Report Information: Participants: Brief Summary of Minutes of Annual Meeting: Station Reports

Transcription

1 Annual/Termination Reports (SAES-422): Date of Annual Report: Report Information: apple Annual Meeting Dates: 21 st -22 nd March 2009 apple Period the Report Covers: 10/2007 to 09/2008 Participants: Name Affiliation State Allen, Stan Virginia Institute of Marine Sciences VA Auyong, Jan Oregon State University OR Camara, Mark USDA ARS - Oregon OR Cordes, Jan Virginia Institute of Marine Sciences VA Davis, Joth Taylor Shellfish Farms WA Frank-Lawale, Anu Virginia Institute of Marine Sciences VA Gomez-Chiarri, Marta University of Rhode Island RI Guo, Ximing Rutgers University NJ Jensen, Gary USDA CSREES DC Kube, Peter CSIRO AUSTRALIA Langdon, Chris Oregon State University OR LaPeyre, Jerome Louisiana State University LA Proestou, Dina University of Rhode Island RI Rawson, Paul University of Maine ME Reece, Kim Virginia Institute of Marine Sciences VA Roberts, Steven University of Washington WA Scarpa, John Habour Branch Florida Atlantic University FL Silverstein, Jeff USDA DC Spikers, Tom Shellfish Culture AUSTRALIA Stick, David USDA ARS - Oregon WA Wilbur, Ami University of North Carolina, Wilmington NC Brief Summary of Minutes of Annual Meeting: Station reports were brief at this meeting in order to make time for group discussions and a presentation on the application of quantitative genetic principles to oyster breeding by Dr. Peter Kube from CSIRO, Australia. The 2010 annual meeting will take place in conjunction with the NSA meeting in San Diego, California. Dr. Steven Roberts will be chair for the 2009 meeting assisted by Dr. Allen. Station Reports University of Maine and Marine Biological Laboratories. Paul Rawson reported on the completed 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 growth and survival. There appeared to be a significant site x genotype interactions in mortality. Mortality was higher for NEH and NEH hybrid in all three Maine sites whereas almost all the UMFS animals died at New Jersey site. Growth did not vary among lines within sites. However, it

2 did vary between Maine sites and New Jersey site with performance lower at the New Jersey site. The hybrid line (UMFS x NEH) exhibited highest performance in growth and survival at Maine sites University of Rhode Island. The Gomez-Chiarri laboratory also reported on the evaluation of oyster lines in Rhode Island. They presented results from the testing of three oyster lines (i-rutgers NEH, ii- GHP a wild stock from Green Hill Pond in Rhode Island where disease pressure is high and iii-a hybrid cross of the two) in 4 Rhode Island farms, two in Narragansett Bay and two in coastal ponds. The NEH line grew significantly better at the 2 Bay sites while the GHP line seemed to grow better in coastal ponds, however this was not statistically significant. By the end of summer, NEH line experienced significantly lower mortality than the two other lines at the Bay sites. The same was true at the coastal pond sites as well, but this was not significantly different. Overall mortality was significantly higher at one of the Narragansett Bay sites (site 2) for all lines. In order to determine the cause of mortality tests were done. Conchiolin was detected at the coastal ponds sites in late August, but very small percentage of oysters examined had it. It was also detected in both bay sites with and incidence level of 14% and 37% in both sites. They are also gave a report on their project looking at the role of Matrix Metalloproteinases (MMPs) in innate immunity. They isolated a novel MMP in oysters-cv1mmp and immunohistochemistry of this MMP shows that it is produced in epithelial barrier tissues (mantle, digestive) and hemocytes. An activity assay suggested that protease production increases in response to challenge with P. marinus. They have also found 41 SNPs in coding region of the Cv1MMP gene; on average 1 every 40 bp. Rutgers University. Ximing Guo reported on several molecular and selective breeding projects at Rutgers University. The line NEH and the FMF line are now highly inbred therefore crossing them should result in some beneficial heterosis. When tested the cross between NEH and FMF resulted in a higher performing hybrid (with respect to yield) than either parental line. Triploids outperformed diploids and hybrid triploids from more than one line outperformed triploids of pure stock, suggesting that the selective advantage in diploid line gets transferred to the triploid line. His group has also identified 341 Co-dominant markers for C.virginica 239 SSRs (Simple sequence repeats) -93 from genomic, 146 from ESTs (express sequence Tags). In addition they have SNPs (Single Nucelotide polymorphisms) from 102 ESTs. They are using these markers to map genes for disease resistance using family-based association. The current resolution of the linkage maps is 1.15 cm and they have identified 5 chromosome regions associated with Dermo resistance using data from 3 families. There is now a marker assisted breeding project under way at Rutgers using 81 families from 3 lines (NEH, NEF and NEL) in a 9x9 matrix. Preliminary results show that the NEF line is the best. Dr. Guo also gave an update on the oyster genome project in China aimed at producing a draft sequence using next generation sequencing (50X Solexa pair-end reads, additional 454 coverage if needed). Results so far indicate that there are too many SNPs (even in 1 individual) and current computer programs cannot distinguish between the two chromosome copies for proper assembly. Louisiana State University. Jerome LaPeyer made his debut presentation at the WERA099 meeting and gave a report on current oyster research in his lab at LSU. His lab focuses on mechanisms of disease resistance in the Louisiana (LA) oyster, which is primarily resistance to dermo. They compared survival between native LA oysters, the Crossbreed oyster (an MSX resistant line developed at

