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a International Plant Genetic Resources Institute (IPGRI), c/o CIAT, A.A. 6713, Cali, Colombia
b Facultad de Agronomía, Universidad de San Carlos de Guatemala, A.P. 1545, Ciudad de Guatemala, Guatemala
c Centro Internacional de Agricultura Tropical (CIAT), A.A. 6713, Cali, Colombia
* Corresponding author (c.devicente{at}cgiar.org)
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Abbreviations: AFLP, amplified fragment length polymorphism
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Home gardens offer complex microenvironments in which farming families maintain populations of many useful plant species close to their dwellings (Hodgkin, 2002), where they can be closely observed and managed. They thus represent an important option for promoting in situ conservation of genetic resources, especially in the tropics where home gardens are significant reservoirs of genetic diversity (Knupffer, 2002). In Guatemala, home gardens contribute significantly to the sustainability of local production systems as a whole and represent an opportunity for conservation efforts in a region where deforestation continues to exacerbate genetic erosion (Leiva et al., 2002). However, thorough genetic diversity studies of the plant species found in these microenvironments are scarce, a situation that hinders the development of conservation strategies that explicitly include home gardens as in situ repositories of plant genetic resources.
A global project was undertaken to study plant genetic resources in home gardens in several countries—Guatemala, Cuba, Ghana, Nepal, Venezuela, and Vietnam—to evaluate their potential as a component for in situ conservation of agrobiodiversity (Eyzaguirre and Watson, 2002). In Guatemala, the project described the richness of plant species in home gardens in the northern (Department of Alta Verapaz) and the western (semiarid) parts of the country. Chili peppers (Capsicum spp.) were among the 500 species documented in Guatemalan home gardens. Inventories of species found in tropical home gardens in the Americas, Africa, and Asia reveal that chili pepper is a key home garden crop in all regions. Home gardens are thus a good site for the study of the global spread and distribution of genetic diversity in chili peppers from tropical America (Williams, 2003).
Four cultivated Capsicum species are found in Guatemala: C. annuum, scattered all over the country; C. chinense, mainly distributed in the department of Peten; C. frutescens, found in the northern parts; and C. pubescens, in the northern, central and western highlands of the country. In addition, C. annuum var. annuum and C. annuum var. glabriusculum (Dunal) Heiser & Pickersgill are found, as semicultivated varieties, in marginal areas throughout the country. Among these four species, several fruit types are identified with different local names, mostly on the basis of their shape, taste, pungency, or cultivation area (Azurdia et al., 1995).
The purpose of the work presented here was to compare by AFLPs the genetic variation of Capsicum found in home gardens of Guatemala with that conserved in the national ex situ collection and to determine the potential importance of home gardens for in situ conservation of plant genetic resources in the future.
Using molecular markers, allows a rapid and efficient assessment of genetic diversity (Hammer, 2003). The choice of AFLPs was based on the demonstrated success of using them to evaluate genetic diversity for studies in numerous species [e.g., soybean, Glycine max (L.) Merr., Maughan et al., 1996; Phaseolus vulgaris L., Tohme et al., 1996; Arabidopsis, Breyne et al., 1999; Limonium, Palacios and González-Candelas, 1999; Azadirachta, Singh et al., 1999; Morus, Sharma et al., 2000; and Stylosanthes, Sawkins et al., 2001).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The total sample studied was composed of 24 semicultivated accessions [C. annuum var. annuum (Diente de Perro and Pico de Gallina) and C. annuum var. glabriusculum (Chiltepe and Chiltepe Grande)], three C. pubescens accessions, one accession of C. frutescens, one of C. chinense, and 45 accessions of cultivated C. annuum. The semicultivated accessions were almost evenly divided between the home gardens sample (13) and the gene bank collection (11).
The distribution of samples in terms of altitude, both in the home garden group and in the gene bank collection, ranged from 50 m (Department of Escuintla) to 1908 m (Department of Huehuetenango) above sea level.
