Plasma proteomic profiling of bacterial cold water disease-resistant and -susceptible rainbow trout lines and biomarker discovery.

Genetic variation for disease resistance is present in salmonid fish; however, the molecular basis is poorly understood, and biomarkers of disease susceptibility/resistance are unavailable. Previously, we selected a line of rainbow trout for high survival following standardized challenge with Flavobacterium psychrophilum (Fp), the causative agent of bacterial cold water disease. The resistant line (ARS-Fp-R) exhibits over 60 percentage points higher survival compared to a reference susceptible line (ARS-Fp-S). To gain insight into the differential host response between genetic lines, we compared the plasma proteomes from day 6 after intramuscular challenge. Pooled plasma from unhandled, PBS-injected, and Fp-injected groups were simultaneously analyzed using a TMT 6-plex label, and the relative abundance of 513 proteins was determined. Data are available via ProteomeXchange, with identifier PXD041308, and the relative protein abundance values were compared to mRNA measured from a prior, whole-body RNA-seq dataset. Our results identified a subset of differentially abundant intracellular proteins was identified, including troponin and myosin, which were not transcriptionally regulated, suggesting that these proteins were released into plasma following pathogen-induced tissue damage. A separate subset of high-abundance, secreted proteins were transcriptionally regulated in infected fish. The highest differentially expressed protein was a C1q family member (designated complement C1q-like protein 3; C1q-LP3) that was upregulated over 20-fold in the infected susceptible line while only modestly upregulated, 1.8-fold, in the infected resistant line. Validation of biomarkers was performed using immunoassays and C1q-LP3, skeletal muscle troponin C, cathelcidin 2, haptoglobin, leptin, and growth and differentiation factor 15 exhibited elevated concentration in susceptible line plasma. Complement factor H-like 1 exhibited higher abundance in the resistant line compared to the susceptible line in both control and challenged fish and thus was a baseline differentiator between lines. C1q-LP3 and STNC were elevated in Atlantic salmon plasma following experimental challenge with Fp. In summary, these findings further the understanding of the differential host response to Fp and identifies salmonid biomarkers that may have use for genetic line evaluation and on-farm health monitoring.

Identification of Haplotypes Associated With Resistance to Bacterial Cold Water Disease in Rainbow Trout Using Whole-Genome Resequencing.

Bacterial cold water disease (BCWD) is an important disease in rainbow trout aquaculture. Previously, we have identified and validated two major QTL (quantitative trait loci) for BCWD resistance, located on chromosomes Omy08 and Omy25, in the odd-year Troutlodge May spawning population. We also demonstrated that marker-assisted selection (MAS) for BCWD resistance using the favorable haplotypes associated with the two major QTL is feasible. However, each favorable haplotype spans a large genomic region of 1.3-1.6 Mb. Recombination events within the haplotype regions will result in new haplotypes associated with BCWD resistance, which will reduce the accuracy of MAS for BCWD resistance over time. The objectives of this study were 1) to identify additional SNPs (single nucleotide polymorphisms) associated with BCWD resistance using whole-genome sequencing (WGS); 2) to validate the SNPs associated with BCWD resistance using family-based association mapping; 3) to refine the haplotypes associated with BCWD resistance; and 4) to evaluate MAS for BCWD resistance using the refined QTL haplotypes. Four consecutive generations of the Troutlodge May spawning population were evaluated for BCWD resistance. Parents and offspring were sequenced as individuals and in pools based on their BCWD phenotypes. Over 12 million SNPs were identified by mapping the sequences from the individuals and pools to the reference genome. SNPs with significantly different allele frequencies between the two BCWD phenotype groups were selected to develop SNP assays for family-based association mapping in three consecutive generations of the Troutlodge May spawning population. Among the 78 SNPs derived from WGS, 77 SNPs were associated with BCWD resistance in at least one of the three consecutive generations. The additional SNPs associated with BCWD resistance allowed us to reduce the physical sizes of haplotypes associated with BCWD resistance to less than 0.5 Mb. We also demonstrated that the refined QTL haplotypes can be used for MAS in the Troutlodge May spawning population. Therefore, the SNPs and haplotypes reported in this study provide additional resources for improvement of BCWD resistance in rainbow trout.

Aquaculture Reuse Water, Genetic Line, and Vaccination Affect Rainbow Trout (Oncorhynchus mykiss) Disease Susceptibility and Infection Dynamics.

