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The Journal of Heredity 2002:93(4)
© 2002 The American Genetic Association 93:287-290

Brief Communication

Multiplexed Systems of Microsatellite Markers for Genetic Analysis of Mahogany, Swietenia macrophylla King (Meliaceae), a Threatened Neotropical Timber Species

M. R. Lemes, R. P. V. Brondani, and D. Grattapaglia

From the Laboratório de Genética e Biologia Reprodutiva de Plantas, Instituto Nacional de Pesquisas da Amazônia, C.P. 478, Manaus, AM, 69011-970, Brazil (Lemes); Department of Biological Sciences, University of Stirling, Stirling, FK9 4LA, Scotland, UK (Lemes); Laboratório de Genética de Plantas, EMBRAPA-Recursos Genéticos e Biotecnologia, C. P. 02372, Brasília, DF, 70770-900, Brazil (Brondani and Grattapaglia); and Departamento de Biologia Celular, Universidade de Brasília, Brasília, DF, 70910-900, Brazil (Brondani). Dario Grattapaglia is currently at the Laboratório de Biotecnologia, Universidade Católica de Brasília, SGAN 916 Mod. B, Brasília, DF, 70790-160, Brazil.

Address correspondence to Maristerra R. Lemes. Coordenação de Pesquisas em Ecologia, Instituto Nacional de Pesquisas da Amazônia, C.P. 478, Manaus, AM, 69011-970, Brazil, or e-mail: mlemes{at}inpa.gov.br.


   Abstract
Top
 Abstract
Introduction
 Material and Methods
 Results
 Discussion
 References
 
Mahogany (Swietenia macrophylla King [Meliaceae]) is the most valuable hardwood species in the neotropics. Its conservation status has been the subject of increasing concern due to overexploitation and habitat destruction. In this work we report the development and characterization of 10 highly variable microsatellite loci for S. macrophylla. Twenty-nine percent of the 126 sequenced mahogany clones yielded useful microsatellite loci. Three high-throughput genotyping systems were developed based on polymerase chain reaction (PCR) multiplexing of these mahogany loci. We identified a total of 158 alleles in 121 adult individuals of S. macrophylla, with an average of 15.8 alleles (range 11–25) per locus. All loci showed Mendelian inheritance in open-pollinated half-sib families. The mean expected heterozygosity was 0.84 and the mean observed heterozygosity was 0.73. The combined probability of identity—the probability that two individuals selected at random from a population would have identical genotypes—was 7.0 x 10-15, and combined probability of paternity exclusion was 0.999998 overall loci. These microsatellite loci permit precise estimates of parameters such as gene flow, mating system, and paternity, thus providing important insights into the population genetics and conservation of S. macrophylla.


   Introduction
Top
 Abstract
 Introduction
Material and Methods
 Results
 Discussion
 References
 
Mahogany (Swietenia macrophylla [Meliaceae]) is the most valuable hardwood species in the neotropics and is seriously threatened owing to overexploitation and habitat destruction. The species has a wide geographic range extending from Mexico to the southern Amazon of Bolivia and Brazil (Lamb 1966; Pennington 1981). It is a canopy emergent (30–50 m tall) monoecious tree pollinated by bees and moths (Styles and Khosla 1976). The seeds are wind dispersed (Whitmore 1983). Mahogany tends to occur in widely scattered patches and the average density in natural forests is less than one tree per hectare (Gullison and Hardner 1993; Verissimo et al. 1995).

In recent years, the conservation status of S. macrophylla has been the subject of increasing concern (CITES 1996; Rodan and Campbell 1996). Mahogany is considered highly threatened throughout Central America and in some regions of Bolivia and Brazil (Collins 1990; Gullison et al. 1996; Verissimo et al. 1992). The selective and intensive logging of S. macrophylla populations may compromise their viability as evolutionary units by reducing population sizes and levels of genetic polymorphism.

Despite the economic importance of mahogany, studies on the extent and distribution of genetic variation in natural populations are scarce and have only been carried out in Central America. Gillies et al. (1999) used random amplified polymorphic DNA (RAPD) markers to investigate the patterns of genetic variation among S. macrophylla populations from Mexico south to Panama. In a study of a fragmented population of the congeneric Swietenia humilis, found only in Central America, White et al. (1999) used microsatellite markers to show the occurrence of extensive gene flow among populations, probably reflecting the species' continuous distribution prior to habitat destruction.

