ã Anita Ahlawat, Meenu Katoch, Gandhi Ram and Ashok Ahuja (2010) Genetic diversity in Acorus calamus L. as revealed by RAPD markers and its relationship with B-asarone content and ploidy level,Scientia Horticulturae 124:294-297

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  b-Asarone content in Acorus calamus is a paramount issue because it limits the usage of plant for medicinal purpose. In the present study A. calamus L. accessions based on RAPDmarker, ploidy level and b-asarone content were characterized and
  This article appeared in a journal published by Elsevier. The attachedcopy is furnished to the author for internal non-commercial researchand education use, including for instruction at the authors institutionand sharing with colleagues.Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third partywebsites are prohibited.In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further informationregarding Elsevier’s archiving and manuscript policies areencouraged to visit:http://www.elsevier.com/copyright  Author's personal copy Short communication Genetic diversity in  Acorus calamus  L. as revealed by RAPD markers and itsrelationship with  b -asarone content and ploidy level Anita Ahlawat a , Meenu Katoch b, *, Gandhi Ram a , Ashok Ahuja a a Biodiversity and Applied Botany Division, Indian Institute of Integrative Medicine (CSIR), Jammu 180001, India b Biotechnology Division, Indian Institute of Integrative Medicine (CSIR), Jammu 180001, India 1. Introduction  Acorus calamus  L. or ‘Sweet Flag’ (Araceae) is a reed like semi-aquatic perennial plant with a stout aromatic rhizome havingmedicinalproperties(Agarwaletal.,1956;Motley,1994).InIndiait growsinwildinabundanceupto2200 minHimalayas.Therhizomecontains active ingredients possessing insecticidal, antifungal,antibacterial, larvicidal, antitermite, larval and insect repellantproperties (Raina et al., 2003; Bertea et al., 2005). In the Ayurvedic system of medicine, the rhizomes are considered to possessantispasmodic, antidiarrhoeic, carminative and anti-helminthic,antidepressant,CNS,anxiolyticproperties(Mcgawetal.,2002;Rainaet al., 2003; Bertea et al., 2005). It is used to treat insomnia,melancholia,neurosis,epilepsy,hysteria,lossofmemory,remittentfever, rheumatism, toothache, and respiratory ailments (Mcgawet al., 2002; Mehrotra et al., 2003; Raina et al., 2003).The essential oil of   Acorus  contains various constituents. Theproportion of each chemical constituent of the oil particularly  b -asaronevariesbetweenthevarietiesof   A.calamus andcorrespondstotheploidylevel(Mcgawetal.,2002).Fromakaryotypicpointofview,sweet flag includes four ploidy levels: diploid (2x =24), triploid(3x = 36),tetraploid (4x = 48) and hexaploid (6x= 72) (Berteaetal.,2005).DiploidkaryotypesgrowinNorthAmericaandinpartsofAsiaand are characterized by the absence of   b -asarone, whereasEuropean,NorthAmericanandKashmiritriploidkaryotypescontain3–19% of  b -asarone. The Indian, Indonesian and Taiwan tetraploidkaryotypes contain up to 96% of   b -asarone (Mcgaw et al., 2002;Bertea et al., 2005), whereas tetraploid of Far East Russia arecharacterized by 10–40% b -asarone (Raina et al., 2003). b -Asarone (z)-1,2,4-trimethoxy-5-prop-1-enyl-benzene is atoxicant causing chromosomal aberrations, mutations and cancer(GoggelmanandSchimmer,1983;Abel,1987).Becauseofvarying b -asaronecontent,preciseidentificationof   A.calamus chemotypesisaprerequisiteforcommercialapplication.Conventionally,identifica-tionofherbalswasbasedonmorphological,anatomicalandchemicalanalysis but these could be influenced by environmental factors.IdentificationofDNAmarkersthatcancorrelateDNAfingerprintingdatawithquantityofselectedphytochemicalmarkerassociatedwiththat particular class of plant would have extensive application inquality control of raw materials. Among various moleculartechniques,RAPDisasimple,largelyautomatabletechniquerequireonlysmallamountofDNAandcanbeperformedwithouttheuseof radioactivity(Williamsetal.,1990).Thistechniqueisanefficientandinexpensive method of generating molecular data has beenemployed in many taxonomic and phylogenetic studies (Keil andGriffin, 1994; Khan et al., 2000). Although RAPD technique isoutdated technique, still it offers several advantages in species ortaxon or chemotype identification. It was also used in prediction of phytochemicals in plants (Chen et al., 2009). Since the correlation of RAPD data with  b -asarone content of these accessions and their ploidy level would be useful for plantbreeding,qualitycontrol,intellectualpropertyrightsandeventually Scientia Horticulturae 124 (2010) 294–297 A R T I C L E I N F O  Article history: Received 31 July 2009 Received in revised form 23 December 2009 Accepted 26 December 2009 Keywords: Acorus calamus  L.RAPD b -AsaroneTriploidTetraploidPloidy level A B S T R A C T b -Asarone content in  Acorus calamus  is a paramount issue because it limits the usage of plant formedicinalpurpose.In thepresentstudy  A. calamus  L.accessions based onRAPD marker, ploidylevel and b -asarone content were characterized and correlated on the basis of  b -asarone content/ploidy level. Of the40randomprimersused,6primersgeneratedpolymorphism.Geneticrelatednessamongaccessionsevaluated by a similarity matrix based on Dice’s coefficient ranged from 0.72 to 0.97. A pheneticdendrogram based on UPGMA analysis grouped accessions into two clusters.  