Progress 10/01/03 to 09/30/04
Outputs
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 (genetic maps, expressed sequence tag information, etc.) will be made available to the international research community and improved germplasm will be made available to the US rainbow trout industry. CRIS project 1930-31000-006-00D was initiated in FY2004 and the project plan is currently under review. 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 curate and analyze 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. As the milestones from the project plan are under review, the current objectives of the CRIS are: 1) Construction of a microsatellite genetic map for use in the identification of genes affecting production traits 2) Development of resources for functional genome research in rainbow trout 3) Identification and characterization of genes affecting production traits (candidate genes) through comparative genomics. 4) Utilization of molecular genetic data in the NCCCWA broodstock program. 3. Milestones: A. List the milestones that were scheduled to be addressed in FY 2004. How many milestones did you fully or substantially meet
in FY 2004 and indicate which ones were not fully or substantially met, briefly explain why not, and your plans to do so. 1. Construction of a microsatellite genetic map. The construction of a microsatellite genetic map will allow for the association of chromosome regions with production traits such as disease resistance, stress tolerance, and growth characteristics by tracking traits and genetic markers through generations. Map construction has been conducted in two stages: 1) marker development; and 2) genotyping of a two generation set of reference families. In FY2004 we terminated the development of new random markers and focused on markers associated with genes or chromosomal regions of interest. The current total of random microsatellite markers available for use in rainbow trout is 940. Development of non-random markers will continue indefinitely, the current total number of available microsatellite markers for genotyping to over 1200. We have begun genotyping these marker
sets on the NCCCWA reference families, to date 250 markers have been completed. 2. Functional genome research. Functional genome research allows for high-throughput studies of gene expression. NCCCWA has supported functional genome research in rainbow trout by sequencing expressed sequence tags (ESTs). This involves partial sequencing of mRNA sequences to identify genes expressed in specific tissues, specific times in development, or as a result of a specific treatment. FY2003 saw the completion of Phase I of a rainbow trout EST in which NCCCWA collaborated with The Institute for Genome Research (TIGR) to develop the rainbow trout gene index (RTGI). In FY2004, these efforts continued as phase II of the project was initiated by sequencing ESTs from specific tissues including testis, pituitary, leukocytes from Yersinia ruckeri or vaccinated fish, whole juvenile, and a subtraction of previous NCCCWA and INRA libraries to produce a new library. To date 17,000 new ESTs have been
identified. 3. Candidate gene identification and characterization. In addition to supporting functional genome research, the EST projects and the NCCCWA 10X bacterial artificial chromosome library allow for the identification and characterization of genes of interest based on functional annotation from other species. In FY2004 we focused on characterizing the following gene families; 1) the inhibitors of DNA binding/differentiation (Id) proteins (4 newly identified) which are important to growth and development); and 2) the Toll-Like Receptors (>3 newly identified) which are important in disease resistance. We also continued efforts to sequence the MHC class II region. 4. Utilization of molecular genetic data in the NCCCWA broodstock program. In FY2004 we genotyped 9 microsatellite loci for all incoming broodstock for the 2003 year class and quantified the amount of genetic variation contained both within and between populations. B. List the milestones that you expect to address
