Genetic Basis Of Novel Germplasm Of Somatic Hybrid Introgression Lines Between Common Wheat And Tall Wheatgrass

We obtained various fertile introgression lines through asymmetric somatic hybridization between protoplast of common wheat (Triticum aestivum) cv. JN177 and UV-irradiated protoplast of tall wheatgrass (Thinopyrum ponticum) and these lines have inherited to F10 generation. Some of these introgression lines showed stable superior agronomic traits compared to JN177, including salt and/or drought tolerance, dwarf habit, disease resistance, high yield and good processing quality etc. SSR analysis conducted by Chen et al.(2004) indicated that genetic materials of Tall wheatgrass have been introgressed into the genome of JN177, and the SSR sequences of JN177 was altered by asymmetric somatic hybridization. Cloning and sequence analysis of HMW-GS/LMW-GS genes of various introgression lines and the two parents JN177 and tall wheatgrass showed that novel glutenin genes not discovered in JN177 appeared in somatic hybrid lines.These novel glutenin genes appeared in the introgression lines come directly from tall wheatgrass or from the recombination between HMW-GS/LMW-GS gene sequences of the two parents or gene sequence alterations of JN177 glutenin genes.14 of 37 cloned JN177 HMW-GS/LMW-GS genes were altered by asymmetric somatic hybridization. Further studies on the manner and mechanisms of genetic variation induced by asymmetric somatic hybridization are the basis for understanding these variations and will be helpful for wheat improvement utilizing these hybrid lines. To explore genomic variation derived from "genomic shock" via parental genome merging and donor genome introgression, a genetic analysis was performed with two asymmetric somatic introgression lines (released cultivars) involving bread wheat(Triticum aestivum), and its relative tall wheatgrass(Thinopyrum ponticum Podp). The two cultivars have proven to be phenotypically stable through a number of selfing generations. A spectrum of cytogenetic assays and DNA profiling techniques was applied to understand the nature of the genetic and epigenetic changes which had been induced by somatic hybridization. At the chromosome level the cultivars appeared very similar to their wheat parent but containing introgressed chromatin. The molecular profiling revealed many genetic and epigenetic differences, including the elimination of genomic DNA, the altered regulation of gene expression, changed patterns of cytosine methylation, and the reactivation of retrotransposons. Repetitive sequences combined with the epigenetic regulation of gene expression and/or retrotransposon transposition appear to be largely responsible for the phenotypic differences between the introgression cultivars and their wheat parent. These results confirmed that asymmetric somatic hybrid intogression lines provide useful materials to explore the nature of the genetic and epigenetic variation of the derivatives induced by genomic shock.The results of my research are listed as follows:1. Genetic alterations induced by asymmetric somatic hybridizationWe utilizd various molecular markers, including SSR/AFLP/EST-SSR/IRAP/ REMAP to analyse the genomic sequences of the introgression lines and the two parents, so as to obtain the data about the alterations of repetitive sequences/ restrotransposons and gene sequences of JN177 genome induced by asymmetric somatic hybridization, and then analyse underlying mechanisms.An examination of the genomic SSR profiles of cv. JN177, SR1 and SR3 revealed no polymorphism between SR1 and SR3, but at 40 of the 116 genomic SSR loci, the cv. JN177 profile differed from that of R2 SR1 and SR3 individuals. At two loci, SR1, SR3 and tall wheatgrass shared the same allele. No variation in SSR profile between the R2 and R10 generations of SR1 or SR3 was observed (data not shown). Given the genotypic stability of SR1 and SR3 over many generations, the remaining fingerprinting analysis was restricted to the R2 generation.Analysis of EST-SSR alterations between JN177 and the two introgression lines indicated that 22(20.37%) of 108 loci were altered in SR3 when compared with JN177, a percentage much lower than that of genomic SSR alterations(34.48%).AFLP profiling revealed 904 cv. JN177 and 777 tall wheatgrass fragments, of which 388 were common to both templates. The comparisons of cv. JN177 with SRI and SR3 revealed 872 monomorphic fragments. Eighteen cv. JN177 fragments were absent from both SRI and SR3, while another 4 and 10 fragments were eliminated from SRI and SR3 respectively; except for the eliminated fragments, SRI and SR3 showed respectively 16 and 20 novel bands.IRAP and REMAP were used to analyze retrotransposon movement in response to somatic hybridization. Of 116 fragments detected,10 were polymorphic between cv. JN177 and SR3. Five of the 10 fragments were absent, and other 5 fragments were present in SR3 but not in cv. JN177. Eleven bands were polymorphic between cv. JN177 and SRI, with 6 bands unique to JN177 and 5 unique to SRI. There were 3 polymorphisms between SRI and SR3. In an attempt to identify the genes associated with retrotransposon activation, some of the unexpected fragments were sequenced, but the analysis of these sequences only recognized homology related to one (present in cv. JN177, absent in SR3), which identified a Ty3-gypsy subclass retrotransposon (ABA97230.1). A second fragment present in SR1/SR3, but absent from cv. JN177 was highly homologous to a wheat pore-forming toxin-like protein Hfr-2 (AAW48295.1), involved in plant defence.SSCP analysis was utilized to detecte the alterations of JN177 gene sequences. Gene loss and insertion were detected in the two introgression lines and gene sequence alterations effects corresponding gene expression. SNPs between JN177 and the two introgression lines were also a commonly observed phenomeon. Novel gene bands in the introgression lines were a unique feature of asymmetric