Study On Agronomic Traits And DNA Methylation In Isonuclear Alloplasmic Rice

Cytoplasmic male sterility makes the three-lines commercial hybrid seed production with CMS, maintainer and restorer possible, which is the main type used in the production of hybrid rice currently. Cytoplasmic male sterility and nuclear gene control fertility restorer, which are genetic tools widely used in crop heterosis breeding. however, the mechanism underlying this kind of cytoplasmic-nuclear interaction remains unclear. In a long-term field breeding work, rice breeders found that sterile cytoplasm would affect many phenotypes of plants, but the results were not the same, indicating that there are significant limitations to just investigate the phenotype of agronomic traits. As an important epigenetic modification, DNA methylation can alter gene expression, and then affect the phenotype. In this study, a methylation sensitive amplified polymorphism (MSAP) technique is used to characterize the differences of nuclear genome methylation among cytoplasmic male sterile line (A) and its corresponding maintainer line (B), and variation of the difference when bring in the same restorer lines. In order to explore the effects of sterile cytoplasm and the impact of restorer on the level of DNA methylation.In this study, representatives of four kinds of important cytoplasmic male sterility, Jin23A (WA-type), G46A (G-type), D62A (D-type) and Ⅱ-32A (ID-type) were used as cytoplasm donor, the maintain line D62B and Ⅱ-32B as nuclear donor to bred two sets of isonuclear alloplasmic materials. The 8 isonuclear alloplasmic CMS lines and 2 maintain lines were crossed with restorers Shuhui527(R527), respectively. The effects of different cytoplasmic in same nuclear background and F1 hybrids in same restorer background were investigated by phenotypes and genomic methylation levels. Results are as follows:1) Field observation and agronomic traits survey of isonuclear alloplasmic CMS lines and corresponding maintainer lines showed that phenotypic differences is not significant during vegetative growth; after the initial heading stage, isonuclear alloplasmic CMS lines and corresponding maintainer lines began to show significant differences, mainly in the long growth period, pollen sterility, low plant height, and the phenomena of serious Panicle Enclosure.2) Field observation and agronomic traits survey of F1 hybrids crossed between isonuclear alloplasmic materials and restorer line R527 showed that the differences in pollen fertility were the most significant between A/R (isonuclear alloplasmic CMS line/R527) and the corresponding B/R (maintainer lines/R527), A/R were lower than B/R 10%. Besides, there were differences in plant height, panicle length, growth period and other traits, too. Reflects in slightly shorter plant height, shorter panicle length, longer growth period. The differences on other agronomic traits were not significant. Analysis of phenotypic data between parents and corresponding F1 showed the phenotypic differences between isonuclear alloplasmic CMS lines and corresponding maintainer lines still continues on in F1, but the restorer R527 significantly reduced this difference.3) Comparing the levels of methylation between isonuclear alloplasmic CMS lines and corresponding maintainer lines, found that different cytoplasm effect on the DNA methylation. In particular in:significant differences were observed in type-Ⅰ, type-Ⅱ, type-Ⅲ methylation between the maintainer lines D62B and corresponding isonuclear alloplasmic CMS lines, D62B was more than corresponding isonuclear alloplasmic CMS lines, led to higher total methylation level and full methylation level of D62B. In F1, the total methylation level and full methylation levels of D62B/R were slightly higher than the corresponding isonuclear alloplasmic F1 with sterile cytoplasm. While in type-Ⅰ, type-Ⅱ, type-Ⅲ methylation the performance were different from the parents. Compared to four groups of F1 with sterile cytoplasm, D62B/R did not show a clear advantage. Indicated that sterile cytoplasm may reduce genome methylation compared to fertile cytoplasm; the reduction of DNA methylation of nuclear genome by sterile cytoplasm got a certain recovery after the introduction of genes to restore.4) Compared the methylation bands between isonuclear alloplasmic CMS lines and corresponding maintainer lines to find the polymorphism bands, which all of the 4 isonuclear alloplasmic CMS lines or corresponding F1 were different from that with maintainer cytoplasm. Polymorphism site between parents and hybrids were 14 and 21, respectively. The rate of polymorphism were 2.82% and 4.02%, respectively. This directly showed that the different cytoplasms resulted in the differences of nuclear genome DNA methylation.5) Compared the DNA methylation between parents and F1,found that the same type bands between F1 and parents with sterile cytoplasm are significantly less than that with the maintainer line cytoplasm; both of the bands only same with female parent and only same with male parent were that with sterile cytoplasm are more than that with the maintainer line cytoplasm. Inferred that sterile cytoplasm may affect the interaction between parents and the inheritance of methylation from the parents to F1.