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Atlantic salmon, SNP discovery, whole genome re-sequencing, aquaculture stocks, continent of origin, North American. [Extracted from the article]

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. [ABSTRACT FROM AUTHOR]

Genetic line-by-environment (G×E) interactions were determined for growth and processing traits of five genetic lines of Rainbow TroutOncorhynchus mykissreared in four environments. Genetic lines included (1) a mixed pool of 109 families selectively bred for improved growth (Growth Line) at the U.S. Department of Agriculture (USDA) National Center for Cool and Cold Water Aquaculture (NCCCWA; Kearneysville, West Virginia), (2) a mixed pool of 52 randomly bred families (Synthetic Control) and fish from three commercial sources, identified as (3) Commercial A, (4) Commercial B, and (5) Commercial C. Two study locations (NCCCWA and The Conservation Fund’s Freshwater Institute [FI], Shepherdstown, West Virginia) contained all five genetic lines of fish while the remaining two sites (USDA National Cold Water Marine Aquaculture Center, Franklin, Maine, and Casta Line Trout Farm, Goshen, Virginia) contained only the Growth Line and Commercial A fish. Fish were PIT-tagged and allowed to comingle at all locations except at Casta Line where Growth Line and Commercial A fish were stocked into separate raceway sections. Performance of the Growth Line and Commercial A fish across all locations were used to detect G×E interactions. Body weight of the Growth Line fish was similar to or exceeded that of Commercial A fish, and the G×E interaction for growth was beyond 1 year of age (P< 0.05). A G×E interaction occurred for condition factor (CF), and the Growth Line fish generally exhibited a higher CF than did the Commercial A fish. There was a G×E interaction for fillet yield in fish harvested at 500 g or less, with no G×E effect for butterfly yield, viscera yield, or final mortality. Data from all five genetic lines at NCCCWA and FI indicate that large differences in genetic potential for growth and processing traits are not significantly affected by G×E interactions Received February 3, 2016; accepted December 2, 2016 Published online February 16, 2017 [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Bacterial cold water disease (BCWD) causes significant economic loss in salmonid aquaculture. Using microsatellite markers in a genome scan, we previously detected significant and suggestive QTL affecting phenotypic variation in survival following challenge with Flavobacterium psychrophilum, the causative agent of BCWD in rainbow trout. In this study, we performed selective genotyping of SNPs from restriction-site associated DNA (RAD) sequence data from two pedigreed families (2009070 and 2009196) to validate the major QTL from the previous work and to detect new QTL. The use of RAD SNPs in the genome scans increased the number of mapped markers from ~300 to ~5,000 per family. The significant QTL detected in the microsatellites scan on chromosome Omy8 in family 2009070 was validated explaining up to 58% of the phenotypic variance in that family, and in addition, a second QTL was also detected on Omy8. Two novel QTL on Omy11 and 14 were also detected, and the previously suggestive QTL on Omy1, 7 and 25 were also validated in family 2009070. In family 2009196, the microsatellite significant QTL on Omy6 and 12 were validated and a new QTL on Omy8 was detected, but none of the previously detected suggestive QTL were validated. The two Omy8 QTL from family 2009070 and the Omy12 QTL from family 2009196 were found to be co-localized with handling and confinement stress response QTL that our group has previously identified in a separate pedigreed family. With the currently available data we cannot determine if the co-localized QTL are the result of genes with pleiotropic effects or a mere physical proximity on the same chromosome segment. The genetic markers linked to BCWD resistance QTL were used to query the scaffolds of the rainbow trout reference genome assembly and the QTL-positive scaffold sequences were found to include 100 positional candidate genes. Several of the candidate genes located on or near the two Omy8 QTL detected in family 2009070 suggest potential linkages between stress response and the regulation of immune response in rainbow trout. [ABSTRACT FROM AUTHOR]

