Isolation and characterization of microsatellite loci in Sorbus porrigentiformis and cross-amplification in S. aria and S. rupicola (Rosaceae)1

This is an open access article distributed under the terms of the Creative Commons Attribution License (CC-BY-NC-SA 4.0), which permits unrestricted noncommercial use and redistribution provided that the original author and source are credited and the new work is distributed under the same license as the original. British whitebeams (Sorbus aria aggr.; Rosaceae) are an em-blematic case study of polyploid evolution in natural tree populations. Southwestern Britain is a " Sorbus hotspot, " with ca. 30 polyploid species (3x, 4x, and even 5x), many of them occurring at just a few localities and therefore highly valuable in regard to conservation. Recent studies have provided detailed knowledge of the morphology and ploidy levels in British populations of Sorbus L. (Rich et al., 2010; Pellicer et al., 2012). Despite this effort, some essential questions regarding the evolution of the complex remain unsolved. In addition to the sexual diploid species S. aria (L.) Crantz s. str., current evidence points at the polyploid S. porrigentiformis E. F. Warb., an endemic to the United Kingdom that shows a distribution significantly larger than the other highly endemic polyploids of the complex, as a parental species of many of these polyploid endemics. The apo-mictic tetraploid S. rupicola (Syme) Hedl., widely distributed in northwestern Europe, including the United Kingdom, may have also been involved. To provide diagnostic alleles for the three species, we isolated and characterized the first set of microsatellites for S. porrigentiformis and tested cross-amplification in S. aria and S. rupicola. Previous studies on Sorbus in southwestern Britain have used two nuclear microsatellites from apple (Malus Mill. sp.) and three from S. A DNA library was generated for one sample of S. porrigentiformis and se-quenced on a Roche/454 GS FLX platform (454 Life Sciences, a Roche Company , Branford, Connecticut, USA). From the 35,638 reads, 10,872 microsatellite loci were detected. Primer pairs were designed with the software QDD (Meglécz et al., 2010) using default parameters (90–320 bp PCR products, with more than five repeats of 2–6 bp motifs, 18–27 bp primer length, 57–63°C annealing temperature). We tested 20 of the primers on seven geographically separated individuals of S. porrigentiformis (Appendix 1). Fluorescent labeling was performed using three primers per locus: a reverse primer, a forward primer with a universal linker sequence (M13) at the 5′ end, and a third primer consisting of the same universal M13 sequence, labeled with 6-FAM or JOE (Schuelke, 2000). We added …