Plant & Animal Genome IX Conference

Poster: Aquaculture
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ABUNDANCE OF MICROSATELLITE-CONTAINING CLONES IN A CHANNEL CATFISH (Ictalurus punctatus) BRAIN cDNA LIBRARY

USDA/ARS Catfish Genetics Research Unit, Thad Cochran National Warmwater Research Center, P.O. Box 30, Stoneville, MS 38776

The first generation linkage map of the channel catfish genome was constructed using 244 random genomic microsatellite (short tandem repeat) markers and 20 type I (expressed) markers. To identify more type I markers, a directional cDNA library was constructed from catfish brain to obtain expressed sequences (ESTs) useful for development of single nucleotide polymorphisms. Approximately 900 cDNA clones were sequenced, and 12% of the clones contained short tandem repeat sequences in the non-coding regions. However, only 10% of these clones were known genes (~1% of total) as determined by sequence similarity search (BLAST). The brain cDNA library was enriched for dinucleotide repeat sequences using single-pass hybridization to biotinylated CA(9) or GT(9) oligonucleotides coupled with streptavidin-coated magnetic beads. PCR analysis using a tandem repeat primer and an M13 sequencing primer demonstrated that 88% of the clones contained potential tandem repeats. Six hundred seventy-two clones of the CA/GT-enriched libraries were sequenced from both ends with 30% clone redundancy, and 299 non-redundant clones (64%) contained microsatellites in their sequences. Gene identification was possible for 138 (29%) of the non-redundant clones. These enriched libraries produced 90 (19%) genes and 209 (45%) ESTs with microsatellites, and 48 (10%) genes and 123 (26%) ESTs without microsatellites. Enrichment of the brain cDNA library for CA or GT repeats increased the number of microsatellite-containing ESTs 5-fold, and genes containing microsatellites by 15-fold. Library enrichment will enhance our capacity to add type I loci to the catfish genetic linkage map to increase map density and permit comparative mapping.