Isolation and Enrichment of Abundant Microsatellites from a Channel Catfish (Ictalurus punctatus) Brain cDNA Library

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 noncoding 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 1644 clones from seven repeat-enriched captures (CA, GT, CT, GA, MTT, TAG, and TAC) were sequenced from both ends, and 795 nonredundant clones were assembled. Thirty-seven 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 0 -UTR, 72% contained repeats in the 3 0 -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 two-fold for repeat sequences and increase the ability to link these ESTs with known sequences greater than six-fold.

Development of novel EST-SSR markers in common carp by data mining from public EST sequences

“…In addition, polymorphism was not entirely dependent on repeat length, since repeats of five dinucleotides were polymorphic while some of six or seven ones were not (Nonneman and Waldbieser, 2005). Therefore, when compared with the Type II microsatellites in the same species, the level of polymorphism of EST-SSRs is normally slightly lower (Cho et al, 2000;Pérez et al, 2005).…”

Assembly of 500,000 inter-specific catfish expressed sequence tags and large scale gene-associated marker development for whole genome association studies

“…This project represents one of the major milestones in catfish research, and brings the catfish EST resources to almost a half million sequences in GenBank [21-26]. This EST resource will prove useful for gene discovery, molecular marker development, and genetic linkage and comparative mapping, and it will help facilitate whole genome sequencing and annotation.…”

“…Many of the required resources have been developed in catfish, including a large number of polymorphic markers [10,11], linkage maps [12-14], bacterial artificial chromosome (BAC) libraries [15,16], physical maps [17,18], and BAC end sequences (BES) [19,20]. However, expressed sequence tag (EST) resources were low from catfish [21-26], hindering both functional and comparative genome analysis in catfish. Large numbers of ESTs have been produced for most model species as well as a number of agriculturally important species [27-32], including cattle (1.5 million), swine (1.4 million), chicken (600,000), Atlantic salmon (471,000), and rainbow trout (281,000).…”

A first generation BAC-based physical map of the channel catfish genome

“…Briefly, framework genetic linkage maps have been produced based on microsatellite loci [2] and Amplified Fragment Length Polymorphism (AFLP) loci [3]. Channel catfish Expressed Sequence Tags (EST) have been identified from several tissues [4,5] and clustered and annotated in the Gene Index Project [6]. A new EST project is underway through the Community Sequencing Program to sequence an additional 300,000 cDNA clones [7].…”