Swartzia glazioviana (Taub.) Glaz. (Fabaceae) is an endemic legume tree species that occurs on the slopes of coastal hills with sandbanks in the Região dos Lagos, Rio de Janeiro, Brazil (Mansano and Lima, 2007; Bohrer et al., 2009). Swartzia glazioviana is considered a threatened species (Ministério do Meio Ambiente, 2014) due to its rare distribution and limited occurrence in very disturbed and fragmented habitats, which may compromise the viability of their populations as evolutionary units. To our knowledge, there are no detailed studies on the ecology, reproductive biology, and population genetics for this species that could contribute effectively to its conservation. Microsatellites (or simple sequence repeats [SSRs]) are considered the marker of choice for population genetic studies due to their codominant nature, high information content (polymorphisms), and reproducibility (Litt and Luty, 1989; Selkoe and Toonen, 2006). They are ideal markers to investigate the genetic diversity and structure, mating system, and gene flow of tree populations (Ashley, 2010), including those considered endangered species, such as S. glazioviana. Here we present the development and characterization of a set of highly polymorphic microsatellite markers developed for S. glazioviana for use in population genetic and conservation studies of this endemic species from the Brazilian Atlantic Forest.
METHODS AND RESULTS
For the library construction, total genomic DNA was extracted from dried leaves previously collected from a single adult of S. glazioviana. The genomic library was enriched for the repeat motifs CT and GT using biotinylated probes with streptavidin-coated magnetic beads according to Billotte et al. (1999). The total genomic DNA extraction followed a modified cetyltrimethylammonium bromide (CTAB) method (Doyle and Doyle, 1990; Ferreira and Grattapaglia, 1998).
The total genomic DNA was cleaved using AfaI restriction enzyme (Invitrogen, Carlsbad, California, USA), and the fragments were ligated to Afa21 (5′-CTCTTGCTTACGCGTGGACTA-3′) and Afa25 (5′-TAGTCCACGC GTAAGCAAGAGCACA-3′) adapters. The linked fragments were amplified via PCR and purified using QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany). Selected fragments were reamplified using primer Afa21, cloned using pGEM-T Easy Vector (Promega Corporation, Madison, Wisconsin, USA), and transformed into Escherichia coli (XL1-Blue) competent cells by electroporation. The transformed cells were grown in Luria–Bertani medium containing ampicillin and IPTG/X-Gal and incubated. Recombinant-positive clones were selected and grown on Circlegrow (MP Biomedicals, Santa Ana, California, USA) and plasmid DNA extracted. Sequencing reactions used T7 or Sp6 universal primers and BigDye Terminator Cycle Sequencing Kit (version 3.1; Life Technologies, Grand Island, New York, USA) and sequencing was performed on an AB1377XL sequencer (Applied Biosystems, Waltham, Massachusetts, USA) following the manufacturer's instructions. Primer pairs complementary to the flanking regions of the repeats were designed using Primer3 software (Rozen and Skaletsky, 1999). Of 96 positive sequenced clones, 38 (∼40%) showed repetitive elements of which ca. 70% were dinucleotide CT or GA. Of these, 20 (52.6%) sequences were appropriate for primer design. The loci were initially amplified using six DNA samples of S. glazioviana from the three populations. PCRs were performed in a final volume of 13.0 µL containing: 2.0 µL of ultrapure water, 1.4 µL of 10× reaction buffer (NH4), 1.0 µL MgCl2 (50 mM), 1.2 µL bovine serum albumin (BSA; 2.5 mg/mL), 0.8 µL dNTPs (250 mM), 4.3 µL of unlabeled primers (0.9 mM of each), 0.3 µL Taq Polymerase (5 U/µL), and 2.0 µL of DNA template (2.5 ng/µL) in a Veriti thermocycler (Life Technologies). The following PCR program was used: one cycle at 95°C for 5 min; 30 cycles at 94°C for 1 min, at primer annealing temperature for 1 min, and at 72°C for 1 min; and a final extension cycle at 72°C for 10 min. Ten out of 20 markers that showed specific and robust PCR products in agarose gels were chosen, and the forward primer of each pair was fluorescently labeled (6-FAM, VIC, and NED). The PCR mixture contained a final volume of 10.0 µL with: 1.6 µL of ultrapure water, 1.0 µL of 10× reaction buffer (NH4), 2.0 µL of MgCl2 (50 mM), 2.0 µL BSA (2.5 mg/mL), 0.8 µL dNTPs (250 mM), 0.3 µL of primers (10 mM), 0.3 µL of Taq DNA Polymerase (5 U/µL), and 2.0 µL of DNA template (2.5 ng/µL). The PCR program was as follows: one cycle at 94°C for 5 min; 30 cycles at 94°C for 1 min, at annealing temperature of each primer pair for 1 min, and at 72°C for 1 min; and a final cycle at 72°C for 60 min.