Infectious hematopoietic necrosis virus (IHNV) and Flavobacterium psychrophilum are major pathogens of farmed rainbow trout. Improved control strategies are desired but the influence of on-farm environmental factors that lead to disease outbreaks remain poorly understood. Water reuse is an important environmental factor affecting disease. Prior studies have established a replicated outdoor-tank system capable of varying the exposure to reuse water by controlling water flow from commercial trout production raceways. The goal of this research was to evaluate the effect of constant or pulsed reuse water exposure on survival, pathogen prevalence, and pathogen load. Herein, we compared two commercial lines of rainbow trout, Clear Springs Food (CSF) and Troutex (Tx) that were either vaccinated against IHNV with a DNA vaccine or sham vaccinated. Over a 27-day experimental period in constant reuse water, all fish from both lines and treatments, died while mortality in control fish in spring water was <1%. Water reuse exposure, genetic line, vaccination, and the interaction between genetic line and water exposure affected survival (P<0.05). Compared to all other water sources, fish exposed to constant reuse water had 46- to 710-fold greater risk of death (P<0.0001). Tx fish had a 2.7-fold greater risk of death compared to CSF fish in constant reuse water (P ≤ 0.001), while risk of death did not differ in spring water (P=0.98). Sham-vaccinated fish had 2.1-fold greater risk of death compared to vaccinated fish (P=0.02). Both IHNV prevalence and load were lower in vaccinated fish compared to sham-vaccinated fish, and unexpectedly, F. psychrophilum load associated with fin/gill tissues from live-sampled fish was lower in vaccinated fish compared to sham-vaccinated fish. As a result, up to forty-five percent of unvaccinated fish were naturally co-infected with F. psychrophilum and IHNV and the coinfected fish exhibited the highest IHNV loads. Under laboratory challenge conditions, co-infection with F. psychrophilum and IHNV overwhelmed IHNV vaccine-induced protection. In summary, we demonstrate that exposure to reuse water or multi-pathogen challenge can initiate complex disease dynamics that can overwhelm both vaccination and host genetic resistance.

Comparisons among rainbow trout, Oncorhynchus mykiss, populations of maternal transcript profile associated with egg viability.

BACKGROUND: Transcription is arrested in the late stage oocyte and therefore the maternal transcriptome stored in the oocyte provides nearly all the mRNA required for oocyte maturation, fertilization, and early cleavage of the embryo. The transcriptome of the unfertilized egg, therefore, has potential to provide markers for predictors of egg quality and diagnosing problems with embryo production encountered by fish hatcheries. Although levels of specific transcripts have been shown to associate with measures of egg quality, these differentially expressed genes (DEGs) have not been consistent among studies. The present study compares differences in select transcripts among unfertilized rainbow trout eggs of different quality based on eyeing rate, among 2 year classes of the same line (A1, A2) and a population from a different hatchery (B). The study compared 65 transcripts previously reported to be differentially expressed with egg quality in rainbow trout. RESULTS: There were 32 transcripts identified as DEGs among the three groups by regression analysis. Group A1 had the most DEGs, 26; A2 had 15, 14 of which were shared with A1; and B had 12, 7 of which overlapped with A1 or A2. Six transcripts were found in all three groups, dcaf11, impa2, mrpl39_like, senp7, tfip11 and uchl1. CONCLUSIONS: Our results confirmed maternal transcripts found to be differentially expressed between low- and high-quality eggs in one population of rainbow trout can often be found to overlap with DEGs in other populations. The transcripts differentially expressed with egg quality remain consistent among year classes of the same line. Greater similarity in dysregulated transcripts within year classes of the same line than among lines suggests patterns of transcriptome dysregulation may provide insight into causes of decreased viability within a hatchery population. Although many DEGs were identified, for each of the genes there is considerable variability in transcript abundance among eggs of similar quality and low correlations between transcript abundance and eyeing rate, making it highly improbable to predict the quality of a single batch of eggs based on transcript abundance of just a few genes.

Identification of High-Confidence Structural Variants in Domesticated Rainbow Trout Using Whole-Genome Sequencing.

Genomic structural variants (SVs) are a major source of genetic and phenotypic variation but have not been investigated systematically in rainbow trout (Oncorhynchus mykiss), an important aquaculture species of cold freshwater. The objectives of this study were 1) to identify and validate high-confidence SVs in rainbow trout using whole-genome re-sequencing; and 2) to examine the contribution of transposable elements (TEs) to SVs in rainbow trout. A total of 96 rainbow trout, including 11 homozygous lines and 85 outbred fish from three breeding populations, were whole-genome sequenced with an average genome coverage of 17.2×. Putative SVs were identified using the program Smoove which integrates LUMPY and other associated tools into one package. After rigorous filtering, 13,863 high-confidence SVs were identified. Pacific Biosciences long-reads of Arlee, one of the homozygous lines used for SV detection, validated 98% (3,948 of 4,030) of the high-confidence SVs identified in the Arlee homozygous line. Based on principal component analysis, the 85 outbred fish clustered into three groups consistent with their populations of origin, further indicating that the high-confidence SVs identified in this study are robust. The repetitive DNA content of the high-confidence SV sequences was 86.5%, which is much higher than the 57.1% repetitive DNA content of the reference genome, and is also higher than the repetitive DNA content of Atlantic salmon SVs reported previously. TEs thus contribute substantially to SVs in rainbow trout as TEs make up the majority of repetitive sequences. Hundreds of the high-confidence SVs were annotated as exon-loss or gene-fusion variants, and may have phenotypic effects. The high-confidence SVs reported in this study provide a foundation for further rainbow trout SV studies.