Efforts to obtain a comprehensive genome sequence for rainbow trout are ongoing and will be complemented by transcriptome information that will enhance genome assembly and annotation. Previously, transcriptome reference sequences were reported using data from different sources. Although the previous work added a great wealth of sequences, a complete and well-annotated transcriptome is still needed. In addition, gene expression in different tissues was not completely addressed in the previous studies. In this study, non-normalized cDNA libraries were sequenced from 13 different tissues of a single doubled haploid rainbow trout from the same source used for the rainbow trout genome sequence. A total of ~1.167 billion paired-end reads were de novo assembled using the Trinity RNA-Seq assembler yielding 474,524 contigs > 500 base-pairs. Of them, 287,593 had homologies to the NCBI non-redundant protein database. The longest contig of each cluster was selected as a reference, yielding 44,990 representative contigs. A total of 4,146 contigs (9.2%), including 710 full-length sequences, did not match any mRNA sequences in the current rainbow trout genome reference. Mapping reads to the reference genome identified an additional 11,843 transcripts not annotated in the genome. A digital gene expression atlas revealed 7,678 housekeeping and 4,021 tissue-specific genes. Expression of about 16,000–32,000 genes (35–71% of the identified genes) accounted for basic and specialized functions of each tissue. White muscle and stomach had the least complex transcriptomes, with high percentages of their total mRNA contributed by a small number of genes. Brain, testis and intestine, in contrast, had complex transcriptomes, with a large numbers of genes involved in their expression patterns. This study provides comprehensive de novo transcriptome information that is suitable for functional and comparative genomics studies in rainbow trout, including annotation of the genome. [ABSTRACT FROM AUTHOR]

Resistance against diseases affects profitability of rainbow trout. Limited information is available about functions and mechanisms of teleost immune pathways. Immunogenomics provides powerful tools to determine disease resistance genes/gene pathways and develop genetic markers for genomic selection. RNA-Seq sequencing of the rainbow trout spleen yielded 93,532,200 reads (100 bp). High quality reads were assembled into 43,047 contigs. 26,333 (61.17%) of the contigs had hits to the NR protein database and 7024 (16.32%) had hits to the KEGG database. Gene ontology showed significant percentages of transcripts assigned to binding (51%), signaling (7%), response to stimuli (9%) and receptor activity (4%) suggesting existence of many immune-related genes. KEGG annotation revealed 2825 sequences belonging to "organismal systems" with the highest number of sequences, 842 (29.81%), assigned to immune system. A number of sequences were identified for the first time in rainbow trout belonging to Toll-like receptor signaling (35), B cell receptor signaling pathway (44), T cell receptor signaling pathway (56), chemokine signaling pathway (73), Fc gamma R-mediated phagocytosis (52), leukocyte transendothelial migration (60) and NK cell mediated cytotoxicity (42). In addition, 51 transcripts were identified as spleen-specific genes. The list includes 277 full-length cDNAs. The presence of a large number of immune-related genes and pathways similar to other vertebrates suggests that innate and adaptive immunity in fish are conserved. This study provides deep-sequence data of rainbow trout spleen transcriptome and identifies many new immune-related genes and full-length cDNAs. This data will help identify allelic variations suitable for genomic selection and genetic manipulation in aquaculture. [ABSTRACT FROM AUTHOR]

Salmonid genomes are considered to be in a pseudo-tetraploid state as a result of a genome duplication event that occurred between 25 and 100 Ma. This situation complicates single-nucleotide polymorphism ( SNP) discovery in rainbow trout as many putative SNPs are actually paralogous sequence variants ( PSVs) and not simple allelic variants. To differentiate PSVs from simple allelic variants, we used 19 homozygous doubled haploid ( DH) lines that represent a wide geographical range of rainbow trout populations. In the first phase of the study, we analysed SbfI restriction-site associated DNA ( RAD) sequence data from all the 19 lines and selected 11 lines for an extended SNP discovery. In the second phase, we conducted the extended SNP discovery using PstI RAD sequence data from the selected 11 lines. The complete data set is composed of 145 168 high-quality putative SNPs that were genotyped in at least nine of the 11 lines, of which 71 446 (49%) had minor allele frequencies ( MAF) of at least 18% (i.e. at least two of the 11 lines). Approximately 14% of the RAD SNPs in this data set are from expressed or coding rainbow trout sequences. Our comparison of the current data set with previous SNP discovery data sets revealed that 99% of our SNPs are novel. In the support files for this resource, we provide annotation to the positions of the SNPs in the working draft of the rainbow trout reference genome, provide the genotypes of each sample in the discovery panel and identify SNPs that are likely to be in coding sequences. [ABSTRACT FROM AUTHOR]

