Location: Cool and Cold Water Aquaculture Research
2017 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
Sub-objective 1.a: Previously we developed a genotyping assay with 96 genetic markers for parentage and population-of-origin assignments in rainbow trout aquaculture and we also demonstrated that it can be used with nearly 100% accuracy for those two applications. This year, we conducted statistical analyses in eight commercial populations to assess association between the 96 genetic markers and body size at 10 months post hatching, but none of the markers from this assay were found to be significantly associated with this important growth trait.
Sub-objective 1.b: Characterization of fillet yield – the proportion of the whole fish that is edible fillet – in a commercial breeding program using automated filleting equipment resulted in a heritability estimate similar to our population, thus suggesting similar genetic gain if selection were to be practiced in that population. Regression analyses to predict fillet yield using non-lethal measures are ongoing.
Sub-objective 1.c: Divergent selection was practiced in a pedigreed population for fillet yield to develop high-yield, control (no selection), and low-yield lines of rainbow trout. After one generation of selection, families (n = 100) from the high-yield line averaged 0.65 percentage points higher, and families (n = 23) from the low-yield line averaged 0.91 percentage points lower, than families (n = 34) from the unselected control line when adjusted to a standard harvest body weight of approximately 2 kilograms. High-yield families had a numerically larger mean body weight than low-yield and control-line families when harvested at the same age, suggesting that selection for increased fillet yield did not adversely affect growth performance. These results confirm our previous claim that fillet yield in rainbow trout is a heritable trait that can be improved through selective breeding, and provide an empirical estimate of the genetic gain that can be made for this economically-important trait using traditional family-based selection.
Because fillet yield is a lethally-measured trait that cannot be measured directly on breeding candidates, we also used phenotypic and genotypic (Affymetrix 57K SNP array) data from two generations of our pedigreed population to conduct a genome-wide association study (GWAS) to understand the genetic architecture of fillet yield. The GWAS did not detect any large-effect quantitative trait loci for fillet yield, suggesting a polygenic architecture for the trait. The use of genotypic data increased the accuracy of breeding value estimates, and thus genomic selection is expected to expedite genetic improvement by accurately exploiting within-family genetic variation.
Sub-objective 1.d: Previously, we determined that rainbow trout selectively bred for improved growth by ARS scientists exhibit better or similar growth rates and fillet yield than commercially-available rainbow trout. This year, we found that while there is considerable variation in growth performance between genetic lines, indices of fillet quality and nutrient composition showed minimal differences. These results emphasize the need for farmers to consider the genetics of their source stock if they require fish with optimal growth performance. In contrast, genetic background becomes less critical if achieving ideal fillet quality is the production goal. Additional progress included analyzing global RNA expression among the three fastest-growing genetic lines, thereby identifying the biological basis for variations in economically-important production traits.
Furthermore, rainbow trout fillets are valued as an excellent source of protein with high concentrations of heart-healthy omega-3 fat. To determine how these qualities differ in rainbow trout at various market weights, fat profiles were analyzed at various stages of development in fertile (diploid) and sterile (triploid) rainbow trout. Triploid rainbow trout continued to deposit fat in fillets throughout development, including the omega-3 fats, while fat accumulation essentially ceased in sexually-maturing diploids. We also examined how fats vary across different regions of the fillet; findings indicate the middle and lower portions of the fillet have the absolute greatest concentration of fats. Although rainbow trout harvested at any age can be considered a high omega-3 food, fillets from larger triploids represent the product with the greatest omega-3 concentrations and therefore could be viewed as the product with optimal benefits for human health.
Objective 2: Columnaris disease (CD) is distributed around the world, and recently it has been identified as an emerging problem for the U.S. rainbow trout aquaculture industry. As a first step in developing selective breeding strategies for improving the resistance to CD, we scanned the genomes of two important domestic rainbow trout breeding populations for chromosome segments that contain genes that significantly affect resistance to CD. Chromosome segments that explained more than 1% of the additive genetic variance were considered associated with CD resistance. A total of 13 segments located on six chromosomes were found to be associated with CD resistance in the USDA-NCCCWA population, including one segment on chromosome 17 with large effect. A total of 16 segments located on nine chromosomes were detected in the commercial breeding population. However, no large-effect segments were identified in the commercial population and only three segments appear to overlap between the two populations. The results suggest that different approaches may be appropriate for using genomic information for selective breeding for CD resistance in the two populations.
