2006 Annual Report 1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? Aquaculture is the most rapidly growing segment of US agriculture and the only sustainable option for satisfying the globally increasing demand for seafood products. Although demands in domestic and international markets remain high, the growth of the US rainbow trout aquaculture industry has leveled off. In order to compete, US trout farmers must overcome losses due to diseases and improve the growth characteristics of this species. Although many strains of rainbow trout have been characterized for traits associated with hatchery production, the US industry lacks well-characterized germplasm which has been genetically improved for aquaculture production efficiency. One approach to genetic improvement is through selective breeding with the aid of molecular genetic technologies. To this end, NCCCWA has initiated a multidisciplinary approach to selective breeding of rainbow trout. The aims of our research are:. 1)to develop molecular genetic tools for rainbow trout;. 2)utilize these tools to identify genes affecting aquaculture production traits; and. 3)to implement molecular genetic data into the NCCCWA long term selective breeding program. Ultimately, molecular genetic tools for conducting research in salmonids (chromosome maps, expressed sequence tag information) will be made available to the international research community and improved germplasm will be made available to the US rainbow trout industry. CRIS project 1930-31000-008-00D was initiated in FY2005. Activities primarily fall within the scope of the Genetic Improvement component (#3) of the Aquaculture National Program Action Plan (106) including: a-Conserve, characterize, and utilize genetic resources; b-Selective breeding for economically important traits; c-Genomic resources; and e-Bioinformatics and statistical analysis tools. 2.List by year the currently approved milestones (indicators of research progress) 2005 1A.1 Genotype the 2005 year class with nine microsatellite loci and estimate population genetic parameters. 1A.2 Construct a multiplex genotyping system to increase efficiency of genotyping. 1B Genotype 500 microsatellite markers and conduct first linkage analysis. 1C Identify superior and inferior germplasm with respect to stress response and feed efficiency for P1 generation of breeding scheme. 1D Identify susceptible/resistant germplasm from the Flavobacterium psychrophilum challenge studies. 2E Complete EST sequencing including oocyte and embryonic libraries. 2F Sequencing, expression analysis and marker identification for the inhibitor of DNA binding (ID) genes. 2G.1 Isolate microsatellite markers for MHC haplotyping. 2G.2 Complete sequencing and characterization of TLR genes. 2G.3 Collect samples and extract DNA from Yersinia ruckeri family challenges. 3H Hire Bioinformaticist, construction of a data pipeline for linkage analysis including evaluation and implementation of software. 2006 1A.1 Genotype the 2006 year class with nine microsatellite loci and estimate population genetic parameters. 1A.2 Evaluate multiplex genotyping system for estimating population genetic parameters. 1B Genotype an additional 300 microsatellites. 1C Breed P1 generation. 1D Identification of siblings from challenge studies to be P1s. 2E Analysis of EST data for the construction of a high-density oligonucleotide microarray. 2F Sequencing, expression analysis and marker identification for uncoupling proteins (UCP), transforming growth factor beta (TGF-beta) receptors. 2G.1 Genotype Yersinia ruckeri resistant/susceptible families with MHC microsatellites. 2G.2 Isolate microsatellites linked to TLRs. 2G.3 Collect samples and extract DNA from Flavobacterium psychrophilum challenges. 3H Conduct genetic linkage analysis for the construction of a genetic map. 2007 1A.1 Genotype the 2007 year class with nine microsatellite loci or multiplex system and estimate population genetic parameters. 1A.2 Conduct a four chromosome characterization of linkage disequilibrium parameters in broodstock. 1B Identify microsatellite markers for conducting genome scans from the genetic map. 1C Identify germplasm for the F1 generation of breeding scheme. 1D Breed P1. 2E Determine suitability of microarrays to identify genes differentially expressed during embryonic development and following growth hormone administration. 2F Conduct microarray experiments to identify genes differentially expressed transcripts with respect to growth and disease traits. 2G.1 Genotype Yersinia ruckeri resistant/susceptible families with TLR microsatellites. 2G.2 Genotype Flavobacterium psychrophilum resistant/susceptible families with MHC and TLR microsatellites. 