DNA Sequence And Methylation Changes In The Introgression Lines Derived From Oryza Sativa L. Mating With0. Rufipogon Griff

Rice is an important food crop in the world, and its narrow genetic basis limits theimprovement of rice yields, so it’s an important way to utilize wild resources forgenetic improvement of rice by wide hybridization. In recent years, some reportsshowed that wild resources have been successfully improved cultivated species, butextensive genetic and epigenetic changes would be induced during the exogenousDNA introgression, which may hinder the successful utilization of the wild genes tosome extent. In addition, these unstable genetic and epigenetic phenomena alsooccurred in the forming process of natural species. In our study, the introgression lines(ILs) deriving from cultivated rice (Oryza sativa L., hereafter referred to as XB)mating with Dongxiang wild rice (O. rufipogon Griff., hereafter referred to as DWR)were used. We analyzed genetic changes and genetic diversity in239BC1F6ILs usingSSR method. In addition, excellent ILs in BC1F9population were analyzed by SSR,AFLP and methylation-sensitive restriction enzymes, which contributed to clarify themolecular mechanisms of genomic genetic changes and DNA methylation changes inthe advanced generation ILs, and provide a theoretical basis for the geneticimprovement and evolution of rice. The main results of this study were displayed asfollows:(1)131SSR makers were used to analyze genetic variation and genetic diversityfor a set of239BC1F6ILs derived from cultivated rice (XB) mating with Dongxiangwild rice (DWR). The results showed that ILs inherited majority parental genomicsequences (homozygous or heterozygous bands) and performed non-Mendelianvariations including the parental sequence elimination and new bands appearance;Principal coordinate analysis (PCA) and the program Structure. revealed that most ofILs were clustered together with XB individual and a few were clustered together withDWR individual. The result from PCA also displayed that some intermediategenotypes between XB and DWR were also found. The gene diversity of ILs washigher than that of cultivated rice, suggesting that the genetic base could be broadenedby wide hybridization.(2) In our study, BC1F9excellent ILs were used by SSR analysis, and the resultsbased on131SSR markers were similar to that of BC1F6population. In BC1F9ILs,most of loci (73.7%) inherited genes from recipient XB while a small part (17%)inherited the genes from donor (DWR). The results of comparative analysis between different generations of ILs revealed that genome of advanced ILs had basicallystabilized and homozygous accompanying with the trend of losing genes from DWRand inheriting genes from recipient XB.(3) The data based on131SSR and18AFLP markers showed that BC1F9excellent ILs performed a high level of genomic sequences changes, includingparental sequence elimination and new sequence emergence. Twenty-one bandsamplified by4primers in the ILs and DWR were sequenced, which revealed that ILscan amplified bands equaled to that of DWR, but sequences of ILs mainly showedhigh similarity to that of DWR. These sequence variations contained a few basestransition, transversion and increased scattered in different locations.(4) In order to analyze changes of methylation levels and patterns,85pairs ofmethylation-specific primers distributed in12chromosomes were used to amplifygenomic DNA which was digested with McrBC enzyme in5BC1F9ILs and parents.The results showed that XB, DWR and ILs had high levels of frequency ofmethylation (61.2%,44.3%and55.5%, respectively). Majoyrity of Ils inheritedmethylation patterns of XB, and only18.6%loci changed in the ILs, includingdemethylation (12%) and hypermethylation (6.6%). The results based on primersaiming at the loci near rough location of intervals and transposons areas (Houba andOsr14) revealed that changes of methylation levels and patterns had happened inexcellent ILs.