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 National Cold Water Marine Aquaculture Center: Orono, ME
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ARS Home Northeast Area Orono, Maine National Cold Water Marine Aquaculture Center Research





- Peterson, Brian
- Proestou, Dina
- Polinski, Mark
- May, Samuel
- 2023
- 2022
- 2021
- 2020
- Publications
- Aquaculture (106)

Research Project: Genetic Improvement of North American Atlantic Salmon and the Eastern Oyster for Aquaculture Production **Location:
                            National Cold Water Marine Aquaculture Center** 2022 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 **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 third annual report for Project 8030-31000-005-000D. With respect to Atlantic salmon, carcass weight, fillet fatty acid levels, and astaxanthin were measured and analyzed from fish cultured in net pens. A multi-trait selection index was implemented for growth and sea lice resistance using genotypic and phenotypic estimated breeding values. Broodstock were selected and offspring produced. The improved germplasm was released to industry stakeholders through a material transfer research agreement.
A new genotyping array (a 50K single nucleotide polymorphism, SNP chip) was developed and utilized to improve chromosomal mapping of Atlantic. A complete chromosome level assembly has been submitted and deposited at the National Center for Biotechnology Information genome database (Accession GCA_021399835.1.) The high-quality genome assembly is composed of 27 chromosome sequences with a scaffold N50 of ~16 million base-pairs. This reference genome map is now an essential resource for conducting genome association studies in Atlantic salmon and detecting genomic markers during selective breeding. 
The new genotyping array has been made commercially available and has attracted much interest from breeding, biotechnology, and diagnostic companies associated with the aquaculture industry. It has been used to genotype over 4,000 fish from the National Cold Water Marine Aquaculture Center (NCWMAC) breeding program and approximately 75% of the genotyping targets are putative high-quality markers for trait selection.
We also genotyped ~1,000 of our 2014 year class (YC) fish phenotyped for sea lice resistance traits in 2015 and ~200 parents of the 2018 YC fish to generate genomic breeding value (GBV) predictions for the parents. The GBV accuracy was 0.41 and did not differ significantly from traditional pedigree-based breeding values for this one generation, possibly due to insufficient sample size. We subsequently genotyped ~1,000 2018 YC fish phenotyped for sea lice resistance and ~2,000 potential breeders to generate GBVs and improve the accuracy of selection for improved resistance in the 2021-2022 breeding season. No major quantitative trait loci (QTL) were found in genome wide association analyses of families from the 2014 and 2018 YC), indicating that many genes of small effect control sea lice resistance trait in this salmon breeding population.
In anticipation of examining genotype X environment interactions, two strains of Atlantic salmon (St. John River and Gaspe) are being raised at the Conservation Fund’s Freshwater Institute located in West Virginia, the Northern Aquaculture Demonstration Facility in Wisconsin, and at the NCWMAC in Maine. Juvenile salmon were pit tagged at all three locations, stocked into recirculated aquaculture systems, and will be evaluated for growth and fillet characteristics next year.
The NCWMAC Shellfish Genetics Laboratory in Kingston, Rhode Island, continued its integration of laboratory disease challenge protocols in family-based breeding by measuring Dermo resistance traits and genetic parameters in forty, 2020 YC eastern oyster families from the Virginia Institute of Marine Science (VIMS) breeding program. 2400 oysters were evaluated. Significant differences in resistance phenotypes across families (e.g. survival ranged from 38 to 97 %) and modest heritability for survival in response to challenge (h2 = 0.147) were observed. A similar experiment, using material from forty families from the 2021 YC is currently underway. In addition, 1120 Dermo-challenged samples from the 2019 YC and 2365 samples from the 2020 YC were genotyped at 60K Single Nucleotide Polymorphism (SNP) loci using the eastern oyster SNP chip developed in collaboration with the Eastern Oyster Breeding Consortium (EOBC). A genome-wide association study (GWAS) will be performed using the phenotype and genotype data from the 2019, 2020, and 2021 YCs to identify genomic regions/markers associated with Dermo-resistance traits. Genotype data will also be used to calculate genomic estimated breeding values for Dermo resistance within the VIMS breeding population.
