Introduction

Microsatellites are short DNA stretches in which a one- to six-nucleotide motif is tandemly repeated. Found in all pro- and eukaryotic genomes, microsatellites show high mutation rates and intraspecific length polymorphisms, making them a powerful marker for population genetics. Development of universal, cross-species-amplifying primers for microsatellites is often not possible and the necessity of de novo isolation for taxa yet to be analyzed is a drawback in the applicability of microsatellites (Zane et al. 2002). Most new microsatellites are isolated using enriched genomic libraries. Marker development consists of (i) enrichment of microsatellite-containing fragments of genomic DNA, (ii) construction of a partial genomic library, (iii) screening of the library by colony hybridization, (iv) sequencing of candidate clones, (v) development of flanking primers, and (vi) exclusion of nonamplifying and monomorphic loci. Poor yields of suitable loci may occur due to technical problems in any of the steps but also due to peculiarities of the analyzed organism. Low abundance of microsatellites in the genome (Fagerberg et al. 2001) and occurrence of multicopy microsatellite families with similar flanking regions (Meglécz et al. 2004) have been considered reasons for such difficulties. We suspect, however, that in most cases isolation failure is not reported.

We evaluated the impact of taxon-specific traits on microsatellite development following the FIASCO protocol (Zane et al. 2002). Applying identical laboratory procedures to a beetle, Pityogenes chalcographus (Scolytinae), and an ant, Lasius austriacus (Formicinae), we address whether (i) numbers of micrsosatellite harboring clones in the enriched libraries, (ii) loss of candidate clones due to subsequent difficulties in amplification, and (iii) rate of polymorphism are comparable between the two species. We discuss explanations for the contrasting findings and identify critical steps of the isolation process. Finally, we offer advice on some avoidable pitfalls.

Materials and Methods

DNA Preparation and Microsatellite Enrichment

Genomic DNA of 30 individuals was extracted with the GenElute Mammalian DNA kit (Sigma, St. Louis, MO, USA) and quantified photometrically, and 150 ng was used for a one-step digestion-ligation reaction with MseI and AFLP adaptors (Zane et al. 2002). Optimal cycle number for amplification with adaptor primers was determined by gel electrophoresis of 5-μl aliquots of the amplicon after 14, 17, …, 29 cycles. To avoid overamplification leading to high clone redundancy, a cycle number where a fine smear became visible for the first time was selected for the production of >1 μg of amplicon. Products were purified with the QIAquick PCR purification kit, then quantified photometrically, and integrity of the smear was tested on a minigel.

One microgram of purified PCR product was diluted with sterile water to 250 μl, denatured for 5 min in a boiling water bath, and cooled on ice. Twenty picomoles of each (AC)8 and (GA)8 biotinylated oligoprobe and 13 μl of 20× SSC were added and the final volume was set to 500 μl. The mixture was hybridized for 1 h at 50°C under constant agitation. Three hundred microliters of Streptavidine MagneSphere Paramagnetic Particles (PMPs; Promega, Madison, WI, USA) were washed three times with 0.5× SSC, resuspended in 100 μl of 0.5× SSC, and, after addition of the DNA-probe mixture, incubated for 20 min at room temperature under constant agitation. Afterward, PMPs were washed four times with 300 μl 0.1× SSC for high stringency or two times with 0.5× SSC and two times with 0.2× SSC for low stringency. DNA was eluted with 100 μl pre-warmed sterile water at 50°C. Recovery PCR was performed using adaptor primers.

Library Construction and Screening

DNA from recovery PCR was ligated into the pGEM vector (Promega) and used for transformation of competent JM109 cells. White colonies were transferred to master plates and, after 16 h, probed with sterile nylon membranes (Roche, USA). Membranes were hybridized overnight at 60°C in Church buffer containing 0.01% denatured salmon sperm DNA (Roche) and 1 μg of each digoxigenin labeled (AC)x and (GA)x oligoprobes. After washing, the presence of probe-target hybrids was detected using the DIG luminescent detection kit (Roche) following the manufacturer’s instructions. Colonies showing the darkest signals on film were transferred to liquid culture for plasmid extraction.

PCR Pretest for Microsatellite Inserts

Two-tenths microliters plasmid DNA was used as template in a PCR containing 1.5 mM magnesium chloride, 50 μM dNTPs (Fermentas, Lithuania), 0.2 μM of each (AC)8 and (GA)8 oligonucleotide, 0.2 μM SP6 vector primer, 0.4 U Biotherm Taq DNA polymerase (Genecraft, Germany), and the reaction buffer provided by Genecraft. Amplification was performed with an initial denaturation at 94°C (3 min) followed by 32 cycles at 94°C (30 s), 59°C (45 s), and 72°C (45 s). Plasmids showing no bands were subjected to PCR with SP6 replaced by T7 vector primer. Plasmids with positive reaction in any of the two PCRs were classified as potential microsatellite isolates and sequenced.

Population Screening

Flanking primers of microsatellite carrying inserts were designed and tested on plasmid and genomic DNA. DNA of single individuals was extracted with the Sigma GenElute Mammalian DNA kit and used for screening. For loci yielding strong bands of expected size, forward primers were labeled with either 5’-FAM, 5’-HEX, or 5’-TET fluorophores. Retrieved microsatellite amplicons were supplemented with GeneScan 500-TAMRA size standard (Applied Biosystems, USA) and separated on an ABI 310 analyzer (Applied Biosystems). Allele sizes were determined using Genotyper software (Applied Biosystems).

