Research Project: Genetic Improvement of North American Atlantic Salmon and the Eastern Oyster for Aquaculture Production

Location: National Cold Water Marine Aquaculture Center

2024 Annual Report

Objectives
Objective 1: Expand and optimize approaches for multi-trait selection in Atlantic salmon. Component 2: Problem Statement 2A Subobjective 1A. Develop a multi-trait selection index in Atlantic salmon germplasm selected for carcass weight, fillet fatty acid levels, and sea lice resistance. Subobjective 1B. Generate a reference genome assembly and genomic research resources for North American Atlantic salmon. Subobjective 1C. Evaluate and validate genome-enabled selection strategies for resistance to sea lice in North American Atlantic salmon. Subobjective 1D. Characterized genetic × environment interactions of Atlantic salmon selected for performance in net pens through evaluations in recirculating aquaculture systems. Objective 2: Advance genetic improvement technologies for the eastern oyster. Component 4: Problem Statement 4A Subobjective 2A. Integrate laboratory disease challenge protocol for measuring Dermo resistance in a family-based breeding program. Subobjective 2B. Discover and validate candidate SNP markers for Dermo resistance from RNA-seq data. Objective 3: Improve Fish Health in Atlantic salmon aquaculture by determining the susceptibility of North American Atlantic salmon selected for performance to new and emerging pathogens and develop strategies to improve fish health. Component 2: Problem Statement 2A Objective 4: Optimize production efficiency in recirculating aquaculture systems. This new objective will focus on developing breeding strategies and management practices that ensure the productivity and profitability of raising fish in recirculating aquaculture systems while maintaining high standards of animal well-being.

Approach
U.S. marine aquaculture industries, which consist primarily of molluscan shellfish and Atlantic salmon were valued at $192 million in 2016. Cold water marine aquaculture production has great potential for expansion, and both Atlantic salmon and eastern oysters are widely accepted as seafood by American consumers. Due to increased demand for high quality seafood and advances in genomic and breeding technologies, the East Coast marine aquaculture industry is projected to double in value over the next five years. Commercial salmon and oyster producers predominantly utilize stocks that are not many generations removed from wild, unselected stocks, so there is a need for continuous support to this industry through breeding programs. The NCWMAC is the only Federal research program supporting the U.S. cold water marine aquaculture industry by developing genetically improved salmon which are optimized for aquaculture production efficiency. Aquaculture of the eastern oyster is a large segment of shellfish aquaculture in the US, and minimal selective breeding has been accomplished in this species. In both species, there is a need to improve the performance of existing stocks. This project plan proposes to meet this need through the following objectives: 1) expand and optimize approaches for multi-trait selection in Atlantic salmon and 2) advance genetic improvement technologies for the eastern oyster. Research accomplished during this project will result in the development of genetically improved Atlantic salmon for release to U.S. producers. Experimental protocols and genomic tools developed for the selectively breeding eastern oysters will facilitate and accelerate the development of high-performing, disease resistant oyster lines and will support the East Coast shellfish aquaculture industry.

