2005 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? What 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). Section c (Genomic resources, goals 1-3) applies as genetic mapping, physical mapping, and functional genomics will be the major focus of this project. Section e (Bioinformatics, goals 1 and. 2)applies as relational databases have been developed to analyze and curate genomic data. The development of bioinformatic tools is required to integrate and implement existing genomic data into the selective breeding program. 2.List the milestones (indicators of progress) from your Project Plan. 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-¿) 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. 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.What was the single most significant accomplishment this past year? To complement the ongoing construction of a genetic map for rainbow trout, a project was initiated to develop a physical map of the rainbow trout genome using the NCCCWA Swanson 10X bacterial artificial chromosome library. The integration of a physical chromosome map with the genetic map will greatly facilitate our ability to identify genes which affect aquaculture production traits. To this end we successfully completed a pilot project to determine the best strategy for physical map development. In collaboration with researchers at West Virginia University and the University of California at Davis, we employed a recently introduced DNA fingerprinting technique previously used for plant species. Protocols were optimized for rainbow trout DNA and parameters for data analyses established. 4b.List other significant accomplishments, if any. The Inhibitor of DNA Binding/Differentiation (ID) proteins play a role in determining the number of muscle cells that develop in an organism. Such an impact on growth is of great interest to the aquaculture community, therefore we have characterized the ID genes in rainbow trout. Two additional ID genes have been identified and all six known ID genes characterized with respect to their expression throughout embryonic development and in adult tissues. Antiviral immunity in fish is not well understood. In mammals, Toll-like receptor 3 (TLR3) is involved in double-stranded RNA recognition and host immune response activation. A rainbow trout TLR3 gene was identified and characterized based on the similarity of its genomic organization to mammalian and other fish TLR3 genes. In healthy trout, TLR3 is highly expressed in the liver, pyloric ceca, intestine, spleen, and anterior and trunk kidney tissues. In fish challenged with the Infectious Hematopoietic Necrosis Virus (IHNV) increased levels of TLR3 were observed 3 days post infection. Fish challenged with the bacterial pathogen Yersinia ruckeri showed no change in levels of TLR3, therefore TLR3 seems to be specific for antiviral activity in rainbow trout. Construction of a genetic map requires the development of molecular markers suitable for mapping. Marker development strategies include the identification of markers from random sequences in the genome and/or the identification of markers within genes. These two strategies were compared by developing 249 markers in random genomic regions and 179 from genes. Both types of microsatellites proved to be suitable for mapping. Those microsatellites identified in genes were more variable and could be found more often in other salmonid species than the random microsatellites, making them useful for comparison of genomes between salmonid species. Random microsatellites provided better information when conducting evolutionary analyses between members of the family salmonidae. DNA based technologies designed to assist in selective breeding for genetic improvement are often not available for species of interest to aquaculturists. To this end, three hundred and eighteen microsatellite genetic markers were optimized for conducting genetic analyses in striped bass. 4c.List any significant activities that support special target populations. None 4d.Progress report. 1930-31000-008-01R: This serves to document research conducted under a reimbursable agreement between ARS and the University of Maryland. We previously determined that the trout major histocompatibility (MH) genes are located on at least four different chromosomes and assembled sets of overlapping BAC clones that spanned the two class I regions and the region where TAP1 resides. In FY 2005 the Transporter Associated Protein 1 (TAP1) region was sequenced and annotated. It spans 3 overlapping BACs that combined to 320,000 bp. A manuscript describing the genes, gene order and a comparative analysis of MH architecture in other vertebrates is in preparation. 