Functional Annotation of All Salmonid Genomes (FAASG): an international initiative supporting future salmonid research, conservation and aquaculture.

We describe an emerging initiative - the 'Functional Annotation of All Salmonid Genomes' (FAASG), which will leverage the extensive trait diversity that has evolved since a whole genome duplication event in the salmonid ancestor, to develop an integrative understanding of the functional genomic basis of phenotypic variation. The outcomes of FAASG will have diverse applications, ranging from improved understanding of genome evolution, to improving the efficiency and sustainability of aquaculture production, supporting the future of fundamental and applied research in an iconic fish lineage of major societal importance.

Transcriptome profiling in fast versus slow-growing rainbow trout across seasonal gradients.

BACKGROUND: Circannual rhythms in vertebrates can influence a wide variety of physiological processes. Some notable examples include annual reproductive cycles and for poikilotherms, seasonal changes modulating growth. Increasing water temperature elevates growth rates in fishes, but increases in photoperiod regime can have similar influences even at constant temperature. Therefore, in order to understand the dynamics of growth in fish it is important to consider the background influence of photoperiod regime on gene expression differences. This study examined the influence of a declining photoperiod regime (winter solstice) compared to an increasing photoperiod regime (spring equinox) on white muscle transcriptome profiles in fast and slow-growing rainbow trout from a commercial aquaculture strain. RESULTS: Slow-growing fish could be characterized as possessing transcriptome profiles that conform in many respects to an endurance training regime in humans. They have elevated mitochondrial and cytosolic creatine kinase expression levels and appear to suppress mTOR-signaling as evidenced by elevated TSC2 expression, and they also have elevated p53 levels. Large fish display a physiological repertoire that may be consistent with strength/resistance physiology having elevated cytoskeletal gene component expression and glycogen metabolism cycling along with higher PI3K levels. In many respects small vs. large fish match eccentric vs. concentric muscle expression patterns, respectively. Lipid metabolic genes are also more elevated in larger fish, the most notable being the G0S2 switch gene. M and Z-line sarcomere remodelling appears to be more prevalent in large fish. Twenty-three out of 26 gene families with previously reported significant SNP-based growth differences were detected as having significant expression differences. CONCLUSIONS: Larger fish display a broader array of genes showing higher expression, and their profiles are more similar to those observed in December lot fish (i.e., an accelerated growth period). Conversely, small fish display gene profiles more similar to seasonal growth decline phases (i.e., September lot fish). Overall, seasonal timing was coupled to greater differences in gene expression compared to differences associated with fish size.

Mapping and Expression of Candidate Genes for Development Rate in Rainbow Trout (Oncorhynchus mykiss).

Development rate has important implications for individual fitness and physiology. In salmonid fishes, development rate correlates with many traits later in life, including life-history diversity, growth, and age and size at sexual maturation. In rainbow trout (Oncorhynchus mykiss), a quantitative trait locus for embryonic development rate has been detected on chromosome 5 across populations. However, few candidate genes have been identified within this region. In this study, we use gene mapping, gene expression, and quantitative genetic methods to further identify the genetic basis of embryonic developmental rate in O. mykiss Among the genes located in the region of the major development rate quantitative trait locus (GHR1, Clock1a, Myd118-1, and their paralogs), all were expressed early in embryonic development (fertilization through hatch), but none were differentially expressed between individuals with the fast- or slow-developing alleles for a major embryonic development rate quantitative trait locus. In a follow-up study of migratory and resident rainbow trout from natural populations in Alaska, we found significant additive variation in development rate and, moreover, found associations between development rate and allelic variation in all 3 candidate genes within the quantitative trait locus for embryonic development. The mapping of these genes to this region and associations in multiple populations provide positional candidates for further study of their roles in growth, development, and life-history diversity in this model salmonid.

Cloning and characterization of a novel oocyte-specific gene encoding an F-Box protein in rainbow trout (Oncorhynchus mykiss).

