DNA Methylation Studies On The Pacific Oyster Crassostrea Gigas

Epigenetic variation refers to phenotype variation in the absence of DNA sequence polymorphism. DNA methylation is the most well studied epigenetic modification which plays an important role in genome defense and transcription regulation. Previous studies usually focus on mammals and plant, little epigenetic information is known in aquatic animals. The Pacific oyster(Crassostrea gigas) is an economically important bivalve not only in China but also around the word. C. gigas is regarded as a model aquatic species considering its dramatic morpho-physiological changes and stressful habitat. Investigation of DNA methylation in C. gigas would contribute to the understanding of DNA methylation mechanisms and function in aquatic animals. This study aims to explore the inheritance and variation of DNA methylation in tissue differentiation, genome doubling, and artificial selection to provide fundamental understanding for future study.1. Genomic DNA methylation inheritance in C. gigasThe fluorescent labeled methylation-sensitive amplified polymorphism (F-MSAP) analysis was used to detect segregation ratio of DNA methylated/unmethylated loci in an oyster linage. The result shows that 90.6% methylated loci segregated following Mendelian expectation with approximately 10% methylated loci deviated significantly from Mendelian expectation. Methylated loci deviated from Mendelian expectation more significantly than unmethylated loci. Compared with parents,22 demethylation fragments,7 hypermethylation fragments and 30 de novo methylation loci was observed in offspring.2. DNA methylation variation in different tissues of C. gigasThe DNA methylaiton extent and patterns of CCGG sites in the genome of C. gigas was investigated among six different tissues using F-MSAP method. The DNA methylation level was higher in digestive glands, labial palp and adductor than the other three tissues with a significant difference between digestive glands and gill/mantle. Both tissue specific (bands methylated or unmethylated in only one of the six tissues) and tissue nonspecific differentially methylated regions (bands methylated or unmethylated in more than one of the six tissues) were found in C. gigas, but these two kinds of bands only occurred in one or few individuals. No tissue specific/nonspecific differentially methylated regions were found in all individuals analyzed and this might correlate with the high polymorphism in the Pacific oyster.3. Genomic DNA methylation variation in diploid and triploid C. gigasGenomic DNA methylation variation was compared in two tissues (adductor and gonad) between diploid and triploid oysters induced by cytochalasin B (CB,0.5 mgL-1) using F-MSAP. No significant difference was observed in total methylation level between diploid and triploid oysters with only a few ploidy-specific loci. However, in triploid offspring, both methylated and unmethylated loci deviated more significantly from Mendelian expectation than diploids, indicating more variation in triploid oyster. PCA analysis showed significant epigenetic structure between diploid and triploid both in adductor and gonad. These results implied that different methylation pattern exists at several loci but cannot influence total methylation level.4. Gene expression and DNA methylation variation in reproduction-related genes between diploid and triploid C. gigasReal time PCR was conducted in 8 reproduction-related genes to investigate the gene expression in late developing and ripe gonads in diploid and triploid oysters. Only putative Vg in late developing female gonads and caER in ripe female gonads displayed significant difference in gene expression between diploid and triploid oysters. The DNA methylation pattern of CpG island in promoter and coding sequence of these two genes were investigated by bisulfite sequencing. Putative Vg was not methylated in all six CpG islands while 2 CpG islands in caER were methylated. The CpG island in exon 6 of caER showed no difference in diploid and triploid while the other one in exon 10 displayed slight difference which might be related with gene expression regulation.5. DNA methylation variation in mass selection population of C. gigasAmplified fragment length polymorphism (AFLP) analysis and methylation-sensitive amplified polymorphism (MSAP) methodology were used to monitor genetic and epigenetic variation in two populations (the base stock and the third selected generation) from a mass selection line in C. gigas. No significant difference was observed on the average state of methylation, but a few bands showed different frequencies between the two populations. Co-Inertia Analysis revealed a significant association between the genetic and epigenetic profiles, but there were some individuals having similar genetic structure with distinct epigenetic structure. Both genetic and epigenetic diversity was a little reduced in the selected line.This study showed that most of methylated loci segregate following Mendelian expectation. DNA methylation varies slightly in different tissue, ploidy and mass selection progress. DNA methylation variation might be related with gene expression.