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On March 23, 2023, Hainan University Sanya Nanfan Research Institute/Tropical Crops Ph. The roles of DNA methylation in tomato domestication and metabolic diversity" research paper, the study carried out whole-genome bisulfite sequencing (WGBS), transcriptome sequencing (RNA-seq ) and metabolomics analysis, analyzed the relationship between tomato population metabolic diversity and DNA methylation changes during breeding, constructed a multi-omics association network and improved the synthesis pathways of tomato polyphenols and other metabolites.
Title: Population analysis reveals the roles of DNA methylation in tomato domestication and metabolic diversity (population analysis reveals the role of DNA methylation in tomato domestication and metabolic diversity)
Time: 2023-03-23
Journal: Sci China Life Sci
Impact factor: IF 10.372
Technology platform: WGBS, RNA-seq, metabolomic analysis, etc.
Research summary:
DNA methylation is an important epigenetic mark, but its diversity and its role in tomato breeding at the population level remain largely unknown. In this study, whole-genome bisulfite sequencing (WGBS), RNA-seq, and metabolomics analyzes were performed on a population including wild varieties, landraces, and cultivars of tomato, and a total of 8375 differentially methylated regions were identified ( DMR), its methylation level gradually decreased during domestication and improvement, and the results showed that more than 20% of DMR overlapped with selective sweep. More than 80% of DMRs in tomato were not significantly associated with single-nucleotide polymorphisms (SNPs), while DMRs were strongly associated with adjacent SNPs. In addition, the study also performed metabolomic analysis on 339 metabolites from 364 different germplasms, and further conducted metabolic association studies based on SNP and DMR, and detected 971 and 711 large effect sites by SNP and DMR markers, respectively . A total of 13 candidate genes were identified and the polyphenol biosynthesis pathway was updated by combining multi-omics. Our results demonstrate that DNA methylation changes can complement SNP profiles of metabolite diversity. In conclusion, this study draws a comprehensive DNA methylation map for different germplasms through omics studies such as WGBS sequencing, and suggests that DNA methylation changes may be the genetic basis of plant metabolic diversity.
Project Design:
(1) Sample selection:
A total of 96 tomato samples of different varieties were included in wild varieties (S.pimpinellifolium), landrace varieties (S.lycopersicum var cerasiforme), and cultivated varieties (S.lycopersicum). At least 20 plants of each variety were collected, and the leaves were randomly collected 40 days after the seeds germinated and quickly placed in liquid nitrogen for DNA, RNA and metabolite extraction and subsequent sequencing analysis.
(2) Project design flow chart:
result graphics
(1) DNA methylation profiles of different tomato varieties
Figure 1: DNA methylation changes during domestication and improvement
- Whole Genome Bisulfite Sequencing (WGBS) data.
- Distribution of DMRs on tomato chromosomes and their relationship to other genomic characterizations. From ① to ⑤ are chromosomes, genes, TE, Dos DMR and Imp DMR of tomato.
- DMR during tomato domestication and improvement. The inner circle indicates the ratio of Hyper-DMR and Hypo-DMR, and the outer circle indicates the DMR corresponding to CG and CHG sites.
- Comparison of DMR lengths in different breeding processes and in different DMR backgrounds. (***P<0.001, Wilcoxon test)
- Comparison of DMR signals between populations during domestication and improvement. (***P<0.001, Wilcoxon test, NS means not significant)
(2) Overlap analysis of DMR and selective sweep to analyze phenotypic diversity
Figure 2: Overlap analysis of differentially methylated regions (DMRs) and selective sweeps.
- DMR ratios overlap with selective removal during domestication and improvement. Inside and out are DMR numbers, different backgrounds and statistics to varying degrees. DNS indicates non-overlapping DMR and selective clearance, and DIS indicates overlapping DMR and selective clearance areas.
- Upper and middle panels show methylation regions and levels around a DMR named cg01g78771878 in the PIM, CER, and BIG populations. The red highlight indicates the cg01g78771878 site. The bottom panel is SlMYB12 located in the selective sweep region, and the nucleotide diversity in BIG is much lower than that in CER or PIM.
- Association analysis of cg01g78771878 methylation level and SlMYB12 transcript level.
- LUC/REN expression ratios of three biological replicates. (Error bars represent SD. t-test *P<0.05.)
(3) meQTL analysis between DMRs and SNPs
Figure 3: Genetic basis of DMR.
- Overview of the genetic basis of DMR in different backgrounds. The red line represents Dos-DMR, and the blue line represents Imp-DMR.
- Distribution of significant SNPs for each DMR.
- DMR distribution for each SNP marker.