BACKGROUND: Oocyte-specific genes play critical roles in oogenesis, folliculogenesis and early embryonic development. The objectives of this study were to characterize the expression of a novel oocyte-specific gene encoding an F-box protein during ovarian development in rainbow trout, and identify its potential interacting partners in rainbow trout oocytes. METHODS: Through analysis of expressed sequence tags (ESTs) from a rainbow trout oocyte cDNA library, a novel transcript represented by ESTs only from the oocyte library was identified. The complete cDNA sequence for the novel gene (named fbxoo) was obtained by assembling sequences from an EST clone and a 5'RACE product. The expression and localization of fbxoo mRNA and protein in ovaries of different developmental stages were analyzed by quantitative real time PCR, immunoblotting, in situ hybridization and immunohistochemistry. Identification of Fbxoo binding proteins was performed by yeast two-hybrid screening. RESULTS: fbxoo mRNA is specifically expressed in mature oocytes as revealed by tissue distribution analysis. The fbxoo cDNA sequence is 1,996 bp in length containing an open reading frame, which encodes a predicted protein of 514 amino acids. The novel protein sequence does not match any known protein sequences in the NCBI database. However, a search of the Pfam protein database revealed that the protein contains an F-box motif at the N-terminus, indicating that Fbxoo is a new member of the F-box protein family. The expression of fbxoo mRNA and protein is high in ovaries at early pre-vitellogenesis stage, and both fbxoo mRNA and protein are predominantly expressed in early pre-vitellogenic oocytes. Several proteins including tissue inhibitor of metalloproteinase 2 (Timp2) were identified as potential Fbxoo protein binding partners. CONCLUSIONS: Results suggest that the novel oocyte-specific F-box protein may play an important role in early oocyte development by regulating other critical proteins involved in oogenesis in rainbow trout.

The generation of the first chromosome-level de novo genome assembly and the development and validation of a 50K SNP array for the St. John River aquaculture strain of North American Atlantic salmon.

Atlantic salmon (Salmo salar) in Northeastern US and Eastern Canada has high economic value for the sport fishing and aquaculture industries. Large differences exist between the genomes of Atlantic salmon of European origin and North American (N.A.) origin. Given the genetic and genomic differences between the 2 lineages, it is crucial to develop unique genomic resources for N.A. Atlantic salmon. Here, we describe the resources that we recently developed for genomic and genetic research in N.A. Atlantic salmon aquaculture. Firstly, a new single nucleotide polymorphism (SNP) database for N.A. Atlantic salmon consisting of 3.1 million putative SNPs was generated using data from whole-genome resequencing of 80 N.A. Atlantic salmon individuals. Secondly, a high-density 50K SNP array enriched for the genic regions of the genome and containing 3 sex determination and 61 putative continent of origin markers was developed and validated. Thirdly, a genetic map composed of 27 linkage groups with 36K SNP markers was generated from 2,512 individuals in 141 full-sib families. Finally, a chromosome-level de novo genome assembly from a male N.A. Atlantic salmon from the St. John River aquaculture strain was generated using PacBio long reads. Information from Hi-C proximity ligation sequences and Bionano optical mapping was used to concatenate the contigs into scaffolds. The assembly contains 1,755 scaffolds and only 1,253 gaps, with a total length of 2.83 Gb and N50 of 17.2 Mb. A BUSCO analysis detected 96.2% of the conserved Actinopterygii genes in the assembly, and the genetic linkage information was used to guide the formation of 27 chromosome sequences. Comparative analysis with the reference genome assembly of the European Atlantic salmon confirmed that the karyotype differences between the 2 lineages are caused by a fission in chromosome Ssa01 and 3 chromosome fusions including the p arm of chromosome Ssa01 with Ssa23, Ssa08 with Ssa29, and Ssa26 with Ssa28. The genomic resources we have generated for Atlantic salmon provide a crucial boost for genetic research and for management of farmed and wild populations in this highly valued species.

A conserved haplotype controls parallel adaptation in geographically distant salmonid populations.