Characteristics of 10 microsatellite markers developed for Swartzia glazioviana.
In total, 96 individuals were sampled (32 per population) from three populations of S. glazioviana located in Arraial do Cabo (22°58′07″S, 42°01′00″W) and Cabo Frio (22°52′21″S, 41°58′53″W; 22°51′20″S, 41°59′38″W), Rio de Janeiro, Brazil (voucher numbers in Appendix 1). A batch of 96 adult individuals (32 per population) of S. glazioviana was used for SSR loci screening (Table 1). The products were analyzed in a 3500XL DNA sequencer (Life Technologies) following the manufacturer's instructions. Alleles were sized using GeneScan 600 LIZ Size Standard (Life Technologies) and GeneMapper 4.0 software (Life Technologies). We estimated total and average number of alleles per locus (A) and observed (H o) and expected (He ) heterozygosities according to the proportions of Hardy–Weinberg equilibrium, for each and overall loci. Inbreeding was quantified by estimating the coefficient of inbreeding (F), and the statistical significance of the F values was tested by Monte Carlo permutation resampling alleles among individuals. We also tested the occurrence of genotypic disequilibrium between pairwise loci using a Bonferroni correction. All estimates were performed using FSTAT (Goudet, 1995). We also inferred the discriminatory power of the SSR loci by estimating the probabilities of genetic identity (I) for each locus and combined (IC) overall loci according to Taberlet and Luikart (1999), and the probabilities of paternity exclusion (Q) for each locus and the combined probability (QC) for all loci (Weir, 1996).
Genetic diversity and coefficient of inbreeding estimated for three populations of Swartzia glazioviana in the central coast of Rio de Janeiro, Brazil, based on the analysis of 10 microsatellite loci.
Cross-species amplification analysis for 10 microsatellite markers developed for Swartzia glazioviana and tested in six congeneric species.a
The number of alleles per locus ranged from four to 12 (mean 8.5). Mean H o was 0.642 (0.290–0.969) and H e ranged from 0.424 to 0.894 (mean 0.776). Mean F was significantly greater than zero for two of the populations (Table 2). QC was 0.999995 (0.213–0.477), and IC was 2.245 × 10-14 (0.022–0.120), across the 10 loci, clearly showing the high individual discrimination power of the loci (Table 2). After Bonferroni corrections, significant genotypic disequilibrium was detected between only two pairwise loci (Sgl15xSgl19 and Sgl15xSgl20).
Amplification of the 10 SSR loci developed for S. glazioviana was tested for six congeners: S. apetala Raddi var. apetala, S.flaemingii Raddi, S. langsdorffii Raddi, S. macrostachya Benth., S. myrtifolia Sm. var. elegans (Schott) R. S. Cowan, and S. simplex (Sw.) Spreng. var. continentalis Urb. We found a high cross-species transportability rate (68%) of the 10 loci across the six Swartzia species. The success amplification rates of the loci for each species were as follows: S. apetala var. apetala (90%), S. flaemingii (45%), S. simplex var. continentalis (40%), S. langsdorffii and S. macrostachya (80%), and S. myrtifolia var. elegans (60%) (Table 3).
The 10 SSR loci isolated for S. glazioviana showed high levels of genetic diversity. The IC and QC estimates also indicate the highly informative and discriminatory power of these loci, which are important for their use in parentage analysis. The results also highlight the potential of the developed microsatellite markers for estimating population genetic parameters in six other Swartzia species.
The 10 polymorphic markers reported here will be useful tools for estimating pollen dispersal, mating system, genetic diversity, and population genetic structure and will provide relevant information for the effective population preservation of this threatened tree species.
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 The authors thank Tarlile Barbosa Lima, Cassio Flavio Fonseca de Lima, Elton John de Lírio, and Vivian Almeida de Assunção for help with field collections and Ricardo de Oliveira Manoel, Karina Hmeljevski, and Eline Martins for laboratory assistance. This study was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES; 562280/2010-3), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq; 203439/2014-7), and Fundaçao de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ; E26/111.351/2010).