Location: Cool and Cold Water Aquaculture Research
2023 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
Progress towards Sub-objective 1a: Fourth-generation nucleus families from the ARS-FY-H (n = 98), ARS-FY-L (n = 22), and ARS-FY-C (n = 30) genetic lines were grown to market body weight (1.8 kg) and approximately 5 fish per family were assigned to one of 5 harvest groups to characterize fillet yield, color, and texture. Phenotyping of 2 of the 5 harvest groups has been completed and phenotyping of the remaining 3 harvest groups will be completed by the end of July 2023. Analysis of selection response data through 3 generations of selection demonstrated that, compared to the randomly-mated ARS-FY-C line, fillet yield has increased in the ARS-FY-H line by 0.70 (SE = 0.16) percentage points per generation due to upward selection and decreased in the ARS-FY-L line by 0.35 (SE = 0.15) percentage points per generation due to downward selection. Preliminary data from the current fourth-generation nucleus families suggests that phenotypic divergence among the genetic lines has continued to increase, with fillet yield in the ARS-FY-H, -C, and -L lines currently averaging 58.2%, 56.5%, and 54.9%, respectively.
Progress towards Sub-objective 1b: A study was conducted to assess the genetic architecture of resistance to infectious hematopoietic necrosis virus (IHNV) in two commercial breeding populations of rainbow trout 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. Several moderate-large effect quantitative trait loci (QTL) were detected in the previous year and this year SNP haplotypes were identified in the QTL regions for further genetic analyses using whole-genome re-sequencing that will allow for enrichment and refining of the QTL regions. If better markers are identified from refinement efforts, they will be evaluated for their efficacy for marker assisted selection and for pinpointing the putative causative mutations and genes.
Progress toward 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 genotyping and DNA sequence analyses, two chromosome segments that span the disease resistance genes were identified in one commercial breeding population. This year the same two QTL were confirmed and validated in another genetic line from a commercial breeding company. In addition, germplasm was prepared for gene expression and gene-editing studies to evaluate candidate genes from the two QTL regions to assess if they are the causative genes for the large effect of the two chromosomal regions on BCWD resistance in the rainbow trout commercial lines.
Progress toward Sub-objective 1e: The aim of this sub-objective is to generate multiple chromosome-level genome assemblies as a pan-genome reference for rainbow trout that will better represent the genetic diversity in this species. Previously we generated a high-quality reference genome assembly from Arlee rainbow trout genetic line, which is now the default annotated genome map for rainbow trout in the NIH-NCBI official genome database. In the past year, additional three chromosome-level de-novo genome assemblies of high quality from the Swanson, Whale-Rock and Keithly Creek genetic lines of rainbow trout were completed and submitted to the NIH-NCBI public genome database.
Progress toward Sub-objective 2a: A genotype by environment study was completed in FY2022 that determined whether the High Yield line of rainbow trout (ARS-FY-H) retained its improved fillet yield trait when consuming three commercially available diets that vary in fat content. As previously reported, findings indicated that the high yield trait is preserved among the different diets, indicating that farmers can obtain benefits from the improved germplasm, regardless of dietary fat content. Subsequent research in FY2023 analyzed muscle fiber characteristics to characterize whether increased fillet yield is a result of increased muscle fiber hyperplasia or hypertrophy. Muscle histology indicated that the ARS-HY-H line exhibits a higher percentage of larger diameter muscle fibers in the 150 – 250 micron range, indicating hypertrophy is at least partially driving the increased muscle yield phenotype. Gene expression analysis is currently being evaluated in muscle and liver from the High Yield and Low Yield lines to characterize mechanisms contributing to differences in muscle growth.
Progress toward Sub-objective 2b: A study was previously conducted to characterize the phenotype of an F1 generation of rainbow trout mutants with truncated Lamp2a genes, originally produced using gene editing. We had previously reported that mutants ate more and grew faster than control fish when consuming a high-carbohydrate diet, suggesting that Lamp2a disruption increased fish adiposity. Subsequent analyses have demonstrated that Lamp2a mutants exhibit unique gene and protein expression profiles, particularly for genes and proteins related to lipid metabolism and appetite control. A second study in F1 fish provided preliminary data that Lamp2a mutants may also be more feed efficient than controls. Since these studies, an F2 generation of homozygous Lamp2a mutants were produced to use in additional studies that characterize nutrient utilization and partitioning and disease resistance in mutants and controls.
Progress toward Sub-objective 2c: Our previous work suggests egg quality is dependent upon proper activation of maternal transcripts stored in the egg. Short tails indicate the transcripts are stored and not active whereas longer tails indicate the transcripts have been activated for translation. Previously we used Oxford Nanopore Sequences to measure mRNA transcript poly-adenosine nucleotide (poly(A)) tail lengths in the eggs of rainbow trout. Last year we found average overall transcript length and the ratio of short to long tails were similar for ~700 genes identified as possible markers of egg quality and ~5,500 genes not associated with differences in egg quality partially meeting this year’s milestones. This year we examined in more detail expression profiles of 27 differentially expressed genes (DEGs) that we had identified as the most consistent markers of egg quality. The ratio of long to short reads varied among the genes suggesting differences in activation among the genes in unfertilized eggs. Unfortunately, the sequencing only yielded 50 or more reads (tail length measurements) for 8 of the genes, which is considered the minimum for expression profiling for comparison among treatments. Thus, a Nanopore Sequencing approach cannot cost effectively provide the number of reads required per gene. We evaluated and validated a new procedure for poly(A) tail measurement in rainbow trout, Palso-Seq, and it appears to provide about 10-fold more reads at the same cost. We sequenced the samples for unfertilized eggs, eggs at 24 hrs post fertilization, and 5 days post fertilization using the new approach and obtained about 4.5 million reads per stage compared with 350,000 using the old approach. Data analysis will continue in FY2023 and into FY2024.
