Carlsson J., S.K. Allen Jr, and K.S. Reece TRACKING BREEDING SUCCESS OF DEPLOYED OYSTERS THROUGH MICROSATELLITE VARIATION Supported by grants from: NOAA Oyster Restoration Program through the Chesapeake Bay Program Office

The problem RESTORATION EFFORTS HAVE BEEN FOCUSED ON CONSTRUCTING REEFS SEEDED WITH DOMESTICATED AQUACULTURE STOCKS (MOSTLY DISEASE-RESISTANCE SELCTED STOCKS-DEBY AND CROSBREED LINEs)

The Question ARE THE DEPLOYED DOEMSTICATED OYSTERS CONTRIBUTE SIGNIFICANTLY TO SPAT PRODUCTION? DEBY DEPLOYMENT – USE mtDNA RFLP (note this also works for Gulf of Mexico strains) PRIOR TO DEPLOYMENT DEPLOYED DEBY PRODUCED SPAT AA   BB   Rare   Data from Great Wicomico 2002-2006

The Problem 1 CROSBREED DEPLOYMENT – USE? Hinf I digest of mtDNA coIII in Crosbreed strain Hinf I digest of mtDNA coIII in natural population The coI and coIII mitochondrial genes do not allow for discriminating among non-DEBY or non-Gulf of Mexico strains

The Problem 2 AA   BB   Rare   The frequency of the BB haplotypes is not 100% in the DEBY line or Gulf strains An oyster showing the AA haplotype could still be a DEBY or Gulf of Mexico oyster An oyster showing the BB haplotype has a high probability of being a DEBY or Gulf of Mexico oyster DEBY WILD mtDNA is good for assigning proportion of DEBY/Gulf of Mexico strains but not for assigning individuals to strains

The Solution? Microsatellites +High power +Highly variable +High throughput +Nuclear -Time consuming to develop -Expensive Today the most widely used genetic marker for population genetic studies Microsatellite ATCTATATATATATATATATATATCGTGG TCGATATATATATATATATATATAGCACC Chromosome from ♀ (TA)10 ATCTATATATATATATATATCGTGG TCGATATATATATATATATAGCACC Chromosome from ♂ (TA)8

Example 1 4 microsatellites 2 mtDNA genes Over 85% did significantly not assign to DEBY 1 individual assigned to DEBY Example 1 DEBY – yellow Wild pop2 – white Wild pop1 – blue Note! Test pop – grey Factorial Correspondence Analysis (Genetix)

Example 2 4 microsatellites 2 mtDNA genes Over 90% did significantly not assign to DEBY No individuals assigned to DEBY Test pop A – pink DEBY – yellow Wild pop 2 - white Wild pop 1 – blue Test pop B - grey Factorial Correspondence Analysis (Genetix)

Microsatellite Work in Progress Spat-on-shell – deployment of domesticated oysters Six sites in the Chesapeake Bay Six samples of wild oysters collected prior to deployment (n=100/site) Sampling of deployed spat on shell (n=50 per deployment, 3 deployments/site) Sampling of 8 domesticated aquaculture lines (n=50/line) Analyze samples with a suite of 8 microsatellite loci

Example Rappahannock River, Drumming Ground Deployed spat-on-shell - blue Rappahannock wild – yellow Factorial Correspondence Analysis (Genetix)

Example Rappahannock River, Drumming Ground Deployed spat-on-shell - blue Rappahannock wild – yellow Hybrids - white Factorial Correspondence Analysis of simulated hybrid progeny (Genetix and Hybridlab)

Hybrid partitioning of simulated hybrid progeny based microsatellite multilocus genotypes (8 loci) F1 hybrids Rappannock Deployed Hybrid partitioning (Structure)

Hybrid partitioning of simulated hybrid progeny based microsatellite multilocus genotypes (8 loci) Rappannock Deployed F1 hybrids Hybrid partitioning (BAPS)

Concluding remarks -mtDNA RFLP has provided and will continue to provide essential information about the contribution of DEBYs on a group level -Microsatellites can clearly discriminate between wild and domesticated oysters on a group and on an individual level -Microsatellites can also be utilized to detect hybrids and to determine the amount of genetic introgression between wild and domesticated oysters (both current and future)

Thanks! SAMPLING Tommy Leggett LABORATORY Jim Wesson Sharon Furiness Mark Luckenbach Wes Heydeck P.G. Ross Elizabeth Francis Missy Southworth Georgeta Constantin Mike Congrove