Transcriptomic Response to Selective Breeding for Fast Growth in Rainbow Trout (Oncorhynchus mykiss).

Genetic improvement for faster growth is a conventional approach to increase growth rates in aquaculture species; however, the genetic and physiological factors regulating growth performance in fish are not fully characterized. The objective of this study was to identify physiological mechanisms associated with faster growth rates by comparing the liver and muscle transcriptome of a rainbow trout line selectively bred for fast growth (growth line, GL) and a contemporary randomly mated control line (synthetic control, SC) from the same selective breeding program. A third genetic line from a commercial egg supplier (commercial A, CA) was also included to characterize differences in gene expression profiles between populations. Body weight of the GL at harvest was approximately 20% and 8% heavier (p < 0.05) than SC and CA, respectively. There were 145 and 36 differentially expressed genes (DEG) in liver and white muscle, respectively, between the GL and SC that were enriched for the growth hormone/insulin-like growth factor axis (GH/IGF) and PI3K-Akt, JAK-STAT, MAPK, and cAMP signal transduction pathways. A greater concentration of plasma IGF-I was detected in the GL compared with SC (p < 0.05). A unique gene profile was detected in CA, with 11 and 210 DEG in liver and white muscle; these genes associated with innate immunity, complement systems, and metabolic pathways. Collectively, these findings provide a more extensive characterization of the fast-growth phenotype in fish that furthers knowledge of the physiological basis for genetic variation in growth performance in selectively bred rainbow trout.

Variance and covariance estimates for resistance to bacterial cold water disease and columnaris disease in two rainbow trout breeding populations1.

Family-based selective breeding can be an effective strategy for controlling diseases in aquaculture. This study aimed to estimate (co)variance components for resistance to bacterial cold water disease (BCWD) and columnaris disease (CD) in two unrelated rainbow trout nucleus breeding populations: the USDA, ARS, National Center for Cool and Cold Water Aquaculture odd-year line (ARS-Fp-R), which has been subjected to five generations of selection for improved resistance to BCWD, and the Troutlodge, Inc., May-spawning odd-year line (TLUM), which has been selected for improved growth performance but not for disease resistance. A total of 46,805 and 27,821 pedigree records were available from both populations, respectively. Between 44 and 138 families per generation and population were evaluated under controlled BCWD and CD challenges, providing 32,311 and 17,861 phenotypic records for BCWD resistance, and 13,603 and 9,413 for CD resistance, in the ARS-Fp-R and TLUM populations, respectively. A two-trait animal threshold model assuming an underlying normal distribution for the binary survival phenotypes was used to estimate (co)variance components separately for each population. Resistance to BCWD (h2 = 0.27 ± 0.04 and 0.43 ± 0.08) and CD (h2 = 0.23 ± 0.07 and 0.34 ± 0.09) was moderately heritable in the ARS-Fp-R and TLUM populations, respectively. The genetic correlation between the resistance to BCWD and CD was favorably positive in the ARS-Fp-R (0.40 ± 0.17) and TLUM (0.39 ± 0.18) populations. These findings suggest that both disease resistance traits can be improved simultaneously even if genetic selection pressure is applied to only one of the two traits.

Genome-Wide Association Analysis With a 50K Transcribed Gene SNP-Chip Identifies QTL Affecting Muscle Yield in Rainbow Trout.

