DNA sampling between the shells: evaluating non-lethal DNA sampling methods in oysters for high-throughput genotyping

With the development of cost-effective high-throughput shellfish genotyping tools, the application of genomic selection in oyster breeding is quickly becoming a reality. A critical, but laborious step in this process is the non-lethal DNA sampling of candidate broodstock for genotyping. Benignly sampling oysters is complex, requiring anesthesia to induce incomplete adductor muscle paralysis, followed by DNA sampling via biopsy, hemolymph or tissue extraction which can take significant time per individual and physically damage valuable breeding candidates. In this study genotype concordance of multiple non-lethal, and less injurious DNA sampling methods in Eastern ( Crassostrea virginica ) and Pacific ( C. gigas ), oysters were evaluated. Genotypes obtained via swabbing, eDNA collection, and mantle tissue clipping were compared to a reference adductor muscle-derived genotype, which was obtained lethally. Eastern oyster ( Crassostrea virginica ) samples were evaluated on a 66K Single Nucleotide Polymorphism (SNP) array and Pacific oyster ( Crassostrea gigas ) samples were evaluated using PCR-based genotyping-by-sequencing. Retention of glue-on tags and oyster survival were also evaluated for 27 days in Pacific oysters in the field, after non-lethal sampling. Overall SNP genotype concordance rates were high in both oyster species (> 99% in samples passing quality control) with DNA quality having little impact on genotype concordance rate. Using a microhaplotype sequence workflow in two Pacific oyster genotyping panels resulted in a higher non-concordance rate (approx. 4%), however the missing (non-called) allele was found in raw sequencing data in the vast majority of cases, suggesting that rudimentary genotype calling parameters are likely driving the higher error rate. This is not surprising given the nascent implementation of microhaplotypes in aquaculture research and ongoing panel development. Post-swab survival was high, and all animals retained physical tags for subsequent identification. Overall, non-lethal swab DNA collection provided high quality genotype data similar to mantle or adductor tissue-derived genotype data; swab sampling was also faster and easier to implement and may be less stressful. The use of non-lethal swabbing for genotyping will be an important step for implementing genomic selection and using advanced genetic tools in research supporting oyster restoration and production aquaculture.