- LD levels of DMR methylation at different distances.
- Distribution of meQTL signals in the tomato genome.
F&G. KEGG enrichment analysis of pure DMR-related genes in CG and CHG environments.
(4) Constructing the association network between metabolome diversity and methylation changes
Figure 4: Integration and construction of multi-omic association networks.
A&B. Genomic distribution of mEWAS and mGWAS. Metabolites are classified into seven classes and marked with different colors and labels (AG).
C. Multi-omic network for association analysis between metabolites, SNPs, and DMRs. Dots indicate metabolites of similar color as in A and B. SNPs and DMRs are shown as small red and blue triangles, respectively.
(5) SNP and DMR Analysis of Metabolic Diversity in Tomato
Figure 5: Tomato polyphenol biosynthesis pathway drawn based on mGWAS and mEWAS.
- Known and candidate genes of the polyphenol biosynthetic pathway identified in this study. Genes in red, blue, and green represent genes associated only with mGWAS, mEWAS, or significantly associated with both methods, respectively.
- EWAS Manhattan plot of Kaempferol-3-O-glucoside. Dashed lines indicate thresholds.
- Unrooted phylogenetic tree of flavonoid glycosyltransferase genes, genes in red are candidate genes (bar: 0.1 amino acids per site).
- Association analysis of DMR (chg7g56698981) methylation level and kaempferol 3-O-glucoside metabolite level.
- Analysis of DNA methylation level, relative expression of UGT71AV3, and relative content of kaempferol 3-O-glucoside in leaves treated with mock and 5'-Aza (error bars represent SD. *P<0.05, **P<0.01,** *t-test P<0.001).
- In vitro activity chromatograms of UGT71AV3 and kaempferol as substrates.
Figure 6: Genetic variation and epigenetic variation synergistically promote flavonoid diversity.
- GWAS (upper panel) and EWAS (lower panel) Manhattan plot of luteolin 7-O-glucoside (luteolin 7-O-glucoside). Dashed lines indicate thresholds for GWAS and EWAS. GWAS and EWAS overlapping signals are highlighted by red shaded columns. Strongly associated SNPs (rs0757317315) and DMRs (cg07g57323266) are marked with red arrows.
- Pairwise r values (a measure of linkage disequilibrium) between polymorphic sites in UGT73L8.
- Box plot of luteolin O-glc content in low methylation level (LM) and high methylation level (HM) (***P<0.001, Student's t-test).
- Box plot of luteolin 7-O-glucoside content in LM and HM with different haplotypes
- Analysis of DNA methylation level, relative expression of UGT73L8 and relative content of luteolin 7-O-glucoside in leaves treated with mock and 5'-Aza (error bars indicate SD. *P<0.05, **P<0.01 , ***P<0.001 by t-test).
- In vitro activity chromatograms of UGT73L8 and luteolin as substrates.
About Yigen Whole Genome Bisulfite Sequencing (WGBS) Technology
Whole-genome bisulfite methylation sequencing (WGBS) can accurately detect the methylation level of all single cytosine bases (C bases) in the whole genome, and is the gold standard for DNA methylation research. WGBS can provide important technical support for the study of temporal and spatial specific modification of genomic DNA methylation, and can be widely used in the study of the mechanism of life processes such as individual development, aging and disease, and is also the preferred method for the study of methylation maps of various species.
The genome-wide methylation sequencing technology provided by Yigene uses T4-DNA ligase to break the junction sequence at both ends of the genomic DNA fragment by ultrasonic waves, and the ligation product is treated with bisulfite to convert the unmethylated cytosine C Uracil U is transformed into uracil U, and then uracil U is transformed into thymine T through the linker sequence-mediated PCR technique.
Application direction:
WGBS is widely used in various species and requires a whole genome scan (does not miss key loci)
- Genome-wide methylation mapping project
- Marker Screening Topics
- Small-Scale Research Topics
Technical advantages:
- Wide range of applications: applicable to methylation studies of all species with known reference genomes;
- Whole-genome coverage: Maximize the acquisition of complete genome-wide methylation information and accurately draw methylation maps;
- Single base resolution: It can accurately analyze the methylation status of each C base.
Yigene Technology provides a comprehensive overall solution for DNA methylation research.
Crossref: Guo H, Cao P, Wang C, Lai J, Deng Y, Li C, Hao Y, Wu Z, Chen R, Qiang Q, Fernie AR, Yang J, Wang S. Population analysis reveals the roles of DNA methylation in tomato domestication and metabolic diversity. Sci China Life Sci. 2023 Mar
Related Reading:
Technology Introduction | Whole Genome Bisulfite Methylation Sequencing (WGBS)