3 Rutgers) and the native Chesapeake Bay oysters. Results showed that the LA oysters had lower parasite levels in their tissues suggesting that they are more resistant. There is now a small breeding population in their lab, now in their fourth generation, that has been selected for resistance to dermo. These animals are going to be compared with wild LA animals. In another experiment they looked at hemocyte activity in susceptible (Maine) and resistant (Louisiana) oysters. They observed a higher hemocyte density/proliferation in infected oysters and resistant (LA oysters). These oysters were able to clear infection better. They have also identified proteins (CvSI-1 and CvSI-2) that may be involved in response to Perkinsus, specifically proteinase inhibitors. They were found in plasma, epithelia, and digestive tubules of oysters. The expression level of CvSI-1 is higher in resistant oysters and thus the capacity to inhibit Perkinsus is much higher in selected oysters. The goal is to develop research tools (antibodies, clone isolation, recombinant, etc.) to study their role in response to infection Virginia Institute of Marine Sciences (VIMS) I. Jan Cordes presented work on the genetic evaluation of recruitment success of deployed C. virginica on man-made reef in Chesapeake Bay conducted in the Reece Lab in VIMS. They deployed DEBY (a disease resistant line developed at VIMS) oysters on a shell Bar Reef and monitored recruitment using spat collectors. Spat were genotyped to distinguish between wild, DEBY and hybrid animals. They used two mitochondrial genes (COI and COIII); and only2% of oysters genotyped possessed DEBY haplotype. In 2006 and 2007 about 5% of spat can be attributed to DEBY-these years represented larger set of wild oysters as well. When microsatellite markers where used, 6% of spat were identified as DEBY in 2006 and 1% in These results pose questions about the success of planting domesticated animals in restoration exercises as no DEBY signals were found in neighboring sites either. Dr. Cordes also mentioned the advisory role that the lab plays. They deal with the verification of sighting of C. ariakensis in Virginia Rivers. So far they have confirmed two sightings- Coan River and York River-, but the animals were triploid and thus sterile. VIMS (II). Stan Allen reported on the line performance and future direction of the line breeding programme at Aquaculture and Breeding Technology Center. The goal of the center is to provide genetically improved broodstock to industry. A total of 11 lines were developed, including DEBYs and Crossbreed (XB), as well as several Louisiana-derived lines and crosses between the three. Lines are tested at 4 different locations and best performers are saved for broodstock (mass selection). Results indicate selection for disease resistance has been successful; all best of lines perform better than wild control, however, there were no observed advantages over the control in size and weight at harvest. The total yields of lines were much higher than the control due to the highly significant difference in survival. These lines are now being coalesced into three main Super lines based on the origin of their parental line. These are DEBY, CROSSBREED and LOUISIANA. The Louisiana line has been diverged into high salinity (hana) and low salinity (LOLA). Efforts are now going to be concentrated on improving growth rate. A new challenge for the center is how to produce and disseminate the large numbers of broodstock that the industry requires. A new breeding plan has been developed using larger numbers of animals to meet this need. Selection will also be done annually to produce odd and even year cohorts. As the industry develops new problems emerge and there appears to be an issue with low fecundity. This could be caused by chronic disease (dermo), pea crabs and inbreeding. The center also