To better understand the genomic basis for tolerance in the eastern oyster response to Dermo, samples from tolerant and susceptible oyster families challenged with increasing doses of the parasite were subject to global transcriptomic analyses. Expression profiles showed significant clustering of samples by treatment and phenotype. Over 7,000 transcripts were differentially expressed between control and injected groups across families and doses; however, only 139 and 156 transcripts were unique in tolerant and susceptible phenotypes respectively. Transcripts correlated with phenotype are associated with immune response, signaling, and programmed cell death. This work identified candidate genes for Dermo tolerance that can be investigated further.
The NCWMAC Shellfish Genetics Laboratory is also working on an improved eastern oyster genome assembly with cooperators at the University of Missouri and Rutgers University to facilitate more precise genome association studies, QTL analyses, locus identification for marker-assisted selection, genomic selection, and gene expression studies. New long read and positional sequence data were generated and assembled according to the Hifiasm bioinformatic pipeline. Additional polishing steps were applied to correct sequencing errors, purge haplotigs, and remove duplicated sequence. Preliminary results suggest the new genome assembly is 13X more contiguous, more complete, and in much better agreement with the existing eastern oyster high-density linkage map.
Foundational work has begun for the establishment of an eastern oyster breeding program for the Northeast region. Wild and cultured oyster populations from the region are being sampled and genotyped on the 60K SNP chip for a population genomic survey that will inform formation of the breeding population. Methods also are being developed to improve hatchery protocols. 100 local broodstock oysters were non-destructively sampled for RNAseq and Whole Genome Bisulfite Sequencing at different time points throughout and sacrificed and sexed at the end of conditioning to identify biomarkers associated with sex and determine how early during the process the markers are expressed. Selective breeding programs and hatchery operators will benefit greatly from advanced knowledge of each animals’ sex prior to spawning.
A low-density genotyping panel for genotype imputation is needed for cost-effective genomic selection. Simulations of breeding programs informed by the genomic architecture and diversity of the eastern oyster determined that genotyping phenotyped individuals and selection candidates with a panel containing 250 SNP loci resulted in optimum accuracy of imputation and GBVs. Results of the simulations were validated with empirical data in collaboration with the ARS Pacific West Area and Texas A&M Corpus Christi.
Low-density genotyping panels are also used for pedigree inference in breeding programs that culture families in mixed groups. SNP loci are typically used for pedigree inference, but microhaplotypes (short regions of the genome containing multiple SNPs) have been demonstrated to be more informative for this purpose. We have developed bioinformatic methods of identifying microhaplotypes and estimating allele frequencies from pool-seq and low-depth whole genome sequencing data to design microhaplotype panels and reduce genotyping costs for pedigree inference.
The NCWMAC developed a Non-Assistance Cooperative Agreement (NACA) with the University of Maine (UMaine). This year, the University of Maine Aquaculture Experimental Station (AES) continued to build on research infrastructure equipment and personnel to address the USDA ARS Northeast Strategic Initiatives. 
University of Maine students and faculty contributed to research on the performance of genomically-selected lines of Eastern oysters with the Eastern Oyster Breeding Consortium under a grant from the Atlantic States Marine Fisheries Commission. This has involved the propagation and monitoring of field performance of two genetic lines of oyster selected for improved growth in northern New England.
The University of Maine’s Aquaculture Research Institute (ARI) in collaboration with the NCWMAC scientists established new methods to analyze water and fish tissue samples for geosmin and 2-methylisoborneol. These compounds are the primary cause for off-flavor tastes in recirculating aquaculture systems and pond-raised fish but can also impart off-flavors in drinking water. We collaborated with Markes International to validate new methods for detection for geosmin and 2-methylisoborneol that improve accuracy, reduce sample preparation time, and increase total sample throughput allowing us to provide this service to more aquaculture farmers at a significantly reduced cost compared to existing methods.