Progress toward Sub-objective 2d: Samples from a study on the effects of overripening on egg quality, and another on maternal water quality parameters on egg quality were awaiting analysis as protocols to cost effectively measure transcript poly(A) tail lengths are being developed. Unfortunately, the samples were lost when a -80C freezer malfunctioned and warmed to ambient temperature over the holidays in December 2022. This resulted in the complete loss of samples from the aforementioned studies. The overripening study was repeated using eggs collected from the 2023 spawning season, but it was too late in the season to repeat the water quality study. Furthermore, earlier data from the water quality study indicated that water quality parameters did not have a large impact on egg quality, bringing into the question the value of repeating the study for transcriptomic analysis. As mentioned in objective 2C, we have recently established Palso-Seq as a viable approach for transcript poly(A) tail length analysis. Custom libraries for poly(A) tail analysis for the samples from the overripening study have been constructed and sent for sequencing.
Accomplishments
1. Hybridizing winter and summer spawning lines of trout yields fall spawning broodstock. Rainbow trout lines have been bred and selected to spawn at four different times of the year, necessitating the maintenance and selective breeding of each population to provide year-round egg production of genetically improved eggs. Rainbow trout females will not release their eggs in captivity, requiring hatchery staff to anesthetize and handle female fish once a week to squeeze the abdomen and determine whether eggs have ovulated and can be collected through manual expression. A spawning season with a 3-month window is manageable, but longer spawning seasons (more than 3 months) require more handling events which can be stressful for fish and is labor intensive for hatchery staff. ARS researchers in Leetown, West Virginia, discovered that eggs from summer spawning females, fertilized with frozen sperm from winter spawning males, yields females that will in turn spawn within a manageable 3-month window in the fall, instead of over a 6 to 12-month window as might be expected. This hybridization option eliminates the need to maintain and select upon a fall breeding population and suggests hatcheries with a single population can efficiently extend their egg production season with cryopreserved sperm from males from a population with an alternate spawn time.
2. The high fillet yield trait persists across different commercial diets. There is considerable variation in how much fat is found in commercial rainbow trout diets; some producers desire a lean diet (16-20% fat) while others feed a diet containing up to 35% fat. ARS scientists in Leetown, West Virginia, used selective breeding to produce a line of rainbow trout exhibiting high fillet yield (ARS-FY-H), but it is unknown if this trait persists across the range of fat levels commonly found in commercial feeds. A study was performed to determine how growth performance, fillet yield, and fillet quality respond in the ARS-FY-H line compared to a low fillet yield line (ARS-FY-L) while consuming a low fat (18%), moderate fat (24%) or high fat (33%) diet. While growth of the two lines was similar, the improved fillet yield trait persisted in the high yield line, regardless of dietary fat content, indicating that trout farmers can continue feeding their preferred dietary fat levels without losing the high fillet yield trait. Additionally, the high yield line exhibited a slightly firmer fillet, providing evidence that selection for fillet yield does not compromise fillet quality.
Review Publications
L. S. Garcia, A., Tsuruta, S., Gao, G., Palti, Y., Lourenco, T., Leeds, T.D. Genomic selection models substantially improve the accuracy of genetic merit predictions for fillet yield and body weight in rainbow trout using a multi-trait model and multi-generation progeny testing. Genetics Selection Evolution. 55:11 (2013). https://doi.org/10.1186/s12711-023-00782-6.
Vallejo, R.L., Evenhuis, J., Cheng, H., Fragomeni, B.O., Gao, G., Liu, S., Long, R., Shewbridge, K., Silva, R.O., Wiens, G.D., Leeds, T.D., Martin, K.E., Palti, Y. 2022. Genome-wide mapping of quantitative trait loci that can be used in marker-assisted selection for resistance to bacterial cold water disease in two commercial rainbow trout breeding populations. Aquaculture. 560(738574). https://doi.org/10.1016/j.aquaculture.2022.738574.
Weber, G.M., Martin, K.E., Palti, Y., Liu, S., Beach, J.N., Birkett, J.E. 2023. Effects of fertilizing eggs from a summer-spawning line with cryopreserved milt from a winter-spawning line on spawning date and egg production traits in rainbow trout. Aquaculture Reports. 29(101495). https://doi.org/10.1016/j.aqrep.2023.101495.
Gao, G., Waldbieser, G.C., Ramey, Y.C., Zaho, D., Pietrak, M.R., Stannard, J.A., Buchman, J.T., Scheffler, B.E., Peterson, B.C., Palti, Y., Rexroad III, C.E., Long, R., Burr, G.S., Milligan, M.T. 2023. The generation of the first chromosome-level de-novo genome assembly and the development and validation of a 50K SNP array for the St John River aquaculture strain of North American Atlantic salmon. G3, Genes/Genomes/Genetics. jkad138. https://doi.org/10.1093/g3journal/jkad138.
Cleveland, B.M., Radler, L.M., Leeds, T.D. 2023. Growth, fillet yield, and muscle quality traits are not affected by a genotype by environment interaction in rainbow trout consuming diets that differ in lipid content. Journal of the World Aquaculture Society. 1-15. https://doi.org/10.1111/jwas.12979.
Ahmed, R.O., Ali, A., Al-Tobasei, R., Leeds, T.D., Kenney, B., Salem, M. 2022. Weighted single-step GWAS identifies genes influencing fillet color in rainbow trout. Genes. 13(8),1331. https://doi.org/10.3390/genes13081331.
