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United States Department of Agriculture

Agricultural Research Service

Title: Isolation and Enrichment of Abundant Microsatellites From A Channel Catfish (Ictalurus punctatus) Brain CDNA Library

Authors
item Nonneman, Danny
item Waldbieser, Geoffrey

Submitted to: Animal Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 15, 2005
Publication Date: December 1, 2005
Citation: Nonneman, D., Waldbieser, G.C. 2005. Isolation and Enrichment of Abundant Microsatellites from a Channel Catfish (Ictalurus punctatus) Brain cDNA Library. Animal Biotechnology. 16:103-116.

Interpretive Summary: A comprehensive genetic linkage map of the catfish genome will allow researchers to identify genes that control economically important traits. In order to place more comparative genes on the catfish linkage map, a brain cDNA library was enriched for clones containing polymorphic sequences termed microsatellites. The enrichment process doubled the number of unique clones that contained microsatellites, and reduced by 1/6 the number of clones needed to identify microsatellites in known genes. Eighty six percent of the clones could be placed on the catfish genetic linkage map.Identification of genes important for catfish production traits will be more efficient when the catfish genome can be compared with more highly resolved genomes such as the fully sequenced zebrafish genome.

Technical Abstract: Efforts to construct a genetic linkage map of channel catfish have involved identification of random genomic microsatellite markers, as well as, anchored Type I loci (expressed genes) from channel catfish. To identify Type I markers we constructed a directional cDNA library from brain tissue to obtain expressed catfish sequences that could be used for single nucleotide polymorphism (SNP) marker development. These cDNA sequences surprisingly contained a high proportion of microsatellites (about 14%) in non-coding regions of expressed sequence tags (ESTs), many of which were not associated with known sequences. To further identify cDNAs with microsatellites and reduce the number of sequencing reactions needed for marker development, we enriched this library for repeat sequences and sequenced clones from both directions. A total of 2054 clones from 7 repeat-enriched captures (CA, GT, CT, GA, MTT, TAG and TAC) were sequenced from both ends and 806 non-redundant clones were assembled. Thirty-five percent of the clones contained microsatellites in the trimmed sequence. After assembly in the TIGR Catfish Gene Index (CfGI), 154 contigs matched known vertebrate genes and 92 contigs contained microsatellites. When Blast-matched orthologues were available for similarity alignments, 28% of these contigs contained repeats in the 5'-UTR, 72% contained repeats in the 3'-UTR and 8% contained repeats at both ends. Using biotinylated repeat oligonucleotides coupled with streptavidin-coated magnetic beads, and rapid, single-pass hybridization, we were able to enrich our plasmid library greater than 2-fold for repeat sequences and increase the ability to link these ESTs with known sequences greater than 6-fold.

Last Modified: 9/20/2014
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