1. 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

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. 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

4. 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

5. 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

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

7. 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)

8. 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)

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

11. 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)

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

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

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