Salmonid fishes exhibit extensive local adaptations owing to abundant environmental variation and precise natal homing. This extensive local adaptation makes conservation and restoration of salmonids a challenge. For example, defining unambiguous units of conservation is difficult, and restoration attempts often fail owing to inadequate adaptive matching of translocated populations. A better understanding of the genetic architecture of local adaptation in salmonids could provide valuable information to assist in conserving and restoring natural populations of these important species. Here, we use a combination of laboratory crosses and next-generation sequencing to investigate the genetic architecture of the parallel adaptation of rapid development rate in two geographically and genetically distant populations of rainbow trout (Oncorhynchus mykiss). Strikingly, we find that not only is a parallel genetic mechanism used but that a conserved haplotype is responsible for this intriguing adaptation. The repeated use of adaptive genetic variation across distant geographical areas could be a general theme in salmonids and have important implications for conservation and restoration.

QTL affecting stress response to crowding in a rainbow trout broodstock population.

BACKGROUND: Genomic analyses have the potential to impact selective breeding programs by identifying markers that serve as proxies for traits which are expensive or difficult to measure. Also, identifying genes affecting traits of interest enhances our understanding of their underlying biochemical pathways. To this end we conducted genome scans of seven rainbow trout families from a single broodstock population to identify quantitative trait loci (QTL) having an effect on stress response to crowding as measured by plasma cortisol concentration. Our goal was to estimate the number of major genes having large effects on this trait in our broodstock population through the identification of QTL. RESULTS: A genome scan including 380 microsatellite markers representing 29 chromosomes resulted in the de novo construction of genetic maps which were in good agreement with the NCCCWA genetic map. Unique sets of QTL were detected for two traits which were defined after observing a low correlation between repeated measurements of plasma cortisol concentration in response to stress. A highly significant QTL was detected in three independent analyses on Omy16, many additional suggestive and significant QTL were also identified. With linkage-based methods of QTL analysis such as half-sib regression interval mapping and a variance component method, we determined that the significant and suggestive QTL explain about 40-43% and 13-27% of the phenotypic trait variation, respectively. CONCLUSIONS: The cortisol response to crowding stress is a complex trait controlled in a sub-sample of our broodstock population by multiple QTL on at least 8 chromosomes. These QTL are largely different from others previously identified for a similar trait, documenting that population specific genetic variants independently affect cortisol response in ways that may result in different impacts on growth. Also, mapping QTL for multiple traits associated with stress response detected trait specific QTL which indicate the significance of the first plasma cortisol measurement in defining the trait. Fine mapping these QTL can lead towards the identification of genes affecting stress response and may influence approaches to selection for this economically important stress response trait.

Identification and expression of Smads associated with TGF-ß/activin/nodal signaling pathways in the rainbow trout (Oncorhynchus mykiss).

The Smad proteins are essential components of the TGF-ß/activin/nodal family signaling pathway. We report the identification and expression of transcripts representing three receptor Smads (Smad2a, Smad2b, and Smad3), two common Smads (Smad4a and Smad4b), and one inhibitory Smad (Smad7). Phylogenetic analysis suggests this gene family evolved through the combination of ancient and more recent salmonid genome duplication events. Tissue distribution, embryonic expression, and expression in growth hormone (GH) treated fish were assessed by reverse transcription PCR or qPCR. All six Smad transcripts were ubiquitously expressed in adult tissues. We observed the highest expression of the receptor Smads in unfertilized eggs, generally decreasing during early embryonic development and slightly increasing around 11 days post-fertilization (dpf). Smad7 expression was low for most of embryonic development, with a dramatic increase at the onset of muscle development (6 dpf), and at hatch (24 dpf). Smad4 expression was low during early embryonic development and increased after 14 dpf. The increased expression of Smad4 and Smad7 during late embryonic development may indicate modulation of gene expression by GH axis, which initiates activity during late embryonic development. These data were supported by the modulation of these Smads in the gill filament, stomach, and muscle following a GH treatment. Additionally, these changes are concurrent with the modulation of expression of TGF-ß family members. Most significantly, the increased expression of Smad7 in the muscle is simultaneous with increased expression of MSTN1A and not MSTN1B during both embryonic development and following GH treatment. These data indicate a promyogenic role for Smad7 as previously identified in other non-fish species.

Generation of a reference transcriptome for evaluating rainbow trout responses to various stressors.

