Completion report. Development of JOD-Resistant Lines and Markers for Eastern Oyster Aquaculture

Transcription

1 Completion report Development of JOD-Resistant Lines and Markers for Eastern Oyster Aquaculture PROJECT CODE: SUBCONTRACT/ACCOUNT NO: Q321501/Q Grant Number: and PREPARED BY: Marta Gómez-Chiarri Project Coordinator 6/25/2010 Date

2 Development of JOD-Resistant Lines and Markers For Eastern Oyster Aquaculture PROJECT TITLE: Aquaculture PROJECT CODE: Development of JOD-Resistant Lines and Markers For Eastern Oyster SUBCONTRACT/ACCOUNT NO: Q321501/ Q Subaward Number: and REPORTING PERIOD: October 2007 May 2010 FUNDING LEVEL: $209,268 (total award) PARTICIPANTS: Marta Gómez-Chiarri, University of Rhode Island (URI); Ximing Guo, Rutgers University (RU); Dale Leavitt, Roger Williams University (RWU); Perry Raso, Ocean State Aquaculture (OSA). PROJECT OBJECTIVES: The identification and mapping of genetic markers associated with disease resistance is an important step in the investigation of mechanisms of disease resistance and in the development of breeding programs (Yu and Guo, 2006). The goal of this project is to identify markers associated with resistance to Juvenile Oyster Disease (JOD) in Eastern oysters, C. virginica through population-based association studies. The specific objectives of this NRAC-funded project are to: 1) Collect spat from wild populations from two locations in the Northeast subject to different environmental conditions and disease pressures (URI, RWU, and RU, year 1). 2) Experimentally challenge spat with JOD, collect samples for genotyping and association analysis, and select for survivors of the disease (URI, Year 2). 3) Identify markers associated with disease-resistance to JOD by association analysis of a large number of genetic markers (RU, Years 1 & 2). 4) Create new lines of JOD-resistant oysters by breeding survivors from the JOD challenges (RWU and RU, Year 2). ANTICIPATED BENEFITS: This research will: a) Identify markers associated with resistance to JOD; b) improve our basic knowledge of the genetic mechanisms of resistance to JOD; c) provide genetic tools to be used in the identification and creation of resistant strains through marker-assisted selection (MAS); and d) create new lines resistant to JOD and well-adapted to local growing conditions in Southern New England. This knowledge will benefit the scientific community by providing new tools for oyster disease research and ultimately the oyster industry by providing means to efficiently and rapidly develop oyster strains resistant to diseases. PROGRESS AND PRINCIPAL ACCOMPLISHMENTS: Wild oyster spat (less than 30 mm in shell height) were collected by Rutgers University from

