BackgroundDNA methylation is an epigenetic mechanism with important regulatory functions in animals. While the mechanism itself is evolutionarily ancient, the distribution and function of DNA methylation is diverse both within and among phylogenetic groups. Although DNA methylation has been well studied in mammals, there are limited data on invertebrates, particularly molluscs. Here we characterize the distribution and investigate potential functions of DNA methylation in the Pacific oyster (Crassostrea gigas).ResultsMethylation sensitive PCR and bisulfite sequencing PCR approaches were used to identify CpG methylation in C. gigas genes and demonstrated that this species possesses intragenic methylation. In silico analysis of CpGo/e ratios in publicly available sequence data suggests that DNA methylation is a common feature of the C. gigas genome, and that specific functional categories of genes have significantly different levels of methylation.ConclusionsThe Pacific oyster genome displays intragenic DNA methylation and contains genes necessary for DNA methylation in animals. Results of this investigation suggest that DNA methylation has regulatory functions in Crassostrea gigas, particularly in gene families that have inducible expression, including those involved in stress and environmental responses.

There is a significant amount of variation in DNA methylation characteristics across organisms. Likewise, the biological role of DNA methylation varies across taxonomic lineages. The complexity of DNA methylation patterns in invertebrates has only recently begun to be characterized in-depth. In some invertebrate species that have been examined to date, methylated DNA is found primarily within coding regions and patterning is closely associated with gene function. Here we provide a perspective on the potential role of DNA methylation in these invertebrates with a focus on how limited methylation may contribute to increased phenotypic plasticity in highly fluctuating environments. Specifically, limited methylation could facilitate a variety of transcriptional opportunities including access to alternative transcription start sites, increasing sequence mutations, exon skipping, and transient methylation.

Characterization of DNA methylation patterns in the Pacific oyster, Crassostrea gigas, indicates that this epigenetic mechanism plays an important functional role in gene regulation and may be involved in the regulation of developmental processes and environmental responses. However, previous studies have been limited to in silico analyses or characterization of DNA methylation at the single gene level. Here, we have employed a genome-wide approach to gain insight into how DNA methylation supports the regulation of the genome in C. gigas. Using a combination of methylation enrichment and high-throughput bisulfite sequencing, we have been able to map methylation at over 2.5 million individual CpG loci. This is the first high-resolution methylome generated for a molluscan species. Results indicate that methylation varies spatially across the genome with a majority of the methylated sites mapping to intra genic regions. The bisulfite sequencing data was combined with RNA-seq data to examine genome-wide relationships between gene body methylation and gene expression, where it was shown that methylated genes are associated with high transcript abundance and low variation in expression between tissue types. The combined data suggest DNA methylation plays a complex role in regulating genome activity in bivalves.

While the goal of most conservation hatchery programs is to produce fish that are genetically and phenotypically indistinguishable from the wild stocks they aim to restore, there is considerable evidence that salmon and steelhead reared in hatcheries differ from wild fish in phenotypic traits related to fitness. Some evidence suggests that these phenotypic differences have a genetic basis (e.g., domestication selection) but another likely mechanism that remains largely unexplored is that differences between hatchery and wild populations arise as a result of environmentally-induced heritable epigenetic change. As a first step toward understanding the potential contribution of these two possible mechanisms, we describe genetic and epigenetic variation in hatchery and natural-origin adult steelhead, Oncorhynchus mykiss, from the Methow River, WA. Our main objectives were to determine if hatchery and natural-origin fish could be distinguished genetically and whether differences in epigenetic programming (DNA methylation) in somatic and germ cells could be detected between the two groups. Genetic analysis of 72 fish using 936 SNPs generated by Restriction Site Associated DNA Sequencing (RAD-Seq) did not reveal differentiation between hatchery and natural-origin fish at a population level. We performed Reduced Representation Bisulfite Sequencing (RRBS) on a subset of 10 hatchery and 10 natural-origin fish and report the first genome-wide characterization of somatic (red blood cells (RBCs)) and germ line (sperm) derived DNA methylomes in a salmonid, from which we identified considerable tissue-specific methylation. We identified 85 differentially methylated regions (DMRs) in RBCs and 108 DMRs in sperm of steelhead reared for their first year in a hatchery environment compared to those reared in the wild. This work provides support that epigenetic mechanisms may serve as a link between hatchery rearing and adult phenotype in steelhead; furthermore, DMRs identified in germ cells (sperm) highlight the potential for these changes to be passed on to future generations.