3H Conduct association analysis of molecular genetic variation with phenotypic variation in NCCCWA Broodstock. 2008 1A Genotype the 2008 year class with nine microsatellite loci or multiplex system and estimate population genetic parameters. 1B Continue to add markers of interest to the genetic map. 1C Breed F1 generation. 1D Identify germplasm for the F1 generation of breeding scheme. 2E Genomic characterization of candidate genes identified through microarray experiments. 2F Isolate and map markers associated with candidate genes identified in microarray experiments. 2009 1A Genotype the 2009 year class with nine microsatellite loci or multiplex system and estimate population genetic parameters. 1B Continue to add markers of interest to the genetic map. 1C Phenotype (Measure stress response and feed efficiency) and genotype F2 generation. 1D Breed F1. 2E Genomic characterization of candidate genes identified through microarray experiments. 2F Isolate and map markerse associated with candidate genes identified in microarray experiments. 3H Data analysis for Stress Response and Feed Efficiency QTL experiments. 4a.List the single most significant research accomplishment during FY 2006. National Program 106, Genetic Improvement, c. Genomic resources. A microsatellite genetic map for rainbow trout. A genetic map for rainbow trout composed of >1000 microsatellite genetic markers has been constructed and analytical methods for analyzing mapping data for outbred crosses of rainbow trout designed. This map will be useful for identifying genes which affect traits associated with aquaculture production efficiency. 4b.List other significant research accomplishment(s), if any. National Program 106, Genetic Improvement, a-Conserve, characterize, and utilize genetic resources Development of a trout microsatellite multiplex. To increase the efficiency of genotyping broodstock, a single-step method of co-amplifying microsatellite markers for genetic studies was developed and optimized to reduce the high costs associated with these analyses. This technique is termed multiplexing and has shown great success in a variety of species. The multiplex provides quality data equal to or better then previously published methods and is expected to be widely used to identify parentage or determine population genetic parameters. National Program 106, Genetic Improvement, c. Genomic resources. Identification of genes expressed in the trout pituitary. The pituitary, a small gland located at the base of the brain, is responsible for the secretion of factors that act in the regulation of numerous physiological processes. We identified 1780 unique gene sequences from male and female rainbow trout pituitaries to facilitate further investigation into the actions of this organ. National Program 106, Genetic Improvement, c. Genomic resources. Identification of genes expressed in response to infection. RNA from spleen and anterior kidney of Yersinia ruckeri infected fish was collected 1,3,5,7 and 10 days post-infection. To understand the biological response to infection, gene sequencing was conducted which resulted in the identification of 6,691 transcripts which were deposited in GenBank. 4c.List significant activities that support special target populations. None. 4d.Progress report. Identification and Characterization of trout uncoupling protein genes. Uncoupling proteins affect various physiological processes such as control of body weight, fatty acid metabolism, control of reactive oxygen species, and negative regulation of insulin secretion. Two rainbow trout uncoupling protein 2 genes were identified and characterized their expression throughout embryonic development and in response to fasting. Genetic mapping of Toll-like receptors. Toll-like receptors (TLRs) are a family of transmembrane proteins that recognize pathogens and initiate immune response. Three previously described TLRs were mapped in rainbow trout in addition to the discovery and mapping of three new TLRs. Detailed mapping and gene annotation of TLR genes in rainbow trout and the development of genetic markers for the different TLR genes will provide useful tools for genetic improvement of disease resistance in rainbow trout and other salmonids. Identification of a putative trout leptin gene. The leptin gene is known to affect feed intake, energy expenditure and body weight in mice and humans. To characterize the effects of leptin in rainbow trout, we determined the DNA sequence of a putative leptin gene and 8 other genes located on the same chromosome. Knowledge of this DNA sequence will help determine how this protein functions. Association of the trout major Histocompatibility genes with pathogen resistance. Two disease challenges were conducted to identify genes associated with resistance to the