Research Project: Integrated Research Approaches for Improving Production Efficiency in Salmonids

Location: Cool and Cold Water Aquaculture Research

2015 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
In salmonids aquaculture, family-based selective breeding programs rely on accurate pedigree information for estimating genetic merits and selecting the best breeding animals. DNA-based markers offer a non-terminal approach for pedigree tracking that has been successfully used in many aquaculture species. Progress to improve DNA markers assay for parentage assignments and pedigree tracking in rainbow trout included the development of a panel of 95 single nucleotide polymorphism (SNP) genotyping assays. Using the new panel for genotyping of offspring and parents from known pedigrees has demonstrated that it can rapidly be used with 100% accuracy for parentage analysis in rainbow trout populations that are typically used in aquaculture breeding programs. Yield of saleable fillet per fish is an economically-important trait for rainbow trout processors, but this trait is difficult to measure and thus has not traditionally been included as a breeding objective. Previous research suggests that fillet yield is a heritable trait and might be improved through selective breeding. Our aim is to estimate heritability of fillet yield in a commercial population of rainbow trout when processed using a high-throughput, automated filleting machine. Progress towards this aim included harvesting market-size fish from 56 pedigreed families of a commercial producer and characterizing their fillet yield. This research is expected to identify genetic and processing-related factors affecting fillet yield, and will inform approaches through selective breeding and/or processing optimization. Previously, USDA-ARS National Cool and Coldwater Aquaculture (NCCCWA) scientists characterized fillet yield in rainbow trout from a pedigreed population over multiple generations and determined that fillet yield is a heritable trait that has potential to be improved through selective breeding. The current research aims to test this hypothesis, and provide estimates of selection response, by developing three lines of fish that are either selected for increased fillet yield, decreased fillet yield, or randomly mated (selection control). To date, the base population from which these selection or control lines has been established and characterized for fillet yield. Families within the base population have been assigned as progenitors of one of the three selection lines based on their yield and pedigree data. Long-term goals of this research are to develop a high-yielding, commercially-relevant rainbow trout line that can be transferred to industry stakeholders, and develop divergently-selected resource populations for use by scientists to identify biological mechanisms affecting fillet yield. The NCCCWA has been selecting rainbow trout for improved growth performance (growth line) for five generations but there has been no controlled study that compares growth performance of these fish to that of commercially available rainbow trout. Rainbow trout from the NCCCWA growth line, a randomly mated selection control, and domestic and international suppliers (commercially available) were stocked into four unique rearing environments. Growth of rainbow trout from the NCCCWA growth line and the domestic supplier was similar up to approximately one year of age, after which growth of NCCCWA was greater. Growth rates of the NCCCWA growth line were 15% and 35% greater than the selection control and international producers. Fillet yield of NCCCWA fish exhibit similar or greater fillet yield percentages compared to all commercially available lines. These findings indicate that NCCCWA fish selected for improved growth exhibit similar or improved growth performance and fillet yield than commercially available rainbow trout across a variety of environmental conditions. The NCCCWA growth line may be especially valuable to producers who market fish with larger body weights (1+ kg) and processors who value high fillet yield. Using genome-based estimated breeding values for selective breeding for disease resistance in aquaculture holds a great promise as it provides individual genetic merit estimate for potential breeders compared to family-average estimates in traditional selective breeding schemes. Progress included genotyping of 2,500 fish from Troutlodge, Inc., the largest U.S. rainbow trout breeding company and egg producer for the aquaculture industry. The genotype data enabled predicting genome breeding values for bacterial cold water disease resistance in 1,000 potential breeders and designing a mating scheme that produced pedigreed families with high and low estimated genome breeding values. The progeny of the families produced in 2015 will be challenged with bacterial cold water disease to evaluate the accuracy of the genome-based breeding values against the true survival performance with the expectation that offspring of breeders with high genome-based values will perform significantly better than the offspring of low genome value breeders. Progress towards our effort to improve the rainbow trout reference genome included generating a high-density linkage map in collaboration with researchers from Norway. High-density linkage maps generated by single nucleotide polymorphism (SNP) array genotype data have proven to be crucial for the accurate assembly of genome sequence scaffolds and anchoring of the scaffolds data onto chromosomes. A total of 47,839 SNPs were mapped to the rainbow trout 29 chromosomes, with an average of 1,650 SNPs per chromosome. The number of SNPs assigned to each group ranged from 754 to 2,934. Identification of genes involved in fish resistance to bacterial cold water disease (BCWD) is an important component of our approach for studying the response to infectious diseases in rainbow trout aquaculture. Progress towards achieving this objective included the identification of genetic markers that are located on or near a single chromosome region with large effect on BCWD resistance and several other chromosome regions with moderate effects in the experimental population of the National Center for Cool and Cold Water Aquaculture. Three of the chromosome regions affecting BCWD resistance in this population were also found to affect stress level in response to handling and confinement in a separate rainbow trout population. Several candidate genes that may be involved in immune or stress response on those chromosomal regions were identified through alignment of the genetic markers and the trout reference genome sequence. The NCCCWA selective breeding program has been selecting rainbow trout for improved growth performance for five generations. While each family is thoroughly characterized for morphometric phenotypes like growth, condition factor, fillet yield, and viscera yield, the physiological basis for variations in these traits is largely unknown. Identifying how physiological mechanisms regulating growth vary between individuals and families will further define the growth and fillet yield phenotypes. To date, concentrations of insulin-like growth factor (IGF-I) have been measured in plasma from 500 fish sampled from the BY2010 population. Liver and muscle tissues are being processed to analyze mechanisms involved in regulation of growth and protein retention. Preliminary results from the muscle analysis suggest an increased capacity for muscle-specific growth mechanisms are associated with improved fillet yield. Subsequent analysis of liver tissue will indicate what endocrine pathways may be responsible for this relationship. In combination with the morphometric analyses, gene expression data will provide additional information useful for identifying markers and developing strategies for selective breeding. In an effort to improve reproduction success in rainbow trout we study the genetics and molecular physiology of egg quality. Egg quality data of eyeing rate were analyzed from two distinct commercial populations to estimate heritability. For each population, approximately 875 records collected over a period of 6 successive generations were available, along with complete pedigree data. Heritability of eyeing rate was also estimated in a pedigreed population maintained at the NCCCWA using three generations of data (566 records). Eyeing rate was found to be moderately heritable in all three populations, which suggests that variation in eyeing rate is affected, at least in part, by the genetics of the female. The application of genomic technologies can be used to address the basis of egg quality. In an effort to characterize variation in the transcriptome responsible for differences in eggs quality; and to evaluate these transcript differences as markers for the basis for diagnostics for why or when these aberrations developed, unfertilized egg and early embryo samples were collected for 192 families from the largest commercial rainbow trout hatchery in the USA, for genomic analysis. From each family, frozen samples of unfertilized eggs were collected for transcript analysis, fixed unfertilized eggs were collected for visual evaluation of egg quality, fixed 24hr old embryos were collected for confirmation of fertilization capacity and early embryonic competence, and survival at eyeing was recorded. Evaluation of all samples for egg quality has been completed and egg lots have been identified for initial RNA-Seq analysis. The transcriptomes, including mRNA and microRNA, of unfertilized eggs from 10 females exhibiting low egg quality and 10 exhibiting high egg quality are being compared by RNA-Seq analysis.