3 Cape May, New Jersey, in October 2007 and January 2008, and sent to the University of Rhode Island for challenge experiments. In addition to the wild oyster spat, Rutgers University also produced a synthetic population of spat for this study. The synthetic population, F2L, is a F2 population produced from F1 hybrids between Rutgers NEH (Northeastern high survival line) and wild oysters from Louisiana. The NEH has shown some resistance to JOD (see below), while the Louisiana oysters had no known history of JOD exposure. In addition, two more synthetic populations produced by RU in 2009 as part of their ongoing breeding program and sent to URI. Unfortunately, no wild spat was available from Rhode Island sites in 2008 or 2009, due to lack of successful sets. Instead, we have collected samples of oyster tissue from natural JOD outbreaks that occurred in Rhode Island farms in July 2007 and August Mortalities due to JOD varied from 70 90% depending on the strain used at the farm. These natural challenges show that the Rutger s NEHY strain shows increased tolerance to JOD when compared to a local strain from Green Hill Pond, Rhode Island. Five experimental challenges with Roseovarius crassostreae, causative agent of Juvenile Oyster Disease (JOD), were performed at URI using the different populations sent by Rutgers University. Statistical differences in survival were observed between infected and control oysters. Oyster survival time ranged from 4 to more than 90 days (survivors), indicating strong differences in susceptibility to JOD between different populations and oysters within a population. We collected more than 3000 oyster DNA samples to be used on genetic analysis and mapping. This collection includes samples collected pre, during, and post-mortality, allowing us to determine shifts in the frequency of genetic markers due to JOD mortality and use that information in identifying genetic regions involved in JODresistance. The challenge using the F2L oysters showed the largest statistical difference between the challenged and control populations, so it was used to identify putative regions of JOD resistance. Rutgers University researchers selected 60 oysters before the challenge (B), 60 survivors after the challenge (S), and 60 oysters died during the challenge for genotyping (D). RU selected and genotyped 257 genetic markers in the 180 F2L oysters from the challenge experiment. The markers included 90 microsatellites, 2 single-nucleotide polymorphism (SNP) markers, and 155 amplified fragment length polymorphism (AFLP) markers. Most of the microsatellites are genic markers that were developed from expressed sequence tags (ESTs), while many of the ESTs are derived from challenging experiment or related to host-defense. Differences in genotype frequency among the three samples (B = before, S = survivors, and D = dead) were tested and 28 of the 257 loci showed significant association with JOD resistant. The markers included 11 microsatellites, 1 SNP and 16 AFLP markers. Twenty-two of the 28 candidate resistance markers were mapped onto a genetic map. The distribution of the candidate resistance markers is clearly not random among chromosomes and chromosome regions. The markers formed 8 well-defined clusters, providing strong evidence that these regions contain JOD-resistance genes. Interestingly, four loci were also identified as MSX/Dermo resistance loci. Survivors from the natural and experimental challenges are currently being kept by farmers at 2 different locations in Rhode Island. Some of these survivors were spawned at Roger Williams University in March May The seed is currently kept in upwellers to be

4 distributed to interested farmers. This potentially JOD disease-resistant broodstock will be available to interested hatcheries. WORK PLANNED (POTENTIAL FUTURE WORK): - As part of an ongoing project funded by NE SARE, we will determine the field performance (growth and survival) of the resulting broodstock in locations that experience periodic outbreaks of JOD and moderate dermo disease pressure. This will indicate if the broodstock shows improved resistance to JOD. We will also perform experimental challenges using Roseovarius crassostreae (causative agent of JOD) as part of a project on marker development funded by the Agricultural Research Services to the East Coast Shellfish Breeding Consortium. - We will involve farmers and the RWU and RU hatcheries in maintaining this broodstock for future studies and line development. - The large collection of oyster samples gathered in this study would be useful to us and other researchers in further studies needed to fine-map and identify the causal genes of JOD disease resistance. IMPACTS: - We have identified 8 genomic regions or major genes are involved in JOD resistance in the eastern oyster. The identification of candidate resistance markers and genomic regions that harbor resistance genes is a significant first step toward the identification of resistance genes and marker assisted selection. - We have optimized the conditions for bath challenge experiments with Roseovarius crassostreae using wild oyster spat, which can be used in future studies identifying genetic mechanisms of disease resistance. - We have collected more than 3000 samples of oysters with different levels of susceptibility/resistance to JOD for genotyping. This will hopefully lead to the identification of markers associated with resistance with JOD that could be used in Marker-Assisted-Selection for breeding. - This knowledge will benefit the scientific community by providing new tools for oyster disease research and ultimately the oyster industry by providing means to efficiently and rapidly develop oyster strains resistant to diseases.

5 SUPPORT: NRAC- OTHER SUPPORT TOTAL YEAR USDA UNIVERSITY INDUSTRY OTHER OTHER TOTAL SUPPORT FUNDING FEDERAL 2008 $64,257 $1,500* $65, $116,841 $1,500* $116, $28,170 $28,170 TOTAL $209,269 $3,000* $209,269 *Funding from the RI Agricultural Experiment Station in support an undergraduate research Fellow working on the project.

