Comparing Minimally Lethal Sampling Methods for Genomics in the Eastern Oyster (Crassostrea virginica).

Understanding how selection acts on individual genotypes often requires genotyping at different life stages and tracking their survival under experimental conditions. However, in mollusks, such as the Eastern oyster (Crassostrea virginica), collecting sufficient high-quality DNA without causing mortality remains challenging. While prior studies have explored different noninvasive genotyping methods, an optimal method hasn't been identified. Here, we tested different techniques by combining two minimally invasive accession techniques (relaxation, shell notching), four types of cell sampling (swab, mantle biopsy, hemolymph, and extrapallial fluid), and three preservation techniques (flash freezing, ethanol, and FTA card) using nine different treatment groups. We monitored mortality for 11 days after cell sampling and quantified the effects of each treatment on mortality rates, DNA quality, and DNA quantity. Our results show that mantle biopsies from relaxed oysters preserved in ethanol yielded DNA quantities comparable to the control groups and significantly higher than either of the liquid samples, while maintaining low post-sampling mortality. Logistic model regression demonstrated that oysters from this treatment that are longer than 61 mm have a greater than 90% chance of survival. These findings establish a viable method for genotyping juvenile oysters while minimizing post-sampling mortality, and will facilitate future studies on genomic selection, individual-level survival responses to stress and disease, and repeated measures of gene expression.