Genetic Relationships among Accessions of Four Species of Desmodium and Allied Genera ( Dendrolobium Triangulare,Desmodium Gangeticum,Desmodium Heterocarpon,and Tadehagi Triquetrum )

Abstract

French

Random amplified polymorphic DNA markers (RAPD) were used to assess the genetic relatedness among accessions of four species of Desmodium and allied genera (Dendrolobium triangulare, Desmodium gangeticum, Desmodium heterocarpon ssp. heterocarpon, and Tadehagi triquetrum) originating from Northeast Vietnam. Since information on the genetic diversity of these species is deficient, the creation of baseline data is an important means for the development of more sustainable and cost-efficient conservation approaches which eventually result in more comprehensive ex situ germplasm collections. The species analyzed are native to tropical and subtropical Asia, Australia, and Oceania and possess a potential as forage and/or medicinal plants. Moderate levels of inter-accession diversity represented by 37.5% and 33.3% of polymorphic fragments (P%) and average Jaccard's similarity coefficients (JSCs) of 0.60 and 0.64 were found in D. heterocarpon and T. triquetrum, respectively, while moderate to high levels were detected in D. triangulare (P% = 52.9 and JSC = 0.61) and D. gangeticum (P% = 34.5 and JSC = 0.49). Mantel tests failed to reveal a correlation between geographic and genetic distances. Based on the results of this study, baseline data for further marker-assisted research are generated and future collecting and ex situ conservation strategies for the species studied are discussed.

Keywords

cover crop forage genetic relatedness medicinal plants Northeast Vietnam RAPD

Introduction

Within the Leguminosae, the tribe Desmodieae (Benth.) Hutch. is recognized for its substantial contribution to forage production in the tropics [43]. It comprises several species of agronomic interest such as Dendrolobium triangulare (Retz.) Schindl. (syn. Desmodium triangulare [Retz.] Merr.), Desmodium gangeticum (L.) DC., Desmodium heterocarpon (L.) DC. ssp. heterocarpon, and Tadehagi triquetrum (L.) H. Ohashi (syn. Desmodium triquetrum (L.) DC.) (Fig. 1). Due to their ability to adapt to low-fertility soils and to tolerate water drought, these species represent valuable plant genetic resources for forage production, soil protection and improvement in marginal smallholder farming systems of subhumid and humid tropical regions [10, 20, 41]. Moreover, the four legume species included in this study have a long history in traditional Asian health care [30, 33, 46].

Fig. 1.

Images of four species of Desmodium and allied genera analyzed with RAPDs: (a) Dendrolobium triangulare (Retz.) Schindl., (b) Desmodium gangeticum (L.) DC., (c) Desmodium heterocarpon (L.) DC. ssp. heterocarpon, (d) Tadehagi triquetrum (L.) H. Ohashi. Photos (a, d) by R. Schultze-Kraft, (b) by A. Ciprián (CIAT), and (c) by B. Heider.

With the exception of D. triangulare, a woody, perennial shrub, all analyzed species are woody herbs or subshrubs that mainly occur in fallow areas, thickets, forest borders, or along roadsides [4, 29]. The genus Desmodium and its allied genera, members of the subtribe Desmodiinae, are mainly native to the tropics and subtropics. Ohashi [28] records two centers of geographic distribution and differentiation: Southeast Asia and Mexico. According to Williams [47] Southeast Asia is a center of legume diversity. There, systematic exploration of native legume genetic resources started only about two decades ago when a series of collecting missions under the auspices of CIAT (International Center for Tropical Agriculture) was carried out in Malaysia, Indonesia, tropical China, Thailand, Papua New Guinea, and Vietnam. However, the mountainous and highly diverse north of Vietnam was not visited during previous collecting missions [37, 38]. At the same time, plant genetic resources of the North Vietnamese mountains are increasingly threatened due to ecosystem transformation and growing demand of natural resources in the wake of high population pressure [45]. For these reasons the area was chosen as the collection target in this study.

Molecular marker techniques have become increasingly important in conservation biology. Information about the underlying genetic diversity of species is essential for sustainable use and efficient conservation efforts of plant genetic resources [21, 40]. Unfortunately, such information is still inadequate concerning species of Desmodium and allied genera, especially at the DNA level. The traditional methodological approach to analyzing genetic diversity is based on morphological and/or agronomic traits. However, results of such studies might be biased by confounding environmental factors that influence the phenotype. Apart from D. heterocarpon, all Desmodium species so far analyzed were merely subjected to taxonomic, agronomic or, in the case of D. gangeticum, chemical studies [9, 22, 28, 41]. Isozyme electrophoresis allowed distinguishing D. heterocarpon ssp. ovalifolium from D. heterocarpon ssp. heterocarpon [27] but failed to discriminate the former at genotype level [17]. In a recent study, genetic diversity of a germplasm collection of D. heterocarpon ssp. ovalifolium held by CIAT was analyzed and a tentative core collection built up on the basis of random amplified polymorphic DNA (RAPD) marker data [6].

