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Single primer amplification reaction (SPAR) reveals inter- and intra-specific naturalgenetic variation in five species of Cymbidium (Orchidaceae)

Santosh Kumar Sharma a, Suman Kumaria b, Pramod Tandon b, Satyawada Rama Rao a,⁎a Department of Biotechnology and Bioinformatics, North-Eastern Hill University, Shillong (Meghalaya), Indiab Centre for Advanced Studies in Botany, North-Eastern Hill University, Shillong (Meghalaya), India

a b s t r a c ta r t i c l e i n f o

Article history:Accepted 17 May 2011Available online 26 May 2011

Received by A.J. van Wijnen

Keywords:RAPDISSRDAMDCymbidiumGenetic variationResolving power

A total of 53 primers belonging to three SPAR methods, viz. RAPD, ISSR and DAMD, collectively produced 456polymorphic amplicons with 96.6% polymorphism at inter-specific level in five species of Cymbidium, viz. C.aloifolium, C. mastersii, C. elegans, C. eburneum and C. tigrinum, whereas at intra-specific level, the observedpolymorphism ranged from 51.2% to 77.1% among them. Three SPARs collectively revealed 25 unique species-specific amplicons; most of them were amplified with RAPD and DAMD primers besides few bands whichwere either missed (absent) or lost (heterozygosity). UPGMA clustering evidently distinguished therepresentatives of C. aloifolium and C. tigrinum, with distinct genetic distance, which may be due to theirentirely different habitats as well as discrete morphological characteristics. Upon analysis of the datagenerated, all the three SPAR methods, either independently and/or in combination, revealed wide range ofgenetic variation between and within five species of Cymbidium. Comparison of matrix of individual SPARmethod revealed that analysis of natural genetic variation using combination of SPARmethods, rather than anisolated approach, is highly effective. The critical analyses of the amplicon data are indicative of DAMD as themost powerful SPAR method by showing highest resolving power (Rp) followed by ISSR and RAPD.Alternatively, the total polymorphic information content was highest in case of RAPD followed by other twoSPAR methods. Thus, the present investigation for the first time provides a valuable baseline data for geneticvariation at inter- and intra-specific levels in horticultural Cymbidiums and also addresses conservationconcerns.

© 2011 Elsevier B.V. All rights reserved.

1. Introduction

Orchidaceae is among the most species-rich plant families, havingan estimated 25,000 species belong to more than 800 genera. Itcomprises several such representatives capable of occupying almostevery conceivable ecological situation, apart from marine environ-ments and habitats characterized by extreme cold throughout theyear. Cymbidium, or boat orchids, is a genus of 44 evergreen orchidspecies subfamily Epidendroideae, tribe Cymbidieae and subtribeCymbidiinae. It is distributed in tropical and subtropical Asia(viz.northeast India, China, Japan, Malaysia, the Philippines, andBorneo) and north Australia, usually growing in cooler climates athigh elevations. To assess the relationships among various species ofCymbidium, the conventional method based on morphological traits

of leaf and flower (Jin and Yao, 2006) is used generally. Problemsassociated with variability, plant growth conditions, and individualbiases have caused confusion in novel cultivar and/or speciesidentification. Therefore, understanding the genetic resources anddiversity is very important for the breeding programs andimprovement of the genus Cymbidium (Wang et al., 2009).

In recent years, genetic diversity and identification of Cymbidiumcultivars has been achieved by various molecular markers, includingisozymes (Obara-Okeyo et al., 1998), random amplified polymorphicDNA (RAPD) (Obara-Okeyo and Kako, 1998;Wang et al., 2004; Choi etal., 2006), inter-simple sequence repeat (ISSR) (Xiaohong et al., 2007;Wang et al., 2009), amplified fragment length polymorphism (AFLP)(Wang et al., 2004), EST-SSR (Huang et al., 2010), extended RAPD(ERAPD) (Jian et al., 2010), and polymorphisms of nrITS and matKsequences (Van den Berg et al., 2002). Besides these, reports are trulydependent on application of single DNAmarker system and restrictedto only one or two representative species, mostly from China andJapan. Sharma et al. (2010a) reported the karyo-morphologicalcharacterization to analyze natural genetic variation in threethreatened Cymbidium species, viz. C. eburneum, C. hookerianum, andC. mastersii, from northeast India. Lack of information, ambiguitiesrelated to morphological, cytogenetical, and taxonomical data, and

Gene 483 (2011) 54–62

Abbreviations: DAMD, directed amplification of minisatellite DNA regions; FISH,fluorescent in situ hybridization; ISSR, inter simple sequence repeat; PCR, polymerasechain reaction; kb, kilobase(s) or 1000 bp; nrITS, nuclear ribosomal internal transcribedspacer; PIC, Polymorphic information content; Rp, Resolving power; RAPD, randomamplified polymorphic DNA; SPAR, single primer amplification reaction; UPGMA,unweighted pair-group method with arithmetic averages.⁎ Corresponding author. Tel.: +91 364 2722404; fax: +91 364 2550076.

E-mail addresses: srrao22@yahoo.com, srrao@mail.com (S.R. Rao).