6 PUBLICATIONS, MANUSCRIPTS, OR PAPERS PRESENTED: Markey, K., Proestou, D., Korun, J., Leavitt, D., and Gomez-Chiarri M Roseovarius oyster disease outbreaks in Rhode Island coastal salt ponds. 100 th Meeting of the National Shellfisheries Associations, Providence, RI. Markey, K., Leavitt, D., Tammi, K., and Gómez-Chiarri M Growth and survival of three strains of Crassostrea virginica in Rhode Island the 2008 season. Northeastern Aquaculture Conference and Exposition. Portland, Maine. Markey, K., Leavitt, D., Tammi, K., and Gómez-Chiarri M Juvenile Oyster Disease, a (manageable) curse for Rhode Island s oyster aquaculture. Abstract submitted for presentation at the 101th meeting of the National Shellfisheries Association, Savannah, Georgia. Gómez-Chiarri, M., Markey, K., Leavitt, D., and Guo X. Development of a line of Eastern oysters (Crassostrea virginica) with resistance to JOD, MSX disease, and dermo disease. Abstract in preparation for the Northeastern Aquaculture Conference and Exposition, December Guo, X., Zhang, L., Gómez-Chiarri, M. Identification of QTLs associated with disease resistance to Juvenile Oyster Disease in the Eastern Oyster, Crassostrea virginica (in preparation).

7 FINAL TECHNICAL REPORT Development of JOD-Resistant Lines and Markers for Eastern Oyster Aquaculture PROJECT CODE: SUBCONTRACT/ACCOUNT NO: Q321501/Q Grant Number: and PREPARED BY: Marta Gómez-Chiarri Project Coordinator 6/25/2010 Date

8 Development of JOD-Resistant Lines and Markers For Eastern Oyster Aquaculture PROJECT TITLE: Aquaculture PROJECT CODE: Development of JOD-Resistant Lines and Markers For Eastern Oyster SUBCONTRACT/ACCOUNT NO: Q321501/ Q Subaward Number: and (year 1) REPORTING PERIOD: October 2007 May 2010 FUNDING LEVEL: $209,268 (total award) PARTICIPANTS: Marta Gómez-Chiarri, University of Rhode Island (URI); Ximing Guo, Rutgers University (RU); Dale Leavitt, Roger Williams University (RWU); Perry Raso, Ocean State Aquaculture (OSA). PROJECT OBJECTIVES: The identification and mapping of genetic markers associated with disease resistance is an important step in the investigation of mechanisms of disease resistance and in the development of breeding programs (Yu and Guo, 2006). The goal of this project is to identify markers associated with resistance to Juvenile Oyster Disease (JOD) in Eastern oysters, C. virginica through population-based association studies. The specific objectives of this NRAC-funded project are to: 1) Collect spat from wild populations from two locations in the Northeast subject to different environmental conditions and disease pressures (URI, RWU, and RU, year 1). 2) Experimentally challenge spat with JOD, collect samples for genotyping and association analysis, and select for survivors of the disease (URI, Year 2). 3) Identify markers associated with disease-resistance to JOD by association analysis of a large number of genetic markers (RU, Years 1 & 2). 4) Create new lines of JOD-resistant oysters by breeding survivors from the JOD challenges (RWU and RU, Year 2). ANTICIPATED BENEFITS: This research will: a) Identify markers associated with resistance to JOD; b) improve our basic knowledge of the genetic mechanisms of resistance to JOD; c) provide genetic tools to be used in the identification and creation of resistant strains through marker-assisted selection (MAS); and d) create new lines resistant to JOD and well-adapted to local growing conditions in Southern New England. This knowledge will benefit the scientific community by providing new tools for oyster disease research and ultimately the oyster industry by providing means to efficiently and rapidly develop oyster strains resistant to diseases. METHODS AND PROCEDURES Oysters: Wild oyster spats (less than 30 mm in shell height) were collected by Rutgers