Detection of coding/functional SNPs that change the biological function of a gene may lead to identification of putative causative alleles within QTL regions and discovery of genetic markers with large effects on phenotypes. This study has two-fold objectives, first to develop, and validate a 50K transcribed gene SNP-chip using RNA-Seq data. To achieve this objective, two bioinformatics pipelines, GATK and SAMtools, were used to identify ~21K transcribed SNPs with allelic imbalances associated with important aquaculture production traits including body weight, muscle yield, muscle fat content, shear force, and whiteness in addition to resistance/susceptibility to bacterial cold-water disease (BCWD). SNPs ere identified from pooled RNA-Seq data collected from ~620 fish, representing 98 families from growth- and 54 families from BCWD-selected lines with divergent phenotypes. In addition, ~29K transcribed SNPs without allelic-imbalances were strategically added to build a 50K Affymetrix SNP-chip. SNPs selected included two SNPs per gene from 14K genes and ~5K non-synonymous SNPs. The SNP-chip was used to genotype 1728 fish. The average SNP calling-rate for samples passing quality control (QC; 1,641 fish) was ≥ 98.5%. The second objective of this study was to test the feasibility of using the new SNP-chip in GWA (Genome-wide association) analysis to identify QTL explaining muscle yield variance. GWA study on 878 fish (representing 197 families from 2 consecutive generations) with muscle yield phenotypes and genotyped for 35K polymorphic markers (passing QC) identified several QTL regions explaining together up to 28.40% of the additive genetic variance for muscle yield in this rainbow trout population. The most significant QTLs were on chromosomes 14 and 16 with 12.71 and 10.49% of the genetic variance, respectively. Many of the annotated genes in the QTL regions were previously reported as important regulators of muscle development and cell signaling. No major QTLs were identified in a previous GWA study using a 57K genomic SNP chip on the same fish population. These results indicate improved detection power of the transcribed gene SNP-chip in the target trait and population, allowing identification of large-effect QTLs for important traits in rainbow trout.

Effects of rearing triplet lambs on ewe productivity, lamb survival and performance, and future ewe performance.

Increasing prolificacy has been proposed to be the most effective way to increase the biological efficiency and profitability of sheep production. However, use of prolific breeds and genes with major effects on ovulation rate can increase prolificacy to levels that may not be desirable or sustainable in extensive rangeland production systems. This study thus evaluated effects of triplet births on ewe productivity and ewe and lamb performance. An initial study used 666 purebred Polypay litters to compare ewes with triplet litters that were required to raise all the lambs (Treatment A) with those whose triplet litters were reduced to 2 lambs (Treatment R). Adult Polypay ewes had an average litter size of 2.35 lambs per litter. The frequency of litters of 3 or more lambs was 43.2%; 56.0% of lambs were born in litters of 3 or more lambs. Ewes that had singles weaned fewer lambs and less body weight (BW) of lambs (P < 0.001; 0.94 lambs and 40.4 kg, respectively) than ewes that had twins or triplets. Ewes with triplet litters in Treatment A weaned more lambs (P < 0.01) and more BW of lambs (P < 0.05) than ewes that had triplets in Treatment R (2.13 lambs and 62.9 kg, respectively, vs. 1.79 lambs and 55.0 kg, respectively), and weaned more lambs than ewes that had twins (1.77 lambs; P < 0.01). However, neither group of triplet-bearing ewes weaned more BW of lambs than ewes that had twins (58.9 kg; P ≥ 0.34). In 2 sets of data involving 442 purebred Polypay litters and 987 litters from Polypay or Romanov‒White Dorper × Rambouillet ewes mated to terminal sires, ewes were required to raise all triplet-born lambs. Death losses for triplets in these studies (39.6 and 31.6%, respectively) were higher than those in Treatment A of the initial study (26.2%), resulting in greater numbers of lambs weaned for triplet, compared to twin, litters (1.79 vs. 1.68, respectively; P = 0.02) but no greater weight of lambs weaned (54.3 vs. 55.4 kg, respectively; P = 0.17). Based on these 3 sets of data, ewes that were required to rear triplet lambs weaned 0.20 more lambs per litter than ewes that had twins but also had 0.75 additional dead lambs per litter, and thus a lamb mortality overhead of 3.75 additional dead lambs for each additional weaned lamb. We conclude that there is an intermediate optimum prolificacy level for extensive rangeland production systems. If optimum prolificacy is exceeded, removal and artificial rearing of surplus lambs are necessary to avoid increased lamb death losses.

Retrospective Evaluation of Marker-Assisted Selection for Resistance to Bacterial Cold Water Disease in Three Generations of a Commercial Rainbow Trout Breeding Population.

Bacterial cold water disease (BCWD), caused by Flavobacterium psychrophilum, is an endemic and problematic disease in rainbow trout (Oncorhynchus mykiss) aquaculture. Previously, we have identified SNPs (single nucleotide polymorphisms) associated with BCWD resistance in rainbow trout. The objectives of this study were (1) to validate the SNPs associated with BCWD resistance in a commercial breeding population; and (2) to evaluate retrospectively the accuracy of MAS (marker-assisted selection) for BCWD resistance in this commercial breeding program. Three consecutive generations of the Troutlodge May breeding population were evaluated for BCWD resistance. Based on our previous studies, a panel of 96 SNPs was selected and used to genotype the parents and ten offspring from each of the 138 full-sib families of the 2015 generation, and 37 SNPs associated with BCWD resistance were validated. Thirty-six of the validated SNPs were clustered on chromosomes Omy3, Omy8 and Omy25. Thus, at least three QTL (quantitative trait loci) for BCWD resistance were validated in the 2015 generation. Three SNPs from each QTL region were used for haplotype association analysis. Three haplotypes, Omy3TGG, Omy8GCG and Omy25CGG, were found to be associated with BCWD resistance in the 2015 generation. Retrospective analyses were then performed to evaluate the accuracy of MAS for BCWD resistance using these three favorable haplotypes. The accuracy of MAS was estimated with the Pearson correlation coefficient between the total number of favorable haplotypes in the two parents and the family BCWD survival rates. The Omy8 and Omy25 haplotypes were positively correlated with the family BCWD survival rates across all three generations. The accuracies of MAS using these two haplotypes together were consistently around 0.5, which was equal or greater than the accuracy of the conventional family-based selection in the same generation. In conclusion, we have demonstrated that MAS for BCWD resistance is feasible in this commercial rainbow trout breeding population.

