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

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[…] A standard cetyltrimethylammonium bromide (CTAB) procedure () was used for extracting genomic DNA from fresh leaf tissue of one individual V. minor plant of garden origin (VM_454_01; see ). Library preparation and shotgun pyrosequencing of a 5-μg DNA aliquot on a 454 GS-FLX Titanium instrument (Roche Diagnostics, Rotkreuz, Switzerland) were performed as described in . 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 (). SciRoKo 3.4 software () was applied to search for perfect SSRs, accepting minimum thresholds of seven repeat units for di-, six for tri-, five for tetra-, and four for penta- and hexanucleotide repeats, respectively. A total of 1371 nonredundant SSRs were present in 24,886 unique sequences, with di- and trinucleotide repeats being almost equally abundant (47.4% and 46.9%, respectively). In a complementary approach, we applied the same SSR search criteria to 723,230 publicly available cDNA sequences (average length = 536 bp) derived from 454 sequencing of the V. minor transcriptome (deposited in GenBank by January 2011; accession number SRX039641). After assembly, a total of 25,253 perfect SSRs were detected within 267,199 unigenes. Trinucleotide repeats were most abundant within the assembled cDNA collection (63.4%), with (ACT)n being the most common motif (22.0%).Thirty-five SSR loci from the genomic 454 data (ngVm01–ngVm35) as well as 60 SSR loci from the cDNA collection (Vimi01–Vimi60), all specifying single, perfect di-, tri-, tetra-, penta-, or hexanucleotide repeats, were arbitrarily selected for primer design using the BatchPrimer3 interface (). For primer construction, we used the following criteria: length ranging from 18 to 23 nucleotides (20 as the optimum), PCR product size ranging from 100 to 300 bp, annealing temperature from 50°C to 70°C (55°C as the optimum), and GC content between 30% and 70% (50% as the optimum). PCR amplifications were performed in 10-μL final volumes using a T-Gradient thermocycler (Biometra, Göttingen, Germany), following the indirect labeling procedure described by . Each assay contained approximately 20 ng of DNA in 1× PCR MangoTaq buffer (Bioline, Taunton, Massachusetts, USA), 5 μg bovine serum albumin (BSA), 1.5 mmol/L MgCl2, 0.2 mmol/L of each dNTP, 0.1 units of Taq DNA polymerase (MangoTaq, Bioline), 0.04 μM forward or reverse primer carrying a 5′-M13 tail, 0.16 μM of M13 forward or reverse primer labeled with fluorescent 5′-IRDye700 or 5′-IRDye800 (Metabion, Martinsried, Germany), and 0.16 μM unlabeled forward or reverse primer, respectively. The cycling conditions described by were used for all PCRs.All primer pairs were initially tested for successful PCR amplification in five V. minor individuals (including accession VM_454_01 as a positive control and one sample each from four different populations; ) on 0.8% agarose gels. Thirty-two primer pairs yielded distinct bands on agarose, and PCR fragments from these loci were separated on denaturing 6% polyacrylamide gels in 1× TBE buffer, using an automated sequencer (Li-Cor 4300 DNA Analyzer; Li-Cor Biosciences, Lincoln, Nebraska, USA). Fragment sizes were scored manually as previously described (). Eighteen primer pairs yielded distinct polymorphic single or double bands within the expected size range. Locus characteristics, primer sequences, and GenBank accession numbers are summarized in . They were used for genotyping 40 V. minor plants from four populations, each with n = 10 (). Total DNA was extracted from dried leaf material using the CTAB procedure described above. Two populations were from the native range in northern Italy, and two from the introduced range in Germany.Allele numbers and observed and expected heterozygosity values were determined with Arlequin 3.5.1.2 (). Results are summarized in . All 18 loci proved to be polymorphic, exhibiting two to 11 alleles per locus among the 40 V. minor plants. In the Italian samples, observed and expected heterozygosities ranged from 0.1 to 1 and from 0.189 to 0.868, respectively (). Extremely low levels of genotypic diversity and a pronounced heterozygote excess were found in the two populations from the introduced range, indicating a high degree of clonality (). Overall, 105 alleles were detected with a strongly uneven distribution between the native and the introduced range (): 62 alleles were only found in the Italian populations, whereas 17 alleles were restricted to Central Europe. Twenty-six alleles were shared between the two regions.The potential for cross-species amplification of the 18 SSR primer pairs was determined with one accession each of V. major L., V. herbacea Waldst. & Kit., and V. difformis Pourr. (Appendix 1). Primer transferability was considered successful when either one or two distinct bands in the expected size range were detected after polyacrylamide gel electrophoresis. Following these criteria, success rates ranged from zero to 100% with a mean of 35.2%. Eight loci (ngVm05, ngVm21, ngVm24, Vimi25, Vimi33, Vimi34, Vimi39, and Vimi43) amplified in one to three species included in the sample set (). […]

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