Establishment Of An Efficient And Cost-effective Method For Genome-wide DNA Methylation Profiling (MethyIRAD-Seq) And Its Application In Marine Bivalves

As an important epigenetic modification factor in eukaryotic genomes, DNAmethylation plays a vital role in many biological processes including maintenance ofnormal cellular function, regulation of imprinted genes, embryonic development andcancers in higher organisms. In invertebrates, DNA methylation also plays a role inregulation of transcription and involved in epigenetic regulation in mechanisms ofadapting to environmental stressors. So far, there has limited work on bivalvesepigenetics. Investigation of DNA methylation in bivalves would contribute to theunderstanding of DNA methylation mechanisms and function in invertebrate andprovide basic epigenetic data for the breeding of bivalves.1、MSAP analysis of genomic DNA methylation level in three species of scallopsIn order to investigate the genome-wide DNA methylation level in bivalves,methylation-sensitive amplified polymorphism (MSAP) technique was applied toanalyze the methylation pattern at CCGG sites in three speices of scallops (Chlamysfarreri，Argopecten irradians，Mizuhopecten yessoensis) and “Haida golden scallop”,a new variety of M. yessoensis recently developed by our group. The results providedsuitable MSAP reaction system for further epigenetic studies on bivalves DNAmethylation. The methylation levels of C. farreri, A. irradians, M. yesoensis and“Haida golden scallop” were32.08%,25.99%,32.88%,34.97%, respectively. Thefully-methylated sites were more than the hemi-methylated sites in these scallopspecies and our data indicated that the major form of DNA methylation in scallop wasCpG methylation.2、Establishment of MethylRAD-Seq method for genome-wide DNA methylationprofilingSince the current high-throughput DNA methylation analysis method has limitation in the application to non-model organimsms, we established a cost-effectivemethod—MethylRAD-Seq (Methylation-dependent restriction-site associated DNAsequencing) for profiling genome-wide DNA methylation which combined thefeature of methylation dependent restriction enzymes and2b-RAD method with noneed for genome sequence. We provided methodological verification forMethylRAD-Seq in Arabidopsis and the results showed that MethylRAD-Seqtechonolgy could accomplish rapid and high-thoughput detection of methylated siteson a genome scale with false positive rate was about0.5%.It could also be used toreflect the whole genome-wide methylation distribution.3、Application of MethylRAD-Seq for investigation of carotenoid accumulationmechanisms in “Haida golden scallop”In this study, MethylRAD-Seq was applied to compare the genome-wide DNAmethylation profiles between “Haida golden scallop” and Mizuhopecten yessoensis..Approximately9.3G data quantities were obtained from twenty-four adult individualsof “Haida golden scallop” and Mizuhopecten yessoensis. The highly technicalreproducibility of the parallel library confirmed that MethylRAD-Seq provided a fast,stable and effective method for genomic DNA methylation study in non-modelbivalves. We achieved234,241methylated sites on a genome scales and236siteswhich were different in methylation level,and this sites may be involved in theregulation of the carotenoid accumulation. GO classification and KEGG pathwayanalysis for the7591methylated genes were found among “Haida golden scallop” andYesso scallop and these genes were involved in various biological functions andmetabolic pathways, such as metabolism, growth, reproduction and immune-relatedpathways. In summary, MethylRAD-Seq is an encouraging technology for DNAmethylation investigation and the variation analysis of the DNA methylation inbivalves will facilitate the understanding of molecular mechanism of importantgrowth traits such as carotenoid accumulation in “Haida golden scallop”.