Development and characterization of microsatellite markers in the African deciduous tree Terminalia superba (Combretaceae).

PREMISE OF THE STUDY: Microsatellites were designed and characterized in the African timber forest tree Terminalia superba (Combretaceae). Due to their high variability, these markers are suitable to investigate gene flow patterns and the structure of genetic diversity. METHODS AND
RESULTS: From a genomic library obtained by next-generation sequencing, seven monomorphic and 14 polymorphic microsatellite loci were developed. The polymorphic microsatellites displayed two to 27 alleles (mean 11.4; expected heterozygosity range 0.283-0.940, mean 0.736) in one population from southeastern Cameroon. Genotypes were typical of an outbreeding diploid species, although null alleles explain a significant heterozygote deficit in three loci. Cross-amplification in three congeneric species (T. ivorensis, T. avicennioides, and T. mantaly) failed, suggesting that T. superba is rather divergent.
CONCLUSIONS: This set of newly developed microsatellite markers will be useful for assessing the genetic diversity, population structure, and demographic history of T. superba in tropical African forests.

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 low-cost 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 (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 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).

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.

Table 1.

Characterization of 14 polymorphic and seven monomorphic nuclear microsatellite loci isolated from Terminalia superba.

Locus	Primer sequences (5′–3′)	Fluorescent label	Repeat motif	Allele size range (bp)	GenBank accession no.
Multiplex 1
TS-08	F: TGTAAAACGACGGCCAGTTTGACAAGTGTTGCAGGAGA	Q1-6-FAM	(CT)36	179–248	KT222788
	R: GTTTGACTGGGATGGCTGAG
TS-25	F: TAGGAGTGCAGCAAGCATCGTTTGAAGTGGGAGACCAT	Q2-NED	(AC)15	281–301	KT222793
	R: TTCCCTCTTGATTCTTCCTAGA
TS-30	F: CACTGCTTAGAGCGATGCAGATCACCCGAGATCACCC	Q3-VIC	(GT)17	202–302	KT222795
	R: TTCTCTTCCAACTGGTCCTCTT
TS-37	F: CTAGTTATTGCTCAGCGGTGTGGTGGGTGCTTGAATTG	Q4-PET	(GA)15	177–250	KT222798
	R: TAGCAGCTAAGGAAGCTGGG
Multiplex 2
TS-5	F: TGTAAAACGACGGCCAGTGGGCATTCAGGGACTACCTT	Q1-6-FAM	(GA)24	174–272	KT222787
	R: ACCACTCAGCCACCACTACC
TS-12	F: TGTAAAACGACGGCCAGTTCACGGACGAGAAATCTCAA	Q1-6-FAM	(AG)13	308–310	KT222789
	R: GCATCATTGGACAAGGAAGAA
TS-17	F: TAGGAGTGCAGCAAGCATGGCCCATATCGAATGAGTTT	Q2-NED	(TA)14	157–213	KT222790
	R: GTTCATCGAACATGGGCTG
TS-28	F: CACTGCTTAGAGCGATGCTAACGAGCAGCAACCATGTC	Q3-VIC	(AT)10	181–197	KT222794
	R: TGCACATCAAAGACCCATCT
TS-42	F: CTAGTTATTGCTCAGCGGTTCCCATGATTAGCTGTCATCC	Q4-PET	(TA)10	214–220	KT222799
	R: TGAGTCCAGTAGTTTAGGAGTGGT
Multiplex 3
TS-22	F: TAGGAGTGCAGCAAGCATCGAGCTTAGCACTTTCCTCC	Q2-NED	(GA)10	224–240	KT222791
	R: GCTCAGTTCGTTTGCACCTT
TS-24	F: TAGGAGTGCAGCAAGCATATCACCTCGCCTCACTGTCT	Q2-NED	(AC)12	303–321	KT222792
	R: TTTACCTTCCGCTACACGAA
TS-32	F: CACTGCTTAGAGCGATGCCAAGTTTGATTCCTGCCCTT	Q3-VIC	(AG)16	222–254	KT222796
	R: ATCCCGTTTGGTTGTTTGAA
TS-34	F: CACTGCTTAGAGCGATGCACCTCAATGGGTGGAATTGA	Q3-VIC	(AG)23	243–286	KT222797
	R: GCTCTCCTCGCCTGAGTAAA
TS-44	F: CTAGTTATTGCTCAGCGGTCCATATTCAACTTCAAGCTCCAC	Q4-PET	(TC)16	196–271	KT222800
	R: AAGCTAGGAGCTTCGTCGG
Monomorphic loci
TS-03	F: TGTAAAACGACGGCCAGTTTGGTGAAATTGAGAGCCAA	Q1-6-FAM	(TA)10	167	KT250550
	R: GCATATGAACATAAGGTGTTAGGG
TS-06	F: TGTAAAACGACGGCCAGTCAAAGACCCTTATTTCTAGTCCTCA	Q1-6-FAM	(AT)10	187	KT250551
	R: TACTTCGCCCAATTCCACCT
TS-10	F: TGTAAAACGACGGCCAGTTTGTTATGGTTATCCTTTCGGG	Q1-6-FAM	(TG)10	225	KT250552
	R: TGCTCGCATGTCATACAAATG
TS-20	F: TAGGAGTGCAGCAAGCATCGATTAGTGGGCAAGGGTTA	Q2-NED	(AT)12	205	KT250553
	R: TCTGATGCTTGTGTTCGCTC
TS-33	F: CACTGCTTAGAGCGATGCGCATTCATTTCTCTACCTTGAGC	Q3-VIC	(AT)12	218	KT250554
	R: AGACCTGCTCGCTCCATAAA
TS-43	F: CTAGTTATTGCTCAGCGGTCGCACCAAATTTCACTCCTT	Q4-PET	(TC)17	228	KT250555
	R: TCATTAAGTCCATCTCACGGC
TS-47	F: CTAGTTATTGCTCAGCGGTTGAAGGAGGAAGAAGATGAATG	Q4-PET	(GA)21	373	KT250556
	R: CAGTTGCAATGTTTCACGAT

Annealing temperature for all reactions was 56°C. The linkers (Q1, Q2, Q3, Q4) attached to the forward primers are underlined.

