The Brazilian rosewood, Aniba rosaeodora Ducke (Lauraceae), is a slow-growing hardwood tree that occurs in nonflooded forests of the Amazon rainforest. The species has its highest population density in central Amazonia, Brazil. It is considered one of the most valuable nontimber forest products (NTFP) in Brazilian Amazonia, and is mainly used as source for essential oil (linalool) in perfumes and aromatherapy (May and Barata, 2004). The oil is extracted almost entirely from the wood, and current extraction methods are destructive to the tree. Due to its high economic value and low-cost unsustainable oil extraction methods, natural stocks of A. rosaeodora have been severely depleted. The species is considered endangered and was listed in Appendix II of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) in 2010. Effective strategies for the conservation and management of this resource should be urgently implemented, taking into consideration ecological and genetic data, among other factors. DNA microsatellite markers (simple sequence repeats [SSRs]) have been widely recognized as highly informative, and are frequently used in population and conservation genetics studies of tropical forest tree species. However, despite its high social and economic value, no microsatellite markers are available for A. rosaeodora or its congeners. Here, we report on the isolation and characterization of 11 highly variable microsatellite loci for A. rosaeodora, aiming to characterize population genetic diversity and structure, gene flow, and mating system of this threatened species. These are important factors in determining how genetic variation is distributed among populations and should be considered in long-term conservation and management strategies for this valuable tropical tree species.
METHODS AND RESULTS
For the construction of an enriched library, total genomic DNA was extracted from silica gel–dried leaves collected from an adult tree (voucher no.: INPA 208904) sampled in a natural population of A. rosaeodora located at Ducke Forest Reserve, Manaus, Amazonas (2°53′18″S, 59°58′18″W). For population genetic analysis, total genomic DNA was extracted from 68 adult trees sampled from this population and a second location at Maués, Amazonas (3°22′82″S, 57°43′13″W) (voucher no.: INPA 237807), both in central Amazonia, Brazil. A standard cetyltrimethylammonium bromide (CTAB) protocol (Doyle and Doyle, 1987) was used for DNA extraction.
The microsatellite loci were isolated and identified from the library enriched for CA repeats following the protocol described in Farias et al. (2003). Approximately 10 µg of extracted genomic DNA was digested with Sau3AI and the appropriate lengths of 200–900 bp were ligated to double-stranded linkers, Er1Bh1Blunt-5′-CGGAATTCAGTGGATCCTGCC-3′ and Er1Bh1 GATCSticky-5′-GCCTTAAGTCACCTAGGACGGCTAG-3′. Magnetic beads linked to streptavidin (Dynal, Life Technologies, Grand Island, New York, USA) were used to select fragments containing microsatellites hybridized to the 5′ biotinylated probe (GA)10. Enriched DNA was ligated into pGEM-T Easy Vector (Promega Corporation, Madison, Wisconsin, USA) and transformed into Escherichia coli strain DH5α. Transformed cells were grown and screened according to the manufacturer's recommendation, with PCR insert screening based on the T7 and Sp6 primers. PCR products were purified and sequenced with T7 and Sp6 primers using the BigDye Terminator version 3.1 Cycle Sequencing Kit (Life Technologies). The amplified products were electrophoresed on an ABI Prism 377 sequencer (Life Technologies) following the manufacturer's instructions.
Microsatellite repeats were found in 72 of 196 sequenced clones using TROLL software (Castelo et al., 2002). Primer pairs complementary to regions flanking microsatellite repeats were successfully designed for 40 clones using Primer3Plus version 1.1.4 (Untergasser et al., 2007). The remaining 32 sequences containing microsatellites did not allow optimal primer design. Optimization of PCR conditions was performed for each locus using total genomic DNA extracted from four adult trees of A. rosaeodora. These individuals were sampled from the same population selected for SSR loci development. PCRs contained 10 ng of DNA template, 0.9 µM of each primer, 1 U Taq DNA polymerase, 200 µM of each dNTP, 0.25 µg of bovine serum albumin (BSA), and 1× reaction buffer (10 mM Tris-HCl [pH 8.3], 50 mM KCl, 1.5 mM MgCl2) in a total volume of 13 µL. PCRs were performed on a Veriti Thermal Cycler (Life Technologies) with the following cycling conditions: 96°C for 2 min; followed by 30 cycles of 94°C for 1 min, 44°C to 58°C (according to the annealing temperature of the primer) for 1 min (Table 1), 72°C for 1 min; and a final elongation step of 72°C for 10 min. The PCR products were visualized in 3% agarose gel containing 0.1 µg/mL of ethidium bromide in TBE buffer (89 mM Tris-borate, 2 mM EDTA [pH 8.3]) and sized with a 1-Kb DNA ladder (Life Technologies).
