Project : USDA ARS

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Research Project: Integrated Research Approaches for Improving Production Efficiency in Rainbow Trout

Location: Cool and Cold Water Aquaculture Research

2021 Annual Report

Objectives
Objective 1: Improve performance of aquaculture production traits in rainbow trout by developing enhanced selective breeding strategies and genomic technologies: 1a: Selective breeding, evaluation of genomic selection, and development of improved germplasm with superior fillet yield; 1b: Analysis of the genetic architecture and evaluation of the accuracy of genomic selection for resistance to infectious hematopoietic necrosis virus (IHNV) in commercial rainbow trout breeding populations; 1c: Identification of candidate genes for bacterial cold water disease (BCWD) resistance in rainbow trout using pool-seq and improvement of marker-assisted selection for BCWD resistance in multiple rainbow trout breeding populations; 1d: Detection and characterization of genomic signature and selective sweeps associated with phenotypic selection for improved resistance to BCWD in rainbow trout; and 1e: Improvement of the rainbow trout reference genome assembly and analysis of structural variations. Objective 2: Characterization of reproductive and metabolic mechanisms affecting production traits to better define phenotypes and improve selective breeding and management practices: 2a: Characterize attributes of fillet quality and feed utilization efficiency in rainbow trout selectively bred for divergent fillet yield phenotypes; 2b: Utilize gene editing technology to better understand and improve growth performance and nutrient utilization; 2c: Characterization of maternal transcript processing; and 2d: Identification of molecular markers for changes in egg quality in response to hatchery conditions and practices.

Approach
Rainbow trout (Oncorhynchus mykiss) are farmed in over half of US states and represent the second most valuable domestic finfish aquaculture product. Although production has increased, the US still imports approximately 50% of the rainbow trout sold for food, so the potential exists to increase domestic production to meet current demand. Increasing production efficiency, product quality, and fish health is central for industry expansion. This project contributes to industry expansion by integrating genomic technologies and enhanced phenotypes with selective breeding strategies that maximize genetic improvements in fillet yield, disease resistance, and reproductive success. Previously, NCCCWA scientists determined that integrating genomic selection with conventional breeding strategies improved genetic gains for resistance to bacterial cold water disease. This project aims to 1) refine genomic selection protocols to support commercial implementation of this breeding technology and 2) develop and evaluate genomic selection tools to (independently) increase fillet yield and improve resistance to infectious hematopoietic necrosis and bacterial cold water disease. Accompanying selective breeding for fillet yield will be an analysis of economically important traits such as growth, feed efficiency, and fillet quality to determine whether selection has indirect effects on performance, nutrient utilization, and product quality. Using gene editing and functional genomics to investigate the physiological mechanisms regulating nutrient metabolism and egg quality will better define these phenotypes, improve understanding of their response to selective breeding, and identify husbandry strategies that optimize performance. Collectively, this project will provide the rainbow trout industry with improved germplasm, genomic selection technologies to accelerate genetic gains, and physiological insights towards improving fish culture.