0378-1119/$ – see front matter © 2011 Elsevier B.V. All rights reserved.doi:10.1016/j.gene.2011.05.013

Contents lists available at ScienceDirect

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the phylogenetical aspects of Cymbidiums are still unclear to date.Other than these reports, more or less squat information is availableabout inter- and intra-specific genetic variability in various Cymbid-ium species, especially at the DNA level and other geographical areas.Despite having rich diversity, natural genetic variation at DNA levelhas not yet been reported in various Cymbidiums from India, which ismost horticulturally valued orchid of family.

In the recent years, the PCR based single primer amplificationreaction (SPAR)methods are gaining prominence as effective tools forgenetic diversity studies in plants and they collectively provide acomprehensive description of the nature and the extent of plausiblediversity (Bhattacharya et al., 2005; Ranade et al., 2009; Sharma et al.,2010b). Three methods involving single primer for amplificationreactions are commonly used in genetic variation analysis of higherplants. These three SPAR methods include (A) Directed Amplificationof Minisatellite DNA regions (DAMD) (Heath et al., 1993); (B) InterSimple Sequence Repeat (ISSR) (Gupta et al., 1994) and (C) RandomAmplified Polymorphic DNA (RAPD) (Welsh and McClelland, 1990;Williams et al., 1990). Therefore, in the present investigation, thesethree SPAR methods are used to determine the natural geneticvariation at inter- and intra-specific levels among the 25 collections,which are representatives of five Cymbidium species (five collectionsof each species), viz. Cymbidium aloifolium (L.) Sw., C. elegans Lindl., C.eburneum Lindl., C. mastersii Griff. ex Lindl. and C. tigrinum Parish exHook. All the five species were collected from northeast India and aremostly threatened in their natural habitat. Further, the efficiency ofthese three SPAR methods has been attempted, individually as well ascollectively.

2. Materials and methods

2.1. Plant material

The plant materials, belonging to five species of Cymbidium,comprised of five individuals of each species viz. C. aloifolium (CA-1 toCA-5), C. elegans (CEL-1 to CEL-5), C. eburneum (CEB-1 to CEB-5), C.mastersii (CM-1 to CM-5) and C. tigrinum (CT-1 to CT-5), respectively,were collected from Sikkim and Meghalaya province of northeasternregion of India. The plants were grown in greenhouse of PlantBiotechnology Laboratory, Department of Botany of North-EasternHill University, Shillong. For each species, a minimum of fiveindividuals and more than one population were analyzed. Leafsamples of 25 individual plants belong to five species (five individualsof each species collected randomly from different areas), were usedfor genomic DNA extraction.

2.1.1. DNA isolationFresh leaves were ground and powdered in a pre-chilled mortar

using liquid nitrogen, and the DNA was then extracted by the methoddescribed by Murray and Thompson (1980) with some minormodification. The extraction buffer was supplemented with 3%CTAB, 3 M NaCl and 1% polyvinylpyrrolidone (PVP). The DNAextracted from the plant material, purified for protein fraction,treated with RNase A, was re-precipitated with pre-chilled absoluteethanol and subsequently dissolved in Tris–EDTA (TE) buffer. Theextracted DNA was further purified with QIAquick columns (Qiagen)and the quality of DNA was checked by mupid gel electrophoresiswith 0.85% (w/v) agarose in 1× TAE.

2.2. RAPD, ISSR, DAMD primers

Six RAPD kits (OPA, OPB, OPC, OPD, OPAA and OPBA) comprised of20 decamer random primers per kit (total 120 primers) wereprocured form Operon Technologies, Alameda, CA, USA. A total of 26ISSR primers and 31 DAMD primers were custom synthesized fromMetabion Inc. Ltd., Germany.

The details of protocols followed for PCR optimization, primersurvey, final amplification, bands scoring and cluster analysis aredescribed elsewhere (Sharma et al., 2010b). Polymorphic informationcontent (PIC; the probability of detection of polymorphism by aprimer/primer combination between two randomly drawn geno-types) and resolving power (Rp) that determines the ability of aprimer/technique to generate optimally informative bands werecalculated as per Sehgal et al. (2009) individually for each SPARmethod to determine their efficacies.

3. Results

3.1. Analysis of inter-specific natural genetic variation and clusteringpattern

A total of 53 primers belonging to three single primer-basedmethods, viz. RAPD, ISSR and DAMD, produced 472 amplificationproducts with 8.9 amplicons per primer collectively. Out of which, 456bands were found to be polymorphic in nature with 8.6 averagenumbers of amplicons per primer among the five Cymbidiumsanalyzed in the present investigation (Table 1). Percentage ofpolymorphic bands ranged from 25% to 100% with overall 96.6%polymorphism. Thirty-nine out of 53 primers exhibited 100% poly-morphism among the samples (Table 1). A minimum of four ampliconproductswere scoredwith ISSR andRAPDprimer, viz. I-832 andOPBA-3, respectively, whereas a maximum of 15 amplicons were yielded byDAMD primers, viz. D-26, D-31 and D-32. The number of polymorphicfragments per sample ranged from 1 (I-832) to 11 (D-26) in all thesamples analyzed. The size of the fragments varied from0.2 to 3.5 kb insize. Fig. 1 and Table 1 are illustrative of the extent of polymorphismobserved among the 25 collections belonging to five Cymbidiums.

