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

Location: Cool and Cold Water Aquaculture Research

2019 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
Subobjective 1.c.: Two generations of divergent selection of a rainbow trout population for fillet yield have been completed and second-generation families from the high-yield (ARS-FY-H; n = 99 families), randomly-mated control (ARS-FY-C; n = 23 families), and low-yield (ARS-FY-L; n = 23 families) lines were phenotyped for fillet yield at approximately 1.9 kilograms body weight (~15 months of age). Through two generations of family-based selection, the average fillet yield (i.e., weight of skin-on, trimmed fillets divided by total body weight) of fish from the ARS-FY-H line (54.3%) was approximately 2.2 percentage points greater compared to fillet yield from the ARS-FY-C (52.0%) and ARS-FY-L (52.1%) lines. Interestingly, and currently unresolved, the 0.8 percentage point difference in fillet yield between the ARS-FY-C and -S lines in the first generation was not maintained in the second generation as expected. Average harvest body weight of the ARS-FY-H line (1,956 grams) was numerically greater compared to the ARS-FY-C (1,788 grams) and -L (1,799 grams) lines, suggesting that selection for increased fillet yield will not adversely affect growth performance. Viscera yield, which includes the intestines and waste abdominal fat, was smaller in the ARS-FY-H line (8.8%) compared to the ARS-FY-C (9.9%) and -L (10.3%) lines. An additional study was conducted to compare long-term feed efficiency between the ARS-FY-H and -L lines. Twenty representative second-generation families from each line were used in the study, and five fish from each family were stocked into each of five replicate tanks per line at a mean body weight of 175 grams. Each tank was fed a standard ration using automated feeders and fish were grown for a period of approximately 4.3 months until the mean body weight reached approximately 1 kilogram. Feed efficiency, expressed as total body weight gain divided by total feed fed, averaged 2.3% better in tanks stocked with ARS-FY-H fish compared to tanks stocked with ARS-FY-L fish. Collectively, these studies suggest: 1) that fillet yield can be increased in a population via traditional family-based selective breeding; 2) the increased fillet yield results in a favorable, decreased yield of viscera waste; 3) growth performance is not adversely affected by selection for increased fillet yield; and 4) feed efficiency is improved as a result of selection for increased fillet yield. Sub-objective 3.a.: Previously we generated a high-quality reference genome assembly from a single rainbow trout fish that originated from a lake in Alaska using short-reads DNA sequencing technology. In less than two years from the release of the genome it has been widely used by the research community for genetic, genomic and physiological research. With the advent of new sequencing technologies and emergence of more affordable long-reads sequencing, it is becoming feasible to follow the human genome model to develop a pan-genome reference for rainbow trout that is composed of multiple high-quality genome assemblies of fish from diverse genetic stocks and geographic origin. The new pan-genome will provide a more complete resource for identifying and evaluating the effects of genomic and gene diversity on traits that are important for rainbow trout aquaculture. To that end we have sequenced the genome of a single fish from the Arlee line, which is a distinct domesticated rainbow trout hatchery strain, using long-reads sequencing technology. Using tested bioinformatic pipelines, we completed the first step of a high-quality assembly that will be superior to the current reference assembly. We have also developed additional genome assembly resources to aid in further improvement of the assembly that we will eventually release as a public resource for research. We will also use comparative genomic analyses in rainbow trout and other salmonid species for improving the rainbow trout reference genome. Sub-objective 3.b.: Previously we reported that selective breeding for bacterial cold-water disease resistance in rainbow trout can be improved not only through the use of whole-genome markers information, but also through the use of a much smaller number of genetic markers from narrow regions on chromosomes 8 and 25. Those select markers from regions with large effects on disease resistance can be used for marker assisted selection in rainbow trout breeding. However, the two regions we have previously identified span more than 1.5 million base-pairs. To refine those regions and improve the utility of markers for effective selection of fish with better disease resistance we conducted whole genome re-sequencing of parents from resistant and susceptible families. The sequencing data were then aligned to the reference genome to identify single nucleotide polymorphisms in the population and to contrast the polymorphism between the resistance and susceptible fish. Overall, we identified 15 million new polymorphisms throughout the rainbow trout, and additional markers were identified with potential for greater association with cold-water disease resistance. Those new markers will be validated further in a larger sample size from multiple genetic stocks and neighboring genes will be examined for their involvement in physiological and immune pathways that may affect resistance to cold-water disease in rainbow trout. Sub-objective 3.c. Previously, the CRISPR/Cas9 gene editing approach was used to produce approximately 400 fish with mutations in insulin-like growth factor-2b (IGFBP-2b) genes and near complete knockout of a functional IGFBP-2b protein. A study was performed to determine the physiological effect of IGFBP-2b disruption during continuous feeding, feed deprivation, and refeeding. Control fish and fish with gene disruption exhibited similar weight gain and weight loss responses, but there were differences in expression of igfbp genes in liver and muscle. These findings suggest that insulin-like growth factor (IGF) signaling in mutants was adjusted to a level similar to controls, due in part to compensatory regulation of IGFBPs that affect the sequestration and binding of IGFs to IGF receptors. Results also suggest that IGFBP families have complementary functions and can be co-regulated to maintain homeostasis or alter IGF signaling to accommodate an appropriate physiological response. In addition to analyzing effects of IGFBP-2b gene disruption, a novel gene target was identified for future studies to investigate mechanisms of nutrient utilization and partitioning. Genome editing protocols were optimized to excise an exon from this gene; the result was a truncated splice variant that inactivates the protein. Approximately 500 rainbow trout eggs were treated to generate fish with the truncated splice variant; approximately half of these eggs hatched. Genomic DNA from these fish will be harvested in September 2019 to identify individuals with gene disruption. These fish will then be evaluated to determine the effects of gene disruption on growth performance and the nutrient utilization/partitioning response. Sub-objective 3.d. Early embryos cannot transcribe mRNA and are therefore dependent on maternal mRNA stored in the egg. Maternal transcripts are stored with short poly(A) tails and the tails are then elongated when they are to be activated for use in translation of proteins. Previously we used RNA-seq analysis to compare eggs of different quality from the largest U.S. trout egg supplier in the country and showed that few mRNA transcripts differed among the highest and lowest quality eggs at ovulation if we looked at total mRNA, which overwhelmingly includes the stored mRNAs with short poly(A) tails. On the other hand, ~1000 mRNA transcripts were differentially expressed if we used poly(A) enriched libraries which likely only captured the activated transcripts with elongated tails. Unfortunately, when we tried to develop multiplex assays for these differentially expressed transcripts results were inconsistent among different platforms. We have now compared transcript levels in the same samples before, during, and after different approaches of poly(A) enrichment, including constructing cDNA libraries using random hexamer primers for measuring total RNA and oligo(dt) primers for measuring poly(A) mRNA. The inconsistent results were likely due to differences in the poly(A) tail lengths of the transcripts, combined with the efficiency with which different methods capture or amplify transcripts with different tail lengths. Nevertheless, we evaluated a 70 gene multiplex based on Nanostrings technology and were able to identify differentially expressed transcripts between low- and high-quality eggs in three broodstock populations of rainbow trout. The transcripts differed among the populations suggesting there may have been some differences in the causes of poor egg quality among the populations. We also used RNA-seq analysis to identify 49 differentially expressed microRNAs among eggs of different quality based on early embryo survival.

