Dalechampia scandens L. (Euphorbiaceae) is a neotropical twining vine native to Mexico, Central America, and South America (Webster and Armbruster, 1991). The species presents a complex hermaphroditic pseudanthial blossom (clusters of female and male flowers forming flowerlike structures). Attached to the male flowers is a resin-producing gland, which secretes resin for pollinator reward (Armbruster, 1984, 1985). Blossoms are self-compatible and often self-pollinate during a bisexual phase. The main floral visitors are species of resin-collecting bees from the Apidae or Megachilidae family. Because of the attractive floral complex and specialized pollination system, Dalechampia L. species have been used to study the evolution and selection of pollination systems and floral characters (e.g., Armbruster, 1985; Armbruster et al., 2009; Bolstad et al., 2010; Pélabon et al., 2012).
Molecular data provide invaluable information to address many ecological and evolutionary questions. Nuclear ribosomal DNA and cpDNA have previously been used to investigate the phylogenetic relationships among Dalechampia species (Armbruster et al., 2009). Inter-simple sequence repeat (ISSR) markers have also been used to analyze the effects of genetic distances between parents on progeny fitness-related traits in D. scandens interpopulation crosses (Pélabon et al., 2005). DNA sequence data may be difficult to apply on intraspecific studies in plants due to low levels of polymorphisms, and ISSR markers have sometimes been shown to yield low reproducibility. Microsatellites (simple sequence repeats [SSRs]) are highly reproducible genetic markers often showing appreciable levels of polymorphisms that have been successfully applied to study a broad spectrum of biological questions. The aim of this study is to identify genomic regions harboring microsatellite loci and to develop appropriate sequence-tagged site markers in D. scandens.
METHODS AND RESULTS
Four natural populations of D. scandens—Ciudad del Carmen (CC: n = 20, 18°56′29″N, 91°18′01″W), Cozumel (CO: n = 10, 20°22′10″N, 86°59′40″W), Puerto Morelos (PM: n = 11, 20°51′11″N, 86°53′43″W), and Valladolid (V: n = 13, 20°42′31″N, 88°15′06″W)—covering large parts of the species range in Mexico were used in this study (see Appendix 1 for voucher information). The Ciudad del Carmen and Puerto Morelos populations differ from the Cozumel and Valladolid in blossom size, the former populations being characterized by large blossoms compared to the latter. Leaf tissue from the Ciudad del Carmen population was used to identify microsatellite loci and to develop primers for the amplification of these loci. The library construction was performed using restriction enzymes BsaAI and HincII following Hamilton et al. (1999). A double-stranded SNX linker was simultaneously ligated to the ends of these fragments with XmnI. The enrichment of DNA fragments containing microsatellite loci was conducted by different 3′-biotinylated oligonucleotides (dimers, trimers, and tetramers), and streptavidin-coated magnetic beads were used to capture the enriched fragments. PCR of genomic DNA fragments enriched for microsatellites was conducted using SNX forward primer (5′-CTAAGGCCTTGCTAGCAGAAGC-3′). PCR reactions were performed in a total volume of 50 µL containing Platinum Taq polymerase (Invitrogen, Carlsbad, California, USA), 10× PCR buffer, 50 mM MgCl2, 10 mM dNTPs, and 10 µM SNX primer. The thermal profile was set at 94°C for 50 s, 55°C for 45 s, and 72°C for 1 min, for 35 cycles. The PCR products were purified using a QIAquick PCR purification kit (QIAGEN, Hilden, Germany) and were digested with NheI and ligated to plasmid pUC19 (New England Biolabs, Ipswich, Massachusetts, USA). The purified ligation was transformed to Electromax DH5α-E cells (Invitrogen) using an Eppendorf Electroporator 2510 (Eppendorf AG, Hamburg, Germany). After electroporation, 300 µL S.O.C. medium (Invitrogen) at room temperature was added to the mixture and incubated in a 37°C water bath for 1 h, followed by incubation at 4°C for 30 min. Two microliters of each library was mixed with 98 µL S.O.C. medium, and 30 and 60 µL of each mixture were spread on the 90-mm Luria–Bertani (LB)/ ampicillin/X-gal plates followed by incubation at 37°C overnight. A total of 700–900 colonies were plated onto 150-mm LB/agar plates with 50 µg/mL of ampicillin and left to grow at 37°C overnight. Hybridization was performed by placing a Magna Lift (137-mm) nylon transfer membrane (Osmonics, Westboro, Massachusetts, USA) on a 150-mm LB/agar plate using 5× saline sodium citrate (SSC)/0.5% sodium dodecyl sulfate (SDS)/1 mM EDTA/0.1% bovine serum albumin (BSA) buffer and incubated at 50°C for at least 2 h. The radiolabeled probes were hybridized to a membrane, incubated at 50°C, and rotated overnight. The membrane was exposed to Kodak BioMax MR35 × 43 cm single-emulsion film (Carestream Health Inc., Rochester, New York, USA), and the developed autoradiograph was aligned to an LB plate to localize the positive colonies. In total, 109 positive clones containing microsatellite repeats were sequenced with an ABI 3730×l DNA Analyzer using a BigDye Terminator version 3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, California, USA).