Results

We constructed three libraries for P. chalcographus (P1, high stringency; P2 and P3, low stringency) and one for L. austriacus (L1, high stringency). After first adaptor-primed PCR, smears were comparable for all libraries, without bands indicative of accumulation of preferentially amplified loci. Three hundred two to 410 white colonies per library were picked for screening (Table 1). Sixty-two and one-half to 92.7% of the plasmids positive in both hybridization and PCR pretest harbored microsatellite inserts. No significant differences in the number of microsatellite-containing clones between the libraries were found. Sequencing revealed extreme redundancy in library P1, where all 15 plasmids carried in total five different alleles of a locus with the motif (TC)16-TTCT-(TC)4. In L1, only 1 of 26 motifs was isolated twice; P2 and P3 showed intermediate levels of redundant clones. Again, the above motif appeared in 40.8% and 44.0% of the plasmids, respectively. In total this locus was isolated 37 times.

Table 1 Microsatellite isolation by screening of genomic libraries enriched for (AC)x and (GA)x motifs of Lasius austriacus L1 and Pityogenes chalcographus P1–P3
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After development of 20 primer pairs for L. austriacus 65.0% of the loci genotyped were polymorphic. Of 17 primer pairs developed for P. chalcographus, 23.5% gave polymorphic amplicons. The overall number of informative loci per screened colony was 0.032 for L. austriacus and 0.004 for P. chalcographus, respectively.

The informative loci of both species were further compared for quality traits (Table 2): PCR failure over all samples, repeat count rc of the cloned allele, and allelic richness A over all samples as estimated by using a sample coverage method (Huang and Weir 2001). The beetle microsatellites differ significantly from their ant counterparts concerning rc and A. PCR failure rate was comparable for both species.

Table 2 Quality traits of informative loci isolated from the ant L. austriacus and the beetle P. chalcographus
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Discussion

We modified the FIASCO protocol by adapting enrichment stringency and included a PCR pretest before plasmid sequencing, which excluded almost all false-positive clones. The frequency of microsatellite-containing clones across all libraries indicates comparable enrichment efficiency with both species.

Repeated isolation of identical motifs is a common feature of enriched libraries (Meglécz et al. 2004), with low microsatellite abundance in the genome (Fagerberg et al. 2001) hypothesized as the cause. Combined with high enrichment efficiency, it may dramatically increase redundancy. While there were almost no redundant sequences in the ant library, repeated isolation of identical motifs frequently occurred with the beetle. Finding different allelic forms of redundant loci in the libraries indicates that redundancy is due to enrichment bias rather than multiple ligation of identical PCR products.

Requirements necessary to turn unique microsatellite sequences into functional markers include suitability of the flanking regions for primer development, availability of sufficient single-specimen PCR product, and interpretable, polymorphic patterns in fragment analysis. The ant met these demands. Thirteen informative loci resulted from one enrichment procedure after excluding five loci because of short, unpromising motifs. As redundancy was negligible (1 of 26 motifs), we expect a scaled-up library screening to result in a multiple number of informative loci. In contrast, in the beetle the 19 unique loci were reduced by flanking regions unsuitable for primer development (2 loci), insufficient yield of PCR product (8 loci), multibanded products (3 loci), and monomorphism (2 loci).

The two species differ not only in the availability of polymorphic microsatellites, but also in their repeat count and allelic richness. While the latter is influenced by population structure, only in the ant was it possible to isolate long, uninterrupted motifs. Hymenopterans have a microsatellite-rich genome (Thoren et al. 1995). This contrasts with the situation in noncarabid coleopterans, where short motifs containing <10 repeat units as well as low allele numbers are common (e.g., Sallé et al. 2003) and loci variability often is insufficient for population genetics (e.g., Sallé et al. 2007). Similar results are reported for lepidopterans (Meglécz et al. 2004; Zhang 2004).

In addition to higher-level systematic differences, also closely related species may differ in microsatellite content (Ross et al. 2003). Microsatellites probably originate from substitutions (Zhu et al. 2000) and evolve by replication slippage (Rose and Falush 1998). Microsatellite content may mirror an equilibrium between slippage events increasing allele size and point mutations breaking up repeat sequences and ultimately leading to loci losses (Ross et al. 2003). While the role of genome size—microsatellites are frequent in noncoding regions and large genomes offer plenty of space to harbor them (Dieringer and Schlötterer 2003)—is not consistent for ants and beetles which show comparable C-values, base composition might be important. Equal abundance of the four bases should produce few microsatellites (Dieringer and Schlötterer 2003). Base composition can differ even between congeners (Lockhart et al. 1994), so that differences between species may reflect changes in mutational processes over a short evolutionary time.

Multicopy microsatellite families with similar flanking regions are characteristic of taxa refractory for microsatellite development (Meglécz et al. 2004). Zhang (2004) suggested a genomic dispersal of new microsatellites via transposition of mobile elements followed by accumulation of point mutations in the flanking regions leading toward single-copy loci. Low isolation efficiency and multibanded PCR amplicons support the hypothesis of a recent propagation of early-stage microsatellites in the apparently microsatellite-poor genome of P. chalcographus.

We demonstrate that poor results in microsatellite isolation are not necessarily a methodology problem. While a protocol might perform well for one group, switching to a new organism may cause problems. Based on the literature and our own experiences, the following critical steps in isolation are of particular importance. (a) Using a standard species with a high microsatellite content may help calibrating isolation efficiency. (b) Scaling-up will only compensate for low yield in the case of high rates of negative clones with some unique positives but not when low yield is caused by clone loss due to redundancy. Controlling redundancy might include stepwise adaptations of stringency as well as careful choice of capture probes for enrichment. (c) Attention often focuses on the outcome of plasmid sequencing, but awareness that as many as half of the isolated loci might never become functional markers is also important. Causes for difficulties include unsuitable primer binding sites, library contamination, suboptimal PCR amplification, and lack of polymorphism.