Progress Report
Sub-objective 1a: Nucleus families from the ARS-FY-H (n = 100), ARS-FY-L (n = 23), and ARS-FY-C (n = 24) were grown to market bodyweight, and approximately 5 fish per family were assigned to one of 5 harvest groups to characterize fillet yield and texture. Harvest and phenotyping of the fish are ongoing and will be completed in August. In addition, a study was conducted to compare the accuracy of breeding value predictions for fillet yield done with or without genomic information to determine the added genetic value of using genomics for this trait. To enable retrospective evaluation of the accuracy of predicted breeding values, we collected fillet yield and body weight phenotypes for 500 fish from year-class 2018 and genotyped fish with known fillet yield and bodyweight from the year- classes 2010, 2012, 2014, and 2016 for a total of approximately 2,000 fish. In addition, we have genotyped the ~200 fish from year-class 2016 that were used as breeders in 2018. The heritability for both traits was moderate, at 0.41 and 0.33 for Fillet Yield and Body Weight, respectively. Both traits were lowly but positively correlated (r = 0.24), which suggests the possibility for favorable correlated genetic gains. Genomic Selection models increased prediction accuracy compared to the traditional pedigree-based model by up to 50% for Fillet Yield and 28% for Body Weight, which suggests that using genomic selection can substantially enhance genetic improvement for the trait of fillet yield in rainbow trout aquaculture. In support of Sub-objective 1b was a study to assess the genetic architecture of resistance to infectious hematopoietic necrosis virus (IHNV) in two commercial aquaculture breeding populations that were not previously exposed to the pathogen or selected for disease resistance and therefore have a very different selective breeding history from the population that we previously studied. In the past year, the disease challenges of the two populations were completed, and nearly 4,000 fish that were phenotyped for resistance to IHNV were genotyped with the rainbow trout 57K SNP array. Analysis of the data from one of the two populations revealed a significant association between markers genotypes in two separate chromosomal regions and the trait of resistance to the disease. Analysis of the data from the second population is currently ongoing. Sub-objective 1c: Bacterial cold water disease (BCWD) causes significant economic losses in rainbow trout, and selection for BCWD resistance is one of the major goals of commercial aquaculture breeding programs. Based on sequence analysis of pooled DNA samples, two chromosome segments that span the disease resistance genes were identified. A shortlist of candidate genes for BCWD resistance was determined after examination of the predicted function of all the genes located in those two chromosome segments. Genetic markers were developed and used to genotype fish with known levels of resistance or susceptibility from a commercial breeding program, and new markers associated with BCWD resistance were identified. Those new markers were used to genotype the breeding candidates for the 2021 year-class of the commercial research partner, and 12 crosses were made to develop experimental germplasm with known genotypes and phenotypes that will be used for further studies aimed at identifying and characterizing the function of the genes involved in BCWD resistance. In support of Sub-objective 1d, a study was performed to identify genomic regions with selection signatures in the USDA broodstock population that was selected over five generations for resistance to BCWD. Our previous analysis used marker genotype data from 191 fish genotyped with 33,439 informative SNPs for selective sweep analysis (SSA). In the past year, we generated whole-genome resequencing data from 123 fish representing the three core populations from the USDA selective breeding program for improved disease resistance. The new whole-genome sequence data were used to identify about 10 million SNPs that will be used to repeat the SSA and examine the effect of the large increase in SNP density on the genome-wide detection of selection signatures that may be associated with theartificial selection for resistance to BCWD in this rainbow trout breeding population. Progress toward Sub-objective 1e in the past year included DNA preparations and sequencing of fish from three additional homozygous genetic lines to generate altogether four high contiguity reference genome maps from four genetic lines that represent large components of the geographic distribution and genetic diversity of rainbow trout. In support of Subobjective 2a, a genetics-by-environment (GxE) study is currently underway to evaluate growth performance, fillet yield, and fillet quality in the High Fillet Yield (ARS-FY-H) and Low Fillet Yield (ARS-FY-L) lines of rainbow trout consuming three commercially available diets that vary in fat content (18%, 24%, 30% fat). Very high-fat diets, such as the 30% fat diet, benefit feed conversion ratio but also partition excess energy into viscera fat which can negatively affect fillet yield. The goal of this study was to determine whether the improved fillet yield trait in the ARS-FY-H line is affected by the nutrient content of the fish diet. Thus far, the fish have been harvested at 500 g and 1 kg body weights. The 2 kg analysis is estimated for August 2021. Early data supports that growth performance is similar between the ARS-FY-H and ARS-FY-L lines, but fillet yield is about 2.3 percentage points higher in the ARS-FY-H line when fish consume the 18% and 24% diets. Fillet yield increases by 3.1 percentage points in the ARS-FY-H line when fish consume the 30% fat diet. These findings are encouraging as they indicate that the fillet yield trait is retained in the ARS-FY-H line, regardless of which diet the trout farmer feeds the fish. Progress towards Subobjective 2b is supported by a study to analyze the phenotype of fish with disrupted lamp2a genes (mutants). Compared to controls, lamp2a mutants exhibited increased condition factor, although similar growth performance, suggesting higher viscera fat stores in the mutants. However, gene expression analysis supported that incomplete lamp2a disruption occurred, resulting in some expression of a functional LAMP2a protein which prevented analysis of the complete lamp2a knockout phenotype. Therefore, the male and female mutants exhibiting the greatest lamp2a mutation were used to produce an F1 generation. Early analysis of offspring indicated approximately 20% were homozygous mutants with complete lamp2a knockout. These fish will be used for extensive phenotyping studies in FY2022. In support of Sub-objective 2c, we have previously used RNA-Seq to identify over 1000 transcripts that are differentially expressed between high- and low-quality eggs. However, these differences were only seen when the libraries were made using poly(A) enrichment which may not capture mRNAs with short poly(A) tails, and not seen when the libraries were made using rRNA removal, which captures mRNAs of all tail lengths. These results suggest it is the correct activation of transcripts and not the total number of transcripts that underlies differences in egg quality. What has yet to be determined are the lengths of the poly(A) tails of stored and activated transcripts which are necessary to study transcript activation. Preliminary samples were sent for long-read sequencing using Oxford Nanopore Sequencing, followed by data analysis using Tailfindr to estimate polyA tail length. We determined the nuclear transcripts in the egg have a wide distribution of polyA tail lengths with a minor peak around ten nucleotides, and individual genes also have transcripts with a wide distribution polyA tail lengths. This means the long-read sequencing approach is not viable due to the high volume of RNA that would be required to discriminate effects of egg quality on polyA tail profiles, and therefore alternative methods are being evaluated. In support of Sub-objective 2d, mRNA and DNA have been extracted and purified from 5 batches of freshly ovulated eggs and 5 batches of overripe eggs, but procedures for final separation of stored and activated eggs may need to be revised (see sub-objective 2C).

