The genus Entandrophragma C. DC. (Meliaceae) includes emblematic African trees, growing in both humid and dry African forests. It is one of the most economically important African genera, comprising 11 species among which five are intensively exploited for their wood. Known under the commercial names kosipo and sipo, E. candollei Harms and E. utile (Dawe & Sprague) Sprague are distributed from Sierra Leone to Uganda and from the Democratic Republic of Congo to Angola. They are pollinated by insects, and their seeds are dispersed by wind. They have undergone extreme logging in many African countries since 1970 and are now registered as vulnerable species on the IUCN Red List (Hawthorne, 1998). The sustainable management of these timber species is therefore urgent. To this end, we developed for each species highly polymorphic nuclear microsatellite markers (nSSRs), which will be used to study patterns of spatial genetic diversity and gene flow (mating system, pollen and seed dispersal).
METHODS AND RESULTS
Microsatellite development—Next-generation sequencing is a rapid method for acquiring a large quantity of genomic data, allowing the identification of nSSRs. Due to the low transferability of E. cylindricum (Sprague) Sprague nSSRs in other Entandrophragma species (Garcia et al., 2004), we isolated new nSSRs for E. candollei and E. utile separately. The total genomic DNA was extracted from leaf tissue of one specimen from each species (GEM09 and GEM11, Appendix 1) using the cetyltrimethylammonium bromide (CTAB) protocol (Doyle and Doyle, 1987). Following Rohland and Reich (2012) and Mariac et al. (2014), nonenriched genomic libraries were constructed after shearing, sizing, DNA end-repair, tagging by blunt ligation, real-time PCR, and Illumina MiSeq sequencing (GIGA platform, Liège, Belgium). The resulting 144 ± 2-bp-long paired-end reads were aligned using PANDAseq (Masella et al., 2012), providing 419,184 and 528,740 reads, respectively, for E. candollei and E. utile. Microsatellite motifs were identified using a QDD pipeline (Meglécz et al., 2009). We obtained 58,113 (13.86%) and 52,216 (10.99%) sequences containing at least one nSSR motif, including 1234/671 di- and 201/53 trinucleotide repeats for E. candollei and E. utile, respectively. We then selected 112 candidate loci for E. candollei and 67 for E. utile from QDD output files using the following criteria: (i) a minimum 20-bp distance between the primers and the microsatellite motif, (ii) a minimum of seven microsatellite repetitions, (iii) only one microsatellite motif present in the fragment, (iv) GC content of the fragment between 20% and 60%, and (v) expected PCR product size between 100 and 300 bp. For each species, amplification tests were then done on 48 loci, using the distribution size of the candidate loci as a subselection criterion to facilitate multiplex definition in the next steps.
Characterization of 16 polymorphic nuclear microsatellite loci isolated from Entandrophragma candollei.
Microsatellite marker selection and simplex reactions—The amplification of these 48 loci was tested on two individuals per species (GEM09 and FM1355 for E. candollei, GEM11 and FM1818 for E. utile) using the following PCR conditions: 1.5 µL buffer (10×), 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 brought to a total volume of 15 µL with purified water. Thermal cycler conditions were: 94°C for 3 min; 30 PCR cycles of 94°C for 30 s, 57°C and 55°C for 45 s (respectively for E. candollei and E. utile), and 72°C for 1 min; and a final extension at 72°C for 10 min. PCR products were mixed with 9 µL of TE 1× and visualized using the QIAxcel DNA Screening Kit (method AL420; alignment markers 15–5000 bp; size marker 100–2500 bp; QIAGEN). We obtained 34 positive amplifications for E. candollei and 44 for E. utile. This set of markers was tested individually (using the above-described PCR conditions) to exclude all unreadable and bad amplification loci. Finally, 16 and 22 (respectively for E. candollei and E. utile) readable loci were retained to define nSSR multiplexes and test polymorphism. In this aim, we added one of the four fluorochrome linkers (Q1–Q4; Micheneau et al., 2011; Tables 1, 2) to the 5′ end of the forward primer of each locus.