somatic hybridization, different from that of polyploidization.These results indicated that asymmetric somatic hybridization induced large scale DNA sequence alterations of JN177, but different components of JN177 genome showed largely different tendency to alternation. Highly mutantable sequences, genomic SSR sequences showed an alternation percentage of 34.4%, while only 4.17% gene sequence of JN177 was altered in somatic hybrids.We supposed that different chromosome configurations between different sequence types were the main cause of different alternation tendencies. Different epigenetic modifications, including cytosine methylation/histone modifications/chromatin remodeling, around different sequences prabably contribute to this process. We also found that the genetic alterations induced by somatic hybridization showed different characters when compared with that of ployploidization. Asymmetric somatic hybrid intogression lines provide useful materials to explore the nature of the genetic and epigenetic variation of the derivatives induced by genomic shock.2. Epigenetic alterations induced by asymmetric somatic hybridizationThirteen pairs of selective primers were used to determine methylation status at 360 CCGG loci. Of these, SR3 and cv. JN177 differed for 84 fragments, with 35 appearing to be hypermethylated and 49 hypomethylated in SR3; while SRI and cv. JN177 differed for 77 fragments, with 31 appearing to be hypermethylated and 46 hypomethylated in SRI. Although rarely happened, the hypermethylation from H to M banding pattern or hypomethylation from M to H banding pattern was detected in the introgression lines. There were less MSAP polymorphisms between SRI and SR3 (25,6.9%) than between cv. JN177 and the two introgression cultivars, of which 12 were hypermethylated and 13 hypomethylated in SRI. Sequence analysis of a number of differentially methylated fragments showed that three of the five hypomethylated fragments in SR3 were highly repeated retrotransposons (TREP acession number: TREP255, TREP1418, TREP3251), and two showed no significant similarity to any known sequence. While the five fragments hypermethylated in SR3 also showed no significant similarity to any known sequence.We also analysed the cytosine methylation pattern of JN177 in gene promoter and coding sequences using bisulfite sequencing, in order to investigate the cytosine methyaltion alterations and its effects on gene expression. The results indicated that the gene promoter sequences of wheat were highly methylated, and cytosine methylation in gene promoter negatively affects corresponding gene expression. Only one of three analysed gene body sequence was methylated. The cytosine methylation level in wheat gene promoter was altered by asymmetric somatic hybridization. The percentage of cytosines with altered methyaltion pattern was different between different genes, from 0.00%(TaWRKY1-7) to 25%(TaCHP). The average alternation percentage was 12.26%. The alternation of cytosine methylation level between JN177 and SR3 was not always correlates with gene expression alteration, except for TaFLS2. We suppose that epigenetic modifications except for cytosine methylation, such as histone acetylation/methylation and chromatin remodeling also contributes to gene expression alterations in the somatic hybrids. Further study on histone modification/chromatin remodeling alterations between JN177 and introgression lines is needed to fully understand the mechanism of gene expression alterations.We suppose that homologous sequences between JN177 and tall wheatgrass may experience complex interactions and induce various genetic and epigenetic varitions in the introgression lines. And genetic and epigenetic alterations all contribute to gene expression alteration and superior agronomic traits of the somatic hybrids.3.Gene expression alterations induced by asymmetric somatic hybridizationTo survey the extent of gene sequence alterations and homoeologous gene silencing or activation induced by somatic hybridization, SSCP analysis was performed using genomic DNA from cv. JN177, SR1 and SR3 and RNA from the shoots and roots of cv. JN177, SR1 and SR3 seedlings. Of the 80 SSCP amplicons,11 were informative between cv. JN177 and SR1/SR3. In four of six missing cDNA fragments, the corresponding genomic amplification products were also missing. Other two missing cDNA was absent from one or both of SR1/SR3 compared to cv. JN177. In two activated cDNAs, products were present in SR1/SR3 but not in cv. JN177. For other three of these genes, the migration of the corresponding cDNAs was also affected. Similar to genomic SSR analysis, we did not find any difference between SR1 and SR3 in genomic sequences in SSCP analysis. However, SR1/SR3 shoot showed qualitatively different gene expression in two genes tested. It is likely that these two genes were differently regulated in SRI and SR3.In conclusion, somatic hybridization between wheat and tall wheatgrass has generated a series of introgression lines displaying a variety of novel genetic and epigenetic changes relative to their wheat parent. The genetic alterations induced by somatic hybridization showed different characters when compared with that of ployploidization. Asymmetric somatic hybrid intogression lines provide useful materials to explore the nature of the genetic and epigenetic variation of the derivatives induced by genomic shock. Moreover, we found repetitive sequences, epigenetic regulation of gene expression and/or retrotransposon transposition mainly relative to the novel phenotypic and agricultural traits. Therefore, somatic hybrid introgression lines provide a unique means to explore the nature of the genetic and epigenetic variation induced by genomic shock. An understanding of these mechanisms should improve our understanding of the genetic basis of agriculturally important phenotypic variation and may shed light on the variation generated by sexual wild hybridization.