey8, Aey9 and Aey10 showed more similarity with x-type genes than with other seven y-type genes, which demonstrated that the structure of the three y type subunits is not as classical as the other y type subunits. In all the known y type subunits, only Aey4, Aey8, Aey9, Aey10 from A. elongatum and some y type subunits encoded by E^e, K, St and Ta genome contain 105 amino acid residues in the N-terminal domain, but all the other known y type subunits contain 104 residues. The subunits contain 105 residues in N-terminal domain are older than those contain 104 residues, so Aey8,Aey9 and Aey10 may represent a kind of state that appeared in the divergence between x- and y-type genes in the HMW-GS evolution. Sequence alignment showed that one gene Aey4 of A. elongatum may be a chimeric gene come from recombination between another two genes Aey5 and Aey10.6. The HMW-GS genes from wheat related speciesFor further understanding the evolutionary process of HMW-GS genes of Triticeae, we cloned four HMW-GS genes from diploid Lophopyrum elongatum, three y-type subunit genes from Pseudoroegneria libanotica and one gene from Australopyrum retrofractum, respectively. The N-terminal domain of the five y type subunits from L. elongatum and P. libanotica all contain 105 residues, one glutamine residue more than most known y type subunits. Besides, this additional glutamine residue also existed in the W2.5 subunit of Aus. retrofractum. Through sequence alignment and phylogenetic analysis of Glu-1 genes from these three grasses and some published typical HMW-GS genes, we found that the E, K, St, Ta and W genome differentiated earlier than other genomes of Triticeae species; meanwhile, the W genome may be older than the other four genomes, it may be separated from the ancestor genome before the differentiation of x and y type subunit genes.The subunit we obtained in Aus. retrofractum is different from both x and y type subunits, it is a new type of HMW-GS; we found a new type of repeat motif---duodecapeptide, which is the fourth type of repeat motif found in HMW-GS except for tripeptide, hexapeptide and nonapeptide. This indicates that the HMW-GS gene of W genome showed different evolutionary process from those genes of other Triticeae genomes.7. Significance of HMW-GS genes for wheat quality improvementInvestigation of HMW-GS genes from hybrid introgression lines and wheat related species may provide new genes for wheat quality breeding. Many HMW-GS genes we cloned show correlation with good grain-processing quality. For example, the HMW-GS genes H1Dx5, HlDy12 and HlBy8 cloned from hybrid lines have been proved to be correlated with good dough quality; the HMW-GS genes Aex4 from A. elongation and W2.5 from Aus. retrofractum contain extra cysteine residues in their coding peptide, W2.5 also contain long central repetitive domain, which have been proved to relate with good processing quality. These genes are potential for improving the flour processing quality of wheat We are using them in gene transformation and marker-assistant quality breeding of common wheat, from which a lot of germplasms and strains with high qualities will be produced.