The UPGMA phenogram (Fig. 2) was constructed by using thecumulative data of three SPAR methods to analyze the genetic distancein all the samples using Jaccard's (1901) coefficients of similarity,respectively. Remarkably, the dendrogram generated through both themethods had at least three major clusters marked A, B, and C with largeparenthesis as detailed in Fig. 2. Cluster A includes five collectionwhichall were representatives of C. aloifoium, i.e., CA-1 to CA-5. Cluster Bfurther sub-divided into three sub-clusters, viz. B1, B2 and B3. Sub-cluster B1 comprisedof six collections, out ofwhich four representativesofC. elegans (CEL-1, 3, 4 and5, respectively), one collectionofC.mastersii(CM-4) and one of C. eburneum (CEB-2). B2 comprises rest fourcollections of C. mastersii except CM-4. Notably, collections CM-2 andCM-3 showed ideal genetic similarity values. Sub-cluster B3 comprisedoffive collections comprising four of C. eburneum (CEB-1, 3, 4 and 5) andone of C. elegans (CEL-2). The third cluster C consists of fiverepresentatives of C. tigrinum (CT-1 to CT-5). The interesting factwhich lies with both cluster A and C is that it comprises representativesof single species, viz. C. aloifoium and C. tigrinum, respectively, henceshowing discrete clustering which may identify different species ofCymbidium. Cluster B also showed more or less similar tendency todifferentiate the remaining three Cymbidium species with moderategenetic similarity. The combined dataset of all the 53 primers belongingto three SPARs revealed identical rooted trees as calculated by Jaccard'scoefficient resulting values between 0.33 and 0.88 (Table 2) which isprecisely supported by 1000 replicate bootstrapping values (Fig. S9).

3.2. Analysis of intra-specific natural genetic variation and clusteringpattern

3.2.1. C. aloifoliumA total of 53 SPARs produced 313 amplification products with 5.9

amplicons per primer collectively. Out of which, 228 bands werefound to be polymorphic in nature with an average of 4.3 ampliconsper primer and showed 72.8% polymorphism among the fiveindividuals of this species analyzed (Table 2). The dendrogram

55S.K. Sharma et al. / Gene 483 (2011) 54–62

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constructed through Jaccard's similarity coefficient that revealed thevalues between 0.41 and 0.84 with mean similarity coefficient of 0.62(Table 2). Out of five representatives of this species, two (CA-1 and 2)showed identical values with highest similarity coefficient (Fig. S1).

3.2.2. C. elegansAs a whole 330 amplicons were produced with 6.2 amplification

products per primer. Out of which 248 amplicons were found to bepolymorphic with an average of 4.6 bands per primer. An average of75.1% polymorphismwas estimated among the five individuals of this

species. The dendrogram revealed the Jaccard's similarity coefficientvalues between 0.42 and 0.76 with a mean value of 0.59 among thefive individuals. Collections CEL-3 and CEL-4 were found identical byshowing highest similarity where as CEL-2 demonstrated minimumsimilarity, possessing different root of the tree and behaved as out-group (Fig. S2).

3.2.3. C. eburneumTotally 321 amplicons were produced with 6.0 amplification

products per primer. Among all amplified bands, 228 amplicons were

Table 1RAPD, ISSR and DAMD primers used in the present study and the extent of polymorphism along with information about unique and/or lost banding pattern obtained in specificspecies.

S. N. Primername

Primer sequence Total bandsamplified

Polymorphicbands

Percentagepolymorphism

Name of the species showing unique /specific bands (no. of unique bands)

Lost bands in species(no. of lost bands)

RAPD1 OPA-01 5′-CAGGCCCTTC-3′ 8 8 100 CT(3) CA(1), CT(1)2 OPA-03 5′-AGTCAGCCAC-3′ 12 12 100 CEL(1), CEB(1)3 OPA-04 5′-AATCGGGCTG-3′ 10 9 904 OPA-05 5′-AGGGGTCTTG-3′ 9 9 1005 OPA-13 5′-CAGCACCCAC-3′ 8 8 100 CA(1)6 OPA-20 5′-GTTGCGATCC-3′ 10 10 100 CT(2)7 OPAA-6 5′-GTGGGTGCCA-3′ 10 10 1008 OPAA-8 5′-TCCGCAGTAG-3′ 12 12 100 CA(1)9 OPAA-12 5′-GGACCTCTTG-3′ 8 8 10010 OPAA-13 5′-GAGCGTCGCT-3′ 9 9 10011 OPAA-18 5′-TGGTCCAGCC-3′ 8 8 10012 OPAA-19 5′-TGAGGCGTGT-3′ 8 8 10013 OPAA-20 5′-TTGCCTTCGG-3′ 11 11 10014 OPB-10 5′-CTGCTGGGAC-3′ 9 9 100 CA(1), CT(1), CA(1), CT(1)15 OPBA-3 5′-GTGCGAGAAC-3′ 4 3 7516 OPBA-7 5′-GGGTCGCATC-3′ 11 11 100 CEL(1), CEB(1)17 OPBA-9 5′-GGAACTCCAC-3′ 6 5 83.318 OPBA-10 5′-GGACGTTGAG-3′ 8 8 10019 OPBA-14 5′-TCGGGAGTGG-3′ 7 6 85.720 OPBA-15 5′-GAAGACCTGG-3′ 8 8 10021 OPBA-16 5′-CCACGCATCA-3′ 7 7 10022 OPBA-17 5′-TGTACCCCTG-3′ 7 7 10023 OPBA-18 5′-CTCGGATGTC-3′ 8 8 10024 OPD-1 5′-ACCGCGAAGG-3′ 7 7 100 CT(1)25 OPD-7 5′-TTGGCACGGG-3′ 10 10 10026 OPD-8 5′-GTGTGCCCCA-3′ 8 8 100 CT(1)27 OPD-18 5′-GAGAGCCAAC-3′ 7 7 100 CT(1), CA(1)28 OPD-20 5′-ACCCGGTCAC-3′ 12 12 100

