Isolation and enrichment of abundant microsatellites from a channel catfish (Ictalurus punctatus) brain cDNA library.
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'-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 two-fold for repeat sequences and increase the ability to link these ESTs with known sequences greater than six-fold.
Nonneman
Dan
D
USDA, ARS, U.S. Meat Animal Research Center, Clay Center, Nebraska 68933-0166, USA. nonneman@email.marc.usda.gov
Waldbieser
Geoffrey C
GC
D000818:Animals; D001483:Base Sequence; D001923:Brain Chemistry; D002874:Chromosome Mapping / Q000662:veterinary; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D020224:Expressed Sequence Tags; D015723:Gene Library; D005819:Genetic Markers; D007059:Ictaluridae / Q000235:genetics*; D018895:Microsatellite Repeats / Q000235:genetics*; D008969:Molecular Sequence Data; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
D018076:DNA, Complementary; D005819:Genetic Markers; D012333:RNA, Messenger