Location: Cool and Cold Water Aquaculture Research
2020 Annual Report
Objectives
1: Improving performance of salmonids using selective breeding and genetic markers.
• Sub-objective 1.a. Develop SNP-based assays for parentage assignments and strains identification in rainbow trout.
• Sub-objective 1.b. Estimate genetic parameters of fillet yield in the Clear Springs Foods, Inc. commercial population.
• Sub-objective 1.c. Divergently select for fillet yield to estimate selection response, develop resource populations for physiological and genomics studies, and develop improved germplasm for release to industry stakeholders.
• Sub-objective 1.d. Assessment of genetic x environmental interactions in the NCCCWA growth line.
2: Evaluate accuracy of selection using within-family genome enabled breeding value (GEBV) predictions in rainbow trout family-based selective breeding program for bacterial cold water disease (BCWD) resistance.
3: Identification of mechanisms affecting production traits to better define phenotypes for selective breeding or to improve management practices.
• Sub-objective 3.a. Improve the rainbow trout reference genome assembly.
• Sub-objective 3.b. Identify positional candidate genes for BCWD resistance.
• Sub-objective 3.c. Determine how factors affecting nutrient partitioning and nutrient retention regulate growth performance traits and fillet yield.
• Sub-objective 3.d. Identification of mechanisms affecting egg quality and development of a transcript array to identify mechanisms impacted in poor quality eggs to suggest means of mitigation.
Approach
Rainbow trout (Oncorhynchus mykiss) are the most widely farmed cold freshwater species and the second most valuable finfish aquaculture product in the United States. The application of genomic technologies towards the genetic improvement of aquaculture species is expected to facilitate selective breeding and provide basic information on the biochemical mechanisms controlling traits of interest. In the previous project, a suite of genome tools and reagents for rainbow trout was developed to identify and characterize genes affecting aquaculture production traits. Projects concurrent with the previous project characterized the genetic variation of the National Center for Cool and Cold Water Aquaculture (NCCCWA) broodstock with respect to resistance to Bacterial Cold Water Disease (BCWD) and response to crowding stress. Specific crosses were identified that will facilitate the identification of chromosome regions and genes affecting these traits through genetic mapping and functional genomic approaches. The current project will continue the genome scans of these crosses with new sets of markers to identify positional candidate genes affecting these traits. In addition, possibilities for developing informative crosses and functional genomic approaches which target the identification of genes affecting carcass quality traits will be determined. We will also continue to identify and characterize genes in the oocyte which impact embryonic development and egg quality traits important to breeders. This information is important to gain a better understanding of the genetics of production traits and for transferring genetic information and improved germplasm from the NCCCWA selective breeding program to customers and stakeholders.
Progress Report
Over the course of the five-year project 8082-31000-012-00D, significant progress was made on all three objectives and their subobjectives, all of which aligned with Component 1 of the 2015-2019 NP 106 Aquaculture Action Plan: Selective Breeding, Directed Reproduction, and Development of Genomic Tools.
Progress towards Objective 1: Improving performance of salmonids using selective breeding and genetic markers, included the development of a fast-growth rainbow trout line. Fast-growth is one of the most important economic traits in rainbow trout aquaculture that can be improved by selective breeding of the fastest-growing fish. Our scientists have selectively bred a pedigreed, commercial-scale rainbow trout population to market weight for five generations. Compared to the unselected control line, body weight in the growth-selected line increased by approximately 12% per generation through 13 months of age. Thus, selection breeding resulted in a line that grows approximately 60% faster to and beyond standard market weight. Furthermore, the improvement in growth over commercial lines was consistent when fish were reared in different environments. This genetically-improved and highly characterized rainbow trout germplasm is available for release to stakeholders for commercial propagation. In the next project plan the NCCCWA selective breeding program will continue selection for improved performance traits (fillet yield).
After five generations of fast growth selection, our scientists began divergent selection for fillet yield in this population to create contemporary high-yield (ARS-FY-H), randomly-mated control (ARS-FY-C), and low-yield (ARS-FY-L) selection lines. Through two generations of selection, improvement in fillet yield was estimated to be 0.6 to 1.1 percentage points per generation. Importantly, selection for increased fillet yield resulted in fish with less waste fat in the viscera, and did not have adverse effects on growth or reproduction traits. Preliminary data also suggest that the ARS-FY-H line has better long-term feed conversion efficiency compared to the ARS-FY-L line. Scientists will continue this divergent fillet yield selection program in the next project plan.
