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ARS Home » Northeast Area » Leetown, West Virginia » Cool and Cold Water Aquaculture Research » Research » Publications at this Location » Publication #154016
Title: CHARACTERIZATION OF A NEW BAC LIBRARY FOR RAINBOW TROUT

Author
item Palti, Yniv
item Gahr, Scott
item Rexroad, Caird

Submitted to: Animal Genetics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/1/2004
Publication Date: 4/1/2004
Citation: Palti, Y., Gahr, S.A., Rexroad III, C.E. 2004. Characterization of a new bac library for rainbow trout. Animal Genetics.35(2):130-133.

Interpretive Summary: Genome research for species of interest is facilitated by the development of species-specific tools such as large insert genomic library of bacterial artificial chromosomes (BACs). Interest in the utilization of rainbow trout as a model species for research is coupled with the need for genetic improvement for aquaculture. This report describes in depth characterization of a new and extensive BAC library for rainbow trout. This library is demonstrated to be a useful tool for physical mapping, isolation and sequencing of genes and chromosomal regions that influence economically important traits.

Technical Abstract: A 10X rainbow trout bacterial artificial chromosome (BAC) 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. The source DNA was extracted from a single male (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 bp (PFGE) or 118,700 bp (DNA fingerprints). The clones were gridded onto 10 large nylon membranes to produce high-density arrays for screening the library by hybridization. The library was probed with 11 cDNAs from the NCCCWA EST project chosen for interest in EST BLAST match, 7 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. FPC analysis of HindIII and EcoRV DNA fingerprints and insert end sequencing were used to estimate the level of redundancy in the library, to construct BAC contigs that span the chromosomal regions that harbor the above genes and ESTs, and to detect duplicated loci in the semi-duplicated rainbow trout genome. A good correlation (R2 = 0.7) was found between the number of positive hits per probe and the number of contigs that were assembled from those BACs. The average number of BACs per contig was 9.6, which confirmed 10X genome coverage of the library. Two thirds of the loci screened were predicted to be duplicated since the positive BACs for those genes were assembled into 2 or 3 contigs. This suggests that most of the rainbow trout genome is still duplicated.