bacterial pathogens Flavobaterium psychrophilum or Yersinia ruckeri. Significant associations were detected between the MHIa region and resistance to Yersinia ruckeri and between MHIb genes and resistance to Flavobacterium psychrophilum. Breeding for bacterial disease resistance. Multiple survival analysis pipelines were evaluated for use in estimating genetic parameters for rainbow trout survival to diseases inflicted by bacterial pathogens such as Flavobacterium psychrophilum and Yersinia ruckeri. The results of these analyses were used to estimate breeding values for disease survival in association studies with disease resistance candidate genes. Response of high growth fish to growth hormone. Growth hormone is a central regulator of a wide variety of physiological functions, such as muscle and bone growth, metabolic status and osmoregulation. One-hundred genes were identified which are differentially expressed in the liver and muscle of high-growth rate fish following growth hormone treatment. 1930-31000-008-06N: This report serves to document research conducted under a non-funded cooperative agreement between ARS and The University of Victoria, Victoria, British Columbia, Canada entitled, "Transcript Analyses in Salmonids." This agreement has the goal of identifying representatives for all salmonid genes by coordinating expressed sequence tag projects for Atlantic salmon and rainbow trout. New tissues for gene discovery including stages of embryonic development have been targeted to increase the probability of identifying novel genes. In FY2006 the first phase of this project was completed, involving partial sequencing of 100,000 genes from rainbow trout. Future analyses will be conducted within and between species to identify common genes and estimate the amount of representation for each transcriptome. 1930-31000-008-03R: This report serves to document research conducted under a reimbursable agreement between ARS and Kent Sea Tech Corporation of San Diego, CA entitled, "Molecular Markers for Genome Mapping and Selective Breeding of Striped Bass" was completed in FY2005. Our objective for the three-year project was to design 100 genetic markers for use in selective breeding programs on Striped Bass, Morone saxatilis. In FY2005, we added 324 microsatellite markers to 177 which we previously identified. A subset of these makers has been evaluated for use in broodstock management in collaboration with North Carolina State University. 1930-31000-008-01R: This report serves to document research conducted under a reimbursable agreement between ARS and the University of Maryland entitled, “Sequencing and Characterization of the Rainbow Trout MHC Class I and II.” The trout major histocompatibility (MH) genes, which play a role in disease resistace, are located on at least four different chromosomes in rainbow trout. A set of overlapping fragments of DNA has been identified which span the two class I regions and the region where TAP1 resides. In FY 2005 320,000 bas pairs of DNA including the TAP1 region was sequenced and annotated. 1930-31000-008-05S: This report serves to document research conducted under a specific cooperative agreement between ARS and the University of California at Davis entitled, "Production of a Physical Map for the Rainbow Trout Genome Using High Throughput DNA Fingerprinting." A total of 9,216 Bacterial Artificial Chromosome clones (0.5X genome coverage) from the Swanson rainbow trout BAC library were fingerprinted using the SNaPshot fingerprinting method to develop resources for an integrated physical and genetic map for the rainbow trout genome. 1930-31000-008-08R: This report serves to document research conducted under a reimbursable cooperative agreement between ARS and Troutlodge, Inc entitled, "Genetic and Diet Effects on Growth Rate and Reproduction in the Rainbow Trout Strains of Troutlodge, Inc." The goal of this study is to test for the effects of genetics and diet on growth and reproductive performance in the rainbow trout strains of Troutlodge, Inc. To this end, 20,000 fish from 95 families were reared on two different diets, one based on traditional fishmeal and the other on a plant protein supplement. To date, the parents have been screened with genetic markers and 1000 progeny from each diet have been sampled. In FY2007 the 2000 progeny will be genotyped to determine parentage and identify correlations between family and performance. 