Terminalia superba Engl. & Diels (Combretaceae) is a Guineo-Congolese African tree naturally distributed from Sierra Leone to the Democratic Republic of the Congo. This pioneer tree is representative of tropical secondary semideciduous forests with an average annual rainfall exceeding 1500 mm and a dry season of less than four months (Orwa et al., 2009). Commonly called limba or fraké, T. superba has been planted for economic purposes in several other countries in Africa, the Americas, and Asia.
With its widespread distribution, T. superba is well suited for studying the response of the African rainforest to past climate changes, especially during the Pleistocene glacial oscillations, during which the rainforest underwent cycles of range contraction and expansion (Plana, 2004). Investigating the phylogeographic pattern, genetic diversity, and demographic history of natural populations requires the development of molecular markers with a high level of polymorphism, such as microsatellites (Frankham et al., 2004). However, to date, no microsatellite resources have been developed in T. superba to decipher its evolutionary history. In this paper, we describe the isolation and properties of 14 new polymorphic microsatellite markers, developed with a lowcost procedure and next-generation sequencing on T. superba. We also attempted cross-amplification in other Terminalia L. species, but without success.
METHODS AND RESULTS
Microsatellite development—Using a cetyltrimethylammonium bromide (CTAB) method (Fu et al., 2005), total DNA was extracted from 30 mg of the leaf of one individual of T. superba (BoD597), from Ghana (Kakum National Park, collected in 2014) (Appendix 1). A DNA genomic library was then prepared, without enrichment, following the protocol of Mariac et al. (2014) and using the Illumina MiSeq platform (San Diego, California, USA) to generate 150-bp paired-end reads (Centre de coopération internationale en recherche agronomique pour le développement [CIRAD] facilities, Montpellier, France). A total of 472,566 reads were obtained, assembled by pair with PANDAseq (Masella et al., 2012), and then analyzed with the software QDD (Meglécz et al., 2014), which detected 7146 loci containing a microsatellite motif, of which 1397 loci had a microsatellite of at least 10 repeats and surrounded by flanking regions suitable to define pairs of PCR primers. From these, we selected 48 loci containing pure (i.e., not compound) microsatellites with at least 10 dinucleotide repeats, with primers situated at least 20 bp from the microsatellite region, and with PCR product length longer than 150 bp. We added to the 5′ end of the forward primer of each locus one of the four possible linkers (Q1–Q4) to label PCR products with distinct fluorochromes (FAM, NED, VIC, and PET; M13-like protocol [Micheneau et al., 2011]).
First amplification tests were performed for each locus on three samples of T. superba: BoD459 (Togo), BoD556 (Ghana), and BoD713 (Nigeria) (Appendix 1). PCR reactions (15 µL) were performed using 1.5 µL buffer (l0×), 0.6 µL MgCl2 (25 mM), 0.45 µL dNTPs (10 mM each), 0.3 µL of each primer (0.2 µM), 0.08 µL TopTaq DNA Polymerase (5 U/µL; QIAGEN, Venlo, The Netherlands), 1.5 µL of template DNA (of ca. 10–50 ng/µL), and H2O. PCR conditions were: 94°C (4 min); 30 cycles of 94°C (30 s), 55°C (45 s), and 72°C (1 min); and a final extension at 72°C (10 min). PCR products were visualized on a 1% agarose gel and stained with SYBR Safe (Invitrogen, Merelbeke, Belgium). Forty-four loci amplified consistently. Their polymorphism was then assessed on seven individuals of T. superba from the Democratic Republic of the Congo, Gabon, Cameroon, Nigeria, Benin, Togo, and Ghana (Appendix 1). Fluorescent labeling was performed by PCR amplification in a total volume of 15 µL, combining: 0.3 µL of the reverse (0.2 µM) and 0.1 µL of the forward (0.07 µM) microsatellite primers with a Q1–Q4 universal sequence at the 5′ end, 0.3 µL of Q1–Q4 labeled primer (0.2 µM each), 3 µL of Type-it Microsatellite PCR Kit (QIAGEN), H2O, and 1.5 µL of DNA. PCR program conditions were: 5-min initial denaturation at 95°C, followed by 30 cycles of denaturation/annealing/extension (95°C for 30 s, 55°C for 90 s, 72°C for 1 min), and a final elongation step at 60°C for 30 min. For each PCR product, 1.5 µL were directly added to 12 µL of Hi-Di Formamide (Life Technologies, Carlsbad, California, USA) and 0.3 µL of MapMarker 500 labeled with DY-632 (Eurogentec, Seraing, Belgium) and genotyped on an ABI3730 sequencer (Applied Biosystems, Lennik, The Netherlands).
