Targeted oligonucleotide-mediated microsatellite identification (TOMMI) from large-insert library clones
© Chen et al; licensee BioMed Central Ltd. 2005
Received: 24 August 2005
Accepted: 15 November 2005
Published: 15 November 2005
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. Calculations showed that a multi-allelic marker system always has more power to detect Linkage Disequilibrium (LD) than does a di-allelic 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 eleven PAC clones by t argeted o ligonucleotide-m ediated m icrosatellite i dentification (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.
The 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.
Primers used for selecting the PAC clones from TAIGP714 large-insert library
Forward Primer (5'-3')
Reverse Primer (5'-3')
Results and discussion
Forward and reverse sequencing primer
Single reverse sequencing primer
Initial sequencing primer (5'-3')
Primer sequence (5'-3')
Primer location (GenBank)
Characteristics of TOMMI-microsatellites
Primer pair sequence (5'-3')
Due to their isolation from partial genomic libraries selected for small insert sizes most of the publicly available porcine microsatellites lie within DNA-fragments of about 80 to 200 bp. Their potential combination in multiplex assays – also considering different annealing temperatures and technical limitations of the automated sequencers (limited number of available fluorescent dyes) – is therefore hampered. Hence, an enhanced number of genotypes per run can only be achieved by the integration of STS-markers covering a larger allelic spectrum. Thus, we intended and focused on the development of large amplicons for microsatellites by utilizing as much sequence information as possible for primer design. Indeed, fourteen STS-markers had allele sizes of at least 200 bp and for five of the isolated microsatellites, sequence information proved to be good enough to amplify allele sizes of at least 300 bp (Table 3).
MARC marker information and linkage mapping results
Forward Primer (5'-3')
Reverse Primer (5'-3')
Number of Alleles
Allele Size Range (bp)
Number of Meioses
Linkage Position (Chr: cM)
The sequencing strategy described in this study provides a targeted, inexpensive and fast method to develop microsatellites from large-insert libraries. It is also well suited to generate polymorphic markers for selected chromosomal regions and contigs of overlapping clones and yielded sufficient high quality sequence data to develop marker amplicons greater than 250 bases.
PAC clone isolation and physical mapping
Prior to STS development, a total of 11 clones were isolated from the porcine PAC library TAIGP714  by a three-dimensional PCR screening strategy. PAC-DNA preparations were done according to the manufacturer's protocol (Qiagen, Hilden, Germany). The physical assignment of the PAC clones was performed by Fluorescence in situ Hybridization (FISH) as described in  or alternatively by analysis of the INRA-UMN porcine radiation hybrid (IMpRH) panel . Microsatellite primers (Table 3) were used to RH map S0703, S0704 and S0708 – S0715. Marker assignment of S0701, S0702, S0705 – S0707, S0766 and S0767 was performed with primers from further sequence segments of the PAC clones.
Microsatellite generation and characterization
Evaluation of microsatellites and size determination of alleles were done with appropriate ABI-softwares GENESCAN (3.7) and GENOTYPER (3.6) using GENESCAN™-500ROX™ as internal size standard. Oligonucleotides were designed with the Oligo Selection Program  and synthesized by MWG Biotech (Ebersberg, Germany). To characterize size range, number of alleles, polymorphism information content (PIC), average heterozygosity (HT) and effective allele number (NE) of the microsatellites, STS-markers were separately amplified. PCR assays were performed at 54°C for S0706, S0708, S0712, S0713, S0714, and S0767, at 56°C for S0701, S0702, S0703, S0705, S0707 and S0715, and at 58°C for S0704, S0709, S0710, S0711, and S0766 in a RoboCycler Gradient 96® (Stratagene, LaJolla, USA) using PURE Taq Ready-To-Go PCR Beads® (Amersham Biosciences, Freiburg, Germany), along with the respective oligonucleotides (one labeled at the 5'-end alternatively with fluorescent dyes FAM, JOE or NED) and 50 ng of genomic porcine DNA in a volume of 12.5 μl (the concentration of each dNTP is 100 μM in 10 mM Tris-HCl (pH 9.0 at room temperature), 50 mM KCl and 1.5 mM MgCl2). In total, 336 unrelated pigs representing nine European breeds (9 Angeln Saddleback, 18 Bunte Bentheimer, 9 German Edelschwein, 15 German Landrace, 30 Hampshire, 27 Göttingen Minipig, 31 Pietrain, 12 Swabian-Haellian Swine, and 7 European Wild Boar), and six Chinese breeds (30 Chinese Jiangquhai, 28 Chinese Luchuan, 30 Chinese Minpig, 30 Chinese Rongchang, 30 Chinese Tibetan, and 30 Chinese Yushanhei) were investigated. The standard PCR profile was as follows: pre-denaturation at 92°C for 2 min, followed by 35 cycles of 92°C for 30 s, the optimal annealing temperature for 30 s, and 72°C for 30 s. The final cycle had an extension at 72°C for 10 min. PIC, HT and NE were estimated based on algorithms as introduced by Botstein and colleagues , Nei , and Kimura and Crow .
Linkage mapping of STS-markers on the USDA-MARC linkage map
Seven families of the MARC Swine Reference Population were genotyped as described . Amplified DNA was radioactively labeled, separated by denaturing polyacrylamide gel electrophoresis and visualized with autoradiography. To ensure accurate sizing and discrimination of alleles, amplification primers were redesigned to yield smaller products for all markers except S0706, S0707 and S0709. S0767 was not tested in this population. Four markers were not informative in the MARC Swine Reference Population (S0702, S0706, S0709 and S0714) and four primer sets failed to produce reliable products (S0703, S0704, S0708 and S0710). Genotypes were determined and entered into the MARC Genome Database. Each marker was initially assigned to a chromosome based on TWOPOINT results of CRIMAP , then multipoint linkage analyses determined the final location of each marker. Genotypic data were evaluated with CHROMPIC and corrections made if necessary. The final position reported is based on the current MARC swine linkage map. Amplification primers for the eight successfully mapped markers are presented in Table 4.
The authors would like to thank A. Siebels for expert technical assistance. This research project was supported by a grant of the Erxleben Research & Innovation Council to B. Brenig (ERIC-BR1959-2001-06).
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