: Fish under intensive rearing conditions experience various stressors which have negative impacts on survival, growth, reproduction and fillet quality. Identifying and characterizing the molecular mechanisms underlying stress responses will facilitate the development of strategies that aim to improve animal welfare and aquaculture production efficiency. In this study, we used RNA-seq to identify transcripts which are differentially expressed in the rainbow trout liver in response to handling and confinement stress. These stressors were selected due to their relevance in aquaculture production. Total RNA was extracted from the livers of individual fish in five tanks having eight fish each, including three tanks of fish subjected to a 3 hour handling and confinement stress and two control tanks. Equal amount of total RNA of six individual fish was pooled by tank to create five RNA-seq libraries which were sequenced in one lane of Illumina HiSeq 2000. Three sequencing runs were conducted to obtain a total of 491,570,566 reads which were mapped onto the previously generated stress reference transcriptome to identify 316 differentially expressed transcripts (DETs). Twenty one DETs were selected for qPCR to validate the RNA-seq approach. The fold changes in gene expression identified by RNA-seq and qPCR were highly correlated (R2 = 0.88). Several gene ontology terms including transcription factor activity and biological process such as glucose metabolic process were enriched among these DETs. Pathways involved in response to handling and confinement stress were implicated by mapping the DETs to reference pathways in the KEGG database. Accession Numbers: Raw RNA-seq reads have been submitted to the NCBI Short Read Archive under accession number SRP022881. Customized Perl Scripts: All customized scripts described in this paper are available from Dr. Guangtu Gao or the corresponding author. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Selective breeding of animals for increased disease resistance is an effective strategy to reduce mortality in aquaculture. However, implementation of selective breeding programs is limited by an incomplete understanding of host resistance traits. We previously reported results of a rainbow trout selection program that demonstrated increased survival following challenge with Flavobacterium psychrophilum, the causative agent of bacterial cold water disease (BCWD). Mechanistic study of disease resistance identified a positive phenotypic correlation between post-challenge survival and spleen somatic-index (SI). Herein, we investigated the hypothesis of a genetic correlation between the two traits influenced by colocalizing QTL. We evaluated the inheritance and calculated the genetic correlation in five year-classes of odd- and even-year breeding lines. A total of 322 pedigreed families (n = 25,369 fish) were measured for disease resistance, and 251 families (n = 5,645 fish) were evaluated for SI. Spleen index was moderately heritable in both even-year (h2 = 0.56±0.18) and odd-year (h2 = 0.60±0.15) lines. A significant genetic correlation between SI and BCWD resistance was observed in the even-year line (rg = 0.45±0.20, P = 0.03) but not in the odd-year line (rg = 0.16±0.12, P = 0.19). Complex segregation analyses of the even-year line provided evidence of genes with major effect on SI, and a genome scan of a single family, 2008132, detected three significant QTL on chromosomes Omy19, 16 and 5, in addition to ten suggestive QTL. A separate chromosome scan for disease resistance in family 2008132 identified a significant BCWD QTL on Omy19 that was associated with time to death and percent survival. In family 2008132, Omy19 microsatellite alleles that associated with higher disease resistance also associated with increased spleen size raising the hypothesis that closely linked QTL contribute to the correlation between these traits. To our knowledge, this is the first estimation of spleen size heritability and evidence for genetic linkage with specific disease resistance in a teleost fish. [ABSTRACT FROM AUTHOR]

To examine the behavior of the estrogenic biomarker vitellogenin (VTG) under the combined impact of estrogens and pathogens, parasite-infected or noninfected rainbow trout were exposed to two doses of 17β-estradiol (E2). Infected and E2-exposed fish showed significantly lower hepatic VTG mRNA levels than healthy fish. Transcriptome data suggest that this was due to energetic constraints. Reduced responsiveness of the VTG biomarker in parasitized fish might obscure detection of low-level field exposure. Environ. Toxicol. Chem. 2012; 31: 2318-2323. © 2012 SETAC [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are a class of endogenous small non-coding RNA molecules that regulate post-transcriptional expression of target genes and play important roles in animal development. The objectives of this study were to characterize the egg miRNA transcriptome and identify novel egg-predominant miRNAs in rainbow trout. Small RNAs isolated from mature unfertilized rainbow trout eggs were subjected to deep sequencing using an Illumina Genome Analyzer. The massive sequencing produced 24,621,741 quality reads, among which, 266 known miRNAs were identified and 230 putatively novel miRNAs were predicted. The most abundantly known miRNAs are let-7 and miR-21, accounting for 24.06% and 18.71% of the known miRNAs, respectively. Other known miRNAs which are abundantly present in eggs include miR-24, miR-202, miR-148, miR-30, miR-10, miR-146, miR-25, and miR-143. Real time PCR analysis using cDNAs derived from 10 tissues validated 87 out of 90 selected putative miRNAs and identified three novel miRNAs predominantly expressed in rainbow trout eggs. Each of these novel egg-predominant miRNAs is predicted to target a significant number of genes, most of which are significantly down-regulated in naturally ovulated rainbow trout eggs based on analysis of publicly available microarray data sets. Quantitative real time PCR analysis also demonstrated low expression of a selected number of target genes in eggs relative to liver and muscle tissues. This study represents the first complete survey of miRNAs in fish eggs and provides a starting point for future studies aimed at understanding the roles of miRNAs in controlling egg quality and early embryogenesis in rainbow trout. [ABSTRACT FROM AUTHOR]