BACKGROUND: Fish under intensive culture conditions are exposed to a variety of acute and chronic stressors, including high rearing densities, sub-optimal water quality, and severe thermal fluctuations. Such stressors are inherent in aquaculture production and can induce physiological responses with adverse effects on traits important to producers and consumers, including those associated with growth, nutrition, reproduction, immune response, and fillet quality. Understanding and monitoring the biological mechanisms underlying stress responses will facilitate alleviating their negative effects through selective breeding and changes in management practices, resulting in improved animal welfare and production efficiency. RESULTS: Physiological responses to five treatments associated with stress were characterized by measuring plasma lysozyme activity, glucose, lactate, chloride, and cortisol concentrations, in addition to stress-associated transcripts by quantitative PCR. Results indicate that the fish had significant stressor-specific changes in their physiological conditions. Sequencing of a pooled normalized transcriptome library created from gill, brain, liver, spleen, kidney and muscle RNA of control and stressed fish produced 3,160,306 expressed sequence tags which were assembled and annotated. SNP discovery resulted in identification of ~58,000 putative single nucleotide polymorphisms including 24,479 which were predicted to fall within exons. Of these, 4907 were predicted to occupy the first position of a codon and 4110 the second, increasing the probability to impact amino acid sequence variation and potentially gene function. CONCLUSION: We have generated and characterized a reference transcriptome for rainbow trout that represents multiple tissues responding to multiple stressors common to aquaculture production environments. This resource compliments existing public transcriptome data and will facilitate approaches aiming to evaluate gene expression associated with stress in this species.

A first generation integrated map of the rainbow trout genome.

BACKGROUND: Rainbow trout (Oncorhynchus mykiss) are the most-widely cultivated cold freshwater fish in the world and an important model species for many research areas. Coupling great interest in this species as a research model with the need for genetic improvement of aquaculture production efficiency traits justifies the continued development of genomics research resources. Many quantitative trait loci (QTL) have been identified for production and life-history traits in rainbow trout. An integrated physical and genetic map is needed to facilitate fine mapping of QTL and the selection of positional candidate genes for incorporation in marker-assisted selection (MAS) programs for improving rainbow trout aquaculture production. RESULTS: The first generation integrated map of the rainbow trout genome is composed of 238 BAC contigs anchored to chromosomes of the genetic map. It covers more than 10% of the genome across segments from all 29 chromosomes. Anchoring of 203 contigs to chromosomes of the National Center for Cool and Cold Water Aquaculture (NCCCWA) genetic map was achieved through mapping of 288 genetic markers derived from BAC end sequences (BES), screening of the BAC library with previously mapped markers and matching of SNPs with BES reads. In addition, 35 contigs were anchored to linkage groups of the INRA (French National Institute of Agricultural Research) genetic map through markers that were not informative for linkage analysis in the NCCCWA mapping panel. The ratio of physical to genetic linkage distances varied substantially among chromosomes and BAC contigs with an average of 3,033 Kb/cM. CONCLUSIONS: The integrated map described here provides a framework for a robust composite genome map for rainbow trout. This resource is needed for genomic analyses in this research model and economically important species and will facilitate comparative genome mapping with other salmonids and with model fish species. This resource will also facilitate efforts to assemble a whole-genome reference sequence for rainbow trout.

Characterization of the rainbow trout transcriptome using Sanger and 454-pyrosequencing approaches.

