The development and characterization of a 57K single nucleotide polymorphism array for rainbow trout.

In this study, we describe the development and characterization of the first high-density single nucleotide polymorphism (SNP) genotyping array for rainbow trout. The SNP array is publically available from a commercial vendor (Affymetrix). The SNP genotyping quality was high, and validation rate was close to 90%. This is comparable to other farm animals and is much higher than previous smaller scale SNP validation studies in rainbow trout. High quality and integrity of the genotypes are evident from sample reproducibility and from nearly 100% agreement in genotyping results from other methods. The array is very useful for rainbow trout aquaculture populations with more than 40 900 polymorphic markers per population. For wild populations that were confounded by a smaller sample size, the number of polymorphic markers was between 10 577 and 24 330. Comparison between genotypes from individual populations suggests good potential for identifying candidate markers for populations' traceability. Linkage analysis and mapping of the SNPs to the reference genome assembly provide strong evidence for a wide distribution throughout the genome with good representation in all 29 chromosomes. A total of 68% of the genome scaffolds and contigs were anchored through linkage analysis using the SNP array genotypes, including ~20% of the genome assembly that has not been previously anchored to chromosomes.

Genotype-by-environment interaction of growth traits in rainbow trout (Oncorhynchus mykiss): a continental scale study.

Rainbow trout is a globally important fish species for aquaculture. However, fish for most farms worldwide are produced by only a few breeding companies. Selection based solely on fish performance recorded at a nucleus may lead to lower-than-expected genetic gains in other production environments when genotype-by-environment (G × E) interaction exists. The aim was to quantify the magnitude of G × E interaction of growth traits (tagging weight; BWT, harvest weight; BWH, and growth rate; TGC) measured across 4 environments, located in 3 different continents, by estimating genetic correlations between environments. A total of 100 families, of at least 25 in size, were produced from the mating 58 sires and 100 dams. In total, 13,806 offspring were reared at the nucleus (selection environment) in Washington State (NUC) and in 3 other environments: a recirculating aquaculture system in Freshwater Institute (FI), West Virginia; a high-altitude farm in Peru (PE), and a cold-water farm in Germany (GER). To account for selection bias due to selective mortality, a multitrait multienvironment animal mixed model was applied to analyze the performance data in different environments as different traits. Genetic correlation (rg) of a trait measured in different environments and rg of different traits measured in different environments were estimated. The results show that heterogeneity of additive genetic variances was mainly found for BWH measured in FI and PE. Additive genetic coefficient of variation for BWH in NUC, FI, PE, and GER were 7.63, 8.36, 8.64, and 9.75, respectively. Genetic correlations between the same trait in different environments were low, indicating strong reranking (BWT: rg = 0.15 to 0.37, BWH: rg = 0.19 to 0.48, TGC: rg = 0.31 to 0.36) across environments. The rg between BWT in NUC and BWH in both FI (0.31) and GER (0.36) were positive, which was also found between BWT in NUC and TGC in both FI (0.10) and GER (0.20). However, rg were negative between BWT in NUC and both BWH (-0.06) and TGC (-0.20) in PE. Correction for selection bias resulted in higher additive genetic variances. In conclusion, strong G × E interaction was found for BWT, BWH, and TGC. Accounting for G × E interaction in the breeding program, either by using sib information from testing stations or environment-specific breeding programs, would increase genetic gains for environments that differ significantly from NUC.

Evidence of major genes affecting resistance to bacterial cold water disease in rainbow trout using Bayesian methods of segregation analysis.

Bacterial cold water disease (BCWD) causes significant economic loss in salmonid aquaculture. We previously detected genetic variation for BCWD resistance in our rainbow trout population, and a family-based selection program to improve resistance was initiated at the National Center for Cool and Cold Water Aquaculture (NCCCWA). This study investigated evidence of major trait loci affecting BCWD resistance using only phenotypic data (without using genetic markers) and Bayesian methods of segregation analysis (BMSA). A total of 10,603 juvenile fish from 101 full-sib families corresponding to 3 generations (2005, 2007, and 2009 hatch years) of the NCCCWA population were challenged by intraperitoneal injection with Flavobacterium psychrophilum, the bacterium that causes BCWD. The results from single- and multiple-QTL models of BMSA suggest that 6 to 10 QTL explaining 83 to 89% of phenotypic variance with either codominant or dominant disease-resistant alleles plus polygenic effects may underlie the genetic architecture of BCWD resistance. This study also highlights the importance of polygenic background effects in the genetic variation of BCWD resistance. The polygenic heritability on the observed scale of survival status is slightly larger than that previously reported for rainbow trout BCWD resistance. These findings provide the basis for designing informative crosses for QTL mapping and carrying out genome scans for QTL affecting BCWD resistance in rainbow trout.

