Location: Cool and Cold Water Aquaculture Research
2022 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 Subobjective 1a: Third-generation ARS-FY-H, ARS-FY-L, and ARS-FY-C families were harvested at approximately 16 months post-hatch and characterized for fillet yield. The resulting fillet yield data, along with pedigree data, were analyzed using an animal model to estimate breeding values and selection response. Mean fillet yield (57.1%) of third-generation ARS-FY-H families (n = 100) was 1.48 percentage points greater compared to ARS-FY-C families (n = 24) and 2.89 percentage points greater compared to ARS-FY-L families (n = 23). Family-based selection was practiced to select 51 high genetic merit families from the ARS-FY-H line for use as sires and dams of fourth-generation families. Random mating was practiced within each of the contemporary ARS-FY-L and ARS-FY-C lines to produce fourth-generation families. At the time of this report, fourth-generation families from the ARS-FY-H (n = 100), ARS-FY-C (n = 30), and ARS-FY-L (n = 22) are in the process of being PIT-tagged for individual identification and will be grown for subsequent fillet yield characterization and spawning to produce fifth-generation families.
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. In the past year, we used genome wide association analyses to identify an oligogenic architecture for genetic resistance to IHNV with several moderate-large effect quantitative trait loci (QTL) and detected large effect QTL that may be used for marker assisted selection following further validation. A total of 1,867 and 1,772 offspring from two commercial aquaculture strains were phenotyped for resistance to IHNV and genotyped with the rainbow trout 57K SNP array. Overall, 21 moderate-large effect QTL were detected in the two strains. Only two QTL were mapped to the same chromosomal regions in both strains. Seven of the QTL were previously mapped to the same chromosomal regions in a third aquaculture strain.
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 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 that were developed and used to demonstrate the effectiveness of marker assisted selection in one commercial breeding population were also demonstrated to be effective for selective breeding in another unrelated commercial breeding population.
Progress toward Sub-objective 1d: A study is 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). To conduct more in-depth analysis, 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 the artificial selection for resistance to BCWD in this rainbow trout breeding population.
Progress toward Sub-objective 1e: 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 we included bioinformatics analysis using long sequence reads technology to complete high quality chromosome level de-novo genome assemblies for rainbow trout from two diverse genetic lines and draft assembly for a third line. Overall, this project when completed will generate four chromosome-level de-novo genome assemblies from the Arlee, Swanson, Whale-Rock and Keithly Creek genetic lines of rainbow trout. In addition, DNA preparations and sequencing of fish from three cutthroat trout genetic lineages was completed this year to generate altogether four high contiguity reference genome maps. The Bonneville, Yellowstone and Coastal cutthroat trout lineages that were sequenced in the past year represent a large component of the geographic distribution and genetic diversity of cutthroat trout in the Northwest U.S.
Progress towards Sub-objective 2a: A genetics-by-environment (GxE) study was conducted to evaluate whether the High Fillet Yield line of rainbow trout (ARS-FY-H) retains its improved fillet yield trait while consuming three commercially available diets that vary in fat content (18%, 24%, 33% dietary fat). At final harvest (average body weight, 2.2 kg), the ARS-FY-H line exhibited improved fillet yield (+2.7%) across all diets compared to the Low Fillet Yield line (ARS-FY-L). The ARS-FY-H line also demonstrated slightly higher muscle lipid content (+0.7%), lower viscera fat (-4.8%), and minor increases in shear force. These results suggest that fat is directed to viscera waste tissue in the AYS-FY-L line, while it is directed more to muscle storage in the HY line. These findings are beneficial to both farmers, processors, and consumers, as they indicate that the high fillet yield trait of the ARS-FY-H line will be achieved regardless of dietary fat content, and provide evidence that selective breeding for higher fillet yield does not compromise fillet quality.
Progress towards Sub-objective 2b: Previously we produced an F1 population of mutant rainbow trout with truncated lamp2a genes on chromosomes 14 and 25. Progress towards this objective included genotyping all F1 individuals to classify fish as single mutants (only one truncated gene) or double mutants (two truncated genes), the latter of which should exhibit complete loss of lamp2a function. A subset of single and double mutants were retained to produce an F2 generation of homozygous knockouts, while the remaining were utilized in a phenotyping study that fed a high-carbohydrate diet to control and mutant fish. Both single and double mutants grew faster than control fish, and exhibited higher hepatosomatic index and visceral somatic index, suggesting that lamp2a disruption increases adiposity and affects carbohydrate utilization. Tissue samples are being analyzed for differential gene expression and histological evaluation to characterize the physiological mechanisms affected by lamp2a disruption.
Progress towards Sub-objective 2c: Last year Oxford Nanopore Sequences were used to measure the length of poly(A) tails for mRNA transcripts within rainbow trout eggs. Short tails (<25 poly(A)s) indicate the transcripts are stored and not active whereas longer tails indicate the transcripts have been activated for translation into proteins. Preliminary analysis of egg transcripts revealed a wide distribution of poly(A) tail lengths with a minor peak around 10 nucleotides, and individual genes exhibit high variation in poly(A) tail lengths. Nanopore sequencing of good versus poor quality eggs yielded ~305,000 transcript tail length reads among 18,000 genes, of which 6,213 genes exhibited 10 or more reads. Among these 6,213 genes, 710 were genes we previously identified as having differential expression in good vs poor quality eggs. In good-quality eggs, these transcripts had and an average tail length of 45 nucleotides with 30% of these genes exhibiting short poly(A) tails. These values were similar to the values for all genes which had an average length of 51 nucleotides and 26% has short poly(A) tails.
