Progress 01/01/07 to 12/31/09
Outputs
OUTPUTS: 1) All the clones from the Swanson YY doubled haploid male HindIII BAC library (10X coverage; 184,704 clones) were fingerprinted and edited using FPMiner software. Approximately 16% of the clones' fingerprints did not pass our editing criteria and were removed from the project. The remaining clones were assembled into physical contigs using the finger-printing contig (FPC) program with a tolerance of 5, an initial cutoff of 1E-70 and extensive manual editing. The current version of the map is composed of 154,439 clones of which 145,060 are assembled into 4,173 contigs and 9,379 are singletons. The total number of unique fingerprinting fragments (consensus bands) in contigs is 1,185,157, which corresponds to an estimated physical length of 2.0 Gb (75% - 80% of the rainbow trout genome). The assembly was validated by 1) comparing it to the agarose gel fingerprinting contigs of Palti et al. (2004; Animal Genetics, 35:130-133), and 2) anchoring large contigs to the microsatellites genetic linkage map. The BAC contigs assembly can be viewed online via WebFPC: http://www.genome.clemson.edu/activities/projects/rainbowTrout. The physical map is described in a manuscript that was published in BMC Genomics (see below). 2) BAC end sequencing (BES) of 100,000 clones from the same library was completed by Genoscope and INRA collaborators in France. We screened BES data from 184 fingerprinting contigs for microsatellites and selected 207 microsatellites for PCR optimization and genotyping. Approximately 65% of the markers were informative for genetic mapping and were added to the NCCCWA genetic map. A manuscript describing the integrated map is in preparation. 3) Two 5X BAC libraries (110,592 clones each) from the same Swanson clonal line were prepared using BamHI and EcoRI partial genomic digestion to complement the current 10X HindIII library. DNA fingerprinting and BES of 5,000 clones from each library was conducted. The fingerprinted BACs were added to the physical map and approximately 60 ne microsatellite markers from the new BES were genotyped for mapping in the NCCCWA genetic mapping panel. The new libraries appear to be very useful for closing gaps in the physical map and for integration with the genetic map. A manuscript describing the evaluation of the two new libraries and preliminary assembly of a 2nd generation integrated map of the rainbow trout genome is in preparation. 4) The 10X BAC library super-pools were screened by PCR using 137 markers that represent 25 of the 29 chromosomes in the NCCCWA genetic map. Those markers were mapped at LOD 4. The results of the library screening were analyzed together with similar screening that was done at INRA to anchor BAC contigs from the physical map to linkage groups of the genetic map and were combined with the BES microsatellites to produce a first generation integrated physical and genetic map (manuscript in preparation). PARTICIPANTS: Collaborations: List new interactions made possible from project support: This CSREES-NRI project support encouraged the French government to support the BAC end sequencing project of INRA and Genoscope and an ongoing effort for de-novo sequencing of the rainbow trout genome using next generation sequencing technologies. We currently work closely with INRA scientists and coordinate our efforts to produce an integrated physical and genetic map, which will provide a first tiling path for the rainbow trout genome sequence and a frame-work for future de-novo genome sequencing projects. Training: Please list the names of undergraduate, graduate, postdoctoral and technical personnel trained under this project. Please highlight their contributions to items in the "deliverables" section above. Contributed to BES microsatellites genotyping and BAC library super-pools PCR screening: Brian Smith, Research Technician, NCCCWA-ARS-USDA Kristy Shewbridge, Research Technician, NCCCWA-ARS-USDA M. Renee Fincham, Research Technician, NCCCWA-ARS-USDA Lindsey Pierce, Graduate Student, NCCCWA-ARS-USDA and Virginia Tech Contributed to the BAC fingerprinting effort at UC Davis: Frank M. You, Bioinofrmatician Yuqin Hu, Staff Research Associate Naxin Huo, Postdoctoral Research Associate Yu-Ting Chien, Junior Specialist Yaqin Ma, Postdoctoral Research Associate Undergraduate Students: Sean Marrow, Jennifer Pham, David Hua, Allen Liu, Frances Chen, Nicolas Tom TARGET AUDIENCES: Scientists and fish breeders who study rainbow trout and pacific salmon biology for aquaculture production, fisheries management, genome evolution, environmental ecology, comparative immunology, toxicology, physiology, human and fish nutrition and diet and many other basic research studies that use rainbow trout as a model research organism. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
1) The integrated map will enable positional cloning of previously mapped QTL in rainbow trout and will facilitate the identification of genes affecting economically important traits. 2) The new map will also facilitate comparative mapping with the sequenced genomes of model fish species which will enable rapid mapping of candidate genes for economically important traits. 3) This project prepares the trout genome to the post Atlantic salmon genome sequence era. Anchoring the BAC contigs to the salmon genome sequence will enable rapid filling of gaps to improve the map and provide a first tiling path for sequencing of the trout genome with more affordable next generation sequencing techniques and a frame-work for future de-novo genome sequencing projects. Community resources generated: BAC physical map for rainbow trout The BAC contigs assembly can be viewed online via WebFPC: http://www.genome.clemson.edu/activities/projects/rainbowTrout.
