Development of 18 polymorphic microsatellite markers for Vinca minor (Apocynaceae) via 454 pyrosequencing1

Ap Applicati tions ons in in Pl Plant t Scien Sciences ces The lesser periwinkle (Vinca minor L.; Apocynaceae) is an evergreen subshrub that is native to Southern Europe but has become naturalized in wider parts of Central Europe and North America (Meusel et al., 1978 ; Swearingen et al., 2010). In Germany, V. minor is nowadays mainly found in the surroundings of ancient Roman remains, medieval castle ruins, and abandoned settlements, but is also cultivated (and propagated asexually) in a number of horticultural varieties (Labhart, 2005). It is commonly assumed that V. minor had been introduced to Germany as an ornamental, symbolic, and/or medicinal plant with the expansion of the Roman Empire. The species is therefore considered as a so-called " relic of cultivation " (Prange, 1996 ; Celka, 2011). However, little is known about the origin of the Central European populations and their colonization history. The ability of V. minor to form stolons often results in the formation of compact carpet-like mats (Hegi, 1966). Because this growth form is often an indicator for clonal growth, vegeta-tive reproduction by the expansion of stolons is frequently considered to be the predominant means of propagation for V. minor (Prange, 1996), especially because mature fruits and seeds are rarely observed in populations north of the Alps (Hegi, 1966). However, the relative importance of asexual vs. sexual propagation in V. minor has never been assessed by molecular methods. Microsatellite or simple sequence repeat (SSR) markers are among the most sensitive tools for the evaluation of in-traspecifi c variation and population structure. Here, we present 18 polymorphic SSR loci developed for V. minor using 454 pyrosequencing technology. These markers are important tools for analyzing genetic diversity, population structure , and clonality of V. minor in its native and introduced ranges. METHODS AND RESULTS A standard cetyltrimethylammonium bromide (CTAB) procedure (Weising et al., 2005) was used for extracting genomic DNA from fresh leaf tissue of one individual V. minor plant of garden origin (VM_454_01; see Appendix 1). Library preparation and shotgun pyrosequencing of a 5-μ g DNA aliquot on a 454 GS-FLX Titanium instrument (Roche Diagnostics, Rotkreuz, Swit-zerland) were performed as described in Wöhrmann et al. (2012). A total of 43,565 sequence reads with an average length of 431 bp were obtained, and assembled into unique sequences using Geneious 5.4 (Drummond et al., 2010). SciRoKo 3.4 software (Kofl er et al., 2007) was applied to …