4 faces the challenge of conditioning the large number of broodstock needed to propagate the lines successfully. This new breeding and dissemination strategy will be online by University of Washington. Steven Roberts reported on genetic studies of Vibrio tubiashi, a major problem for the shellfish industry. A disease challenge of C.gigas with Vibrio was conducted to identify genes involved in disease response (Interluekin, Prostaglandin 7 receptor). They identified ESTs containing microsatellites. Disease challenges were also conducted with three other shellfish species. A challenge with C. virginica yielded potential indicators using resistant and susceptible lines. Lab trials with H. cracherodii using resistant/susceptible suggest different levels of gene expression and raised a question about gene expression being used as a phenotypic marker for marker-assisted breeding? With M. mercenaria they observed indicators of resistance to QPX. They are sequencing the genome (short reads) to find some potential genes to look at, but they are currently using Sole and there may be problems with assembly. Molluscan Broodstock Program (MBP). Chris Langdon reported on the problems faced by oyster hatcheries in the Pacific Northwest and MBP s role in helping the industry. Last year, larval production at hatcheries in the region was extremely low. They looked at conditions leading to lack of natural set of oysters in Willapa Bay and performed experiments in lab. They concluded that the source of the problems were Vibriosis, deceased PH and higher water temperatures. They increased water treatment capabilities in their hatchery (filtration, uv sterilization, and foam fractionation) and found that although better water quality improved larval growth the mortality still remained. They found that the lower ph in the ocean water decreased CaCO3 saturation levels and negatively affects larval shell deposition. Furthermore warmer water decreases solubility of dissolved oxygen by about 30%, resulting in lower larval survival. This problem was addressed by aeration. Chris Langdon also gave an update on the performance of MBP families. The MBP adopted a rotational breeding design aimed preventing inbreeding. Results from cohort 18 from this scheme showed that the line was superior to wild and unselected controls in terms of yield (survival and body weight). Families were also tested for increased resistance to possible stressors in hatchery. Results from evaluation of families after two generations of selection suggested better performance relative to non-selected broodstock. The MBP has established a commercial repository to supply hatcheries with inbred parental lines. It is hoped that commercial hatcheries will use MBP lines for production and pass on their superior performance as seed to growers. So far the process has been extremely successful. Finally after two years of quarantine and $16,000 worth of disease testing Kumamoto oysters from Japan were seen to be free of diseases for 2 generations, and so have been planted in Yaquina Bay. There were no differences observed between Japan oysters and West Coast US oysters with respect to growth. This new Japan stock will be available through Taylor Shellfish. USDA ARS Shellfish Genetics Program (Oregon). Mark Camara reported on several ongoing projects. On the first project he presented information on the population genetics of pacific oysters using material from Japan, New Zealand, Pacific Northwest and MBP lines. He used AFLP markers to generate a tree of the relationships between different stocks and found that the tree did not agree with history as the MBP populations sorted with Japan populations. In the second project he gave details of