1930-31000-008-02S: This report serves to document research conducted under a Specific Cooperative Agreement between ARS and West Virginia University entitled, "Development of Genetic Markers for Rainbow Trout." One benefit of genome research is the ability to identify genes of interest based on information from other species. To this end, several genes have been identified and characterized in rainbow trout which may impact fillet characteristics. The cDNAs for rainbow trout calpain catalytic subunits of the ¿-and m- calpains have been identified and their expression in muscle wasting during fasting has been determined. Also, a novel gill-specific calpain catalytic subunit has been identified and characterized. Finally, two calpastatin isoforms (long and short) have been identified and their activity evaluated in three strains of rainbow trout differing in growth rate. 1930-31000-008-04s: This report serves to document research conducted under a Specific Cooperative Agreement between ARS and West Virginia University entitled, "Functional Genomics Research for Rainbow Trout Aquaculture Production." Significant progress has been made in the identification of genes which may affect aquaculture production traits. With respect to identifying and characterizing genes important in embryonic development, a novel oocyte-specific gene (OORP-T) encoding a protein with a conserved oxysterol binding protein domain has been identified. Also, a molecular characterization of caspase-9 was conducted including observation of expression patterns in spawning-associated muscle atrophy. Finally, the anabolic effects of feeding beta2-adrenergic agonists (clenbuterol and ractopamine) on rainbow trout muscle proteases and proteins were determined. 5.Describe the major accomplishments to date and their predicted or actual impact. National Program 106, Genetic Improvement, c. Genomic resources. Genome research is facilitated by the development of species specific molecular tools constructed from the species of interest. One such molecular tool is the expressed sequence tag, or EST. ESTs are identified by sequencing RNA molecules from organisms’ tissues. The sequence gives clues to what proteins are being made which possibly affect a trait of interest. Researchers at National Center for Cool and Cold Water Aquaculture and West Virginia University have collaborated to generate ~125,000 ESTs which are deposited for public access in GenBank. A Rainbow Trout Gene Index was established in collaboration with The Institute for Genome Research to provide in-depth analyses of these data to the public. An update of the original gene index was released in 2006 revealing over 83,000 unique sequences. This resource openly provides a very large quantity of molecular DNA information to the scientific community and combines gene sequence data from many international projects to enhance functional genomic research in this species. 6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? NCCCWA continues to share its genomic resources with academic researchers around the globe. This year 8 material transfer agreements were established to transfer clones from the NCCCWA bacterial artificial chromosome libraries and/or cDNA libraries. 7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Rexroad C. - October 9, 2005 presentation at Virginia Tech University entitled, “Observations of an evolutionarily recent genome duplication in salmonids: Consequences for genetic research.” Rexroad C. - October 21, 2005 presentation at West Virginia State University entitled, “Rainbow Trout Genomics: Applications for Aquaculture.” Palti Y. – October 25, 2006 presentation at the Atlantic salmon Genome Sequencing Workshop in Aas, Norway entitled, “Genomic resources needed in rainbow trout and other salmonid species to fully exploit an Atlantic salmon genome sequence.” Rexroad C. - May 17, 2006 presentation at Washington State University/University of Idaho entitled, “NCCCWA Genome Research for the Selective Breeding of Rainbow Trout.” Rexroad C. - May 22, 2006 presentation on NCCCWA molecular genetics research to representatives of Aquabounty. Rexroad C. - May 22, 2006 presentation on NCCCWA molecular genetics research to representatives of BARD/MARD. Rexroad C. - June 13, 2006 presentation to Jefferson County Adult Education Science Class. Palti Y. - March 15, 2006 presentation at Virginia Tech University entitled, “Genomics implementation into the NCCCWA rainbow trout breeding program.” Review Publications Couch, C., Garber, A., Abrams, J., Rexroad III, C.E., Westerman, M., Sullivan, C. 2005. Microsatellite development for a hybrid striped bass (morone chrysops x m. saxatilis) selective breeding program. Meeting Abstract. International Marine Biotechnology Conference, June 7 - 12, 2005, St. Johns, New Foundland, Canada. Nagler, J., Cavileer, T., Cyr, D., Rexroad III, C.E. 2005. Estrogen receptor alpha-2: a novel isoform from the rainbow trout. Society for the Study of Reproduction Annual Meeting. 