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 Cool and Cold Water Aquaculture Research: Leetown, WV
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ARS Home Northeast Area Leetown, West Virginia Cool and Cold Water Aquaculture Research Research





- Cleveland, Beth
- Palti, Yniv
- Vallejo, Roger
- Liu, Sixin
- Leeds, Timothy - Tim
- Weber, Gregory - Greg
- 2023
- 2022
- 2021
- 2020
- Publications
- Aquaculture (106)

Research Project: Integrated Research Approaches for Improving Production Efficiency in Rainbow Trout **Location:
                            Cool and Cold Water Aquaculture Research** 2020 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 toward Sub-objective 1a includes characterization of fillet yield in second-generation families from the high (ARS-FY-H), randomly-mated control (ARS-FY-C), and low fillet yield (ARS-FY-L) genetic lines and selection and spawning of broodstock to produce third-generation families. A total of 100 ARS-FY-H, 24 ARS-FY-C, and 23 ARS-FY-L third-generation nucleus families were produced and retained for grow-out. These families will be phenotyped for fillet yield at approximately 14 months of age. Also in support of Sub-objective 1a was a study is 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 have completed fillet yield phenotyping for 500 fish from year-class 2018 and genotyped fish with known fillet yield from 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 data we have collected and tabulated are currently being analyzed in collaboration with researchers from the University of Georgia.
In support of Sub-objective 1b, a study was completed to compare between the accuracy of breeding value predictions done with or without genomic information for viral disease resistance to infectious hematopoietic necrosis virus (IHNV) to determine the added genetic value of using genomics for this trait.  The study was conducted in a commercial breeding population from a stock that has been selected for IHNV resistance for more than 10 years. Adding genomics information increased the accuracy of the genetic merit prediction by 15% (from 0.33 to 0.38), which is a meaningful improvement, but smaller than what we have found in a previous study on bacterial cold-water disease resistance in another commercial breeding population. We have identified differences in the genetic architecture of the two disease resistance traits and aspects of the study designs and breeding schemes that likely contributed to the smaller improvement from genomic selection in this study. Also in support of Sub-objective 1b was a study to assess the genetic architecture of IHNV resistance in two additional 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 by an external collaborator and we have begun to extract DNA samples from the nearly 4,000 fish that were phenotyped for resistance to IHNV to enable genotyping with the 57K SNP chip.
Progress towards Sub-objective 1c included sequencing DNA of fish from 60 families from one of the nucleus breeding populations of our industry research partner. Families were selected because they were previously found to have intermediate BCWD survival rates within that population. DNA from one random fish that did not survive the disease challenge was pooled per family to make a susceptible DNA pool (S-pool), and DNA from one random survivor was pooled to make a resistant DNA pool (R-pool). In total, we sequenced four biological replicates from each pool. SNP DNA markers associated with BCWD resistance were identified after the pooled DNA sequencing analysis. To validate the association of the SNPs, genotyping assays were developed from these putative markers and were used to initiate genotyping of a larger sample size of individual fish from all the families that were phenotyped for resistance to BCWD in that nucleus breeding population. The detection of DNA markers and genes with major effects on resistance to the disease can be used for marker assisted selection, which is less costly than the whole-genome enabled selective breeding approach.
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 data analysis included quality control for marker genotype data from six rainbow trout lines (n= 670 fish) genotyped with the 57K SNP chip. After quality control, we identified the fish that provide the best representation of each genetic line and used marker genotype data from 191 fish genotyped with 33,439 informative SNPs for selective sweep analysis (SSA). We performed SSA with three methods and identified the four most informative genetic lines for future SSA that we plan to conduct with genotype data with higher density DNA markers. We also found four genomic regions with very strong signatures of selection on four chromosomes (Omy5, 9, 12 and 17). These regions will be re-assessed using higher density SNP genotype data from a SNP array or from whole-genome resequencing in the next phase of the study.