Publications
- Palti Y, Luo M-C, Hu Y, Genet C, Vallejo RL, Rexroad III CE (2009). A First Generation BAC Physical Map of the Rainbow Trout Genome. Plant & Animal Genome XVII, the international conference on the status of plant and animal genome research, January 10-14, 2009, San Diego, CA, USA.
- Genet C, Dehais P, Palti Y, Gavory F, Wincker P (2009). Generation of BAC-end sequences for rainbow trout genome analysis. Plant & Animal Genome XVII, the international conference on the status of plant and animal genome research, January 10-14, 2009, San Diego, CA, USA.
- Palti Y, Luo M-C, Hu Y, Genet C, You, FM, Vallejo RL, Thorgaard, GH, Wheeler, PA, Rexroad III, CE (2009). A First Generation BAC-Based Physical Map of the Rainbow Trout Genome. BMC Genomics 2009, 10:462.
- Palti, Y., Luo, M., Genet, C., Hu, Y., You, F., Vallejo, R.L., Rexroad III, C.E. (2010). A first generation integrated physical and genetic map of the rainbow trout genome. Plant & Animal Genome XVIII, the international conference on the status of plant and animal genome research, January 9-13, 2010, San Diego, CA, USA.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: 1) All the clones from the Swanson YY doubled haploid male HindIII BAC library (10X coverage; 184,704 clones) were fingerprinted and edited using FPMiner software. Approximately 16% of the clones' fingerprints did not pass our editing criteria and were removed from the project. The remaining clones were assembled into physical contigs using the finger-printing contig (FPC) program with a tolerance of 5, an initial cutoff of 1E-70 and extensive manual editing. The current version of the map is composed of 154,439 clones of which 145,060 are assembled into 4,173 contigs and 9,379 are singletons. The total number of unique fingerprinting fragments (consensus bands) in contigs is 1,185,157, which corresponds to an estimated physical length of 2.0 Gb (75% - 80% of the rainbow trout genome). The assembly was validated by 1) comparing it to the agarose gel fingerprinting contigs of Palti et al. (2004; Animal Genetics, 35:130-133), and 2) anchoring large contigs to the microsatellites genetic linkage map. The BAC contigs assembly can be viewed online via WebFPC: http://www.genome.clemson.edu/activities/projects/rainbowTrout/index. shtml. 2) BAC end sequencing (BES) of 100,000 clones from the same library was completed by Genoscope and INRA collaborators in France. We screened BES data from 184 fingerprinting contigs for microsatellites and selected 195 microsatellites for PCR optimization and genotyping. Approximately 80% of the markers were optimized and are being genotyped and added to the NCCCWA genetic map. 3) Two 5X BAC libraries (110,592 clones each) from the same Swanson clonal line were prepared using BamHI and EcoRI partial genomic digestion to complement the current 10X HindIII library. DNA fingerprinting and BES of 5,000 clones from each library are currently under way to evaluate the quality of the libraries and their utility for closing gaps in the physical map and for integration with the genetic map. 4) The 10X BAC library super-pools were screened by PCR using 137 markers that represent 25 of the 29 chromosomes in the NCCCWA genetic map. Those markers were mapped at LOD 4. The results of the library screening are currently being analyzed together with similar screening that was done at INRA to anchor BAC contigs from the physical map to linkage groups of the genetic map. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
1) The integrated map will enable positional cloning of previously mapped QTL in rainbow trout and will facilitate the identification of genes affecting economically important traits. 2) The new map will also facilitate comparative mapping with the sequenced genomes of model fish species which will enable rapid mapping of candidate genes for economically important traits. 3) This project prepares the trout genome to the post Atlantic salmon genome sequence era. Anchoring the BAC contigs to the salmon genome sequence (if and when available) will enable rapid filling of gaps to improve the map and provide a first tiling path for sequencing of the trout genome with more affordable new generation sequencing techniques.
