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Mol Biol Rep (2011) 38:50255036 DOI 10.

1007/s11033-010-0649-2

Estimation of genetic diversity and evaluation of relatedness through molecular markers among medicinally important trees: Terminalia arjuna, T. chebula and T. bellerica
Maryam Sarwat Sandip Das Prem S. Srivastava

Received: 27 July 2010 / Accepted: 4 December 2010 / Published online: 15 December 2010 Springer Science+Business Media B.V. 2010

Abstract Terminalia trees are being over-exploited because of their medicinal and economical importance leading to loss of valuable genetic resources. For sustainable utilization and conservation, assessment of genetic diversity therefore becomes imperative. We report a comprehensive rst study on estimation and analysis of genetic variation through Amplied fragment length polymorphism (AFLP), inter simple sequence repeat polymorphism (ISSR) and random amplication of polymorphic DNA (RAPD) across three species of Terminalia. The study included (i) characterization of genetic diversity at interspecic level, and (ii) comparison of efciency of the marker systems. That the three species are genetically distinct was revealed by all the three marker systems as unique DNA ngerprints were obtained. This led to identication of several species-specic amplication products. Further analysis helped in species-wise clustering. The species specic bands obtained from the present investigation can be used as diagnostic markers to identify the raw materials for herbal drug preparations for authentication purposes.

Keywords Terminalia (Combretaceae) AFLP ISSR RAPD Genetic diversity Conservation Abbreviations AFLP Amplied Fragment Length Polymorphism ISSR Inter-simple Sequence Repeats RAPD Random Amplied Polymorphic DNA

Introduction Terminalia, well known for medicinal properties is a genus of mostly deciduous trees. In the Indian subcontinent, natural forests of Terminalia are reported in West Bengal, Madhya Pradesh, Uttar Pradesh, Maharashtra, Assam, Tamil Nadu, Rajasthan, Karnataka, Kerala and Punjab. The medicinal utility of Terminalia is far-reaching. T. arjuna, T. bellerica and T. chebula have achieved utmost importance because of varied number of ailments they treat. The three species show potent antibacterial activity against the multi-drug resistant Salmonella typhi [1]. Triphala a combination of T. chebula, T. bellerica alongwith Emblica ofcinalis, extensively used in Indian System of Medicine, is anticancerous [2], radio-protective [3], anti-mutagenic [4], anti-diabetic [5] and purgative [6]. Subasini et al. [7] have shown the hepatoprotective activity of T. arjuna on mice. The leaves of Terminlia are used in a number of industries like Tasar silk (silkworm rearing). The tannins from bark help curing of leather. In addition, the genus is an important source of timber and pharmaceutically valuable compounds. Over-exploitation for economic activity is causing immense pressure on Terminalia and in fact, populations of some of

M. Sarwat S. Das P. S. Srivastava Department of Biotechnology, Faculty of Science, Jamia Hamdard (Hamdard University), Hamdard Nagar, New Delhi, India M. Sarwat (&) Department of Pharmaceutical Biotechnology, Amity Institute of Pharmacy, Amity University, Noida, Uttar Pradesh, India e-mail: maryam21_7@yahoo.com; msarwat@amity.edu S. Das Department of Botany, Delhi University, Delhi, India

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the species are now highly threatened. Pither et al. [8] have already described loss of genetic diversity in long-term fragmented populations of T. amazonia. The fast depletion of this genus is also likely to disturb the ecological balance of the tropical forests. For the effective conservation practices, detailed understanding of the extent and distribution of natural genetic diversity is a pre-requisite. Hence, a concerted effort to conserve existing genetic diversity of the genus Terminalia is urgently needed. We employed three different molecular markers (AFLP, ISSR and RAPD) to catalogue, estimate and analyze the extent of genetic diversity between the three species of Terminalia T. arjuna, T. bellerica and T. chebula. These marker systems differ widely in their properties, need no prior sequence information, scan the large segments of the genome and have been used extensively in carrying out genetic diversity studies; e.g., RAPDs for Andrographis paniculata [9], Panax quinquefolium [10], Jatropha [1113] and others; ISSR for Corchorus species [14], Lupinus angustifolius [15], Jatropha [16], Citrullus lanatus [17], Juglans regia [18] and AFLP for Myricaria laxiora [19], Gardenia jasminoides [20], Chimonanthus spp. [21], Cedrela odorata [22], Punica granatum [23], Warburgia [24], Anacardium occidentale [25], Vigna mungo [26], Viola spp. [27], Jatropha [1113], Prunus lennesiana [28], Typha [29] and others. Besides, genetic diversity these marker systems are widely used for other purposes like elucidation of population genetic structure (like in the endemic medicinal plant Commiphora wightii, [30], identication of toxic and nontoxic varieties (Jatropha curcas, [12]) etc. Markers linked to special characters are then converted into SCAR markers for specialized purposes, like the conversion of AFLP markers linked to bacterial wilt resistance in tomato by Miao et al. [31].