Response to selection for bacterial cold water disease resistance in rainbow trout.

A family-based selection program was initiated at the National Center for Cool and Cold Water Aquaculture in 2005 to improve resistance to bacterial cold water disease (BCWD) in rainbow trout. The objective of this study was to estimate response to 2 generations of selection. A total of 14,841 juvenile fish (BW = 3.1 g; SD = 1.1 g) from 230 full-sib families and 3 randomly mated control lines were challenged intraperitoneally with Flavobacterium psychrophilum, the bacterium that causes BCWD, and mortalities were observed for 21 d. Selection was applied to family EBV derived from a proportional-hazards frailty (animal) model while constraining rate of inbreeding to

Evidence of major genes affecting stress response in rainbow trout using Bayesian methods of complex segregation analysis.

As a first step toward the genetic mapping of QTL affecting stress response variation in rainbow trout, we performed complex segregation analyses (CSA) fitting mixed inheritance models of plasma cortisol by using Bayesian methods in large full-sib families of rainbow trout. To date, no studies have been conducted to determine the mode of inheritance of stress response as measured by plasma cortisol response when using a crowding stress paradigm and CSA in rainbow trout. The main objective of this study was to determine the mode of inheritance of plasma cortisol after a crowding stress. The results from fitting mixed inheritance models with Bayesian CSA suggest that 1 or more major genes with dominant cortisol-decreasing alleles and small additive genetic effects of a large number of independent genes likely underlie the genetic variation of plasma cortisol in the rainbow trout families evaluated. Plasma cortisol is genetically determined, with heritabilities of 0.22 to 0.39. Furthermore, a major gene with an additive effect of -42 ng/mL (approximately 1.0 genetic SD) is segregating in this rainbow trout broodstock population. These findings provide a basis for designing and executing genome-wide linkage studies to identify QTL for stress response in rainbow trout broodstock and markers for selective breeding.

Rainbow trout resistance to bacterial cold-water disease is moderately heritable and is not adversely correlated with growth.

The objectives of this study were to estimate the heritabilities for and genetic correlations among resistance to bacterial cold-water disease and growth traits in a population of rainbow trout (Oncorhynchus mykiss). Bacterial cold-water disease, a chronic disease of rainbow trout, is caused by Flavobacterium psychrophilum. This bacterium also causes acute losses in young fish, known as rainbow trout fry syndrome. Selective breeding for increased disease resistance is a promising strategy that has not been widely used in aquaculture. At the same time, improving growth performance is critical for efficient production. At the National Center for Cool and Cold Water Aquaculture, reducing the negative impact of diseases on rainbow trout culture and improving growth performance are primary objectives. In 2005, when fish averaged 2.4 g, 71 full-sib families were challenged with F. psychrophilum and evaluated for 21 d. Overall survival was 29.3% and family rates of survival varied from 1.5 to 72.5%. Heritability of postchallenge survival, an indicator of disease resistance, was estimated to be 0.35 +/- 0.09. Body weights at 9 and 12 mo posthatch and growth rate from 9 to 12 mo were evaluated on siblings of the fish in the disease challenge study. Growth traits were moderately heritable, from 0.32 for growth rate to 0.61 for 12-mo BW. Genetic and phenotypic correlations between growth traits and resistance to bacterial cold-water disease were not different from zero. These results suggest that genetic improvement can be made simultaneously for growth and bacterial cold-water disease resistance in rainbow trout by using selective breeding.

Genomic organization of the IGF1, IGF2, MYF5, MYF6 and GRF/PACAP genes across Salmoninae genera.

Whole-genome duplication in the ancient ray-finned fish and subsequent tetraploidization in the ancestor to the salmonids have complicated genomic and candidate gene studies in these organisms as many genes with multiple copies are present throughout their genomes. In an attempt to identify genes with a potential influence on growth and development, we investigated the genomic positions of insulin-like growth factors 1 and 2 (IGF1, IGF2), myogenic factors 5 and 6 (MYF5, MYF6) and growth hormone-releasing factor/pituitary adenylate cyclase-activating polypeptide (GRF/PACAP) in three salmonid species: rainbow trout (Oncorhynchus mykiss), Atlantic salmon (Salmo salar) and Arctic charr (Salvelinus alpinus). Our results suggest a tight association between the IGF1, MYF5 and MYF6 genes in all three species. We further localized the duplicated copies of IGF1 to the homeologous linkage groups RT-7/15 in rainbow trout and AC-3/24 in Arctic charr, and the two copies of MYF6 to homeologous linkage groups AS-22/24 in Atlantic salmon. Localization of GRF/PACAP to RT-7, AS-31 and AC-27 and IGF2 to RT-27, AS-2 and AC-4 in rainbow trout, Atlantic salmon and Arctic charr respectively is consistent with previously reported homologies among these chromosomal segments identified using other genetic markers. However, localization of the second copy of GRF/PACAP to RT-19 and AC-14 and the duplicated copy of IGF2 to AC-19 suggest a possible new homology/homeology between these chromosomes. These results might also be an indication of a more ancient polyploidization event that occurred deep in the ray-finned fish lineage.

