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United States Department of Agriculture

Agricultural Research Service

2009 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 U.S. 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 U.S. rainbow trout aquaculture industry has leveled off. In order to compete, U.S. 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 information which will allow for increased understanding of the biological mechanisms controlling these disease resistance, growth, stress, tolerance, and embryonic development and exploitation of positive genetic variation on the NCCCWA selective breeding program.

3.Progress Report
In collaboration with scientists at the USDA/ARS MARC, we are developing enhanced protocols for analyzing large volumes of rainbow trout DNA sequence data using clonal fish lines. Current analysis protocols do not account for the recent trout genome duplication, which causes incorrect assembly of DNA sequences. Clonal fish do not contain genetic variation. Therefore, sequence variation in the clonal lines is caused by gene duplication rather then allelic variation. To this end, we initiated sequencing of the Swanson clonal line. Fish response to stress is an important factor in aquaculture production, having impacts on growth, feed efficiency, immune response, and reproductive characteristics. In collaboration with the Genetics and Physiology Project, we determined that there is a major gene segregating in the NCCCWA broodstock which impacts response to handling stress as measured by cortisol concentration in the blood. Efforts to identify the gene responsible for this effect include cortisol measurements for three generations of a broodstock pedigree combined with genetic marker analyses. Spleen size index was previously shown to be correlated with resistance to Flavobacterium psychrophilum, a major concern for trout aquaculture. In collaboration with the Fish Health Project, we determined that there is a major gene segregating in the NCCCWA broodstock which impacts spleen size. To identify genes affecting this trait we measured spleen size in two generations of a broodstock pedigree and genetic marker analysis is underway. Developing species-specific molecular tools such as chromosome maps is critical for identifying genes affecting production traits. In an effort to integrate the rainbow trout genetic and physical maps, the NCCCWA mapping reference families were genotyped with approximately 200 new genetic markers to integrate existing maps and facilitate gene identification and characterization. The development of robust lab protocols for bulk extraction of DNA samples is critical for using DNA marker technologies for pedigree monitoring in commercial performance trials. We are evaluating the capabilities of several commercially available kits for bulk DNA extraction of trout DNA. Bacterial cold water disease caused by Flavobacterium psychrophilum is a major concern for trout aquaculture. In collaboration with the Fish Health Project, we determined that there is a major gene segregating in the NCCCWA broodstock which impacts resistance to F. psychrophilum as measured by 21-day survival post exposure to the pathogen. To identify genes affecting this trait we measured post exposure survival in three generations of a broodstock pedigree and identified informative families for conducting genetic marker analyses. A relational database integrating performance and molecular information will facilitate the incorporation of data from diverse scientific disciplines and will contribute to improved methods for selective breeding in rainbow trout. We have identified and obtained hardware and software, generated a relational model for the broodstock data and loaded the records of 1,825 families and 18,683 tagged fish into the database system.

1. Genetic Analysis of the Rainbow Trout Genome Reveals Need for additional Genetic Markers: The National Center for Cool and Cold Water Aquaculture (NCCCWA) has established a breeding program for rainbow trout. The use of molecular genetic technologies in this program is expected to enhance capabilities for the selective breeding of important aquaculture production traits. To this end we have worked within international collaborations to develop genomic tools and technologies for rainbow trout while concurrently initiating and characterizing our broodstock population with respect to genetic and phenotypic variation relevant to aquaculture production. In FY 2009, we completed genetic analysis of four chromosomes in 96 individuals from the NCCCWA rainbow trout broodstock population using 49 genetic markers. Our results indicate that many more molecular markers than currently exist are required to effectively implement the use of molecular genetics technologies in rainbow trout aquaculture breeding programs. Therefore, the NCCCWA must continue to develop robust markers system such as single nucleotide polymorphisms (SNPs) for use in identifying genes that affect important aquaculture production traits.

2. The Development of a Robust Genetic Markers System for Genetic Analyses in Rainbow Trout: Molecular genetic technologies and the use of marker assisted selection strategies have the potential of increasing the rate of genetic gain over traditional selective breeding schemes. The application and implementation of molecular technologies requires a large suite of polymorphic genetic markers to enhance capabilities for genetic analyses in rainbow trout. Single Nucleotide Polymorphisms (SNPs) are highly abundant markers which are evenly distributed throughout the trout chromosomes. In FY 2009, we employed a high throughput strategy to discover SNPs in rainbow trout. Over twenty thousand putative SNPs were identified and 384 were tested resulting in a 48% validation rate. Of those new markers, 167 were placed on the rainbow trout genetic map. Based on the validation results, we anticipate that at least 10,000 putative SNPs from the original data set will be useful for implementing molecular genetic technologies into rainbow trout selective breeding for improved production efficiency and sustainability.