further use. DNA was quantied using agarose gel electrophoresis and serial dilutions of uncut k DNA size marker served as standard. Qualitative and quantitative assessment was done by agarose gel electrophoresis. RAPD analysis RAPD assay was carried out in 15 ll reaction volume containing 50 ng DNA, 25 mM MgCl2, 2 mM each dNTP (MBI Fermentas), 10 lM random decamer primer (Microsynth) and 0.6U Taq polymerase (Invitrogen). Amplication was performed in thermal cycler (Eppendorf Mastercycler Gradient, Germany) using our earlier protocols [3335]. The amplied products were resolved on 1.2% agarose gel (0.5 9 TBE) and photographed under UV light. ISSR analysis Reagents of ISSR assay were assembled in the same manner as RAPD, except with lower amounts of DNA (20 ng) and primer concentration (7.5 lM). Annealing temperature of primers was according to their Tm. All the components of ISSR analysis were standardized by using various concentrations. The sequence of ISSR primers are listed in Table 2. AFLP analysis AFLP analysis was performed as per the instructions of the manufacturer (Invitrogen, Life Technologies), and according to our earlier reports [34, 35]. 300 ng of highly puried genomic DNA was restricted simultaneously with 2.5 units of EcoRI and MseI, each in a 5 9 reaction buffer (50 mM Tris HCl, pH 7.5, 50 mM Mg- acetate, 250 mM K-acetate) in 25 ll reaction volume. MseI and EcoRI adapters were subsequently ligated to restricted fragments. The adapter ligated DNA was diluted and preamplied using primers based on the sequences of adapters including one additional selective nucleotide at the 30 end of the MseI primer (MseI ? C) and EcoRI primer (EcoRI ? A). The cycling parameters were: 20 cycles of 94C30 s, 56C60 s, 72C60 s. The pre-amplied DNA was diluted 50-fold and used as a template for selective amplication using EcoRI and MseI primers with three selective nucleotides at 30 end (EcoRI ? 3 and MseI ? 3). The EcoRI primers were end-labelled with c-P32-ATP using T4 PNK (Invitrogen). The cycling parameters were 94C for 30 s, touch-down with lowering of annealing temperature by 1C in every cycle, 72C for 30 s, followed by 23 cycles of 94C for 30 s, 56C for 30 s, 72C for 60 s. Amplication reaction was stopped by addition of 109 formamide dye, and products were electrophoresed on a 6% denaturing polyacrylamide gel on a

Materials and methods Leaf samples of fully grown trees and saplings of Terminalia arjuna, T. bellerica and T. chebula were collected from natural forests at the outskirts of Dehradun (Uttaranchal). Species identication was done on the basis of morphological characters of leaf and fruit and further conrmed by our taxonomist colleague Professor Dr. M.P. Sharma (Department of Botany, Jamia Hamdard). A minimum distance of 30 m was kept between samples. DNA extraction Genomic DNA was extracted from lyophilized leaf material of Terminalia bellerica and T. chebula following CTAB procedure [32], and from T. arjuna following our modied CTAB protocol [33] and stored at -20C till

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sequencing apparatus (BioRad) and autoradiographed as per protocol of Sambrook et al. [36]. Each assay was repeated at least thrice and only clear and unambiguous bands were utilized for data analysis. Data analysis The data was scored for presence (1) and absence (0) of bands across genotypes to generate a binary matrix. Co-migrating bands were assumed to be originating from the same locus. Only clearly amplied bands were taken for analysis. The binary matrix thus obtained was used for calculating pair-wise similarity between operational taxonomic units (OTUs) according to Jaccard coefcient [37]; GS(ij) = a/a ? b ? c] where GSij represents genetic similarity between two individuals i & j. a is the number of polymorphic bands shared by i & j, b is the number of bands present in i and absent in j, and c is the number of bands present in j and absent in i. The statistical analysis was carried out using NTSYS-pc (version 2.11 w). Phenograms were created by employing the Unweighted Pair Group Method of Arithmetical Averages (UPGMA) in order to group genotypes into clusters. Bootstrap analysis was performed using WINBOOT software to evaluate the degree of condence of the clustering [38]. To summarize the relationships among the genotypes principal correspondance analysis was carried out based on the similarity matrix. To assess the correspondance of results obtained with different marker systems Mantel matrix correspondence test [39] was conducted on the similarity matrices obtained from data. This test also helped in determining the significance of the correlation coefcient between pairs of
Table 1 AFLP analysis for interspecies genetic diversity in Terminalia Primer combination E-ACG ? M-CTG Name of species T. arjuna T. bellerica T. chebula E-AAG?M-CTA T. arjuna T. bellerica T. chebula E-AAG ? M-CAG T. arjuna T. bellerica T. chebula E-AGC ? M-CAG T. arjuna T. bellerica T. chebula E-AAC ? M-CTG T. arjuna T. bellerica T. chebula

similarity matrices and cophenetic correlation values to determine the efciency of UPGMA in estimating genetic relationships between samples.

Results AFLP analysis Five different primer combinations were employed that generated 509 amplicons across all the three species; of these only three amplicons were common across all the three species. The number of markers obtained from each primer varied between 87 (E-ACG ? M-CTG) to 137 (E-AAC ? M-CTG). On an average each primer generated 101 informative markers (Table 4). The species-wise distribution of polymorphism is given in Table 1. The most informative primer combination was E-AAC ? M-CTG which produced 137 amplicons (137) and maximum percentage of polymorphism i.e. 94.2 in T. arjuna, 62.7 in T. bellerica, and 70.2 in T. chebula. Among the three species, T. bellerica was least polymorphic (68.7%), followed by T. arjuna and T. chebula at 72 and 73%, respectively. A representative autoradiogram obtained with primer combination E-AGC ? M-CAG is shown in Fig. 1a. Out of 107 amplied products generated, only two are shared by the three species (marked as S). In addition, amplication products that discriminate the three species can also be observed. Such products, indicated as A, B and C, unique to T. arjuna, T. bellerica and T. chebula, respectively can be deemed as species-specic. Amplied products indicated as D are shared between two species (T. arjuna and T. chebula) and absent from T. bellerica. Some rare
Source of accession Dehradun Dehradun Dehradun Dehradun Dehradun Dehradun Dehradun Dehradun Dehradun All over India Dehradun Dehradun All over India Dehradun Dehradun Polymorphic bands/total no. of bands 36/42 29/40 45/61 14/38 47/68 41/56 23/45 22/33 39/57 70/78 38/50 39/52 114/121 47/75 59/84 % Polymorphism Species specic bands 2 2 10 8 9 9 3 3 3 2 3 2 1

85.71 72.50 73.77 36.84 69.12 73.21 51.11 66.66 68.42 89.74 76.00 75.00 94.21 62.66 70.24

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Fig. 1 Fingerprint proles showing interspecic genetic diversity in Terminalia. a AFLP prole with primer E-AGC ? M-CAG. Lane C: Control (tomato DNA), 110: T. arjuna genotypes, 1120: T. bellerica and 2128: T. chebula. Smonomorphic bands. A, B and Cspecies specic bands for T. arjuna, T. bellerica and T. chebula, respectively. Dthe shared bands between T. arjuna and T. chebula. R-1 and R-2rare polymorphic bands of T. arjuna and T. chebula, repectively. b ISSR prole with primer UBC-08. Lane M: Standard (mix of k DNA digested with HindIII and u 9 174 DNA digested with HaeIII), 19: T. arjuna,1018: T. bellerica and 1925:

T. chebula genotypes from Dehradun. Bands Amonomorphic to T. arjuna. Shared bands between T. arjuna, and T. bellerica. T.bellerica and T.chebula, T. arjuna and T. chebula are D, E and F, respectively. Stutter bands are labelled C#. Two T. arjuna-specic bands A* of 1.0 and 1.1 kb and one T. chebula-specic band C* of less than 0.6 kb. c RAPD prole with primer A-01. Lane M: Standard, 112: T. arjuna, 1319: T. bellerica, 2025: T. chebula. A, B and Cmonomorphic bands in T. arjuna, T. bellerica and T. chebula, respectively. B* is a 1.8 kb band specic to T. bellerica and C* of 0.75 kb is specic to T. chebula

polymorphic bands can also be visualized which are exclusive for a given sample and altogether absent from the rest. For example, bands R-1 and R-2 of T. arjuna and T. chebula, respectively. ISSR analysis Of 28 microsatellite based primers only 16 produced clear and unambiguous bands. A total of 211 amplied products, in the range of 0.3 to 2.2 kb were obtained. An average of 13 bands per primer were amplied, though primer UBC-8 produced 18 bands with no bands being shared across all the three species. Primers S-6 and UBC-6 generated only eight bands each (Table 2). Figure 1b represents an ISSR ngerprint produced by the primer UBC-8. This primer amplied 18 bands, all polymorphic across the three

species. At species level, 9 out of 16 bands were polymorphic in T. arjuna (56.25%), 7 out of 9 in T. bellerica (77.8%) and 6 out of 11 in T. chebula (54.54%). Bands A are monomorphic in T. arjuna. Shared bands between T. arjuna, and T. bellerica, T. bellerica and T. chebula, and T. arjuna and T. chebula are D, E and F, respectively. Stutter bands, a characteristic of only microsatellite ngerprints could also be observed in T. chebula (C#, Fig. 1b. Unique feature of this ISSR ngerprint include the species specic bands observed in T. arjuna (A*, 1.0 and 1.1 kb) and T. chebula (C*, \0.6 kb). RAPD analysis Eighteen random decamers produced a total of 224 amplicons between 0.75 and 2.0 kb among the three

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Mol Biol Rep (2011) 38:50255036 Table 2 Estimation of interspecic genetic diversity in Terminalia through ISSR Sequence (50 30 ) Polymorphic bands/ Species-specic bands total no. of bands (CA)5(GA)3G (GT)4G(AG)4A (TG)7(TA)2T (GACA)4 (CAG)5 (CA)6GT (CA)6AG (CA)6GG (GA)8A (CA)8A (TC)8A (AC)8G (TC)8G (AG)8C (GA)8YT 12/12 11/11 7/8 15/15 17/17 12/12 13/13 12/12/ 14/15 9/9 17/17 8/8 16/16 18/18 14/14 14/14 209/211 T. bellerica1.7 kb T. bellerica1.0 kb, T. chebula1.35 kb T. arjuna0.8 kb, T. bellerica0.65 kb, T. chebula0.55 kb

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Primer S-4 S-3 S-6 S-8 S-9 S-12 S-13 S-14 UBC-2 UBC-4 UBC-5 UBC-6 UBC-7 UBC-8 UBC-9 Total

T. bellerica1.35 and 1.07 kb T. arjuna0.4 kb and 0.68 kb, T. bellerica0.73 kb T. chebula0.5 kb T. arjuna0.9 kb, T. bellerica0.77 kb, T. chebula1.25 kb T. arjuna1.0 and 1.1 kb, T. chebulaless than 0.6 kb T. arjuna1.45 kb, T. bellerica0.62 kb