Families from the growth-selected line were used in in genome wide association studies to elucidate the genetic architecture of the fillet yield trait. Genotype data from two different 50-K SNP chip identified mostly polygenic architecture for the trait with many genome loci having small effects on the overall genetic variation in the population, but potentially a couple of loci with moderate effect on the variation for the trait. We also found moderate heritability (h2 = 0.36) for the trait in this population. These findings suggest that genomic selection can substantially improve the accuracy of genetic merit predictions for this trait in rainbow trout. In the next project plan we will use progeny testing for retrospective evaluation of the accuracy of genomic selection predictions for fillet yield in the NCCCWA breeding population.
Also supporting Objective 1 was the development of a SNP panel for parentage assignment and genetic assignments in rainbow trout. Parentage assignment and population assignment are essential for selective breeding of rainbow trout. A 96-SNP panel for parentage assignment was developed and validated in rainbow trout. This SNP panel can also be used for assessment of genetic differentiation and genetic assignment of unknown individuals to reference populations. It has also has been used by commercial rainbow trout breeders and is also offered as a commercial product by aquaculture biotech companies. In the next project plan we will continue to use this assay for pedigree assignments in support of mating designs and other aspects of breeding management.
Progress towards Objective 2: Evaluate accuracy of selection using within-family genome enabled breeding value predictions in rainbow trout family-based selective breeding program for bacterial cold water disease (BCWD) resistance, included a genome-wide association study to identify SNPs associated with BCWD resistance in research and commercial rainbow trout breeding populations. Collectively, we found several moderate to large effect quantitative trait loci (QTL) on chromosomes Omy3, 5, 8, 10 and 25 which explained up to 45% of the genetic variance for BCWD resistance in these populations. We have reported that both genomic selection (GS) and marker-assisted selection (MAS) for BCWD resistance are feasible in research and commercial rainbow trout breeding populations. Following our demonstration of the effectiveness of GS for BCWD resistance, Troutlodge, Inc. has begun implementing this approach in their selective breeding program. Using the methods we developed in the BCWD study, in the next project plan we will continue to evaluate the accuracy of GS models predictions for other economically important traits in rainbow trout aquaculture.
Additional progress toward understanding the genetic architecture of disease resistance was achieved as a collaborative effort with Clear Springs Foods, Inc. We detected 10 moderate-effect quantitative trait loci (QTL) associated with resistance to infectious hepatic necrosis virus (IHNV) that jointly explain up to 42% of the additive genetic variance. The heritability estimated for the phenotype of survival status was 0.25 (± 0.07). The accuracy of genomic breeding value predictions was substantially better than the accuracy of the pedigree-based estimated breeding values (EBV). These findings indicate that implementation of genomic selection in the Clear Springs Foods selective breeding program for IHNV resistance will enhance the rate of genetic improvement of their stock. In the next project plan we will use progeny testing for retrospective evaluation of the accuracy of genomic selection predictions for IHNV resistance in two major US commercial rainbow trout breeding programs.
Progress toward Objective 3: Identification of mechanisms affecting production traits to better define phenotypes for selective breeding or to improve management practices, included characterization of proteolytic mechanisms in rainbow trout white muscle. The autophagy-lysosome system (ALS) was identified as a major mechanism responsible for 30-50% of muscle protein degradation. Results also indicated that effects of IGF-I on protein degradation are attributed to down-regulation of the ALS and not the ubiquitin-proteasome system. Determination of the ALS as a major proteolytic mechanism in fish muscle identified this system a novel target for gene editing studies that will be performed in the next project plan.
Gene editing capabilities were developed during this project plan that provided the first proof-of-concept that CRISPR/Cas9 technology can induce gene editing in rainbow trout and produce fish with a complete loss-of-function phenotype. We also confirmed that genetic modifications are transmitted to sterile offspring, thereby preventing the unintended propagation of genetically modified germplasm. The CRISPR/Cas9 technique was also used to target genes important for endocrine regulation of growth. As gene editing technologies continue to progress, the capacity to manipulate the rainbow trout genome can be useful for understanding protein function and complement ongoing selective breeding efforts to accelerate the rate of genetic gain. In the next project plan we will continue to use gene editing to manipulate production-relevant traits and evaluate the potential for gene editing as a novel breeding tool.
Also in support of Objective 3 were studies that characterized the effect of hatchery practices on egg quality. Findings indicated that rearing conditions in a recirculating aquaculture system can have negative impacts on gonadal development but still allow females to develop large oocytes and high-quality fillets. These findings support optimal conditions for growth can be maintained until ~22 months without reproduction impacting growth and fillet quality, and possibly still allow time for select females to be rescued for egg production. In addition, we worked with a commercial hatchery to define effects of incubation temperature treatments used to control the timing of hatching, on larval development. These studies showed incubation at 5 degrees C within the first 24hrs of fertilization should be avoided. In addition, we showed eggs can be instantly switched between 5 and 10 degrees C without impacting eyeing rate, simplifying protocols for altering hatch date. In related research, we identified changes in egg transcripts with egg quality. Changes in transcript levels of nuclear genomic mRNAs, microRNAs, mitochondrial mRNAs, and mitochondrial genome-encoded small RNAs were all identified to be associated with egg quality in rainbow trout. The diversity of molecular systems altered with egg quality revealed the complexity of disruption associated with reduced egg quality in rainbow trout. In the next project plan we will continue to investigate how regulation of transcripts contribute to differences in egg quality.