Multiplex reactions and polymorphism tests—PCR amplification was performed using the QIAGEN Multiplex PCR Kit in a 15-µL volume of 0.3 µL of the reverse (0.2 µM) and 0.1 µL of the forward (0.07 µM) primers with a Q1– Q4 universal sequence at the 5′ end, 0.15 µL of Q1–Q4 labeled primer (0.2 µM each), 7.5 µL of Type-it Microsatellite PCR Kit, H2O, and 1.5 µL of DNA. PCR program conditions were: initial denaturation at 95°C for 3 min; followed by 30 PCR cycles of 95°C for 30 s, with 57°C or 55°C for 90 s (respectively for E. candollei and E. utile), and 72°C for 1 min; and a final elongation at 60°C for 30 min. The nSSR polymorphism was investigated in three populations of E. candollei and four populations of E. utile (Appendix 1). The 16 polymorphic loci for E. candollei and the 22 polymorphic loci for E. utile were combined in three and four multiplexes, respectively (Tables 1, 2) using Multiplex Manager 1.0 software (Holleley and Geerts, 2009). We added 3 µL of 5× Q-solution and adjusted the volume of the reverse primer labeled by Q-tailed fluorescent Q1 to Q4 based on the number of loci containing the corresponding tail in the final multiplex.
Using 1.5 µL of PCR product, 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), all individuals were genotyped using an ABI3730 sequencer (Applied Biosystems, Lennik, The Netherlands; ULB-EBE platform). Microsatellite profiles of each individual were analyzed with Peak Scanner software version 1.0 (Applied Biosystems). One or two alleles per individual and per locus were found, suggesting that E. candollei and E. utile are diploids. For each locus, we estimated the number of alleles (A), observed heterozygosity (Ho), expected heterozygosity (He), inbreeding coefficient (F), and null allele frequency (r) using INEst 1.0 (Chybicki and Burczyk, 2008). Deviations from Hardy–Weinberg equilibrium (HWE) were measured with SPAGeDi (Hardy and Vekemans, 2002).
Microsatellite marker data analysis in E. candollei and E. utile—In E. candollei, the 16 polymorphic loci exhibited up to 17 alleles per locus and population, with mean A and He per population ranging from 7.3 to 8.9 and 0.67 to 0.75, respectively (Table 3). Significant deviation from HWE was observed in all populations for two loci (EnC-ssr9, EnC-ssr13), and in at least one population for five other loci, in part due to the presence of null alleles (Table 3). Nevertheless, null allele frequencies were always below 0.20 for 14 loci.
Characterization of 22 polymorphic nuclear microsatellite loci isolated from Entandrophragma utile.
For E. utile, the 22 loci showed up to 19 alleles per locus and population, with mean A and He per population ranging from 7.09 to 8.5 and 0.69 to 0.73, respectively (Table 4). Significant deviation from HWE was observed in all populations for six loci, here also in part due to null alleles (Table 4). Nevertheless, null allele frequencies were always below 0.20 for 15 loci.
Cross-amplification in E. congoense and E. angolense—These sets of markers were also tested with the same PCR conditions on E. congoense (Pierre & De Wild.) A. Chev. and E. angolense (Welw. ex C. DC.) C. DC. (Appendix 1). A total of eight and six loci developed on E. utile amplified on E. angolense and E. congoense, respectively; at least five of these loci were monomorphic (Table 5). For primers developed in E. candollei, four and three successfully amplified on E. angolense and E. congoense, respectively, and two were monomorphic (Table 5). The developed markers have been deposited in GenBank (Tables 1, 2), and the physical specimens from which markers were developed have been deposited in BioSample (submission ID SAMN06009795 [E. candollei] and SAMN06009796 [E. utile]).
In this paper, we developed 16 and 22 polymorphic nSSR markers for E. candollei and E. utile, respectively. These markers will be useful to study intraspecific diversity and gene flow within both species, allowing the implementation of sustainable conservation programs for populations of these vulnerable species (Hawthorne, 1998).
Genetic characterization of the 16 polymorphic microsatellite loci for four populations of Entandrophragma candollei.a
Genetic characterization of the 22 polymorphic microsatellite loci for three populations of Entandrophragma utile.a
Cross-amplification results (allele size ranges) of microsatellite loci isolated from Entandrophragma utile and E. candollei tested in two congeneric species.
The laboratory work was financed by the Fonds pour la Formation à la Recherche dans l'Industrie et l'Agriculture (FRIA, Belgium) and the Fonds de la Recherche Scientifique (F.R.S.-FNRS, grants T.0163.13, J014315F). The authors thank Emmanuel Kasongo and Nils Bourland for the sampling in Yangambi.