Regulation of animal biotechnology: research needs.

Livestock that result from biotechnology have been a part of agricultural science for over 30 years but have not entered the market place as food or fiber. Two biotechnologies are at the forefront as challenges to the world's systems for regulating the market place: animal clones and transgenic animals. Both technologies have come before the Food and Drug Administration in the United States and it appears that action is imminent for clones. The FDA has asserted principles for evaluation of clones and asserts that "... remaining hazard(s) from cloning are likely to be subtle in nature." The science-based principles recognize that in some areas related to developmental biology and gene expression in clones, additional scientific information would be useful. The role of science then is to use the genomic tools that we have available to answer questions about epigenetic regulation of development and reprogramming of genes to the state found in germ cells. Transgenics pose additional challenges to regulators. If the transgenics are produced using cloning from modified cells then the additional scientific information needed will be related to the effects of insertion and expression of the transgenes. Other approaches such as retrovirally vectored transgenesis will elicit additional questions. These questions will be challenging because the science will have to be related to the expression and function of each gene or class of genes. For the promises of animal biotechnology to be fulfilled, scientists will have to resolve many questions for regulators and the public but tools to answer those questions are rapidly becoming available.

A polymerase chain reaction screening method for rapid detection of microsatellites in bacterial artificial chromosomes.

Standard protocols aimed at identifying subclones of interest from bacterial artificial chromosomes (BACs) include the use of hybridization methods that are time consuming and often require the use of radioactive isotopes. Through our efforts to identify microsatellites in BACs from rainbow trout (Oncorhynchus mykiss) we have developed a nonradioactive polymerase chain reaction (PCR)-based screening technique to select microsatellites containing subclones for marker development. Two BACs were subcloned and screened by PCR using a vector-specific primer and a mix of microsatellite repeat primers. The subclones were then sequenced to evaluate the efficiency of the PCR screening method. Correlation between positive PCR amplification and presence of microsatellites varied between the two BACs (21.9% and 71.4%), but still a sufficient number of subclones were identified to enable design and optimization of microsatellite markers.

Identification of a novel gill-specific calpain from rainbow trout (Oncorhynchus mykiss).

Calpains are calcium-dependent neutral proteases responsible for many cellular functions. The two forms of calpain ubiquitously expressed in mammalian tissues are known as mu-calpain and m-calpain. We report here the identification of a novel calpain that is similar to but distinct from the mu- and m-calpains in rainbow trout. The cDNA of the novel gene is 2623 bp in length with a single open reading frame. The predicted protein (676 amino acids) contains the conserved calpain characteristic domains that include: domain I (pro peptide), II (cysteine catalytic site), III (electrostatic switch), and IV (calmodulin-like) with five Ca(2+)-binding EF hands. Northern blot and RT-PCR analyses demonstrated that the novel calpain gene is predominantly expressed in rainbow trout gills. Comparison of the novel protein with the ubiquitously expressed calpains and several mammalian tissue-specific calpains revealed that the novel calpain is an orthologue of the mammalian digestive tract specific calpain (calpain 9).

Characterization and comparison of microsatellites derived from repeat-enriched libraries and expressed sequence tags.

The construction of high-density linkage maps for use in identifying loci underlying important traits requires the development of large numbers of polymorphic genetic markers spanning the entire genome at regularly spaced intervals. As part of our efforts to develop markers for rainbow trout (Oncorhynchus mykiss), we performed a comparison of allelic variation between microsatellite markers developed from expressed sequence tag (EST) data and anonymous markers identified from repeat-enriched libraries constructed from genomic DNA. A subset of 70 markers (37 from EST databases and 33 from repeat enriched libraries) was characterized with respect to polymorphism information content (PIC), number of alleles, repeat number, locus duplication within the genome and ability to amplify in other salmonid species. Higher PIC was detected in dinucleotide microsatellites derived from ESTs than anonymous markers (72.7% vs. 54.0%). In contrast, dinucleotide repeat numbers were higher for anonymous microsatellites than for EST derived microsatellites (27.4 vs.18.1). A higher rate of cross-species amplification was observed for EST microsatellites. Approximately half of each marker type was duplicated within the genome. Unlike single-copy markers, amplification of duplicated microsatellites in other salmonids was not correlated to phylogenetic distance. Genomic microsatellites proved more useful than EST derived microsatellites in discriminating among the salmonids. In total, 428 microsatellite markers were developed in this study for mapping and population genetic studies in rainbow trout.