BACKGROUND: Rainbow trout are important fish for aquaculture and recreational fisheries and serves as a model species for research investigations associated with carcinogenesis, comparative immunology, toxicology and evolutionary biology. However, to date there is no genome reference sequence to facilitate the development of molecular technologies that utilize high-throughput characterizations of gene expression and genetic variation. Alternatively, transcriptome sequencing is a rapid and efficient means for gene discovery and genetic marker development. Although a large number (258,973) of EST sequences are publicly available, the nature of rainbow trout duplicated genome hinders assembly and complicates annotation. RESULTS: High-throughput deep sequencing of the Swanson rainbow trout doubled-haploid transcriptome using 454-pyrosequencing technology yielded ~1.3 million reads with an average length of 344 bp, a total of 447 million bases. De novo assembly of the sequences yielded 151,847 Tentative Consensus (TC) sequences (average length of 662 bp) and 224,391 singletons. A combination assembly of both the 454-pyrosequencing ESTs and the pre-existing sequences resulted in 161,818 TCs (average length of 758 bp) and 261,071 singletons. Gene Ontology analysis of the combination assembly showed high similarities to transcriptomes of other fish species with known genome sequences. CONCLUSION: The 454 library significantly increased the suite of ESTs available for rainbow trout, allowing improved assembly and annotation of the transcriptome. Furthermore, the 454 sequencing enables functional genome research in rainbow trout, providing a wealth of sequence data to serve as a reference transcriptome for future studies including identification of paralogous sequences and/or allelic variation, digital gene expression and proteomic research.

Identification, characterization and genetic mapping of TLR1 loci in rainbow trout (Oncorhynchus mykiss).

Induction of innate immune pathways is critical for early anti-microbial defense but there is limited understanding of how teleosts recognize microbial molecules and activate these pathways. In mammals, Toll-like receptors (TLR) 1 and 2 form a heterodimer involved in recognizing peptidoglycans and lipoproteins of microbial origin. Herein, we identify and describe the rainbow trout (Oncorhynchus mykiss) TLR1 gene ortholog and its mRNA expression. Two TLR1 loci were identified from a rainbow trout bacterial artificial chromosome (BAC) library using DNA sequencing and genetic linkage analyses. Full length cDNA clone and direct sequencing of four BACs revealed an intact omTLR1 open reading frame (ORF) located on chromosome 14 and a second locus on chromosome 25 that contains a TLR1 pseudogene. The duplicated trout loci exhibit conserved synteny with other fish genomes that extends beyond the TLR1 gene sequences. The omTLR1 gene includes a single large coding exon similar to all other described TLR1 genes, but unlike other teleosts it also has a 5' UTR exon and intron preceding the large coding exon. The omTLR1 ORF is predicted to encode an 808 amino-acid protein with 69% similarity to the Fugu TLR1 and a conserved pattern of predicted leucine-rich repeats (LRR). Phylogenetic analysis grouped omTLR1 with other fish TLR1 genes on a separate branch from the avian TLR1 and mammalian TLR1, 6 and 10. omTLR1 expression levels in rainbow trout anterior kidney leukocytes were not affected by the human TLR2/6 and TLR2/1 agonists diacylated lipoprotein (Pam(2)CSK(4)) and triacylated lipoprotein (Pam(3)CSK(4)). However, due to the lack of TLR6 and 10 genes in teleost genomes and up-regulation of TLR1 mRNA in response to LPS and bacterial infection in other fish species we hypothesize an important role for omTLR1 in anti-microbial immunity. Therefore, the identification of a TLR2 ortholog in rainbow trout and the development of assays to measure ligand binding and downstream signaling are critical for future elucidation of omTLR1 functions.

Single nucleotide polymorphism discovery in rainbow trout by deep sequencing of a reduced representation library.

BACKGROUND: To enhance capabilities for genomic analyses in rainbow trout, such as genomic selection, a large suite of polymorphic markers that are amenable to high-throughput genotyping protocols must be identified. Expressed Sequence Tags (ESTs) have been used for single nucleotide polymorphism (SNP) discovery in salmonids. In those strategies, the salmonid semi-tetraploid genomes often led to assemblies of paralogous sequences and therefore resulted in a high rate of false positive SNP identification. Sequencing genomic DNA using primers identified from ESTs proved to be an effective but time consuming methodology of SNP identification in rainbow trout, therefore not suitable for high throughput SNP discovery. In this study, we employed a high-throughput strategy that used pyrosequencing technology to generate data from a reduced representation library constructed with genomic DNA pooled from 96 unrelated rainbow trout that represent the National Center for Cool and Cold Water Aquaculture (NCCCWA) broodstock population. RESULTS: The reduced representation library consisted of 440 bp fragments resulting from complete digestion with the restriction enzyme HaeIII; sequencing produced 2,000,000 reads providing an average 6 fold coverage of the estimated 150,000 unique genomic restriction fragments (300,000 fragment ends). Three independent data analyses identified 22,022 to 47,128 putative SNPs on 13,140 to 24,627 independent contigs. A set of 384 putative SNPs, randomly selected from the sets produced by the three analyses were genotyped on individual fish to determine the validation rate of putative SNPs among analyses, distinguish apparent SNPs that actually represent paralogous loci in the tetraploid genome, examine Mendelian segregation, and place the validated SNPs on the rainbow trout linkage map. Approximately 48% (183) of the putative SNPs were validated; 167 markers were successfully incorporated into the rainbow trout linkage map. In addition, 2% of the sequences from the validated markers were associated with rainbow trout transcripts. CONCLUSION: The use of reduced representation libraries and pyrosequencing technology proved to be an effective strategy for the discovery of a high number of putative SNPs in rainbow trout; however, modifications to the technique to decrease the false discovery rate resulting from the evolutionary recent genome duplication would be desirable.