RAPDs provide a simple, fast, and cost efficient methodology to primarily screen the level of genetic variation of a great number of yet unknown genotypes [48]. Even though RAPDs are frequently criticized for unreliable reproducibility [15], dominance [25], and comigration [35], they have proven to be a useful tool in studies analyzing genetic variation in a substantial number of tropical legume species (e.g., [8], [16], [23]).

For these reasons, RAPD markers were used in the present study to determine the genetic relatedness among accessions of four Desmodium and allied genera collected in Northeast Vietnam. The RAPD methodology applied will also be useful for screening duplication and establishing core collections in gene banks.

Methods

Plant material - Germplasm of the four species was collected in 1999 and 2000 from native populations within their natural habitats in Bac Kan province, Northeast Vietnam (Fig. 2). At each collecting site ecogeographic and plant specific passport data were recorded including geographic position, elevation, habitat, topography, soil, and site characteristics as well as plant-related information (e.g., plant vigor, occurrence of pests and diseases). A total of 35 Dendrolobium triangulare, 37 Desmodium gangeticum, 66 Desmodium heterocarpon, and 24 Tadehagi triquetrum accessions were included in this study. All accessions are combined (bulked) seed samples in order to represent the natural population at the sites where they were collected.

Fig. 2.

Collecting sites of Desmodium and allied genera in Bac Kan province, Northeast Vietnam.

Ten plants of each accession were propagated from scarified seeds and maintained under greenhouse conditions. Fresh leaf material was sampled from six-month-old plantlets. In the case of T. triquetrum and D. heterocarpon, genetic analyses were performed on a maximum number of 10 individual plants per accession in order to obtain sufficient leaf material for DNA extraction. Growth of D. gangeticum and D. triangulare was more vigorous, yet few individuals failed to survive. In order to standardize methodology, the number of individuals per bulk was limited to five plants per accession forming the a bulk. The surplus was placed in the b bulk. DNA of a and b bulks was separately isolated and analyzed. The division into an a and b bulk was also used to approximate the ideal number of plants per bulk.

DNA isolation and amplification - Genomic DNA was extracted from 100 mg of fresh leaf tissue. Samples were frozen in liquid nitrogen and subsequently homogenized. Three out of the four species (D. gangeticum, D. heterocarpon, and T. triquetrum) were analyzed using a small-scale DNA isolation method (Plant DNA Mini Kit EZNA from Peqlab). In the case of D. triangulare, the established method had to be modified since several alternative extraction protocols were tested but failed to extract good quality DNA. Therefore DNA extraction and purification were finally performed with the DNeasy Plant Mini Kit from Qiagen which provided the best DNA extraction results. DNA concentrations were measured (Gene Ray photometer; Biometra) and standardized against known concentrations of Λ-DNA (Roche Diagnostics) on agarose gels. After quantification DNA was diluted in TE buffer to a final concentration of 5 μg DNA μl¹.

Polymerase chain reaction (PCR) amplifications were performed in reaction mixtures containing 10 ng template DNA, 0.2 μM primer, 100 μM dNTP's, 10 mM PCR buffer, 1.5 mM MgCl₂, and 1 unit Taq-polymerase in a total volume of 10 μl. Amplifications were carried out in a thermo-cycler (T Gradient, Biometra) with the following program: initial denaturation step of 3 min at 94 °C followed by 34 cycles of 1 min at 94 °C, 1 min at 36 °C, 2 min at 72 °C, and a final extension step of 5 min at 72 °C. DNA fragments were resolved by electrophoresis in 1.5% agarose gels run at 4 V cm¹. Gels were stained with ethidium bromide, visualized under UV-light, and photographed by a CCD video camera (Raytest). To ensure reproducibility of banding patterns, each amplification reaction was repeated twice. Four primer kits (L, M, N, and U; Roth), each comprising 20 decamer primers, were screened for each of the four analyzed species. Only those primers were selected that showed well-defined and reproducible banding patterns (Table 1).

Table 1:

Oligonucleotide primers employed in RAPD analysis, their sequence, number of bands obtained per primer, size range of bands, and specific size of polymorphic (polym.) fragments.

Primer code	Sequence (5′ to 3′)	Number of bands		Size (bp)	Specific size (bp) of polym. fragments
		Ploym.	Monom.	min-max
Dendrolobium triangulare
L-15	AAGAGAGGGG	4	1	500–1000	700, 800, 900, 1000
L-16	AGGTTGCAGG	1	3	490–1000	490
M-10	TCTGGCGCAC	1	3	450–1300	1300
N-10	ACAACTGGGG	3	1	350– 700	500, 600, 700
Desmodium gangeticum
L-01	GGCATGACCT	2	2	450– 900	520, 750
L-15	AAGAGAGGGG	2	3	500–1300	620, 750
L-18	ACCACCCACC	2	2	250–1200	700, 1200
M-06	CTGGGCAACT	1	5	200–1500	480
M-10	TCTGGCGCAC	1	2	750–1400	1100
M-12	GGGACGTTGG	2	5	320–1300	600, 650
Desmodium heterocarpon
L-11	ACGATGAGCC	1	5	290–1300	900
L-15	AAGAGAGGGG	3	2	400–1100	650, 750, 1100
L-19	GAGTGGTGAC	2	1	400– 900	750, 900
N-01	CTCACGTTGG	2	4	400–1000	700, 900
N-09	TGCCGGCTTG	1	3	500–1000	900
Tadehagi triquetrum
L-13	ACCGCCTGCT	1	4	350–1200	450
M-12	GGGACGTTGG	2	3	450–1200	900, 1100
M-18	CACCATCCGT	2	3	350–1250	1200, 1250
N-10	ACAACTGGGG	3	1	350– 650	520, 650, 700
U-09	CCACATCGGT	1	3	400–1000	1000
U-20	ACAGCCCCCA	1	6	350–1300	700