DNA Extraction
Five individuals per accession were germinated in the greenhouse. Ten days after germination, a uniform amount of fresh leaf tissue (25 mm2) was harvested from each individual. Leaf tissue harvested from all individuals of an accession was pooled for DNA extraction. Leaf tissue was crushed with 400 µL extraction buffer [200 mM Tris-HCl pH 7.5; 250 mM NaCl; 25 mM EDTA, 0.5% (w/v) SDS] in 1.5 mL tubes. After shaking, tubes were left at room temperature for 1 h and then spun down in a centrifuge at medium speed for 10 min. Supernatant was transferred to a clean tube, isopropanol was added (4:3 v/v) and the tube was gently inverted to help blending. The mixture was centrifuged at medium speed for 10 min at room temperature and the supernatant discarded. The DNA pellet was rinsed with ethanol 70% (v/v), centrifuged for 5 min more and resuspended in 100 µL TE. Each DNA extract was diluted to a final concentration of 25 ng/µL and stored at 4°C for future use.
Amplified Fragment Length Polymorphisms
Amplified fragment length polymorphisms were obtained with the Analysis System I and the AFLP Starter Primer kit of GIBCO-BRL (Life Technologies) with adapters and primers on the basis of the EcoRI and MseI restriction enzymes sites. All steps in DNA restriction digestion, adapters' ligation, preselective and selective PCR amplifications were performed following the directions provided by the manufacturer.
Out of 40 combinations of +3/+3 selective bases, two were selected on the basis of the number of amplified fragments and the polymorphism detected in a set of four samples randomly taken. Given the high number of bands obtained per combination, an additional nucleotide was added to the selective primers to further restrict the last amplification and to increase the reliability of band scoring. A test was made with the 16 new combinations of selective primers and on the basis of the number of amplified fragments, the polymorphism observed in a set of four random samples and the clarity of bands, three primer combinations were finally chosen: EcoRI+AGC/MseI+CAGC, EcoRI+AGG/MseI+CAGT, and EcoRI+AGAG/MseI+CAG. All PCR amplifications were performed in a PTC-100 programmable thermocycler (MJ Research, Inc., Watertown, MA).
Electrophoresis of AFLP products was performed in 6% (w/v) denaturing polyacrylamide gels containing 7 M urea and TBE 0.5x buffer. Six microliters of buffer [95% (v/v) formamide, 20 mM EDTA pH 8.0, 0.05% (w/v) bromophenol blue and xylene cyanol FF] were added to each PCR product and 5µL of each was loaded in the gel. Gels were left to migrate for approximately 2 h (110 W, 50°C), and immediately afterwards, stained with silver nitrate.
To check reproducibility of results and to ensure the comparison of similar bands in the two gels required for the analysis of each primer combination, a sample of control DNA was included in the gels. In addition, several accessions, randomly selected, were reprocessed and run in separate gels.
Data Analyses
Clear polymorphic bands were manually scored and a binary matrix produced (1 = presence, 0 = absence). The calculation of the heterogeneity or total genetic diversity was done according to Nei (1987). For this estimation, each band was considered as a diallelic locus, such that the band present is one allele and its absence is the alternative allele. Because AFLP bands detect dominant loci, Nei's coefficient may not be interpreted as the estimation of heterozygosis, but rather heterogeneity within groups. Then, total diversity (H = Hs + Dst) is the average locus heterogeneity (hi) over the total number of evaluated loci (i = 1, ..., n); within group diversity (Hs) is the weighted average of diversity within each group and the diversity among groups (Dst) is the difference between H and Hs. The ratio Dst/H corresponds to the genetic differentiation coefficient (Gst), which measures the proportion of total variation explained by the difference among groups.
A multiple correspondence analysis (MCA) was performed with the binary matrix. The output of this test allows the representation of the distribution of the accessions in a multidimensional metric space in such a way that it reflects the relationships among samples based on their similarity in banding profiles. Its graphical representation shows the spatial location of the accessions, facilitating the visualization of their dispersion and also a possible structuring of the populations. For the present study, three dimensions were chosen. Calculations were done by the CORRESP procedure of the SAS statistical package v. 8.2 and clustering was performed by applying the average linkage method (UPGMA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Only two (133 and 269) of the 74 accessions gave similar AFLP genotypes for the 68 polymorphic bands scored. The other 72 accessions showed different band profiles, which means that the AFLP primer combinations selected had good discriminatory power, and that the sample under study was highly variable.