1930-31000-008-02S: This report serves to document research conducted under a Specific Cooperative Agreement between ARS and West Virginia University. A normalized cDNA library from rainbow trout oocyte and a subtracted library from embryos of various developmental stages have been successfully constructed. A total of 20,160 EST sequences from the oocyte library has been generated and analyzed. Over 10,000 unique oocyte-expressed sequences have been identified and annotated. In addition, over 5,000 sequences have been generated from the subtracted embryo library. The information of these sequences provides a global picture of genes expressed in trout oocyte and embryos and is important for identification of key genes essential for oocyte development, maturation, and embryogenesis. The cDNAs for rainbow trout calpain catalytic subunits of the calpains have been successfully cloned and characterized. The expression of these genes in muscle wasting during starvation has been determined. A novel gill-specific calpain catalytic subunit has been identified and characterized. cDNAs encoding 2 calpastatin isoforms (long and short) have been identified. The expression of calpastatin mRNA and the calpain activities in 3 rainbow trout strains with different growth rates have been determined. 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. This project 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. 5.Describe the major accomplishments over the life of the project, including their predicted or actual impact. 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 115,864 ESTs which are deposited for public access in GenBank, ~10,000 more are soon to be added. 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 January 2005. 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. Clones from the NCCCWA bacterial artificial chromosome libraries (n=26) have been distributed to two laboratories in the US, clones from the NCCCWA cDNA libraries (n=564) have been distributed to eight laboratories in 3 countries. 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). White Paper Warr G., Liu J., Rexroad C. E., Trumble B., Gross P., Benzie J., Wilson M., Gaffney P., Yant R., Kocher T., Mileham A., Waldbieser G., Hedgecock D., and Westerman M. (2005). The Genomic Enablement of Aquacultured Species. white paper to funding agencies, a second version to USDA NRI. Posters Coulibaly, I, Yao, J, Rexroad, C. (2005). Comparisons of microsatellites derived from repeat-enriched libraries and ESTs in rainbow trout (Oncorhynchus mykiss). Plant and Animal Genome XIII, January 15-19, San Diego, CA, USA. Gahr, S, and Rexroad, C. (2005). Tissue distribution and embryonic expression of the Inhibitor of Differentiation/DNA Binding (ID) family of proteins in the rainbow trout (Oncorhynchus mykiss). Plant and Animal Genome XIII, January 15-19, San Diego, CA, USA. Palti, Y, Fincham, R, Rodriguez, F, Gahr, S, Rexroad, C, Hansen, J. (2005). Genomic sequencing of a rainbow trout Major Histocompatibility Region that harbors Class II and III genes. Plant and Animal Genome XIII, January 15-19, San Diego, CA, USA. Phillips, R, Noakes, M, DeKoning, J, Morash, M, Keatley, K, Rexroad, C, Palti, Y, Danzmann, R, Nichols, K, Drew, R, Thorgaard, G (2005). Assignment of the rainbow trout linkage groups to specific chromosomes. Plant and Animal Genome XIII, January 15-19, San Diego, CA, USA. Rexroad, C, Rodriguez, MF, Danzmann, R, Palti, Y. (2005). Development of comparative maps using microsatellites developed from rainbow trout ESTs. Plant and Animal Genome XIII, January 15-19, San Diego, CA, USA. Yao, J, Higgins, A, and Rexroad, C. (2005). Transcriptome analyses of rainbow trout oocytes. Plant and Animal Genome XIII, January 15-19, San Diego, CA, USA. Couch, C, Garber, A, Abrams, J, Rexroad, C, Westerman, M, Sullivan, C. Microsatellite development for a hybrid striped bass (Morone chrysops x M. saxatilis) selective breeding program. (2005). Aquaculture America, January 17-20, New Orleans, LA, USA. Salem, M, Yao, J, Rexroad, C, Kenney, B, Semmens, K, Killefer, J, Nath, J. (2005). Molecular characterization of calpastatins from rainbow trout: their roles in muscle growth and fillet texture development. Aquaculture America, January 17-20, New Orleans, LA, USA. Gahr, SA, Rexroad, CE, and Weber GM. (2005). Changes