Genetic And Epigenetic Variation In Rice Introgression Lines Induced By Introgressive Hybridization Between Rice (Oryza Sativa L.) And Zizania Latifolia Griseb.

We have reported previously that introgression by Zizania latifolia resulted in extensive genetic and epigenetic changes in the recipient rice genome. In this study, using the methylation-sensitive amplified polymorphism (MSAP) method, we globally assessed the extent and pattern of cytosine methylation alterations in three typical introgression lines (RZ1, RZ2 and RZ35) relative to their rice parent at ~2,700 unbiased genomic loci each representing a recognition site cleaved by one or both of the isoschizomers, HpaII/MspI. Based on differential digestion by the isoschizomers, it is estimated that 15.9% of CCGG sites are either fully methylated at the internal Cs and/or hemi-methylated at the external Cs in the rice parental cultivar Matsumae. In comparison, a statistically significant increase in the overall level of both methylation types was detected in all three studied introgression lines (19.2, 18.6, 19.6%, respectively). Based on comparisons of MSAP profiles between the isoschizomers within the rice parent and between parent and the introgression lines, four major groups of MSAP banding patterns are recognized, which can be further divided into various subgroups as a result of inheritance of, or variation in, parental methylation patterns. The altered methylation patterns include hyper- and hypomethylation changes, as well as interconversion of hemi- to full-methylation, or vice versa, at the relevant CCGG site(s). Most alterations revealed by MSAP in low-copy loci can be validated by DNA gel blot analysis. The changed methylation patterns are uniform among randomly selected individuals for a given introgression line within or among selfed generations. Sequencing on 31 isolated fragments that showed different changing patterns in the introgression line(s) allowed their mapping onto variable regions on one or more of the 12 rice chromosomes. These segments include protein-coding genes, transposon/retrotransposons and sequences with no homology.At the same time, 98 microsatellites markers that mapped to all 12 rice chromosomes and the two cytoplasmic genomes were seleceted for detecting the variation in those specific sequences. Microsatellites, also called simple sequence repeats (SSRs), are widely dispersed in eukaryotes and have a high evolutional rate. In this study, we found that microsatellite variated extensively and rapidly in all three introgression lines. All of nuclear noncoding region located microsatellites extensively variated (100% in intergenic sequences, 66.7% in 5' regulatory regions and 83.3% in introns), but no change was detected in coding regions. And more surprisingly, 50% chloroplast DNA loci and 14.3% mitochondrial DNA loci showed variation and the variation even happened in coding regions of cytoplamic DNA. According to electrophoresis in denaturing polyacrylamide sequencing gels, five classes of polymorphic bands were found, and sequence comparison of selected loci between introgression lines and its rice parent showed that the high degree of variability in the microsatellite containing sequences were mainly due to variation in the repeated regions, while variation in the flanking regions of the microsatellite also happened. As methylation variation, the changed microsatellite patterns are uniform among randomly selected individuals for a given introgression line within or among selfed generations. We also found expression of mismatch repair (mismatch repair,MMR) genens in introgression lines changed, considering the relation of MMR genes with genome stability, this may be one of the reason for microsatellite stability in introgression lines. Finally, issues about possible causes for the introgression-induced methylation changes and their implications for genome evolution and crop breeding as well as introgression lines can be very good materials for microsatellite function analysis are discussed.