Brief Summary of Minutes of Annual Meeting

The 2009 annual meeting will take place in conjunction with the NSA meeting in Savannah, Georgia. Dr. Frank-Lawale will be chair for the 2009 meeting. Station Reports University of Maine and Marine Biological Laboratories: Paul Rawson reported on a collaborative project between institutions in the Northeast and oyster farmers, on the crossbreeding and field trials for disease resistant oysters. Two oyster lines were crossed, the MSX and Dermo-resistant NEH and the University of Maine/Industry Cooperative Program UM Flowers select (UMFS), with putative resistance to Juvenile Oyster Disease. Parental lines, a UMFS x NEH F1 hybrid, as well as two backcrosses (NEH F1 x UMFS and NEH F1 x NEH) were deployed in locations in the Northeast and tested for performance. The hybrid line (UMFS x NEH) showed fastest growth at Rhode Island, Connecticut, and Massachusetts sites through the first season. The backcross line F1 X NEH has relatively high survival at sites where Dermo disease (New Jersey), MSX disease (Connecticut and Massachusetts) and Juvenile oyster disease (Rhode Island) occur with no apparent difference in growth performance. Although results are only preliminary and disease testing needs to be performed, the results from the first season indicate the promise of using hybrid or backcross lines for enhanced resistance to multiple diseases. University of Rhode Island. The Gomez-Chiarri laboratory has been working on the evaluation of the performance of three oyster lines/stocks (Rutgers NEHY, Frank M. Flowers from Long Island, and a local stock from Green Hill Pond in Rhode Island) in Rhode Island farms. The FMF stock showed the highest mortality in both field and experimental challenges. No statistical significances were seen in performance between the stocks, due to variations in performance at the different farms. Mortality happened throughout the year and dermo disease prevalence and intensity were low in both farms. A hybrid between the NEHY line and the local stock from Green Hill Pond will be produced in 2008 and performance tested in several farms. A second project, related to the search of markers for disease resistance, involves the investigation of the role of a candidate molecule, a matrix metalloproteinase (MMP) from the Eastern oyster, in immune responses. Tissue patterns of production of MMP in oysters showed that the protein is induced in mucosal surfaces and hemocytes in response to infectious challenge, feeding, and shell damage, suggesting potential roles in immune defenses, shell formation, and digestion. Rutgers University. Ximing Guo reported on the selective breeding efforts at Rutgers University. The line NEHY, selected for 48 years for disease resistance, shows strong resistance to MSX disease and some resistance to dermo disease. A hybrid between NEHY and the Flowers line (resistant to juvenile oyster disease - JOD) shows improved yield for the hybrid compared to parental lines and improved resistance to JOD in field trials. Triploids of NEHY and the NEHY x Flowers hybrid tested in field conditions in NJ and MA showed improved resistance to JOD and dermo disease and higher yield. Triploids outperformed diploids, and hybrid outperformed pure stock, showing that the selective advantage in diploid line gets transferred to the triploid line. Triploids may have performed better against JOD because got to refuge size (more than 25 mm in shell height) faster. Dr. Guo also reported on the progress of the projects on the mapping of genes involved in disease resistance. Markers developed by the Rutgers laboratory is up to 239 microsatellite loci (93 genomic, 146 from ESTs) and 102 SNPs from ESTs, most corresponding to genes involved in host defense (over 300 co-dominant markers). The current resolution of the linkage maps is 2-6 cM. Oyster families have been developed to map genes involved in disease resistance by family-based association. Several QTLs that may be involved in resistance to dermo disease have been identified. The laboratory is now developing 81 families (9 x 9 crosses) for marker-assisted breeding. University of Delaware. Pat Gaffney reported on the global population genetic structure in Crassostrea virginica. Single nucleotide polymorphism (72 loci) allele frequency variation was detected across the species range, showing large scale structure (Gulf, S Atlantic, N. Atlantic) and localized heterogeneity/ structure. Null alleles are common. Primers for the amplification of a few SNP markers have been developed that work in different oyster species. A bacterial artificial chromosome (BAC) library (from an individual, highly inbred C. gigas oyster) has been constructed and has been fingerprinted. The resulting physical map will be combined with genetic maps, allowing for targeted analysis of selected genes and selection of evenly spaced high-density markers for QTL mapping. The library can also be used to facilitate a whole genome sequencing project. Virginia Institute of Marine Sciences (VIMS) I. Kim Reece reported in the development or genetic markers for Crassotrea virginica, Crassostrea ariakensis, and Mercenaria mercenaria, and on the application of the markers in different project. Genetic markers for C. virginica, which allow distinguishing between the DEBY line (used in this restoration project) and local wild oyster stocks have been used to track the success of restoration projects in Chesapeake Bay. Data suggested that DEBY contribution has been low, maybe due to predation, poor survival and/or poor reproduction. Large deployments of DEBY seed (12 million) were performed in 2005-07 with efforts at protection of the seed with netting. However, genetic signal from DEBY line at the restoration site is still not above background. Models indicate that the lack of genetic signal may be due to physical characteristics of the site leading to low larval retention. In another project, the utility of 8 microsatellite loci for evaluating hatchery-selected lines of Crassostrea virginica