over the next 3 years (FY 2005, 2006, & 2007). What do you expect to accomplish, year by year, over the next 3 years under each milestone? 1. Construction of a microsatellite genetic map for use in the identification of genes affecting production traits. We anticipate genotyping all publicly available reference families will continue through FY2005 with linkage analyses being conducted in FY2006. Additional mapping will continue indefinitely and involve mapping specific genes or chromosomal regions of interest. A set of markers for conducting genome scans for quantitative trait loci analyses is expected to be identified in FY2006. 2. Development of resources for functional genome research in rainbow trout. In FY2005 we will complete sequencing of expressed sequence tags, submit the data to GenBank, and cooperate with The Institute for Genome Research to update the Rainbow Trout Gene Index. This data will be used to construct high-density microarrays in FY2006 and FY2007 in
coordination with West Virginia University. 3. Identification and characterization of genes affecting production traits (candidate genes) through comparative genomics. Genomic characterization of the Inhibitor of Differentiation/DNA binding proteins and the Toll-Like Receptors is expected to be completed in FY2005, functional genomic studies will continue through FY2006. Characterization of the MHC Class II region will continue through FY 2007. 4. Utilization of molecular genetic data in the NCCCWA broodstock program. In FY2005 we anticipate developing a multiplex set of microsatellite markers in FY2005 for use in genotyping broodstock and other populations of interest. Broodstock from each year class will be genotyped (FY2005- FY2009) to evaluate the effects of selection on inbreeding and population structure. 4. What were the most significant accomplishments this past year? A. Single most significant accomplishment during FY 2004: Genome research requires the development of
species-specific molecular genetic resources for organisms of interest. Unfortunately, these resources are very expensive to construct and maintain, thus they are not available for every species. NCCCWA characterized a rainbow trout bacterial artificial chromosome library for use in genetic and physical mapping and sequencing DNA segments of interest. The library, made up of 135,000 base pair DNA segments retained in bacterial cells, has the equivalent of 10 times the amount of DNA of the rainbow trout genome. This means that for any DNA segment of interest, there is a 95% probability of identifying at least one clone retaining that segment. On average, we observed that each DNA segment is retained 10 times as expected. As rainbow trout have undergone and evolutionarily recent genome duplication, some DNA segments were retained 20 times. Further DNA fingerprinting analyses revealed that these were actually two sets of clones retaining recently diverged duplicates of a single
gene. Mapping these copies has revealed homeologous segments of the genome which would be expected to harbor similar genes. These results have had an impact on strategies for constructing physical maps and the eventual sequencing of the rainbow trout genome. The NCCCWA library is publicly available to the research community. B. Other significant accomplishments: A study was conducted to test for genetic and diet effects on growth in a rainbow trout brood strain of Trout Lodge, Inc. Fish from 20 half-sib families (20 dams crossed to 10 sires) were pooled together prior to hatching and divided into two groups. One group was fed fishmeal diet and the other was raised on a plant based diet. At the end of the growth trial the fastest and slowest growing fish were genotyped with microsatellite markers for parentage identification. The family assignment demonstrated strong genetic effect on growth, and also indicated interaction between diet and family (i.e. some families that
performed well when fed fishmeal diet did not perform well on plant diet and vice versa). C. Significant activities that support special target populations. None. D. Progress Report: 1. Construction of a microsatellite genetic map for use in the identification of genes affecting production traits. In FY2004 NCCCWA genotyped 250 microsatellite markers on reference families towards the goal of a 1000 marker genetic map. Map construction was initiated by genotyping all publicly available randomly generated markers including those from NCCCWA and the WVU collaboration and markers associated with candidate genes. Many NCCCWA markers were also genotyped on other reference families, contributing to a comparative map between salmonid fishes and an ongoing effort to integrate rainbow trout cytogenetic and genetic maps. Project number 1930-31000-006-02S is a cooperative agreement between ARS and West Virginia University entitled 'Development of Genetic Markers for Rainbow Trout'. In
