Characterization of eighteen novel microsatellite markers and multiplex PCR protocol for Fagus sylvatica
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TECHNICAL NOTE
Characterization of eighteen novel microsatellite markers and multiplex PCR protocol for Fagus sylvatica Andrea R. Pluess • Kirsti Ma¨a¨tta¨nen
Received: 21 September 2012 / Accepted: 28 September 2012 / Published online: 6 October 2012 Ó Springer Science+Business Media Dordrecht 2012
Abstract Eighteen novel microsatellite markers for European Beech, Fagus sylvatica, were developed using next-generation sequencing technique. Subsequently, four multiplex PCRs were established for the fast and costeffective use of the primers. In 60 individuals, we found 3–12 alleles per locus, an expected and observed heterozygosity of 0.445–0.821 and 0.250–0.867, respectively. Fixation index was significant in three loci. Yet, if these measures were jointly estimated with the probability of null alleles, these loci and an additional two indicated low occurrence of null alleles while the overall fixation index was non-significant. The loci were not in linkage disequilibrium. Overall, these markers will be useful for population genetic research to support management decisions for the preservation of this species in changing environmental conditions. Keywords European beech Fagaceae nSSR Population genetics
A. R. Pluess (&) K. Ma¨a¨tta¨nen Department of Environmental Systems Science, Swiss Federal Institute of Technology (ETH Zurich), Universitaetstrasse 16, 8092 Zurich, Switzerland e-mail: [email protected]
Introduction The distribution range of Fagus sylvatica (Fagaceae), a nowadays widespread forest tree, is expected to shrink drastically due to climate change (Meier et al. 2011) as this species is vulnerable to long drought periods, a forecasted weather change (European Environmental Agency 2004). Recent studies showed adaptive variation to water availability (Rose et al. 2009; Pluess and Weber 2012) suggesting that pre-adaptive genetic variation might be available for a continuation of beech stands. To assess its micro-evolutionary potential, a thorough understanding of its gene exchange across landscapes is needed. Therefore, we established 18 novel microsatellite loci while other authors recently re-evaluated loci of the literature and also developed some new ones (Lefevre et al. 2012). Fagus sylvatica is a diploid, wind-pollinated, mainly outcrossing (Merzeau et al. 1994) tree with primarily gravity dispersed nutlets. The generation time is [40 year (Hess et al. 1967) limiting its evolutionary potential to rapid changes. For its conservation, there is a call for the development of seed transfer guidelines and gene reserves (von Wuehlisch 2008). Leaf genomic DNA was extracted from 60 individuals originating from three regions of Switzerland with two stands per region (Table 1) using DNeasy-Plant Mini and 96 Kits (Qiagen, Venlo, The Netherlands). A microsatellite enrichment library was created by ‘‘ecogenics’’ (Zu¨rich, Switzerland) on a Roche 454 Genome Sequencer FLX using the Titanium Sequencing Kit (Roche Life Sciences, Branford, CT, USA). A total of 842 reads with microsatellite inserts were
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