Data analysis - Presence or absence of amplified products was scored as discrete characters (1 = presence and 0 = absence) across all accessions and for each primer. The resulting binary matrices were used to calculate Jaccard's similarity coefficients (JSCs) with the aid of SIMQUAL (Similarity for Qualitative Data) routine. Based on JSCs, dendrograms were constructed by means of the unweighted pair group method with arithmetic averages (UPGMA) employing SAHN (Sequential, Agglomerative, Hierarchical, and Nested clustering). These analyses were performed using the software package NTSYS-pc, version 1.80 [36]. Based on the calculation of pairwise binary genetic distances for dominant data [5, 14] a Mantel test (999 permutations) was applied to analyze correlations between genetic and geographical distances for each accession using GenAlEx V5 [31].

Results

Dendrolobium triangulare (Retz.) Schindl.

RAPD polymorphism - In the analysis of 35 D. triangulare accessions, four primers were used that generated a total of 17 RAPD fragments of which 52.9% were polymorphic. Each primer amplified four (L-16, M-10, and N-10) to five (L-15) markers, an average of 4.25 markers per primer and 2.25 polymorphic markers per primer, which ranged in size from 350 to 1300 basepairs (bp; Table 1). Examples of RAPD profiles due to primer L-15 are shown in Fig. 3.

Fig. 3.

Examples of RAPD profiles among accessions of Dendrolobium triangulare, Desmodium gangeticum, D. heterocarpon, and Tadehagi triquetrum obtained with the respective primers. Numbered lanes display the RAPD profile of different accessions; lane M: molecular weight marker (100 bp DNA-ladder, Roche Diagnostics); lane C: negative control (H₂O_dest. instead of template DNA). ^*Polymorphism at 1200 bp not shown on this gel.

JSCs based on RAPD variation - A similarity matrix based on JSCs was constructed to estimate the relatedness among accessions. Generally, JSCs of all analyzed species ranged from 0 to 1. In the case of D. triangulare, the lowest value (JSC = 0) was found in comparisons of accession 493a to accession 744a and 873b, respectively. Mean JSC was 0.61 indicating that a medium to high level of genetic similarity was identified among D. triangulare accessions. Out of a total of 35 accessions, 23 were divided into a and b bulks yielding 12 accessions, in which a and b bulks proved to be identical. However, 65.7% were divergent.

UPGMA cluster analysis - The dendrogram (Fig. 4) depicts a diverse and complex clustering. Accessions of D. triangulare were classified into seven main clusters (I to VII). A and b bulks of the same accessions were frequently located in different subclusters with very few exceptions positioned within the same main cluster. The first and second cluster comprised 73% of all accessions. No tendency to group in accordance to collecting regions was detected. A Mantel test resulted in a coefficient of correlation between genetic and geographic distances of R = 0.095 (P = 0.042).

Fig. 4.

UPGMA dendrogram illustrating genetic relationships among 35 accessions of Dendrolobium triangulare. The numerical scale of JSCs indicates increasing similarity.

Desmodium gangeticum (L.) DC.

RAPD polymorphism - A total of six primers were used to screen 37 accessions of D. gangeticum. These selected primers amplified 29 bands, of which ten were polymorphic (34.5%). The number of amplified fragments varied from three (M-10) to seven (M-12) with an average of 4.83, and fragment size from 200 to 1500 bp (Table 1). The typical RAPD banding pattern obtained with primer L-18 is presented in Fig. 3.

JSCs based on RAPD variation - RAPD-based JSCs ranged from 0 to 1. Average JSC was 0.49, the lowest value among all species analyzed in this study. Out of 37 accessions, 34 were split into a and b bulks. Fewer than half (43.2%) of all b bulk results confirmed the respective a bulk data. A moderate to high level of genetic diversity was found among the analyzed D. gangeticum accessions.

UPGMA cluster analysis - A complex dendrogram visualizes how the D. gangeticum accessions are grouped on the basis of JSCs (Fig. 5). Seven main clusters shared more than 30% of all alleles. The clustering does not allow concluding whether there is a correspondence to geographical boundaries or origin. A Mantel test showed no correlation between genetic and geographic distances (R = 0.032; P = 0.186).

Fig. 5.

UPGMA dendrogram illustrating genetic relationships among 37 accessions of Desmodium gangeticum. The numerical scale of JSCs indicates increasing similarity.

