Carpotroche brasiliensis (Raddi) A. Gray (Flacourtiaceae), an endemic species from the Brazilian Atlantic Forest and popularly known as sapucainha, produces nutritious fruits that are rich in medicinal substances. The fruits, which are high in fat and mineral residues (Pinto et al., 2012), are dispersed by important forest fauna such as paca (Agouti paca) and agouti (Dasyprocta agouti) (Zucaratto et al., 2010). The oil extracted from its seeds, known as chaulmoogra oil, is of high economic value because it is used for medicinal and cosmetic purposes. In Camamu-Maraú County, State of Bahia, Brazil, farmers grow C. brasiliensis and Theobroma cacao L. (cocoa) in the shade of native canopy tree species. Therefore, C. brasiliensis is an important economic agricultural crop in this agroforestry system. Furthermore, it is a valuable species for maintaining diversity and species richness in the agricultural-ecological landscape (Bhagwat et al., 2008).
In this study, we developed microsatellite (i.e., simple sequence repeat [SSR]) markers because they are considered to be the most polymorphic class of markers, and can usually be treated as neutral (Tautz, 1989; Weber, 1990). As a result, they are often useful for population genetic studies. The advent of next-generation sequencing (NGS) allows whole genomes to be mined very efficiently to identify SSRs. Here, we report on the first set of microsatellites for C. brasiliensis, which is important for understanding the genetic behavior for breeding purposes of this commercially and ecologically valuable species.
METHODS AND RESULTS
Fresh C. brasiliensis leaves were collected from two populations (Pau Coco and Santa Rita) in Camamu-Maraú County, State of Bahia, Brazil. To design and characterize the developed markers, we sampled a total of 32 trees (16 individuals from each population). Sampling coordinates, voucher numbers, and Universidade Estadual de Santa Cruz (UESC) DNA Bank codes are provided in Appendix 1. The DNA was isolated using the cetyltrimethylammonium bromide (CTAB) extraction protocol (Doyle and Doyle, 1990). The company ecogenics GmbH (Zurich, Switzerland) enriched libraries for AG and AC motifs and performed next-generation sequencing (Roche 454 pyrosequencing platform [454 Life Sciences, a Roche Company, Branford, Connecticut, USA]) with high-quality DNA that was extracted from the leaves of a single specimen. After sequencing, we designed microsatellite primers using the software Primer3 (Rozen and Skaletsky, 1999).
We performed PCR using 7.5 ng of genomic DNA, 1.3 µL of 10× buffer (10 mM Tris [pH 9.0], 50 mM KCl, and 1.5 mM MgCl2), 20 mM MgCl2, 3.25 mM each dNTP, 3.6 mg bovine serum albumin (BSA), 1 unit of Taq DNA polymerase (Phoneutria, Belo Horizonte, Minas Gerais, Brazil), 3.9 mM primers (forward, reverse, and M13 tail [CACGACGTTGTAAAACGA]), and 1.43 mM tail-complementary primer labeled with fluorochromes (6-FAM, VIC, PET, or NED [Applied Biosystems, Foster City, California, USA]). The amplification reactions were carried out under the following conditions: an initial step at 94°C for 5 min; followed by 30 cycles of 94°C for 45 s, annealing temperature (54–58°C, see Table 1 for details of each primer) for 45 s, and 72°C for 1 min; followed by eight cycles of 72°C for 1 min, 53°C for 1 min, and 72°C for 1 min; with a final extension step at 72°C for 10 min. In this study, all of the amplification reactions were verified on 1.5% agarose gels before capillary electrophoresis, which was performed on an ABI 3500 Genetic Analyzer (Applied Biosystems).
Characteristics of 30 Carpotroche brasiliensis microsatellite loci.
SSR allele peaks were detected using GeneMarker version 1.95 (SoftGenetics, State College, Pennsylvania, USA). The number of alleles (A), observed heterozygosity (Ho), expected heterozygosity (He), fixation index (F), and gametic disequilibrium were calculated using FSTAT version 220.127.116.11 (Goudet, 1995). The combined probability of exclusion (Pe) and combined probability of identity (PID) were calculated using CERVUS 3.0.3 (Marshall et al., 1998). Departure from Hardy-Weinberg equilibrium (HWE) was estimated using GenAlEx 6.5 (Peakall and Smouse, 2012).
Number of designed primers and next-generation sequencing results for Carpotroche brasiliensis library synthesis.
A total of 380 sequences were generated, of which 33.4% had microsatellites. Thirty primer pairs were designed, 25 of which were polymorphic, with expected fragment sizes and patterns of amplification. NGS results, including the number of designed primers, information about amplicon size, and repeat length, are available in Table 2. The description of primers and characterization of each locus are provided in Table 1. Six types of repeat motifs were identified in the primer set. The dinucleotide motif AC/TG had the highest percentage of occurrence, followed by AG/TC (Fig. 1); this indicates that, unexpectedly (Morgante and Olivieri, 1993), the AG motif seems to be the second most common in the C. brasiliensis genome.
The 25 studied loci had a maximum of 12 alleles per locus. Estimates of Ho consistently exceed those of He, indicating an excess of heterozygotes as shown by a negative fixation index. We found significant differences in number (193 alleles in the Pau Coco population and 174 alleles in the Santa Rita population) and frequency of alleles between the analyzed populations. Additionally, we discovered private alleles in both populations (Fig. 2). For CA_3 and CA_21 (Fig. 2B and 2D), all of the alleles from the Santa Rita population were different from those that were found in the Pau Coco population. The allele frequency distribution was different for most of the loci. The Pau Coco population showed deviation from HWE in four loci (CA_1, CA_13, CA_21, and CA_32). In contrast, in the Santa Rita population, we observed deviations from HWE for three SSR loci (CA_7, CA_10, and CA_12) (Table 3). We did not observe gametic disequilibrium between pairs of loci, indicating that the entire set can be used for population and quantitative genetic studies. The average combined Pe was greater than 0.999, valorizing the developed molecular markers as well as the values obtained for the combined (1.4 × 10−29 and 7.3 × 10−27 for the Pau Coco and Santa Rita populations, respectively) (Table 3).
The economic and ecological interest in C. brasiliensis makes studies related to population and quantitative genetics imperative for future advances in conservation and breeding programs for this species. The genetic characterization of these 25 new microsatellite loci indicates the accuracy of these new genetic tools for paternity tests, which can be used in studies of gene flow. Our results based on the distribution of allelic frequencies highlight the importance of selecting the best loci for each analytical purpose. For example, in ongoing research we are comparing the distance of gene flow occurring in natural and crop populations (agroforestry) to propose a management design to maximize the admixture between these populations. The planned distribution of C. brasiliensis plants in an agroforestry population will help both in the conservation of local fauna and in genetic diversity, which is important to start a breeding program for this species. Additionally, the microsatellite primers developed here can be applied to investigations of genetic structure and can also be transferred to other Carpotroche species. In short, these molecular markers will be useful for improving the knowledge of this important forest resource, which provides food for midsize fauna and economic support for families reliant on farming in southern Bahia, Brazil.
Genetic diversity statistics of 25 polymorphic Carpotroche brasiliensis microsatellite loci.a
 The authors thank Jose Lima, Fernando Santana, and Holei Silva for field and laboratory assistance, and Dr. Kent E. Holsinger (Associate Editor) and an anonymous reviewer for valuable suggestions on an earlier version of this paper. This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq; grant no. 487030/2012-5), by Natura Inovação e Tecnologia de Produtos, and by fellowships from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for F.B. and from CNPq for F.A.G.