UBC-13 (CA)8RG

Terminalia species. A summary of the results is presented in Table 3. Of these 224 amplicons, only one product (of 0.87 kb obtained with primer G-10) was common to all the three species. A representative prole obtained with primer A-1 is shown in Fig. 1c. A, B and C are monomorphic bands in T. arjuna, T. bellerica and T. chebula, respectively. B* is a 1.8 kb band specic to T. bellerica and C* of 0.75 kb is specic to T. chebula. Statistical analysis Phenograms prepared from the binary data obtained from all the three marker systems are shown in Fig. 2. All the three marker systems clearly differentiated the three species, T. arjuna, T. chebula and T. bellerica into distinct clusters. The similarity coefcient obtained from AFLP data for T. arjuna is much lower (0.430.62), in comparison to those obtained from ISSR and RAPD data, 0.630.74, and 0.61.0, respectively (Cluster I, Fig. 2ac). Whereas, in case of T. bellerica, the similarity coefcient obtained from AFLP data (Cluster II, Fig. 2a) and ISSR data are equivalent, and much higher to RAPD data (Cluster II, Fig. 2b, c). In case of T. chebula, the similarity coefcients obtained from RAPD data was much lower (0.460.66; Cluster II, Fig. 2c), followed by AFLP (0.530.73; Cluster III, Fig. 2a) and ISSR (0.50.8; Cluster III, Fig. 2b). The data utilized for two dimensional Principal Correspondence Analysis (PCA, NTSYS-pc, version

2.11), also grouped the three species in three clusters. The result of the PCA is similar to that obtained from the phenogram constructed using UPGMA (data not shown). Comparison of molecular markers We have compared the molecular marker techniques for their relative efciency in detecting polymorphism, average heterozygosity revealed (Hav), multiplex ratio (MR) and marker index (MI). Subsequently, we have also ` -vis from checked the concordance of our results vis-a others and the goodness-of-t of each marker system derived by their cophenetic correlations by employing Mantel test. The relative efciencies of AFLP, ISSR and RAPD markers utilized for interspecic genetic diversity analysis are given in Table 4. The total number of bands (n) produced by AFLP were highest (509) as compared to ISSR (211) and RAPD (224) which resulted in highest multiplex ratio (MR) of AFLP markers (101.8), though the number of assays performed for AFLP (5) were lesser than those for ISSR (16) and RAPD (18). The percent polymorphism produced by all the three markers, however, were all in the range of 99%. The average heterozygosity (Hav) too which is a function of polymorphic information content (PIC) does not differ signicantly. The overall efciency of a marker system is detected by marker index (MI = Hav 9 MR) which is highest for AFLP (36.65) than for ISSR (5.14) and

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5030 Table 3 Estimation of Interspecic genetic diversity in Terminalia through RAPD Sequence (50 30 ) CAG GCC CTT C TGC CGA GCT G AAT CGG GCT G AGG GGT CTT G GAA ACG GGT G GTG ACG TAG C GGG TAA CGC C GGG TAA CGC C GGT GAC GCA G GGT CAC CTC A GAG CGC CTT G GAT GAC CGC C GTC GCC GTC A CCG CGT CTT G GGA CCT GCT G AGG TGA CCG T TGT TCG CAG A TGA GCG GAC A

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Primer A-1 A-2 A-4 A-5 A-7 A-8 A-9 A-10 G-2 G-4 G-5 G-6 G-8 G-9 G-10 G-11 G-13 G-15 Total

Polymorphic bands/ total no. of bands 16/16 6/6 10/10 14/14 13/13/ 9/9 16/16 16/16 10/10 15/15 9/9 7/7 15/15 13/13 15/16 14/14 13/13 12/12 223/224

Species-specic bands T. bellerica1.8 kb, T. chebula0.8 and 1.35 kb T. arjuna0.6 and 0.7 kb, T. chebula1.2 kb T. arjuna1.2 kb T. bellerica1.2 kb T. bellerica1.9 kb, T. chebula0.8 kb T. arjuna0.55 kb T. arjuna1.36 kb, T. bellerica -1.07 and 1.1 kb, T. chebula0.87 and 1.2 kb T. arjuna0.8 kb T. arjuna0.6, 0.9 and 1.1 kb T. arjuna1.7 kb T. chebula1.87 kb T. chebula1.0 kb T. arjuna0.59 kb, T. bellerica1.4 kb T. arjuna0.97 kb, T. bellerica1.8 kb, T. chebula0.75 kb T. bellerica0.65 kb T. arjuna0.8 kb, T. chebula0.75 kb