Desmodium heterocarpon (L.) DC. ssp. heterocarpon

RAPD polymorphism - The five primers selected to detect polymorphism among 66 D. heterocarpon accessions yielded a total of 24 RAPD markers of which nine (37.5%) were polymorphic. The total number of scored products per primer ranged from three (L-19) to six (L-11) with an average of 4.8. Molecular mass of bands varied from 290 to 1300 bp in size (Table 1). Examples of the polymorphism detected with primer N-01 are shown in Fig. 3.

JSCs based on RAPD variation - Among the investigated D. heterocarpon accessions an intermediate level of genetic similarity was found. RAPD based JSCs varied from 0 to 1 with a mean of 0.60.

UPGMA cluster analysis - Two main clusters (I and II) can be distinguished that deviate at a JSC of 0.27 (Fig. 6). Accessions within each of the two clusters share between 61% (lower division) and 67% (upper division) of all alleles. Genetic differentiation among accessions of the two genetically distinct main clusters (I and II) is substantial though it is not related to geographic distances (Mantel test: R = 0.05; P = 0.187).

Fig. 6.

UPGMA dendrogram illustrating genetic relationships among 66 accessions of Desmodium heterocarpon. The numerical scale of JSCs indicates increasing similarity.

Tadehagi triquetrum (L.) H. Ohashi

RAPD polymorphism - The 24 T. triquetrum accessions were analyzed using six primers that produced 30 RAPD markers ranging in size between 350 and 1300 bp. The total number of amplified bands per primer varied between four (N-10 and U-09) and seven (U-20) with an average of five bands per primer (Table 1). Mean percentage of polymorphic bands of all primers was 33.3%. Of the polymorphic fragments, three were unique and occurred only in accession 918. An example of the amplification products obtained with primer U-09 is shown in Fig. 3.

JSCs based on RAPD variation - Jaccard's similarity coefficients ranged from 0 to 1 with a mean of 0.64. The lowest similarity value was exclusively observed in comparisons of accession 918 with other accessions.

UPGMA cluster analysis - The UPGMA dendrogram divided into four main clusters (I–IV, Fig. 7). The first branch separated the outlier accession 918 from the remaining samples at a JSC of 0.04. Accession 918 formed an isolated cluster (IV), most distinct from all other accessions. The division of the main branch detached into an upper (I and II) and a lower cluster (III) at a JSC of 0.45. The UPGMA dendrogram visualises an intermediate level of similarity among T. triquetrum accessions. Clustering, however, did not coincide with geographic distances. A Mantel test rendered a coefficient of correlation of R = 0.135 (P = 0.107).

Fig. 7.

UPGMA dendrogram illustrating genetic relationships among 24 accessions of Tadehagi triquetrum. The numerical scale of JSCs indicates increasing similarity.

Discussion

RAPD analyses were useful to determine genetic relatedness among accessions of four species of Desmodium and allied genera collected in North Vietnam. Moderate levels of diversity existed among accessions of D. heterocarpon and T. triquetrum while moderate to high levels were found in D. gangeticum and D. triangulare.

The level of polymorphism observed among accessions of D. gangeticum (34.5%), D. heterocarpon (37.5%), and T. triquetrum (33.3%) was among the lowest compared to many studies of tropical herbaceous legume species. Liu [23, 24] reported 68.9% of polymorphism among Lablab purpureus and 86.6% among Stylosanthes scabra genotypes. RAPD analyses of Vigna angularis (49%), Pueraria montana var. lobata (54.3%), and P. phaseoloides (45.5%) yielded slightly higher levels of polymorphism [13, 49]. Comparisons of D. triangulare (52.9%) with RAPD marker studies of other tropical legume trees and shrubs resulted in a relatively high level of polymorphic loci for this species. While higher levels of polymorphism were reported for Cratylia argentea (91.8%) [1], and Flemingia macrophylla (95%) [12], lower levels were found in Argentinian Acacia species (19.1%) [3] and Prosopis species (28.3%) [16]. However, it should be recognised that the chosen primer selection approach may not have resulted in unbiased estimates of levels of polymorphism. Preliminary primer screens were run on a selection of 10 accessions per species and only those primers that generated polymorphic fragments were selected. In the case of uniform amplification, primers were discarded and not used for full screening of all accessions which might have interfered with data accuracy.

Average JSCs obtained in this study (D. heterocarpon, 0.60, T. triquetrum, 0.64, and D. gangeticum, 0.49) were comparable to previous RAPD marker studies of herbaceous legumes. Xu et al. [49] found JSCs of 0.73 in cultivated forms and 0.60 in wild and weedy populations of V. angularis, while Yee et al. [50] and Heider et al. [13] recorded JSCs of 0.52 in V. angularis and P. phaseoloides, respectively. Results of a recent molecular marker study showed a comparatively low JSC in Pueraria montana var. lobata (0.35) [13]. RAPD analyses of tropical tree and shrub legumes revealed average JSCs that were slightly higher in F. macrophylla (0.67) [12] than in D. triangulare (0.61). However, the fact that a large number of previous studies on genetic diversity of tropical legumes did not disclose the overall JSC, used different diversity indices, or employed different molecular marker techniques, represents an obstacle to direct comparisons.