Integrated analysis of lncRNA and mRNA expression in rainbow trout families showing variation in muscle growth and fillet quality traits.

Muscle yield and quality traits are important for the aquaculture industry and consumers. Genetic selection for these traits is difficult because they are polygenic and result from multifactorial interactions. To study the genetic architecture of these traits, phenotypic characterization of whole body weight (WBW), muscle yield, fat content, shear force and whiteness were measured in ~500 fish representing 98 families from a growth-selected line. RNA-Seq was used to sequence the muscle transcriptome of different families exhibiting divergent phenotypes for each trait. We have identified 240 and 1,280 differentially expressed (DE) protein-coding genes and long noncoding RNAs (lncRNAs), respectively, in fish families exhibiting contrasting phenotypes. Expression of many DE lncRNAs (n = 229) was positively correlated with overlapping, neighboring or distantly located protein-coding genes (n = 1,030), resulting in 3,392 interactions. Three DE antisense lncRNAs were co-expressed with sense genes known to impact muscle quality traits. Forty-four DE lncRNAs had potential sponge functions to miRNAs that affect muscle quality traits. This study (1) defines muscle quality associated protein-coding and noncoding genes and (2) provides insight into non-coding RNAs involvement in regulating growth and fillet quality traits in rainbow trout.

Accurate genomic predictions for BCWD resistance in rainbow trout are achieved using low-density SNP panels: Evidence that long-range LD is a major contributing factor.

Previously accurate genomic predictions for Bacterial cold water disease (BCWD) resistance in rainbow trout were obtained using a medium-density single nucleotide polymorphism (SNP) array. Here, the impact of lower-density SNP panels on the accuracy of genomic predictions was investigated in a commercial rainbow trout breeding population. Using progeny performance data, the accuracy of genomic breeding values (GEBV) using 35K, 10K, 3K, 1K, 500, 300 and 200 SNP panels as well as a panel with 70 quantitative trait loci (QTL)-flanking SNP was compared. The GEBVs were estimated using the Bayesian method BayesB, single-step GBLUP (ssGBLUP) and weighted ssGBLUP (wssGBLUP). The accuracy of GEBVs remained high despite the sharp reductions in SNP density, and even with 500 SNP accuracy was higher than the pedigree-based prediction (0.50-0.56 versus 0.36). Furthermore, the prediction accuracy with the 70 QTL-flanking SNP (0.65-0.72) was similar to the panel with 35K SNP (0.65-0.71). Genomewide linkage disequilibrium (LD) analysis revealed strong LD (r2  ≥ 0.25) spanning on average over 1 Mb across the rainbow trout genome. This long-range LD likely contributed to the accurate genomic predictions with the low-density SNP panels. Population structure analysis supported the hypothesis that long-range LD in this population may be caused by admixture. Results suggest that lower-cost, low-density SNP panels can be used for implementing genomic selection for BCWD resistance in rainbow trout breeding programs.

Similar Genetic Architecture with Shared and Unique Quantitative Trait Loci for Bacterial Cold Water Disease Resistance in Two Rainbow Trout Breeding Populations.

Bacterial cold water disease (BCWD) causes significant mortality and economic losses in salmonid aquaculture. In previous studies, we identified moderate-large effect quantitative trait loci (QTL) for BCWD resistance in rainbow trout (Oncorhynchus mykiss). However, the recent availability of a 57 K SNP array and a reference genome assembly have enabled us to conduct genome-wide association studies (GWAS) that overcome several experimental limitations from our previous work. In the current study, we conducted GWAS for BCWD resistance in two rainbow trout breeding populations using two genotyping platforms, the 57 K Affymetrix SNP array and restriction-associated DNA (RAD) sequencing. Overall, we identified 14 moderate-large effect QTL that explained up to 60.8% of the genetic variance in one of the two populations and 27.7% in the other. Four of these QTL were found in both populations explaining a substantial proportion of the variance, although major differences were also detected between the two populations. Our results confirm that BCWD resistance is controlled by the oligogenic inheritance of few moderate-large effect loci and a large-unknown number of loci each having a small effect on BCWD resistance. We detected differences in QTL number and genome location between two GWAS models (weighted single-step GBLUP and Bayes B), which highlights the utility of using different models to uncover QTL. The RAD-SNPs detected a greater number of QTL than the 57 K SNP array in one population, suggesting that the RAD-SNPs may uncover polymorphisms that are more unique and informative for the specific population in which they were discovered.