ISSR29 ISSR-12 5′-CATGGTGTTCATCATTGTTCC-3′ 5 4 80 CT(1)30 ISSR-15 5′-GTGTGTGTGTGTGG-3′ 8 8 100 CT(1)31 ISSR-16 5′-CACCACCACGC-3′ 7 7 10032 ISSR-25 5′-ACACACACACACACACGA-3′ 8 8 10033 ISSR-27 5′-ACACACACACACACAACG-3′ 11 11 100 CA(3), CT(2)34 I-17898A 5′-CACACACACACAAC-3′ 6 5 83.335 I-815 5′-CTCTCTCTCTCTCTCTG-3′ 5 4 8036 I-821 5′-GATTGTGTGTGTGTGTT-3′ 6 5 83.337 I-832 5′-ATATATATATATATATCC-3′ 4 1 2538 I-841 5′-GAGAGAGAGAGAGAGACC-3′ 6 6 100

DAMD39 D-26 5′-ATCCAAGGTCCGAGACAACC-3′ 15 15 10040 D-27 5′-GTGTGCGATCAGTTGCTGGG-3′ 8 8 10041 D-28 5′-CCCAGCAACTGATCGCACAC-3′ 10 10 100 CA(1)42 D-29 5′-AGGACTCGATAACAGGCTCC-3′ 11 11 100 CA(1)43 D-31 5′-GGCAAGCTGGTGGGAGGTAC-3′ 15 14 93.344 D-32 5′-ATGTGTGCGATCAGTTGCTG-3′ 15 13 86.6 CT(1)45 D-33 5′-TACATCGCAAGTGACACAGG-3′ 6 6 10046 D-35 5′-GATGTGTTCTTGGAGCCTGT-3′ 12 12 10047 D-38 5′-AAGAGGCATTCTACCACCAC-3′ 7 6 85.7 CA(2)48 DB-2 5′-CCTCCTCCCTCCC-3′ 5 5 10049 DB-3 5′-GGAGGTTTTCA-3′ 14 14 10050 DB-4 5′-GAGGGTGGCGGTTCT-3′ 10 10 10051 DB-5 5′-CTCTGGGTGTCGTGC-3′ 18 18 100 CM(1), CT(1) CT(1)52 DB-7 5′-CCCGTGGGGCCGCCG-3′ 12 11 91.6 CM(1) CA(2), CEL(1), CEB(5), CT(1)53 DB-8 5′-GACNGGNACNGG-3′ 6 6 100

Total 472 456 96.6

Abbreviations: CA, Cymbidium aloifolium; CEL, C. elegans; CEB, C. eburneum; CM, C. mastersii; CT, C. tigrinum.

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found polymorphic in nature with an average of 4.3 bands per primerand polymorphism of 71% among individuals. The dendrogramrevealed the Jaccard's similarity coefficient values between 0.44 and0.79 with mean similarity of 0.61 among the five individuals. Thisspecies also revealed more or less same clustering pattern as C.elegans. Collections CEB-3 and CEB-4 were found identical by showinghighest similarity, whereas collection CEB-2 demonstrated minimumsimilarity possessing different root of the tree and behaved as out-group (Fig. S3).

3.2.4. C. mastersiiTwo hundred and eighty-five amplicons were produced with 5.3

amplification products per primer. Out of which 220 amplicons werefound to be polymorphic with an average of 4.1 bands per primer. Anaverage of 77.1% polymorphism was estimated among the fiveindividuals of this species analyzed. The dendrogram revealed theJaccard's similarity coefficient values between 0.40 and 0.88 withmean similarity coefficient of 0.64 among the five individuals.Clustering pattern reveled that out of five representative, twocollections (CM-2 and CM-3) showed highest similarity ( Fig. S4).

3.2.5. C. tigrinumOverall 242 amplicons were produced with 4.5 amplification

products per primer. Among all amplified bands, only 124 amplicons

were found polymorphic in nature with an average of 2.3 bands perprimer and comparatively very low (51.2%) polymorphism. Thedendrogram revealed the Jaccard's similarity coefficient valuesbetween 0.70 and 0.83 with mean similarity coefficient of 0.76among the five individuals. Clustering pattern reveled that collectionsCT-2 and CT-3 were found identical by showing highest similarity inthe dendrogram (Fig. S5). This species showed minimum distance inthe form of natural genetic variation among five representativescompared to other four species investigated presently.

