Lady’s slipper orchid (Cypripedium calceolus L.) is one of the largest and the most spectacular elements of European flora. This Euro-Asiatic species has suffered a marked decrease of localities and area occupied (Terschuren 1999). It is legally protected in all European countries, and listed in various national conventions and directives (Terschuren 1999). Studies of genetic diversity and fine-scale spatial genetic structure in relation to habitat management will improve management strategies of this vulnerable species. Two, out of four recently published microsatellite loci for C. calceolus, may pose scoring problems (Pedersen et al. 2012). Therefore a higher number of variable loci is needed to estimate genetic structure and distinguish close relatives. Here we report the isolation and characterization of polymorphic loci that will be useful in future population genetic studies.

Seeds of C. calceolus were collected from population Bukówki (Poland) and asymbiotically germinated in in vitro culture. Total genomic DNA was isolated from three seedlings using procedure described by Bekesiova et al. (1999). Extracted DNA was used for a library preparation with a NEBNext® DNA Library Prep Master Mix Set for Illumina. The sequencing was performed on the MiSeq Benchtop Sequencer (Illumina) using the 2 × 250 bp read mode. The obtained data (10,19 Mega reads) was assembled using CLCGenomicWorkbench (CLCBio) into 513,225 contigs and the microsatellites were then detected using QDD 2.1 Beta (Meglecz et al. 2010). A total of 22,162 contigs contained at last one microsatellite of which 53 loci were selected for initial screening. We screened 32 plants from two Polish populations (Bukówki and Prokowo) for polymorphisms at these loci. All forward primers were tagged with M13(-21) (5′-TGTAAAACGACGGCCAGT-3′) at the 5′ end. The 10 µL PCR volume contained: 4.5 µL MyTaq™ HS Mix (Bioline), 0.4 µM of both forward and reverse primers, 0.2 µM dye labelled primer, 0.3 % DMSO, 1.2 µL water and 1 µL DNA template (~50 ng). The following PCR conditions were used: 2 min initial denaturation at 96 °C, followed by 35 cycles of 95 °C for 30 s/primer specific annealing temperature for 30 s/72 °C for 45 s. Next, the reaction was paused at 72 °C and fluorescently labelled (6-FAM, NED, PET or VIC) M13 primer was added. Amplification was then continued for the next 10 cycles according to Arruda et al. (2010) with 15 min of final extension at 72 °C. Samples were run on an ABI 3730 DNA Analyzer and analyzed with GeneMarker 2.2.0 (SoftGenetics) using GS-500 (LIZ) as a size standard.

The number of alleles per locus, departures from Hardy–Weinberg equilibrium (HWE), and heterozygosity for two Polish populations were calculated in GenAlEx (Peakall and Smouse 2012). The presence of linkage disequilibrium (LD) was tested in Arlequin (Excoffier and Lischer 2010). Presence of null alleles and scoring errors were checked using Micro-Checker version 2.2.3 (Van Oosterhout et al. 2004). The statistical significances in multiple statistical tests were adjusted by the Bonferroni corrections (Rice 1989). A set of 18 SSR was polymorphic. Three of them showed evidence for presence of null alleles and were excluded. Remaining loci showed no evidence of LD after sequential Bonferroni correction. Number of alleles per locus for remaining 15 SSR ranged from 2 to 8 (Table 1) with mean of 4.3. In the studied populations, the mean NA ranged from 3.27 to 3.6. Observed (H o) and expected (H e) heterozygosity ranged from 0.19 to 0.89 and 0.12 to 0.71 respectively. None of the loci showed deviation from Hardy–Weinberg equilibrium. The 15 polymorphic SSR loci reported in this study will be useful for assessing genetic diversity, population structure and parentage analysis.

Table 1 Characteristics of 15 loci for Cypripedium calceolus
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