Location: Cool and Cold Water Aquaculture Research
2016 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, tools are needed to enable assigning fish from the farm back to the breeding strain of origin. This type of management tool can improve quality assurance in the aquaculture industry and help support or refute claims of growers against the seed provider. Our research partner, one of the largest commercial rainbow trout breeders in the world, maintains 8 distinct breeding populations. Using a new panel for rainbow trout genotyping and publically available computer programs we have shown that 97% of our research partner’s fish can be assigned correctly back to their strain of origin, while 98% of fish from four different strains could be correctly excluded from being Troutlodge fish.
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. Progress toward evaluating this trait in a commercial environment included collecting body weight, ultrasound images, digital images, fillet weight, head weight, and fin clips from 596 pedigreed fish from the breeding families of a commercial producer. The preliminary estimate of fillet yield heritability in this commercial population was similar to that reported in our USDA experimental breeding population, suggesting that automated filleting equipment provides sufficient accuracy for measuring variation in fillet yield and improving this trait through selective breeding in a commercial setting.
Previously, ARS 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. Progress toward developing and evaluating a select line for improved fillet yield included producing 100 upward selected, 24 downward selected, and 34 randomly-mated control families. Weighted parental family mean fillet yield was 53.1% for upward selected, 51.8% for randomly-mated control, and 50.2% for downward selected families, yielding upward and downward selection differentials of 1.3 and 1.6 percentage points, respectively. Approximately 3,320 first-generation fish were tagged for growth performance and fillet yield evaluation at market size and use as sires and dams to produce second-generation families.
The ARS experimental selective breeding program has been breeding rainbow trout for improved growth for five generations. Growth and processing traits of the Growth Line, Control fish, and commercially available rainbow trout were analyzed in four unique production environments. Growth performance of the Growth Line was similar to or exceeded all other genetic lines at each environment. Fillet yield and viscera yield were also improved in the Growth Line compared to commercial lines, regardless of rearing environment. These results indicate that improved rainbow trout lines selectively bred by the ARS program retain their superior traits across different production environments and may be of value to domestic egg producers. Progress has been made toward identifying how physiological mechanisms differ between lines that contribute to variations in growth performance. Concentrations of plasma insulin-like growth factor-I (IGF-I) differed between genetic lines and were positively correlated with fillet yield. Global RNA expression experiments were completed for the top performing lines and data analyses are expected to identify additional factors and pathways that may affect production traits.
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. Last year we used disease resistance phenotypes and genotype data to predict the genetic merits of ~1,000 potential breeders from a commercial research partner. Progress in FY2016 included generating 138 families from selected breeders with predicted genetic merits and phenotyping ~10,000 offspring from those 138 families to empirically assess the accuracy of the genetic merit predictions. The study results confirmed our expectations as offspring of breeders with high genetic merit values performed significantly better than the offspring of low genome value breeders.
In an effort to improve the rainbow trout reference genome assembly, we used recent improvements in DNA sequencing technology and sophisticated new bioinformatics pipelines to generate a new and larger 2.17Gb reference genome assembly, containing 139,726 scaffolds with N50 greater than 1.7Mb. To generate a dense genetic map for each chromosome, we genotyped 5,716 fish from 146 full-sib families with the Affymetrix 57K SNP array. Using the information from this genetic map and two other SNP–based linkage maps, and from comparative sequence alignment with the Atlantic salmon genome, we anchored and ordered ˜88% of the new assembly scaffolds within chromosomes to generate contiguous chromosome sequences. The new Rainbow trout genome assembly and chromosome sequences provide major improvements over the current rainbow trout reference genome in all categories; including genome coverage, scaffold size, precision, and percent of the genome that is in chromosome sequences.
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 chromosome regions with large to moderate effects on BCWD resistance in the experimental population developed by ARS and in a commercially important breeding population of a commercial research partner. We then developed a focused assay of 96 genetic markers representing the chromosomal regions and we are currently genotyping the two populations of origin to validate the assay and assess its utility for markers assisted selection in rainbow trout aquaculture.
Growth performance traits and fillet yield are controlled by physiological mechanisms regulated by 1) genetic and 2) environmental factors. Genetic factors were investigated by analyzing variations in physiological mechanisms in families from the ARS experimental breeding program that exhibit differences in fillet yield. Family variation in plasma IGF-I was identified and future analysis will determine if family differences correlate with fillet yield and muscle-specific growth mechanisms. Findings will provide information useful for identifying markers and developing strategies for selective breeding. Diet composition is an environmental factor affecting growth and fillet yield, with dietary protein being the most important and expensive ingredient. Protein is composed of amino acids, but the functional ability of each amino acid to directly regulate muscle growth is unknown. Research findings determined that among all of the essential amino acids, dietary deficiencies in leucine and methionine reduce capacity for muscle growth and a dietary excess in valine and lysine have negative effects on muscle protein retention. This research progress is critical to optimize dietary nutrient profiles for maximal growth rates and fillet yield.