9 University from Cape May, New Jersey, in October 2007 and January 2008, and sent to the University of Rhode Island for challenge experiments. In addition to the wild oyster spat, Rutgers University also produced a synthetic population of spat in The synthetic population, F2L, is a F2 population produced from F1 hybrids between Rutgers NEH (Northeastern high survival line) and wild oysters from Louisiana. The NEH has shown some resistance to JOD (see below), while the Louisiana oysters had no known histories of JOD exposure. In addition, two more synthetic populations produced by RU in 2009 as part of their ongoing breeding program (F309, LABC, and GX09) and were sent to URI. Unfortunately, no wild spat was available from Rhode Island sites in 2008 or 2009, due to lack of successful sets. Instead, we have collected samples of oyster tissue from natural JOD outbreaks that occurred in Rhode Island farms in July 2007 and August Mortalities due to JOD varied from 70 90% depending on the strain used at the farm. These natural challenges show that the Rutger s NEHY strain shows increased tolerance to JOD when compared to a local strain from Green Hill Pond, Rhode Island. Experimental challenges: Five experimental challenges with Roseovarius crassostreae, causative agent of Juvenile Oyster Disease (JOD), were performed at URI in November 2008 (wild spat from NJ), January March 2009 (F2L population), and January March 2010 (F309, LABC, and GX09), using the different populations sent by Rutgers University. For each challenge experiment, spat per tank (18 ± 5 mm for F2L challenge) were placed in 40 liter aquaria filled with artificial seawater at 18 C and 20 psu and slowly acclimated in the period of a week to a salinity of 28 psu and C. Oysters were maintained in a temperature-controlled room at 25 C with a light/dark cycle of 12h/12h. Oysters (2 tanks) were experimentally challenged by immersion in 10 5 colony forming units per liter of water of Roseovarius crassostreae strain CV T. A control set of 2 tanks received no bacteria. Oysters were fed every other day with algal paste (Reed Mariculture) and 20% water changes were performed. Water quality (ammonia, DO, nitrites, and nitrates) was checked twice per week and stayed within optimal parameters throughout the experiment. Tanks were checked for mortalities every 2 3 days for 28 days, and oyster meats from recently dead and gaping oysters were collected for DNA analysis. Challenges were run for 3 months or until mortality reached 80%. A sample of 100 oysters was also collected before challenge and from the survivors for the challenge. Significant differences in mortality between control and challenged oysters and between populations were determined using log-rank survival. Genotyping: Oysters from the F2L 2009 challenge (60 collected prior to the challenge B; 60 survivors S, and 60 oysters that died during the challenge D) were used for genotyping. The goal was to identify loci that show significant differences in genotype or allele frequency among the three samples. Genotypes that have significant increases in frequency among the survivors and significant decrease among the dead or vice visa are considered as possible markers for JOD-resistance. The position on the genetic map, such as clustering with each other, should provide further evidence for their association with JOD-resistance. We selected and genotyped 257 genetic markers in the 180 F2L oysters from the challenge experiment. The markers included 90 microsatellites, 2 single-nucleotide polymorphism (SNP) markers, and 155 amplified fragment length polymorphism (AFLP) markers. Most of the microsatellites are genic markers that were developed from expressed sequence tags (ESTs)