Accomplishments
1. Gene expression within eggs can predict reproductive success. Variation in egg quality within a rainbow trout breeding population is quite extensive and unpredictable. As a result of this variation, a single female may produce anywhere from zero to several thousands of offspring. To understand what is contributing to egg quality and reproductive success, ARS researchers in Leetown, West Virginia, compared the gene expression profile in rainbow trout eggs exhibiting good and poor eyeing rate and found over 1,000 differences in gene levels between eggs of varying quality. This new way of predicting the eyeing rate will help producers develop husbandry strategies for improved egg quality and reproductive success.

2. Improved growth performance in triploid rainbow trout. Rainbow trout are diploids (possess two copies of each chromosome) like terrestrial livestock, but unlike terrestrial livestock they are tolerant to triploidy (three copies of each chromosome). Triploid rainbow trout are sterile, just like seedless watermelons, and are used extensively to avoid negative impacts of sexual maturation on performance and to avoid their breeding with native populations. However, this sterility complicates selective breeding programs because genetically-superior triploids cannot be used to produce offspring, and scientists have been uncertain if breeding for improved diploid growth performance also results in improved triploid performance. ARS researchers in Leetown, West Virginia, evaluated long-term growth performance of diploids and triploids from a growth-selected line and an unselected control line. They demonstrated that selection on diploid growth performance is effective for improving triploid growth performance, thereby simplifying commercial breeding programs that market triploid rainbow trout.