in the expression of the Inhibitor of DNA Binding genes following fasting and re-feeding in the Rainbow Trout (Oncorhynchus mykiss). International Conference of Comparative Endocrinology, May 23-28, Boston, MA, USA. Nagler, J, Cavileer, T, Cyr, D, Rexroad, C. (2005). Estrogen receptor alpha-2: a novel isoform from the rainbow trout. Society for the Study of Reproduction, July 24-27, Quebec City, QC, Canada. Presentations Rodriguez, F, Wiens, G, Hovatter, K, Fincham, R, Gahr, S, Rexroad, C, Palti, Y. Genomic characterization of rainbow trout (Oncorhynchus mykiss) Toll-Like Receptor genes. Plant and Animal Genome XIII, January 15-19, 2005, San Diego, CA, USA. Gahr, S, and Rexroad, C. Gene expression of the Inhibitor of DNA Binding (ID) proteins during embryonic development in the rainbow trout (Oncorhynchus mykiss). Aquaculture America, January 17-20, 2005, New Orleans, LA, USA. Rexroad, C, Gahr, S, and Palti, Y. Identifying candidate genes for rainbow trout (Oncorhynchus mykiss) aquaculture research. Aquaculture America, January 17-20, 2005, New Orleans, LA, USA. Rexroad, C. Careers in Biotechnology. Aquaculture America, January 17-20, 2005, New Orleans, LA, USA. Rexroad, C. Selective breeding and genome mapping of striped bass. Aquaculture America, January 17-20, 2005, New Orleans, LA, USA. Silverstein, J, Parsons, J, Rexroad, C, and Palti, Y. Heterosis and genetic distance in strain crosses of rainbow trout. Aquaculture America, January 17-20, 2005, New Orleans, LA, USA. Rexroad, C. Presentation to advanced placement science and Future Farmers of America Students from Philip Barbour High School, March 18, 2005. Rexroad, C, presentation at NCCCWA to Future Farmers of America students from Jefferson County High School, March 23, 2005. Rexroad, C. Presentation to federal judges and lawyers attending a course entitled, "Exploring the Ecology, Economics, and Public Policy of Freshwater Resources and Fisheries" hosted by the Foundation for Research on Economics and the Environment, April 23, 2005. Coulibaly, I. Characterization of molecular markers and candidate genes in rainbow trout. NCCCWA Cooperators Conference May 11 and 12, 2005. Palti, Y. Quantitative Trait Loci: in search of a fugitive. NCCCWA Cooperators Conference May 11 and 12, 2005. Rexroad, C. Tools for Genome Research in Rainbow Trout. NCCCWA Cooperators Conference May 11 and 12, 2005. Rexroad, C. Exhibit at the West Virginia State Fair, August 13 and 14, 2005. Review Publications Coulibaly, I., Gharbi, K., Danzmann, R., Yao, J., Rexroad III, C.E. 2005. Characterization and comparison of microsatellites derived from repeat-enriched libraries and expressed sequence tags. Animal Genetics 36, 309-315. Gahr, S.A., Rodriguez, M.F., Rexroad III, C.E. 2005. Identification and expression profile of the id gene family in rainbow trout (oncorhynchus mykiss). Biochimica et Biophysica Acta 1729:64-73. Pruett, C., Saillant, E., Renshaw, M., Patton, J., Rexroad Iii, C.E., Gold, J., Microsatellite dna markers for population-genetic studies and parentage assignment cobia, rachyentron canadum. Molecular Ecology Notes 5: 84-86. Rexroad III, C.E., Rodriguez, M.F., Gharbi, K., Danzmann, R., Dekoning, J., Phillips, R., Palti, Y. 2005. Comparative mapping of expressed sequence tags containing microsatellites in rainbow trout (oncorhynchus mykiss). Genomics. BMC Genomics 6:54. Krasnov, A., Koskinen, H., Rexroad III, C.E., Afanasyev, S., Molsa, H., Oikari, A. 2005. Transcriptome responses to carbon tetrachloride and pyrene in the kidney and liver of juvenile rainbow trout (oncorhynchus mykiss). Aquatic Toxicology 74: 70-81. Salem, M., Nath, J., Rexroad III, C.E., Killefer, J., Yao, J. 2005. Identification and molecular characterization of the rainbow trout calpains (capn1 and capn2)their expression in muscle wasting during starvation. Comparative Biochemistry and Physiology 140:63-71. Mohamed, S., Yao, J., Rexroad III, C.E., Kenney, B., Semmens, K., Killefer, J., Nath, J. 2005. Characterization of the calpastatin gene in fish: its potential role in muscle growth and fillet quality. Comparative Biochemistry and Physiology 141: 488-97. Wagner, J.L., Palti, Y., Didario, D., Faraco, J. 2005. Sequence of the canine major histocompatibility complex region containing non-classical class 1 genes. Tissue Antigen 65: 549-55. Krasnov, A., Koskinen, H., Petri, P., Rexroad Iii, C.E., Afanasyev, S., Molsa, H. 2005. Gene expression in the brain and kidney of rainbow trout in response to handling stress. Biomed Central (BMC) Genomics 6:3.