Sub-objective 3.a: This year, we generated an optical genome map that can be used for bridging gaps in the current chromosome sequences and improve the ordering and orientation of sequence fragments on each of the 29 rainbow trout chromosomes to further improve the reference genome assembly.
Sub-objective 3.b: Using genome-based estimated breeding values for selective breeding for disease resistance holds great promise as it provides individual genetic merit estimates for potential breeders compared to family-average estimates in traditional selective breeding schemes. Previously we demonstrated that whole-genome genotype data from 50,000 markers in a commercial breeding population yielded genetic merit predictions for resistance to bacterial cold water disease that are substantially more accurate than traditional pedigree-based predictions. This year, we identified 70 markers that are strongly associated with resistance or susceptibility to the disease in the same breeding population. We then found that genetic-merit predictions using only those 70 markers are as accurate as predictions we generated in the previous year with 50,000 markers. This information can be used to reduce the cost of generating superior and highly-accurate genome-based predictions for commercial rainbow trout breeders in the aquaculture industry.
Sub-objective 3.c: Insulin-like growth factor-I (IGF1) is a hormone that stimulates growth, although it is bound to IGF binding proteins (IGFBP) that may increase or decrease its level of activity. ARS scientists are using a novel gene editing technique called CRISPR to reduce or eliminate IGFBP expression in rainbow trout and identify effects on growth and muscle development to determine the functional role of each IGFBP. Progress has included screening candidate gene sequences to determine the most effective sites to target for gene editing; six demonstrated capacity to genetically modify IGFBP genes in live fish. These sequences will be used to produce experimental animals.
Furthermore, little is known regarding the effect of maternal diet on offspring growth performance in fish. We investigated whether supplementing rainbow trout broodstock diets with choline or methionine will affect growth rates in the offspring. Results indicated that supplementing a commercial broodstock diet with additional choline increased body weight in offspring by approximately 17% at the 13-month harvest weight. Future analyses will indicate whether this effect is driven by genetic modifications during egg development or an enhanced nutrient profile of the egg that aids development. These findings will indicate the potential for nutritional intervention of broodstock diets as a management strategy to improve economically-important traits in aquaculture species.
Sub-objective 3.d: Early embryos cannot transcribe mRNA and are therefore dependent on maternal mRNA stored in the egg. It is not known to what extent differences in levels of these stored transcripts affect egg quality and if there are profiles that are characteristic to different causes of reduced egg quality. Using RNA-seq analysis to compare eggs of different quality from a domestic rainbow trout egg producer, we have previously shown that few transcripts differ in total mRNA expression level but thousands differ when only polyadenylated, or activated, mRNAs are compared among the highest- and lowest-quality eggs at ovulation. For the current reporting year, we established a multiplex assay that can simultaneously measure 25 genes that have been shown to be differentially expressed in high- versus low-quality eggs from the RNA-seq analysis or from available literature. We used the assay to compare ~150 egg lots from each of 2 year classes from a commercial egg producer and found few to be consistently differentially expressed, although only a few egg lots of those available were considered poor quality. Only genes identified from the RNA-seq study were found to be differentially expressed. Therefore, we expanded our egg collection to include egg lots from the USDA-NCCCWA population which vary considerably in egg quality, and are developing an additional multiplex assay based solely on differentially-expressed genes identified by the RNA-seq study.
Accomplishments
1. A reference genome assembly for rainbow trout. A high-quality reference physical genome map is important for facilitating meaningful genetic analyses and enhancing research on the physiology of fish. ARS researchers at Leetown, West Virginia, have provided leadership and worked closely with national and international cooperators to generate a reliable and high-quality reference genome for rainbow trout. In an effort to improve the rainbow trout reference genome assembly, we used recent improvements in DNA sequencing technology and sophisticated new bioinformatics. This new reference genome assembly aligns with 88% of the assembly chromosome sequences of rainbow trout. The NIH National Center for Biotechnology Information (NCBI) has made the new reference genome coding available for browsing through the NCBI online interactive databases. The new rainbow trout genome assembly and chromosome sequences provide major improvements for rainbow trout aquaculture genetics research, and for all aspects of fish quality improvement.
2. 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. ARS researchers at Leetown, West Virginia 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, the selection breeding practiced by ARS researchers resulted in a line that grows approximately 60% faster to and beyond standard market weight. The improvement 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.
Review Publications
Nezafatiana, E., Zadmajid, V., Cleveland, B.M. 2017. Short-term effects of genistein on gamete quality, steroidogenesis and histological changes in gonads in gibel carp, Carassius auratus gibelio. Journal of the World Aquaculture Society. doi:10.1111/jwas.12399.