10 (Wang and Guo, 2007; Wang et al., 2009). Many of the ESTs are derived from challenging experiment or related to host-defense (Tanguy et al., 2004; Quilang et al., 2007). DNA was extracted using the Qiagen DNeasy Tissue DNA extraction kit. SSR primers were labeled with fluorescence dyes (FAM, NED, VIC and PET, ABI) for genotyping using an ABI 3130xl genetic analyzer. PCR was carried out in 10 µl solution containing 1X PCR buffer with mm MgCl2, 1.0 mg/ml BSA, 0.2 mm dntps, 25 U of Taq DNA polymerase, 1 M of each primer, and ng of oyster genomic DNA. PCR was conducted on an ABI GeneAmp 9700 system with the following protocol: an initial denaturing for 2 min at 95 C followed by cycles of 95 C for 30 s, C for 1 min, and 72 C for 2 min; and a final extension at 72 C for 10 min. SNPs were genotyped using the Tm-shift assay with real-time PCR (Wang et al., 2005). Data analysis: Genotype frequencies were calculated for each sample of 60 oysters at each locus. Differences in genotype frequency among the three samples (B = before, S = survivors, and D = dead) were tested with chi-square test. For loci with more than 4 genotype classes, allele frequency was determined and tested. Possible JOD-resistant markers were identified with the following criteria: 1) the markers have significant (p < 1) differences in genotype frequency between the S and D oysters after JOD challenge; 2) the genotype frequency should be in the order of S > B > D or S < B < D; and 3) the differences between S and B, and between D and B do not need to be highly significant, but the average p-value of the three chi-square tests (S vs, D, S vs. B and D vs. B) must be smaller than To determine if these markers linked to each other (evidence for linkage to resistance gene) or randomly distributed (random variation in frequency), we mapped all possible loci to a genetic map. Progeny belonging to a single F2 family were identified by pedigree analysis and used for linkage mapping under the cross-pollen model of JoinMap 4.0 (Van Ooijen, 2006). Breeding: Survivors from the NEH line (deployed during the summer of 2008) that experienced a natural outbreak of JOD during the summer of 2008 at two farms located in Rome Point and Portsmouth (RI) were collected in February 2010 and brought to the Roger Williams Hatchery for spawning using routine procedures. Oysters were set on microcultch and kept in the hatchery until a size of 1-3 mm. Oyster spats will be distributed to farmers for evaluation of field performance in locations that experience high JOD pressure and moderate dermo/msx pressure during the summer of RESULTS AND DISCUSSION Statistical differences in survival were observed between infected and control oysters for all challenge experiments (not shown). Oyster survival time ranged from 4 to more than 90 days (survivors), indicating strong differences in susceptibility to JOD between different populations (F2L versus F2L, p<01 log rank survival analysis) and oysters within a population (Figure 1). We collected more than 3000 oyster DNA samples to be used on genetic analysis and mapping from all the different challenges (not shown). This collection includes samples collected pre, during, and post-mortality, allowing us to determine shifts in the frequency of

11 genetic markers due to JOD mortality and use that information in identifying genetic regions involved in JOD-resistance. The challenge using the F2L oysters showed the largest statistical difference between the challenged and control populations, so it was used to identify putative regions of JOD resistance. We selected 60 oysters before the challenge (B), 60 survivors after the challenge (S), and 60 oysters died during the challenge for genotyping (D). The goal is to identify loci that show significant differences in genotype or allele frequency among the three samples. Genotypes that have significant increases in frequency among the survivors and significant decrease among the dead or vice visa are considered as possible markers for JODresistance. The position on the genetic Fig 1: Cumulative percent mortality (y axis) of oysters from synthetic populations F2L and F2M after exposure to Roseovarius crassostreae. Date (month/day) is in the x axis. Cumulative mortality in control, non-challenged oysters was 30% at the end of experiment. map, such as clustering with each other, should provide further evidence for their association with JOD-resistance. At the defined criteria described in the methods, 28 of the 257 loci showed significant association with JOD resistant (Table 1). The markers included 11 microsatellites, 1 SNP and 16 AFLP markers. Twenty-two of the 28 candidate resistance markers were mapped on the genetic map. The genetic map consisted of 181 loci in 10 linkage groups (LGs) in accordance with the haploid chromosome number (Figure 2). The distribution of the candidate resistance markers is clearly not random among chromosomes and chromosome regions. For example, LGs 1, 6 and 10 had 17 of the 22 mapped candidate markers, while LGs 4, 5 and 8 had none. The markers formed well-defined clusters on LG 1, 6, 7 and 10. Eight clusters (or pairs of markers) were identified on the genetic map, providing strong evidence that these regions contain JOD-resistance genes. Four loci were also identified as MSX/Dermo resistance loci: RUCV395, RUCV066/131, RUCV397/423, and SPI-224 suggesting that some of the mechanisms of resistance against bacterial (JOD) and parasitic (Dermo and MSX) diseases may be common. Survivors from the natural and experimental challenges are currently being kept by farmers at 2 different locations in Rhode Island. Some of these survivors were spawned at Roger Williams University in March May The seed is currently kept in upwellers to be distributed to interested farmers. This potentially JOD disease-resistant broodstock will be available to interested hatcheries. CONCLUSIONS We have genotyped 257 genetic markers in 180 oysters from a challenge experiment. Analysis of genotype frequency changes identified 28 candidate resistance markers. Mapping