FY2004 we developed 249 new random microsatellite markers from microsatellite repeat enriched libraries and 179 from expressed sequence tag data. These markers were developed in cooperation with NCCCWA and will be included in the genetic map. Project number 1930-31000-006-04S is a specific cooperative agreement between ARS and the University of New Hampshire entitled 'Extraction of Comparative Mapping Information from the NCCCWA BAC Library'. The University is actively distributing copies and high-density filters available to the international research community. 2. Development of resources for functional genome research in rainbow trout. NCCCWA conducted Phase II of an Expressed Sequence Tag project in collaboration with West Virginia University, INRA, and GRASP. Clones from cDNA libraries were sequenced from both the 3' and 5' ends. In all, 77,000 sequences were obtained from 7 libraries and are in a bioinformatic pipeline for analyses and submission to public databases. 20,000
sequences remain to be completed including transcripts from embryonic development stages and vaccinated/challenged leukocytes. Project number 1930-31000-006-02S is a cooperative agreement between ARS and West Virginia University entitled 'Development of Genetic Markers for Rainbow Trout'. A high-quality cDNA library from rainbow trout oocytes has been successfully constructed and normalized. A total of 20,160 EST sequences from the normalized library have been generated. These sequences provide a global picture of gene expression in trout oocytes and are important for the identification of key genes essential for oocyte development, maturation, and early embryogenesis. 3. Identification and characterization of genes affecting production traits (candidate genes) through comparative genomics. Genome research aids in the selection of 'candidate genes' to be targeted for study with respect to traits of interest. NCCCWA made significant progress characterizing a number of candidate
genes in FY2004. a. Myogenic Regulatory Factors: We have attempted to identify all the myogenic regulatory genes by in silico analyses of the rainbow trout gene index. This investigation has lead to the identification of 64 transcripts with significant homologies to myogenic regulatory factors from other species. We are currently sequencing the transcripts for gene identification and characterization. b. TGF-ss Superfamily: To gain a more full understanding of the role of the myostatin genes in the regulation of trout muscle development, a search of the rainbow trout gene index for sequences with significant homologies to myostatin and related genes revealed 83 transcripts that will be used in the genomic and functional characterization of the myostatin regulatory pathway. Two of the clones identical to the two trout myostatin genes have been used to screen the NCCCWA BAC library. We have been able to identify several clones containing homologous sequence to each of the rainbow
trout myostatin genes. c. Inhibitors of DNA binding/differentiation (ID genes): In silico analysis of the rainbow trout gene index revealed the presence of a number of cDNA clones with significant homologies to the ID genes. Further analysis identified six unique transcripts. We have finished the full length sequence of a representative from each group and used these clones to screen the BAC library for genomic characterization. To date, genomic sequence has been obtained for four of the six ID genes and is in progress for the other two. For both ID1 and ID2, we have identified two genes (Classified as ID1A and ID1D and ID2A and ID2B, respectively) with greater than 90% homology, suggesting they resulted from the duplication. An additional pair of the genes (ID1B and ID1C) has been identified with 90% homology to the ID1 genes. Genetic and physical mapping of the ID1 homologue genes showed that at least three of the four genes occupy unique loci in the rainbow trout genome. d.
Toll-like Receptors (TLG genes): Using in silico analysis we identified six rainbow trout EST sequences with high homology to TLR genes from other species, none of which showed similarity to the published rainbow trout TLR gene sequence. The cDNA library inserts were sequenced and used as probes to identify rainbow trout BAC clones containing homology to TLR genes. Banding patterns (DNA fingerprinting) were generated by restriction digestion of the BACs positives for the putative TLR genes and the results suggest that at least two of the six genes are duplicated in the rainbow trout genome. We have completed three of the six putative TLR gene sequences, one of which shows higher similarity to the zebrafish TLR5, and the other two to TLR3 of different species. Reverse transcriptase PCR was used to determine tissue expression profile in 22 different tissues of adult rainbow trout for one of the putative TLR genes and will be used to characterize the remaining five genes. These