Publications
- Genet C, Dehais P, Palti Y, Gavory F, Wincker P (2009). Generation of BAC-end sequences for rainbow trout genome analysis. Plant & Animal Genome XVII, the international conference on the status of plant and animal genome research, January 10-14, 2009, San Diego, CA, USA.
- Palti Y, Luo M-C, Hu Y, Genet C, Vallejo RL, Rexroad III CE (2009). A First Generation BAC Physical Map of the Rainbow Trout Genome. Plant & Animal Genome XVII, the international conference on the status of plant and animal genome research, January 10-14, 2009, San Diego, CA, USA.
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: 1)All the clones from the Swanson 10X genome coverage BAC library (184,704 clones) will be fingerprinted by the end of January 2008. As of December 3, 2007 we have fingerprinted 75% of the library (~135,000 clones). 2)BAC end sequencing of 100,000 clones from the same library was completed by Genoscope and INRA collaborators in France and data will be available for microsatellites design and genetic mapping in 2008. 3)Collaborative arrangements were made to construct additional BAC libraries from the same Swanson clonal line, which will complement the current 10X library. 4)The 10X BAC library super-pools were screened by PCR using 52 microsattelites that were previously mapped at high stringency (LOD 4). A total of 137 markers that represent 25 of the 29 chromosomes in the NCCCWA genetic map will be screened by project end date.
PARTICIPANTS: BAC DNA fingerprinting was conducted in the Lab of Dr. Ming-Cheng Luo (Co-PD) at the Dept. of Plant Sciences, University of California, Davis. Individuals that contributed to the fingerprinting effort at UC Davis and obtained professional training in molecular genetics techniques were: Yuqin Hu, Staff Research Associate, Yu-Ting Chien, Junior Specialist, Yaqin Ma, Postdoctoral Research Associate. Undergraduate Students: Sean Marrow, Jennifer Pham, David Hua, Allen Liu, Frances Chen, Nicolas Tom. Individuals that contributated to the BAC library super-pools PCR screening at the NCCCWA molecular genetics lab in Leetown, West Virginia were: Kristy Shewbridge, Research Technician, NCCCWA-ARS-USDA, and Lindsey Pierce, Graduate Student, NCCCWA-ARS-USDA and Virginia Polytechnic Institute and State University.
TARGET AUDIENCES: The trout and salmon research community and aquaculture genetics researchers are the immediate target audiences for this project. Rainbow trout breeders, growers, hatchery managers and fisheries biologists are the target audiences for downstream applications that are likely to be developed using the genomics research tools that are being produced by this project
PROJECT MODIFICATIONS: In the original project plan we proposed to start with BAC end-sequencing and DNA fingerprinting of the new CHORI-220 library (5X genome coverage), however, our initial quality controls detected serious quality issues with that library that have made it un-useful for high throughput DNA fingerprinting. As a result we decided to complete the DNA fingerprinting of the 10X HindIII BAC library and allocate funds from this grant for the constraction of two new 5X Sanson BAC libraries that will complement the current 10X library. BAC end sequencing and DNA fingerprinting of 5,000 clones from each library will be conducted upon completion of the new libraries' construction to evaluate their efficiency in closing gaps in the physical map. The project and budget amendments were reported to and approved by the National Prorgram Leader of the CSREES-NRI Animal Genomics Program.
Impacts
1)The integrated physical and genetic map will enable positional cloning of previously mapped QTL in rainbow trout and will facilitate the identification of genes affecting economically important traits. 2)The new map will also facilitate comparative mapping with the sequenced genomes of model fish species which will enable rapid mapping of candidate genes for economically important traits. 3)This project prepares the trout genome to the post Atlantic salmon genome sequence era. Anchoring the BAC contigs to the salmon genome sequence (if and when available) will enable rapid filling of gaps to improve the map and provide a first tiling path for sequencing of the trout genome. 4)This CSREES-NRI project support encouraged the French government to support the BAC end sequencing project of INRA and Genoscope and enhanced international collaboration to produce a physical map for the rainbow trout genome.
Publications
- No publications reported this period
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