Vallejo, R.L., Leeds, T.D., Gao, G., Parsons, J.E., Martin, K.E., Evenhuis, J., Fragomeni, B.O., Wiens, G.D., Palti, Y. 2017. Genomic selection models double the accuracy of predicted breeding values for bacterial cold water disease resistance compared to a traditional pedigree-based model in rainbow trout aquaculture. Genetics Selection Evolution. 49(17):1-33. doi: 10.1186/s12711-017-0293-6.
Gonazlez-Pena, D., Gao, G., Baranski, M., Moen, T., Cleveland, B.M., Kenney, B., Vallejo, R.L., Palti, Y., Leeds, T.D. 2016. Genome-wide association study for identifying loci that affect fillet yield, carcass, and body weight traits in rainbow trout (Oncorhynchus mykiss). Frontiers in Genetics. 7(203):1-14. doi:10.3389/fgen.2016.00203.
Kutyrev, I., Cleveland, B.M., Leeds, T.D., Wiens, G.D. 2016. Proinflammatory cytokine and cytokine receptor gene expression kinetics following challenge with Flavobacterium psychrophilum in resistant and susceptible lines of rainbow trout (Oncorhynchus mykiss). Fish and Shellfish Immunology. 58:542-553. doi: 10.1016/j.fsi.2016.09.053.
Koganti, P., Wang, J., Cleveland, B.M., Ma, H., Weber, G.M., Yao, J. 2016. Estradiol regulates expression of miRNAs associated with myogenesis in rainbow trout. Molecular and Cellular Endocrinology. 443:1-14. doi:10.1016/j.mce.2016.12.014.
Kutyrev, I., Cleveland, B.M., Leeds, T.D., Wiens, G.D. 2017. Dataset of proinflammatory cytokine and cytokine receptor gene expression in rainbow trout (Oncorhynchus mykiss) measured using a novel GeXP multiplex, RT-PCR assay. Data in Brief. 11:192-196.
Cleveland, B.M., Weber, G.M., Raatz, S.K., Rexroad III, C.E., Picklo, M.J. 2017. Fatty acid partitioning varies across fillet regions during sexual maturation in female rainbow trout (Oncorhynchus mykiss). Aquaculture. 475:52-60.
Koganti, P., Wang, J., Cleveland, B.M., Yao, J. 2017. 17ß-Estradiol increases non-CpG methylation in exon 1 of the rainbow trout (Oncorhynchus mykiss) MyoD gene. Marine Biotechnology. 19:1-7. doi:10.1007/s10126-017-9756-6.
Leeds, T.D., Vallejo, R.L., Weber, G.M., Gonzalez-Pena, D., Silverstein, J. 2016. Response to five generations of selection for growth performance traits in rainbow trout (Oncorhynchus mykiss). Aquaculture. 465:341-351.
Gatrell, S.K., Silverstein, J., Barrows, F., Grimmett, J., Cleveland, B.M., Blemings, K.P. 2016. Effect of dietary lysine and genetics on growth and indices of lysine catabolism in rainbow trout. Aquaculture Nutrition. doi: 10.1111/anu.12459.
Weber, G.M., Martin, K., Kretzer, J.D., Ma, H., Dixon II, D. 2016. Effects of incubation temperatures on embryonic and larval survival in rainbow trout, Oncorhynchus mykiss. Journal of Applied Aquaculture. 28:285-297. doi:10.1080/10454438.2016.1212447.
Cleveland, B.M., Leeds, T.D., Rexroad III, C.E., Summerfelt, S., Good, C., Davidson, J., May, T., Crouse, C., Wolters, W.R., Plemmons, B., Kenney, P. 2017. Genetic line by environment interaction on rainbow trout growth and processing traits. North American Journal of Aquaculture. 79:140-154. doi:10.1080/15222055.2016.1271846.
Liu, S., Palti, Y., Martin, K.E., Parsons, J.E., Rexroad, III, C.E. 2016. Assessment of genetic differentiation and genetic assignment of commercial rainbow trout strains using a SNP panel. Aquaculture. 468(1):120-125. doi: 10.1016/j.aquaculture.2016.10.004.
Ma, H., Weber, G.M., Gao, G., Wei, H., Yao, J. 2016. Identification of mitochondrial genome-encoded small RNAs related to egg deterioration caused by postovulatory aging in rainbow trout. Marine Biotechnology. 18(5):584-597. doi:10.1007/s10126-016-9719-3.
