Integrative Methylome and Transcriptome Analysis Reveals Epigenetic Regulation of Pigmentation in Oujiang Color Common Carp

Abstract

Oujiang color common carp display four striking varieties of pigmentation, but their epigenetic basis is unclear. We integrated genome-wide DNA methylation (MBD-seq) and transcriptomes (RNA-seq) from dorsal skin of four Oujiang color common carp varieties with three biological replicates. Black-spotted groups (RB, WB) showed approximately 6% higher global methylation than non-black-spotted groups (WR, WW), with differential methylation enriched in introns (>23%) and intergenic regions (>47%). Integrative analyses revealed a strong inverse association between promoter methylation and gene expression; 96 pigmentation-related genes were identified, spotlighting genes such as ASIP and frmA as key epigenetically silenced regulators in black-spotted carp. RT-qPCR confirmed directional concordance with RNA-seq for ASIP, frmA, DGAT2, SCARB1, and FOSB. Pathway enrichment implicated melanogenesis metabolism, tyrosine metabolism, lipid metabolism, and fatty acid metabolism, suggesting an interplay between pigment deposition and metabolic regulation. Collectively, the findings present an exploratory view of epigenetic control of coloration and underscore promoter methylation as a core layer influencing color diversity in Oujiang color common carp.

Keywords: MBD-seq; epigenetic regulation; methylation; pigmentation; spot formation.

Figure 1

Genome-wide DNA methylation profiling across four pigmentation morphs. (a) The bar plot shows genome-wide methylation coverage. (b) The distribution of MBD-seq reads in the region around the gene body. (c) Bar plots display predicted methylation peaks across pigmentation morphs. (d) The distribution of methylation peaks across different genomic features. Asterisks indicate statistical significance between groups (* p < 0.05; one-way ANOVA followed by Tukey’s HSD). Letters with different a and b indicate significant difference between groups at p < 0.05 (one-way ANOVA followed by Tukey’s HSD; multiple-comparison p values adjusted as specified).

Figure 2

Differential methylation analysis. (a) Chromosomal distribution of DMRs identified in four pairwise comparisons. (b) DMRs, hypermethylated and hypomethylated in four pigmentation variants. The enrichments shown in the heatmap were calculated within a ±5 kb window around each DMR. (c) The distribution of DMRs across CPG and genomic regions. (d) Bar plots showing the proportion of hypermethylated regions across genomic features.

Figure 3

Differential gene expression analysis between groups with different colors. (a) The volcano plot shows the differentially expressed genes screened out through transcriptome analysis, and some genes related to body color are listed on the right side. (b) Venn diagram showing the overlap of DEGs among the four pairwise comparison groups. (c) Enriched pathways of DEGs in each of the four comparisons. (d) Heatmap showing the expression patterns of pigmentation-related genes across the four varieties.

Figure 4

The relative expression levels of 5 DEGs were verified by RT-qPCR compared with RNA-seq, respectively. With β-actin as an internal reference gene. gray bars, qRT-PCR; black line, RNA-seq.

Figure 5

Integrated transcriptome methylome analysis. (a) The venn diagram of the numbers of overlapping DEGs-DMGs in the four group comparisons. (b) DMR overlapped ratio in the four groups of comparisons. (c) The correlation of methylation level with expression in different varieties. (d) Pigmentation terms associated genes epigenetically downregulated via methylation. Asterisks indicate statistical significance between groups (**: p < 0.01; one-way ANOVA followed by Tukey’s HSD).

Figure 6

Candidate gene methylation and expression associated with body color. (a) Methylation peak profile of ASIP promoter across four varieties, with boxplot showing gene expression. (b) Methylation peak profile of the frmA promoter and boxplots showing gene expression in four varieties. Asterisks indicate statistical significance between groups (*: p < 0.05; **: p < 0.01; one-way ANOVA followed by Tukey’s HSD).