The objective was to conduct an RNA-seq comparison of good and poor quality eggs from a commercial breeder to identify mRNA that might serve as indicators for egg quality, and then use those differentially expressed genes as markers for egg quality in development of a multiplex array. Since eggs can’t make mRNA, all the mRNA in the egg is stored and referred to as maternal mRNA, which is stored as non-polyadenylated mRNAs. We therefore conducted the RNA-seq analysis using total mRNA, polyadenylated and non-polyadenylated mRNAs combined. Very few differentially expressed transcripts were identified. We also ran the RNA-seq looking specifically at polyadenylated mRNAs which are maternal mRNAs that are activated. We identified over 1000 differentially expressed polyadenylated mRNAs but only when comparing the worst quality eggs (<20 eyeing rate) to either the medium quality eggs (30-80% eyeing rate) or high quality eggs, (>80% eyeing rate). The results suggest differences in egg quality are due to the ability to activate or deactivate stored mRNAs as opposed to accumulation of the maternal mRNAs during egg development. The second year eggs have been collected and only 2 out of 143 crosses were of the lowest quality, <20% eyeing. Transcripts of interest from both our analyses and the literature have been incorporated into the design of a multiplex GeXP assay. To identify alternative RNA markers, we have expanded our search to mitochondrial mRNAs and small RNAs. In addition, we collected samples for examining effects of egg aging and rate of maturational development on egg quality.
Genome editing is a useful approach to characterize functional role of specific proteins. A new gene editing technique can modify an organism’s DNA without introducing foreign genetic material into the genome. The technique was successfully applied in rainbow trout to reduce and eliminate expression of a functional tyrosinase protein, which resulted in fish with variations in eye and skin pigmentation. This proof-of-concept study determined that the new technology is a viable approach to reduce or knockout gene expression in rainbow trout. Additional studies can target negative regulators of muscle growth to assess their effect on fillet yield and nutrient partitioning.
Accomplishments
1. Genome-based selection has a great potential to enhance genetic improvement in rainbow trout aquaculture. Using genome-based estimated breeding values for selective breeding for disease resistance in aquaculture holds a great promise as it provides individual genetic merit estimates for potential breeders compared to family-average estimates in traditional selective breeding. ARS researchers in Leetown, West Virginia, are studying how to improve resistance to an economically devastating pathogen that is causing bacterial cold water disease in rainbow trout. They used genome-based selection models to predict the genetic merit of potential breeders in terms of resistance to the disease in a commercial rainbow trout population, and then compared the accuracy of the genome-based genetic merit predictions to those estimated with a traditional pedigree-based model. Using progeny performance data, they have shown that the accuracy of the genome-based predictions was substantially better than the traditional pedigree-based predictions. The implication of their findings is that genome selection can substantially improve the genetic gains in traits that cannot be measured directly on the potential breeders in rainbow trout aquaculture. Therefore, using genomic selection will likely reduce that number of generations, time, labor and the number of fish that are currently needed for achieving the same level of performance improvement. The positive impacts include increased farm productivity, improved animal welfare, and reduced environmental impact of aquaculture production and improved overall sustainability of rainbow trout aquaculture production systems.
2. Development of a genotyping-based assay for pedigree and population assignments in rainbow trout. In salmonids aquaculture, family-based selective breeding programs rely on accurate pedigree information for estimating genetic merits and selecting the best breeding animals. In addition, tools are needed to enable assigning fish from the farm back to the breeding population of origin as this type of management tool can improve quality assurance in the aquaculture industry and help support or refute claims of growers against the seed provider. ARS researchers at Leetown, West Virginia developed a new DNA markers genotyping assay for parentage assignments and pedigree tracking in rainbow trout using a new panel of 95 single nucleotide polymorphism (SNP) genotyping assays. Using the new panel for genotyping of offspring and parents from known pedigrees they have 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 and have also shown that 97% of the fish can be assigned correctly back to their breeding population of origin. The new assay provides a highly accurate and reliable approach for pedigree and population assignments that can be applied rapidly to samples collected on the farm and is relatively inexpensive. The genotyping assay has already been accepted and used by the largest trout breeding company in North America and is currently offered as a commercial product by an aquaculture biotechnology company.
None.
Review Publications
Danzmann, R., Kocmarek, A., Notman, J., Rexroad III, C.E., Palti, Y. 2016. Transcriptome profiling in fast versus slow-growing rainbow trout across seasonal gradients. Biomed Central (BMC) Genomics. 17:1-18. doi: 10.1186/s12864-016-2363-5.
Vallejo, R.L., Leeds, T.D., Fragomeni, B.O., Gao, G., Hernandez, A.G., Misztal, I., Welch, T.J., Wiens, G.D., Palti, Y. 2016. Evaluation of genome-enabled selection for bacterial cold water disease resistance using progeny performance data in Rainbow Trout: Insights on genotyping methods and genomic prediction models. Frontiers in Livestock Genomics. doi: 10.3389/fgene.2016.00096.
Cleveland, B.M., Weber, G.M. 2016. Effects of steroid treatment on growth, nutrient partitioning, and expression of genes related to growth and nutrient metabolism in adult triploid rainbow trout (Oncorhynchus mykiss). Domestic Animal Endocrinology. 56:1-12.
Liu, S., Palti, Y., Gao, G., Rexroad III, C.E. 2015. Development and validation of a SNP panel for parentage assignment in rainbow trout. Aquaculture. 452:178–182. doi: 10.1016/j.aquaculture.2015.11.001.
Palti, Y., Vallejo, R.L., Gao, G., Liu, S., Hernandenz, A., Rexroad III, C.E., Wiens, G.D. 2015. Detection and validation of QTL affecting bacterial cold water disease resistance in rainbow trout using restriction-site associated DNA sequencing. PLoS One. 10(9):e0138435. doi: 10.1371/journal.pone.0138435.
Liu, S., Vallejo, R.L., Palti, Y., Gao, G., Marancik, D.P., Hernandez, A.G., Wiens, G.D. 2015. Identification of single nucleotide polymorphism markers associated with bacterial cold water disease resistance and spleen size in rainbow trout. Frontiers in Genetics. 6:298. doi:10.3389/fgene.2015.00298.
