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. Here, we introduce an improved approach called TOMMI (

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Fifteen of the seventeen microsatellites (Table 2) were developed with sequencing primers containing one selective nucleotide at the 3'-end: (CA)₈T (S0701, S0703, and S0767), (CA)₈A (S0702, S0704, and S0710), (CA)₈G (S0705, S0706, S0712, and S0766), (AC)₈C (S0709), (AC)₈G (S0707 and S0715), (AC)₈T (S0708 and S0711). 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 (for S0713) and TAIGP714I23038Q (for S0714) contained additional (CA)₈A or (CA)₈G primer binding regions or motifs. Contrary, a further extension with three nucleotides at the 3'-ends of the primers did not result in additional microsatellites in any of the PAC clones or was not required. Therefore, we conclude that repeat primers with two 3'-nucleotides next to the repeat motif are sufficient to detect and sequence all repeats potentially present on a large-insert library clone. The results of our isolation strategy also indicate that two sequencing reactions (the reverse sequencing primer was designed based on the obtained sequences) seem to be sufficient in most cases to gain sequence information of high quality to amplify microsatellites (Table 2). Usage of sequencing primers degenerated at the 3'-end proved, however, to be inadequate as no sequence information at all was achieved. Also, to avoid overlapping primary sequences, oligonucleotides that basically extend the dinucleotide repeat at the 3'-end – such as (CA)₈C and (AC)₈A – are not recommended. TOMMI proved to be an efficient and reliable isolation strategy. Besides new STS-markers, six previously described microsatellites were also detected. Three of these loci, microsatellites S0111 10, SW742 11, and SW813 12, were initially used as probes for the isolation of clones TAIGP714L02061Q, TAIGP714I23038Q, and TAIGP714F10061Q. The other three already described microsatellite sequences reside on TAIGP714C09004Q [GenBank: AJ440949 (repeat location: 3172–3231) and GenBank: AJ440950 (repeat location: 15831–15860 and 16007–16038)]. They were not further considered in this study as they were not regarded as novel. Independently of our effort, two other groups 1314 introduced similar sequencing approaches to generate microsatellites from large-insert libraries. There are, however, several differences between our approach and the ones of the other groups in terms of sequence generation and selective amplification of microsatellites. Here, contrary to Waldbieser and colleagues 14 – who used trinucleotide repeat containing primers for sequencing – both gene-specific primers are not 5'-tailed with extra nucleotide stretches to enable either product labeling or to promote alleged non-template adenylation. Fujishima-Kanaya's group 13 used larger repeat compounds contributing to the primer [(CA/GT)₍₁₀₎ instead of (CA/GT)₍₈₎]. Secondly, the sequencing primers consisted generally of three selective nucleotides at the 3'-end adjacent to the repeat motif (e.g. CNA/GVG). There, the first of the three terminal nucleotides was always identical with the starting nucleotide of the dinucleotide repeat primer used. In addition, primers contained a degenerated base according to the International Union of Biochemistry (IUB) codes at the second position from or directly at the 3'-end. Thirdly, determination of the double-stranded primary DNA sequence stretch was achieved by four sequencing reactions using both a CA-repeat containing primer plus a GT-repeat containing primer heading in the opposite direction and two reverse primers were developed based on the obtained sequence. Finally, they always designed an additional primer pair for the specific amplification of the microsatellite. In contrast, we used the single reverse sequencing primer in combination with a newly developed sequence specific primer (S0766 and S0767) or designed a new primer pair to amplify the microsatellite (S0701 to S0715).

The observed number of alleles per locus (monomorphic locus S0709 is not included in this calculation) in the heterogeneous sampling was as low as 2 (S0702) and as high as 22 (S0713), leading to an average number of 9.94 alleles, N_E ranged from 1.05 to 11.54 and both H_T and PIC from 0.05 to 0.91 (Table 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).

By the guided isolation of STS-markers S0709 to S0715 from three SSC16q derived PAC clones (relative position 0 cM to 9.3 cM 7; 2.33 STS-markers per clone), the marker density in this chromosomal region was improved remarkably. An average of 1.55 new microsatellites was isolated from PAC clones harboring functional candidate genes (S0701-S0708; S0766 and S0767). Considering all used PAC clones and developed STS-markers, 1.55 microsatellites per clone were isolated. As the PAC clones had an average length of 80 kb (as shown by pulsed-field-gel electrophoresis) the frequency of dinucleotide repeats every (30 to) 50 kb 5 was more or less confirmed. TOMMI holds therefore the potential to identify existing STS-markers linked/adjacent to e.g. candidate genes on large-insert library clones. Thus, in combination with a genome scan, respective putative candidate genes could either be transformed to or excluded as positional candidate genes prior to their complete structural characterization including SNP detection. Linkage mapping results for S0701, S0705, S0707, S0711, S0712, S0713, S0715, and S0766 are presented in Table 4. A comparison of their mapping positions with QTL positions (Pig Quantitative Trait Loci (QTL) database 15 reveal that S0705 (64.22 cM), S0707 (43.19 cM), and S0766 (102.50 cM) reside on the respective chromosomes exactly at QTL locations (S0705: backfat between the last 3^th and 4^th rib; S0707: early growth rate and water holding capacity; S0766: backfat thickness at first rib and intra-muscular fat). The other STS-markers are located in QTL spans of ± 5 cM. This indicates their immediate potential to further dissect these respective QTL regions.

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.

KFC conducted the lab work to isolate and characterize S0701 to S0715 and CK to isolate and characterize S0766 and S0767. CK shared manuscript preparation and editing with KFC, supervised KFC's Ph.D. thesis, evaluated microsatellite data, and organized and provided DNA of the European pig breeds. KBK optimized and conducted fragment analysis and was responsible for evaluation of microsatellite data. JR assisted KFC in the beginning of the project. LSH organized DNA of the Chinese pig breeds. GAR conducted linkage mapping of the markers and edited the manuscript. BB proposed the idea, supervised and commented on the project, was responsible for funding and manuscript editing, and acts as head of the research group in Göttingen.