Several authors experimented with the optimal number of individual plants that were pooled in order to reduce the sensitivity of RAPDs to DNA sequence variation and thus to ensure a representative estimation of genetic diversity in heterogeneous populations [7, 18, 19]. The recommended number of individuals per bulk ranged between three and 20. Based on these references, bulk sizes in the study of D. heterocarpon and T. triquetrum was adjusted to 10 individuals in each bulk. Due to low germination rates and low seedling vigor, the number of surviving greenhouse individuals restricted the bulk sizes of D. triangulare and D. gangeticum to five individual plants each (a bulk). Any additional plants formed b bulks that consisted of up to five individuals. The fact that 70.4% of a and b bulks of D. triangulare and 43.2% of D. gangeticum were not identical and therefore did not coincide in the same cluster, illustrates clearly that bulk sizes were too small to dilute rare alleles. In general, Desmodieae are assumed to be predominantly self-pollinated, but outcrossing rates, ranging from 0.01% to 10%, are also likely to occur as in other tropical legumes [26, 32]. Given the rudimentary knowledge available on breeding systems of the analyzed species, the degree of heterozygosity inherent in accessions of D. triangulare and D. gangeticum seems to be much higher than originally assumed. Seeds were sampled as accessions representing the natural population from which they were collected. The aim of collecting accessions is to capture the entire range of genetic variation, as much as possible, in a given population [2]. Thus individual seedlings were bulked for RAPD analysis to represent the genetic constitution of the original accession. The exclusive use of bulk samples, however, does not allow the use of statistical parameters of population genetics and impedes the assessment of the species' breeding system. There was no substantial information available on the reproduction mode of the four studied species. However, such information is decisive for the amount and partitioning of genetic variation in and among populations [11]. Therefore, in order to draw firm conclusions on intra-accession diversity, individual plants composing an accession should additionally be analyzed in future studies, a measure that might aid in improving data quality and thereby contribute to optimizing collection and conservation management.

Implications for conservation

Desmodium and allied genera play an important role in different research and development endeavors to address declining soil fertility, erosion, overgrazing, and dry-season forage constraints in marginal areas of the tropics and subtropics. Desmodium heterocarpon ssp. heterocarpon attracted research interest due to its ability to persist in highly productive grass/legume mixtures in humid subtropical and tropical regions, while tolerating heavy grazing, acid soils, drought, temporary water-logging, and moderate frost periods [10]. The carpon desmodium cultivar “Florida” of this species obtained economic significance as a forage legume in peninsular Florida [39]. Tadehagi triquetrum (L.) H. Ohashi is agronomically interesting because of its capacity to provide crude protein for livestock feeding during dry seasons and for its good adaptation to acid, low-fertility soils [42]. Dendrolobium triangulare (Retz.) Schindl. provides fuelwood and is suitable for soil protection and improvement. It has minor forage potential due to variable levels of condensed tannins [22]. Desmodium gangeticum (L.) DC. was found to be highly productive in shady environments such as under plantation crops [41]. Particularly, D. gangeticum is considered a plant resource of pharmaceutical importance which serves as a remedy for diarrhea, dysentery, and several eye diseases. These curative properties are due to a number of alkaloids, a pterocarpanoid (gangetin), flavons, and isoflavanoid glycosides as active compounds with antifungal, antileishmanial, antioxidant, antiradical, and anticholinesterase activities [e.g., 9].

The mountainous north of Vietnam experienced dramatic environmental and social changes during the last decades threatening the biological diversity of the region [45]. Desmodieae as well as other wild-growing plant species are subjected to genetic erosion because of habitat destruction and resource overexploitation as a consequence of increasing demographic pressure. Since the vast majority of tropical forage legume cultivars are unimproved selections from wild populations [34], the enormous yet untapped legume diversity of the North Vietnamese mountainous regions should be explored in order to avoid reliance on a narrow germplasm base. This includes further germplasm collecting as well as efficient ex situ conservation measures. Conservation of plant genetic diversity in gene banks requires balancing carefully between numerically extensive collections and the feasibility of maintaining and managing germplasm storage. Knowledge about genetic diversity in a target taxon is also indispensable when considering the establishment of a core collection or eliminating redundancy in collections [21, 40].

The heterogeneous clustering of a and b bulks in D. triangulare (Fig. 4) and D. gangeticum accessions (Fig. 5) imposes difficulties to the establishment of an ex situ conservation approach for these species. But generally, genotypes that are redundant may be removed from ex situ storage while outliers and particularly distinct accessions should be included. Clusters in which numerous extremely related accessions occur may be condensed to subsamples. However, such selection must be carried out with circumspection as RAPD diversity does not necessarily reflect diversity at quantitative trait loci, and important genotype characteristics might become lost [44]. Thus morphological plant characteristics should also be considered before discarding germplasm. In the case of D. triangulare and D. gangeticum, each cluster VII certainly encompasses the most distinct accessions (Figs. 4 and 5). Accessions that did not represent unique genotypes, i.e., those that formed clusters at a JSC of 1, are candidates for removal. The D. triangulare dendrogram contains several of these, particularly in clusters I and II. The first three clusters of D. gangeticum could also be reduced.