Progress Report
This is the final annual report for Project 8030-31000-005-000D. With respect to Atlantic salmon research, the objective to expand and optimize approaches for multi-trait selection was met by developing genomic tools such as a reference genome assembly for the NA SJR strain and a 50k SNP microarray. These tools have been made publicly available in the form of peer-reviewed publications and have enabled: (1) cost effective high-throughput genotyping of program animals, (2) increased genetic selection efficiency through a transition from traditional EBVs to GEBVs, and (3) characterization of genomic architecture for novel traits. Specifically, the genomic architecture of sea lice resistance was described in a peer reviewed publication, demonstrating its potential for incorporation into a multi-trait selection index. This multi-trait, genomic selection index has now been adopted into the NA Atlantic salmon breeding program for two traits: growth and sea lice resistance. In the future, we aim to use the genomic tools, collaborations, and analysis pipelines developed here to investigate additional novel traits for inclusion in the selection index. Growth performance of fish in any production system is affected by environment and genetics. The St. John River (SJR) strain of Atlantic salmon Salmo salar has been selected for growth in net pens at the National Cold Water Marine Aquaculture Center (NCWMAC), Franklin, Maine, for five generations while the Gaspe strain was previously selected for growth in tanks for two generations. It was previously shown the Gaspe strain outperformed the SJR strain in a recirculating aquaculture system (RAS). Eyed eggs from both strains were shipped separately to The Conservation Fund’s Freshwater Institute (TCFFI; Shepherdstown, West Virginia) and the Northern Aquaculture Demonstration Facility (NADF; Bayfield, Wisconsin). Eyed eggs were also maintained and grown at the NCWMAC. All studies were conducted under a range of RAS environments. Once the fish reached ~50 g/fish, they were pit tagged, weighed, and combined into tanks at each location until they reached approximately 4 kg/fish. After approximately three years, results clearly demonstrated the growth superiority of the SJR strain when compared to the Gaspe strain when reared in RAS. With respect to eastern oyster research, the objective of advancing genetic improvement technologies was met by implementing large-scale laboratory challenge experiments to characterize and quantify Dermo disease resistance within a family-based selection framework. Disease response phenotypes and high-density genotypes were obtained from ~ 6000 pedigreed individuals from three consecutive year classes. The data collected from the challenged samples were used to assess whether survival in response to challenge is a heritable trait that can be targeted for selection and to evaluate the potential of applying marker-assisted or genomic selection to this trait. To facilitate eastern oyster genetic improvement for the Northeast U.S., individuals from ~40 wild populations throughout the region were sampled and surveyed for genetic diversity. Genotype data were run through a novel mathematical algorithm to determine which populations and in what proportion to include in the base population used for breeding. Adult oysters from eight populations were collected, reproductively conditioned, and spawned. Resulting seed was deployed at a partner farm for performance evaluation in June. Low-cost genotyping techniques are key to improving selection accuracy and efficiency through genomic selection methods. Using existing eastern oyster sequence and genotype data, simulation studies were performed to test the utility of low-density marker panels and pedigree-based imputation for estimating genomic breeding values. Among the low-density panels tested (random SNPs, optimized SNPs, microhaplotypes), microhaplotypes best approximated breeding values obtained using the more costly high-density panels. The NCWMAC has a Non-Assistance Cooperative Agreement (NACA) with the University of Maine (UMaine). With respect to finfish, the Aquaculture Research Institute (ARI) worked with the USDA NCWMAC on a study looking at the genomic contribution towards phenotypic differences in ISAV resistance in select Atlantic salmon families. The study indicated a wide range in family-specific susceptibility to the virus suggesting selective breeding strategies may prove effective in promoting ISAV resistance. University of Maine Cooperative Extension and ARI staff also developed a new technique for sequencing the HPR of ISAV strains from samples with very low viral loads. This new technique will allow researchers to determine the ISAV HPR strain type from samples with infection levels too low for traditional Sanger sequencing, or those that are suspected of being infected with multiple ISAV strains. ARI also continues to explore methods to detect geosmin and 2-methylisoborneol in water and fish tissue samples as a method for assessing off flavor development in recirculating or pond-based aquaculture. The oyster group at the University of Maine continued to monitor the performance of two commercial industry lines at three locations in Maine. On farm studies also evaluated the effect of gear type (traditional vs. intertidal fixed longline systems) on shell shape. The development of tools to optimize site selection of oyster farms also continued. High resolution temperature, chlorophyll, and turbidity products from the Landsat suite of satellites and Sentinel 2A/2B were validated for Maine coastal waters. These data were incorporated into many farmer-focused workshops. Additionally, a mechanistic growth model for northeast C. virginica based in DEB theory was validated and is in the final stages of being coupled to satellite products for wide scale time-to-market estimates. The NCWMAC developed a Non-Assistance Cooperative Agreement (NACA) with Auburn University (AUSL). To gain insight on the effects of ploidy and salinity on eastern oyster performance in the Gulf of Mexico, two tetraploid lines and their reciprocal crosses were created and deployed at three field sites representing a range of salinity environments. Significant line by site interactions were detected, prompting additional field trials aimed at investigating if and how the interaction effects transfer to triploid offspring. The effects of thermal and salinity stress on performance were also examined by measuring physiological and behavioral endpoints in a series of laboratory experiments. Lab experiments also examined how gametogenesis and gamete quality of diploid and tetraploid oysters are affected by salinity. Because oysters are filter feeders and human pathogens are common in the water column, behavioral feeding experiments were run observe the relationship between shell valve gaping and Vibrio counts. Diploid and triploid oysters were included in the experiments. Successful oyster spawns for breeding depend on the reproductive condition of broodstock. Reproductive condition is difficult to ascertain without time consuming and expensive histological techniques. An inexpensive method for assessing female oyster reproductive condition based on egg size during spawning was developed to aid in generating a more consistent and higher quality seed supply.