3.3. Comparison of different SPAR methods

To determine the efficiency of the threemethods, distance range ofeach SPAR was calculated for five species of Cymbidium by Jaccard'scoefficient, both individually and collectively for all SPAR methodused (Table 2). The distance values at inter-specific level for fivespecies of Cymbidium cumulatively revealed values of Jaccard'scoefficient ranged from 0.25 to 0.96, 0.42 to 0.97 and 0.32 to 0.86with RAPD, ISSR and DAMD method, respectively (Figs. S6–S8).Consensus values of SPARs pairs, viz. RAPD+ISSR+DAMD,arecalculated which showed genetic distance ranging from 0.10 to 0.90for all the species investigated. Consensus values for each SPARindividually were also calculated using two different similaritymatrixes generated through Jaccard and Dice coefficient which

Fig. 1. Typical RAPD, ISSR and DAMD profiles obtained with primer OPD-20, I-27 and D-31 primer, respectively, with 25 collections of five Cymbidium species. Individual's collectionnumbers are marked above the lanes [C. aloifolium (CA-1 to CA-5), C. elegans (CEL-1 to CEL-5), C. eburneum (CEB-1 to CEB-5), C.mastersii (CM-1 to CM-5) and C. tigrinum (CT-1 to CT-5)]. All profiles were resolved in 1.5% agarose gels in TAE buffer. The lanes marked as A and B represents known DNA fragment, i.e., 500 and 100 bp size marker, respectively.

57S.K. Sharma et al. / Gene 483 (2011) 54–62

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showed moderate values of genetic distances and illustrated inTable 2. Likewise, the co-phenetic value matrix was used to test thegoodness of fit of a cluster analysis to the data by using the MXCOMPmodule to compare the original similarity or dissimilarity matrix thatwas clustered with the co-phenetic value matrix for all Cymbidiumspecies investigated at inter-specific levels. The degree of fit can beinterpreted as matrix correlation (r-value) for each pair and it showedthe good fit values as 0.83. 0.90 and 0.85 for pairs, viz. RAPD+ISSR,RAPD+DAMD and ISSR+DAMD, respectively.

Effectiveness of each SPAR method individually was also deter-mined at intra-specific levels for each Cymbidium species studied.Three SPARs viz. RAPD, ISSR and DAMD when used individually,generated more or less moderate values for genetic distances for eachspecies (Figs. S1–S5). The values obtained using individual SPARmethod is illustrated in Table 2. Consensus values of SPARs pairs viz.RAPD+ISSR+DAMD is calculated which showed moderate resultsfor all the species investigated (Table 2). The degree of fit which isdescribed as matrix correlation (r-value) for each pair which showedthe good fit values (0.8≤rb0.9) for each SPARmethod collectively. Rpand PIC values calculated as (6.68 and 0.792), (6.20 and 0.685) and(9.57 and 0.736) for RAPD, ISSR and DAMD, respectively.

3.4. Identification of species-specific unique banding pattern along withlost and missed bands

In the present investigation, three SPARs collectively revealed 25unique species-specific amplicons which were present in specific

species uniformly and absent in subsequent other species (Table 1).Out of 25 unique bands, 17, 2 and 6 bands were produced throughRAPD, ISSR and DAMD methods, respectively. Highest (11) bandswere specific to C. tigrinum, whereas only 2 bands were specific to C.elegans, C. eburneum and C. mastersii, respectively. Six bands provedthe explicitly towards C. aloifolium only (Table 1). The existence of thespecies- specific ISSR-PCR markers was confirmed by re-amplificationof the template DNAs. Alternatively, the monomorphic ampliconsdominantly visualized in four species and their representatives weretypically found lost in one species. A total of 21 bands were regardedas lost bands, out of which highest (9) bands were lost in C. aloifolium,whereas only one in C. elegans. Five and six bands were considered aslost bands in C. eburneum and C. tigrinum, respectively. Notably, not asingle band was lost in C. mastersii (Table 1).

The amplicons which were present in four species and mostlypolymorphic in nature but subsequently absent in rest one specieswere considered as missed bands for that particular species. Incomparison of 472 bands, which were amplified through three SPARs,viz. RAPD, ISSR and DAMD, at inter-specific level, a number of bandswere missed in particular species at intra-specific level (Table 2). In C.tigrinum, highest number of bands (230) band were missed, out ofwhich 126, 32, and 72 bands related to RAPD, ISSR and DAMD,respectively. In C. elegans, lowest number (142) of bands was missed,out of which 81, 22 and 39 had reference with RAPD, ISSR and DAMD,respectively. Similarly, a total of 187, 159 and 151 bands were missed,respectively, in C. mastersii, C. aloifoilum, and C. eburneum, out of whichhighest number of missed bands were correspondent to RAPD only.

