© 2004 The American Genetic Association
Brief Communication |
Genotyping of Mature Trees of Entandrophragma cylindricum with Microsatellites
From Centre de Coopération Internationale en Recherche Agronomique pour le Développement-Forêt, Campus International de Baillarguet, TA 10/B, F34398, Montpellier Cedex 5, France (Garcia and Chevallier), and Centre de Coopération Internationale en Recherche Agronomique pour le Développement, UMR, 1096/PIA, TA 40/03, F34398, Montpellier Cedex 5, France (Noyer and Risterucci). M.-H. Chevallier is currently at Centre d'Ecologie Fonctionelle et Evolutive, 1919 route de Mende, F34293, Montpellier Cedex 5.
Address correspondence to M.-H. Chevallier at the above address, or e-mail: chevallier{at}cefe.cnrs-mop.fr.
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Abstract |
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We have characterized 10 microsatellite loci for the tropical tree Entandrophragma cylindricum (Sprague) Sprague (sapelli) in order to genotype individuals in forest stands for estimation of the genetic diversity of the species. We used the technique of building a (GA)n microsatellite–enriched library by capture with streptavidin-coated magnetic beads. We assessed the polymorphism of seven microsatellites in 186 mature trees in a selectively logged stand (Dimako) and an unlogged stand (Ndama), both in Cameroon. All the loci were polymorphic, and the number of alleles was high, ranging from eight to 36, with a mean of 22.1. Both stands showed the same high level of genetic diversity (mean HE = 0.85) and a low genetic differentiation (FST = 0.007), indicating that genetic diversity was within rather than among populations. Five and three out seven loci in Dimako and Ndama, respectively, showed a deficit of heterozygotes. The seven loci enabled more than 97% of the mature trees in each stand to be identified. It was concluded that these markers can be efficiently used for gene flow studies.
We have characterized microsatellites (SSRs) in order to genetically identify trees of Entandrophragma cylindricum (Sprague) Sprague, common name sapelli, within two forest stands in Cameroon. The identification of individual trees is necessary both for studying the genetic diversity of a species and its spatial organization, and for evaluating gene flow within and among populations. With such knowledge, we can quantify how selective logging alters genetic and demographic processes such as loss of genetic variability for adaptative evolution, random fixation of deleterious mutations or alleles by genetic drift, and inbreeding depression (Alvarez-Buylla et al. 1996; Barrett and Kohn 1991). We can also assess the impact of logging on pollen dissemination and seed dispersal of this species for which logging removes mature trees, which changes the spatial distribution of reproductive trees and the mating system, including the behavior of pollinators.
Sapelli is one of the most important hardwoods in the rain forests of the Congo Basin. It is a heliophilous species of the canopy, hermaphrodite and insect pollinated. Its fruits are produced when the trees reach about 50 cm diameter at breast height (dbh), and its seeds are wind dispersed. The selective logging in East Cameroon consists of extracting stems of certain species when dbh is larger than the prescribed cutting limit fixed at 100 cm for sapelli. As a consequence, logging reduces the density of the reproductive trees and locally threatens regeneration. Obviously, understanding the effects of selective logging on the levels of genetic diversity and gene flow is crucial to providing recommendations for in situ conservation of the species and to developing management practices that ensure its sustainable use.
In this study, we describe the isolation of a set of microsatellite loci by building an enriched library, and we use seven loci for a first assessment of the genetic diversity of two populations of sapelli from Cameroon. Microsatellite loci are well adapted to genotype-unrelated individuals and to pursuit of genetic studies, because they are codominant, highly polymorphic, abundant, and uniformly distributed over the genome (Lefort et al. 1999).
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Materials and Methods |
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A (GA)n microsatellite enriched–library was built according to the methodology of Billotte et al. (1999), optimized by Dutech et al. (2000), using a biotin-labeled microsatellite oligoprobe and streptavidine-coated magnetic beads.