Highly polymorphic microsatellites, also called simple sequence repeats (SSRs), are the most genetically informative class of molecular markers for studies of genetic variation in natural populations (Rafalski et al. 1996). The variability observed at SSR loci allows precise individual identification and parentage studies in natural populations, as well as the estimation of fundamental genetic parameters for the genetic conservation and management of heavily exploited tropical trees such as S. macrophylla. In the last few years microsatellite markers have been developed and characterized for a number of tropical tree species (Aldrich et al. 1998; Chase et al. 1996; Collevatti et al. 1999; Dayanandan et al. 1999; Dick and Hamilton 1999; Dutech et al. 2000; Hufford et al. 2000; Miwa et al. 2000; Rodriguez et al. 2000; White and Powell 1997b). Although batteries of microsatellite markers have been made available, significant further work is typically needed to optimize more efficient analytical procedures based on multiplexed genotyping systems to allow the analysis of large numbers of samples such as those commonly used for humans (e.g., Urquhart et al. 1995). Furthermore, although successful amplification of S. humilis SSR markers was reported for 11 species of Meliaceae, including S. macrophylla (White and Powell 1997a), our unpublished data indicated that only two of the 12 loci described were in fact polymorphic in S. macrophylla. These results indicate that although flanking regions of SSR are often conserved among the Meliaceae species, the SSRs themselves may be lost, making necessary the specific development of microsatellites for our target Swietenia species.

This study reports the development and characterization of 10 highly variable microsatellite loci for S. macrophylla. In order to facilitate the large-scale analysis of natural populations, we optimized a multilocus genotyping system based on fluorescent detection of multiplexed SSR loci. Our main objective was to develop a robust system that could be immediately used for detailed characterization of S. macrophylla genetic structure, gene flow, and mating system.


   Material and Methods
Top
 Abstract
 Introduction
 Material and Methods
Results
 Discussion
 References
 
Plant Material and DNA Isolation
Total genomic DNA was extracted from leaves of a single individual of S. macrophylla and used to develop an (AG) genomic-enriched library. Characterization of SSR loci was based on 121 adult individuals of S. macrophylla collected from four natural populations in the southern Amazon region of Brazil. Genomic DNA extraction followed standard CTAB procedure (Doyle and Doyle 1987).

Microsatellite Marker Development
Genomic library construction, selection and sequencing of positive clones, and primer design was performed following protocols developed at Du Pont (Rafalski et al. 1996) and optimized for tropical tree genomes in our laboratory (Brondani et al. 1998). An anchored-PCR strategy was performed to determine the presence of the SSR repeat and its position within the cloned insert (Rafalski et al. 1996; Taylor et al. 1992). PCR conditions and steps for screening of microsatellite loci for marker quality and polymorphism were carried out as described earlier (Collevatti et al. 1999) using eight individuals of S. macrophylla. Selected loci were further screened on 16 individuals on 4% PAGE stained with silver nitrate (Bassam et al. 1991) and sized by comparison to a 10 bp DNA ladder (Gibco, MD).

Multiplexed Fluorescence-Based Analysis
The microsatellite markers that showed clearly interpretable polymorphisms in silver-stained denaturing PAGE were selected for fluorescent-dye labeling. One primer of each pair was labeled at the 5' end with a fluorescent dye (6-FAM, TET, or HEX). The choice of the fluorescent-dye label for each microsatellite primer was based on its observed allelic size range. Loci with overlapping allele sizes were multiplexed by labeling them with different dyes, whereas amplified products with nonoverlapping allelic range sizes were labeled with the same fluorescent dye. Three multiplexed systems of microsatellite markers were developed.

PCR was performed in a final volume of 25 µl for multiplexed and 10 µl for nonmultiplexed reactions containing 1.25–2.0 µM of each forward and reverse primers (Table 1), 1 unit of Taq DNA polymerase, 200 µM of each dNTP, 1x reaction buffer (10 mM Tris-HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl2), BSA (2.5 mg/ml), 5.0 ng of template DNA, and dH2O. PCR amplifications were performed with an initial denaturation at 96°C for 2 min followed by 30 cycles at 94°C for 1 min, 56°C for 1 min, and 72°C for 1 min, and a final elongation step at 72°C for 7 min. Following PCR, 1.0 µl of 1:10 diluted reaction of each multiplex was added to 0.25 µl of GeneScan 500 ROX internal lane size standard (ABI), 0.45 µl of loading buffer (25 mM EDTA and 50 mg/ml Blue-Dextran) and 2.3 µl of deionized formamide. The reactions were heated to 95°C for 3 min, chilled on ice, and electrophoresed in 5% denaturing polyacrylamide gel on a ABI 377XL sequencer. GeneScan and Genotyper software were used for data collection and analysis. To check the accuracy and reproducibility of allele sizing, we evaluated intra- and intergel variation. DNA of eight individuals at five loci was amplified, and run four times in the same gel and in three different gels. An analysis of variance (ANOVA) of the results was performed using the program Statistica (StatSoft 1995).


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  		Table 1.. Characteristics of the 10 microsatellite loci developed for Swietenia macrophylla: SSR locus name, repeat motif, forward and reverse primer sequences, primer concentration (µM), allelic size range (bp), number of alleles detected per locus (A), observed heterozygosity (Ho), expected heterozygosity (He), probability of paternity exclusion (Q), probability of genetic identity (I), and GenBank accession number

 
Inheritance and Characterization of Microsatellite Loci
Mendelian inheritance was evaluated in two S. macrophylla open-pollinated half-sib progeny arrays of 16 individuals and characterization carried out with 121 adult individuals from four populations. Descriptive locus statistics were estimated using Genetic Data Analysis software (GDA; Lewis and Zaykin 1999). Genetic information content was estimated by the single-locus and multilocus probability of genetic identity (I) (Paetkau et al. 1995) and the paternity exclusion probability (Q) (Weir 1996).


   Results
Top
 Abstract
 Introduction
 Material and Methods
 Results
Discussion
 References
 
Microsatellite Marker Development
Digestion of S. macrophylla DNA with three restriction enzymes revealed that Sau 3AI produced the most adequate digestion profile for library construction, with a range of fragments between 280 and 600 bp. After the enrichment step, approximately 300 clones were screened, from which 168 were scored as positive (56%). From these positive clones, 126 (75%) were selected for sequencing following anchored-PCR analysis. A total of 43 (34%) sequences contained repeats suitable for primer design. We used a single PCR program to screen the 43 SSR loci. A total of 6 primer pairs (14%) did not amplify any product at all, 17 pairs of primers (39%) amplified clearly interpretable PCR products but also showed nonspecific amplification of secondary bands, and 20 loci (46%) showed very clean and easily interpretable PCR products in agarose. In total, 85% of the primer pairs yielded clearly interpretable PCR products under a single set of PCR conditions.

The 20 loci that showed clean and clearly interpretable PCR products were selected to evaluate polymorphisms in 16 individuals of S. macrophylla in silver-stained denaturing PAGE. Two of the loci appeared monomorphic. Of the 18 polymorphic loci, 12 that did not show spurious bands were selected for fluorescent-dye labeling.

Multiplexed Fluorescence-Based Analysis
Three multiplex systems for S. macrophylla were optimized using 10 of the 12 fluorescent dye-labeled primer pairs. Multiplex 1 comprised a triplex PCR reaction (sm01-TET, sm34-FAM, and sm47-FAM) and a biplex PCR (sm22-TET and sm40-HEX). The triplex and biplex PCRs were combined before electrophoresis and loaded onto a single gel lane to form a pentaplex system. Multiplex 2 included two single-locus PCRs (sm31-FAM and sm46-FAM) that were combined before electrophoresis and loaded in a single gel lane. Multiplex 3 comprised a biplex PCR (sm45-TET and sm51-HEX) plus a single-locus reaction (sm32-FAM) that were combined before electrophoresis to form a triplex set. The annealing temperature of the 10 primers was 56°C. Variations in allele size estimation were not significant, either within (F = 0.00038, P = .99999) or between (F = 0.001016, P = .974578) gels, indicating that the analysis of the 10 S. macrophylla loci is accurate and reproducible.

Inheritance and Characterization of Microsatellite Loci
All repeat sequences found were simple and uninterrupted, with repeat motifs ranging in size from 18 to 31 (Table 1). For the 10 microsatellite loci, we identified a total of 166 alleles among the 121 adult individuals of S. macrophylla and the 32 progeny, plus mother trees representing the two opened-pollinated half-sib families. Considering only the 121 adults, we detected an average of 15.8 alleles per locus. All 10 loci were hypervariable, with the least and most variable loci showing 11 and 25 alleles, respectively. The mean expected heterozygosity was 0.84 and the mean observed heterozygosity was 0.73. The allele frequency distributions for the 10 loci are shown in Figure 1. For some loci, two or three alleles were particularly frequent. At other loci, for example, sm31, sm32, sm45, and sm46, the alleles were distributed more uniformly.



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  		Figure 1.. Patterns of allele frequency distribution for 10 microsatellite loci of S. macrophylla. x axis, allele size in base pairs; y axis, allele frequency.

 
All loci exhibited segregation patterns expected under Mendelian inheritance. Each of the 16 sibs shared at least one allele with the mother tree for all 10 loci. No significant deviation from the expected 1:1 Mendelian ratio for the maternal alleles was observed for the 10 loci tested after a chi-square test.