Genetic Diversity of Gene Bank Accessions versus Accessions Obtained from Home Gardens
The comparison of genetic diversity of AFLP bands obtained with the Capsicum accessions from home gardens and those from the national gene bank collection showed that the amount of diversity in the two samples was similar; that is, their divergence (Gst) accounted for only 4% of the total diversity (Table 2). Because the gene bank collection also held six accessions from the department of Alta Verapaz, the source of the home garden samples, the analysis was redone after removing these accessions and similar results were obtained, which means that the contribution of these six samples to the total diversity of the Capsicum gene bank collection was not significant (H = 0.280, Hs = 0.265, Gst = 0.052).
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The semicultivated accessions made up 38% of the home gardens sample and 27% of the gene bank sample. An analysis was performed comparing the diversity of these accessions with that of the cultivated types, and no significant differences were obtained (Table 2). In addition, the analysis of diversity performed between the semicultivated accessions from home gardens and those from the gene bank revealed no significant differences (H = 0.248, Hs = 0.233, Gst = 0.060).
Likewise, an analysis was performed to evaluate the genetic diversity among the samples collected in different climatic or altitude conditions considering two groups, from 50 to 950 and from 951 to 1908 m above sea level. Some 15% of the home garden accessions and 32% of the gene bank accessions were from high altitude. The results again showed no significant difference in the genetic diversity of these two groups (H = 0.280, Hs = 0.280, Gst = 0.000). Once more, the comparison of the genetic diversity differences for the low altitude and high altitude accessions respectively, both in the home gardens and the gene bank samples, were detected to be nonsignificant (low altitude: H = 0.274, Hs = 0.258, Gst = 0.076; high altitude—without correction factor because n was small: H = 0.281, Hs = 0.269, Gst = 0.045).
The lack of significant differences in all genetic diversity comparisons explains the uniform genetic diversity values in the home gardens and the gene bank samples in spite of the fact that they differ in their composition, both in terms of type of materials and the collecting site altitude of the accessions.
Multiple Correspondence Analysis
The clustering procedure applied on the coordinates generated by the MCA distinguished a structure of six groups, whose presence explains 93% of the total variation in the sample (Fig. 2)
. The third dimension allowed the separation of the three accessions belonging to C. pubescens (group E) from the rest of the materials included in the study. The first and second dimensions differentiated the remaining 71 accessions of C. annuum, C. frutescens, and C. chinense.
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The configuration of groups resulting from the MCA of the AFLP data matrix successfully discriminated the C. pubescens accessions. The single accessions of C. frutescens and C. chinense grouped with C. annuum in Groups C and F, respectively. Groups A, B, and D were composed exclusively of C. annuum accessions. It is noteworthy that Group B was composed mainly of the semicultivated variety C. annuum var. glabriusculum. Morphological characterization of the accessions in Group A could help to understand the reason why they significantly separated from B and D.
In terms of genetic diversity, Groups D and F brought together low numbers of accessions (6 and 4, respectively) yet were the most diverse. In contrast, Group A was the least diverse despite the fact that its accessions originated from seven different departments. The value of Gst, the genetic differentiation coefficient, indicated that the differences among the six distinct groups resulting from the MCA explained 45% of the total diversity of the sample (Table 4).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It is important to note that, although the amount of genetic diversity in both samples was similar, our results showed that the polymorphism detected in the two sets of accessions, independently of their provenance, was different because we were able to discriminate all but two of the accessions in the samples. In addition, the accessions collected in home gardens showed a higher number of unique AFLP bands than those from the ex situ collection, which could indicate the fact that Alta Verapaz maintained a higher frequency of rare genetic variants. Alta Verapaz is the Department in Guatemala where consumption of pepper is highest, a fact likely because of the presence of the Ketchi and Pocomchi indigenous cultures which use pepper both as a basic component of their diet as well as for religious rituals. Furthermore, new Capsicum genetic types may have arrived from other regions. Lately, the northern part of Alta Verapaz has received a significant number of immigrants moving from other parts of the country, carrying seeds from their original locations.