5 a mixed family selection strategy using molecular markers to retrospectively assign pedigree after pooling offspring from several parents in order to remove common environmental effects in the hatchery. The idea is to answer the question when is the best time to mix larvae? Straight hinge, pediveliger, d-larvae, post-larvae? Results were compared from mixing at each of the larval stages and it was found that earlier mixing resulted in higher genetic variability. In a third project he conducted heat tolerance test of all MBP families. The idea was to identify tolerant and susceptible families and correlate survival in the field to survival in the laboratory test. This was done to develop a laboratory test that could provide an indication of susceptibility to summer mortality. Further molecular tests using microarray profiling were done unfortunately, polymorphism lead to qpcr artifacts. An alternative approach using reverse microarray is being considered, however, it is not yet optimized. Dr. Camara then talked about future projects. His first project will be looking at QTLs for gene expression when summer mortality is simulated in a laboratory and the second will be to identify superior genotypes for larval survival in order to help alleviate the current problems faced by hatcheries with poor larval performance. Habour Branch Florida Atlantic University. John Scarpa reported on the Molluscan Research Program in Florida Atlantic University. Their apple snail breeding program is in it s fourth generation and is thriving. There is also work being done on conch reproductive stock enhancement. In bivalves the sunray Venus clam can now be cultured using methods developed for hard clam culture. The next step is to identify methods of brood stock handling and spawning, establish hatchery methods, evaluate growth and survival during grow out, test market at wholesalers. With Mercenaria mercenaria there continues to be increased clam mortality in SE Florida. Hybrids have been created and their performance is currently being tested in the field. So far, hybrids show superior growth. They are also looking at larval dispersal of C. virginica along Eastern Florida Industry Participation: Taylor Shellfish Farms. Joth Davis gave a first hand view of the problems being faced by the oyster industry in the Pacific Northwest. He then talked about the line evaluation and development process at Taylor Shellfish. Taylor run a 4-phase rearing schedule to evaluate hybrid lines and measure additive and non-additive contribution to yield. Larval rearing systems have been changed to low volume, high density flow through system. This increased ability to test more families (50 to 100 lines per cohort), they aim to optimize seed testing methods to minimize time and reduce cost. They found that cage culture produces poor results and rotating seed bag worked better; cylinder worked best. They are also working on Manila clam breeding. There is now a need to develop molecular tools for genetic typing and pedigree analysis Invited speaker Dr. Peter Kube CSIRO, Australia: Peter Kube gave a presentation titled Genetic Improvement of the Pacific Oyster- A quantitative genetics approach down under. He gave an overview of the oyster industry in Australia. Two species are farmed-the Pacific and Sydney rock oysters. The industry generates approximately $50M annually. 20% of the production is from genetically improved seed. The breeding program began in 1997 and to date there has been 10 year classes with families per year class but because the breeding objective was defined by farmers preferences progress was slow and not cumulative. A new direction was taken based on an economic model and the economic values of traits were used to identify the breeding goals. He described an

6 economic model based on economic weights. The economic weight of a trait is the dollar value of a change in the trait and it can be expressed as change in production cost. He then went on to describe the ways in which quantitative genetics was used to select potential broodstock. Data is combined from families across all year classes and analyzed using ASREML, a statistics program that allows multivariate models while at the same time combining information on all traits and all animals in a pedigree (up to 14,000 individuals). This program is used to generate EBVs (estimated breeding values) for all traits between families, the higher the EBV the better the family. The trait EBVs are then combined into a single index using economic weights and families are selected for breeding based on this index. Future work on the program included setting up a database to streamline the process, understand the genetics of survival and increase the number of families tested each year. Opportunities for collaborative research Gary Jensen USDA CSREES, and Jeff Silverstein USDA. Gary Jensen and Jeff Silverstein gave a joint presentation on ways in which grant applications can be strengthened by incorporating various disciplines and including outreach activities. The key is to keep stakeholders in view and talk to program managers before applying. USDA and CSREES priority areas were identified. Accomplishments: WERA-099 provided a forum for US and international molluscan geneticists, physiologists and pathologists to exchange ideas and information on quantitative and molecular genetics, reproduction, diseases, chromosome and genetic manipulation techniques, broodstock management and breeding programs. Highlights of the 2008 meeting included: 1) A one day workshop titled From Falconer to the Field- the practical application of quantitative genetics theory to existing oyster breeding programmes & Gene maps to field maps- a synergy of molecular and quantitative tools targeted at improved oyster performance 1) This increased the awareness of US scientists about the need to design their breeding schemes with additive genetic variance in view and has resulted in a reassessment of our breeding program designs 2) Highlighted the areas of collaborative research as this is essential to tackle the disease problems faced by the aquaculture industries across the US; 3) intensified efforts towards developing multidisciplinary projects ; 4) increased knowledge about the relative performance of selectively bred strains, hybrids, and triploids in farms in the Eastern US; 5) intense efforts towards marker development; 6) development of molecular diagnostic tools and laboratory disease challenges for diseases affecting the bivalve industry; 7) efforts towards the identification of molecules involved in bivalve immune responses and resistance to stress; and 8) 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, particularly in quantitative genetics. The meeting also provided a forum to stimulate and develop collaborations between US and Australia. Impact Statements:

7 1. At the meeting it became obvious that funding required to conduct fundamental breeding experiments that generate parameters and information needed to optimize breeding programs in severely lacking. 2. Discussions between researchers at the workshop showed that a significant amount of work is needed to modify current breeding programs in order to make them more efficient 3. Conversations between researchers should result in the development of markers for markerassisted selection that could be used in more efficient breeding programs to produce diseaseresistant strains. 4. Research efforts are geared to provide industry with stocks, families, and lines that show improved performance. 5. International efforts to map the genome of the Pacific and Eastern oyster and to sequence the genome of the Pacific oyster are moving forward. Exchange of information on the development of new maps and mapping techniques will lead to a better understanding of the genetic basis of disease resistance in Eastern oysters as well as growth and survival in farmed Pacific oysters. Publications: Batista FM, Arzul I, Pepin JF, Ruano F, Friedman CS, Boudry P, Renault T (2007) Detection of ostreid herpesvirus 1 DNA by PCR in bivalve molluscs: a critical review. J Virol.Methods 139:1-11 Burge CA, Judah LR, Conquest LL, Griffin FJ, Cheney DP, Suhrbier A, Vadopalas B, Olev PG, Renault T, Friedman CS (2007) Summer seed mortality of the Pacific oyster, Crassostrea gigas thunberg grown in Tomales Bay, California, USA: The influence of oyster stock, planting time, pathogens, and environmental stressors. J.Shellfish Res 26(1): Camara MD, Evans S, Langdon C (2007) Inbreeding effects on growth and survival in a naturalized population of the Pacific oyster (Crassostrea gigas) revealed using molecular marker-based estimates of parental relatedness. Aquaculture 272:S247 Curole JP, Hedgecock D (2007) Bivalve Genomics: Complications, Challenges, and Future Perspectives. Aquaculture Genome Technologies. Jul 2007, pp , Gomez-Leon J, Villamill L, Salger SA, Sallum RH, Remacha-Trivino A, Leavitt DF, Gomez-Chiarri M (2008) Survival of eastern oysters Crassostrea virginica from three lines following experimental challenge with bacterial pathogens. Dis Aquat Organ 79: Jenny MJ, Chapman RW, Mancia A, Chen YA, McKillen DJ, Trent H, Lang P, Escoubas JM, Bachere E, Boulo V, Liu ZJ, Gross PS, Cunningham C, Cupit PM, Tanguy A, Guo X, Moraga D, Boutet I, Huvet A, De GS, Almeida JS, Warr GW (2007) A cdna microarray for Crassostrea virginica and C. gigas. Mar Biotechnol.(NY) 9:

8 Moss JA, Burreson EM, Cordes JF, Dungan CF, Brown GD, Wang A, Wu X, Reece KS (2007) Pathogens in Crassostrea ariakensis and other Asian oyster species: implications for non-native oyster introduction to Chesapeake Bay. Dis Aquat Organ 77: Moss JA, Xiao J, Dungan CF, Reece KS (2008) Description of Perkinsus beihaiensis n. sp., a new Perkinsus sp. parasite in oysters of Southern China. J Eukaryot.Microbiol 55: Quilang J, Wang S, Li P, Abernathy J, Peatman E, Wang Y, Wang L, Shi Y, Wallace R, Guo X, Liu Z (2007) Generation and analysis of ESTs from the eastern oyster, Crassostrea virginica Gmelin and identification of microsatellite and SNP markers. BMC Genomics 8:157 Roberts S, Gueguen Y, de LJ, Goetz F (2008) Rapid accumulation of an interleukin 17 homolog transcript in Crassostrea gigas hemocytes following bacterial exposure. Dev Comp Immunol 32: Taris N, Lang RP, Camara MD (2008) Sequence polymorphism can produce serious artefacts in realtime PCR assays: hard lessons from Pacific oysters. BMC Genomics 9:234 Wang Y, Guo X (2007) Development and characterization of EST-SSR markers in the eastern oyster Crassostrea virginica. Mar Biotechnol.(NY) 9: