2007 Annual Report 1a.Objectives (from AD-416) 1) Continued development of a rainbow trout genetic map to reveal qualitative and quantitative trait loci affecting traits associated with aquaculture production. 2) Identification and characterization of genes affecting disease resistance, growth, and embryonic development through functional genomic technologies. 3) Development of strategies to implement functional genomic and genetic mapping information into the NCCCWA rainbow trout selective breeding program using bioinformatics. 4) Develop databases and programs to assimilate and coordinate genomic data on the rainbow trout broodstock developed at the NCCCWA. 5) Develop methodology to utilize rainbow trout genomic information in comparative analyses to identify potential critical functional genes and genetic pathways. 1b.Approach (from AD-416) 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. A major constraint to increasing the production efficiency is the lack of genetically improved aquaculture stocks. One approach to address these problems is genetic improvement through selective breeding. NCCCWA has initiated a multidisciplinary approach to selective breeding of rainbow trout. Coordination of the activities proposed in this Project Plan with ongoing pathogenic disease research being conducted by project "Host, Pathogen, and Environmental Interactions in Cool and Cold Water Aquaculture" and growth, stress, and reproduction research being conducted by project "Utilizing Gentic for Enhancing Cool and Cold Water Aquaculture Production", will allow for the identification of genes affecting important aquaculture production traits. The result will be genome mapping information which will allow for an understanding of the biological mechanisms controlling these phenotypes and exploitation of positive genetic variation in the NCCCWA selective breeding program. 3.Progress Report This 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” (1930-31000-008-04S). The focus of this project continues to be functional genomic characterization of biological processes including oocyte and spermatocyte maturation, embryonic development, and improved protein accretion in muscle as related to aquaculture production traits. Currently sixteen genes, including those annotated in other species and several not previously identified, are being characterized for their roles in these process by determining their DNA sequence, genetic map location, and patterns of gene expression. The ADODR is in frequent contact with the cooperator through phone calls, email, and annual site visits in addition to receipt of written reports. This serves to document research conducted under a specific cooperative agreement between ARS and the University of California, Davis entitled, “Production of a physical map for the rainbow trout genome using high throughput DNA fingerprinting” (1930-31000-008-10G) and a reimbursable agreement between ARS and CSREES-NRI, entitled “Production of an integrated physical and genetic map for rainbow trout” (1930-31000-008-09R). Over 100,000 bacterial artificial chromosome clones (5.5X genome coverage) were evaluated by DNA fingerprinting to initiate construction of a rainbow trout physical map to be integrated with the current genetic map. The ADODR is in frequent contact with the cooperator through phone calls and email in addition to receipt of written reports. This serves to document research conducted under a reimbursable agreement between ARS and Troutlodge, Inc. entitled, “Genetic and diet effects on growth rate and reproduction in the rainbow trout strains of Troutlodge, Inc.” (1930-31000-008-08R). Ten thousand fish from 95 full-sib families were each grown on a traditional fish meal diet or a plant protein/oil based diet. A significant genetic x diet effect was detected which accounted for 5% of the random variation, the genetic correlation for growth was 73%. The genetic variation we identified can be explored to identify and select for genes involved in improved utilization of plant based diets. The ADODR is in frequent contact with the cooperator through phone calls and email in addition to receipt of written reports. 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, “Transcriptome Analyses in Salmonids" (1930-31000-008-06N). This agreement has the goal of identifying representatives for all salmonid genes by coordinating expressed sequence tag projects for Atlantic salmon and rainbow trout. A set of cDNA clones from Atlantic salmon and rainbow trout representing unique transcripts were identified for construction of a new high-density microarray representing over 28K transcripts for use in characterizing gene expression in salmonid species. The ADODR is in frequent contact with the cooperator through email and phone calls. 