data in combination with functional analysis of the TLR genes will provide a better understanding of host-pathogen interactions and the evolution of innate immunity. e. Major Histocompatibility Complex: Project number 1930-31000-006-01R is a cooperative agreement between ARS and the University of Maryland entitled 'Mapping and Sequencing of the Rainbow Trout Major Histocompatibility Regions I and II'. We have assembled three sets of overlapping BAC clones that span the two class I regions and the region where TAP1 resides. We have completely sequenced and annotated the core clone for each of the 3 regions (approximately 4 million base pairs of raw sequence). f. Calpains: Project number 1930-31000-006-02S is a cooperative agreement between ARS and West Virginia University entitled 'Development of Genetic Markers for Rainbow Trout'. Calpain proteins from rainbow trout muscle have been isolated and characterized. The roles of these proteins in texture development have been
determined. The cDNAs for calpain regulatory subunit, the u calpain and the m calpain have been successfully cloned and characterized. The expression of these genes in muscle during starvation has been determined. 4. Utilization of molecular genetic data in the NCCCWA broodstock program. NCCCWA continued to genotyped parental broodstock in FY2004 with nine microsatellite markers. Analyses revealed estimates of genetic diversity and population structure. Project number 1930-31000-006-03R entitled 'Molecular Markers for Genome Mapping and Selective Breeding of Striped Bass' is a cooperative agreement between ARS and Kent Sea Tech Corporation in collaboration with North Carolina State University. In FY2004 177 microsatellite markers were developed for striped bass and sent to North Carolina State University for genotyping analyses on broodstock that will determine the amount of genetic variation and population structure. In addition, four new microsatellite repeat enriched libraries
were obtained for additional marker development. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Development of resources for functional genome research in rainbow trout. A rainbow trout EST library developed by scientists at the National Cool and Cold Water Aquaculture Research Center contains 45,000 sequences and defines the functional identity of these sequences essential for using this information to assist in producing genetically improved strains. Completion of such an undertaking requires the input of large numbers of researchers. A Rainbow Trout Gene Index was established in collaboration with The Institute for Genome Research and was initially made available to the public in April 2003; an update of the original submission was conducted in June 2003. This provides a very large quantity of molecular DNA information to the scientific community and combines gene sequence data with that from many international
projects. This collaborative information will greatly enhance the functional definition of the sequences and speed-up progress on rainbow trout functional genomics. This accomplishment primarily falls within the scope of the Genetic Improvement component (#3) of the Aquaculture National Program 106 Action Plan. Section c (Genomic resources, goals 1-3) applies as functional genomics was the focus of this project. Section e (Bioinformatics, goals 1 and 2) applies as a relational database was developed to curate and analyze data. 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? Clones from the NCCCWA BAC library and EST libraries have been distributed to researchers all over the globe. Two recipients of these clones have constructed microarrays (Dr.
Paul Coussens, Michigan State University and Dr. Aleksei Krasnov, University of Kuopio, Finland) for use in functional genome research. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. Publications in the Popular Press: Rexroad, C.E. Salmonid Genomics: Applications in Salmonid Aquaculture. April 2004 Global Aquaculture Advocate, p.46-47. Presentations to Organizations: October 2004, presentation at NCCCWA to US Trout Farmers Association on genome research in rainbow trout. April 23, 2004 presentation at NCCCWA to advanced science and FFA students from Philip Barbour High School, WV. May 9, 2004 presentation at NCCCWA to participants of the summer 2004 West Virginia Farm Bureau 'Agriculture Awareness Tour'. Presentation Abstracts: Coulibaly, I., Rexroad, C.E. 2004. Identification Of Anonymous Microsatellite Markers For Mapping In Rainbow Trout. Plant & Animal Genome XII, the international conference
