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

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

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

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

5. 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 The Problem 2 DEBY mtDNA is good for assigning proportion of DEBY/Gulf of Mexico strains but not for assigning individuals to strains WILD Rare BB AA

6. 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 ATCTATATATATATATATATATATCGTGG TCGATATATATATATATATATATAGCACC ATCTATATATATATATATATCGTGG TCGATATATATATATATATAGCACC Chromosome from ♀ (TA) 10 Chromosome from ♂ (TA) 8 Microsatellite

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

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

9. 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

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

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

12. RappannockF 1 hybridsDeployed Hybrid partitioning of simulated hybrid progeny based microsatellite multilocus genotypes (8 loci) Hybrid partitioning (Structure)

13. RappannockF 1 hybridsDeployed Hybrid partitioning (BAPS) Hybrid partitioning of simulated hybrid progeny based microsatellite multilocus genotypes (8 loci)

14. 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)

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