Development and characterization of microsatellite markers for Tibouchina hatschbachii ( Melastomataceae ) , an endemic and habitat-restricted shrub from Brazil

Tibouchina hatschbachii Wurdack (Melastomataceae) is an autogamous shrub restricted to granite (GO) and sandstone (SO) rock outcrops from subtropical Brazil. We designed primers for the amplification of microsatellite regions for T. hatschbachii, and characterized these primers to estimate genetic diversity parameters and contemporary genetic structure patterns. Eight loci were successfully amplified and were characterized using 70 individuals from three natural populations. Polymorphic information content ranged from 0.200 to 0.772 per locus. All loci were polymorphic, with allele numbers ranging from two to eight. The low degree of polymorphism may be explained by the fact that T. hatschbachii has disjunct populations and a recent genetic bottleneck, and also that it is self-pollinated. The observed and expected heterozygosities ranged from 0.115 to 1.000 and from 0.112 to 0.800, respectively. We observed private alleles in all loci. These are important features that enable us to identify population differentiation and help to us understand gene flow patterns for T. hatschbachii in subtropical Brazil. Eight microsatellite loci from other species of Tibouchina amplified positively in T. hatschbachii.


Introduction
Plant population genetic studies have increased substantially in the last years (Collevatti, Castro, Lima, & Telles, 2012;Pinheiro et al., 2014;Reis, Ciampo-Gullardi, Bajay, Souza, & Santos, 2015), mainly with studies on microsatellite loci (Sunnucks, 2000), which are thought to be selectively neutral (Schlötterer, 1998) and display considerable levels of polymorphism and variation.Tibouchina hatschbachii (Melastomataceae) is a shrub endemic to subtropical grassland areas in Brazil.It occurs in granite (GO) and sandstone (SO) rock outcrops, respectively in the Atlantic rain forest and in the southern portions of the Brazilian "Cerrado" (Wurdack, 1963;1984;Meyer, Guimarães, & Goldenberg, 2009).These plants produce many flowers that offer only pollen for their visitors, playing a crucial role as a pollen source for bees in these habitats (Maia, Varassin, & Goldenberg, 2016).A previous study found high levels of autogamy in one population, as a result of a self-compatible reproductive system (Maia et al., 2016).Phylogeographic studies using cpDNA markers have showed genetic and geographical structure with genetically distinct lineages, and a recent reduction of effective population size.However, the contemporary genetic structure patterns that may still be limiting gene flow between lineages remain to be assessed.Moreover, it is still necessary to identify differences in the interpopulation gene flow patterns related to geographical and ecological barriers in the Brazilian subtropical region; and to propose conservation measures considering that T. hatschbachii could undergo drastic habitat reduction due to global climatic changes.The development of primers to evaluate microsatellite loci (SSR) will be useful to address these evolutionary and ecological questions involving contemporary gene flow (Ellegren, 2004).
At present, 24 microsatellite loci are available for the genus Tibouchina (12 loci for T. pulchra, Brito, Vigna, & Souza, 2010; 12 loci for T. papyrus, Telles et al. 2011).Nevertheless, polymorphisms found for one species do not necessarily corresponds to a polymorphism found in loci from other species, even among phylogenetically related species (Ellegren, Primer, & Sheldon, 1995;Oliveira, Pádua, Zucchi, Vencovsky, & Vieira, 2006).This shows how important is the development of specific microsatellite loci for species focused in a study.
In this study, for the first time we isolated and characterized eight pairs of specific primers that amplify polymorphic microsatellite loci of Tibouchina hatschbachii, in order to study population genetic structure and gene exchange among populations.Furthermore, we tested crossamplification (transferability) of 24 microssatelites loci developed for other Tibouchina species in T. hatschbachii.A microsatellite-enriched genomic library was constructed following specific protocol (Billotte, Lagoda, Risterucci, & Baurens, 1999), with a few modifications, using DNA from one individual from PS population.Genomic DNA was digested with Rsa I enzyme (Invitrogen, Carlsbad, California, USA) in incubation at 37°C for 3 hours, and the digested fragments were enriched in microsatellite fragments using (CT) 8 and (GT) 8 motifs.Digested fragments were ligated to the double-stranded AfaI adapters Afa21 (5′-CTCTTGCTTACGCGTGGACTA-3′) and Afa25 (5′-TAGTCCACGCGTA AGCAAGAGCACA-3′) and incubated for 2 hours at 20°C.Hybridized DNA was captured by streptavidin-coated magnetic probe beads (MagneSphere Magnetic Separation Products, Promega Corporation, Madison, Wisconsin, USA).The enriched fragments were amplified by polymerase chain reaction (PCR), the product was cloned into pGEM -T Easy Vector (Promega Corporation), and ligation products were used to transform Epicurian Coli XL1-Blue Escherichia colicompetent cells (Stratagene, Agilent Technologies, Santa Clara, California, USA).