Within the D. heterocarpon (Fig. 6) collection accessions 491, 512, 544, and 743 were the most distinct accessions and therefore suggest themselves for ex situ conservation. Apart from these, a representative subsample of each cluster, eliminating the numerous duplicates, would suffice.

A comparison of passport data between the two main clusters of D. heterocarpon revealed that accessions forming the upper division were collected from humid and frequently shady habitats growing in heavy soils characterized by increased nitrogen, calcium, magnesium, and organic matter contents while accessions from the lower division grew in poorer soils and in more marginal, sun-prone, and therefore drier environments (data not shown). Thus it appears that the two main divisions consist of two different ecotypes.

High genetic differentiation was found among three of 24 T. triquetrum accessions (944, 945, and 918), while the remaining accessions harbored high similarity values among them. The outlier (918) of T. triquetrum exhibited three unique markers and as a consequence may contain unique genetic characteristics, which indicates that significant distinctiveness was captured here. However, neither taxonomic plant characteristics nor collecting site-related passport data could explain this distinctiveness. In general, data show that T. triquetrum represented the least diverse collection among the four species analyzed but also the smallest. Therefore, it seems advisable to conduct further, more extensive collecting missions at larger distances in order to collect more genetic diversity that would qualify for ex situ conservation. As for the samples collected in Bac Kan province, the outlier, accessions comprising cluster III, and a representative subsample of clusters I and II (Fig. 7) should be included if aiming at efficient ex situ conservation.

Future collecting missions should target populations at large distances covering altitudinal gradients in order to ensure that the ecological and geographic ranges of the respective species are included [2]. Outcrossing must be assumed in all four species studied. Hence, individuals within distant populations should be comprehensively collected instead of extracting a few individuals from many populations at short distances. A manageable and efficient ex situ conservation approach aims at capturing maximum genetic diversity within stored material, comprising outliers and genetically distinct germplasm, and representative subsamples of very similar or even redundant genotypes. Ideally such ex situ conservation would be based on both molecular and morphological data in order not to lose valuable adaptive variation.

Concluding remarks

Generated data showed that RAPD markers, despite their widely discussed disadvantages, are suitable tools to scan anonymous DNA of species with high secondary metabolite content such as Desmodium and allied genera. However, limited reliability of RAPD molecular markers should be improved by increasing the number of RAPD fragments. As no correlation between the pattern of genetic clustering and geographic distance or passport data was detected, further data analyses will be necessary in order to relate genetic diversity to greater geographic distance. Increasing emphasis is being placed on empirical data of genetic diversity to assess and manage genetic resources in a sustainable way. Therefore this study may contribute to provide germplasm conservationists with data in order to facilitate conservation efforts.

Footnotes

Acknowledgements

Research was financially supported by the Vater-und-Sohn-Eiselen-Stiftung, Ulm, Germany. The authors are also thankful to the Vietnam Agricultural Science Institute, Hanoi, Vietnam, especially to Prof. Dr. Tran Dinh Long and Mr. Ha Dinh Tuan for their advice und cooperation during germplasm collection.

References

Andersson

M.S.

Schultze-Kraft

Peters

Duque

M.C.

Gallego

2007. Extent and structure of genetic diversity in a collection of the tropical multipurpose shrub legume Cratylia argentea (Desv.) O. Kuntze as revealed by RAPD markers. Electronic Journal of Biotechnology 10(3): 386–399. www.ejbiotechnology.info/content/vol10/issue3/full/2/index.html

Brown

A.H.D.

Marshall

D.R.

1995. A basic sampling strategy: Theory and practice. In: Collecting Plant Genetic Diversity - Technical Guidelines. Guarino

Rao

V. Ramanatha

and Reid

(Eds.), pp. 75–91. CAB International, Wallingford, UK.

Casiva

P.V.

Saidman

B.O.

Vilardi

J.C.

Cialdella

A.M.

2002. First comparative phenetic studies of Argentinean species of Acacia (Fabaceae), using morphometric, isozymal, and RAPD approaches. American Journal of Botany 89: 843–853.

Dy Phon

Ohashi

Vidal

J.E.

1994. Leguminosae (Fabaceae), Papilionoideae, Desmodieae. Révision de la Flore Générale de l'Indochine. Flore du Cambodge, du Laos et du Viêtnam, Vol. 27, Muséum National d'Histoire Naturelle, Paris, France.

Excoffier

Smouse

P.E.

Quattro

J.M.

1992. Analysis of molecular variance inferred from metric distances among DNA haplotypes: Application to human mitochondrial DNA restriction sites. Genetics 131: 479–491.

Fischer

Berndl

Schultze-Kraft

2004. Identification of a core collection in Desmodium ovalifolium based on marker data. Book of Abstracts, Deutscher Tropentag 2004, pp. 281. Humboldt-Universität zu Berlin, Germany.

Forster

J.W.

Jones

E.S.

Kölliker

Drayton

M.C.

Dupal

M.P.

Guthridge

K.M.

Smith

K.F.

2001. Application of DNA profiling to an outbreeding forage species. In: Plant Genotyping: The DNA Fingerprinting of Plants. Henry

R.J.

