National Shellfisheries Association Annual Meeting

1. DNA Methylation Patterns & Epigenetic Regulation in the Pacific Oyster Mackenzie Gavery & Steven Roberts University of Washington School of Aquatic and Fishery Sciences

3. Results: characterization of DNA methylation in Pacific oysters

4. Implications

6. Background: color disease resistance growth TRAITS pathogens toxins nutrition EPIGENOME (DNA methylation) ENVIRONMENT GENES (DNA)

7. Background: color disease resistance growth TRAITS pathogens toxins nutrition EPIGENOME (DNA methylation) ENVIRONMENT GENES (DNA)

9. regulates gene expression

10. influenced by the environment Me C G G C

11. DNA methylation Effects of disruptions: tumor promotion alteration of development inhibition of reproduction Compounds that impact normal epigenetic functions: Endocrine disruptors: estrogen, BPA, pesticides

12. Characterization of DNA methylation in Pacific oysters: describe distribution of methylation elucidate functional significance

13. Results Methylation Specific PCR Bisulfite sequencing In silicoanalysis

14. Results Methylation Sensitive PCR Bisulfite sequencing In silicoanalysis

15. Results: gene-targeted approach Methylation Sensitive PCR 5 stress related genes were examined Identified CpG methylation in heat shock protein 70 Bisulfite sequencing 136 bp fragment: 1 of 7 CpG methylated (homology to neuromedin-u receptor) 93 bp fragment: 1 of 2 CpG methylated (homology to bromodomain adjacent to zinc finger domain)

16. Results Methylation Sensitive PCR Bisulfite sequencing In silicoanalysis

17. Results Methylation Sensitive PCR Bisulfite sequencing In silicoanalysis predicted methylation status of 12,000 C. gigas sequences from GigasBase sequences were grouped by Gene Ontology term an average predicted methylation status was determined

18. Regulation of Gene Expression low mid high Predicted DNA Methylation

19. Regulation of Gene Expression low mid high Predicted DNA Methylation

20. Implications: evidence suggests DNA methylation plays a regulatory role in Pacific oysters implications for immune/stress responses

21. Implications: Environment LowHigh

22. Implications: Selective Breeding Selective breeding can contribute to improved & predictable performance in oysters Understanding geneticand epigenetic influences will increase predictability

23. Implications: Hybrid Vigor Heterosis (hybrid vigor) mechanism not fully understood epigenetic mechanisms have been proposed better understanding will allow for greater control in predicting and manipulating gene expression in oysters X =

25. Future Directions Method evaluation/development: challenges associated with non-model species new approaches: Whole genome bisulfite sequencing (BS-seq) Methylated DNA immunoprecipitation (MeDIP) MeDIP-seq MeDIP-chip

26. Summary Characterization of DNA methylation in Pacific oyster suggests a role in gene regulation, specifically genes with inducible expression DNA methylation could be an important mechanism contributing to phenotypic variation in oysters Important evaluate & develop methods and tools to evaluate epigenetic mechanisms in bivalves

27. Acknowledgements UW, SAFS Dr. Steven Roberts Samuel White Lisa Crosson Emma Timmins-Schiffman Taylor Shellfish Farms Joth Davis NSA-PCS NOAA Aquaculture Program