Genomic characterization of a novel pair of ID genes in the rainbow trout (Oncorhynchus mykiss).

The ID (inhibitors of DNA binding/differentiation) proteins represent a family of dominant negative regulators of the basic helix-loop-helix transcription factors whose activities result in delayed cell differentiation and prolonged proliferation. A pair of expressed sequence tag clones with homologies to the ID proteins were identified and used to screen a rainbow trout bacterial artificial chromosome (BAC) library to identify clones containing homologues sequences. Phylogenetic analysis of the predicted amino acid sequences revealed close similarities to the rainbow trout ID1 protein, the genes were therefore classified as rainbow trout ID1B and ID1C. Genome characterization based on BAC sequencing showed each gene to have two exons separated by a small intron. The genes are 83% similar in their transcribed regions, yet they are only 64 and 65% similar in the upstream and downstream sequences, respectively. Using reverse transcription polymerase chain reaction, we found both genes to be expressed in a variety of tissues in the adult rainbow trout.

Characterization of a new BAC library for rainbow trout: evidence for multi-locus duplication.

A 10X rainbow trout bacterial artificial chromosome (BAC) library was constructed to aid in the physical and genetic mapping efforts of the rainbow trout genome. The library was derived from the Swanson clonal line (YY male) and consists of 184,704 clones with an average insert size of 137,500 bp (PFGE) or 118,700 bp (DNA fingerprinting). The clones were gridded onto 10 large nylon membranes to produce high-density arrays for screening the library by hybridization. The library was probed with 11 cDNAs from the NCCCWA EST project chosen because of interest in their homology to known gene sequences, seven known genes, and a Y-specific sex marker. Putative positive clones identified by hybridization were re-arrayed and gridded for secondary confirmation. FPC analysis of HindIII and EcoRV DNA fingerprinting was used to estimate the level of redundancy in the library, to construct BAC contigs and to detect duplicated loci in the semi-duplicated rainbow trout genome. A good correlation (R2 = 0.7) was found between the number of hits per probe and the number of contigs that were assembled from the positive BACs. The average number of BACs per contig was 9.6, which is in good agreement with 10X genome coverage of the library. Two-thirds of the loci screened were predicted to be duplicated as the positive BACs for those genes were assembled into two or three different contigs, which suggests that most of the rainbow trout genome is duplicated.

Alkaline phosphatase activity in bovine oocytes and preimplantation embryos as affected by removal of the zona pellucida and culture medium constituents.

The aims of the present study were: (1) to characterize alkaline phosphatase (AP) activity in bovine oocytes and embryos with intact or removed zona pellucida (ZP); and (2) to evaluate the effect of culture medium constituents on AP activity. Alkaline phosphatase activity in non-matured and matured oocytes was most evident nearest the plasma membrane and perivitelline space. In more than 90% of two- to 16-cell embryos, AP activity was observed in the perivitelline space and at blastomere contacts. In blastocysts, AP activity was localized to the trophectoderm. Only after immunodissection was AP activity detected in the inner cell mass. Removal of the ZP by pronase or mechanical means reduced AP activity. Alkaline phosphatase activity was detected in evacuated ZP after mechanical removal. Specific constituents comprising the embryo culture medium altered AP activity. Alkaline phosphatase activity was reduced in eight- to 16-cell embryos and evacuated ZP cultured in CR1aa + 0.4% bovine serum albumin compared with embryos cultured in CR1aa alone or embryos co-cultured on a monolayer of Buffalo rat liver cells in the presence of 10% fetal bovine serum. The presence of AP activity at blastomere contacts and in evacuated ZP limits its usefulness as a marker for the differentiation of embryonic cells comprising the early cleavage-stage embryo.

Sequence analysis of a rainbow trout cDNA library and creation of a gene index.

Expressed sequence tag (EST) projects have produced extremely valuable resources for identifying genes affecting phenotypes of interest. A large-scale EST sequencing project for rainbow trout was initiated to identify and functionally annotate as many unique transcripts as possible. Over 45,000 5' ESTs were obtained by sequencing clones from a single normalized library constructed using mRNA from six tissues. The production of this sequence data and creation of a rainbow trout Gene Index eliminating redundancy and providing annotation for these sequences will facilitate research in this species.