on the status of plant and animal genome research, San Diego, CA. Gahr, S.A., Palti Y., Rexroad C.E. 2004. Identification and characterization of two Inhibitor of Differentiation (Id) genes in Rainbow Trout (Oncorhynchus mykiss). Plant & Animal Genome XII, the international conference on the status of plant and animal genome research, San Diego, CA. Hansen J.D., Zimmerman, A., Palti, Y., Landis, E., Noakes, M., Nichols, K, Phillips, R. 2003. Physical and genetic mapping of the trout MHC: the application of MHC functional genomics during acute viral infection. 9th ISDCI congress, St. Andrews, Scotland. Higgins, A.D., Silverstein, J.T., Wilson, M.E., Rexroad, C.E., Blemings, K.P. 2004. The effect of genetics and starvation on lysine catabolism in rainbow trout (Oncorhynchus mykiss) liver. FASEB J. 18(4):A539. Landis, E., Palti, Y., Lohr, J., Eiben, L. Phillips, Hansen, J.D. 2004. Genomic sequencing of the rainbow trout class IA and IB regions: evidence for the duplication of the PSMB
and ABCB3 genes. Plant & Animal Genome XII, the international conference on the status of plant and animal genome research, San Diego, CA. p.251. Palti, Y., Phillips, R., Zimmerman, A., Noakes, M., Rodriguez, F., Nichols, K., Thorgaard, G., Hansen, J.D. 2003. Characterization and mapping of MHC haplotypes in rainbow trout (Oncorhynchus mykiss). American Fisheries Society, 133rd annual meeting, August 10-14, 2003, Quebec City, Quebec, Canada. Phillips, R., Zimmerman, A., Noakes, M., Palti, Y., Morash, M., Eiben, L., Ristow, S.S., Thorgaard, G.H., Hansen, J.D. 2003. Physical and genetic mapping of the rainbow trout major histocompatibility regions: evidence for duplication of the class I region. ARS Immunology Research Workshop, December 1-4, 2003, Bethesda, MD. Rexroad, C.E. 2003. Development of Genetic Markers for Striped Bass and Rainbow Trout. Genetics in Aquaculture VIII, November 9-15, Puerto Varas, Chile. Rexroad, C.E. Applying Genomics To Salmonid Aquaculture. Aquaculture
2004, Honolulu, HI, March 1-5, 2004. Rodriguez,F., Hansen, J.D., Wiens, G., Welch, T., Palti, Y. 2003. Characterization and mapping of immune response genes in rainbow trout (Oncorhynchus mykiss). Genetics in Aquaculture 8, Meeting of the International Association for Genetics in Aquaculture, 9-15 November, 2003, Puerto Varas, Chile. Rodriguez, F., Wiens,G., Palti, Y. 2004. Characterization of toll like receptor genes in rainbow trout (Oncorhynchus mykiss). 12th International Congress of Immunology: Montreal, Canada. July 18-23, 2004. Salem, M., Nath, J., Rexroad, C.E., Killefer, J., and Yao, J. 2004. Identification and molecular characterization of the rainbow trout calpains (Capn1 and Capn2): Their expression in muscle wasting during starvation. ASBMB annual meeting and IUBMB 8th conference. The FASEB J. June 12-16, 2004. 18(8). Abstract No. 158. Stewart, A.B., Dailey, R.A.,Rexroad, C.E. 2004. Identification and use of polymorphic markers in rainbow trout. Aquaculture 2004,
Honolulu, HI, March 1-5, 2004. Stewart, A.B., Soonhag, K., Killefer, J., Rexroad, C.E. 2004. Expressed sequence tags in rainbow trout intestine, liver, and kidney (Oncorhynchus mykiss). Plant & Animal Genomes XII Conference, San Diego, CA, January 10- 14, 2004. Waldbieser, G., Karsi, A., Gahr, S., Rexroad, C.E. 2003. Rapid development of gene-tagged microsatellite markers from BAC clones of channel catfish, Ictalurus punctatus, and rainbow trout, Oncorhynchus mykiss. Genetics in Aquaculture VIII, November 9-15, 2003. Puerto Varas, Chile. Wiens, G., Hovatter, K., Gahr, S., Morrison, C., Palti, Y., Rexroad, C., Rodriquez F., Welch, T. 2004. Genomic Characterization and Expression Analysis of a Tumor Necrosis Factor Superfamily 13b (TNFSF 13b) Homologue from Rainbow Trout (Oncorhynchus mykiss). 12th International Congress of Immunology: Montreal, Canada. July 18-23, 2004. Palti, Y., Gahr, S., Hansen, J., Rexroad, C. 2004. Physical mapping of loci of interest in rainbow trout: Evidence
for genome-wide duplication. Plant & Animal Genome XII, the international conference on the status of plant and animal genome research, San Diego, CA. p.251.
Impacts
(N/A)
Publications
|