Fast growth is an important and highly desired trait, which affects the profitability of food animal production, with feed costs accounting for the largest proportion of production costs. Traditional phenotype-based selection is typically used to select for growth traits; however, genetic improvement is slow over generations. Single nucleotide polymorphisms (SNPs) explain 90% of the genetic differences between individuals; therefore, they are most suitable for genetic evaluation and strategies that employ molecular genetics for selective breeding. SNPs found within or near a coding sequence are of particular interest because they are more likely to alter the biological function of a protein. We aimed to use SNPs to identify markers and genes associated with genetic variation in growth. RNA-Seq whole-transcriptome analysis of pooled cDNA samples from a population of rainbow trout selected for improved growth versus unselected genetic cohorts (10 fish from 1 full-sib family each) identified SNP markers associated with growth-rate. The allelic imbalances (the ratio between the allele frequencies of the fast growing sample and that of the slow growing sample) were considered at scores >5.0 as an amplification and <0.2 as loss of heterozygosity. A subset of SNPs (n = 54) were validated and evaluated for association with growth traits in 778 individuals of a three-generation parent/offspring panel representing 40 families. Twenty-two SNP markers and one mitochondrial haplotype were significantly associated with growth traits. Polymorphism of 48 of the markers was confirmed in other commercially important aquaculture stocks. Many markers were clustered into genes of metabolic energy production pathways and are suitable candidates for genetic selection. The study demonstrates that RNASeq at low sequence coverage of divergent populations is a fast and effective means of identifying SNPs, with allelic imbalances between phenotypes. This technique is suitable for marker development in non-model species lacking complete and well-annotated genome reference sequences. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 preexisting 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. [ABSTRACT FROM AUTHOR]

Thirty-eight microsatellite markers were developed from an enriched genomic DNA library of the cyprinid fish (minnow) Dionda episcopa. The microsatellites include 31 perfect-repeat motifs (29 dinucleotide, 1 trinucleotide, and 1 tetranucleotide) and 7 imperfect-repeat dinucleotide motifs. The microsatellite primers were used to amplify microsatellites from five related congeners: D. argentosa, D. diaboli, D. episcopa, D. nigrotaeniata, and D. serena. One species (D. diaboli) is listed as threatened and critically imperiled and two species (D. argentosa and D. serena) are listed as imperiled; the conservation status of D. nigrotaeniata is unknown. The number of experimentally tractable microsatellite markers varied from 28 for D. diaboli to 34 for D. episcopa. The number of polymorphic microsatellites conforming to Hardy-Weinberg expectations (following Bonferroni correction) ranged from 19 (D. diaboli) to 27 (D. argentosa). One pairwise comparison of microsatellites (in D. nigrotaeniata) deviated significantly from expectations of genotypic equilibrium. The microsatellite markers will be useful for conservation and population-genetic studies of these and other species in genus Dionda. [ABSTRACT FROM AUTHOR]

Thirteen nuclear-encoded microsatellites from a genomic DNA library of Serra Spanish mackerel, Scomberomorus brasiliensis, were isolated and characterized. The microsatellites include 10 perfect repeats (eight tetranucleotide and two dinucleotide) and three imperfect repeats (two tetranucleotide and one dinucleotide). An additional five microsatellites, isolated originally from two congeneric species ( S. cavalla and S. niphonius), were characterized in S. brasiliensis. Serra Spanish mackerel support artisanal fisheries along the Caribbean and Atlantic coasts of Central and South America, from Belize to Brazil. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The article focuses on the polymerase-chain-reaction (PCR) primer pairs for 101 nuclear-encoded microsatellites developed from a genomic library for red drum. Researchers were successful in the amplication and they used to genotype 24 red drum obtained from Galveston Bay, Texas. They noted that 69 microsatellites have an uninterrupted dinucleotide motif while 30 had an imperfect dinucleotide motif.