Fig. 2. UPGMA dendrogram obtained through collective dataset in case of the 25 collections belonging to five Cymbidium species at inter-specific level. The numbers indicated to theright of the tree are individual's collection numbers [C. aloifolium (CA-1 to CA-5), C. elegans (CEL-1 to CEL-5), C. eburneum (CEB-1 to CEB-5), C. mastersii (CM-1 to CM-5) and C.tigrinum (CT-1 to CT-5)]. The branch lengths are based on the distance values computed using Jaccard's coefficient of NTSYS-pc 2.02k software. The large parenthesis to the right sidelabeled with A–C are the major clusters, while all inner parenthesis showing sub-clusters within the respective cluster.

58 S.K. Sharma et al. / Gene 483 (2011) 54–62

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Table2

Theva

riou

sSP

ARmetho

dsan

dde

tails

oftheirresu

ltsan

dan

alysis

compu

tedforfive

speciesof

Cymbidium

atinter-

andintra-sp

ecificleve

ls.

S.no.

Nam

eof

thesp

eciesstudied

SPARmethod

No.

ofpr

imers

used

Totalno.

ofba

nds

Ave

rage

no.

ofba

nds

/primer

Size

range

No.

ofpo

lymor

phic

bands

Ave

rage

no.

ofpo

lymor

phic

bands

/primer

Ave

rage

%po

lymor

phism

Jaccard's

similarity

coefficien

t

Ave

rage

similarity

Consensu

sva

luerange

Inter-specificva

riation

1Five

speciesof

Cymbidium

analyz

edco

llectively

RAPD

2824

28.6

0.2kb

–3kb

238

8.5

98.3

0.25

–0.96

0.60

0.09

–0.91

2ISSR

1066

6.6

0.2kb

–2.5kb

595.9

89.2

0.42

–0.97

0.69

0.12

–0.88

3DAMD

1516

410

.90.2kb

–3.5kb

159

10.6

96.9

0.32

–0.86

0.59

0.10

–0.90

4SP

AR(R

APD

+ISSR

+DAMD)

5347

28.9

0.2kb

–3.5kb

456

8.6

96.6

0.33

–0.88

0.60

0.10

–0.90

Intra-specificva

riation

5C.

aloifoliu

m(C

A-1

toCA

-5)

RAPD

2815

35.4

0.2kb

–3kb

109

3.9

71.2

0.37

–0.96

0.66

0.25

–0.75

ISSR

1045

4.5

0.2kb

–2kb

282.8

62.2

0.44

–0.92

0.68

0.25

–0.75

DAMD

1511

57.6

0.2kb

–3.5kb

916.0

79.1

0.45

–0.68

0.56

0.25

–0.75

SPAR

5331

35.9

0.2kb

–3.4kb

228

4.3

72.8

0.41

–0.84

0.62

0.33

–0.67

6C.

elegan

s(C

EL-1

toCE

L-5)

RAPD

2816

15.7

0.2kb

–3kb

123

4.3

76.3

0.39

–0.80

0.59

0.20

–0.80

ISSR

1044

4.4

0.2kb

–2.5kb

262.6

59.0

0.46

–0.87

0.66

0.20

–0.80

DAMD

1512

58.3

0.2kb

–3.5kb

996.6

79.2

0.43

–0.71

0.57

0.20

–0.80

SPAR

5333

06.2

0.2kb

–3.5kb

248

4.6

75.1

0.42

–0.76

0.59

0.20

–0.80

7C.

eburne

um(C

EB-1

toCE

B-5)

RAPD

2815

55.5

0.2kb

–3kb

115

4.1

74.1

0.40

–0.75

0.57

0.20

–0.80

ISSR

1050

5.0

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343.4

68.0

0.49

–0.86

0.67

0.20

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DAMD

1511

67.7

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795.2

68.1

0.47

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0.66

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5332

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228

4.3

71.0

0.44

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171.7

38.8

0.70

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0.82

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DAMD

1510

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845.6

78.5

0.41

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0.62

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5328

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220

4.1

77.1

0.40

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tigrinum

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CT-5)

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2811

64.1

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702.5

60.3

0.67

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1034

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90.9

26.4

0.83

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0.90

0.25

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DAMD

1592

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453.0

48.9

0.66

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124

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51.2

0.70

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4. Discussion

Classical approaches for the identification and assessment ofvariation among Cymbidiums are based on morphological traits(Wang et al., 2009). The assessment of these traits is difficult and theirevaluation can be subjective considering that most of the species andcultivars are related. Molecular markers and other genomic ap-proaches have been highly successful in characterizing the existinggenetic variation both at inter- and intra-species level and theygenerate new genetic diversity parameters that often extend beyondspecies boundaries (Gepts, 2002) and boast value for diagnosticapplications (Tong-Jian et al., 1991; Nekrutenko et al., 2000). Toovercome the predicament associated with genetic variation analysis,presently a variety of molecular markers such as RAPDs, ISSRs, SSRsand AFLPs have been routinely used for estimating the geneticdiversity of orchids (Hedrén et al., 2001; Li and Ge, 2006; Pillon et al.,2007). Such strategies are useful for conservation of genetic resources,identification of cultivars, and finally the selection of parents forhybridization program (Graner et al., 2004). Therefore, the selectionof trait specific DNAmarker is important and critically depends on theintended use (Gupta et al., 2002).

