Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 1/11/2003
Publication Date: 1/11/2003
Citation: Palti, Y., Rexroad Iii, C.E., Gahr, S.A. 2003. Utilization and characterization of the ncccwa rainbow trout bac library for physical and linkage mapping. Plant and Animal Genome XI. p.241.
Technical Abstract: A 10X rainbow trout bacterial artificial chromosome library was constructed by Amplicon Express for the National Center for Cool and Cold Water Aquaculture (NCCCWA) primarily to aid in the physical and genetic mapping efforts of the rainbow trout genome. Dr. Gary Thorgaard of Washington State University contributed the source DNA from a male fish (total blood cells) of the Swanson strain, a YY clonal line expected to be homozygous at most loci. The library consists of 184,704 clones with an average insert size of 137,500 bps. The clones were gridded onto 10 large nylon membranes to produce high-density arrays for screening the library by hybridization. To date the library has been probed for 11 cDNAs from the NCCCWA EST project chosen for interest in EST BLAST match, 8 known genes, and a Y'specific sex marker. Clones identified as positive by initial hybridization with probe cocktails were re-arrayed and gridded for a secondary single probe hybridization confirmation. When possible, clones were also verified by PCR amplification with gene-specific primers. The average number of verified positive hits per probe confirmed 10X coverage of the library. DNA fingerprinting was used to estimate the level of redundancy in the library, and to construct BAC contigs that span the chromosomal regions of the above genes and ESTs. The BACs of interest are being sub-cloned to identify microsatellites for genetic mapping, used to characterize gene sequences, and as probes for fluorescent in situ hybridization.