RNA-Seq reveals MicroRNA expression signature and genetic polymorphism associated with growth and muscle quality traits in rainbow trout.

The role of microRNA expression and genetic variation in microRNA-binding sites of target genes on growth and muscle quality traits is poorly characterized. We used RNA-Seq approach to investigate their importance on 5 growth and muscle quality traits: whole body weight (WBW), muscle yield, muscle crude-fat content, muscle shear force and whiteness. Phenotypic data were collected from 471 fish, representing 98 families (~5 fish/family) from a growth-selected line. Muscle microRNAs and mRNAs were sequenced from 22 families showing divergent phenotypes. Ninety microRNAs showed differential expression between families with divergent phenotypes, and their expression was strongly associated with variation in phenotypes. A total of 204 single nucleotide polymorphisms (SNPs) present in 3' UTR of target genes either destroyed or created novel illegitimate microRNA target sites; of them, 78 SNPs explained significant variation in the aforementioned 5 muscle traits. Majority of the phenotype-associated SNPs were present in microRNA-binding sites of genes involved in energy metabolism and muscle structure. These findings suggest that variation in microRNA expression and/or sequence variation in microRNA binding sites in target genes play an important role in mediating differences in fish growth and muscle quality phenotypes.

Identification of SNPs associated with muscle yield and quality traits using allelic-imbalance analyses of pooled RNA-Seq samples in rainbow trout.

BACKGROUND: Coding/functional SNPs change the biological function of a gene and, therefore, could serve as "large-effect" genetic markers. In this study, we used two bioinformatics pipelines, GATK and SAMtools, for discovering coding/functional SNPs with allelic-imbalances associated with total body weight, muscle yield, muscle fat content, shear force, and whiteness. Phenotypic data were collected for approximately 500 fish, representing 98 families (5 fish/family), from a growth-selected line, and the muscle transcriptome was sequenced from 22 families with divergent phenotypes (4 low- versus 4 high-ranked families per trait). RESULTS: GATK detected 59,112 putative SNPs; of these SNPs, 4798 showed allelic imbalances (>2.0 as an amplification and <0.5 as loss of heterozygosity). SAMtools detected 87,066 putative SNPs; and of them, 4962 had allelic imbalances between the low- and high-ranked families. Only 1829 SNPs with allelic imbalances were common between the two datasets, indicating significant differences in algorithms. The two datasets contained 7930 non-redundant SNPs of which 4439 mapped to 1498 protein-coding genes (with 6.4% non-synonymous SNPs) and 684 mapped to 295 lncRNAs. Validation of a subset of 92 SNPs revealed 1) 86.7-93.8% success rate in calling polymorphic SNPs and 2) 95.4% consistent matching between DNA and cDNA genotypes indicating a high rate of identifying SNPs with allelic imbalances. In addition, 4.64% SNPs revealed random monoallelic expression. Genome distribution of the SNPs with allelic imbalances exhibited high density for all five traits in several chromosomes, especially chromosome 9, 20 and 28. Most of the SNP-harboring genes were assigned to important growth-related metabolic pathways. CONCLUSION: These results demonstrate utility of RNA-Seq in assessing phenotype-associated allelic imbalances in pooled RNA-Seq samples. The SNPs identified in this study were included in a new SNP-Chip design (available from Affymetrix) for genomic and genetic analyses in rainbow trout.

Genomic selection models double the accuracy of predicted breeding values for bacterial cold water disease resistance compared to a traditional pedigree-based model in rainbow trout aquaculture.