Accomplishments
1. Non-destructive tissue sampling method for eastern oyster. Developed a non-destructive sampling of eastern oyster tissue is needed for physiology, pathology, and genetics research. The USDA ARS NCWMAC developed an anesthesia and biopsy protocol for routine use in experimental work. Two experiments tested the effects of oyster age (size) and sampling frequency on sampling success. Age did not affect the ability to sample or oyster survival; however, multiple biopsies one week apart did reduce survival slightly. This method offers an effective, single-use technique for sampling tissue from live eastern oysters and has important implications for advancing genetic improvement through precision phenotyping and genomic selection.

2. Selection for Dermo disease resistance in cultured eastern oysters.. Dermo disease is an ubiquitous parasitic infection that causes tissue wasting and death among cultured eastern oyster populations. The efficacy of selection for Dermo resistance in aquaculture stocks has not been fully investigated. ARS researchers in Kingston, Rhode Island, in collaboration with partners at the Virginia Institute of Marine Science, performed large-scale disease challenge experiments using pedigreed families to estimate the potential for genetic improvement and identify the best selection approach. Challenge survival was moderately heritable and was associated with a few genetic markers that explained a small percentage of variation in the trait. Genomic selection performed significantly better than traditional methods for estimating breeding values. This work will allow for greater efficiency and precision in the development of Dermo-resistant eastern oyster aquaculture stocks.

3. Reducing the cost of genetic improvement in aquaculture.. A major impediment to aquaculture production is the lack of genetically improved animals. Application of genomic selection can quickly solve this issue, but it is unapplicable to most species due to high genotyping costs. ARS researchers (Northeast Area and Pacific West Area) and collaborators at Texas A&M Corpus Christi developed a new genotyping strategy that utilizes microhaplotype panels and a statistcial algorithm to dramatically lower genotyping costs. This strategy will allow aquaculture breeding programs to implement genomic selection in a wide range of species and provide industry with genetically improved animals.