Central for progress in all objectives was an international collaboration led by our scientists that generated the first chromosome-level reference genome assembly for rainbow trout (NCBI GenBank Accession GCA_002163495). The assembly is composed of a 2.18 GB (~90% of the 2.4 GB estimated genome size) and contains 139,800 scaffolds with N50 of 1.67Mb. A high-density genetic map of over 46K SNPs was used to guide chromosome sequences referenced by anchoring and ordering of the scaffolds to linkage groups. This is the first chromosome level annotated reference genome assembly for rainbow trout. It has been an essential resource for conducting genome association studies, identifying QTL for production traits, and detecting genomic markers valuable for marker assisted selection strategies.
Accomplishments
1. Feeding strategies to maintain heart-healthy omega-3 fats in rainbow trout fed a sustainable diet containing plant oils. Plant-based feed for rainbow trout. Rainbow trout feeds containing plant-based oils are more sustainable than feeds containing fish oil, but lack the heart-healthy omega-3 fats. Collaborative research between ARS scientists in Leetown, West Virginia, and Grand Forks, North Dakota, developed rainbow trout feed formulations using sustainable plant oils that retained omega-3 fat in the fillet. This work developed feeding strategies for rainbow trout that improves sustainability without sacrificing health benefits for trout consumers.
Review Publications
Pearse, D.E., Barson, N.J., Nome, T., Gao, G., Campbell, M.A., Abadia-Cardoso, A., Anderson, E.C., Rundio, D.E., Williams, T.H., Naish, K.A., Moen, T., Liu, S., Matthew, K., Minkley, D.R., Rondeau, E.B., Brieuc, M.S., Sandye, S., Miller, M.R., Cedillo, L., Baruch, K., Hernandez, A.G., Ben-Zvi, G., Shem-Tov, D., Barad, O., Kuzishchin, K., Garza, J., Lindley, S.T., Koop, B.F., Thorgaard, G.H., Palti, Y., Lien, S. 2019. Sex-dependent dominance maintains migration supergene in rainbow trout. Nature Ecology and Evolution. 3:1731–1742. https://doi.org/10.1038/s41559-019-1044-6.
Shapagain, P., Arivett, B., Cleveland, B.M., Walker, D., Salem, M. 2019. Gut microbiome analysis of fast- and slow-growing Rainbow Trout (Oncorhynchus mykiss). Frontiers in Microbiology. 20:788. https://doi.org/10.1186/s12864-019-6175-2.
Silva, R., Evenhuis, J., Vallejo, R.L., Gao, G., Martin, K., Leeds, T.D., Lourenco, D., Palti, Y. 2019. Genomic regions associated with Columnaris disease in two rainbow trout breeding populations. Genetics Selection Evolution. 51:42. https://doi.org/10.1186/s12711-019-0484-4.
Vallejo, R.L., Cheng, H., Fragomeni, B.O., Shewbridge, K., Gao, G., Macmillan, J.R., Towner, R., Palti, Y. 2019. Genome-wide association analysis and accuracy of genome-enabled breeding value predictions for resistance to infectious hematopoietic necrosis virus in a commercial rainbow trout breeding population pserial online]. Genetic Selection Evolution. 51:47. https://doi.org/10.1186/s12711-019-0489-z.
Gao, G., Pietrak, M.R., Burr, G.S., Rexroad III, C.E., Peterson, B.C., Palti, Y. 2020. A new single nucleotide polymorphism database for North American Atlantic salmon generated through whole genome re-sequencing. Frontiers in Genetics. 11:85. https://doi.org/10.3389/fgene.2020.00085.
Ali, A., Al-Tobasei, R., Lourenco, D., Leeds, T.D., Kenney, B., Salem, M. 2019. Genome-wide association study identifies genomic loci affecting fillet firmness and protein content in rainbow trout. Frontiers in Genetics. 10(386):1-17. https://doi.org/10.3389/fgene.2019.00386.
Ali, A., Al-Tobasei, R., Lourenco, D., Leeds, T.D., Kenney, B., Salem, M. 2020. Identification of genomic loci associated with growth in rainbow trout. Biomed Central (BMC) Genomics. 21(209). https://doi.org/10.1186/s12864-020-6617-x.
