DNA methylation mediates differentiation in thermal responses of Pacific oyster (Crassostrea gigas) derived from different tidal levels

Abstract

Epigenetic mechanisms such as DNA methylation have the potential to affect organism acclimatization and adaptation to environmental changes by influencing their phenotypic plasticity; however, little is known about the role of methylation in the adaptive phenotypic divergence of marine invertebrates. Therefore, in this study, a typical intertidal species, the Pacific oyster (Crassostrea gigas), was selected to investigate the epigenetic mechanism of phenotypic plasticity in marine invertebrates. Intertidal and subtidal oysters subjected to one-generation common garden experiments and exhibited phenotypic divergence were used. The methylation landscape of both groups of oysters was investigated under temperate and high temperature. The two tidal oysters exhibited divergent methylation patterns, regardless of the temperature, which was mainly original environment-induced. Intertidal samples exhibited significant hypomethylation and more plasticity of methylation in response to heat shock, while subtidal samples showed hypermethylation and less plasticity. Combined with RNA-seq data, a positive relationship between methylation and expression in gene bodies was detected on a genome-wide scale. In addition, approximately 11% and 7% of differentially expressed genes showed significant methylation variation under high temperatures in intertidal and subtidal samples, respectively. Genes related to apoptosis and organism development may be regulated by methylation in response to high temperature in intertidal oysters, whereas oxidation-reduction and ion homeostasis-related genes were involved in subtidal oysters. The results also suggest that DNA methylation mediates phenotypic divergence in oysters adapting to different environments. This study provides new insight into the epigenetic mechanisms underlying phenotypic plasticity in adaptation to rapid climate change in marine organisms.

Fig. 1. Global methylation landscape.

a Global methylation levels of CpGs of each group. Significant differences calculated from the three biological replicates are indicated by *P value < 0.05 and **P value < 0.01. b Clustering dendrogram of the 12 samples. The red frame denotes the clustered intertidal samples. c Principal component analysis of the 12 samples; the red and blue circles denote the clustered intertidal and subtidal samples. d Variation in DNA methylation plasticity between intertidal and subtidal oysters. The mean methylation changes from control to heat shock in intertidal/subtidal oysters was indicated by the red/blue arrows.

Fig. 2. Assessment of genetic effect on DNA methylation.

a Pairwise correlation analysis and clustering of SNP data from all 12 samples. b Scatterplot of F_ST values and methylation-level difference in genes of InC and SubC oysters.

Fig. 3. Relationship between DNA methylation and gene expression.

a Correlation analysis of methylation level and expression in genes. b Distributions of methylation levels partitioned by different expression levels. Genes with FPKM value < 0.1 were considered nonexpressed.

Fig. 4. Analysis of different genes in two tidal oysters.

a Venn plot of overlapped genes of DEG and DMG in InC vs. SubC. b Heatmap of methylation level and FPKM in overlapped genes in InC and SubC. c Enriched GO terms of overlapped genes in biological process.

Fig. 5. Comparative methylome analysis under heat shock.

a Circos plots of genome scaffolds. The three circles from outer to inner represented the methylation levels of the heat-stress-treated groups (InH and SubH), the differences between heat-shock and control groups (InH vs. InC and SubH vs. SubC), and the control groups (InC and SubC), respectively. The level of methylation is denoted by the darkness of the color, and the degree of the difference is represented by the heatmap. b Distributions of DNA methylation levels among different genomic regions. c Heatmap of methylation levels within different methylation regions. Darker red indicates greater methylation in an individual for that DMR, whereas darker blue indicates lesser methylation in an individual for that DMR.

Fig. 6. Analysis of gene response to heat shock.

a Numbers of hyper- and hypo-DMGs of InH vs. InC and SubH vs. SubC. Unique DMGs represent all DMGs in InH vs. InC, or SubH vs. SubC subtract the common genes between InH vs. InC and SubH vs. SubC. b Venn plot of the common genes of DMGs and DEGs in InH vs. InC and SubH vs. SubC. Functional categories showed the specific genes enriched in InH vs. InC and SubH vs. SubC. c A simple sketch of apoptosis. d ML and FPKM variations of two genes involved in apoptosis.