Review Publications
Delomas, T.A., Willis, S.C. 2023. Estimating microhaplotype allele frequencies from low-coverage or pooled sequencing data. Bioinformatics. https://doi.org/10.1186/s12859-023-05554-z.
Proestou, D.A., Sullivan, M.E., Markey Lundgren, K.R., Ben-Horin, T., Witkop, E.M., Hart, K.M. 2023. Understanding Crassostrea virginica tolerance of Perkinsus marinus through global gene expression analysis. Frontiers in Genetics. https://doi.org/10.3389/fgene.2023.1054558.
Proestou, D.A., Delomas, T.A., Sullivan, M.E., Markey Lundgren, K.R. 2024. Sex-specific gene expression in eastern oyster gonad and mantle tissues. Invertebrate Biology. 143(1):e12418. https://doi.org/10.1111/ivb.12418.
Puritz, J., Guo, X., Hare, M., He, Y., Hillier, L., Jin, S., Liu, M., Lotterhos, K., Minx, P., Modak, T., Proestou, D.A., Rice, E., Tomlison, C., Warren, W., Witkop, E., Zhao, H., Gomez-Chiarri, M. 2023. A second unveiling: haplotig masking of the eastern oyster genome improves population-level inference. Molecular Ecology Resources. https://doi.org/10.1111/1755-0998.13801.
Polinski, M.P., Gross, L., Marty, G.D., Garver, K.A. 2022. Heart inflammation and piscine orthoreovirus genotype-1 in Pacific Canada Atlantic salmon net-pen farms: 2016-2019. BMC Veterinary Research. https://doi.org/10.1186/s12917-022-03409-y.
Cain, K.D., Polinski, M.P. 2023. Infectious Diseases of Coldwater Fish in Fresh Water. In: Woo, P.T.K., Subasinghe, R.P., editors. Climate Change on Diseases and Disorders of Finfish in Cage Culture. 3rd edition. Oxfordshire, London: CAB International. p. 76-124.
Legacki, E.L., Peterson, B.C., Boggs, A. 2023. Using skin mucus for the identification of ovulation biomarkers in North American Atlantic salmon (Salmo salar). Aquaculture. https://doi.org/10.1016/j.aquaculture.2023.739717.
Ott, B.D., Torrans, E.L., Griffins, M.J., Allen, P.J., Duke, M.V., Peterson, B.C., Scheffler, B.E., Hulse-Kemp, A.M. 2024. Hypothalamic Transcriptome Response To Simulated Diel Earthen Pond Hypoxia Cycles In Channel Catfish (Ictalurus punctatus). Physiological Genomics. https://doi.org/10.1152/physiolgenomics.00007.2024.
Vallejo, R.L., Pietrak, M.R., Milligan, M.T., Gao, G., Tsuruta, S., Fragomeni, B.O., Long, R., Peterson, B.C., Palti, Y. 2024. Genetic architecture and accuracy of predicted genomic breeding values for sea lice resistance in the St John River aquaculture strain of North American Atlantic salmon. Aquaculture. 586:740819. https://doi.org/10.1016/j.aquaculture.2024.740819.
Gao, G., Waldbieser, G.C., Ramey, Y.C., Zaho, D., Pietrak, M.R., Stannard, J.A., Buchman, J.T., Scheffler, B.E., Peterson, B.C., Palti, Y., Rexroad III, C.E., Long, R., Burr, G.S., Milligan, M.T. 2023. The generation of the first chromosome-level de-novo genome assembly and the development and validation of a 50K SNP array for the St. John River aquaculture strain of North American Atlantic salmon. G3, Genes/Genomes/Genetics. jkad138. https://doi.org/10.1093/g3journal/jkad138.
Guo, X., Puritz, J., Zhenwei, W., Proestou, D.A., Allen, S., Small, J., Verbyla, K., Zhao, H., Haggard, J., Chriss, N., Zeng, D., Markey Lundgren, K.R., Allam, B., Bushek, D., Gomez-Chiarri, M., Hare, M., Hollenbeck, C., Lapeyre, J., Liu, M., Lotterhos, K., Plough, L., Rawson, P., Rikard, S., Sallient, E., Varney, R., Wikfors, G., Wilbur, A. 2023. Development and evaluation of high-density SNP arrays for the eastern oyster Crassostrea virginica. Marine Biotechnology. https://doi.org/10.1007/s10126-022-10191-3.
Rounsville, T., Polinski, M.P., Marini, A., Turner, S., Vendramin, N., Cuenca, A., Pietrak, M.R., Peterson, B.C., Bouchard, D. 2024. Rapid differentiation of infectious salmon anemia virus avirulent (HPR0) from virulent (HPR) variants using multiplex RT-qPCR. Journal of Veterinary Diagnostic Investigation. 36(3):329-337. https://doi.org/10.1177/10406387231223290.
Delomas, T.A., Hollenbeck, C.M., Matt, J.L., Thompson, N.F. 2024. Microhaplotypes generate higher breeding value accuracy compared to SNPs for imputation-based breeding strategies. Aquaculture. 586:740779. https://doi.org/10.1016/j.aquaculture.2024.740779.
Hargrove, J.S., Delomas, T.A., Powell, J.H., Hess, J.E., Narum, S.R., Campbell, M.R. 2023. Efficient population representation with more genetic markers increases performance of a steelhead (Oncorhynchus mykiss) genetic stock identification baseline. Evolutionary Applications. 17(2):e13610. https://doi.org/10.1111%2Feva.13610.