Abstract
BackgroundIn the last few years, microsatellites have become the most popular molecular marker system and have intensively been applied in genome mapping, biodiversity and phylogeny studies of livestock. Compared to single nucleotide polymorphism (SNP) as another popular marker system, microsatellites reveal obvious advantages. They are multi-allelic, possibly more polymorphic and cheaper to genotype. Calculations showed that a multi-allelic marker system always has more power to detect Linkage Disequilibrium (LD) than does a di-allelic marker system [1]. 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.ResultsSeventeen porcine microsatellite markers were isolated from eleven PAC clones by targeted oligonucleotide-mediated microsatellite identification (TOMMI), an improved efficient and rapid flanking sequence-based approach for the isolation of STS-markers. 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. 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.ConclusionThe sequencing strategy described in this study provides a targeted, inexpensive and fast method to develop microsatellites from large-insert libraries. It is well suited to generate polymorphic markers for selected chromosomal regions, contigs of overlapping clones and yields sufficient high quality sequence data to develop amplicons greater than 250 bases.
Highlights
In the last few years, microsatellites have become the most popular molecular marker system and have intensively been applied in genome mapping, biodiversity and phylogeny studies of livestock
Characterization of microsatellites S0713 and S0714 was only accomplished by an improved discrimination of the PAC clone sequences with sequencing primers further extended at the 3'-end with a second nucleotide [(CA)8AT for S0713 and (CA)8GC for S0714]
The second nucleotide became necessary because the respective clones TAIGP714L02061Q and TAIGP714I23038Q contained additional (CA)8A or (CA)8G primer binding regions or motifs
Summary
In the last few years, microsatellites have become the most popular molecular marker system and have intensively been applied in genome mapping, biodiversity and phylogeny studies of livestock. Compared to single nucleotide polymorphism (SNP) as another popular marker system, microsatellites reveal obvious advantages. They are multi-allelic, possibly more polymorphic and cheaper to genotype. In order to directly generate microsatellites from large-insert libraries a sequencing approach with repeat-containing oligonucleotides is introduced. Almost all of the applied protocols to isolate microsatellites de novo include construction of partial genomic libraries (selected for small insert size) followed by cumbersome screening steps with hybridization probes [2]. The objective of the present study was the selective generation of microsatellites from PAC-clones, which were prior to STS development isolated from the porcine PAC library TAIGP714 [3] by a three-dimensional PCR screening strategy [9]. Eight of the eleven clones harbored functional or positional candidate genes involved in health, reproduction, production, and regulation, whereas the other three clones have been used in the attempt to construct a PAC contig covering SSC16q11-13 (Table 1)
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