Characterization of 14 polymorphic and seven monomorphic nuclear microsatellite loci isolated from Terminalia superba.a
We selected 14 polymorphic and readable loci (seven loci were monomorphic, 17 loci failed to amplify, and six loci were polymorphic but showed uninterpretable amplification patterns). The 14 polymorphic loci were combined in three multiplexed reactions (Table 1) using Multiplex Manager 1.0 software (Holleley and Geerts, 2009). Preliminary population genetic analyses were performed on 42 individuals of T. superba from southeastern Cameroon (Appendix 1). Multiplexed PCRs were carried out using the Type-it Microsatellite PCR Kit (QIAGEN) as follows: 7.5 µL of Multiplex Master Mix, 0.1 µL (0.07 µM) of forward primer and 0.3 µL (0.2 µM) of reverse primer labeled with Q-tailed fluorescent Q1–Q4, 0.3 µL (0.2 µM) of Q1–Q4 primer labeled with 6-FAM, NED, VIC, and PET, 3 µL of 5× Q-solution, H2O, and 1.5 µL of DNA extract. Multiplex PCR conditions were identical to those above, with 27 cycles and annealing temperature of 56°C.
Cross-amplification was also tested on three congeneric species (Appendix 1), including another African forest tree species (T. ivorensis A. Chev., n = 7), an African savanna tree (T. avicennioides Guill. & Perr., n = 2), and a Madagascar native tree (T. mantaly H. Perrier, n = 2), but all of these tests failed (no amplification), indicating that our microsatellites are probably not transferable.
Genetic properties of 14 polymorphic nuclear microsatellite loci developed in Terminalia superba for 42 individuals sampled in southeastern Cameroon.
Microsatellite marker data analysis—Genotypes were analyzed with GeneMapper version 3.7 (Applied Biosystems). Microsatellite profiles were typical of a diploid species, displaying at most two alleles per individual and locus. For each of the 14 loci, allele size range, number of alleles (A) per locus, observed (Ho) and expected (He) heterozygosities, inbreeding coefficient (F), and null allele frequencies (r) were calculated with INEst 1.0 (Chybicki and Burczyk, 2009). Deviation from Hardy-Weinberg equilibrium (HWE) was tested for each locus with SPAGeDi (Hardy and Vekemans, 2002), and linkage disequilibrium tests were performed with GENEPOP 4.1 (Rousset, 2008).
The southeastern Cameroon population of T. superba revealed a high degree of polymorphism, with the number of alleles per locus ranging from two to 27 (mean of 11.43 alleles per locus; Table 2). Regarding the indices of heterozygosity, Ho ranged between 0.143 and 0.902 (Ho was higher than 0.5 for nine of 14 loci) and He ranged between 0.283 and 0.940 (Table 2). Four loci (TS-5, TS-28, TS-42, and TS-44) significantly departed from HWE (Table 2), generally due to the presence of null alleles or a large amount of missing data. After accounting for the effect of null alleles, INEst inferred a global inbreeding coefficient (F = 0.026 ± 0.089) not significantly different from zero, indicative of an outbreeding species. Significant linkage disequilibrium was detected between four pairs of loci (P < 0.05) after Bonferroni correction (TS-17 and TS-22, TS-37 and TS-25, TS-28 and TS-32, TS-42 and TS-32).
We developed the first set of microsatellite markers for the Guineo-Congolese Combretaceae species T. superba. These 14 microsatellite markers display a high level of polymorphism at the intrapopulation scale. These markers will help to estimate a reliable and informative panel of genetic diversity parameters for the reconstruction of the population history of African rainforests, still rarely conducted in tropical timber tree species.