was tested. The markers were able to discriminate among 4 hatchery lines and retrospectively quantify the success of the various crosses involving 1, 2 or more of the lines. Self assignment tests showed greater than 95% assignment for all parental lines. Markers for C. ariakensis were used to inventory germplasm resources. Genetic variation and differentiation among natural populations of C. ariakensis from China, Korea, and Japan was examined, and compared to hatchery stocks in the US. Studies showed temporal stability and population structure (isolation by distance) in natural Asian populations. A reduction in expected heterozygosity and allelic richness was observed in older hatchery stocks of C. ariakensis in the US, while in new stocks (a single generation in hatchery) no significant differences in heterozygosity but a reduction in allelic richness were observed. Eight molecular markers for hard clams, Mercenaria mercenaria, have been developed and tested, and show overall significant difference between Virginia and South Carolina wild populations. VIMS (II). Stan Allen reported in the Aquaculture and Breeding Technology Center. The goal of the center is to provide shellfish broodstock to industry. A total of 11 lines have been developed, including DEBYs and Crosbreed (XB), as well as several Louisiana-derived lines and crosses between the three. Lines are tested in several locations and best performers are saved for broodstock (mass selection). Results indicate a strong interaction between genetics and environment, warranting the development of regional varieties. A line (WTS) derived from natural pops of Chesapeake Bay oysters that might have developed resistance to dermo disease has been developed. University of Washington (I). Kristi Straus reported for the Friedman laboratory. Mitochondrial and nuclear markers indicate that a population of abalone from the San Juan Islands (WA) that visually look like pinto abalone is more genetically similar to flat abalone. The biological relevance of these observations is being evaluated in fertilization studies. Another project plans to evaluate the effect of geoduck aquaculture on wild populations, based on the observation that aquaculture geoducks show high rates of maturation and spawning. The Friedman laboratory is also developing tools for the diagnosis of oyster herpes virus and abalone withering syndrome using quantitative real time PCR. These tools are being used in projects related to the development and evaluation of disease resistance strains. A novel Ricketssia-like bacterium has been detected in abalone, as determined by morphology, susceptibility to antibiotics, and genetic analysis. University of Washington (II). Steven Roberts reported on the development of a real time PCR method for the quantification of Quahog Parasite Unknown in hard clams. He also reported on the identification of molecules involved in disease resistance/ tolerance in different species of shellfish. Genes potentially involved in immunity in oysters have been identified from a EST library from oyster granulocytes. One of the genes is a homolog from the cytokine interleukin 17. A stock of oysters derived from survivors of a dermo disease outbreak in Marthas Vineyard, MA, has been developed and tested in the field in MA. The stock showed improved performance. Differences in patterns of expression of constitutive genes were observed between the resistant and a susceptible stock. The Roberts laboratory, in collaboration with the Friedman laboratory, is starting to mine ESTs for abalone that may be involved in resistance to Withering syndrome. The Roberts lab is also looking at differences in gene expression in lines of hard clams showing differences in resistance to QPX disease. Molluscan Broodstock Program (MBP). Chris Langdon reported on the search for Kumomoto oyster broodstock from Japan that is not inbred and has not been hybridized with the Pacific oyster, C. gigas. The search reveled that there is no species-specific shell morphology for Kumamoto oysters that would allow them to be differentiated from C. gigas, and points to the need of genetic markers that allow to differentiate between different oyster species. Currently available genetic markers do not distinguish between C. angulata and C. gigas. Dr. Langdon also reported on the breeding program. As of 2006, the MBP have produced about 1200 families from 7 original broodstock lines. The MBP relies now on rotational breeding to avoid inbreeding. The response to selection after two generations indicates that, on average, the top 5% are 70% better than progeny from unselected broodstock. Improvements in yield in the Pipestem line are related to improved survival, and this line is being considered for commercial production. MBP is now generating inbred parental lines using an Adam and Eve strategy of crosses for family amplification which show good resistance to summer mortality. Another line of research relates to selection for color pattern in Pacific oysters. The program surveyed 20+ people at two locations and found that people generally prefer mottled or stripey oysters instead of pure black or white. Overall color of the shell is highly heritable and could be manipulated through breeding In most familes there is a continous optical density distribution. But in some families you see a bimodal distribution with about 3:1 ratio or a 1: 1 ratioindicating a single major gene controlling shell color. In collaboration with Camara and Lang, the MBP is searching for genetic markers for marker assisted selection using microarrays to identify differences in ESTs between sensitive and tolerant MBP families to stress (acute heat-shock). The ESTs are verified using qPCR, and differences in expression are correlated with field performance traits. About 20 ESTs are correlated with either oyster growth in field or yield (i.e. galectin). USDA ARS Shellfish Genetics Program (Oregon). Mark Camara reported on several ongoing projects. On the first project, regarding the importation