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. 2024 Apr 5;25(1):87.
doi: 10.1186/s13059-024-03220-y.

Increased DNA methylation contributes to the early ripening of pear fruits during domestication and improvement

Bobo Song #  1   2 , Jinshan Yu #  1   2 , Xiaolong Li #  3 , Jiaming Li #  1   2 , Jing Fan #  4 , Hainan Liu  5 , Weilin Wei  1   2 , Lingchao Zhang  1   2 , Kaidi Gu  6 , Dongliang Liu  1   2 , Kejiao Zhao  1   2 , Jun Wu  7   8
Affiliations

Increased DNA methylation contributes to the early ripening of pear fruits during domestication and improvement

Bobo Song et al. Genome Biol. .

Abstract

Background: DNA methylation is an essential epigenetic modification. However, its contribution to trait changes and diversity in the domestication of perennial fruit trees remains unknown.

Results: Here, we investigate the variation in DNA methylation during pear domestication and improvement using whole-genome bisulfite sequencing in 41 pear accessions. Contrary to the significant decrease during rice domestication, we detect a global increase in DNA methylation during pear domestication and improvement. We find this specific increase in pear is significantly correlated with the downregulation of Demeter-like1 (DML1, encoding DNA demethylase) due to human selection. We identify a total of 5591 differentially methylated regions (DMRs). Methylation in the CG and CHG contexts undergoes co-evolution during pear domestication and improvement. DMRs have higher genetic diversity than selection sweep regions, especially in the introns. Approximately 97% of DMRs are not associated with any SNPs, and these DMRs are associated with starch and sucrose metabolism and phenylpropanoid biosynthesis. We also perform correlation analysis between DNA methylation and gene expression. We find genes close to the hypermethylated DMRs that are significantly associated with fruit ripening. We further verify the function of a hyper-DMR-associated gene, CAMTA2, and demonstrate that overexpression of CAMTA2 in tomato and pear callus inhibits fruit ripening.

Conclusions: Our study describes a specific pattern of DNA methylation in the domestication and improvement of a perennial pear tree and suggests that increased DNA methylation plays an essential role in the early ripening of pear fruits.

