Epigenetic Alterations In Cold Tolerance Introgression Lines Derived From Dongxiang Wild Rice(O.rufipogon Griff.)

Interspecific hybridization and gene introgression are deemed to an importantapproach to use of exogenous excellent genes and increase genetic variability inbreeding programmes and genetic research aimed at creating novel plant forms andimproving the quality of crops. However, extensive genetic and epigenetic variationswould be induced during alien gene introgression, which might delay the sufficientutilization of favorable wild genes in the practical breeding programs. In addition,epigenetic variations like retrotransposons and DNA methylation have an effect ongenome structure and function of organism, and then some phenotypic phenomenonin plant were changed.In our study, the cold tolerance introgression lines IL5335and IL5243derivingfrom Dongxiang Wild Rice (O. rufipogon Griff.) were used. Some unstablephenotypic phenomenon was still observed in IL5335selfing generations. Weanalyzed reverse transcriptase structure of Ty1-copia like retrotransposon and itsexpression activation in two introgressions. Using MSAP and SSAP method, cytosineDNA methylation and genetic diversity were studied. These results provide usefulinformation for ascertain epigenetic changes in rice interspecific hybrids offspringstabilization process and lay the foundation for efficiently exploit and use ofexogenous excellent genes for crop improvement. The main results of this study weredisplayed as follows:1. Cloning and expression analysis of retrotransposons reverse transcriptase inintrogression lines derived from Dongxiang Wild Rice (O. rufipogon Griff.)The cold tolerance introgression lines IL5335、IL5243and their parents wereused in the present study.75sequences of Ty1-copia like retrotransposon reversetranscriptase with highly heterogeneous were cloned. Seven families weredistinguished after cluster and alignment analyses of their nucleotide sequences.65RT clones in family6and family7account for about86.7%of the total clones andsimilarity ranged from44.9%-99.3%. Family1-5contains only10RT sequences.8sequences came from introgression lines and2sequences came from DWR. Thesesequence mainly characterized by deletion or insertion mutation and similarity rangedfrom29.2%-52.8%compared with RT sequences in their parents. Real-Time PCRdetection showed that the expression of houba, osr15and osr17reverse transcriptasein IL5335and IL5243were1.50-5.04times higher than their expression in parents. It also revealed that reverse transcriptase sequences in IL5335and IL5243were changedstructure and activated expression through Introgressive hybridization.2. Alterations of DNA methylation in cold tolerance introgression lines derivedfrom Dongxiang Wild Rice (O. rufipogon Griff.)Levels and patterns of cytosine DNA methylation in introgression lines (IL5335and IL5243) and their parents were researched by methylation sensitive amplifiedpolymorphism (MSAP) method. The results showed that15.84%and11.79%ofCCGG sites are either fully methylated and/or hemi-methylated in the rice parentalXB and DWR, respectively. Compared to their parents, a statistically significantdifference in the overall levels of both DNA methylation types was detected in IL5335and IL5243(14.14%and15.90%, respectively). Analysis of methylation patternsshowed that majoyrity of introgression lines inherited methylation patterns of XB andDWR, and only88sites changed in the ILs, including demethylation (32sites) andhypermethylation (56sites).17differentially methylated sequences were choned andsequenced. The results indicated this sequences similarity to known functional genes,retrotransposon and sequences with no homology. In addition, real-Time PCRanalysis suggested that the expression in demethylation of CHY1and MAPK higherthan their expression in parents, which further supported the hypothesis that DNAmethylation plays a significant role in introgression hybridization. These results laythe foundation for use of exogenous excellent genes for crop improvement.3. Alterations of DNA methylation in cold tolerance introgression lines derivedfrom Dongxiang Wild Rice (O. rufipogon Griff.)46materials including introgression lines (ILs) derived from Dongxiang WildRice and cultivated rice from China and Foreign were used to investigate the geneticdiversity by using sequence specifc amplifcation polymorphism (SSAP) markers. TheSSAP primers were designed according to the LTR region of Ty1-copia likeretrotransposon (houba、osr15and osr17). The result showed that a total of140sepecific bands were amplified. Six families were distinguished after cluster andalignment analyses of the bands. ILs derived from Dongxiang Wild Rice exsit in all ofthe families. The average Nei’s gene diversity (He) of ILs was0.394, which washigher than that of China and Foreign cultivated rice (0.385and0.376, respectively).It suggested that introgression hybridization introgression could broaden the base ofgenetic variation and provided a method in breed improvement of rice.