3. Identification of Immune Response Genes in Rainbow Trout: The U.S. salmonid aquaculture industry suffers severe economic loss to diseases. Every year, viral and bacterial epidemics in farmed Atlantic salmon and rainbow trout have resulted in production losses accounting for millions of dollars of lost revenue. The development of genetic markers for immune response genes is important for improving natural disease resistance in aquaculture fish populations. Toll-like receptors (TLRs) are a family of transmembrane proteins that recognize conserved pathogen structures to induce immune responses in human and in other vertebrates. In FY 2009, we completed the identification, annotation and genetic mapping of nine TLR genes in rainbow trout. Detailed mapping and annotation of TLR genes in rainbow trout and the development of genetic markers for the different TLR genes provide useful tools for genetic improvement of disease resistance in rainbow trout and other salmonids.

6.Technology Transfer

Number of the New/Active MTAs (providing only)2

Review Publications
Renshaw, M., Douglas, K., Rexroad Iii, C.E., Churney, A., Gold, J. 2009. Isolation and characterization of microsatellite markers in the Serra Spanish mackerel, Scomberomorus brasiliensis. Molecular Ecology Resources. 9:830-832.

Rexroad Iii, C.E., Palti, Y., Gahr, S.A., Vallejo, R.L. 2008. A second generation genetic map for rainbow trout (Oncorhynchus mykiss). BioMed Central (BMC) Genetics. 9:74.

Koop, B., Von Schalburg, K., Leong, J., Walker, N., Lieph, R., Cooper, G., Robb, A., Beetz-Sargent, M., Holt, R.A., Moore, R., Brahmbhatt, S., Rosner, J., Mcgowan, C., Rexroad Iii, C.E., Davidson, W. 2008. A salmonid EST genomic study: genes, duplications, phylogeny and microarrays. Biomed Central (BMC) Genomics. 9:545.

Palti, Y. 2009. Aquaculture Genomics. In: Overturf, K. editor. Current Status of Molecular Techniques in Aquaculture. First Edition. Ames, IA: Wiley, John & Sons, Incorporated. 408 p.

Karlsson, S., Renshaw, M., Rexroad III, C.E., Gold, J. 2008. Microsatellite primers for red drum (Sciaenops ocellatus). Fishery Bulletin. 106:476-82.

Salem, M., Kenney, B., Rexroad III, C.E., Yao, J. 2008. Development of a 37K high-density oligo-nucleotide microarray for rainbow trout. Journal of Fish Biology. 72:(9)2187-2206.

Hoghadam, H., Ferguson, M.M., Rexroad III, C.E., Coulibaly, I., Danzmann, R.G. 2007. Genomic Organization of the igf1, igf2, myf5, myf6 and grf/pacap Genes across Salmoninae Genera. Animal Genetics. 38(5):527-532.

Coulibaly, I., Gahr, S.A., Yao, J., Rexroad III, C.E. 2006. Embryonic expression of uncoupling protein 2 genes in rainbow trout. Fish Physiology and Biochemistry Journal. 32:249-253.

Couch, C., Garber, A., Rexroad III, C.E., Abrams, J., Stannard, J., Westerman, M., Sullivan, C. 2006. Isolation and characterization of 149 novel microsatellite dna markers for striped bass, morone saxatilis, and cross-species amplification in white bass, m. chrysops, and their hybrid. Molecular Ecology Notes. 6(3):667-669.

Salem, S., Rexroad Iii, C.E., Yao, J. 2006. Identification of a novel gill specific calpain from the rainbow trout (oncorhynchus mykiss). Fish Physiology and Biochemistry Journal. 32:1-6.

Somer, L., Danin-Poleg, Y., Diamant, E., Palti, Y., Kashi, Y. 2005. Amplified intergenic locus polymorphism as a basis for bacterial typing of listeria ssp. and escherichia coli. Applied and Environmental Microbiology. 71(6):3144-3152.

Last Modified: 11/28/2014
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