and evaluation of new Kumamoto oyster germplasm to address problems with inbreeding and hybridization, two sets of markers allowed identifying pure Kumomoto oysters and hybrids with other oyster species in the Pacific, indicating that while there is no evidence for extensive hybridization in the Ariake Sea, hybridization barriers appear to be lower in US cultured oysters. Furthermore, morphology cannot be reliable used to distinguish between Pacific and Kumomoto oysters. The ARS lab is also involved in the genetic characterization of existing Pacific oyster germplasm resources using AFLP markers. On a third project, the ARS lab has developed software tools to optimize pedigree reconstruction using molecular markers & mixed-family selection. The lab has also identified candidate genes for stress resistance in Pacific oysters using DNA microarrays, cDNA-AFLP differential display, and RT-qPCR, is mapping quantitative trait loci for economically important traits in Pacific oysters; and is characterizing molecular genetic variation within and among remnant populations of native Olympia oysters. University of Southern California. Dennis Hedgecock reported on the centromere mapping for the Pacific oyster, Crassostrea gigas. Expectations from cytology indicate that the Pacific oyster has 10 linkage groups and ~600 cM map units, but linkage studies suggest that the genome is twice as big. Gene centromere mapping was performed based on segregation in chemically-induced triploids (c3N). That most markers (87/93) show 1:1 homozygote ratios in c3N is consistent with the likelihood that paternal allele in these triploid offspring mask the effects of "homozygosity" for any recessive mutations coming from the female. There are also indications of chromosomal rearrangements. Six out of 10 centromeres were marked, facilitating mapping of new markers. Interference is much less than previously believed; explaining why genome size estimated in linkage studies is larger than cytological estimations. International Participation: Shellfish Aquaculture in Australia. Judd Evans and Tony Troup, two farmers sponsored by Australian Seafood Cooperative Research Centre reported on oyster aquaculture industry and research in Australia. There are three separate oyster industries in Australia (Tasmania, New South Wales and Southern Australia), hoping to combine into one cohesive industry. The Australian oyster industry has US 1 billion, 1300 direct jobs, and 3000 indirect jobs invested in the production of Sidney rock oysters, flat oyster, and C. gigas. Research projects include a C. gigas breeding program, which began in 1997 (CSIRO) and was handed to industry in 2001. The selectively bred oysters showed improved growth rate (5% increased growth per generation) at the expense of changes in shape, so the program now selects for increased survivability, shape, and growth rates. The SRO breeding program has developed lines resistant to the disease QX (caused by Marteilia sydneyi) that show improvements in growth and survival. Further breeding development by the Sidney Oyster Company (SOCo) is geared to increased growth, condition, shape, and survival to QX and winter mortality. Efforts also include the development of hatchery production of selectively bred stock, creation of a nursery network, and environmental awareness, best management practices, and quality controls. Current research on QX disease (Marteilia sydneyi) has led to the development of markers for QX resistance, diagnostic probes, and identification of 2 polychaete species as the primary host for the parasite. Gary Jensen USDA CSREES, and Fred Wheaton, Northeast Regional Aquaculture Center, reported on grant opportunities, Dr. Jensen encouraged researchers to consider applying to integrative programs. Oyster Genomics Update (Hedgecock, Gaffney). JGI is moving forward on the EST sequencing project of cDNA libraries that were developed from C. gigas inbred lines 35 and 51. The sequencing of BACs that had some genes of physiological interest is close to being complete. The first BAC that was done had 9-10 genes including cytochrome P450. An annotation workshop will be scheduled sometime in the Fall. Next steps should include: 1) placing markers onto the map; 2) find funding for BAC end sequencing to construct a high quality map Broodstock for restoration projects. The use of selectively bred stocks for restoration projects was briefly discussed, projects should strive to maintain local adaptation and avoid bottlenecks. Issues to consider include number of broodstock, effective population size, etc. The idea of so called genetic restoration has been reconsidered in the favor of trying to persist with wild stocks that may show evidence of development of disease resistance, combined with good hatchery techniques. C. ariakensis update. Environmental impact statement is still being crafted due in May. Nothing is really happening except an occasional deployment of a million or so triploids.

WERA-099 provided a forum for US and international molluscan geneticists, physiologists and pathologists to exchange ideas and information on genetics, reproduction, diseases, chromosome and genetic manipulation techniques, broodstock management and breeding programs. Highlights of the 2008 meeting included: 1) increased knowledge about the relative performance of selectively bred strains, hybrids, and triploids in farms in the Northeast US; 2) intense efforts towards marker development; 3) development of molecular diagnostic tools for diseases affecting the bivalve industry; 4) efforts towards the identification of molecules involved in bivalve immune responses and resistance to stress; and 5) progress towards mapping and genome sequencing. Multi-disciplinary and inter-regional exchanges among participants revealed new directions of scientific enquiry and facilitated formulation of new research questions. The meeting also provided a forum to stimulate collaborations between US and Australia.

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