Novel microsatellite markers for the high-alpine Geum reptans (Rosaceae)1

• Premise of the study: Geum reptans reproduces by outcrossing or by the formation of stolons. Sexual and clonal reproduction are not exclusive and occur mostly simultaneously. We developed novel microsatellite primers for this species, which will be used in a study about local adaptation, phenotypic plasticity, and random molecular divergence of alpine plants. • Methods and Results: Twelve microsatellite primer sets were developed for G. reptans, of which nine were polymorphic. Initially, the forward primers had an M13 tail, and the allelic signals of each locus were amplified using a single fluorescent-labeled M13 forward sequence. In the running phase, a multiplex PCR assay was developed using different fluorophore-labeled forward primers. Two to 11 alleles were found per locus, depending on the studied population. • Conclusions: Identical multilocus genotypes (i.e., clonal offspring) were not found because individuals in our sampling were at least 4 m distant from each other. FST–QST analysis will be applied to detect selection processes in populations of G. reptans across the Alps.

It is generally assumed that alpine plants are locally adapted due to strong selection in habitats characterized by severe climatic conditions and high environmental heterogeneity ( Körner, 2003 ). However, this assumption has rarely been tested. Our research uses reciprocal transplantation experiments (RTE) and sibling analyses to estimate the degree of phenotypic plasticity vs. the degree of local adaptation in populations of several alpine plant species ( Via, 1984 ;Kawecki and Ebert, 2004 ). Because each transplantation site of an RTE is also a common garden, phenotypic differentiation of plant functional traits can be compared to neutral genetic differentiation based on microsatellite data (i.e., F ST -Q ST analysis). This allows the deduction of the strength and direction of past selection on the measured traits and, therefore, allows testing for local adaptation ( Spitze, 1993 ;Scheepens et al., 2013 ).
One of our focal alpine plants is Geum reptans L. (Rosaceae, Rosidae). Geum reptans is a diploid, perennial rosette plant usually found in high-alpine scree fi elds and in glacier forelands. The plant uses sexual reproduction to produce viable seeds, and also expands through vegetative aboveground stolons ( Fig. 1 ; Weppler et al., 2006 ). Selfi ng provides no viable seeds in G. reptans , probably due to gametophytic self-incompatibility as in other species of the Rosaceae (see Rusterholz et al., 1993 ). Cross-amplifi cation of microsatellite primers developed for the lowland rosid G. urbanum L. has been reported in seeds of G. reptans ( Arens et al., 2004 ); however, trans-species amplifi cation was not successful in our laboratory despite our use of the PCR protocol described by Arens et al. (2004) . Hence, we have developed new polymorphic microsatellite loci in G. reptans to analyze neutral genetic differentiation within our study populations.