Characteristics of 37 microsatellite loci in Dalechampia scandens.
Allelic diversity and observed and expected heterozygosities in 37 microsatellite loci in four Mexican populations of Dalechampia scandens.
Primer pairs (n = 42) for flanking regions of microsatellite repeats were designed by BatchPrimer3 (You et al., 2008). Two individuals from each population were used to test for primer amplification. Microsatellite loci were amplified in a 10-µL reaction containing 2× Type-it Microsatellite PCR Kit (containing HotStarTaq Plus DNA Polymerase, MgCl2, and dNTPs) from QIAGEN, 0.2 µM of each primer, and 10 ng DNA PCR amplification was performed on an ABI 9600 thermal cycler (Applied Biosystems) at 95°C for 5 min; 10 initial cycles as touchdown at 94°C for 30 s, 60-50°C for 45 s, 72°C for 45 s; 25 cycles at 94°C for 30 s, 50°C for 45 s, 72°C for 45 s; and final extension at 72°C for 10 min. For successful markers (n = 39), the 5′ end of the forward primer was fluorescently labeled with different fluorophores (6-FAM, HEX, NED, and PET; Table 1) and fragment analysis was conducted with an ABI 3130xl Genetic Analyzer (Applied Biosystems). Nucleotide sequences for microsatellite clones were deposited in GenBank (accession no.: JX668754– JX668790). Two markers, CCtri5 and CCtri12, were removed from data analyses (data not shown). The estimation of the number of alleles (A), observed (Ho) and expected (He) heterozygosities, and test for Hardy-Weinberg equilibrium were performed using Arlequin version 18.104.22.168 (Excoffier and Lischer, 2010).
In total, 37 microsatellite loci were successfully screened for variability across the four populations of D. scandens (Table 1). Locus CCtri14-2 amplified only in CC and PM populations, and CCtri1 was monomorphic across all individuals/populations. Overall, 166 alleles were identified across all loci/ individuals, with an average of 4.49 (SD = 2.47, range 1–11) alleles per locus (Table 1). The proportion of polymorphic loci varied from 0.38 (for PM) to 0.81 (for V). The average number of alleles across all loci varied between 1.51 (SD = 0.73, for PM) and 2.32 (SD = 1.25, for V; Table 2). The average Ho over all loci ranged from 0.06 (for PM) to 0.59 (for V; Table 2). Similarly, the average He across all loci ranged from 0.14 (SD = 0.21, for PM) to 0.40 (SD = 0.26, for V). Overall, lower levels of genetic variation were found in CC and PM compared with CO and V populations.
The microsatellite markers reported here provide a valuable tool for various kinds of genetic and ecological studies in D. scandens. The present set of markers will be useful in addressing questions about causes of genetic structure, demographic history, phylogeography, and mating systems of natural populations of D. scandens. These markers will be attempted for cross-amplification in closely related species of Dalechampia.
 The authors thank Steve Bogdanowicz (Evolutionary Genetics Core Facility, Cornell University) for technical help and Randi Røsbak (Department of Biology, Norwegian University of Science and Technology) for help in fragment analysis. This project was supported by National Science Foundation grants DEB-0444745 to W.S.A. and DEB-0444157 to TFH.