Progress 06/01/03 to 05/31/06
Outputs
The physical mapping project involving BAC end sequencing and BAC fingerprinting was initiated a year ago. The main objective of this project was to demonstrate the technical feasibility of conducting fingerprinting and BAC end sequencing in our laboratory, and produce preliminary data for the application of extramural funding. We have demonstrated BAC DNA isolation, end sequencing, fingerprinting, and contig assembly using FPC. A grant application to USDA NRI was made and was successful this year. The catfish physical mapping project was founded this year through NRI Genome Tools and Resource Program. Therefore, we have successfully completed the project. In the next a few months, manuscripts will be prepared for publications.
Impacts
The seed funding of this project has allowed demonstration of technical feasibility and generation of preliminary data, which was crucial for obtaining extramural funding. The funding of the physical mapping project through USDA set the solid foundation for the construction of the catfish physical map that will be very important for catfish genome research and for genetic improvements of catfish broodstocks.
Publications
- No publications reported this period
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Progress 01/01/04 to 12/31/04
Outputs
Good progress has been made with the project. We have accomplished objective 1, and are in the middle of objective 2. This year we will complete objective 2. Since June 2003, we have obtained the BAC library of channel catfish CHORI 212 through USDA sponsored genomic reagents and tools program. The library was constructed using EcoR1 enzyme by Dr. Piter DeJong with 10.6 X genome coverage, or 72,000 clones with average insert size of 161 kb. We have conducted pilot studies for large-scale BAC DNA isolation using a 96-well VAC system (Eppendorf). Almost 10,000 BAC DNAs have been prepared. Fingerprinting and BAC ends sequencing experiments suggested that the BAC DNA was of good quality and sufficient quantity. The 96-well VAC procedure produced BAC DNA sufficient for both fluorescent fingerprinting and for sequencing. We have used several sets of overlapping clones as well as independent clones for the analysis of contigs using FPC software package. For the demonstration
of contig assembly, we obtained clones containing a specific gene using overgo hybridization. Analysis using FPC properly placed the related clones into contigs, while independent clones were singletons, as expected. This set of preliminary work demonstrated that our laboratories have gained all the necessary experience and proficiency for constructing catfish BAC contigs. We have conducted pilot experiments for large-scale overgo hybridization of genes to BAC filters. In the pilot studies, we used 100 overgo probes in one hybridization. From a filter with 10X genome coverage, over 1,100 positives were observed. Subsequent two-dimensional hybridization allowed identification of BACs containing the genes as represented by the overgo probes. This pilot study indicated that two-dimensional hybridization using overgo probes was both effective and efficient for large-scale assignment of genes to BACs.
Impacts
This is only the start of a very large project. However, the production of the preliminary data will allow us to be successful for grant application to obtain enough funds for completion of the physical map. The construction of the physical map of catfish will be one major achievements in catfish genome studies. The map will be very important for comparative genomic studies. Along with genetic linkage and QTL mapping analysis, such genomic studies will lead to eventual identification and cloning of economically important genes such as genes for disease resistance.
Publications
- Chen, L., He, C., Baoprasertkul, P., Xu, P., Li, P., Serapion, J., Waldbieser G., Wolters, W., Liu, Z.J. 2005. Analysis of a catfish gene resembling interleukin-8: cDNA cloning, gene structure, and expression after infection with Edwardsiella ictaluri. Developmental and Comparative Immunology 29, 135-142.
- Baoprasertkul, P., Peatman, E., He, C., Kucuktas, H., Li, P., Chen, L., Simmons, M., and Liu, Z.J. 2004. Sequence analysis and expression of a CXC chemokine in resistant and susceptible catfish after infection of Edwardsiella ictaluri. Developmental and Comparative Immunology 28, 769-780.
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