The paternity exclusion probability (Q) was high for all loci (Table 1). The combined probability of paternity exclusion (QC) using all 10 loci is 0.999998, indicating a probability of 99.9998% of correctly excluding a random nonparent individual tree in the population. The single-locus probability of genetic identity varied from 0.02 to 0.12, with a combined value of 7.0 x 10-15.


   Discussion
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Discussion
References
 
We described the development, genetic characterization, and optimization of an efficient system of multiplexed analysis of a set of selected hypervariable microsatellite markers for S. macrophylla. We optimized a procedure that permits the semiautomated analysis of 10 microsatellite loci in six PCRs and three gel lanes. Although the description of such detailed analytical procedures has been common in humans (e.g., Urquhart et al. 1995), domestic animals (e.g., Glowatzki-Mullis et al. 1995), and crop plants (e.g. Mitchell et al. 1997), it has not been described in the few reports that describe microsatellite markers for tropical trees. The optimization of such multiplexed systems not only results in the selection of more robust and hypervariable loci, but also effectively allows one to immediately approach population genetics questions that demand the typing of extensive sample sizes with very rigorous allele size calling. In our study the maximum difference in size estimates found for one allele was 0.43 bp (range 0.01–0.43). The size estimates of fragments did not vary significantly, either within or between gels.

The allele diversity of microsatellite loci of S. macrophylla was found to be positively correlated with the number of repeat units of the locus (see Weber 1990). The sm31 locus contained the largest number of (AG) repeats (n = 31) and also had the greatest number of alleles (n = 25). The mean expected (He) and observed (Ho) heterozygosities for S. macrophylla were 0.84 and 0.73, respectively. These are amongst the highest heterozygosity values yet observed for tropical tree species (Aldrich et al. 1998; Collevatti et al. 1999; Dayanandan et al. 1999; Dutech et al. 2000; Gaiotto et al. 2001; Hufford et al. 2000; Miwa et al. 2000; Rodriguez et al. 2000; Stacy et al. 2001; White and Powell 1997b). Both the high number of alleles and the consequent high heterozygosity values most likely derive from the large and genetically diverse origin of the sample of trees used in the characterization. A heterozygote deficiency was observed for some loci, most likely due to the stratified sampling of individuals used for locus characterization. Our unpublished data on the mating system of S. macrophylla with these markers has ruled out the hypothesis of null alleles. Furthermore, no significant deviation from the expected 1:1 Mendelian ratio was observed, although the power of the chi-square test to detect segregation distortions in families of the size examined here was limited.

The combined probability of identity—the probability that two individuals selected at random from a population would have identical genotypes—was estimated at 7.0 x 10-15, clearly demonstrating that the multiplex system allows very precise individual discrimination. This is particularly interesting in the context of enforcing timber harvesting certification laws for mahogany. The microsatellite systems developed in this study could be immediately used in forensic investigation, where there is a need to match a particular tree log to a specific stump in the forest in order to check whether its origin is legal. The probabilities of paternity exclusion were very high, both for single loci (.52–.87) and the combined 10 loci estimate (.999998). Again, the semiautomated genotyping system described herein effectively constitutes an important tool to move toward more refined and challenging questions regarding breeding structure, gene flow, and parentage in natural populations of S. macrophylla. Answers to these questions will certainly allow a more thorough understanding of the impact of timber harvesting methods on the population genetics dynamics of mahogany. To this end, we have been using this genotyping system to carry out a study on the population genetics structure and mating system of a number of S. macrophylla populations in the Brazilian Amazon, the results of which will be the subject of an upcoming report.


   Acknowledgments
 
This study is part of the PhD dissertation of Maristerra R. Lemes. This work was funded by the World Wildlife Fund-Brazil (grant no. CSR 95033 to M.R.L.), the Brazilian Ministry of Science and Technology (CNPq/PADCT grant no. 62.0059/97.4 to D.G. and CNPq/RHAE fellowship no. 260021/94.6 to M.R.L.), and Fundação Botânica Margaret Mee, whose support we gratefully acknowledge. We also acknowledge partial support from the European Commission, DGXII, International Cooperation with Developing Countries Programme (contract no. ERBIC18CT970149), EMBRAPA—Genetic Resources and Biotechnology (Brazilian Ministry for Agriculture), and IBAMA (Brazilian Ministry of Environment). We wish to thank José Ribeiro for his kind help with the field work. We are very grateful to Eldredge Bermingham and Chris Dick, who helped to improve this manuscript.


   Footnotes
 
Corresponding editor: David B. Wagner

Received March 21, 2001
Accepted June 10, 2002


   References
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 Introduction
 Material and Methods
 Results
 Discussion
 References
 

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