RAPD (4.85) for this study. Mantel test [39] was employed to determine the coefcient of correlation between the similarity matrices generated by these markers. The coefcient of correlation between ISSR and RAPD similarity matrices was r = 0.648 (P = 0.01). Mantel test helped evaluate the goodness of t for the UPGMA dendrograms. This was achieved by making cophenetic similarity matrices from each UPGMA phenogram and then comparing these with the original similarity matrices (generated from binary data). The cophenetic correlation was highest for AFLP (r = 0.991, P = 0.01) followed by ISSR (r = 0. 989, P = 0.01) and RAPD (r = 0.978, P = 0.01).

Discussion Till date, the efforts done for unravelling genetic diversity in the genus Terminalia have been negligible. Prior to this work, only Pither et al. [8] has reported the population structure of T. amazonia where ve RAPD assays detected an overall diversity index of 0.38 (Shannon diversity index) among 104 individuals derived from six populations. Various levels of ploidy have been reported in T. chebula (2n to 6n) and T. bellerica (2n to 4n). In T. chebula, fruit varieties have been commercially graded into eight types,

viz. Bhimlies (Tamil Nadu), Jubbulpores (Madhya Pradesh), Rajpores (Maharashtra), Vingorlas (Maharashtra), Madras Coast, Survari harde, Bala harde and Java harde [40]. In the plant populations, genetic structures are mainly governed by their mode of reproduction. Tropical trees are reported to have high levels of intrapopulation genetic diversity [41]. According to Aldrich et al. [42] out-crossing is the main form of reproduction in tropical trees and Srivastava et al. [43] have indeed suggested out-crossing as the primary mode of reproduction in the genus Terminalia. The inter- as well as intra-specic variation in this genus might have resulted because of sexual recombination, segregation, together with mutations, acted on by natural selection. The aim of the present study was to estimate the extent of genetic variation and enumerate taxonomic relationship based on molecular markers between three Terminalia species. We analyzed genetic relationship using AFLP, ISSR and RAPD markers at the interspecic level between samples of T. arjuna, T. bellerica and T. chebula collected from Uttaranchal region of India. We report high level of interspecic genetic diversity in this study, which is comparable to other reports; 99.6% polymorphism reported in

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Mol Biol Rep (2011) 38:50255036 Fig. 2 Genetic relationships among Terminalia species depicted by UPGMA trees based on three different marker systems: AFLP a ISSR, b and RAPD, c. Genetic distances are indicated below each tree, numbers next to the branches indicate levels of bootstrap support (only levels above 50% are indicated)

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Table 4 Relative efciency of molecular markers in determining interspecic genetic polymorphism in Terminalia Type of study AFLP ISSR RAPD No. of Total no. of Polymorgenotypes bands (n) phic bands (p) 26 25 25 509 211 224 506 209 223 Total no. of assays/ primer combinations (T) 5 16 18 Multiplex ratio (n/T) 101.80 13.18 12.44 Percentage polymorphism (%p) 99.60 99.00 99.55 Average heterozygosity (Hav) 0.36 0.39 0.39 Marker index (MI = Hav 9 MR) 36.65 5.14 4.85