Because the Capsicum accessions analyzed from the gene bank collection included material from 13 of the 22 departments of Guatemala, the question arises as to whether the breadth of the gene bank collection of Capsicum is sufficiently comprehensive or if further explorations are needed. Given its high diversity and the possibility of finding new pepper types there, Alta Verapaz may be considered a suitable region both for further collecting for ex situ conservation as well as for establishing in situ conservation priority areas, as already suggested recently for genetic resources of chayote and sapote (Azurdia et al., 2001a; Azurdia et al., 2002). In any case, in situ conservation must be always considered as a complementary approach to that of ex situ conservation. In this regard, increasing the ex situ collection with germplasm of Capsicum genotypes from Alta Verapaz would allow further characterization and evaluation of these materials. Moreover, seed kept in the ex situ collection would then be available for breeders and other users, and would help guarantee the conservation of these valuable genetic resources for the future in the event that they disappeared from home gardens.
Sample Genetic Structure
The configuration of the sample in six main clusters indicates that there is structure among the accessions analyzed. Capsicum pubescens separates significantly from the rest of the accessions, as expected according to results obtained previously with morphological characters (Pickersgill, 1971) and isozymes (Jensen et al., 1979). This species originated in South America, though its introduction in Mesoamerica seems to date from the pre-Hispanic times (Heiser, 1995). The clustering of C. frutescens together with C. annuum reflects the closeness of these two species (McLeod et al., 1979; Loaiza-Figueroa et al., 1989). On the other hand, C. chinense groups also with C. annuum, but it does so in a more differentiated cluster with the highest number of unique bands, which also may point to the fact that C. chinense has a South American origin (Prince et al., 1992, 1995).
In addition to the clusters that included accessions from species other than C. annuum, Groups A and B were particularly interesting because they mainly included the semicultivated accessions. These genetic resources grow either in home gardens or areas of cultivation in close proximity with other wild or semiwild peppers and are tolerated as weeds because of their importance for human consumption and with which introgression is likely to occur.
The combined clusters (C and F) of C. annuum with C. frutescens, C. chinense and the semicultivated types (C. annuum var. annuum and C. annuum var. glabriusculum) confirm the existence of gene flow among all these materials (Pickersgill, 1971; Prince et al., 1992). Capsicum annuum, C. frutescens, and C. chinense are in fact distinct morphologically, but can only be considered as different species from a taxonomic perspective and are not biologically distinct. Moreover, C. annuum var. glabriusculum is thought to be the taxon of origin of all cultivated C. annuum types (Azurdia et al., 1995).
Although more accessions per species would be needed to make firm conclusions with regard to their taxonomy, the presence of gene flow tends to even out the diversity among populations and could explain why, other than C. pubescens, the other species or varieties clustered together with C. annuum.
Molecular and Morphological Polymorphism
The three AFLP primer combinations used (modified with an extra selective base) produced a total of 68 polymorphic bands and 72 different banding patterns that allowed the distinction of all but two of the accessions involved (97%). This is an indication of the high discrimination power of the AFLP technology. Accessions 133 and 269, however, shared the same AFLP banding pattern, and this was so in spite of their morphological differences and the fact that they were collected in quite different regions. Accession 133, known as "San Pedro," was collected in San Antonio, Suchitepequez, a hot and humid environment. Accession 269, known as "Miracielo," is only found in Cuilco, Huehuetenango, with a temperate and dry climate. The extension of the experiment with one or more AFLP primer combinations would likely lead to the molecular differentiation of these materials.
On the other hand, the relative evenness in the AFLP genetic composition of Capsicum accessions collected in home gardens and those maintained in the ex situ collection may reflect the fact that most of them are landraces, which have not been involved in formal breeding programs, so that the relative levels of diversity among themselves and even with semicultivated materials are not great. Morphological differences among these accessions may be the result of just a few genes (Azurdia et al., 1995) and, consequently, are difficult to distinguish with the AFLP analysis of a few bands. To test this hypothesis, another type of molecular marker would need to be used, such as microsatellites, which are more powerful for infraspecific discrimination, but which because there were not publicly available sequences at the time of this research, could not be considered.
In the present work, the importance of home gardens as an agroecosystem to generate and conserve genetic diversity of Capsicum in situ has been proven. This is the first time that a molecular methodology was applied to assess and compare the genetic diversity maintained in two complementary conservation schemes, ex situ and in situ. Therefore, it can be taken as the basis for additional studies that include more species and more accessions per species to elaborate on the substantiation of the importance of home gardens as an attractive option for in situ conservation, especially in centers of origin and diversity of cultivated plants where both the biological and cultural richness are combined.
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ACKNOWLEDGMENTS |
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Received for publication December 5, 2003.
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