(Ed.), pp. 299–319. CAB International, Wallingford, UK.

Gillies

A.C.M.

Abbott

1998. Evaluation of random amplified polymorphic DNA for species identification and phylogenetic analysis in Stylosanthes (Fabaceae). Plant Systematics and Evolution 211: 201–216.

Govindarajan

Rastogi

Vijayakumar

Shirwaikar

Rawat

A.K.S.

Mehrotra

Pushpangadan

2003. Studies on the antioxidant activities of Desmodium gangeticum. Biological and Pharmaceutical Bulletin 26: 1424–1427.

10.

Hacker

J.B.

Kretschmer

A.E.

Jr.

1992. Desmodium heterocarpon (L.) DC. In: PROSEA 4: Forages, 't Mannetje

and Jones

R.M.

(Eds.), pp. 106–108. Backhuys Publishers, Leiden, The Netherlands.

11.

Hamrick

J.L.

Godt

M.J.W.

1996. Effects of life-history traits on genetic diversity in plant species. Philosophical Transactions of the Royal Society of London, Series B 351: 1291–1298.

12.

Heider

Andersson

Schultze-Kraft

2007. RAPD variation among North Vietnamese Flemingia macrophylla (Willd.) Kuntze ex Merr. accessions. Biodiversity and Conservation 16: 1617–1631.

13.

Heider

Fischer

Berndl

Schultze-Kraft

2007. Analysis of genetic variation among accessions of Pueraria montana (Lour.) Merr. and Pueraria phaseoloides (Roxb.) Benth. based on RAPD markers. Genetic Resources and Crop Evolution 54: 529–542.

14.

Huff

D.R.

Peakall

Smouse

P.E.

1993. RAPD variation within and among populations of outcrossing buffalograss (Buchloë dactyloides (Nutt.) Engelm.). Theoretical and Applied Genetics 86: 927–934.

15.

Jones

C.J.

Edwards

K.J.

Castaglione

Winfield

M.O.

Sala

van de Wiel

Bredemeijer

Vosman

Matthes

Daly

Brettschneider

Bettini

Buiatti

Maestri

Malcevschi

Marmiroli

Aert

Volckaert

Rueda

Linacero

Vazquez

Karp

1997. Reproducibility testing RAPD, AFLP and SSR markers in plants by a network of European laboratories. Molecular Breeding 3: 381–390.

16.

Juárez-Muñoz

Carrillo-Castañeda

Arreguin

Rubluo

2002. Inter-and intra-genetic variation of four wild populations of Prosopis using RAPD-PCR fingerprints. Biodiversity and Conservation 11: 921–930.

17.

Klein

1997. Isoenzymcharakterisierung einer Kollektion der tropischen Weideleguminose Desmodium ovalifolium. MSc thesis, University of Hohenheim, Stuttgart, Germany.

18.

Kongkiatngam

Waterway

M.J.

Coulman

B.E.

Fortin

M.G.

1996. Genetic variation among cultivars of red clover (Trifolium pratense L.) detected by RAPD markers amplified from bulk genomic DNA. Euphytica 89: 355–361.

19.

Kraft

Säll

1999. An evaluation of the use of pooled samples in studies of genetic variation. Heredity 82: 488–494.

20.

Kretschmer

A.E.

Jr. Pitman

W.D.

2001. Germplasm resources of tropical forage legumes. In: Tropical Forage Plants: Development and Use. Sotomayor-Ríos

and Pitman

W.D.

(Eds.), pp. 41–57. CRC Press, Boca Raton, Florida, USA.

21.

Lanaud

1999. Use of molecular markers to increase understanding of plant domestication and to improve the management of genetic resources; some examples with tropical species. Plant Genetic Resources Newsletter 119: 26–31.

22.

Lascano

C.E.

Maass

Keller-Grein

1995. Forage quality of shrub legumes evaluated in acid soils. In: Proceedings of the Workshop “Nitrogen Fixing Trees for Acid Soils”. Evans

D.O.

and Szott

L.T.

(Eds.), pp. 228–236. Nitrogen Fixing Tree Association (NFTA) and Centro Agronómico Tropical de Investigación y Enseñanza (CATIE), Turrialba, Costa Rica.

23.

Liu

C.J.

1996. Genetic diversity and relationships among Lablab purpureus genotypes evaluated using RAPD markers. Euphytica 90: 115–119.

24.

Liu

C.J.

1997. Geographical distribution of genetic variation in Stylosanthes scabra revealed by RAPD analysis. Euphytica 98: 21–27.

25.

Lynch

Milligan

B.G.

1994. Analysis of population genetic structure with RAPD markers. Molecular Ecology 3: 91–99.

26.

Maass

L.M.

Torres

A.M.

1998. Off-types indicate natural outcrossing in five tropical forage legumes in Colombia. Tropical Grasslands 32: 124–130.

27.

McKellar

M.A.

1988. Characterization of Desmodium heterocarpon (L.) DC. and Desmodium ovalifolium Wall. PhD thesis, University of Florida, Gainesville, Florida, USA.

