Submitted to: Biomed Central (BMC) Genomics
Publication Type: Peer reviewed journal
Publication Acceptance Date: 11/15/2005
Publication Date: 11/15/2005
Citation: Chen, K., Knorr, C., Bornemann-Kolatzki, K., Ren, J., Huang, L., Rohrer, G.A., Bertram, B. 2005. Targeted oligonucleotide-mediated microsatellite identification (tommi) from large-insert library clones. BMC Genetics 6:54. Interpretive Summary: Microsatellites are a very useful marker system and have intensively been applied in swine genome mapping. Traditional methods for isolation of microsatellites are time-consuming and cost-intensive. In order to directly generate microsatellites from targeted regions of the genome a unique sequencing approach was developed. Seventeen porcine microsatellite markers were isolated from twelve regions by targeted oligonucleotide-mediated microsatellite identification (TOMMI), an efficient and rapid sequence-based approach for the isolation of genetic markers. Sixteen of the microsatellite markers proved to be useful in a sample of industry pigs. In addition, fourteen of the seventeen markers are of appropriate size to be included in multilocus assays. Mapping results of the markers indicate they are located in regions of the pig genome that possess QTL. Thus they could have immediate use in QTL studies to further dissect these important chromosomal regions.
Technical Abstract: In the last few years, microsatellites have become the most popular molecular marker system. Traditional isolation methods using partial genomic libraries are time-consuming and cost-intensive. In order to directly generate microsatellites from large-insert libraries a sequencing approach with repeat-containing oligonucleotides is introduced. Seventeen porcine microsatellite markers were isolated from twelve PAC clones by targeted oligonucleotide-mediated microsatellite identification (TOMMI), an improved efficient and rapid flanking sequence-based approach for the isolation of STS-markers. Prior to microsatellite identification, the PAC clones were physically mapped by Fluorescence in situ Hybridization (FISH) or by radiation hybrid panel analysis and assigned to porcine chromosomes 2, 3, 6, 13, 16, 18, and the X chromosome. With the application of TOMMI, an average of 1.55 (CA/GT) microsatellites per PAC clone was identified. The number of alleles, allele size distribution, polymorphism information content (PIC), average heterozygosity (HT), and effective allele number (NE) for the STS-markers were calculated using a sampling of 336 unrelated animals representing fifteen pig breeds (nine European and six Chinese breeds). Sixteen of the microsatellite markers proved to be polymorphic (2 to 22 alleles) in this heterogeneous sampling. Most of the publicly available (porcine) microsatellite amplicons range from approximately 80 bp to 200 bp. Here, we attempted to utilize as much sequence information as possible to develop STS-markers with larger amplicons. Indeed, fourteen of the seventeen STS-marker amplicons have minimal allele sizes of at least 200 bp, and for five of them minimal alleles of at least 300 bp length are amplified. Thus, most of the generated STS-markers can easily be integrated into multilocus assays covering a broader separation spectrum. Linkage mapping results of the markers indicate their potential immediate use in QTL studies to further dissect trait associated chromosomal regions.