Very few reports dealing with analysis of genetic variation inCymbidium species based on different molecular markers are on hand(Obara-Okeyo and Kako, 1998; Obara-Okeyo et al., 1998; Van denBerg et al., 2002; Wang et al., 2004, 2009; Choi et al., 2006; Xiaohonget al., 2007; Huang et al., 2010). Many of them deal with limited use ofonly one marker system to assess intra-specific genetic variationparticularly in one Chinese Cymbidium species viz. C. goeringii. On thecontrary, the present study is the first report to make use of threedifferent marker systems to generate massive and comprehensivedata to analyze genetic variation both at inter- and intra-specificlevels in five species of Cymbidium collected fromnorth-east India, in amore authentic and convincing manner.

In the present investigation, 53 SPARs belonging to RAPD, ISSR andDAMD, were used both individually and cumulatively to analyzenatural variation at inter- and intra-specific levels. Three SPARs, eithercumulatively or individually, revealed high number of polymorphicamplicons at inter-specific level. However, the cumulative geneticsimilarity ranges among 25 collections belonging to five species ofCymbidium viz. C. aloifolium, C. mastersii, C. elegans, C. eburneum and C.tigrinum as calculated through Jaccard's coefficient that reveleddiscrete values with an average distance of 60% among the species(Table 2). The UPGMA clustering evidently showed that cluster A andC comprises only representatives of C. aloifolium and C. tigrinumrespectively. The reason for such apparent clustering may be due toentirely different climatic conditions as well as morphologicalfeatures of both the species. C. aloifolium is a medicinal, cultivatedtropical cymbidium and a biological indicator of tropical environment(Somashekarappa et al., 1996) with very thick, rigid leaves, whichreminds one of Aloe. Phylogenetic analysis using nuclear ribosomal ITSsequence data (unpublished data) also revealed that the clade ofCymbidium sect. Cymbidium comprising C. aloifolium was also quitedistinct being the base of the trees in the ITS analysis (unpublisheddata). Section Cymbidium presents several typical anatomicalcharacters, such as stomata within an elliptical cover and slit-shapepores and a complete layer of sub-epidermal schlerenchyma cells(Van den Berg et al., 2002). Sequence data of nrITS region alsoconfirmed the apparent clustering of all the three subgenera of genusCymbidum. C. tigrinum (subg. Cyperorchis; section Parishiella) did notreveal any affinity to any of the members of subg. Cyperorchis. Theextended form of hybridization signals as dots of fluorescence, both atinterphases and metaphases of the C. aloifolium and C. tigrinum,evidently represents the transcriptional activity of ribosomal genesthrough FISH technique (Fig. S10, unpublished data). Such transcrip-tional activity of ribosomal genes may be linked with coevolution ofIGS sequence with RNA polymerase I transcription factors which leads

to species specificity of the rDNA transcription machinery (Burtonet al., 2005).

A total of 25 fragments were identified as unique and species-specific in this study; most of themwere generated through RAPD andDAMD and highly specific to C. tigrinum only. These fragments can beused to reliably distinguish the tropical orchid species and theirpossible hybrids. Such data would also be very useful to accuratelydetermine the extent and role of introgression in the evolution of thisspecies as well as development of species-specific markers (Pharma-wati et al., 2005). The elucidation of the relationships among the 25collections belonging to five ornamental Cymbidiums and identifica-tion of species-specific SPARmarkers are important milestones for thebreeding andmanagement of elite Cymbidium germplasm. C. tigrinum,a rare and endangered Cymbidium, exclusively grows in cooler and dryclimate where the temperature raises up to 20 °C only with wideleaves and small, round, clustered pseudo bulbs. It is smallest memberof the genuswhich does not look like a typical cymbidiumwhen not inflower.

Cluster B consists of rest three species, viz. C. mastersii, C. eburneumand C. elegans, in the form of three sub-clusters, which grows in sub-tropical climatic condition and more or less threatened in theirnatural habitat. C. mastersii and C. eburneum are members of sectionEburnea and were morphologically characterized earlier (Zhong-Jianet al., 2006). In an earlier effort, Wang et al. (2004) also concluded thatC. mastersii and C. eburneum has low genetic distance with highsimilarity using RAPD and AFLP dataset. The observation derivessupport from our earlier report on karyo-morphological studies(Sharma et al., 2010a) of C. mastersii and C. eburneum that revealedsymmetrical karyotypes of both the species with only one sub-telocentric/telocentric chromosome pair. The absence of any nucle-olar organizer chromosomes and deviant number without numericaland structural changes suggested more or less stabilized genome of C.mastersii and C. eburneum. Similarly, nrITS sequence data also revealedclose relationship of two members of subg. Cyperorchis, i.e. C. elegans(section: Cyperorchis) and C. mastersii (section: Eburnea), with highbootstrap support (unpublished data), corroborating earlier reports(Van den Berg et al. 2002). Occurrence of representatives of C.mastersii, C. eburneum and C. elegans in the same cluster, i.e., B, may bedue to possible heterozygosity of genome reflected at DNA level asdetected by three SPAR methods.

