Genome-wide Analysis Of DNA Methylation Profile,DNA Methyltransferase And Demethylase Genes In Salvia Miltiorrhiza

Cytosine DNA methylation,a dominating epigenetic modification mechanism,is known to be involved in multiple biological processes,including gene imprinting,plant growth and development,stress response,and secondary metabolism.Due to the important research advancement on DNA methylation in model plants and crops,scientists are gradually realizing that medicinal plant quality could be regulated by DNA methylation.However little is known about the underlying mechanisms.Salvia.miltiorrhiza is emerging as a model plant for tranditional Chinese medicine?TCM?studies due to its specific medicinal active ingredients,short life cycle,simple micropropagation methods and efficient genetic transformation system.S.miltiorrhiza is an ideal material for medicinal plant genetics and epigenetics research.In order to explore the biological function of DNA methylation in S.miltiorrhiza,we analyzed DNA methylation variation between Root₃?roots collected in March?and Root₇?roots collected in July?,DNA methylation variation between Root₇ and Leaf₇?leaves collected in July?.We also identified and characterized cytosine-5 DNA methyltransferase?C5-MTase?genes and DEMETER-like DNA glycosylase?DML?genes.The purpose of this study was to lay a solid foundation for understanding the role of DNA methylation in various biological processes of medicinal plants,such as the growth and development,secondary metabolism and stress response,and to provide theoretical guidance for medicinal plant cultivation and molecular breeding in epigenetics.The main results and conclusions obtained from this study are as follows.1.We obtained single-base resolution methylomes of Root₃,Root₇ and Leaf₇ through whole genome bisulfite sequencing.Genome-wide DNA methylation level and pattern variance of each sequence context mainly existed in promoter regions of genes and repeat regions.The maximum of methylated cytosines were identified for CHH sequence context.Similarly,the majority of differential methylation sites?DMSs?and differential methylation regions?DMRs?were both from CHH sequence context.443245 CG,315015CHG and 1879317 CHH DMSs,and 12539 CG,10879 CHG and 18336 CHH DMRs were identified for two root tissues,were identified from two root tissues.293994 CG,394298 CHG and 3093880 CHH DMSs,13072 CG,13018 CHG and 34873 CHH DMRs were identified between Root₇ and Leaf₇.2 KEGG enrichment analysis of the DMR-related genes between Root₃ and Root₇ showed that the CG DMR-related genes and the CHH DMR-related genes were both significantly enriched in the terpene biosynthesis pathway.The content of tanshinones significantly increased in S.miltiorrhiza hair roots under the treatment of 5Aza-dC?a general DNA methylation inhibitor?.It suggests the involvement of DNA methylation in tanshinone biosynthesis.3.DNA methylation was established and maintained by cytosine-5 DNA methyltransferases?C5-MTases?in plants.Eight SmC5-MTase genes were identified using whole genome sequence and RNA-seq data from S.miltiorrhiza.Based on the phylogenetic tree and the distribution of conserved domains,SmC5-MTases were divided into four subfamilies,including SmMET,SmCMT,SmDRM and SmDNMT2.Comparative analysis of SmC5-MTases and Arabidopsis AtC5-MTases revealed the conservation and divergence of plant C5-MTases.Differential expression of SmC5-MTases were found in plant response to yeast extract,MeJA,and salicylic acid treatments.It implies functional importance of SmC5-MTases in secondary metabolism and stress response in S.miltiorrhiza.4.DEMETER-like DNA glycosylases?DMLs?initiate the base excision repair-dependent DNA demethylation to regulate a wide range of biological processes in plants.Six putative SmDML genes,termed SmDML1-SmDML6,were identified from the genome of S.miltiorrhiza.Integrated analysis of gene structures,sequence features,conserved domains and motifs,phylogenetic analysis and differential expression showed the conservation and divergence of SmDMLs.SmDML1,SmDML2 and SmDML4 were significantly down-regulated under the treatment of 5Aza-dC,suggesting the involvement of SmDMLs in genome DNA methylation change.SmDML1 was predicted and experimentally validated to be a target of Smi-miR7972.Computational analysis of forty whole genome sequences and almost all of RNA-seq data from Lamiids revealed that MIR7972s were only distributed in some species of three orders,including Lamiales,Solanales and Boraginales.The number of MIR7972 genes varied among species.It suggested that MIR7972 genes underwent expansion and loss during the evolution of some Lamiids species.These results provide a valuable resource for elucidating DNA demethylation mechanism in S.miltiorrhiza.5.Two CRISPR/Cas9 binary vectors,which targets SmMET1 and SmDML3,respectively,were constructed.Each of them carries two-gRNA expression cassettes.Mutation analysis revealed that eight SmMET1 mutants and four SmDML3 mutants were obtained.It provids a platform for functional analysis of SmMET1 and SmDML3,and indicates that the CRISPR/Cas9 genome editing system is a promising potential tool for gene function analysis and quality improvement of medicinal plants.