Project : USDA ARS

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Research Project: Integrated Research Approaches for Improving Production Efficiency in Salmonids

Location: Cool and Cold Water Aquaculture Research

2018 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.c: Previously, we demonstrated the efficacy of family-based selection to improve fillet yield in a rainbow trout population. Compared to the randomly-mated control line of similar genetic origin, first-generation families from the upward-selected (high fillet yield) line had 0.65 percentage points higher fillet yield and families from the downward-selected (low fillet yield) line had 0.91 percentage points lower fillet yield. Second-generation families from the high (n = 100), low (n = 23), and randomly-mated control (n = 23) have been produced and will be grown to ~1.5 kilograms to evaluate selection response through two generations. Due to the favorable selection response in the first generation, representative second-generation high- and low-yield families will be pooled within line to confirm the divergent fillet yield phenotype and evaluate differences in long-term feed intake and feed efficiency between lines, with specific emphasis on the efficiency by which a unit of feed is converted into a unit of edible fillet. Sub-objective 1.d: Rainbow trout dorsal and caudal fin condition was assessed on 501 fish (approximately 14.5 months old) from 102 full-sib families from the USDA-National Center for Cool and Cold Water Aquaculture (NCCCWA) nucleus population using subjective quality scoring (0 = good; 5 = poor) and objective indices (fin ray length ÷ fork length; thus, a larger index value reflects better fin condition). Subjective dorsal and caudal fin scores averaged 1.30 and 1.51, respectively, and the fish averaged 44.9 centimeters in length (approximate body weight = 1.7 kilograms). The three objective indices (dorsal fin, top caudal fin, and bottom caudal fin) and fork length were analyzed using a four-trait animal model to derive preliminary estimates of heritabilities for, and genetic correlations among, the four traits. The three fin indices were lowly heritable (0.05 ± 0.15 to 0.18 ± 0.12), whereas fork length was moderately heritable (0.35 ± 0.16). Genetic correlations among the three fin indices were large and positive (0.66 to 0.89), whereas the genetic correlations between fin index and fork length tended to be small and negative (-0.45 to 0.13). These data suggest only limited potential to improve fin condition through selective breeding, and fish with increasing erosion generally have both fins affected. The small and negative correlation between fork length and fin indices gives no indication that smaller fish are more prone to increased erosion due to aggression from larger contemporary fish. Additional Sub-objective 1.d: Previously, scientists at the NCCCWA determined that rainbow trout bred for improved growth as part of their experimental selective breeding program exhibit better or similar growth rates and fillet yield than commercially available rainbow trout. To understand the physiological basis of improved performance, transcriptomic analysis was performed to identify differentially expressed genes between the different lines of rainbow trout. Unique gene expression profiles were identified in the liver and muscle of the growth-selected lines that support tissue-specific mechanisms driving improved performance in the faster growing fish. These differentially expressed genes have been validated and subsequent analyses will cluster these genes into functional groups to characterize the physiological impacts of the expression profiles. These findings are important for identifying genes that play a functional role in growth and identification of genetic markers useful for selective breeding. Objective 2: Infectious hematopoietic necrosis virus (IHNV) is the major viral pathogen in U.S. rainbow trout aquaculture. Our commercial partner has an ongoing selective breeding program for IHNV resistance that is based on pedigree and phenotypes from lab disease challenges. Our recent work with resistance to bacterial pathogens has demonstrated that use of genomics can accelerate the genetic progress achieved through selective breeding. As a first step in evaluating the benefits of genomic selection for IHNV disease resistance in this breeding population we conducted a whole genome scan in 1,000 fish from 100 families. Five chromosome segments that explain more than 1% of the total additive genetic variance for this trait were identified, but the overall oligogenic genetic architecture of the trait in this population indicates that further progress can be achieved through genomic selection. We will continue to work with the commercial producer to evaluate the genetic improvement that can be achieved through one generation of genomic section compared with conventual pedigree-based selective breeding. Sub-objective 3b: Genetic markers that were previously found to be in association with resistance to bacterial cold water disease in one of the rainbow trout breeding population of a commercial research partner, were also confirmed to be in association with disease resistance in a different breeding population of that commercial rainbow trout breeding company. Sub-objective 3c: Insulin-like growth factor-I (IGF1) is a hormone that stimulates growth, although it is bound to IGF binding proteins (IGFBP) that may increase and decrease IGF1 activity. Although expression of IGFBPs is highly regulated, their physiological significance as major regulators of growth and nutrient retention in fish is largely unknown. Pilot studies performed in 2017 indicated that a novel gene editing technique called CRISPR/Cas9 could be used to knockdown IGFBP2b protein abundance by greater than 95%. This success warranted production of additional fish to characterize the functional role of the IGFBP2b gene. In 2018, approximately 400 fish with mutated IGFBP2b genes were produced and will be phenotyped for growth and nutrient partitioning. These fish will indicate whether the IGFBP2b is a significant regulator of economically important traits. Additional Sub-objective 3c: Rainbow trout fillets are valued as an excellent source of protein with high concentrations of heart-healthy omega-3 fat. However, adding high omega-3 fish-oil to trout feeds is expensive so plant-based oils are desirable, but this comes at an expense to fillet omega-3 content. As a compromise, fish can be raised on a plant-oil diet for most of the grow-out period and transitioned to a high omega-3 “fish-oil finishing diet” several weeks prior to harvest to boost fillet omega-3 content. A previous study by scientists at the ARS NCCCWA and cooperators indicated that retention and mobilization of fat varied significantly across different regions of the fillet. Therefore, a study was performed to determine how different regions of the fillet respond during the transition from a plant-oil diet to a fish-oil finishing diet. Results indicated that the central fillet region was most dynamic in its retention of omega-3 fats, with the dorsal and ventral regions responding at similar slower rates. In addition, feed depriving fish for a short period prior to transition to the fish-oil finishing diet did not benefit omega-3 uptake, although how each region exhibited a unique response to feed deprivation. These findings indicate that while the lipid profile of the whole fillet is important from a consumer standpoint, it represents the mean of spatially distinct regions, each of which pose a unique physiological and metabolic response to feeding strategies. Sub-objective 3d: Early embryos cannot transcribe mRNA and are therefore dependent on maternal mRNA stored in the egg. It is not known to what extend 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 the largest U.S. trout egg supplier in the country we have previously shown that ~1000 polyadenylated or activated mRNAs transcripts differed among the highest and lowest quality eggs at ovulation. A multiplex assay that could measure 25 genes was developed using these data and the literature. We used the assay to compare ~200 egg lots from 2 yr classes from the commercial egg producer and our own hatchery, but found few genes to be consistently associated with egg quality. Nevertheless, we did find that mitochondrial gene transcript levels were reduced in many of the poor-quality eggs, and this appears to be the result of a combination of less mitochondria in the poor-quality eggs and lower mitochondrial gene expression in those eggs. We have decided we need to change the format on which the multiplex is based. A new multiplex is in design that can measure 70 genes in a single sample. This year we collected eggs from fish we raised through spawning, exposed to different amounts of reuse water, resulting in different water quality parameters and embryo survival rates. We will use the new multiplex to investigate the effects of these treatments on gene expression in the eggs and compare expression to that of good and poor-quality eggs from the broodstocks mentioned earlier.

