Markers with different amplicon sizes and similar annealing temperature were identified with Multiplex Manager ( Holleley and Geerts, 2009) and combined in the same multiplex. We set up 12 simplex reactions containing one microsatellite marker and four multiplex reactions containing up to three loci ( Table 1). All DNA extractions were performed with the DNeasy Plant Mini Kit (QIAGEN, Valencia, California, USA). Sixteen primer combinations exhibiting robust amplification were selected ( Table 1). PCR products (0.7 µL) were separated on an AB I 3730 sequencer (Applied Biosystems, Lennik, The Netherlands) with 10 µL of HiDi Formamide and 0.15 µL of GeneScan 500 ROX Size Standard (Applied Biosystems). Amplifications were performed as follows: 94☌ (4 min) 25 or 30 cycles of 94☌ (30 s), 55☌ (45 s), 72☌ (1 min) followed by 10 cycles each of 94☌ (30 s), 53☌ (45 s), 72☌ (45 s) and a final extension at 60☌ for 30 min. 10–50 ng/µL), and H 2O up to a final volume of 15 µL. We added 7.5 µL of Multiplex Mix (10×), 0.2 µL of bovine serum albumin (BSA), 0.3 µL of each reverse primer (10 µM), 0.15 µL of dye-labeled and forward primers (10 µM), 1 µL of template DNA (ca. 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 tested 20 of the primers on seven geographically separated individuals of S. 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☌ annealing temperature). From the 35,638 reads, 10,872 microsatellite loci were detected. porrigentiformis and sequenced on a Roche/454 GS FLX platform (454 Life Sciences, a Roche Company, Branford, Connecticut, USA). torminalis (L.) Crantz ( Robertson et al., 2010 Ludwig et al., 2013).Ī DNA library was generated for one sample of S. Previous studies on Sorbus in southwestern Britain have used two nuclear microsatellites from apple ( Malus Mill. porrigentiformis and tested cross-amplification in S. To provide diagnostic alleles for the three species, we isolated and characterized the first set of microsatellites for S. rupicola (Syme) Hedl., widely distributed in northwestern Europe, including the United Kingdom, may have also been involved. 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. str., current evidence points at the polyploid S. In addition to the sexual diploid species S. Despite this effort, some essential questions regarding the evolution of the complex remain unsolved. Recent studies have provided detailed knowledge of the morphology and ploidy levels in British populations of Sorbus L. 30 polyploid species (3 x, 4 x, and even 5x), many of them occurring at just a few localities and therefore highly valuable in regard to conservation. Southwestern Britain is a “Sorbus hotspot,” with ca. Rosaceae) are an emblematic case study of polyploid evolution in natural tree populations.
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