Intra-specific variation detected in the five species presentlyinvestigated revealed low polymorphism compared to inter-specificlevel. Interestingly, C. tigrinum followed the same trend of beingdistinguished from other four species with high genetic similarity(76%), compared to other species. Five representatives of C. tigrinum, arare and endangered Cymbidium, showed identical genetic structurewith less polymorphism (51.2%) within the species and it drawssupport from studies of Xue et al. (2004), who opined that rare andendangered species are susceptible to loss of genetic variationthrough genetic drift in small populations. The rest of four speciesrevealed moderate genetic variation within the species, whichmay bedue to wide spread large population structure. A highly significantnumber of endangered species and their populations had low level ofgenetic variation compared to related non-endangered taxa withlarge population size (Frankham, 1995).

An interesting and significant aspect of the present investigationhas been that RAPDs, ISSR and DAMD, which are all single primer-based amplification strategies, were analyzed and compared not onlyindividually but also pair-wise with each other, both at inter- andintra-specific levels. The values of polymorphic information content(PIC) for all of the three SPARs were significantly high, compared totheoretical maximum of 0.5, indicating high number of informativebands produced with these markers. RAPD gets merit over other twoSPARs, viz. ISSR and DAMD, with comparatively high PIC values(0.792). DAMD marker revealed highest value (9.57) of resolvingpower (Rp), which is an efficient tool to assess the capacity of a given

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primer(s)/marker(s) to distinguish among various genotypes. There-fore, authors are of the opinion that DAMD is the ideal marker systemto analyze natural genetic variation in plants more authenticallycompared to other SPARs viz. RAPD and ISSR.

It has been clearly observed from our studies that the r-value,which reveals the efficacy of these methods, has been best fit for allthe SPARs, especially when DAMD coupled with one of the other two,i.e., RAPD and ISSR marker systems. Similar observations were madeby different workers while analyzing genetic variation in plants, viz.mulberry, neem and pomegranate, respectively (Bhattacharya andRanade, 2001; Ranade and Farooqui, 2002; Ranade et al., 2009). ISSRand DAMD pair when compared to RAPD either individually or incombination reveled that missed bands belonged to the latter, whichshows its less reproducibility and therefore diminished reliability overother two SPARs (Sharma et al., 2010b). Such observations are alsoreported earlier in case of other plants (Saxena et al., 2005; Ranadeet al., 2009; Sharma et al., 2010b) to describe variation. On the otherhand, highest numbers of unique species-specific banding patternswere also obtained by RAPDs alone, a phenomenon which was lessobserved by ISSR and DAMD. It demonstrates the usefulness of RAPDmethod in the generation of new markers for precision genomemapping which can be developed as sequence-characterized ampli-fied regions (SCAR) linked to various traits and their putative genes asreported earlier in certain orchid genera (Handa, 1998; Jin et al., 2010)and other plant species (Arnau et al., 2003; Pharmawati et al., 2005).The presence of discrete variations in certain random sequences ofDNA may be considered as an effort by these genomes to adapt to theextreme environmental conditions in which they grow and propagate(Trivedi, 2003; Hickey and Singer, 2004; Wood et al., 2008).Therefore, it can be authentically concluded that in order to revealthe intrinsic inter- and intra-specific variation, SPAR approachincluding RAPD, ISSR and DAMD reveals more genetic variation inplants like Cymbidium. The sufficient genetic variation whichevidently exists among Cymbidium species, can be further exploitedin designing and framing of breeding programs. The efficacy andreliability of SPAR assays coupled with the observed levels ofpolymorphism with five species of Cymbidium indicate the potentialof the SPARs in identification of Cymbidium species and their possiblerole in genetic and breeding studies. Further applications should bestretched out for using thesemarkers to tag desirable traits and can beused for commercial production of cut flowers and pot plants withnew floral characteristics and other beneficial traits through inter-specific hybridization (Wang et al., 2009).

Management decisions for the conservation of rare/threatenedplants, i.e. orchids, ideally necessitate an understanding of theirbiology and other influencing factors including genetic variability andlevel of threat or endangeredness. Successful management of manyrare plant populations has been greatly improved by genetic data. Thegeneration of knowledge about genetic structure/variation of rarespecies has become a common prelude to conservation planning(Ellstrand and Elam, 1993; Archibald et al., 2001). PCR based singleprimer amplification reaction (SPAR) methods including RAPD, ISSRand DAMD are gaining prominence as effective tools for geneticdiversity analysis of plants since they collectively provide a compre-hensive account of the nature and extent of variation (Ranade et al.,2009; Sharma et al., 2010b). The information thus obtained providesvaluable baseline data for knowing genetic variation at inter- andintra-specific levels in five species of Cymbidium and addressconservation of this horticultural and ornamental orchid genus.

Supplementarymaterials related to this article can be found onlineat doi:10.1016/j.gene.2011.05.013.

Acknowledgements

The present work is supported by a grant from University GrantsCommission, Government of India, New Delhi, through University

with Potential for Excellence (UPE)–Bioscience program. Sincerethanks are due to all members of Plant Biotechnology Laboratories,Department of Botany as well as Department of Biotechnology andBioinformatics, NEHU, Shillong, for their constant encouragement andhelp.

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