Accomplishments

Review Publications
Marancik, D.P., Gao, G., Paneru, B., Ma, H., Hernandez, A.G., Salem, M., Yao, J., Palti, Y., Wiens, G.D. 2015. Whole-body transcriptome of selectively bred, resistant-, control-, and susceptible-line rainbow trout following experimental challenge with Flavobacterium psychrophilum. Frontiers in Genetics. 5(243):1-15. DOI:10.3389/fgene.2014.00453.
Andersson, L., Archibald, A.L., Bottema, C.D., Brauning, R., Burgess, S.C., Burt, D.W., Casas, E., Cheng, H.H., Clarke, L., Couldrey, C., Dalrymple, B.P., Elski, C.G., Foissac, S., Giuffra, E., Groenen, M.A., Hayes, B.J., Huang, L.S., Khatib, H., Kijas, J.W., Kim, H., Lunney, J.K., McCarthy, F.M., McEwan, J.C., Moore, S., Nanduri, B., Notredame, C., Palti, Y., Plastow, G.S., Reecy, J.M., Rohrer, G.A., Sarrapoulou, E., Schmidt, C.J., Silverstein, J., Tellam, R.L., Tixier-Biochard, M., Tosser-Klopp, G., Tuggle, C.K., Vilkki, J., White, S.N., Zhao, S., Zhou, H. 2015. Coordinated international action to accelerate genome-to-phenome with FAANG, The Functional Annotation of Animal Genomes project. Genome Biology. 16:57. DOI:10.1186/S13059-015-0622-4.
Palti, Y., Gao, G., Moen, T., Liu, S., Kent, M., Sigbjorn, L., Miller, M., Rexroad III, C.E. 2015. The development and characterization of a 57K SNP array for rainbow trout. Molecular Ecology Resources. 15:662-672.