Genipa americana L. (Rubiaceae) is widespread throughout Brazil and tropical America, both in plantations and in the wild (Durigan and Nogueira, 1990). It is a dioecious tree species (Mielke et al., 2003) and is used in forest gallery restoration (Salvador, 1986) and in mixed plantings of swampy and degraded permanent preservation areas, because of its high tolerance to flooding (Lorenzi, 2002). As a result of habitat fragmentation and damage caused by human activities, the remaining natural populations of G. americana currently occur in small forest fragments and protected parks. This represents a potentially serious long-term threat to global biodiversity. Thus, it is necessary to understand the effects of forest fragmentation on the genetic diversity and structure, mating system, and gene flow of the species for conservation and sustainable use. Therefore, assessment of genetic studies using molecular markers is urgently needed, particularly for the conservation of genetic resources of the remaining populations. For this purpose, the development of microsatellite markers for the species is useful, due their high level of polymorphism, and will create new opportunities for conservation research that can be used to minimize the negative implications of population fragmentation. In this study, we created a set of 17 polymorphic microsatellite loci for G. americana that were confirmed to be reproducible in assessing its genetic diversity, mating system, and gene flow.
METHODS AND RESULTS
Fresh G. americana leaves were collected from a single individual to be used in the initial DNA extraction, using the protocol proposed by Doyle and Doyle (1990). A microsatellite-enriched genomic library was constructed following the protocol by Billotte et al. (1999), with slight modifications. Genomic DNA was digested with RsaI enzyme (Invitrogen, Carlsbad, California, USA) for 3 h incubation at 37°C, and the digested fragments were enriched in microsatellite fragments using (CT)8 and (GT)8 motifs. Digested fragments were then ligated to the double-stranded Rsal adapters Rsa21 (5′-CTCTT-GCTTACGCGTGGACTA-3′) and Rsa25 (5′-TAGTCCACGCGTAAGCAA-GAGCACA-3′) for 2 h incubation 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 PCR, the product was cloned into pGEM-T Easy Vector (Promega Corporation), and ligation products were used to transform Epicurian Coli XL1-Blue Escherichia coli–competent cells (Stratagene, Agilent Technologies, Santa Clara, California, USA). A total of 192 recombinant colonies were obtained, and 144 were selected and sequenced in an automated ABI 3500xL Genetic Analyzer (Perkin Elmer–Applied Biosystems, Foster City, California, USA) using T7 and SP6 primers and the BigDye Terminator version 3.1 Cycle Sequencing Kit (Perkin Elmer–Applied Biosystems); 65 of these contained microsatellite motifs. Dinucleotide motifs were the most abundant, followed by mono-, tetra-, tri-, and pentanucleotide motifs (approximately 80.49%, 10.97%, 4.88%, 2.44%, and 1.22%, respectively). Oligonucleotides complementary to the genomic sequences flanking the microsatellite region were designed using Primer3Plus (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%. Developed primer pairs were assessed using 40 samples by PCR amplification on a Mastercyler thermocycler (Eppendorf, Hamburg, Germany) in a final volume of 16 µL using GoTaq Colorless Master Mix (Promega Corporation) containing 6.0 µL GoTaq Colorless Master Mix, 10 µM of each primer (F and R), 2.7 µL of nuclease-free water, and 7.5 ng of template DNA. The PCR program for all primers consisted of 5 min of initial denaturation at 95°C followed by 30 cycles of denaturation at 95°C for 45 s, a primer-specific annealing temperature (Table 1) for 1 min, extension at 72°C for 1 min, and a final extension at 72°C for 7 min. The amplification products were checked with 3% agarose gels, separated in 6% denaturing polyacrylamide gels, and visualized by silver-staining. We used a 10-bp DNA ladder (Invitrogen) as a size standard for allele scoring. The number of alleles per locus (A), the observed (Ho) and expected heterozygosities (He), fixation index (F), and genotypic disequilibrium were estimated for each population using the FSTAT program (Goudet, 2002). To test whether the F values and the genotypic disequilibrium were significantly different from zero, we used 1000 Monte Carlo permutations (alleles among individuals) and a Bonferroni correction (95%, α = 0.05).
Characteristics of 32 microsatellite loci developed in Genipa americana.a
Results of initial polymorphic microsatellite marker screening in two populations of Genipa americana.a
For the polymorphism evaluation, we sampled a total of 40 adult G. americana trees from two populations 580 km apart—21 individuals from a small forest fragment in the Ecological Station of Mogi-Guaçu (São Paulo State, Brazil) and 19 individuals from a natural population in Selvíria (Mato Grosso do Sul State, Brazil). Of the 32 designed primer pairs, 17 were polymorphic and 15 were monomorphic in the analyzed populations. In the Selvíria population, among the polymorphic microsatellites, the number of alleles ranged from three (Gam01 and Gam31) to eight (Gam06 and Gam24), with an average of 4.58 alleles per locus (Table 2). In the Mogi-Guaçu population, the number of alleles ranged from two (Gam36) to seven (Gam 11), with an average of 4.58 alleles per locus (Table 2). For the Selvíria population, Ho and He ranged from 0.43 to 0.89 and from 0.56 to 0.75, respectively, and in Mogi Guaçu these ranged from 0.24 to 1.00 and from 0.22 to 0.78, respectively. The Hardy–Weinberg equilibrium was tested in both populations. All loci were in disequilibrium (except the locus Gam06 in the Selvíria population and the average populations), most likely due to genetic drift caused by forest fragmentation.
The molecular markers described here are the first microsatellite loci isolated for G. americana. The microsatellite loci showed median levels of polymorphism in the two populations analyzed. Based on this, our data suggest that the developed microsatellite markers may constitute new tools for population genetic studies, such as genetic diversity, spatial genetic structure, mating system, and gene flow of G. americana. These studies will produce valuable information for managing fragmented populations, including information for breeding, conservation, and reforestation plans.
 This research was supported by the Fundação de Amparo à Pesquisa do Estado da São Paulo (FAPESP; 2010/19613-4) and by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq; 473677/ 2010-5). The author would like to thank FAPESP for financial support provided to R.O.M. (scholarship no. 2011/01518-8).