Typha spp. [29], 95% in Nicotiana [44], 90% in Echinacea [45], 90.2% in Dioscorea rotundata [46], 90.% in Rehmannia glutinosa [47], 82.61% in Anisodus tanguticus, a rare endemic herb of Tibet [48] and between 78 and 86% polymorphism in Magnolia ofcinalis [49]. Other studies indicate somewhat lower levels of polymorphism, e.g., Chao et al. [50] recorded only 67.8% polymorphism across 34 cultivars belonging to 15 species of Calathea. These lower levels of polymorphism could be attributed to the obvious inherent difference in genetic polymorphism in natural populations. One interesting nding of our study is that at individual species level the percent polymorphism was signicantly less, 73.32 among T. arjuna genotypes, 68.8 among T. bellerica and 71.93 among T. chebula. Similar ndings are recorded in Papaver somniferum, P. bracteatum and P. somniferum ssp. setigerum where AFLP markers revealed 91% polymorphism, but exclusively within P. somniferum, it was much lower (73%, [51]). In the present study phenograms based on UPGMA algorithm and PCA plots indicate clear separation of the three species of Terminalia into well-dened groups. These clusters are linked at a very low similarity coefcient of 0.27 and supported by high bootstrap values (78.6). The groupings of genotypes in dendrograms derived from ISSR and RAPD data show T. chebula to be closer to T. arjuna genotypes, although, T. chebula samples share more medicinal properties with T. bellerica than T. arjuna. The dendrogram derived from AFLP data gives a true picture of the relationship among the three species that places T. chebula genotypes closer to T. bellerica. It may be because AFLP analyzes large number of loci and hence is more dependable than ISSR and RAPD. We detected several species-specic bands through the three marker systems in Terminalia species. RAPD primer A-01 amplied a 1.8 kb band specic to T. bellerica and a 0.8 kb band specic to T. chebula genotypes. With primer A-10 a 0.8 kb band specic to T. arjuna genotypes was detected. Primer G-2 brought out 1.1, 0.9 and 0.6 kb bands specic to T. arjuna genotypes. This can be explained on the basis of evolutionary forces like natural selection, drift, and random allele xation; some alleles have become specic to one species or one region. Such highly differentiating markers are also reported in other plants. In

Echinacea RAPD assays detected one marker specic to E. angustifolia [52], e.g., a band of 1800 bp was obtained with random primer OPA-20. Another random primer OPA-10 amplied a 6000 bp band specic to E. pallida. Three markers for E. purpurea were obtained with primers OPA-11 (1250 bp), and OPA-17 (750, 1800 bp). Two markers (IVC134, IVC335) reported by Percield et al. [53] are specic to Hypericum perforatum and may be used for identication of raw material for herbal drug preparation. Similarly, the species specic bands obtained in our study are immensely helpful in discriminating the three species of Terminalia. Sequencing of these markers will provide information on their position in genome. Choi et al. [54] have developed AFLP derived SCAR marker, JG14 for discriminating Panax japonicus from the other three species of Panax, i.e. P. ginseng, P. quinquefolius and P. notoginseng. Jain et al. [55] developed RAPD derived SCAR markers for Phyllanthus ssp. These markers are very useful for authentication purpose and prevention of adulteration and biopiracy. Since, the molecular marker technologies differ from each other in evaluating genetic relationships between different plant species or population, the choice of the marker system depends upon the objectives of the study and careful consideration of factors. For good genome coverage more than one marker system should be used. Dominant markers such as AFLP, ISSR and RAPD were used in this study because of the large number of loci detected per assay, thereby requiring lesser number of assays for a particular study by these markers. For the comparison of various markers, Powell et al. [56] dened two important parameters, namely, level of polymorphism detected and number of loci detected per assay as a measure of effectiveness. Among the three marker systems employed by us, AFLP with highest multiplex ratio (MR) proved best. However, the MR of RAPD is slightly higher than ISSR in Terminalia. Similar results have been demonstrated in garlic [57] as well. High MR of AFLP, than RAPD has also been calculated in Vigna sp. [58]. These results are in accordance with the ndings of Saini et al. [59] who compared the relative efciency of AFLP, ISSR and SSR for rice (Oryza sativa) and concluded that AFLP markers displayed

