Dissection The Epigenetic Variation And Effects In Germplasm Population Of Populus Tomentosa

DNA methylation,as the stable epigenetic marker in plant genome,is very important for tree growth and development,and it is also impacting the phenotypic variation.Until now,DNA methylation is widely considered as an inhibitory epigenetic marker,but little is known about its detailed functional mechanism,especially the epigenetic basis for phenotypic variation and plasticity of forest trees.Therefore,we dissected the genome-wide single base resolution of DNA methylation variation in germplasm population of Populus tomentosa.Then,integration of whole genome bisulfite sequencing,genome re-sequencing,RNA sequencing and metabolome profiling,we firstly used the epigenome wide association study（EWAS）in perennial tree species,dissected the epigenetic effects of DNA methylation contributed to metabolic traits,and identified the candidate epiallele for traits.These results systematically illustrated the putative function of DNA methylation,revealed the epigenetic basis of local adaption of P.tomentosa.The main results and conclusions in this study are as follows:1.We performed the whole genome bisulfite sequencing for germplasm population of P.tomentosa,and obtained 10.35 T raw data.Then,we analyzed the methylation level of cytosine in the whole genome,and identified 83,354,324 single methylation polymorphism（SMPs）in the germplasm population of P.tomentosa,which indicated that about 69.52%of cytosine had DNA methylation modification variation,and revealed that DNA methylation was a widely distributed epigenetic marker in the genome.2.Integration of principal component analysis（PCA）,linkage disequilibrium（LD）decline analysis and population differentiation coefficient(F_ST)analysis showed that SMPs were strongly selected by climatic environment,and the DNA methylation pattern of P tomentosa showed local adaptability to geographical environment.We identified a total of 7,680 population-specific differentially methylation regions（DMRs）covering 1.01%（4.10 MB）of the genome,which were significantly enriched in the selective sweeps determined by genetic variation,indicating that natural selection acted on specific genomic regions to alter the methylation modification.Next,we integrated GWAS and co-expression network analysis that identified the natural variation of Pto MS1 affected the genomic methylation level,and the genetic adaptability of this loci also caused the difference of the methylation level of in different climatic population.3.A total of 684 metabolites were detected in germplasm population of P.tomentosa by metabolome profiling,which showed abundant phenotypic variation within the population.In total,the content of 381 metabolites were significantly different among the three climatic populations,indicating that the metabolites in germplasm population of P.tomentosa were selected by the geographical climate.Then,we used SMPs and metabolite traits to conduct EWAS in the germplasm population of P.tomentosa.Under the threshold of P=1.26×10^-10（0.01/n）,we detected 472 significant SMP-trait associations,representing 324 SMPs and 143 metabolic traits,which indicated that EWAS could enlarge the regulatory network of metabolic pathway from the layer of epigenetic,enrich the function of metabolites and gene annotation,and provide a strategy to explore the function of DNA methylation.Next,mining the results of EWAS and population-specific DMRs,we identified 93 candidate genes located in the DMRs,and most of these genes also showed differential expression patterns in the three climatic populations,indicating that geographical climate affected gene expression by shaping the differential methylation modification in the genome,thus affecting the differential accumulation of metabolites.These results enhance the understanding of local adaptation of P.tomentosa.4.Salicylic acid（SA）is a vital hormone for adaptive responses to biotic and abiotic stresses,which facilitating growth-immunity trade-offs in plants.We first conducted EWAS for nine metabolites in SA biosynthesis pathway,and identified eight SMPs significantly associated with the metabolic contents.We found that the natural selection shaped the epigenetic variation of the promoter of Pto NAC072,and decreased the expression of Pto NAC072 through inhibited the binding of transcription factor SRM1,which caused the high accumulation of SA content.Meanwhile,we also performed GWAS to dissect the genetic basis of SA biosynthesis,and identified 27 SNPs responsible for the variation of metabolites.We focused on Pto WRKY70,an efficient transcription factor,as the key causal gene in the regulatory network,and combined the novel genes coordinating the accumulation of SA.Finally,there are no overlapping genes in the results of EWAS and GWAS,indicating that the epigenetic regulation and genetic regulation are complementary.The causative epiallele and allele improved the understanding of regulatory network of SA biosynthesis from two independent layer,which provide a new idea for dissection the regulatory network of metabolic biosynthesis.This study uncovered the natural variation of DNA methylation and dissected the epigenetic effects of DNA methylation on important traits in the forest tree populations,and provided the theoretical and applied values for protection of germplasm resources and forest molecular breeding.