A first generation BAC-based physical map of the rainbow trout genome.

BACKGROUND: Rainbow trout (Oncorhynchus mykiss) are the most-widely cultivated cold freshwater fish in the world and an important model species for many research areas. Coupling great interest in this species as a research model with the need for genetic improvement of aquaculture production efficiency traits justifies the continued development of genomics research resources. Many quantitative trait loci (QTL) have been identified for production and life-history traits in rainbow trout. A bacterial artificial chromosome (BAC) physical map is needed to facilitate fine mapping of QTL and the selection of positional candidate genes for incorporation in marker-assisted selection (MAS) for improving rainbow trout aquaculture production. This resource will also facilitate efforts to obtain and assemble a whole-genome reference sequence for this species. RESULTS: The physical map was constructed from DNA fingerprinting of 192,096 BAC clones using the 4-color high-information content fingerprinting (HICF) method. The clones were assembled into physical map contigs using the finger-printing contig (FPC) program. The map is composed of 4,173 contigs and 9,379 singletons. The total number of unique fingerprinting fragments (consensus bands) in contigs is 1,185,157, which corresponds to an estimated physical length of 2.0 Gb. The map assembly was validated by 1) comparison with probe hybridization results and agarose gel fingerprinting contigs; and 2) anchoring large contigs to the microsatellite-based genetic linkage map. CONCLUSION: The production and validation of the first BAC physical map of the rainbow trout genome is described in this paper. We are currently integrating this map with the NCCCWA genetic map using more than 200 microsatellites isolated from BAC end sequences and by identifying BACs that harbor more than 300 previously mapped markers. The availability of an integrated physical and genetic map will enable detailed comparative genome analyses, fine mapping of QTL, positional cloning, selection of positional candidate genes for economically important traits and the incorporation of MAS into rainbow trout breeding programs.

Estimates of linkage disequilibrium and effective population size in rainbow trout.

BACKGROUND: The use of molecular genetic technologies for broodstock management and selective breeding of aquaculture species is becoming increasingly more common with the continued development of genome tools and reagents. Several laboratories have produced genetic maps for rainbow trout to aid in the identification of loci affecting phenotypes of interest. These maps have resulted in the identification of many quantitative/qualitative trait loci affecting phenotypic variation in traits associated with albinism, disease resistance, temperature tolerance, sex determination, embryonic development rate, spawning date, condition factor and growth. Unfortunately, the elucidation of the precise allelic variation and/or genes underlying phenotypic diversity has yet to be achieved in this species having low marker densities and lacking a whole genome reference sequence. Experimental designs which integrate segregation analyses with linkage disequilibrium (LD) approaches facilitate the discovery of genes affecting important traits. To date the extent of LD has been characterized for humans and several agriculturally important livestock species but not for rainbow trout. RESULTS: We observed that the level of LD between syntenic loci decayed rapidly at distances greater than 2 cM which is similar to observations of LD in other agriculturally important species including cattle, sheep, pigs and chickens. However, in some cases significant LD was also observed up to 50 cM. Our estimate of effective population size based on genome wide estimates of LD for the NCCCWA broodstock population was 145, indicating that this population will respond well to high selection intensity. However, the range of effective population size based on individual chromosomes was 75.51 - 203.35, possibly indicating that suites of genes on each chromosome are disproportionately under selection pressures. CONCLUSIONS: Our results indicate that large numbers of markers, more than are currently available for this species, will be required to enable the use of genome-wide integrated mapping approaches aimed at identifying genes of interest in rainbow trout.