12 analysis shows that there are at least eight genomic regions or major genes are involved in JOD resistance in the eastern oyster. The identification of candidate resistance markers and genomic regions that harbor resistance genes is a significant first step toward the identification of resistance genes and marker assisted selection. Further studies are needed to fine-map and identify the causal genes. REFERENCES Quilang, J., S. Wang, P. Li, J. Abernathy, E. Peatman, Y. Wang, L. Wang, Y. Shi, R. Wallace, X. Guo, and Z. Liu Generation and analysis of ESTs from the eastern oyster, Crassostrea virginica Gmelin and identification of microsatellite and SNP markers. BMC Genomics, 8:157. Tanguy, A., X. Guo and S.E. Ford Discovery of genes expressed in response to Perkinsus marinus challenge in eastern (Crassostrea virginica) and Pacific (C. gigas) oysters. Gene, 338: Van Ooijen, J.W JoinMap 4, Software for the calculation of genetic linkage maps in experimental populations. Kyazma B.V., Wageningen, Netherlands. Wang J, Chuang K, Ahluwalia M, Patel S, Umblas N, Mirel D, Higuchi R, Germer S (2005) High-throughput SNP genotyping by single-tube PCR with T-m-shift primers. Biotechniques 39: Wang, Y. and X. Guo Development and characterization of EST-SSR markers in the eastern oyster Crassostrea virginica. Marine Biotechnology, 9, Wang, Y., Y. Shi and X. Guo Identification and characterization of 66 EST-SSR markers in the eastern oyster Crassostrea virginica (Gmelin). J. Shellfish Res., 28(2):

13 Table 1. Candidate JOD-resistance markers identified by differences in genotype frequency among dead, survivors and oysters sampled before a JOD-challenge, and their genomic positions. Co-dominant markers are bold. Marker Survivor/Dead (p-value) Survivor/Before (p-value) Dead/Before (p-value) Average (p-value) Linkage Group Position (cm) RUCV RUCV405B F2f F1f F2f F1f F2f RUCV405C C6f F2f H3f RUCV RUCV C6f Cvi2m RUCV SPI H3f RUCV RUCV RUCV Cvi C6f Unassigned F3f Unassigned C6f Unassigned C6f Unassigned F1f Unassigned C6f Unassigned