Keywords: DNA methylation; Domestication and improvement; Early ripening; Pear.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
The distribution patterns of DNA cytosine methylation levels in the pear genome. a The density distribution of DNA methylation levels in three contexts (CG, CHG, and CHH), gene density, and TE density on the 17 pear chromosomes. b The average rates of methylated cytosines (mC) in the three contexts in the pear genome for the three pear populations (wild, landrace, and improved). c Comparisons of methylation levels of the three contexts in the wild, landrace, and improved populations (*P < 0.05; **P < 0.01; ***P < 0.001, two-tailed paired Student’s t-test). d Distribution of DNA methylation levels across the upstream 2 kb, gene body, and downstream 2-kb regions of genes and TEs. e Comparisons of relative gene expression levels (FPKM; fragments per kilobase of transcript per million mapped reads) of PpyDML1.1, PpyDML1.2, and PpyDML1.3 in the wild, landrace, and improved pear populations. PpyDML1.1, PpyDML1.2, and PpyDML1.3 showed continuous decreases in expression during pear domestication and improvement (*P < 0.05; **P < 0.01; ***P < 0.001, differentially expressed analysis using cuffdiff)
Fig. 2
Fig. 2
Comparisons of differentially methylated regions (DMRs) in the wild vs. landrace and landrace vs. improved populations. Principal component analysis (PCA) plots of DNA methylation levels in the CG (a) and CHG (b) contexts. The yellow squares represent the wild pear accessions, the blue points represent the landrace pear accessions, and the green triangles represent the improved pear accessions. c The number of hyper/hypo-DMRs in the wild vs. landrace, landrace vs. improved, and wild vs. improved comparisons. d The total lengths of hyper/hypo-DMRs in the wild vs. landrace, landrace vs. improved, and wild vs. improved comparisons. e Comparisons of the lengths of DNA sequence regions under selection (DSRs) and the CG and CHG context DMRs in the wild vs. landrace and landrace vs. improved comparisons (*P < 0.05; **P < 0.01; ***P < 0.001, two-tailed paired Student’s t-test). f Genomic compositions of the DSRs and DMRs, including TEs, introns, exons, and intergenic regions. g Distribution of the DMRs between the wild and landrace populations for the 17 pear chromosomes. Proceeding from the outer ring to the inner ring, the data represents TE density (I), gene density (II), dom-CG-DMR density (III), dom-CHG-DMR density (IV), imp-CG-DMR density (V), imp-CHG-DMR density (VI), dom-DSR (VII), and imp-DSR (VIII). h Overlap of DMRs in the wild vs. landrace and landrace vs. improved comparisons for the CG and CHG contexts. i Overlap of DMRs in the 2 methylation contexts in the wild vs. landrace (domestication process) and landrace vs. improved (improvement process) comparisons. Correlation analysis between methylation levels of the CG and CHG contexts in o_CG_CHG_DMRs during pear domestication (j) and improvement (k)
Fig. 3
Fig. 3
Genetic diversity changes in the DMRs. Comparison of genetic diversity between DMRs, DSRs, and NSRs in different genomic compositions for all pears (a) and the wild (b), landrace (c), and improved (d) populations (*P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant; two-tailed paired Student’s t-test). The different genomic compositions include intergenic regions, TEs, exons, and introns. e Genetic diversity changes in the hyper DMRs and hypo DMRs during the domestication (Dom-DMRs) (f) and improvement processes (Imp-DMRs). Each black line represents one DMR. g The relationships between DNA methylation levels and genetic diversity in the dom-CG-DMRs, dom-CHG-DMRs, imp-CG-DMRs, and imp-CHG-DMRs
Fig. 4
Fig. 4
The genetic basis of the DMRs. a The chromosomal location distributions of the meQTLs identified for DMRs during pear domestication and improvement. The x-axis represents the genomic positions of significant SNPs, and the y-axis represents the genomic positions of the corresponding DMRs of the SNPs. The colors of the dots represent the P-value in the meQTL analysis. The meQTL significant threshold was set as 1.78 × 10−9 (0.01/N, N=5,618,948), and only significant meQTLs were plotted. Dom-CG-DMR represents the CG DMRs between wild and landrace accessions; Dom-CHG-DMR represents the CHG DMRs between the wild and landrace accessions; Imp-CG-DMR represents the CG DMRs between the landrace and improved pear accessions; Imp-CHG-DMR represents the CHG DMRs between the landrace and improved pear accessions. b Distribution of the number of significant SNPs per DMR. c Distribution of the number of significantly associated DMRs for each SNP. d Summary of the genetic basis for the CG and CHG DMRs during pear domestication and improvement
Fig. 5
Fig. 5
Correlation between DNA methylation and gene expression levels during pear domestication and improvement. a Relationships between CG and CHG methylation levels and expression levels for all genes in the 2-kb upstream, gene body, and 2-kb downstream regions. The genes were divided into four groups (low, mid-low, mid-high, and high) based on expression level. b, c Distribution of Pearson correlation coefficients between gene expression levels in the 2-kb upstream, gene body, and 2-kb downstream regions and methylation levels of the DMRs in the CG and CHG contexts during pear domestication (b) and improvement (c)
Fig. 6
Fig. 6
A hypermethylated DMR in the pear CAMTA2 gene. a GO enrichment analysis (top 15 significant terms) of hyper-CG-DMR-associated genes during pear domestication. b GO enrichment analysis (top 15 significant terms) of hyper-CG-DMR-associated genes during pear improvement. The blue stars represent the GO terms associated with senescence. c The CAMTA2 gene structure is shown at the top of the figure. Exons are represented by yellow-shaded boxes, introns are represented by black lines, and blue-shaded boxes represent the 5′ and 3′ UTRs. The bottom figure shows the CG methylation level of a DMR (Chr13:26,073,542–26,073,668) located in the CAMTA2 gene in the wild, landrace, and improved pear populations. The entire gene (2-kb upstream, gene body, and 2-kb downstream regions) is shown in the upper panel, and the DMR in exon 11 is shown enlarged below. d Comparison of the expression levels (FPKM) of CAMTA2 in the wild (yellow box), landrace (green box), and improved (blue box) pear populations (*P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant; two-tailed paired Student’s t-test)
Fig. 7
Fig. 7
The function of CAMTA2 in transgenic pear callus and transgenic tomato plants. a Relative expression of the CAMTA2 gene in control and 5′-azacytidine (5′-Aza)-treated pear callus. b The CAMTA2-GFP fusion protein is localized to the nucleus of agroinfiltrated Nicotiana benthamiana leaf cells. c Growth of WT and CAMTA2-overexpressing (OE) pear callus. P1 = immediately after subculture, P2 = 14 days after subculture, and P3 = 24 days after subculture. d, e Cross-sections of pear callus stained with Toluidine Blue. The images show the number of cells in the same visual field of cross-sections of WT (d) and transgenic pear callus overexpressing CAMTA2 (e). Scale bars = 100 μm. f Growth status of T1-generation transgenic seedlings before transplanting. Scale bar = 1 cm. g Statistical analysis of root lengths of WT and T1-generation CAMTA2-OE seedlings. h Phenotypes of WT and CAMTA2-OE transgenic tomato plants. Scale bars = 1 cm. i Statistical analysis of plant height in the WT and transgenic tomato plants. j Representative phenotypes of WT CAMTA2-OE transgenic tomato fruits at 43, 46, 49, 52, 55, and 57 days after full bloom (DAFB). k Statistical analysis of fruit firmness in the WT and CAMTA2-OE transgenic tomato fruits harvested at the red stage (*P < 0.05; **P < 0.01; ***P < 0.001, two-tailed paired Student’s t-test)
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