A Vision for Development and Utilization of High-Throughput Phenotyping and Big Data Analytics in Livestock.

Automated high-throughput phenotyping with sensors, imaging, and other on-farm technologies has resulted in a flood of data that are largely under-utilized. Drastic cost reductions in sequencing and other omics technology have also facilitated the ability for deep phenotyping of livestock at the molecular level. These advances have brought the animal sciences to a cross-roads in data science where increased training is needed to manage, record, and analyze data to generate knowledge and advances in Agriscience related disciplines. This paper describes the opportunities and challenges in using high-throughput phenotyping, "big data," analytics, and related technologies in the livestock industry based on discussions at the Livestock High-Throughput Phenotyping and Big Data Analytics meeting, held in November 2017 (see: https://www.animalgenome.org/bioinfo/community/workshops/2017/). Critical needs for investments in infrastructure for people (e.g., "big data" training), data (e.g., data transfer, management, and analytics), and technology (e.g., development of low cost sensors) were defined by this group. Though some subgroups of animal science have extensive experience in predictive modeling, cross-training in computer science, statistics, and related disciplines are needed to use big data for diverse applications in the field. Extensive opportunities exist for public and private entities to harness big data to develop valuable research knowledge and products to the benefit of society under the increased demands for food in a rapidly growing population.

Effects of short-term growth hormone treatment on liver and muscle transcriptomes in rainbow trout (Oncorhynchus mykiss).

Although studies have established that exogenous growth hormone (GH) treatment stimulates growth in fish, its effects on target tissue gene expression are not well characterized. We assessed the effects of Posilac (Monsanto, St. Louis, MO), a recombinant bovine GH, on tissue transcript levels in rainbow trout selected from two high-growth rate and two low-growth rate families. Transcript abundance was measured in liver and muscle with the Genome Research in Atlantic Salmon Project (GRASP) 16K cDNA microarray. A selection of the genes identified as altered by the microarray and transcripts for insulin-like growth factors, growth hormone receptors (GHRs), and myostatins were measured by real-time PCR in the liver, muscle, brain, kidney, intestine, stomach, gill, and heart. In general, transcripts identified as differentially regulated in the muscle on the microarray showed similar directional changes of expression in the other nonhepatic tissues. A total of 114 and 66 transcripts were identified by microarray as differentially expressed with GH treatment across growth rate for muscle and liver, respectively. The largest proportion of these transcripts represented novel transcripts, followed by immune and metabolism-related genes. We have identified a number of genes related to lipid metabolism, supporting a modulation in lipid metabolism following GH treatment. Most notable among the growth-axis genes measured by real-time PCR were increases in GHR1 and -2 transcripts in liver and muscle. Our results indicate that short-term GH treatment activates the immune system, shifts the metabolic sectors, and modulates growth-regulating genes.

Cloning and characterization of microRNAs from rainbow trout (Oncorhynchus mykiss): their expression during early embryonic development.

BACKGROUND: Current literature and our previous results on expression patterns of oocyte-specific genes and transcription factors suggest a global but highly regulated maternal mRNA degradation at the time of embryonic genome activation (EGA). MicroRNAs (miRNAs) are small, non-coding regulatory RNAs (19-23 nucleotides) that regulate gene expression by guiding target mRNA cleavage or translational inhibition. These regulatory RNAs are potentially involved in the degradation of maternally inherited mRNAs during early embryogenesis. RESULTS: To identify miRNAs that might be important for early embryogenesis in rainbow trout, we constructed a miRNA library from a pool of unfertilized eggs and early stage embryos. Sequence analysis of random clones from the library identified 14 miRNAs, 4 of which are novel to rainbow trout. Real-time PCR was used to measure the expression of all cloned miRNAs during embryonic development. Four distinct expression patterns were observed and some miRNAs showed up-regulated expression during EGA. Analysis of tissue distribution of these miRNAs showed that some are present ubiquitously, while others are differentially expressed among different tissues. We also analyzed the expression patterns of Dicer, the enzyme required for the processing of miRNAs and Stat3, a transcription factor involved in activating the transcription of miR-21. Dicer is abundantly expressed during EGA and Stat3 is up-regulated before the onset of EGA. CONCLUSION: This study led to the discovery of 14 rainbow trout miRNAs. Our data support the notion that Dicer processes miRNAs and Stat3 induces expression of miR-21 and possibly other miRNAs during EGA. These miRNAs in turn guide maternal mRNAs for degradation, which is required for normal embryonic development.