One hundred nuclear-encoded microsatellites from a genomic library of red drum ( Sciaenops ocellatus) were isolated and characterized. Eight microsatellites had tetranucleotide motifs; 92 had dinucleotide motifs. The average number of alleles per microsatellite (sample of 22–24 fish) was 17.7 (range = 2–30); gene diversity averaged 0.796 (range = 0.227–1.000). Following Bonferroni correction, genotype frequencies at 90 microsatellites did not deviate significantly from Hardy–Weinberg equilibrium expectations. Occurrence of null alleles was inferred at 15 microsatellites; alleles differing by only a single base were observed at 11 microsatellites. The microsatellites developed should prove useful for population-genetic studies of ‘wild’ red drum and in construction of a genetic map. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Current literature and our previous results on expression patterns of oocytespecific 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 upregulated 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. [ABSTRACT FROM AUTHOR]

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Lysine is the second limiting amino acid in fish meal based diets, second only to methionine. However, little is known about lysine metabolism in rainbow trout (RBT). Therefore, lysine catabolism by the lysine α-ketoglutarate reductase (LKR) pathway was studied. Additionally, since genetically improved strains could influence fish production, these studies were performed in 4 distinct families of RBT. Two full-sibling families, differing in feed efficiency, were selected from each of 2 strains (A and B) of RBT. Eight fish from each of the 4 families were allotted to individual tanks. Fish were fed until satiation for 5 weeks when four fish within each family were randomly selected for 2 weeks of starvation. After the starvation period, all fish were harvested. Hepatic in-vitro LKR activity and lysine oxidation were measured as was LKR mRNA. No effect of family within strain on LKR activity or lysine oxidation was detected. Strain A exhibited a 55% reduction ( p < 0.01) in LKR transcripts compared to strain B pooled across both feeding levels. Within each family, LKR mRNA was decreased ( p < 0.01) in starved vs. fed fish. On average, there was a 68% decrease in LKR transcripts for starved fish. LKR activity averaged 104 ± 33 and 150 ± 31 nmol/min*gm liver ( p > 0.1) in fed and starved fish, respectively. Lysine oxidation averaged 1.2 ± 0.5 and 2.2 ± 0.4 nmol/min*gm liver ( p > 0.1) in fed and starved fish, respectively. LKR transcripts were positively correlated to weight gain ( p < 0.01). These data are consistent with multiple modes of LKR regulation in fish. [ABSTRACT FROM AUTHOR]

Background: Comparative genomics, through the integration of genetic maps from species of interest with whole genome sequences of other species, will facilitate the identification of genes affecting phenotypes of interest. The development of microsatellite markers from expressed sequence tags will serve to increase marker densities on current salmonid genetic maps and initiate in silico comparative maps with species whose genomes have been fully sequenced. Results: Eighty-nine polymorphic microsatellite markers were generated for rainbow trout of which at least 74 amplify in other salmonids. Fifty-five have been associated with functional annotation and 30 were mapped on existing genetic maps. Homologous sequences were identified for 20 of the EST containing microsatellites to identify comparative assignments within the tetraodon, mouse, and/or human genomes. Conclusion: The addition of microsatellite markers constructed from expressed sequence tag data will facilitate the development of high-density genetic maps for rainbow trout and comparative maps with other salmonids and better studied species. [ABSTRACT FROM AUTHOR]