The NCWMAC developed a Non-Assistance Cooperative Agreement (NACA) with Auburn University. This year, funds were used for personnel and project-specific equipment/supply purchases at the Auburn University Shellfish Laboratory (AUSL) in Dauphin Island, Alabama. These investments will expand the ability to evaluate the response of selectively bred lines of oysters with different ploidy to a range of environmental stressors and various culture methods.
AUSL continues to maintain oysters in support of all research programs at its open-water farm site in Grand Bay, Alabama. A regional selective breeding program to develop other diploid lines targeting shell shape characteristics and broadly diverse lines for Gulf-wide use in varying salinity environments is also ongoing. Field trials of AUSL oyster lines continue at several cooperating commercial oyster farms. All sample processing for these trials was completed in Spring of 2022 and data analysis is underway. **Accomplishments**
                                

1. Improved North American Atlantic salmon smolt. Commercial salmon farms are expected to increase 5-fold over the next 3 years and will require a fast-growing fish to compete in a global market. A higher weight at smolt usually results in a faster time to market and a higher chance of survival. When the National Coldwater Marine Aquaculture Center selective breeding program in Franklin, Maine, began in 2007, the average weight at smolt was 65 grams per fish. After four generations of selecting for growth, the average weight at smolt more than doubled at 167 grams per fish. This improved germplasm has been transferred to industry stakeholders and will have an immediate economic impact on reducing the time to market and profitability. 2. New method to detect off-flavor in water and fish tissue. An increase in land-based aquaculture systems in the U.S. to produce Atlantic salmon is expected and will require methods to monitor off-flavor to ensure fish are acceptable to consumers. Previous methods could only process 10 samples a day and cost $120 per sample. University of Maine researchers in collaboration with ARS scientists at Franklin, Maine, developed a new method of detecting geosmin and 2-methylisoborneol, the two compounds that cause off-flavor in water and fish tissues. The new method utilizes a high capacity sorptive extraction with gas chromatography/mass spectrometry detection. The new method can process at least 40 samples per day at a cost of $40 per sample. Industry and stakeholders such as the Institute of Marine and Environmental Technology, the Freshwater Institute, Aquacon, and Superior Fresh are currently sending their water and fish tissue samples to University of Maine. This new technology is reducing the cost to measure off-flavor by two thirds and will save the industry thousands of dollars each year. **Review Publications**
                            *Witkop, E.M., Proestou, D.A., Gomez-Chiarri, M. 2022. The expanded Inhibitor of Apoptosis gene family in oysters possesses novel domain architectures and may play diverse roles in apoptosis following immune challenge. BMC Genomics. https://doi.org/10.1186/s12864-021-08233-6.*
                                *Witkop, E., Wikfors, G.H., Proestou, D.A., Markey Lundgren, K.R., Sullivan, M.E., Gomez-Chiarri 2022. Perkinsus marinus suppresses in vitro eastern oyster apoptosis via IAP-dependent and caspase-independent pathways involving TNFR, NF-kB, and oxidative pathway crosstalk. Developmental and Comparative Immunology. https://doi.org/10.1016/j.dci.2022.104339.*
                                *Bledsoe, J.W., Pietrak, M.R., Burr, G.S., Peterson, B.C., Small, B.C. 2022. Symmetry of tissue-specific immune expression and microbiota profiles across mucosal tissues of Atlantic salmon (Salmo salar) highlight host-microbe coadaptations that are marginally perturbed by functional feeds. Animal Microbiome. https://doi.org/10.1186/s42523-022-00173-0.*
                                *Zhao, J., Vendramin, N., Cuenca, A., Polinski, M.P., Hawley, L., Garver, K. 2021.  Pan-Piscine Orthoreovirus (PRV) detection using reverse transcription quantitative PCR. Pathogens. https://doi.org/10.3390/pathogens10121548.*
                                *Burr, G.S., Peterson, B.C., Gaylord, T.G. 2022. Effects of Histidine on growth performance of North American Atlantic Salmon. Journal of the World Aquaculture Society. 10.1111/jwas.12873.*


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