BACKGROUND: Previously, we have shown that bacterial cold water disease (BCWD) resistance in rainbow trout can be improved using traditional family-based selection, but progress has been limited to exploiting only between-family genetic variation. Genomic selection (GS) is a new alternative that enables exploitation of within-family genetic variation. METHODS: We compared three GS models [single-step genomic best linear unbiased prediction (ssGBLUP), weighted ssGBLUP (wssGBLUP), and BayesB] to predict genomic-enabled breeding values (GEBV) for BCWD resistance in a commercial rainbow trout population, and compared the accuracy of GEBV to traditional estimates of breeding values (EBV) from a pedigree-based BLUP (P-BLUP) model. We also assessed the impact of sampling design on the accuracy of GEBV predictions. For these comparisons, we used BCWD survival phenotypes recorded on 7893 fish from 102 families, of which 1473 fish from 50 families had genotypes [57 K single nucleotide polymorphism (SNP) array]. Naïve siblings of the training fish (n = 930 testing fish) were genotyped to predict their GEBV and mated to produce 138 progeny testing families. In the following generation, 9968 progeny were phenotyped to empirically assess the accuracy of GEBV predictions made on their non-phenotyped parents. RESULTS: The accuracy of GEBV from all tested GS models were substantially higher than the P-BLUP model EBV. The highest increase in accuracy relative to the P-BLUP model was achieved with BayesB (97.2 to 108.8%), followed by wssGBLUP at iteration 2 (94.4 to 97.1%) and 3 (88.9 to 91.2%) and ssGBLUP (83.3 to 85.3%). Reducing the training sample size to n = ~1000 had no negative impact on the accuracy (0.67 to 0.72), but with n = ~500 the accuracy dropped to 0.53 to 0.61 if the training and testing fish were full-sibs, and even substantially lower, to 0.22 to 0.25, when they were not full-sibs. CONCLUSIONS: Using progeny performance data, we showed that the accuracy of genomic predictions is substantially higher than estimates obtained from the traditional pedigree-based BLUP model for BCWD resistance. Overall, we found that using a much smaller training sample size compared to similar studies in livestock, GS can substantially improve the selection accuracy and genetic gains for this trait in a commercial rainbow trout breeding population.

Genome-Wide Association Study for Identifying Loci that Affect Fillet Yield, Carcass, and Body Weight Traits in Rainbow Trout (Oncorhynchus mykiss).

Fillet yield (FY, %) is an economically-important trait in rainbow trout aquaculture that affects production efficiency. Despite that, FY has received little attention in breeding programs because it is difficult to measure on a large number of fish and cannot be directly measured on breeding candidates. The recent development of a high-density SNP array for rainbow trout has provided the needed tool for studying the underlying genetic architecture of this trait. A genome-wide association study (GWAS) was conducted for FY, body weight at 10 (BW10) and 13 (BW13) months post-hatching, head-off carcass weight (CAR), and fillet weight (FW) in a pedigreed rainbow trout population selectively bred for improved growth performance. The GWAS analysis was performed using the weighted single-step GBLUP method (wssGWAS). Phenotypic records of 1447 fish (1.5 kg at harvest) from 299 full-sib families in three successive generations, of which 875 fish from 196 full-sib families were genotyped, were used in the GWAS analysis. A total of 38,107 polymorphic SNPs were analyzed in a univariate model with hatch year and harvest group as fixed effects, harvest weight as a continuous covariate, and animal and common environment as random effects. A new linkage map was developed to create windows of 20 adjacent SNPs for use in the GWAS. The two windows with largest effect for FY and FW were located on chromosome Omy9 and explained only 1.0-1.5% of genetic variance, thus suggesting a polygenic architecture affected by multiple loci with small effects in this population. One window on Omy5 explained 1.4 and 1.0% of the genetic variance for BW10 and BW13, respectively. Three windows located on Omy27, Omy17, and Omy9 (same window detected for FY) explained 1.7, 1.7, and 1.0%, respectively, of genetic variance for CAR. Among the detected 100 SNPs, 55% were located directly in genes (intron and exons). Nucleotide sequences of intragenic SNPs were blasted to the Mus musculus genome to create a putative gene network. The network suggests that differences in the ability to maintain a proliferative and renewable population of myogenic precursor cells may affect variation in growth and fillet yield in rainbow trout.

Evaluation of Genome-Enabled Selection for Bacterial Cold Water Disease Resistance Using Progeny Performance Data in Rainbow Trout: Insights on Genotyping Methods and Genomic Prediction Models.

Bacterial cold water disease (BCWD) causes significant economic losses in salmonid aquaculture, and traditional family-based breeding programs aimed at improving BCWD resistance have been limited to exploiting only between-family variation. We used genomic selection (GS) models to predict genomic breeding values (GEBVs) for BCWD resistance in 10 families from the first generation of the NCCCWA BCWD resistance breeding line, compared the predictive ability (PA) of GEBVs to pedigree-based estimated breeding values (EBVs), and compared the impact of two SNP genotyping methods on the accuracy of GEBV predictions. The BCWD phenotypes survival days (DAYS) and survival status (STATUS) had been recorded in training fish (n = 583) subjected to experimental BCWD challenge. Training fish, and their full sibs without phenotypic data that were used as parents of the subsequent generation, were genotyped using two methods: restriction-site associated DNA (RAD) sequencing and the Rainbow Trout Axiom® 57 K SNP array (Chip). Animal-specific GEBVs were estimated using four GS models: BayesB, BayesC, single-step GBLUP (ssGBLUP), and weighted ssGBLUP (wssGBLUP). Family-specific EBVs were estimated using pedigree and phenotype data in the training fish only. The PA of EBVs and GEBVs was assessed by correlating mean progeny phenotype (MPP) with mid-parent EBV (family-specific) or GEBV (animal-specific). The best GEBV predictions were similar to EBV with PA values of 0.49 and 0.46 vs. 0.50 and 0.41 for DAYS and STATUS, respectively. Among the GEBV prediction methods, ssGBLUP consistently had the highest PA. The RAD genotyping platform had GEBVs with similar PA to those of GEBVs from the Chip platform. The PA of ssGBLUP and wssGBLUP methods was higher with the Chip, but for BayesB and BayesC methods it was higher with the RAD platform. The overall GEBV accuracy in this study was low to moderate, likely due to the small training sample used. This study explored the potential of GS for improving resistance to BCWD in rainbow trout using, for the first time, progeny testing data to assess the accuracy of GEBVs, and it provides the basis for further investigation on the implementation of GS in commercial rainbow trout populations.

