Direct cloning of microsatellite loci from Cottus gobio through a simplified enrichment procedure

Abstract

Applying a simplified enrichment procedure, we have isolated and characterized 177 microsatellite markers for Cottus gobio L., 1758 (Cottidae, Scorpaeniformes, Teleostei). In contrast to using specific probes for the enrichment, we use genomic DNAs of unrelated organisms for cross-hybridization. This takes advantage of the fact that simple sequences are the only repetitive elements that are abundantly found in all eukaryotic genomes and that any genome usually contains all permutations of microsatellite motifs. This cross-hybridization principle was employed to enrich genomic libraries of Cottus DNA to obtain a large number of nonredundant microsatellite markers without further screening procedures

Identification and characterization of microsatellites from the Antarctic isopod Ceratoserolis trilobitoides: nuclear evidence for cryptic species

“…1 was isolated from ethanol preserved muscle tissue using standard spin column protocols (Qiagen DNeasy). A genomic library enriched for microsatellites was created using the reporter genome protocol (Nolte et al 2005) as described in Held and Leese (2007). Hybridization chips (Hybond N+, GE Healthcare) with single stranded genomic DNA from Mus musculus, Drosophila melanogaster (strain Canton S) and Homo sapiens as reporter genomes were used as natural enrichment probes (reporter genomes).…”

The utility of fast evolving molecular markers for studying speciation in the Antarctic benthos

“…For more technical details and an evaluation of the properties of this and other protocols for the isolation of microsatellites, the reader is referred to another paper (Leese et al 2006, submitted) and the original papers describing the method (Nolte et al 2005;Zane et al 2002).…”

“…The use of a modiWed protocol (Nolte et al 2005) shows that many obstacles previously associated with the isolation of microsatellites may no longer be a problem.…”

“…AmpliWed fragments containing microsatellites were selectively enriched by hybridizing 300-500 ng against immobilized single-stranded DNA fragments of the reporter genomes bound to nylon membranes as described in Nolte et al (2005). Three washing steps were performed to keep only those fragments of the target genome that formed stable duplexes with the immobilized reporter genome, indicating high similarity in low-complexity regions.…”

“…Three diVerent hybridization Wlters with single stranded reporter genomes of Mus musculus domesticus, Drosophila melanogaster, and Homo sapiens were prepared according to Nolte et al (2005). DNA from 18 specimens from six populations of the C. trilobitoides species complex was pooled.…”