Begonia L. is a diverse tropical genus with over 1500 species. Evolutionary research has focused on the early-diverging African species (e.g., Hughes and Hollingsworth, 2008) and the more derived Asian species (e.g., Thomas et al., 2011), with the American species largely overlooked. The most recent common ancestor of Central American Begonia is likely to be relatively recent (Miocene; Dewitte et al., 2011), and subsequent speciation has resulted in high species richness (total c. 690 species; Goodall-Copestake et al., 2010). Population studies of Central American Begonia species will shed light on the evolution of species richness in a morphologically diverse group of neotropical herbs; but to date, studies have been limited by the availability of suitable nuclear markers to complement plastid microsatellite markers (Twyford et al., 2013).
In this study, we describe the development of nuclear microsatellite markers to study gene flow within and between Central American Begonia species. This requires markers that amplify over a broad phylogenetic scope, which can then be cross amplified in divergent species.
METHODS AND RESULTS
Microsatellite markers were designed from the transcriptome sequence of vegetative meristem tissue from B. plebeja Liebm., a related species from Begonia sect. Gireoudia (European Nucleotide Archive Sequence Read Archive accession number: ERP001195; Brennan et al., 2012). The QDD bioinformatic pipeline (Meglécz et al., 2010), which integrates microsatellite detection, a redundancy check to avoid amplifying multiple PCR products, and designs primers, was used according to Lepais and Bacles (2011). A FASTA file of the B. plebeja transcriptome sequence assembly was analyzed in QDD version 1.3 using default parameters: selecting only primers that amplify a PCR product between 90 and 320 bp in length, with a repeat motif of 2–6 bp repeats, and a minimum length of four repeat units. To make microsatellite amplification in other species more likely, primers were excluded if they did not have a perfect BLAST match to the transcriptome of B. conchifolia A. Dietr. (sect. Gireoudia; Brennan et al., 2012). Reads from which the primers were designed were BLAST searched against the Arabidopsis Information Resource (TAIR) database ( http://www.arabidopsis.org) to investigate the putative function of each locus.
Thirty-one primer pairs detected in QDD were tested for amplification in B. heracleifolia Cham. & Schltdl. and B. nelumbiifolia Cham. & Schltdl. These species were chosen because they are two of the most widespread Begonia species in a genus of mostly rare endemics (Hughes and Hollingsworth, 2008). The species are known to hybridize (Burt-Utley, 1985), facilitating studies of species boundaries. Primer amplification was tested in seven individuals of the two species (Appendix 1). A subset of polymorphic markers that amplified reliably in both species was then tested for multiplex compatibility by mixing equimolar ratios of each primer. The PCR multiplexes were then tested on a population of each species (20 individuals) to estimate the genetic diversity of the markers. The primer sequences were BLAST searched against the transcriptome sequence of the divergent Asian species B. venusta King (sect. Platycentrum) to test for likely cross-amplification of primers in other Begonia species.
Approximately 15 mg of silica-dried leaf material was extracted using DNeasy 96-sample kit (QIAGEN, Germantown, Maryland, USA). To overcome an Twyford et al.—Begonia microsatellites unknown PCR inhibitor that coelutes with DNA extractions in Begonia, extractions were diluted 100-fold with Millipore dH2O to a final DNA concentration of ∼0.1–1.5 µg/mL. PCR reactions were performed using the M13-tailed primer method (Schuelke, 2000) in a final reaction volume of 10 µL containing: 0.5 µL of 1 mM M13-tailed forward primer (Invitrogen, Grand Island, New York, USA), 1 µL reverse primer (1 mM), 1 µL of 1 mM M13 fluorescently modified primer (6-FAM,VIC, NED, PET), 0.25 µL bovine serum albumin (BSA, 0.4%), 1 µL of 10× reaction buffer, 1 µL of 2 mM dNTPs, 0.6 µL of 25 mM MgCl2, 0.05 µL BIOTAQ polymerase (Bioline, London, United Kingdom), 1 µL dilute DNA template, and made up to the final volume using dH2O. PCR cycles consisted of an initial denaturation of 1 min at 95°C, followed by 40 cycles of denaturation for 1 min at 95°C, annealing for 1 min at 57°C, and extension for 1 min at 72°C. Five microliters of each PCR product labeled with the four fluorescent dye colors was pooled and diluted 2× in Millipore dH2O, and the GeneScan 500 LIZ internal size standard (Applied Biosystems, Foster City, California, USA) was added prior to fragment analysis on the ABI 3730xl analyzer (Applied Biosystems; analysis was performed at GenePool, University of Edinburgh, Edinburgh, United Kingdom). Fluorescent traces were analyzed automatically with manual editing using GeneMapper version 4.0 (Applied Biosystems).
A total of 136 primer pairs were located in the B. plebeja transcriptome using the QDD bioinformatic pipeline (Appendix 2). All 31 of the subset of primers tested for amplification yielded a PCR product (Table 1). Sixteen loci had a significant (<E-5) BLAST match in the TAIR database (Table 1). Of these loci, four loci were monomorphic (BI6701, BC402, BI6294, and BI7247) and one amplified multiple PCR products (BI3377). Two PCR multiplex reactions were designed to amplify a total of 15 polymorphic loci (Table 1). All loci were polymorphic in at least one of the populations tested, and showed moderate genetic diversity, with the number of alleles per species ranging from one to five and the expected within-population heterozygosity between 0 and 0.75 (Table 2). Twenty-one of the 62 primers (34%) had perfect BLAST matches in the transcriptome of the divergent B. venusta, including both the forward and reverse primers for loci BI3348, BC932, and BC552.
CONCLUSIONS
We have described the development of nuclear microsatellite primers that amplify in two divergent Central American Begonia species. Some of the primers have exact BLAST matches in the transcriptome of the Southeast Asian species B. venusta and, therefore, may be transferable more widely across the genus. The transferability of markers is important for the study of natural hybrids, and the development of a multiplexed assay of 15 loci should enable accurate assignment to hybrid classes (e.g., F1, backcross). Future studies will use these loci to estimate the genetic structure of populations, the frequency of hybrids, and the extent of introgression in hybrid swarms.
TABLE 2.
Genetic diversity in population samples of Begonia heracleifolia and B. nelumbiifolia.
