Genomic DNA Methylation Analysis Of A Photoperiod-and Thermo-Sensitive Genic Male Sterile Rice Peiai64S

Methylation, an epigenetic phenomenon with no effect on DNA base sequence, involves in the regulation of gene expression, embryonic development, genomic imprinting, cell differentiation, X-chromosome inactivation, and cell memory. The DNA methylation could be inherited or reversed, depending on the different environmental press. Also, a specific dynamic change pattern exists for this DNA modification. Peiai64S, a photoperiod-sensitive genic male sterility (PGMS) and thermo-sensitive genic male sterile (TGMS) rice resource, is very important in hybrid rice breeding such as the "Super Rice". With the completion of rice whole-genome sequencing and subsequent progress in proteomics, epigenetics and gene annotation, the PGMS/TGMS rice research has impetused for the study of cytology, physiology and molecular biology. Additionally, several genetic sterile genes have been cloned and mapped to corresponding chromosomes. In view of the unclear genetic mechanisms of PGMS/TGMS, the effects of epigenetic modification on rice fertility have been considered. In the present study, differences in DNA methylation level between the male-sterile and fertile plants of Peiai64S were investigated. Results speculated that the DNA methylation level was associated with the TGMS fertility conversion.DNA methylation differences in the young panicles at meiosis stage between the male-sterile and fertile plants of Peiai64S were analyzed by MSAP technology. Additionally,192AFLP primer pairs detected27417polymorphic bands on the PAGE gel, of which1215bands were different between the sterile plants and fertile plants. Totally,346differential bands were recoveryed, cloned, sequenced, and blasted against the NCBI database. Results indicated that there were95homologous sequences in rice, of which10were highly conversed involving in the photosynthetic system, the mitochondrial respiratory transport chain, cytoskeleton and signaling cascade reaction processes. Several genes involving in photosynthesis or energy metabolism, including D2(photosystem II core heterodimer polypeptide), P700(apoproteins of photosystem I reaction), Nad7(the main dehydrogenase of the mitochondrial respiratory chain), VIP2, Cyt f, Ret and MTs were chosed for the real-time PCR analysis, which have higher expression level in male sterile line than in fertile lines. Comparative analysis of MSAP methylation profiling showed that all these genes were highly methylated in the fertile lines, which further participating in the regulation of fertility conversion.Genome-wide DNA methylation level analysis of Peiai64S was carried out by MeDIP-sequencing analysis, and the differential distribution of DNA methylation within the CpG Island,2000bases upstream and downstream,5’-UTR regions, CDS, intron,3’-UTR regions, and other functional elements were also compared. Results inferred that the reads gather the2000bases upstream and downstream regions, with the highest methylation level. Though, low-level methylation existed for the3’-UTR and5’-UTR regions. Differences of DNA methylation distributed widely among all the functional elements of both sterile plants and fertile plants of Peiai64S, though the methylation level was higher in fertile lines than the sterile line. Obviously, average depth of coverage of reads in the CpG Island region was higherr than that of other regions. Peaks, representing the methylation level, were mostly concentrated in the2000bases upstream and downstream region, intron, and CDS components, while the5’-UTR and3’-UTR regions had fewer peaks.1126differentially expressed genes were found among the functional elements based on the difference in coverage of peak between the sterile plants and fertile plants, and most distributed in the2000bases upstream and downstream regions. GO annotation and Pathway functional analysis was also performed to identify the PGMS/TGMS related genes.Genetic mechanism of PGMS/TGMS is not well understood, and studies of its relationship with DNA methylation level are also limited. In this research, we have found that the genome-wide DNA methylation level of sterile line of Peiai64S was lower than that of fertile lines with both MSAP and MeDIP technique. Totally,95differentially expressed genes have been identified with MSAP technique, and1126differentially expressed genes were characterized by MeDIP-sequencing. Our studies may supplement the knowledge of genetic mechanism of PGMS/TGMS, and paved the way for the further research on the fertility alteration of PGMS/TGMS, which could enhance its application in scientific research and breeding program.