Leaves
A total of 95 positive clones were sequenced using the universal T7 primer combination and a BigDye v3.1 terminator kit on an ABI3730 DNA Analyzer automated sequencer (Applied Biosystems, Foster City, CA, USA).The selection of sequences containing microsatellites was performed using WebSat (Martins, Lucas, Neves, & Bertioli, 2009).Primers were designed using PRIMER3 (Untergasser et al., 2007) according to the following criteria: size of primers preferably between 18 and 22 bp, melting temperature (Tm) between 45°C and 60°C, amplified product length between 100 and 300 bp, and GC content between 40% and 60%.
Twelve microsatellite loci developed for T. pulchra (Brito et al., 2010) and other twelve for T. papyrus (Telles et al., 2011) were tested in T. hatschbachii.The PCR conditions were performed as previously described, but with an annealing temperature of 60°C for all tested primers.The amplifications were visualized in 3% agarose gels.The loci were considered successfully amplified when at least one band of the expected size was observed.A 100bp DNA ladder (Promega) was used as molecular size marker.

Results and discussion
From the 95 recombinant colonies sequenced, 16 clones contained simple sequence repeats, of which eight presented proper flanking regions for primer design (Table 1).Polymorphic information content ranged from 0.200 to 0.772 per locus (average 0.492) and all markers were informative (Table 1).The highest PIC value was found in the That02 and That04 loci, which contained the most alleles (Table 1).All eight designed primer pairs were polymorphic at the analyzed populations (Table 2).Within the populations of Tibouchina hatschbachii, the N A per locus ranged from two to eight, the mean N A per locus was 3.812 and H O and H E varied, respectively, from 0.115 to 1.000 and from 0.112 to 0.800 (Table 2).We also suspect that the low heterozygosity levels may be related to high levels of autogamy found in populations of these species (Frankham, Ballou, & Briscoe, 2008;Silva-Arias, Mäder, Bonato, & Freitas, 2015;Maia et al., 2016).In addition, endemic species with an aggregated distribution pattern tend to have low heterozygosities (Gitzendanner & Soltis, 2000), since they suffer greater influence of genetic drift and inbreeding.In fact, the low diversity values were similar to those found for T. papyrus (N A per locus ranged from one to six), a congeneric and endemic species of "rock fields" ("campos rupestres") in the Brazilian Cerrado (Telles et al. 2011).The disjunct distribution can also be another factor acting on the genetic diversity (Telles et al., 2011).
On the other hand, the allelic richness found in microsatellite loci of T. hatschbachii (ranging from two to eight alleles) was smaller than the one found for T. pulchra (ranging from 4 to 31 alleles; Brito et al., 2010).We believe that this may result from different distribution patterns found for these species.While T. hatschbachii has a restrict and disjunt distribution that may reduce genetic diversity (as shown above), T. pulchra has a broad and continuous distribution along the Atlantic Forest (Meyer, Guimarães, & Goldenberg, 2010), which reduces the effects of the genetic drive and endogamy on these populations.
Except for That06 and That07, all loci deviated from HWE in one or all populations, especially in EEB population, in which four out of eight loci showed this deviation, most likely due to deficiency in heterozygotes found here.No LD was detected between pairs of loci.
Eleven private alleles were found in all the loci (four in the PS population and seven in the EEB population) showing that the two populations are differentiating.These results may explain the divergent lineages found for Tibouchina hatschbachii, but this needs to be tested on a larger sample.Anyway, these are important features that enable us to identify population differentiation (Collevatti et al., 2012;Reis et al., 2015) and can help to us understand contemporary gene flow patterns between genetic lineages identified for T. hatschbachii.
Cross-species amplification tests revealed six microsatellite loci (50%) from T. pulchra (TP01; TP03; TP05; TP25; TP27 and TP33) and two loci (16,66%) from T. papyrus (TPAP 16 and TPAP17) that amplified satisfactorily in T. hatschbachii, indicating that these loci can also be useful for studies on population genetics for other Tibouchina species.However, we did not test polymorphisms for these markers, which means that many of them can be monomorphic and consequently with no use in these studies.The transferability success can also be a consequence of low divergence in DNA sequences among these Tibouchina species, especially with species T. pulchra (50% positive amplification).
This suggests that some markers may be readily transferable between species of the same genus or family (Ellegren et al., 1995;Oliveira et al., 2006;Barbará et al., 2007).

Conclusion
The eight polymorphic loci isolated for T. hatschbachii proved to be useful for studying genetic diversity of this species and will be used to test hypotheses regarding the maintenance of barriers to gene exchange among genetic lineages of T. hatschbachii, that were shown in previous phylogeographic studies using cpDNA markers.Such markers will provide not only knowledge on the genetic diversity and population genetic structure for this species, but also genetic conservation of these stocks in the Brazilian subtropical region.

1
All values are based on 70 samples from three populations of Tibouchina hatschbachii; 2 Fragment sizes does not include the M13 tail (5′-CACGACGTTGTAAAACGAC-3′) attached to the forward primer; 3 Ta = annealing temperature; N A = number of alleles per locus; PIC = polymorphic information content;

Table 2 .
Results of initial polymorphic microsatellite marker screening in three populations (70 individuals) of Tibouchina hatschbachii.Note: N A = number of alleles per locus in each population; F = fixation index; H E = expected heterozygosity; H O = observed heterozygosity; N=sample size for each population; BP: Buraco do Padre; PS: Piraí do Sul; EEB: Estação Ecológica da Barreira; * Deviations from HWE were not statistically significant (ns) or were statistically significant at p ≤ 0.001. .