For a preliminary polymorphism analysis, loci with clear and robust amplified products in the agarose gel were later resolved on 4% denaturing polyacrylamide gel electrophoresis (PAGE). The gel was stained with silver nitrate (Creste et al., 2001) and sized by comparison to a 10-bp DNA ladder standard (Life Technologies). Eleven SSR loci produced clearly interpretable and polymorphic bands in PAGE. The forward primer of each pair was fluorescently labeled with 6-FAM, HEX, or TET fluorescent dye for genotyping on an ABI Prism 377 sequencer (Life Technologies). Allele sizes were scored against an internal GeneScan 500 TAMRA size standard (Life Technologies). Individuals were genotyped using GeneScan Analysis version 3.1 and Genotyper version 2.5 softwares (Life Technologies).
Characteristics of 11 microsatellite loci developed for Aniba rosaeodora.
Eleven polymorphic SSR loci were characterized (Table 1) by genotyping 68 adult individuals of A. rosaeodora sampled from the two populations. GenAlEx version 6.1 (Peakall and Smouse, 2006) was used to estimate total number of alleles (A), observed (Ho) and expected (He) heterozygosities, and Wright's F-statistics (i.e., inbreeding within populations [FIS], total inbreeding [FIT], and genetic diversity among populations [FST]) for each locus and overall means. We also tested the statistical power and informative content of the 11 SSR loci by inferring genetic identity (I) (Taberlet and Luikart, 1999) and paternity exclusion (Q) (Weir, 1996). The paternity analysis considers exclusion probabilities to exclude a random individual from paternity, which are based on the allele frequencies of a locus. Considering n independent loci, Qi is the exclusion probability for one locus (i), and the combined probability of exclusion (Q) equals 1 − [Π (1 − Qi)] for the set of loci (Weir, 1996). The linkage disequilibrium between loci was estimated using GENEPOP version 4.0.7 (Raymond and Rousset, 1995).
The number of alleles observed per locus over two populations of A. rosaeodora ranged from six to 14. Ho varied from 0.400 to 0.811 and He ranged from 0.611 to 0.884 (Table 2). All pairwise tests for linkage disequilibrium among the 11 SSR loci were nonsignificant. Three (Ar18, Ar30, and Ar33) and six (Ar05, Ar18, Ar23, Ar29, Ar33, and Ar39) loci deviated from expectations for Hardy–Weinberg equilibrium in the Manaus and Maués populations, respectively, after Bonferroni correction (adjusted P value = 0.0045). The probabilities of genetic identity per locus ranged from 0.012 to 0.080, and the combined probability of genetic identity (I) was 6.38 × 10−17. The paternity exclusion probabilities ranged from 0.594 to 0.843, and the combined probability (Q) was 0.9999995, clearly indicating that these loci allow very precise individual discrimination.
The high allelic variability and individual discrimination power found for the 11 microsatellite loci allow application of these markers as powerful tools for future studies on population genetics of A. rosaeodora. We aim to use these markers to estimate gene flow and to characterize population genetic diversity and structure and mating system of the species. These data will contribute to effective strategies for the conservation and management of this threatened Amazonian forest resource.
Population genetic parameters estimated per SSR locus over two populations of Aniba rosaeodora from central Amazonia, Brazil.
 This work is part of R.C.A.'s Master's thesis at INPA's graduate program in Genetics, Conservation, and Evolutionary Biology. The authors thank Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)/ Brazil (CT-AMAZONIA no. 553225/2005-7 to L.A.S.C., Master's fellowship to R.C.A., and research fellowship to M.R.L.) and the European Commission (SEEDSOURCE Project, contract no. 003708 to M.R.L.) for financial support.