Epigenetic Research Of Rice Drought Resistance After Multi-Generation Drought Impostion

Rice is not only one of the most important foods in the world, but also a crop with largest water requirement. So far, great progress has been made in the study of rice drought resistance on genetic level and breeding. However, drought resistance is a very complicated quantitative trait in rice, and a few genes modification can not significantly improve it. Meanwhile,conventional genetic research methods about drought responsive genes are mainly based on DNA sequence variations or gene expression variations. Epigenetic mechanisms, which could improve long-term plant adaptation to environmental challenges, have received less focus under drought responses in rice plants.In this study, two rice epimutation accumulation lines were derived from 11 generations of single-seed descent under drought stress from two rice varieties with distinct drought resistance levels, Huhan-3(O. sativa L. ssp. japonica, a drought resistance variety) and II-32B(O. sativa L. ssp. indica, a drought sensitive variety). As all samples in each epi-MA lines came from a common ancestor and genetic variation was almost completely absent in them, differences in their morphological and physiological phenotype should be caused by epigenetic modification and gene expression variation. Identification of changes in drought resistance and variations in DNA methylation between drought exerted generations(G6 and G11) and the original generation(G0) allowed a further investigation into the impacts of multi-generation drought exposure on rice drought resistance, DNA methylation pattern and gene expression. The main results were as follows:1. After multi-generation drought imposition, significant reduction of tiller numbers are observed in the offspring of both Huhan3 and II-32 B. However, their seed settings, thousand grain weight and volume and weight of deep root per tiller are increased. These observations imply that two varieties adopt relative tactics to adapt to drought imposition: under drought condition, plants keep alive through reducing tillers but improving water absorbing capacity by deep root. Meanwhile, they try to keep yield by improving seed setting and grain weight.2. A total of 3070 and 4739 MSAP loci were recorded in II-32 B and Huhan-3 using the 256 primer-pairs, respectively. In both varieties, unmethylated sites accounted for more than half of the CCGG sites. The general DNA methylation levels were ~39% and ~31% in II-32 B and Huhan-3, respectively. Statistical results showed that II-32 B has a significantly lower proportion of free methylated loci(P<0.01) and a significantly higher proportion of both hemimethylated loci(P<0.01) and hypermethylated loci(P<0.05).3. II-32 B had ~13.10%(402/3070) differentially methylated loci(DML) between generations or/and between treatments. In II-32 B, the proportion was ~1.77%(84/4739). Huhan3 had less numbers and proportions of DML than II-32 B, indicating that DNA methylome of Huhan3 was much more stable than II-32 B.4. G6 had more variations in DNA methylation between normal and drought condition than G0 in both Huhan3 and II-32 B. DNA methylation variation between G6 and G0 mainly in happened in only normal condition. These observations indicated that G6 had a more unstable DNA methylome than G0, when subject to drought stress again, G6 had more DNA methylation variation to adapt to changed environment.5. In II-32 B, 27.61%(111/402) differentially methylated loci(DMLs) were directly induced by drought, among which 11.71%(13/111) DMLs kept their changed DNA methylation status in offspring. In Huhan3, the proportions were 47.62%(40/84) and 62.50%(25/40). These observations indicated that most of DNA methylation variations in II-32 B occurred randomly and can not be inherited while about half of DNA methylation variations in Huhan3 directly induced by drought stress and most of them were transgenerational, implying that multi-generation had more impact on Huhan3 than on II-32 B.6. DMLs widely distributed on all 12 chromosomes and mainly occurred in genes’ promoter regions and exons. Further analysis revealed that 62.73% and 45.00% genes related to DMLs were functional genes in Huhan3 and II-32 B, respectively. GO analysis revealed that some of these genes directly participated in stress response pathway.To investigate the impact of eleven-generation drought imposition on DNA methylation pattern of the adult plants of two rice varieties, we analyzed the variation in DNA methylation in both Huhan3 and II-32 B using whole genome bisulfite sequencing(WGBS). The main results were as follows:1. DNA methylation variations in both Huhan3 and II-32 B after 11 generations of drought exposure were characterized by using WGBS method. On average, the strandspecific coverage depth reached ~13.45× and ~11.21× per individual for Huhan3 and II-32 B, respectively. The high throughput sequencing data for Huhan3 and II-32 B covered 84.64% and 76.42% of all cytosines in reference genome, respectively.2. In both varieties, the CG methylation had less variation between generations and between treatments than the CHG and CHH contexts(non-CG context), indicating that the CG methylation had much greater stability than the non-CG methylation in both the gene bodies and TEs. What is more, the DNA methylation in Huhan3 had less variation than that in II-32 B, which implied that Huhan3 had more stable methylome than II-32 B.3. Drought-induced epimutations arose non-randomly. Certain positions were “hot spots” and variations in their methylation status were specifically induced by drought stress. In Huhan3, we observed 3.76% hypo-methylated DMPs(differentially methylated positions) and 6.45% hyper-methylated DMPs that occurred more than once, In II-32 B, the frequencies were 2.78% and 5.79%, respectively. These proportions of DMP recurrent events were much higher than the random frequency in both varieties. The same result was observed in SMPs(single-cytosine methylation polymorphisms).4. Hierarchical clustering based on drought induced epimutations in both varieties revealed that multi-generation drought exposure exerted considerable influence effects on rice methylome, and the effects of long-term drought exposure on epimutations were directional processes.5. Considerable amounts of epimutations that accumulated after 10-generation of drought exposure were transgenerational. 9.39% and 7.51% of the accumulated-DMPs could transmit their altered methylation status to their offspring in Huhan3 and II-32 B, respectively. For SMPs, the proportions were 14.86% and 15.60%, respectively.6. High proportion of drought-induced epimutations maintained their changed DNA methylation status in advanced generations under drought stress. For DMPs, the proportions were 68.44% and 71.14% in Huhan3 and II-32 B, respectively. For SMPs, the proportions were 44.80% and 71.14%, respectively.7. There was no significant correlation between the DNA methylation status of promoter DMRs and gene expression, while the DNA methylation status of gene body DMRs had positive correlation with gene expression.The results of both MSAP and WGBS revealed that multi-generation drought imposition induced widespread DNA methylation variation in two rice varieties, and the drought sensitive variety had more variations than the drought resistant variety. In both varieties, DNA methylation variation in certain positions were directly and specifically induced by drought stress, among which, some of them could transmit their changed DNA methylation status to offspring. Considerable proportions of genes that related to transgenerational epimutations directly participated in stress response pathways. These observations indicated that DNA methylation played an important role in plant adaption to drought stress.