We sampled mature trees in two tropical natural forest stands in Cameroon, Dimako, and Ndama, separated by a distance of 150 km. The Dimako stand (400 ha) was exploited between 1958 and 1974, and 80% of the sapelli trees greater than 100 cm in dbh have been removed. Up to the present there has been no logging in the Ndama stand (100 ha). Every mature tree in both stands was tagged and mapped (64 trees in Dimako and 122 in Ndama).
Leaf samples were collected from mature trees greater than 50 cm in dbh and stored in plastic bags with a few crystals of dry silicagel. Total DNA was extracted according to Bousquet et al. (1990) and restricted with the RsaI restriction enzyme, and fragments with a size range from 300 to 1500 bp were recovered from a 2% agarose gel after run and then isolated with an extraction kit (Promega France). Enriched DNA was ligated to pGEM-T vector and transformed into Epicurian-coli XL1-Blue MRF supercompetent cells (Stratagene USA). A total of 384 clones were screened, and 151 gave a positive response. Thirteen clones were sequenced by Eurogentec (Belgium). Eleven nonredundant sequences were obtained. Primers annealing to flanking regions were designed for 10 of them, with use of the OLIGO 4.06 software (Rychlik 1992). One clone could not be used to define primers because of the extreme position of the SSR in the DNA fragment.
Polymerase chain reaction (PCR) temperatures were optimized with a RoboCycler gradient 96 (Stratagene; Table 1). PCR amplifications were carried out in a final volume of 12 µL containing 10 ng DNA, 0.5 U Taq polymerase (Gibco-BRL), 0.1 mM of each dNTP, 67 mM Tris-HCl (pH 8.0), 2 mM MgCl2, 16.6 mM (NH4)2SO4, 0.025% detergent W1 (Gibco-BRL), 0.07% ß-mercaptoethanol, 0.2 mg/ml BSA, and 0.2 µM of each primer. Amplifications were carried out with an MJ Research PTC-100 thermocycler under the following conditions: initial denaturation at 94°C for 4 min and then 30 cycles of 94°C for 30 s, 54°C or 56°C for 30 s, 72°C for 1 min, and a final elongation step at 72°C for 10 min. PCR products were denatured and separated on 6% sequencing polyacrylamide gels in 7 M urea and 1x Tris Borate Edta buffer, stained with silver nitrate (Streiff et al. 1998), and sized by comparison to a 10-bp ladder (Gibco-BRL) standard.
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In order to measure the information content of a given microsatellite, the discrimination power at a locus was calculated as 1 –
(pi)2, where pi is the frequency of each genotype (Kloosterman et al. 1993). The number of alleles per locus (A), the observed heterozygosity (HO), the expected heterozygosity (HE), and the genetic differentiation (FST) were computed with use of Popgen (Yeh and Boyle 1997). Wright's fixation index (FIS) was calculated as 1 – HO/HE.
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Results and Discussion |
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All 10 microsatellites tested with a minimum of five trees were found to be polymorphic (Table 1). A group of seven microsatellites among the most polymorphic loci, pEc-CIR12, pEcCIR22, pEcCIR143, pEcCIR156, pEcCIR227, pEcCIR244, and pEcCIR271, was selected to screen the 186 mature sapelli trees from the Dimako and Ndama sites. For this group, 155 alleles were identified, and the number of alleles per locus ranged from eight (pEcCIR227) to 36 (pEcCIR244); the average was 22.1. Frequencies for individual alleles at all loci were generally low, with only 13% of the alleles having values greater than 0.25 at a locus in Dimako and 9% in Ndama. Rare alleles with frequencies less than 0.10 accounted for 78.2% and 81.8% of the total, respectively, in Dimako and Ndama. Two markers, pEcCIR244 and pEcCIR271, were particularly informative for tree genotyping, with more than 25 alleles per locus and per stand, and a high power of discrimination (Table 2). Using seven loci enabled us to identify 63 genotypes among 64 individuals in Dimako and 119 genotypes among 122 trees in Ndama. Using the three most polymorphic loci (pEcCIR244, 271, and 156) taken two by two allowed 95% of individuals with pEcCIR244-271 and 95.8% with pEcCIR271-156 in Ndama to be identified. For Dimako either of these previous pairs permitted 98.4% of individuals (i.e., 63 individuals) to be identified.
