Scots pine (Pinus sylvestris L.) is a wind-pollinated, wind-dispersed, predominantly outcrossing conifer (Kärkkäinen et al. 1996) and the most widely spread among pines, extending from south of Spain (38°N) to eastern Siberia (140°E). The taxon is best thought of as a complex of different evolutionary units (Moritz 1994) and several subspecies or varieties have been recognized (Farjon 1998). Neutral genetic markers provide powerful tools for identification of differentiated gene pools within widespread species. Many forest trees with large geographical distributions, such as Scots pine, show genetic signatures resulting from recent patterns of colonization from genetically differentiated glacial refugia (Hewitt 2004; see Naydenov et al. 2007; Pyhäjärvi et al. 2008 for Scots pine). When considering neutral nuclear markers, the genetic variation of Scots pine is high and accumulated mainly within populations (e.g., Dvornyk 2001). Soto et al. (2010), in a comparative study across six Iberian native pines, suggested that contemporary high levels of genetic diversity in Scots pine result from higher past effective population sizes boasted by its remarkable cold-tolerance.

Nuclear microsatellites (simple sequence repeats, nuSSRs) have proved to be useful to study phylogeographic and gene flow patterns in conifers (e.g., Bagnoli et al. 2009; González-Martínez et al. 2010) and are increasingly being used to infer demographic history in tree species (e.g., Daïnou et al. 2010). Apart from their interest per se, demographic inferences can also be used to obtain null hypotheses to test for signatures of selection in functional genetic markers (e.g., candidate genes, SNPs), which in turn provide insights on the molecular basis of adaptive evolution in forest trees. Unfortunately, only few nuSSRs are available for Scots pine (Kostia et al. 1995; Soranzo et al. 1998; González-Martínez et al. 2004; Liewlaksaneeyanawin et al. 2004) mainly because reliable nuSSRs are difficult to develop for conifer species due to their large genome size (estimated in 22,474 Mbp for Scots pine; Plant DNA C-values Database, release 5.0, December 2010) and the extensive repetitive nature of their DNA (Scotti et al. 2002).

In order to provide a set of easily scorable nuSSRs, di-, tri- and tetra-nucleotide repeats were isolated from EST libraries and characterized for their level of polymorphism in three natural populations. A total of 6,641 P. sylvestris EST sequences were obtained from cDNA libraries developed within the EU-EVOLTREE project (libraries WZ0APSBA, WZ0APSBB, WZ0APSBC; http://www.evoltree.eu/index.php/elab-start/elab-wizard), which were assembled in 6,107 unique sequences using CodonCode software (CodonCode Corporation, USA). The sequences were screened for the presence of di-, tri- and tetra-nucleotide repeats using Msatfinder v.2.0 software (http://www.genomics.ceh.ac.uk/cgi-bin/msatfinder/msatfinder.cgi). A total of 55 primer pairs were selected for testing. Twelve primer pairs generated easily scorable amplification products of the expected size while the others showed no amplification, multi-banding patterns, or too pronounced stutters. To confirm marker usability and characterize the selected twelve SSR markers for variation and presence of null alleles, a total of 44 individuals from two Russian and one Finnish populations were analysed (Table 1). Ten out of the twelve selected nuSSRs displayed consistent and polymorphic patterns.

Table 1 Primer sequences and characteristics of the 10 selected polymorphic nuclear microsatellite markers in P. sylvestris
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Amplification reactions were carried out following the PCR method by Schuelke (2000) in a final volume of 10 μl containing 30 ng of template DNA, 1× PCR buffer, 0.2 mM of each dNTP, 1 U of GoTaq polymerase (Promega, Madison, WI), 1.5 mM of MgCl2, 0.2 μM of the reverse and the M13 universal primer (the latter labeled with FAM, NED, VIC or PET to the 5′ end), and 0.07 μM of the modified forward primer with the M13 primer sequence (18 bp) added at its 5′ end. The PCR profile was: denaturation at 94°C for 4 min followed by 35 cycles at 94°C (30 s), 55°C (30 s), 72°C (40 s), and a final step at 72°C for 8 min. Amplification reactions were carried out in a GeneAmp PCR system 9700 (Applied Biosystems, Foster City, CA) and amplified products were run in an ABI 3130xl automatic sequencer (Applied Biosystems, Foster City, CA). Electropherograms were analyzed using GeneMapper v4.0 (Applied Biosystems, Foster City, CA). Primer sequences, repeat motifs, and GenBank accession numbers are shown in Table 1.

Standard genetic diversity parameters and departure from Hardy–Weinberg equilibrium (HWE) were estimated using GENALEX, v.6 (Peakall and Smouse 2006). Null allele frequencies were estimated using FreeNA (Chapuis and Estoup 2007), and linkage disequilibrium between pairs of loci, applying Bonferroni corrections, using FSTAT 2.9.3.2 (Goudet 2002).

Considering the three populations separately, the overall number of alleles per locus ranged from 1 to 6 (Table 1). Expected heterozygosity ranged from 0.095 to 0.785 in KAR population, from 0.000 to 0.698 in LAD population, and from 0.000 to 0.772 in PUN population (Table 2). Psyl17 and psyl16 loci showed significant HWE departures in KAR and PUN populations, respectively, probably due to a moderate presence of null alleles. In fact, the estimated frequency of null alleles was very low (<5%, but generally <1%) with the only exceptions of psyl16 (19.7%) in PUN population and psyl17 (17.6%) in KAR population (Table 2). No significant linkage disequilibrium among loci was detected (P < 0.05).

Table 2 Genetic diversity parameters, deviation from HW equilibrium and frequency of null alleles for the three natural populations of P. sylvestris
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Because of the high polymorphism, almost no deviation from HWE due to low null alleles frequency, and amenability to score in multiplex reactions, these markers are likely to be valuable tools for population genetic studies, in particular to elucidate fine-scale population structure and demographic history, and for parentage assignment in P. sylvestris.