Background
• Molluscan shellfish, such as clams, oysters, mussels and scallops, represent around 20% of worldwide aquaculture production
• Wild spat can be negatively impacted by overfishing, environmental or trophic changes and disease outbreaks, none of which are under the control of the producer. There is therefore a trend toward hatchery-based production of juveniles for on-growing in the sea.
• Hatchery-based production allows for genetic improvement of stock via selective breeding, which has the potential to improve economically important traits such as growth and disease resistance by 10–15% per generation
Methods
• Oysters were acclimatized in a 50 L seawater aquarium at 16 °C for 60 days, with 700 L/hour external filtration
• They were derived from native stock from the Argyll area and were fed a maintenance diet of powdered algal biomass
• Each oyster was externally rinsed with reverse osmosis (RO) filtered water and placed into a polypropylene vessel with 500 mL seawater made from artificial salt
• After being introduced, duplicate water samples of 75 mL were taken from each vessel after 72 hours and a 5 mm2 section of mantle was dissected and stored in 100% ethanol
• A 75 mL artificial seawater control sample was taken before filling the vessels and all 75 mL water samples were filtered using a vacuum filtration manifold and 47 mm 0.45 μm cellulose nitrate filters
Genotyping proceeded with the EP1™ platform using the
manufacturer’s protocols.
SNP genotypes were called using k-means clustering under the default settings in the SNP Genotyping Analysis Software.
Results:
• The EP1 platform was used to synthesize 16 SNPs with the highest mean minor allele frequency (MAF) for the discovery of target DNA in parentage assignment. To minimize the amount of potentially
PCR-inhibiting co-purified contaminants from DNA extractions, several different dilutions with RO water were trialed.
• Results showed that the overall SNP call rate was calculated as the proportion of allele calls across the 96 assays (16 SNPs, six replicates per SNP) for the eDNA sample that matched the tissue sample
• Two of the 16 trialed assays failed to produce any identifiable clusters, indicating no polymorphism among the tested oysters or a non- functioning assay, and the tissue samples for the remaining 14 assays gave high-quality clusters reliably identified using k-means clustering (SNP call data)
All three strategies provided the highest accuracy between eDNA and tissue DNA genotypes for the DNeasy® and Chelex® extractions.
Results
Background (continued)
• One particular challenge of molluscan aquaculture is the availability of non-invasive DNA sampling techniques for parentage assignment and advanced marker-assisted or genomic selection strategies
• Recent advances in the isolation of environmental DNA (eDNA) potentially offer a non-invasive alternative to tissue sampling
• Typing accuracy was assessed to produce a practical protocol for the European flat oyster that can be used by researchers and aquaculture professionals as a template to develop viable alternatives to invasive tissue sampling in similar species
• We also demonstrated that eDNA extracted using this protocol
is of sufficient quality and quantity for multi-locus genotyping, which is necessary for most applications in aquaculture breeding programs
• eDNA is used to accurately genotype bivalve mollusks and other aquatic organisms
• A protocol for the European flat oyster was developed to develop viable alternatives to invasive tissue sampling
• The protocol is of sufficient quality and quantity for multi-locus genotyping, which is necessary for most applications in aquaculture breeding programs
• Invasive methods involving the removal of internal tissue or fluid are routinely reported in marine and freshwater mussels
Conclusions