4.Accomplishments Complex segregation analysis of stress response. Experimental strategies to identify genetic variation in regions of chromosomes affecting traits of economic importance are enhanced by specific knowledge of the mode of inheritance of the trait. There is a great deal of diversity in the way that fish respond to stress as measured by the changes in the level of cortisol in their bloodstream. Complex segregation analysis was performed on National Center for Cool and Cold Water Aquaculture broodstock to predict the mode of inheritance of cortisol response to crowding stress so that future breeding studies can be efficiently designed. We determined that this trait is largely affected by genetics, is inherited in a co-dominant fashion, and is significantly affected by at least two major genes. This information will facilitate genetic improvement of this trait through selective breeding and the identification of genes affecting stress tolerance. National Program 106, Genetic Improvement, b. Selective breeding for economically important traits. Response of high growth fish to growth hormone. Although numerous studies have established that exogenous growth hormone (GH) treatment stimulates growth in fish; the effects on target tissue gene expression have not been well characterized. We assessed the effects of a short term GH treatment on muscle and liver gene expression in families characterized as having high and low growth rates. A total of 113 and 67 genes were differentially expressed in muscle and liver, respectively. The majority of these genes have not been previously characterized; a significant number of well-annotated genes included those associated with immune and metabolic functions. Our results indicate that short-term GH treatment activates the immune system, shifts the metabolic sectors and modulates growth regulating genes, providing insights into the physiological processes affected by GH. These genes constitute a wealth of new candidates for characterizing growth in rainbow trout at the molecular level. National Program 106, Genetic Improvement, a-Conserve, characterize, and utilize genetic resources. Identification of associations between major histocompatibility (MH) genes and resistance to Flavobacterium psychrophilum in rainbow trout. The gram-negative bacteria Flavobacterium psychrophilum (Fp) poses major fish health concerns in trout and salmon hatcheries in the US and around the world. Selective breeding of disease resistant lines is a promising solution in the absence of approved, cost-effective vaccination methods. The National Center for Cool and Cold Water Aquaculture has an ongoing breeding program for Fp resistance in its odd year broodstock. Genes encoded by the major histocompatibility complex (MHC) play an important role in immune response to a variety of pathogens. Genetic variation of markers linked to MHC genes in the NCCCWA 2005 brood-year was characterized and associations were detected between Fp resistance and the trout MHC-IB and MHC class II genomic regions. These associations will enable monitoring of the impact of the selection program on MHC variation and facilitate assessment of its impact on resistance to other economically important pathogens. National Program 106, Genetic Improvement, b. Selective breeding for economically important traits. 5.Significant Activities that Support Special Target Populations None 6.Technology Transfer Number of new CRADAs and MTAs 5 Number of active CRADAs and MTAs 20 Number of non-peer reviewed presentations and proceedings 24 Review Publications Palti, Y., Rodriguez, F., Gahr, S.A., Hansen, J.D. 2007. Evolutionary history of the abcb2 genomic region in teleosts. Developmental and Comparative Immunology 31(5):483-498. Rexroad III, C.E., Vallejo, R.L., Coulibaly, I., Couch, C., Garber, A., Westerman, M., Sullivan, C. 2006. Identification and characterization of microsatellites for striped bass from repeat-enriched libraries. Conservation Genetics 7(6) 971-982. Phillips, R., Nichols, K., Dekoning, J., Morasch, M., Keatley, K., Rexroad III, C.E., Gahr, S.A., Danzmann, R., Drew, R., Thorgaard, G. 2006. Assignment of rainbow trout linkage groups to specific chromosomes. Genetics 174(3):1661-1670. Coulibaly, I., Danzmann, R., Palti, Y., Vallejo, R.L., Gahr, S.A., Yao, J., Rexroad III, C.E. 2006. Mapping of genes in a region associated with upper temperature tolerance in rainbow trout. Animal Genetics 37(6):598-599. Renshaw, M.A., Patton, J.C., Rexroad III, C.E., Gold, J.R. 2006. Pcr primers for trinucleotide and tetranucleotide microsatellites in greater amberjack, seriola dumerili. Molecular Ecology Notes 6: 1162-1164. Salem, M., Kenney, B., Rexroad III, C.E., Yao, J. 2006. Microarray gene expression analysis in atrophying rainbow trout muscle: an unique non-mammalian muscle degradation model. Physiological Genomics 28:33-45. Nagler, J., Cavileer, T., Sullivan, J., Cyr, D., Rexroad III, C.E. 2007. The complete nuclear estrogen receptor family in the rainbow trout: discovery of the novel er.2 and both er. isoforms. Gene Vol 392(1-2)164-173. Palti, Y., Rodriguez, F., Vallejo, R.L., Rexroad III, C.E. 2006. Mapping of toll like receptor (TLR) genes in rainbow trout. Animal Genetics 37(6):597-598. Renshaw, M.A., Patton, J., Rexroad III, C.E., Gold, J. 2007. Pcr primers for dinucleotide microsatellites in greater amberjack, seriola dumerili. Conservation Genetics 2007, 8(4):1009-1011. Rexroad III, C.E. 2007. Radiation Hybrid Mapping in Aquatic Species. Aquaculture Genome Technologies Chapter 18. Johnson, N., Rexroad III, C.E., Eric, H., Vallejo, R.L., Palti, Y. 2007. Development and critical evaluation of a new multiplex system for parental allocation and broodstock management in rainbow trout. Aquaculture 266:53-62. Ramachandra, R.K., Lankford, S.E., Weber, G.M., Rexroad III, C.E., Yao, J. 2006. Identification of OORP-T, a novel oocyte-specific gene encoding a protein with a conserved oxysterol binding protein domain in rainbow trout. Molecular Reproduction and Development 74(4) 502-11. Garikipati, D.K., Gahr, S.A., Roalson, E.H., Rodgers, B.D. 2007. Characterization of rainbow trout myostatin-2 genes (rtMSTN-2a & -2b): genomic organization, differential expression and pseudogenization. Endocrinology 148:2106-2115. Garikipati, D.K., Gahr, S.A., Rodgers, B.D. 2006. Identification, characterization, and quantitative expression analysis of rainbow trout myostatin-1a and myostatin-1b genes. Journal of Endocrinology. 190:879-888.