Progress towards Sub-objective 2d: The Oxford Nanopore Sequencing approach used in Objective 2C to analyze poly(A) tail length, as well as alternatives such as Tail-Seq, requires analyzing transcripts with poly(A) tails that are too short to be captured using typical mRNA enrichment methods. Therefore, these approaches require rRNA depletion which is cost prohibitive to obtain the amount of transcript reads required to discriminate between low- and high-quality eggs as planned. For this reason, we have utilized alternative methodologies that do not require rRNA depletion. One approach, mTail-Seq, not only proved to be technically difficult to prepare the libraries and required spike-in standards, but it may soon be outdated. However, a second alternative approach, PAIso-Seq, shows promise and is a methodology we are currently evaluating. Also in support of subobjective 2a, an oocyte aging study was repeated. The previous study used in vitro incubation of ovulated eggs to compare the effects of post-ovulatory ageing on egg quality and the egg transcriptome. Egg survival data was variable among egg lots, suggesting confounding factors deriving from the in vitro incubation procedures. The repeated study used in vivo incubation, leaving the eggs in the females’ body cavity, and periodically collecting aliquots of eggs from the females over a 2-week period which resulted in more consistent effects of time on egg survival.
Accomplishments
1. Marker assisted selection for resistance to bacterial cold-water disease in rainbow trout. Bacterial cold-water disease (BCWD) is one of the most devastating diseases in rainbow trout aquaculture. Improving resistance to BCWD using traditional family-based selective breeding or genomic selection has shown promise but is limited since these methods are labor intensive, costly, and the resistance trait cannot be measured directly in potential breeders. For those reasons marker assisted selection (MAS) is advantageous because it can directly predict the genetic merit of potential breeding animals using just a small number of DNA markers. Therefore, MAS is a cost savings approach for improving disease resistance through selective breeding. ARS researchers in Leetown, West Virginia, have identified a set of six DNA markers that can be used to predict the genetic merit of breeding animals with the same or better accuracy than the traditional family-based selective breeding approach. The effectiveness of this cost-reducing approach was demonstrated in a commercial breeding population, indicating that it can further improve the efficiency and sustainability of rainbow trout aquaculture in the U.S.
2. Improved procedure for sex-reversal in rainbow trout. Most of the rainbow trout industry depends upon production of all-female fish for grow-out. The maintenance of all-female lines depends upon sex reversal of female fry into sperm-producing fish, a process that involves supplementing feed with 17alpha-methyltestosterone steroid for 60 days. Drawbacks of this approach include the need to surgically remove testes for sperm extraction because the sex-reversed fish seldom develop functional sperm ducts, and release of steroid into the environment through effluent water. ARS researchers in Leetown, West Virginia, developed an improved approach to sex reversal by exposing female fry to 17alpha-methyltestosterone through immersion rather than feeding. Researchers have shown that immersion treatment consisting of 7 one-hour weekly immersions in the steroid beginning at 4-7 days post-hatching greatly reduced the number of fish with sperm duct abnormalities, avoiding the need to terminate fish to surgically remove testes to harvest sperm, and prevents environmental contamination by allowing for the steroid to be captured from the immersion bath.
Review Publications
Vallejo, R.L., Cheng, H., Fragomeni, B.O., Silva, R.O., Martin, K.E., Evenhuis, J., Wiens, G.D., Leeds, T.D., Palti, Y. 2021. The accuracy of genomic predictions for bacterial cold water disease resistance remains higher than the pedigree-based model one generation after model training in a commercial rainbow trout breeding population. Aquaculture. 545:737164. https://doi.org/10.1016/j.aquaculture.2021.737164.
Weber, G.M., Ma, H., Birkett, J.E., Cleveland, B.M. 2022. Effects of feeding level and sexual maturation on expression of genes regulating growth mechanisms in rainbow trout (Oncorhynchus mykiss). Aquaculture. 551: 737917. https://doi.org/10.1016/j.aquaculture.2022.737917.
Deck, C.A., Mankiewicz, J.L., Borski, R.J., Cleveland, B.M. 2022. Evidence for a leptin–insulin axis in a fish, the tilapia (Oreochromis mossambicus). Journal of Endocrinology. 253(1):13-25. https://doi.org/10.1530/JOE-21-0139.
Mankiewicz, J.L., Picklo, M.J., Idso, J.P., Cleveland, B.M. 2022. Evidence of hyperphagia and fatty acid mobilization in leptin receptor deficient rainbow trout (Oncorhynchus mykiss). Biomolecules EISSN 2218-273X. 12(4):516. https://doi.org/10.3390/biom12040516.
Weber, G.M., Leeds, T.D. 2022. Effects of duration and timing of immersion in 17alpha-methyltestosterone on sex reversal of female rainbow trout. Aquaculture Reports. 23: 101014. https://doi.org/10.1016/j.aqrep.2022.101014.
Liu, S., Martin, K.E., Gao, G., Long, R., Evenhuis, J., Leeds, T.D., Wiens, G.D., Palti, Y. 2022. Identification of haplotypes associated with resistance to bacterial cold water disease in rainbow trout using whole-genome resequencing. Frontiers in Genetics. 13:936806. https://doi.org/10.3389/fgene.2022.936806.
Bernard, M., Dehaullon, A., Gao, G., Paul, K., Lagarde, H., Charles, M., Prchal, M., Danon, J., Jaffrelo, L., Poncet, C., Patrice, P., Haffray, P., Quillet, E., Dupont-Nivet, M., Palti, Y., Lallias, D., Phocas, F. 2022. Development of a high-density SNP array for rainbow trout genome-wide genotyping. Frontiers in Genetics. 13:941340. https://doi.org/10.3389/fgene.2022.941340.