28.

Ohashi

1973. The Asiatic Species of Desmodium and its Allied Genera (Leguminosae). Ginkgoana. Contributions to the Flora of Asia and the Pacific Region No. 1, Academia Scientific Book, Tokyo, Japan.

29.

Ohashi

1991. Taxonomic studies in Desmodium heterocarpon (L.) DC. (Leguminosae). Journal of Japanese Botany 66: 14–25.

30.

Parrotta

J.A.

2001. Healing plants of peninsular India. CABI Publishing, Wallingford, UK.

31.

Peakall

Smouse

P.E.

2001. GenAlEx V5: Genetic Analysis in Excel. Population genetic software for teaching and research. Australian National University, Canberra, Australia. www.anu.edu.au/BoZo/GenAlEx/

32.

Pearson

C.J.

Ison

R.L.

1997. Agronomy of grasslands systems. 2nd edn. Cambridge University Press, UK.

33.

Perry

L.M.

Metzger

1980. Medicinal plants of East and Southeast Asia: attributed properties and uses. The Massachusetts Institute of Technology, Cambridge, USA.

34.

Peters

Horne

P.M.

Schmidt

Holmann

Kerridge

P.C.

Tarawali

S.A.

Schultze-Kraft

Lascano

C.E.

Argel

Stür

Fujisaka

Müller-Sämann

Wortmann

2001. The role of forages in reducing poverty and degradation of natural resources in tropical production systems. Agricultural Research and Extension Network (AgREN), Paper 117.

35.

Rieseberg

1996. Homology among RAPD fragments in interspecific comparisons. Molecular Ecology 5: 99–105.

36.

Rohlf

F.J.

1993. NTSYS-pc numerical taxonomy and multivariate analysis system. Version 1.8, Exeter Software, Setauket, New York, USA.

37.

Schultze-Kraft

Pattanavibul

Gani

Wong

C.C.

1989. Collection of native germplasm resources of tropical forage legumes in Southeast Asia. Proc. XVI International Grassland Congress, 4–11 October 1989, Nice, France, pp. 271–273. Association Française pour la Production Fourragère, Nice, France.

38.

Schultze-Kraft

Tuan

H.D.

N.P.

1993. Collection of native forage legume germplasm in Vietnam. Proc. XVII International Grassland Congress, 13–16 February 1993, New Zealand and Queensland, Australia, pp. 233–234. New Zealand Grassland Association and Tropical Grassland Society of Australia, New Zealand and Australia.

39.

Sollenberger

L.E.

Kalmbacher

R.S.

2005. Aeschynomene and carpon desmodium: Legumes for bahiagrass pasture in Florida. Tropical Grasslands 39: 227.

40.

Spooner

van Treuren

de Vicente

M.C.

2005. Molecular markers for genebank management. IPGRI Technical Bulletin No. 10. International Plant Genetic Resources Institute (IPGRI), Rome, Italy.

41.

Stür

W.W.

1990. Screening forage species for shade tolerance – a preliminary report. In: Forages for Plantation Crops, Shelton

H.M.

and Stür

W.W.

(Eds.), pp. 58–63. ACIAR Proc. No. 32, Canberra, Australia. 27–29 June 1990, Sanur Beach, Bali, Indonesia.

42.

Thomas

Schultze-Kraft

1990. Evaluation of five shrubby legumes in comparison with Centrosema acutifolium, Carimagua, Colombia. Tropical Grasslands 24: 87–92.

43.

Van der Maesen

L.J.G.

1996. The wealth of forage plants. Wageningen Agricultural University Papers 96: 83–92.

44.

Virk

P.S.

Ford-Lloyd

B.V.

Jackson

M.T.

Pooni

H.S.

Clemeno

T.P.

Newbury

H.J.

1996. Predicting quantitative variation within rice germplasm using molecular markers. Heredity 76: 296–304.

45.

Quy

. 1998. An overview of the environmental problems in Vietnam. Vietnamese Studies 3: 7–32.

46.

WHO. 1990. Medicinal Plants in Vietnam. WHO Regional Publications. Western Pacific Series no. 3, WHO, Manila, Philippines and the Institute of Materia Medica, Hanoi, Vietnam.

47.

Williams

R.J.

1983. Tropical legumes. In: Genetic Resources of Forage Plants. McIvor

J.G.

and Bray

R.A.

(Eds.), pp. 17–31. CSIRO, Melbourne, Australia.

48.

Williams

J.G.K.

Kubelik

A.R.

Livak

K.J.

Rafalski

J.A.

Tingey

S.V.

1990. DNA polymorphism amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Researc. 18: 6531–6535.

49.

R.-Q.

Tomooka

Vaughan

D.A.

Doi

2000. The Vigna angularis complex: Genetic variation and relationships revealed by RAPD analysis, and their implications for in situ conservation and domestication. Genetic Resources and Crop Evolution 47: 123–134.

50.

Yee

Kidwell

K.K.

Sills

G.R.

Lumpkin

T.A.

1999. Diversity among selected Vigna angularis (azuki) accessions on the basis of RAPD and AFLP markers. Crop Science 39: 268–275.