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.

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).

Table 2.

Genetic properties of 14 polymorphic nuclear microsatellite loci developed in Terminalia superba for 42 individuals sampled in southeastern Cameroon.

Locus	N	A	Ho	He	F	r
Multiplex 1
TS-08	40	19	0.850	0.913	0.069	0.022 ± 0.055
TS-25	40	9	0.575	0.586	0.019	0.000 ± 0.000
TS-30	40	17	0.780	0.861	0.094	0.033 ± 0.049
TS-37	41	27	0.902	0.940	0.040	0.010 ± 0.046
Multiplex 2
TS-5	32	15	0.375	0.822	0.544*	0.369 ± 0.093
TS-12	41	2	0.415	0.493	0.158	0.042 ± 0.062
TS-17	42	10	0.595	0.627	0.050	0.013 ± 0.052
TS-28	42	12	0.738	0.841	0.123*	0.055 ± 0.056
TS-42	35	4	0.143	0.570	0.749*	0.407 ± 0.066
Multiplex 3
TS-22	41	6	0.659	0.723	0.089	0.043 ± 0.047
TS-24	30	5	0.267	0.283	0.058	0.000 ± 0.000
TS-32	38	9	0.579	0.753	0.231	0.090 ± 0.058
TS-34	31	17	0.806	0.895	0.099	0.037 ± 0.057
TS-44	28	10	0.393	0.807	0.513*	0.320 ± 0.068

Note: A = number of alleles; F = fixation index; He = expected heterozygosity; Ho = observed heterozygosity; N = number of individuals successfully genotyped on 42 individuals; r = frequency of null alleles.

*Significant deviation from Hardy–Weinberg equilibrium (P < 0.001).

CONCLUSIONS

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.

Appendix 1.

Locality information for the samples used in this study.

Species	Sample no.	Country	Locality	Latitude	Longitude
Terminalia superba	BoD597b	Ghana	Kakum National Park	5.34833	−1.38444
	BoD459c	Togo	Agou Nyogbo	6.87204	0.72110
	BoD556c (BRLU herbarium sample Demenou 63)	Ghana	Sagyimasi	6.20716	−0.52927
	BoD713c	Nigeria	Ondo state	7.11568	4.80733
	MP309d	DRC	Luki	−5.62133	13,10709
	GiD1654d	Gabon	Région du Mayumbe	−3.40982	11.41850
	NB669d	Cameroon	UFA 09-024	2.32537	10.4336
	BoD721d	Nigeria	Bolorunduro, Owena Forest	7.2161	5.01921
	BOD335d	Benin	Pobè	6.98891	2.66840
	BoD476d	Togo	Tomégbé	7.41984	0.60108
	AD815, AD816, AD817, AD818, AD819, AD820, AD821, AD822, AD823, AD824, AD825, AD826, AD827, AD828, AD829, AD830, AD831, AD832, AD833, AD834, AD835, AD836, AD837e	Cameroon	Mindourou	3.27	14.34
	JFG0468, OH3472, OH3473e	Cameroon	Barrière Mbang	3.45230	14.07680
	JFG0504, CM5, OH3778e	Cameroon	Carrefour Bali	3.76350	14.81470
	JFG0406e	Cameroon	Forêt communautaire de Mbol	3.44230	14.62310
	JMR0022, JMR0029e	Cameroon	SFID-Mbang 10.038	3.82336	13.97913
	OH3781e	Cameroon	UFA 09.021	3.52333	14.49806
	JFG0306, JFG0313, JFG0348, JFG0374, JFG0453e	Cameroon	UFA 10.026 Alpicam	3.55030	14.63600
	OH3780e	Cameroon	UFA 10.044-Pallisco	3.69722	13.49667
	JFG0401, JFG0472e	Cameroon	UFA 10.052 Decolvenaere	3.76800	14.56470
	RP0132, RP0278, RP0334e	Cameroon	UFA 10031, Boumba	3.19004	14.25405
	NB779, NB814, NB800, OH3468, OH3469e	Cameroon	UFA Lorema (SFID)	3.31335	13.02998
Terminalia avicennioides	BoD387, BoD388	Benin	Manigri	8.90456	1.77053
Terminalia ivorensis	NB398, NB407, NB411, NB412	Cameroon	UFA 11.005 Wijma	5.49082	8.95034
	NB439	Cameroon	Village d’Ogurang	5.48998	8.95359
	NB457	Cameroon		5.58650	9.05916
	PE15	Liberia	Nimba	7.48599	−8.63453
	OH3197	Ghana	near Yegue	8.2959	0.60137
Terminalia mantaly	BoD00467	Togo	Kpalimé	6.91005	0.63224
	BoD00470	Togo	Badou	7.58249	0.61276

Note: DRC = Democratic Republic of the Congo.

Herbarium sample of individual BoD556 (Demenou 63) is available at the Herbarium of the Université Libre de Bruxelles (BRLU), Brussels, Belgium. Leaf or cambium silica gel samples for all other samples are deposited at BRLU (silica gel collection of Dr. Olivier Hardy).

Individual used for DNA bank enriched in microsatellite markers.

Individuals used for first amplification test.

Individuals used for testing the polymorphism of loci.

Individuals from southeastern Cameroon used to compute population genetic polymorphism.