38:M184. Nam, S., Choi, S., Kang, M., Kozukue, N., Friedman, M. 2005. Antioxidative, antimutagenic, and anticarcinogenic activities of rice bran extracts in chemical tests and in cell cultures. Journal of Agriculture and Food Chemistry.53:516-822. Coulibaly, I., Gahr, S.A., Palti, Y., Yao, J., Rexroad III, C.E. 2005. Genomic structure and expression patterns of uncoupling protein 2 genes in rainbow trout. Plant and Animal Genome Conference. Abstract ID W188, page 50. Rexroad III, C.E., Coulibaly, I., Couch, C., Garber, A., Westerman, M., Sullivan, C. 2005. Microsatellite markers for population genetics and genome mapping in striped bass. Plant and Animal Genome Conference. Abstract ID P638, page 261. Rexroad III, C.E., Danzmann, R., Palti, Y., Vallejo, R.L. 2005. The national center for cool and cold water aquaculture genetic map for rainbow trout. Plant and Animal Genome Conference. Abstract ID P639, page 261. Palti, Y., Rexroad III, C.E., Welch, T.J., Wiens, G.D., Silverstein, J. 2005. Characterization of major histocompatibility (MH) haplotypes and their association with desease resistance in rainbow trout. Plant and Animal Genome Conference. Abstract ID P643, page 262. Landis, E.D., Palti, Y., Dekoning, J., Phillips, R.B., Hansen, J.D. 2006. Mapping and functional genomics of the TAPBP and TAPBP-R genes in rainbow trout. Plant and Animal Genome Conference. Meeting book of Abstracts p. 253. Rexroad III, C.E., Palti, Y., Vallejo, R.L., Silverstein, J. 2006. Genomics and genetics:molecular variation, bioinformatics and functional genomics, implementation into the ncccwa breeding program. BARD Workshop: Aquaculture Genetics-Status and Prospects, Eilat, Israel. Meeting Abstract. Gharbi, K., Coulibaly, I., Rexroad III, C.E., Moghadam, H., Leder, E.H., Ng, S., Davidson, W., Guyomard, R., Ferguson, M., Danzmann, R. 2006. Comparative maps of salmonid genomes: an update. Meeting Abstract. Montpellier, France 6/25 - 30, 2006. Gharbi, K., Coulibaly, I., Rexroad III, C.E., Moghadam, H., Leder, E.H., Ng, S., Davidson, W., Ferguson, M., Danzmann, R. 2006. Progress toward a gene map for rainbow trout (oncorhynchus mykiss). Meeting Abstract International Symposium for Genetics in Aquaculture 6/25-30, 2006 Montpellier, France. Rise, M., Honeyfield, D.C., Devlin, R.H., Rexroad III, C.E., Davidson, W.S., Koop, B.F. 2006. Genomic resources for studying early life stage salmonid health. Meeting Abstract. 7:192 Davidson, W., Chevalet, C., Rexroad III, C.E., Omholt, S. 2006. Salmonid genomic sequencing initiative: the case for sequencing the genomics of atlantic salmon (salmo salar) and rainbow trout (oncorynchus mykiss). Government Publication/Report. p. 1-22. Coulibaly, I., Gahr, S.A., Palti, Y., Yao, J., Rexroad III, C.E. 2006. Genomic structure and expression of uncoupling protein 2 genes in rainbow trout (oncorhynchus mykiss). Biomed Central (BMC) Genomics 7:203. Landis, E.D., Palti, Y., Dekoning, J., Drew, R., Phillips, R.B., Hansen, J.D. 2006. Identification and regulation of rainbow trout tapasin and tapasin-related genes. Immunogenetics 58(1):56-69. Mohamed, S., Keeney, B., Rexroad III, C.E., Yao, J. 2006. Molecular characterization of fish muscle atrophy and proteolysis associated with spawning. Comparative Biochemistry and Physiology 1(2):227-237. Salem, M., Levesque, A., Moon, T., Rexroad III, C.E., Yao, J. 2006. Anabolic effects of feeding beta-2 adrenergic agonists on rainbow trout muscle proteases and myofibrillar proteins. Comparative Biochemistry and Physiology. 1 44(2) 145-154. Rodriguez, M.F., Gahr, S.A., Rexroad III, C.E., Palti, Y. 2006. A pcr screening method for rapid detection of microsatellites in bacterial artificial chromosomes (bacs). Marine Biotechnology DOI: 10.1007/s10126-005-5064-7. Online Publication 5/25/2006. Danzmann, R., Cairney, M., Ferguson, M., Gharbi, K., Guyomard, R., Holm, L., Hoyheim, B., Leder, E.H., Okamoto, N., Ozaki, A., Rexroad III, C.E., Sakomoto, T., Taggart, J., Woram, R. 2005. A comparative analysis of the rainbow trout genome with two other species of fish (arctic charr and atlantic salmon) within the tetraploid derivative salomidae family (subfamily: salmoninae). Genome 2005 Dec;48(6):1037-51. Palti, Y., Rexroad Iii, C.E., Welch, T.J., Wiens, G.D., Silverstein, J., Vallejo, R.L. 2006. Selective breeding and genetic mapping of disease resistance in rainbow trout. Meeting Abstract. Presented at The 9th International Symposium of The International Association for Genetics in Aquaculture, June 26-30, 2006 at Montpellier, France. Meeting Book of Abstracts p. 81. Nichols, K., Gahr, S.A., Rexroad III, C.E., Phillips, R., Thorgaard, G. 2005. Mapping, expression, and molecualr variation of potential candidate genes underlying a major embryonic development rate qtl in rainbow trout. Plant and Animal Genome Conference. Abstract ID W205, page 54.