Preliminary Studies On Epigenetics Of The Rice PTGMS Line Wuxiang S

WuxiangS (WXS), which is a novel strain of the non-pollen type PTGMS with properties of low critical temperature, high combining ability and morphological markers, was generated by our laboratory. In the present study, WXS have been detected via the sterility SNP loci sequence, day length and temperature treatment, and cytological observation. And then MSAP, cDNA-AFLP and high throughput sequencing have been used to research the mechanism of epigenetic regulation in PTGMS rice. The MSAP and cDNA-AFLP have been analyzed for DNA methylation and transcriptions change in the panicles and leaves under the conditions of sterile and fertile pollen during the mother cell formation stage of WXS. Using high throughput sequencing, WXS have been analyzed via miRNAs and lncRNA sequencing under two different conditions of panicles from pollen mother cell formation stage to meiosis stage. Futhermore, we have developed a new method, which use conventional MSAP technique combined with high throughput sequencing. WXS and rice 9311 have been analyzed the DNA methylation sites using the method in panicle, leaf and stem under two different conditions. The main results were as follows:1. MSAP and cDNA-AFLP methods have been adapted to study DNA cytosine methylation and transcriptional change in the fertility transition stage of WXS. In young panicle and leaves,2561 and 2561 CCGG sites were detected respectively. Morever, 1630 and 1409 transcripts were detected in cDNA-AFLP. Relative to the sterile lines, the overall level of methylation declined 0.24%and 0.16%in young panicle and leaf, respectively. The transcription level was increased by 0.53%and 1.44%in young panicle and leaf, respectively. Overall methylation level was negatively correlated to the level of transcription in young panicle and leaf. Twenty-eight bands from eleven types of methylation/demethylation and twenty-three bands from the fifteen classes of transcription patterns were selected for cloning sequencing and gene annotation. GO enrichment analysis showed that these genes were mostly involved in environmental stress. Eight genes were selected for qPCR verification, and the results showed that methylation fragment methylation status was negatively related to gene expression level. These functional genes may be involved in fertility transition.2. The results of miRNA-seq showed that 72 known miRNAs were differentially expressed between WXS-S and WXS-F. There were 55 and 51 differentially expressed miRNA in SP2-FP3 and SP3-FP3 groups, respectively. Among of these miRNAs, twenty-six miRNAs were significance differences both in the two groups, which including 11 known miRNA were down-regulated and 15 miRNAs were up-regulated in the sterile lines. The differential expression miRNA targets contained ribosome inactivating protein, disease-resistant protein, MYB family proteins, transposons, retroposons, zinc finger protein and etc. GO anlaysis shows that the miRNAs were generally involved in the signal transduction, stress response and cell components. Furthmore, we have detected amounts of miRNAs editing events in the fertility transition stage, mainly in the 11 and 18 site of miRNAs. However,1-3 sites of 5’end and core 8-10 sites have been not found editing events in the miRNAs, suggesting that the miRNA editing events might be maintain the miRNA function stable within enriching diversity at the same time.3. Preliminary analysis of lncRNA transcriptome showed that 93%of lncRNAs were originated from genome intergenic regions and antisense strand with uniform distribution on the chromosomes. Two-thirds lncRNA were length ranging from 200 bp to 1000bp with tissue/development-specific expression. Expression level density analysis showed that the expression level of lncRNAs was similar to mRNA. Differential expression analysis revealed that 1596 lncRNAs were up-regulated and 1053 lncRNAs were down-regulated in the pollen mother cell formation stage, In meiosis stage,1195 lncRNAs were up-regulated and 334 lncRNAs were down-regulted. LncRNAs target prediction revealed that there were 238 lncRNA can be used as endogenous target mimics of miRNAs. MiRNA precursor predictions revealed that 26 high-trust lncRNAs were considered as mature miRNAs synthetic precursors. LncRNA-mRNA co-expression analysis showed that 1873 differentially expressed lncRNAs corresponding to 10739 cis-regulation targets and 1100 trans-regulation targets. From above, we have found 32 candidate lncRNAs involved in the regulation of pollen development relevant GO pathway.4. The MSAP-seq were performed by digesting DNA samples with EcoRI/MspI and EcoRI/HapII enzyme combinations, and then we have designed primers compatible with NGS sequencing for ligation reaction. The analysis results showed that our method were suitable for subsequent bioinformatics analysis after the construction libraries basing on the quality data and suitable insert fragment length. As an example, panicles of rice 9311 and WXS were disposed by the standardized anaylsis, producing a total of 14,304 cytosine methylations available for methylaiton analysis. Expression pattern analysis and comparison found that 142 C methylation sites during the fertility transition. In these sites, fourty related genes were located in the specific gene regions or adjacent genes using Annovar software.