Background: Microarray technologies are rapidly becoming available for new species including teleost fishes. We constructed a rainbow trout cDNA microarray targeted at the identification of genes which are differentially expressed in response to environmental stressors. This platform included clones from normalized and subtracted libraries and genes selected through functional annotation. Present study focused on time-course comparisons of stress responses in the brain and kidney and the identification of a set of genes which are diagnostic for stress response. Results: Fish were stressed with handling and samples were collected 1, 3 and 5 days after the first exposure. Gene expression profiles were analysed in terms of Gene Ontology categories. Stress affected different functional groups of genes in the tissues studied. Mitochondria, extracellular matrix and endopeptidases (especially collagenases) were the major targets in kidney. Stress response in brain was characterized with dramatic temporal alterations. Metal ion binding proteins, glycolytic enzymes and motor proteins were induced transiently, whereas expression of genes involved in stress and immune response, cell proliferation and growth, signal transduction and apoptosis, protein biosynthesis and folding changed in a reciprocal fashion. Despite dramatic difference between tissues and time-points, we were able to identify a group of 48 genes that showed strong correlation of expression profiles (Pearson r > ∣0.65∣) in 35 microarray experiments being regulated by stress. We evaluated performance of the clone sets used for preparation of microarray. Overall, the number of differentially expressed genes was markedly higher in EST than in genes selected through Gene Ontology annotations, however 63% of stress-responsive genes were from this group. Conclusions: 1. Stress responses in fish brain and kidney are different in function and time-course. 2. Identification of stress-regulated genes provides the possibility for measuring stress responses in various conditions and further search for the functionally related genes. [ABSTRACT FROM AUTHOR]

Genetic variation fuels selective change in natural and captive populations. In establishing a broodstock for selective improvement, the level of genetic diversity is an important consideration because it provides an indication of the scope for selective progress. Three domesticated strains of rainbow trout, Oncorhynchus mykiss (Walbaum), were examined at nine polymorphic microsatellite loci to assess detectable levels of allelic diversity and heterozygosity within and differentiation among the strains. A total of 126 alleles were observed to segregate into unique multilocus genotypes for each of the 152 individuals assayed. There was an average heterozygosity of 71.5% at these nine loci, and an average of 14 alleles at a locus. Each locus was represented by alleles unique to at least two of the three strains. Deviations from Hardy–Weinberg expectations of genotype frequencies were detected in each strain. Subsequent analysis indicated sub-structuring within strains leading to Wahlund effects that caused these deviations. Significant differences in genotype frequencies and pairwise FST values demonstrated that all strains were unique. The overall FST of 0.089 provides additional evidence of unique genetic diversity present in each strain, and agrees well with the degree of genetic variation found in rainbow trout across broad geographical ranges. The genetic diversity contributed by each population suggests that there is greater scope for selective improvement of numerous traits within a synthetic strain combining these three strains than within any individual strain. [ABSTRACT FROM AUTHOR]

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. Copyright © 2003 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The starting point of the present study was the reported identification of a chromosomal region on bovine Chromosome (Chr) 15 (BTA15) carrying loci affecting meat tenderness. A comparative linkage map of BTA15 and human Chr 11 (HSA11) was constructed to identify potential positional candidate genes and to provide a resource of genetic markers to support marker-assisted selection (MAS). Relative rearrangements between the bovine and human genomes for these chromosomes are the most complex observed in comparative mapping between the two species, with nine alternating blocks of conserved synteny between HSA11 and bovine Chrs 15 and 29. The results of this study were the addition of nine genes to the HSA11/BTA15 comparative linkage map, and development of five microsatellite markers within the quantitative trait locus (QTL) interval. One gene with known effects on muscle development (MYOD1) was mapped to the interval. A second gene (CALCA) involved in regulation of calcium levels, a key factor in postmortem tenderization, also mapped within the interval. Refinement of the comparative map and QTL position will reduce the interval on the human transcription map to be scanned in search of candidates, reducing the effort and resources required to identify the allelic variation responsible for the genetic effect. [ABSTRACT FROM AUTHOR]

We used a comparative mapping approach to identify segments of conserved synteny between human Chromosome 14 (HSA14), bovine Chromosome 21 (BTA21), and the portion of ovine Chromosome 18 (OAR18) that contains the clpg locus. A bovine radiation hybrid map of the region was constructed with available Type II genetic markers and seven candidate genes to establish the comparative interval between BTA21 and HSA14. We developed polymorphic microsatellite and SNP markers associated with five candidate genes and placed them on the ovine and/or bovine genetic maps by multipoint linkage analysis. Three additional genes were mapped by virtue of their physical linkage to genetically mapped makers. Development of integrated linkage and physical maps facilitates the selection of positional candidate genes from the gene rich human map. The physically linked candidate genes PREF-1 and MEG3 map to the interval containing the clpg locus. Comparative biology suggests imprinting of MEG3 and/or the influences of PREF-1 on cellular differentiation, should be examined for their role in the parent-of-origin dependent influence of mutant clpg alleles on sheep muscle characteristics. [ABSTRACT FROM AUTHOR]