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the highest multiplex ratio. The multiplex ratio can be modulated by increasing or decreasing the number of selective nucleotides at the 30 end of the primers [60]. In a particular population, the number of alleles at a locus and their frequency of distribution can be evaluated as polymorphism, and heterozygosity is the probability that two alleles taken at random from a population can be distinguished by the given marker technique. So, both of them are taken as the second criterion for measuring the effectiveness of a marker system. Archak et al. [61] have also measured the efciency of a molecular marker as its capacity to detect polymorphism. Percent polymorphism detected by all the three marker systems AFLP, ISSR and RAPD in our study is near about equal (99) and the average heterozygosity too does not differ signicantly (although, slightly higher in case of ISSR), indicating that similar level of polymorphism was detected by the three marker systems in the given germplasm pool. Similar results have been reported in Trigonella foenum-graecum [62]. But in other studies, the situation is quite different. Likewise, AFLP is reported to be more efcient than RAPD in terms of detection of polymorphism in Saxifraga cernua [63]. In Dioscorea rotundata, Mignouna et al. [46] compared AFLP, RAPD and SSR and claimed that AFLP detected highest percent polymorphism, followed by RAPD and SSR. Between ISSRs and RAPDs, ISSRs have always performed better than RAPDs. For example, in Oryza granulata, Wu et al. [64] demonstrated out-performance of ISSRs over RAPDs in detecting polymorphism. Our study indicates that AFLP has high multiplex ratio while ISSR revealed high average heterozygosity, whereas RAPD markers did not prove as efcient as the other two marker systems. To determine the overall efciency of the marker system, marker index (MI) is calculated. In the present study AFLP has highest marker index than ISSR and RAPD. Similar results were reported in the outcrossing plant cashew (Anacardium occidentale), where MI of AFLP is ten times higher than ISSR and RAPD [61]. Clustering obtained in the present investigation show poor correlation between ISSR and RAPD data and no correlation between these phenograms and that derived from AFLP. Such type of correspondance between different molecular markers have been widely investigated. One of the earliest report [56] who found similar genetic relationships in wild and cultivated soybean using four marker systems. RAPD and AFLP revealed similar relationships among clones of various willow species [65], pepper inbreds [66], apple cultivars [67], and rice genotypes [68]. Dangi et al. [62] found high correlation coefcient for RAPD and ISSR in case of Trigonella foenumgraecum and T. caerulea. Sudheer Pamidiamarri et al. [13] have also reported good correspondance between dendrograms obtained from AFLP and RAPD data of the seven species

of Jatropha. Majority of the reports which show existence of correlation between molecular markers are the studies on autogamous crops or inbred lines where genotypes tend to be homozygous. This is in contrast to the present taxa, which is out-crossed and thus has a high level of heterozygosity. In such cases, the genetic background of accessions are expected to be diverse, and could contribute to the present observation. Archak et al. [61] have also found poor correlation between ISSR and RAPD and absence of correlation between AFLP and the other two techniques. Souframanien and Gopalakrishna [69] have reported poor correlation between ISSR and RAPD. As far as correlation between the cophenetic and similarity matrices is concerned, Mantel test showed signicant correlations (r [ 0.9) between these matrices when obtained from similar data set. AFLP proved superior here also as the cophenetic correlation values were highest when compared to other markers. High cophenetic correlation values of AFLP prove the goodness-of-t of these markers, and hence, the robustness of the technique. On the basis of the criteria of scorability, AFLP markers again came rst, as they produced more clear, unambiguous and distinguishable proles. ISSR and RAPD markers may also generate ambiguous bands of non-homologous origin. Several groups have tested the reproducibility of various marker systems, e.g., Mc Gregor et al. [70] reported AFLP as 99.6% reproducible, ISSR 87% reproducible and RAPD only 84.4%. Kjlner et al. [63] have further provided the evidence of high reproducibility of AFLP markers. Vosman et al. [71] have also reported the high correlation (r = 0.93) in AFLP results from two different laboratories while examining genetic diversity in rose (Rosa sp.). In our study also AFLP proved to be better in terms of scorability and reproducibility.

Conclusions The species specic markers amplied in this study can be converted into simple PCR-based sequence-characterized amplied region (SCAR) markers which will allow the screening of large number of samples and populations from different regions. All markers can be tested against other potential species. A set of highly differentiating markers can be used as diagnostic markers to identify the raw material for herbal drugs of different species and different regions, the data of which would contribute to the authentication of herbal medicines, to avoid batch to batch variations in extraction of standard drugs and to devise strategies for forest conservation and biodiversity.
Acknowledgments The study was supported by a nancial grant from Department of Biotechnology, Govt. of India to SD (grant

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5034 number BT/PR 1969/PB/17/096/2000) and an award of Junior, and Senior Research Fellowship to MS, from DBT, Govt. of India. The help to identify the species provided by our colleague Dr. M.P. Sharma (Dept. of Botany, Jamia Hamdard) is gratefully acknowledged.

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