Accomplishments
1. Gene editing in rainbow trout. Advancements in gene editing technologies have enabled inducing targeted mutations in genes of interest, allowing for precision-level manipulation of the genome and the ability to link a specific mutation with a specific phenotypic response. ARS researchers at Leetown, West Virginia have provided the first proof-of-concept for rainbow trout by showing that this new gene editing technology can produce fish with a complete loss-of-function phenotype. They also confirmed that genetic modifications induced by this technology can be transmitted to the next generation. The genetic modification can even be transmitted into sterile triploids, thereby preventing the unintended propagation of genetically modified germplasm. The researchers also used this technique to target genes important for endocrine regulation of growth and demonstrated that the abundance of proteins encoded by those targeted genes was reduced by more than 95%. By optimizing gene editing technology in rainbow trout, researchers now have a tool to study the function of specific proteins and unravel how genetic mechanisms regulate economically important traits.

2. Incubation temperature impact on embryo survival. Incubation temperature is commonly used to manipulate hatch date in salmonids but there is little information on the effect of such changes in temperature on embryo survival. Working with a stakeholder, ARS researchers at Leetown, West Virginia found that incubation at 5 degrees Celsius within the first day of fertilization reduced embryo survival compared to incubation at 10 degrees Celsius. Also, rapidly transferring embryos between 10 degrees Celsius and 5 degrees Celsius after 100 degree days of incubation did not affect survival. This information cautions stakeholders to end the practice of starting incubation of fertilized eggs at 5 degrees Celsius and supports that hatcheries do not need a complicated system to slowly change water temperatures when development needs to be accelerated or slowed to meet production needs.

U.S. DEPARTMENT OF AGRICULTURE

Cool and Cold Water Aquaculture Research: Leetown, WV