Progress toward Sub-objective 1e included improving the contiguity and annotation of the current reference genome for rainbow trout and to generate a pan-genome reference from several homozygous genetic lines that represent the geographic distribution of rainbow trout in western United States. In the past year we have completed another de-novo genome assembly using DNA from the Arlee homozygous genetic line. The Arlee genome assembly was conducted using long-reads sequencing technology and other advanced sequence scaffolding tools and produced a much greater contiguity with a scaffolds N50 size of 47.5 Mb compared to only 1.6 Mb in the current Swanson genome assembly. The Arlee assembly was submitted to the NCBI public genome database and is currently being annotated for protein coding genes. In addition, we have raised fish from three additional homozygous genetic lines for sampling and purification of high-grade DNA that will be of sufficient quality and quantity for additional de-novo genome assemblies using advanced long-reads DNA sequencing technologies.
In support of Sub-objective 2a, a study was performed to determine whether selection for fillet yield in rainbow trout had an indirect effect on feed efficiency. Twenty families were sampled each within the ARS-FY-H and ARS-FY-L lines (40 total families). Twenty-five fish per family were stocked into five separate tanks; lines were kept separate so a total of 10 tanks were included in the study.  Fish were reared from approximately 175 g to 1 kg and body weight gain and feed intake was recorded. A 2.3 percentage point improvement in feed efficiency was observed in the ARS-FY-H line, indicating that selection for fillet yield also benefits feed utilization efficiency.
Progress toward Sub-objective 2b includes application of the previously developed CRISPR/Cas9-mediated gene editing technology to disrupt expression of a functional lamp2a gene by targeted excision of the 9a exon critical for protein function.  Rainbow trout were produced with partial excision of the lamp2a-9a exon on both chromosome 14 (chr14) and chromosome 25 (chr25). These fish will be retained for breeding to produce homozygous mutants in an F1 offspring generation. In addition, a large number of fish were produced with near-complete excision of lamp2a-9a on chr25, with the gene on chr14 intact. Previous data indicates that lamp2a on chr25 is the only expressed gene (chr14 may be a pseudogene), so fish with only chr25 disruption could represent functional LAMP2a knockouts. These fish are currently being analyzed for growth performance and indices of altered nutrient metabolism.
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 is necessary to study transcript activation. We have collected unfertilized eggs, and 21-hr and 5-day post-fertilization embryos for characterization of tail lengths using Tail-Seq, and in fixative for characterization of developmental stage. Samples from 40 females have been collected and the survival at eyeing for each family was determined as a measure of egg quality. Changes in overall abundance of the different transcripts as well as their tail lengths will be compared during early development. **Accomplishments** **Review Publications**
                            *Cleveland, B.M., Gao, G., Leeds, T.D. 2020. Transcriptomic response to selective breeding for fast growth in rainbow trout (Oncorhynchus mykiss). Marine Biotechnology. https://doi.org/10.1007/s10126-020-09974-3.*
                                *Kumar, V., Lee, S., Cleveland, B.M., Romano, N., Hardy, R., Lalgudi, R., Mcgraw, B. 2020. Comparative evaluation of processed soybean meal (EnzoMealTM) vs. soybean meal as a fishmeal replacement in diets of rainbow trout (Oncorhynchus mykiss). Aquaculture. 516:734652. https://doi.org/10.1016/j.aquaculture.2019.734652.*
                                *Gao, G., Pietrak, M.R., Burr, G.S., Rexroad III, C.E., Peterson, B.C., Palti, Y. 2020. A new single nucleotide polymorphism database for North American Atlantic salmon generated through whole genome re-sequencing. Frontiers in Genetics. 11:85. https://doi.org/10.3389/fgene.2020.00085.*
                                *Weber, G.M., Leeds, T.D. 2020. Sex reversal of female rainbow trout by immersion in 17alpha-methyltestosterone. Aquaculture. 528:735535. https://doi.org/10.1016/j.aquaculture.2020.735535.*
                                *Shapagain, P., Arivett, B., Cleveland, B.M., Walker, D., Salem, M. 2019. Gut microbiome analysis of fast- and slow-growing Rainbow Trout (Oncorhynchus mykiss). Frontiers in Microbiology. 20:788. https://doi.org/10.1186/s12864-019-6175-2.*
                                *Lee, S., Kumar, V., Cleveland, B.M., Tomano, N., Vemuri, G., Yadav, A., Hardy, R. 2020. Fishmeal alternative from renewable CO2 for rainbow trout feed. Aquaculture Research. 261, 357–368. https://doi.org/10.1111/are.14749.*
                                *Caballero-Solares, A., Xue, X., Cleveland, B.M., Beheshti Foroutani, M., Parrish, C., Taylor, R., Rise, M. 2020. Multiplex PCR platforms to evaluate diet-induced gene expression changes in the liver of Atlantic salmon (Salmo salar). Marine Biotechnology.*


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