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These seven microsatellites were also tested with the same PCR conditions on one sample each of two other species of Entandrophragma, E. candollei Harms and E. angolense (F. Welwitsch) De Candolle, in order that the potential applicability of these markers to other Entandrophragma species might be evaluated. Five primers (pEcCIR12, pEcCIR22, pEcCIR143, pEcCIR156, and pEcCIR244) were amplified from the two species, and two primers gave new alleles not seen in E. cylindricum.
The expected heterozygosity ranged from 0.64 (pEcCIR227) to 0.95 (pEcCIR156) with a mean of 0.85 over all loci (Table 2). Intrastand diversity was high, with 128 alleles in Dimako versus 133 in Ndama, and the mean number of alleles per locus of 18.3 (Dimako) and 19 (Ndama). Ten alleles were peculiar to Dimako; 15, to Ndama. High levels of genetic diversity, typical of highly outcrossing species, were observed in both stands, with the same mean observed (HO = 0.77) and expected (HE = 0.85) heterozygosities across loci. The high genetic diversity observed was similar to that found in Ceiba pentadra (HE = 0.85, Brondani et al. 2003) but higher than diversities recorded in other mahoganies in South America as Carapa guianensis (HE = 0.61, Dayanandan et al. 1999), Swietenia humilis (HE = 0.53, White et al. 1999), Swietenia macrophylla (HE = 0.66, Novick et al. 2003) or (HE = 0.78, Lemes et al. 2003). The genetic differentiation is low (FST = 0.007), indicating that the genetic diversity is maintained within rather than among populations. This observation is close to that reported by Hamrick et al. (1992) on tropical forest tree populations. White et al. (1999) also found no differentiation on the neotropical Meliaceae, Swietenia humilis, but over distances much lower (1 to several km) than those investigated in this study (150 km).
Genotypic frequencies showed significant departures from Hardy–Weinberg expectations for five and three out of seven loci in Dimako and Ndama, respectively, with a deficit of heterozygotes. Other tropical forest species have shown such positive fixation indices (Aldrich et al. 1998; Brondani et al. 2003; Chase et al. 1996; Latouche-Hallé et al. 2003; White et al. 1999). The occurrence of null alleles at microsatellite loci, and the population substructuring, as well as mating among relatives or partial selfing, may explain the deficit of heterozygotes found in the two stands of sapelli. Jarne and Lagoda (1996) reported null alleles (mutation in primer annealing sites) at microsatellite loci at frequencies up to 15%. The values of FIS are variable between loci and between stands ranging from –0.057 at locus pEcCIR156 from Dimako, to 0.280 at locus pEcCIR227 from Ndama. This result could indicate the presence of null alleles. Although we cannot exclude this explanation, we consider it unlikely because the blanks observed at some loci on the gels were due to unreadable patterns from few individuals, which had damaged leaves yielding a bad quality of DNA and therefore to a bad amplificated product. Further studies should be done, especially on the flowering phenology and the mating system of sapelli, to corroborate or reject the two other explanations.
The high power of individual discrimination provided by microsatellite markers will allow better understanding of the genetic mechanisms affected by selective logging on tree species (Lemes et al. 2003; Obayashi et al. 2002). For sapelli, the seven microsatellite markers are highly variable, and sufficiently informative to enable further studies to characterize the impact of logging on gene flow. With some adaptation, this set of microsatellite markers could also be used to assess genetic diversity and gene flow of related taxa (Entandrophragma sp.).
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Acknowledgments |
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The authors are very grateful to Adèle Mbenda and anonymous pygmy people who collected leaf material in the Cameroon forests. We are indebted to E. Forni, G. Moynot, A. Vaillant and H. Joly for helpful collaboration. This work was supported by funds from Centre de Coopération Internationale en Recherche Agronomique pour le Développement.
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Footnotes |
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Corresponding Editor: David Wagner
Received March 23, 2003
Accepted April 28, 2004
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References |
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