14 Figure 2. A genetic map of the eastern oyster showing the distribution of candidate JODresistance markers (red) in eight clusters representing genomic region harboring JODresiatance genes. Asterisks designates significant association with JOD-rsistance: *, p < 0.10; **, p < 5; ***, p < 1; and ****, p < RUCV395** RUCV424s RUCV405B**** F1f358 C6f58 RUCV399 F3f367 C7f199 F2f431 RUCV876 H3f148 RUCV409 F2f268** F2f301s F2f435 F1f125** F2f233 F2f367 H3f204 C7f183 F2f436* F1f263* RUCV046 F3f254 C7f361 RUCV113 RUCV061 RUCV374 RUCV287 RUCV119 RUCV051 RUCV RUCV073 F2f505 C6f113 RUCV075 H3H321 C6f198 cvi2k14 C6f301 Cvi2i23 H3f131 F2f293 F2f82 Cv01-03 C7f275 RUCV230 RUCV753 F1f179s F2f197s C6f245s F2f276** C7f119 H3f179 F2f181 H3f130 H3f154 BD-90 C6f169 F2f152 Cvi2j RUCV297 F3f224 F2f147 RUCV003 C7f372 RUCV114 F2f355 H3f195 RUCV851 RUCV769s RUCV405C3** C6f101 RUCV156 C7f H3H311 H3f112 F3f137 H3f111 F2f235 Cvi8 Cvi13 F3f372 F3f223 F2f236 H3f226 C7f278 RUCV013 F3f386 F2f178 RUCV772 RUCV045 F3f134 H3f276 F1f337 H3f274 RUCV183 RUCV027 F1f215 F1f226 H3f RUCV717 H3f267 RUCV108 H3H377 C7f352 C7f452 RUCV373 H3f130 F2f170 C7f175 C7f336 cvi9 C7f cvi2i20 C6f161** F2f274** H3f65* F1f609 C7f329 RUCV274 C6f124 F2f270 Cvi1248 C6f339 RUCV025 RUCV008 F2f239 RUCV Cv01-d18 C6f99 C7f59 C7f RUCV788 RUCV164 RUCV112 RUCV388 RUCV026 H3f233* RUCV370** RUCV423** RUCV397* C7f249 F3f RUCV131* C6f445 RUCV109 cvi6 F1f64 RUCV066* C6f315** Cvi2m10* F3f91 RUCV279 Cvo1-c11 C7f334 F3f C6f411 RUCV834*** spi-224*** 42.7 F3f C6f192 C7f F2f138 RUCV748 C7f246 C7f122 C6f105 RUCV270 Cvi12*** C6f111 H3f448 F2f205 C6f112 F3f273 Cvi2m RUCV834C RUCV256 RUCV018

15 IMPACTS: - We have identified 8 genomic regions or major genes are involved in JOD resistance in the eastern oyster. The identification of candidate resistance markers and genomic regions that harbor resistance genes is a significant first step toward the identification of resistance genes and marker assisted selection. - We have optimized the conditions for bath challenge experiments with Roseovarius crassostreae using wild oyster spat, which can be used in future studies identifying genetic mechanisms of disease resistance. - We have collected more than 3000 samples of oysters with different levels of susceptibility/resistance to JOD for genotyping. This will hopefully lead to the identification of markers associated with resistance with JOD that could be used in Marker-Assisted-Selection for breeding. - This knowledge will benefit the scientific community by providing new tools for oyster disease research and ultimately the oyster industry by providing means to efficiently and rapidly develop oyster strains resistant to diseases. SUPPORT: NRAC- OTHER SUPPORT TOTAL YEAR USDA UNIVERSITY INDUSTRY OTHER OTHER TOTAL SUPPORT FUNDING FEDERAL 2008 $64,257 $1,500* $65, $116,841 $1,500* $116, $28,170 $28,170 TOTAL $209,269 $3,000* $209,269 *Funding from the RI Agricultural Experiment Station in support an undergraduate research Fellow working on the project. PUBLICATIONS, MANUSCRIPTS, OR PAPERS PRESENTED: Markey, K., Proestou, D., Korun, J., Leavitt, D., and Gomez-Chiarri M Roseovarius oyster disease outbreaks in Rhode Island coastal salt ponds. 100 th Meeting of the National Shellfisheries Associations, Providence, RI. Markey, K., Leavitt, D., Tammi, K., and Gómez-Chiarri M Growth and survival of three strains of Crassostrea virginica in Rhode Island the 2008 season. Northeastern Aquaculture Conference and Exposition. Portland, Maine. Markey, K., Leavitt, D., Tammi, K., and Gómez-Chiarri M Juvenile Oyster Disease, a (manageable) curse for Rhode Island s oyster aquaculture. Abstract submitted for presentation at the 101th meeting of the National Shellfisheries Association, Savannah, Georgia. Gómez-Chiarri, M., Markey, K., Leavitt, D., and Guo X. Development of a line of Eastern oysters (Crassostrea virginica) with resistance to JOD, MSX disease, and dermo disease. Abstract in preparation for the Northeastern Aquaculture Conference and Exposition, December 2010.

16 Guo, X., Zhang, L., Gómez-Chiarri, M. Identification of QTLs associated with disease resistance to Juvenile Oyster Disease in the Eastern Oyster, Crassostrea virginica (in preparation).