A salmonid EST genomic study: genes, duplications, phylogeny and microarrays.

BACKGROUND: Salmonids are of interest because of their relatively recent genome duplication, and their extensive use in wild fisheries and aquaculture. A comprehensive gene list and a comparison of genes in some of the different species provide valuable genomic information for one of the most widely studied groups of fish. RESULTS: 298,304 expressed sequence tags (ESTs) from Atlantic salmon (69% of the total), 11,664 chinook, 10,813 sockeye, 10,051 brook trout, 10,975 grayling, 8,630 lake whitefish, and 3,624 northern pike ESTs were obtained in this study and have been deposited into the public databases. Contigs were built and putative full-length Atlantic salmon clones have been identified. A database containing ESTs, assemblies, consensus sequences, open reading frames, gene predictions and putative annotation is available. The overall similarity between Atlantic salmon ESTs and those of rainbow trout, chinook, sockeye, brook trout, grayling, lake whitefish, northern pike and rainbow smelt is 93.4, 94.2, 94.6, 94.4, 92.5, 91.7, 89.6, and 86.2% respectively. An analysis of 78 transcript sets show Salmo as a sister group to Oncorhynchus and Salvelinus within Salmoninae, and Thymallinae as a sister group to Salmoninae and Coregoninae within Salmonidae. Extensive gene duplication is consistent with a genome duplication in the common ancestor of salmonids. Using all of the available EST data, a new expanded salmonid cDNA microarray of 32,000 features was created. Cross-species hybridizations to this cDNA microarray indicate that this resource will be useful for studies of all 68 salmonid species. CONCLUSION: An extensive collection and analysis of salmonid RNA putative transcripts indicate that Pacific salmon, Atlantic salmon and charr are 94-96% similar while the more distant whitefish, grayling, pike and smelt are 93, 92, 89 and 86% similar to salmon. The salmonid transcriptome reveals a complex history of gene duplication that is consistent with an ancestral salmonid genome duplication hypothesis. Genome resources, including a new 32 K microarray, provide valuable new tools to study salmonids.

Identification of RtGST-1, a novel germ cell-specific mRNA-like transcript predominantly expressed in early previtellogenic oocytes in rainbow trout (Oncorhynchus mykiss).

Noncoding mRNA-like transcripts play important roles in a wide range of biological events such as cell differentiation and organogenesis. We report here the identification of a novel germ cell-specific mRNA-like transcript (RtGST-1) from a rainbow trout oocyte cDNA library. The novel transcript of 1,165 bp was confirmed to be full-length based on Northern blot and 5' RACE analyses. The transcript is polyadenylated but does not contain a significant open reading frame (ORF). The presumable ORF (encodes a peptide of 71 amino acids) has poor codon usage for rainbow trout and the AUG codon is in a poor context for translation initiation. RT-PCR showed that the novel gene is specifically expressed in ovaries of various stages and immature testis, whereas no transcripts of this gene were detected in mature testis and somatic tissues. Quantitative real-time PCR analysis revealed that the mRNA level of this novel gene is extremely high in early previtellogenesis stage ovaries relative to vitellogenesis stage ovaries. In situ hybridization analysis further demonstrated that the novel transcript is localized exclusively in early previtellogenic oocytes and spermatocytes. To our knowledge, this study represents the first report of a germline-specific mRNA-like transcript in fish.