We examined the effects of enclomiphene and zuclomiphene, alone and in combination with oestradiol, on basal and gonadotrophin-stimulated progesterone secretion by isolated subpopulations of both large (granulosa-lutein) and small (theca-lutein) ovine luteal cells. Isolated large and small luteal cells derived from intact, enucleated ovine corpora lutea were incubated for 48–120 h with or without 22R-hydroxycholesterol or pregnenolone (2.5 μM) and a range of enclomiphene, zuclomiphene, and/or oestradiol concentrations (3–100 μM), both with and without ovine Iuteinizing hormone (100 ng/ml). Spent media were assayed in duplicate for progesterone content by radioimmunoassay. Enclomiphene, zuclomiphene, and oestradiol exhibited equivalent dose-dependent inhibitory effects on basal and gonadotrophin-stimulated small and large ovine luteal cell progesterone secretion under all substrate conditions. Both cell types became more sensitive to clomiphene inhibition with increasing time in culture. In combined treatments, the effects of oestradiol and either enclomiphene or zuclomiphene became additive in longer-term cultures and were never antagonistic In this model system, (i) clomiphene, like oestradiol, appears to inhibit 3β-hydroxysteroid dehy-drogenase activity, (ii) both stereoisomers act as oestrogen agonists, (iii) neither demonstrates any anti-oestrogenic properties, and (iv) both large and small luteal cells become more sensitive to clomiphene inhibition with increasing duration of exposure. [ABSTRACT FROM PUBLISHER]

Thirteen nuclear-encoded dinucleotide microsatellites were characterized from a genomic DNA library of greater amberjack, Seriola dumerili. The microsatellites include 12 perfect-repeat motifs and one imperfect-repeat motif. The number of alleles at the 13 microsatellites among a sample of 29 fish ranged from 3 to 25; gene diversity (expected heterozygosity) ranged from 0.296 to 0.948, while observed heterozygosity ranged from 0.276 to 0.897. Following Bonferroni correction, genotypes at all 13 microsatellites fit expectations of Hardy-Weinberg equilibrium. One pairwise comparison of microsatellites deviated significantly from expectations of genotypic equilibrium, suggesting that these two microsatellites may be linked. Greater amberjack support commercial and recreational fisheries along both the Atlantic and Gulf coasts of the U.S. and represent a species with potential for worldwide aquaculture. The microsatellites developed will be useful for conservation and population genetic studies of ‘wild’ and domesticated populations of greater amberjack. [ABSTRACT FROM AUTHOR]

Eighteen nuclear-encoded microsatellites from a genomic DNA library of greater amberjack, Seriola dumerili, were isolated and characterized. The microsatellites include 13 perfect (five tetranucleotide and eight trinucleotide) and five imperfect (three tetranucleotide, one trinucleotide and one combination dinucleotide/trinucleotide) repeat motifs. The number of alleles at the 18 microsatellites among a sample of 29 fish ranged from two to 20; gene diversity (expected heterozygosity) ranged from 0.068 to 0.950, whereas observed heterozygosity ranged from 0.069 to 0.966. Following Bonferroni correction, genotypes at all 18 microsatellites fit expectations of Hardy–Weinberg equilibrium, and all pairwise comparisons of microsatellites did not deviate significantly from genotypic equilibrium. Greater amberjack support commercial and recreational fisheries along both the Atlantic and the Gulf coasts of the USA and represent a species with potential for worldwide aquaculture. The microsatellites developed will be useful for population genetic studies of ‘wild’ populations and breeding studies of domesticated populations. [ABSTRACT FROM AUTHOR]

Twenty nuclear-encoded microsatellites from a genomic DNA library of cobia, Rachycentron canadum, were isolated and characterized. The microsatellites include two tetranucleotide, one trinucleotide, three combination tetranucleotide/dinucleotide, nine dinucleotide, and five imperfect (dinucleotide) repeat motifs. Gene diversity ranged between zero to 0.910; the number of alleles among a sample of 24 fish ranged from one to 15. Cobia support an important recreational fishery in the southeastern United States and recently have become of interest to aquaculture. The microsatellites developed will be useful tools for studying both population genetics (e.g. stock structure, effective population size) and inheritance of traits important to aquaculture. [ABSTRACT FROM AUTHOR]