METHODS AND RESULTS
We sampled three geographically distinct populations from the Swiss Alps (Davos: 46 ° 44 ′ 47.46 ″ N,9 ° 56 ′ 47.70 ″ E;Furka: 46 ° 33 ′ 24.28 ″ N,8 ° 24 ′ 45.40 ″ E;Zermatt: 45 ° 59 ′ 16.69 ″ N,7 ° 40 ′ 40.73 ″ E). Tissue samples from young leaves of 20 randomly selected individuals were collected from each population in summer 2012. Sampled individuals were separated by at least 4 m to minimize the risk of resampling identical clones ( Pluess andStöcklin, 2004 , p. 2014). Leaf material was stored in silica in the collection of the University of Basel, section of Population Biology of Plants. Microsatellite marker development was outsourced to Ecogenics GmbH (Zurich-Schlieren, Switzerland). The genome screening technique of Ecogenics has been described previously ( Kesselring et al., 2013 ). The total of 26,603 reads had an average length of 178 bp, and 2222 of these reads contained a microsatellite insert with a tetra-or trinucleotide of at least six repeat units or a dinucleotide of at least 10 repeat units. Suitable primer design was possible in 309 reads.
To fi nd allelic polymorphisms, a test sample of N = 15 individuals was used. For the screening of loci for polymorphism and PCR functionality, a PCR strategy (see details in Kesselring et al., 2013 ) that involved M13-tailing at the 5 ′end of each forward primer was used ( Schuelke, 2000 ). Finally, nine out of 12 loci provided suffi cient allelic polymorphisms and robust PCR characteristics ( Table 1 ) . To facilitate subsequent genotyping, the PCR of the nine loci were combined in three multiplex assays using fl uorophore labeling ( Table 1 ). For each locus, a fraction of the forward primer was labeled with a fl uorophore and complemented with nonlabeled forward primer and reverse primer to a concentration of 10 μ M primer master mix (see Table 1 , footnote 1). Subsequently, 20 individuals from each of the three populations (Davos, Furka, Zermatt) were tested (a total of N = 60 individuals, see Table 2 ) . PCR was done in a fi nal volume of 10 μ L, and contained 1 μ L PCR stock buffer (QIAGEN, Hilden, Germany) with 15 mM MgCl 2 and 200 μ M dNTPs, 0.3 μ L primer master mix • Premise of the study: Geum reptans reproduces by outcrossing or by the formation of stolons. Sexual and clonal reproduction are not exclusive and occur mostly simultaneously. We developed novel microsatellite primers for this species, which will be used in a study about local adaptation, phenotypic plasticity, and random molecular divergence of alpine plants. • Methods and Results: Twelve microsatellite primer sets were developed for G. reptans , of which nine were polymorphic. Initially, the forward primers had an M13 tail, and the allelic signals of each locus were amplifi ed using a single fl uorescentlabeled M13 forward sequence. In the running phase, a multiplex PCR assay was developed using different fl uorophore-labeled forward primers. Two to 11 alleles were found per locus, depending on the studied population. • Conclusions: Identical multilocus genotypes (i.e., clonal offspring) were not found because individuals in our sampling were at least 4 m distant from each other. F ST -Q ST analysis will be applied to detect selection processes in populations of G. reptans across the Alps.
Key words: alpine scree; clonal reproduction; ECO500 size marker; Geum reptans ; multiplex PCR; Rosaceae . of each of the three loci, 0.5 unit HotStar Taq polymerase (QIAGEN), and 2-10 ng DNA. Cycling conditions were: denaturation at 95 ° C for 15 min, followed by start PCR at 94 ° C for 30 s, 56 ° C for 90 s, and 72 ° C for 60 s in 35 cycles. Final elongation was set to 72 ° C for 30 min. After PCR, samples were mixed with ECO500 size standard (provided by Ecogenics) and loaded on an ABI3730 sequencer (Applied Biosystems, Carlsbad, California, USA). This size marker is suitable for accurate sizing in the range of 50-500 bp. ECO500 was labeled with orange DY -630 dye (Dyomics GmbH, Jena, Germany) and comprised the following base pair fragments: 75,102,124,148,171,207,229,260,274,311,321,349,374,395,419,455,473, and 497 bp. Allelic assignment of the electropherograms was done with GeneMarker version 1.80 (SoftGenetics, State College, Pennsylvania, USA). Data were cross-checked for repeatability. Eight of the 60 individuals were retested, starting again with DNA extraction of the silica gel-dried leaves and microsatellite fi ngerprinting. Hence, 72 fi ngerprints (i.e., 8 samples · 9 loci) of the fi rst run were opposed to 72 fi ngerprints of the repetition run. Just one pairwise comparison differed, which is an error rate of 1.4%. The error occurred due to inconsistent allelic assignment of an individual at locus 015615. Instead of being heterozygous, the individual was interpreted in the repetition run as homozygous. Two loci showed background noise (loci 015967 and 013998; Table 1 ), i.e., we interpreted the constant occurrence of an additional peak as a mismatch. Moreover, at locus 013998 an allele of 151 bp was found to occur at a frequency of 5%. This allele was binned with the common allele of 150 bp because of potential stuttering (see Table 1 ). The same was done with the 144-bp allele (frequency of 8%; binning with 143 bp) at locus 002235 ( Table 1 ). The polished data set was written in GENEPOP format ( Rousset, 2008 ). Three software packages were used: GENEPOP on the Web for general index calculations and tests on linkage disequilibrium ( Rousset, 2008 ; http://genepop.curtin.edu.au/), MICRO-CHECKER ( van Oosterhout et al., 2004 ) for tests on potential null alleles with a prior value of maximum allele length of 250 bp and a 95% confi dence limit, and GenAlEx version 6.2 ( Smouse et al., 2008 ;Beck et al., 2008 ) for fi nding identical multilocus clones.
Two to 11 alleles were found per locus, depending on the studied population ( Table 2 ). Observed ( H o ) and expected heterozygosities ( H e ) were in good agreement overall, indicating sexual outcrossing and random mating of alleles. However, three loci showed obvious deviation between H o and H e ( Table 2 ). The presence of null alleles was suggested by MICRO-CHECKER for loci 002235, 007389, and 011721 in some populations ( Table 2 ). The independent evolution of the microsatellite loci was tested with linkage analysis. There was no linkage disequilibrium among pairs of loci across all populations (all P > 0.09). In a further step, we searched for identical multilocus genotypes because of clonal reproduction by stolons. Six of the 60 individuals had to be excluded from the analysis because they had missing values at some loci (000-allele code in GENEPOP). We did not fi nd identical multilocus genotypes in the 54 remaining individuals, although the establishment of clonal offspring by stolons of G. reptans is common and was estimated to range between 53% and 74% ( Weppler et al., 2006 ).

CONCLUSIONS
The new microsatellite markers described here proved to be valuable tools to perform population and landscape genetics studies in the clonal plant G. reptans , for parental analysis or further investigations of its breeding system. Observed and expected heterozygosities were in good agreement, indicating random mating of alleles and sexual outcrossing; however, null alleles might occur at some loci. Given the absence of identical multilocus genotypes, we assume that our sampling design was successful in avoiding clonal individuals and indicate that clonal offspring of G. reptans establish only right next to their 'mother' plants. In the near future, we will examine microsatellite data of G. reptans to identify neutral genetic differentiation across the Alps. Contrasting molecular differentiation with differentiation in fi tness-related phenotypic traits of reciprocally transplanted populations ( F ST -Q ST analysis) should allow detection of selection and local adaptation.