Review Publications
Cleveland, B.M., Yamaguchi, G., Radler, L.M., Shimizu, M. 2018. Editing the duplicated insulin-like growth factor binding protein-2b gene in rainbow trout (Oncorhynchus mykiss). Scientific Reports. 8:16054. https://doi.org/10.1038/s41598-018-34326-6.
Silva, R., Evenhuis, J., Vallejo, R.L., Tsuruta, S., Wiens, G.D., Martin, K., Parsons, J., Palti, Y., Lourenco, D., Leeds, T.D. 2018. Variance and covariance estimates for resistance to bacterial cold water disease and columnaris disease in two rainbow trout breeding populations. Journal of Animal Science. 97(3):1124-1132. https://doi.org/10.1093/jas/sky478.
Cleveland, B.M., Radler, L.M. 2018. Essential amino acids exhibit variable effects on protein degradation in rainbow trout (Oncorhynchus mykiss) primary myocytes. Comparative Biochemistry and Physiology - Part A: Molecular & Integrative Physiology. doi:10.1016/j.cbpa.2018.11.019.
Latimer, M., Reid, R., Bigga, P., Cleveland, B.M. 2019. Glucose regulates protein turnover and growth-related mechanisms in rainbow trout myogenic precursor cells. Comparative Biochemistry and Physiology - Part A: Molecular & Integrative Physiology. 232:91-97. https://doi.org/10.1016/j.cbpa.2019.03.010.
Cleveland, B.M., Leeds, T.D., Picklo, M., Brentesen, C., Frost, J., Biga, P. 2019. Supplementing rainbow trout (Oncorhynchus mykiss) broodstock diets with choline and methionine improves growth in offspring. Journal of the World Aquaculture Society. 50(3):1-16. https://doi.org/10.1111/jwas.12634.
Larson, W.A., Palti, Y., Gao, G., Warheit, K.I., Seeb, J.E. 2018. Rapid discovery of SNPs differentiating hatchery steelhead trout from ESA-listed natural-origin steelhead trout using a 57K SNP array. Canadian Journal of Fisheries and Aquatic Sciences. 75:1160-1168. doi:10.1139/cjfas-2017-0116.
Liu, S., Vallejo, R.L., Evenhuis, J., Martin, K.E., Hamilton, A., Gao, G., Leeds, T.D., Wiens, G.D., Palti, Y. 2018. Evaluation of marker-assisted selection for resistance to bacterial cold water disease in three generations of a commercial rainbow trout breeding population [serial online]. Frontiers in Genetics. 9:286. https://doi.org/10.3389/fgene.2018.00286.
Salem, M., Al-Tobasei, R., Ali, A., Lourenco, D., Gao, G., Palti, Y., Kenney, B., Leeds, T.D. 2018. Genome-wide association analysis with a 50K transcribed gene SNP-chip identifies QTL affecting muscle yield in rainbow trout. Frontiers in Genetics [serial online]. 9:387. doi:10.3389/fgene.2018.00387.
Leeds, T.D., Weber, G.M. 2019. Effects of triploidization on genetic gains in a rainbow trout (Oncorhynchus mykiss) population selectively bred for diploid growth performance. Aquaculture. 505:481-487. https://doi.org/10.1016/j.aquaculture.2019.03.003.
Ali, A., Al-Tobasei, R., Kenney, B., Leeds, T.D., Salem, M. 2018. Integrated analysis of IncRNA and mRNA expression in rainbow trout families showing variation in muscle growth and fillet quality traits. Scientific Reports. 8:12111. https://doi.org/10.1038/s41598-018-30655-8.
Ma, H., Martin, K., Dixon III, D., Hernandez, A.G., Weber, G.M. 2019. Transcriptome analysis of egg viability in rainbow trout (Oncorhynchus mykiss). Biomed Central (BMC) Genomics. 20:319. https://doi.org/10.1186/s12864-019-5690-5.