Biochemical reference intervals and pathophysiological changes in Flavobacterium psychrophilum-resistant and -susceptible rainbow trout lines.

Host genetic resistance against disease-causing pathogens can be enhanced through family-based selective breeding. At present, there is an incomplete understanding of how artificial selection of fish alters host physiology and response following pathogen exposure. We previously reported the generation of selectively-bred rainbow trout Oncorhynchus mykiss lines with either increased resistance (ARS-Fp-R) or susceptibility (ARS-Fp-S) to bacterial cold water disease (BCWD). This study (1) determined baseline reference-range intervals for packed cell volume (PCV) and 18 plasma biochemistry analytes, and (2) examined pathophysiological changes following infection between the genetic lines. PCV and biochemistry reference-range intervals did not significantly differ between genetic lines; thus data were pooled into a single reference-range population (n = 85). ARS-Fp-R and ARS-Fp-S line fish were intraperitoneally challenged with Flavobacterium psychrophilum, and plasma was collected on Days 1, 3, 6, and 9 post-challenge. Splenic bacterial load was measured using an F. psychrophilum-specific qPCR assay. In both genetic lines, changes were observed in mean PCV, total protein, albumin, glucose, cholesterol, chloride, and calcium, falling outside the established reference intervals and significantly differing from phosphate-buffered saline challenged fish, on at least 1d post-challenge. Mean PCV, total protein, and calcium significantly differed between ARS-Fp-R and ARS-Fp-S line fish on Day 9 post-infection, with values in the ARS-Fp-S line deviating most from the reference interval. PCV, total protein, cholesterol, and calcium negatively correlated with bacterial load. These findings identify divergent pathophysiological responses between ARS-Fp-R and ARS-Fp-S line fish following laboratory challenge that are likely associated with differential survival.

Histopathologic Changes in Disease-Resistant-Line and Disease-Susceptible-Line Juvenile Rainbow Trout Experimentally Infected with Flavobacterium psychrophilum.

Abstract A challenge to selectively breeding fish populations for improved disease resistance in aquaculture is an incomplete understanding of how artificial selection affects innate immunity at the host-pathogen level. The objective of this study was to determine whether Rainbow Trout Oncorhynchus mykiss bred for differential susceptibility to bacterial cold-water disease exhibited altered tissue damage and cellular inflammatory response following experimental challenge with Flavobacterium psychrophilum. Fish from disease-resistant (ARS-Fp-R) and disease-susceptible (ARS-Fp-S) lines were experimentally challenged as juveniles, and mortalities, as well as survivors, were sampled for histopathology during the acute phase of the disease. Microscopic lesions were quantified or semiquantified and statistically compared for changes over time and between genetic lines. Significant progression in the degree of perisplenitis, splenic necrosis, splenic inflammatory infiltrates, average splenic ellipsoid area, total splenic ellipsoid area, and peritonitis was present over time in both genetic lines on at least one postinfection time point. No differences were found between renal inflammatory infiltrates and renal hematopoietic cell depletion over time. Perisplenitis was significantly lower in fish from the ARS-Fp-R line on day 9 postinfection than in fish from the ARS-Fp-S line. The ARS-Fp-R line demonstrated a trend towards reduced splenic necrosis compared with the ARS-Fp-S line that approached significance, and fish from the ARS-Fp-S line were 3.6 times more likely than fish from the ARS-Fp-R line to have a higher splenic necrosis lesion score after day 3 postinfection. These findings support the hypothesis that differential survival is a result of divergence in disease magnitude and not altered disease course between genetic lines. Characterization of histopathologic changes between genetic lines and over time helps elucidate mechanisms of disease resistance and contributes to our understanding of disease pathogenesis in fish infected with F. psychrophilum. Received January 7, 2014; accepted March 10, 2014.