Accomplishments
1. Accuracy of genomic selection for improved fillet yield and body weight in rainbow trout aquaculture. In aquaculture the proportion of edible meat (fillet yield) is of major economic importance and breeding animals of superior genetic merit for this trait can improve efficiency and profitability. Fillet yield is a trait that cannot be measured directly in the potential breeding animals. However, genetic gains for fillet yield are possible via family-based selective breeding using information from siblings of the potential breeders. Genomic selection strategies for selective breeding holds great potential for further improving the accuracy of genetic merit predictions. ARS researchers in Leetown, West Virginia, compared the accuracy of genetic merit predictions for fillet yield between the genomic selection approach and family-based selective breeding. The genomic selection model increased the accuracy of genetic merit predictions for fillet yield by 50% compared to the family-based model, indicating that the use of genomic selection can enhance genetic improvement for the fillet yield trait and further enhance the efficiency and sustainability of rainbow trout aquaculture.

2. Accuracy of genomic selection for resistance to infectious hematopoietic necrosis virus in a commercial rainbow trout breeding population. Infectious hematopoietic necrosis (IHN) is a viral disease of salmonid fish that causes significant mortality and economic losses. Improving resistance to IHN using traditional family-based selective breeding has shown promise but is limited since the IHN resistance cannot be measured in potential breeders. For this reason, genome-enabled breeding strategies are advantageous because they predict the genetic merit of the trait of resistance to IHN directly in the potential breeding animals. ARS researchers in Leetown, West Virginia, compared the accuracy of genetic merit predictions among genome-enabled breeding strategies and the traditional family-based selective breeding approach using disease resistance data from a commercial rainbow trout breeding program. Findings demonstrated that genome-enabled breeding strategies resulted in a 15% improvement in prediction accuracy of an individual’s genetic merit for IHN resistance. These results indicate that genome-enabled breeding can be more effective than traditional family-based selection in improving the resistance of rainbow trout to the IHN virus.

3. Identification of novel structural variants in the rainbow trout genome. Genomic structural variants refer to changes in the length or orientation of the DNA sequence at specific locations in the genome. Structural variants were a major source of trait variation in human and plant systems but have not been investigated systematically in rainbow trout. ARS scientists in Leetown, West Virginia, used whole-genome sequence data to identify 13,863 structural variants in the genome of farmed rainbow trout from three U.S. breeding programs. This pioneering study in rainbow trout provides the foundation for studying the role of this important source of variation that is far more abundant in the genome than previously thought. These findings provide a useful resource to investigate further potential associations between structural variants and economically important traits for developing novel breeding strategies in rainbow trout aquaculture.

4. Release of an improved rainbow trout reference genome map. A high-quality reference genome map is vital for facilitating meaningful genetic analyses and enhancing research on the physiology of the organism. ARS scientists from Leetown, West Virginia, used recent improvements in DNA sequencing technology and bioinformatics to generate a new and improved reference genome map for rainbow trout. The number of gaps in the roadmap of chromosome sequences was reduced from over 427,000 in the most recent version of the genome assembly to only 486 in the current assembly. The importance of the improvement in the genome map contiguity was demonstrated by better annotating the genes in the two complex genome regions that harbor the immunoglobulin heavy chain genes that are important for producing antibodies for the adaptive immune response of the fish. The new rainbow trout genome assembly and chromosome sequences provide significant opportunities for rainbow trout aquaculture genetics research and for all aspects of research aimed at a better understanding of the biology of this economically and scientifically important fish.

5. Defining physiological mechanisms through gene editing. Recent advancements in gene-editing technology, specifically CRISPR/Cas9 methodology, increase the efficiency of genomic modification and is helpful to define the molecular regulation of economically important traits. ARS scientists in Leetown, West Virginia, used gene editing to mutate the insulin-like growth factor binding protein-2 (IGFBP2) genes; these genes regulate the action of insulin-like growth factor (IGF), the major growth-promoting hormone in rainbow trout. Findings indicated that other IGFBPs compensated for the loss of IGFBP2, supporting that IGFBPs are coordinately regulated and introducing the novel concept that IGFBPs tightly regulate growth responses through redundant functions. These outcomes define the physiological mechanisms regulating growth in rainbow trout and characterize the genome-to-phenome relationship central for developing novel breeding strategies that improve growth performance.

U.S. DEPARTMENT OF AGRICULTURE

Cool and Cold Water Aquaculture Research: Leetown, WV