Encholirium Mart. ex Schult. & Schult. f., together with Deuterocohnia Mez, Dyckia Schult. & Schult. f., Fosterella L. B. Sm., and Pitcairnia L'Hér., belongs to Bromeliaceae, subfamily Pitcairnioideae (Givnish et al., 2011). Encholirium is an exclusively Brazilian genus, with a distribution closely associated with inselbergs, in caatinga, cerrado, and Atlantic Forest phytogeographic domains. Of the 27 species described in this genus, only E. horridum L. B. Sm. and E. gracile L. B. Sm. are endemic to the Atlantic Forest (Forzza et al., 2013). Encholirium horridum has large populations distributed mainly in Espírito Santo State, extending to southern Bahia, eastern Minas Gerais, and northern Rio de Janeiro (Forzza et al., 2013). In 2008, the species was indicated as Data Deficient in the Official List of the Endangered Species of the Brazilian Flora (Ministério do Meio Ambiente, 2008).
Inselbergs are isolated rock outcrops that can be considered terrestrial islands (Porembski and Barthlott, 2000), where the species usually exhibit spatial isolation and restricted gene flow (Barbará et al., 2008; Boisselier-Dubayle et al., 2010). These features may allow E. horridum populations from these habitats to evolve separately, favoring long-term processes of speciation. To understand possible genetic patterns of these particular habitats, microsatellite markers were developed to assess genetic diversity, genetic structure, and gene flow between populations of E. horridum from different inselbergs.
METHODS AND RESULTS
A microsatellite-enriched library was constructed according to the method described by Billotte et al. (1999). For this purpose, genomic DNA from fresh leaves of an individual of E. horridum (Appendix 1) was extracted following Doyle and Doyle (1990). The DNA sample was digested with the RsaI restriction enzyme (Invitrogen, Carlsbad, California, USA), and the fragments were then linked to RsaI adapters. The library was enriched for dinucleotide sequences, using biotinylated (CT)8 and (GT)8 bound to Streptavidin MagneSphere Paramagnetic Particles (Promega Corporation, Fitchburg, Wisconsin, USA). Enriched fragments were amplified in 100-µL final volume containing 20 µL of selected fragments, 1× Taq buffer, 1.5 mM MgCl2, 200 µM dNTPs, 4 pmol of primer Rsa21, and 2.5 U of Taq DNA polymerase. A PTC-100 thermal cycler (MJ Research, Waltham, Massachusetts, USA) was used for PCR with the following program: 95°C for 1 min, followed by 25 cycles of denaturation at 94°C for 40 s, 60°C for 1 min, extension of 72°C for 2 min, and a final extension of 72°C for 5 min. The amplicons were cloned into a pGEM-T Easy Vector (Promega Corporation) and transformed into Escherichia coli XL1-Blue competent cells. Positive clones were selected by the expression of β-galactosidase gene and sequenced in an automated ABI 377 sequencer (Applied Biosystems, Foster City, California, USA) using T7 and SP6 primers and the BigDye Terminator version 3.1 Cycle Sequencing Kit (Applied Biosystems). Chromatograms were assembled and edited using SEQUENCHER 4.9 (Gene Codes Corporation, Ann Arbor, Michigan, USA). Microsatellites were identified using the Simple Sequence Repeat Identification Tool (SSRIT; Temnykh et al., 2001). Of the 96 clones sequenced, 34 contained simple sequence repeat (SSR) motifs. It was not possible to design suitable primers harboring SSR regions for four of these clones, because these regions were either at the beginning or end of the sequences. Primer pairs were designed using Primer3Plus software (Untergasser et al., 2007).
Characteristics of 10 polymorphic microsatellite markers developed for Encholirium horridum.
Evaluation of 30 primer pairs was performed by PCR in a Veriti Thermal Cycler (Applied Biosystems), in 10-µL final volume containing 10 ng of template DNA, 1× Taq buffer (Bioline, Tauton, Massachusetts, USA), 2.0 mM MgCl2 (Bioline), 100 µM dNTPs (Fermentas, Glen Burnie, Maryland, USA), 2 pmol of each forward and reverse primer, and 0.25 U of Taq DNA polymerase (Bioline). The following program was used: 95°C for 3 min, followed by a first step consisting of 10 cycles of denaturation at 94°C for 30 s, at the specific annealing temperature (Ta; Table 1) for 30 s, extension at 72°C for 30 s, and a second step consisting of 20 cycles of denaturation at 90°C for 30 s, at the Ta (Table 1) for 30 s, extension at 72°C for 30 s, and a final extension at 72°C for 60 min. Eight samples from four populations located at the outermost geographic boundaries of species distribution were used to evaluate amplification success and loci polymorphism, respectively, on 1.5% and 3.0% agarose gels stained with GelRed (Biotium, Hayward, California, USA). Those loci with detected polymorphism were reamplified using forward primers labeled with fluorescence (FAM, NED, VIC, PET; Applied Biosystems; Table 1) and were resolved in an ABI 3500xL Genetic Analyzer (Applied Biosystems) using GSLIZ600 as the size standard (Applied Biosystems). Loci amplifications were made in single PCRs and were then multiplexed for fragment analysis. Fragment size and allele identification were determined using GeneMapper version 4.1 software (Applied Biosystems). For polymorphism evaluation, we used 89 individuals from 11 populations located along the species distribution (Table 2). These samples were dried in silica gel, and the genomic DNA was extracted using a NucleoSpin Plant II Kit (Macherey-Nagel, Düren, Germany). Genetic diversity parameters were estimated using FSTAT version 220.127.116.11 (Goudet, 2002). Out of the 30 microsatellite loci isolated, 18 were successfully amplified by PCR, of which 10 were polymorphic in the tested samples (Table 1). The number of alleles for the polymorphic loci ranged from eight to 20, with an average of 14.7. The observed and expected heterozygosities ranged from 0.000 to 1.000, and from 0.000 to 0.929, respectively, across the populations, with an average of 0.345 and 0.433 (Table 2).
Cross-species amplification was evaluated in the five genera of subfamily Pitcairnioideae (Appendix 1). Genomic DNA from dried leaves in silica gel was extracted using a DNeasy Plant Mini Kit (QIAGEN, Valencia, California, USA). The PCR protocol used was the same as described above. In general, cross-amplification was high (Table 3). All of the 10 primer pairs amplified successfully in E. gracile, while for the other four species the success of amplification varied from 40% to 60%.
The markers presented here will be useful in evaluating genetic diversity, spatial genetic structure, analysis of gene flow by paternity, and characterization of mating system of E. horridum. Results of cross-amplification indicate that this set of primers could be promising chiefly in studies with other species of Encholirium, but also with other Pitcairnioideae.
Results for primer screening of polymorphic loci in samples from 11 populations of Encholirium horridum. a
Transferability of 10 microsatellite markers developed for Encholirium horridum across species of Bromeliaceae, subfamily Pitcairnioideae.a
 The authors would like to thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq process numbers 472146/2009-2) and Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ process number E26/102.206/2009) for financial support, and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Centra Nacional de Conservação da Flora (CNC Flora) for a fellowship granted to K.V.H. M.S.R. and R.C.F. are CNPq research fellows.