A. calamus  L. accessionswerefoundtobetriploidandtetraploidandtheir b -asarone contentwasfoundintworanges 6.92–8.0%and 73–88%. The study clustered the accessions as per their ploidy level,  b -asarone content andgeographical locations. This study would have extensive application in quality control of raw materials.   2010 Elsevier B.V. All rights reserved. * Corresponding author at: Department of Biotechnology, Indian Institute of Integrative Medicine, Canal Road, Jammu, India. Tel.: +91 09419157224. E-mail address:  meenusamiksha@rediffmail.com (M. Katoch). Contents lists available at ScienceDirect Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti 0304-4238/$ – see front matter    2010 Elsevier B.V. All rights reserved.doi:10.1016/j.scienta.2009.12.035  Author's personal copy for pharmacological studies, the present study was made tocharacterize  A. calamus  L. accessions based on RAPD marker, ploidylevel and  b -asarone content and correlate them on the basis of  b -asarone content/ploidy level. 2. Materials and methods  2.1. Plant material Twenty-three accessions of   A. calamus  L. were from four statesin northern and north-east India namely Uttarakhand (UK),Himachal Pradesh (HP), Jammu & Kashmir (J&K) and Manipur(MN) representing four agro-ecological regions (Table 1). Theywere planted in an herbal garden and conserved through clonalpropagation.  2.2.  b -Asarone content analysis The essential oil was obtained from aerial parts of naturallygrown plants by hydro-distillation using a Clevenger-typeapparatus. Triplicate distillations were performed in successionfor each sample. The oil samples were stored at 4  8 C until used forchemicalanalysis.Gaschromatography(GC)analysisoftheoilwascarriedoutonNUCONGasChromatographapparatus,fittedwithafused silica capillary column coated with FFAP and helium ascarrier gas. The compoundswere identifiedby comparisonof theirrelative retention times with those of known compounds rununder similar conditions and by enrichment technique. GC–MSwas recorded on QP-2000 Shimadzu Model fitted with BP-10column. The GC oven temperature was programmed as follows:initial temperature 90  8 C/2 min to 220  8 C with rise at the rate of 7  8 C/min to 220  8 C. The carrier gas was helium with FID detector.Thesampleswereinjectedusingsplitsamplingmethod,ratio1:50.The content of  b -asarone (retention time 18.6 min) in the relativepercentage was computed by the normalization method from GCpeakareas.All 23accessionswereanalyzedfor b -asaronecontent.  2.3. Ploidy analysis For cytological studies growing root tips pretreated with asaturated solution ofp-dichlorobenzenefor 4 h at20  8 C. Followingwashing is distilled water; the material was fixed in mixture of acetic acid and ethanol (1:3) and exposed to a chilling treatment(4  8 C) for 4 h. Subsequently the material was transferred to 70%alcohol for further use. For tissue staining orecein dye was used(orecein 1%, 1N HCl, 18:1). Squash preparations were made inacetocarmine and chromosomal analysis was done at themetaphase/anaphase stage. Data on ploidy level was computedfrom observations of about 100 cells per sample. All 23 accessionswere analyzed for ploidy level.  2.4. RAPD analysis Young and fresh leaves (0.2 g) of randomly selected plants of various accessions were collected and used for genomic DNAextraction (Ahmad et al., 2004). Forty decamer primers wereinitially screened to generate RAPD profiles. RAPD profiles weregenerated as described by Ahmad et al., 2006. A control PCR tubecontainingallcomponents,butnogenomicDNAwasrunwitheachprimer to check for contamination. All the PCR results were testedfor reproducibilityby at least threetimes. Bandsthat did not showfidelity were eliminated for statistical analysis.Discriminating power (Dj) of each primer, i.e., the probabilitythat the two randomly chosen accessions from the sample of 23accessions have different banding pattern and, thus, are distin-guishable from one another, was estimated (Tessier et al., 1999). Genetic diversity was estimated by Shannon index (Lewontin,1972).Toinvestigatepheneticrelationshipsamongaccessions,thebinary matrix was used to cluster individuals using procedure of NTSYS-PC2.1 (Rohlf, 1993). A dendrogram was constructed basedon Dice coefficient’s similarity data applying the unweighted pairgroup method (UPGMA). The robustness and validity of clusteringpattern was tested by Bootstrap analyses of 1000 bootstrapsamples using the software  WINBOOT   (Yap and Nelson, 1996). 3. Results and discussion  3.1.  b -Asarone content and ploidy analysis All 23 accessions analyzed for chemical spectrum and ploidylevel. Accessions AC8 and AC10 belonging to Kashmir (J&K) andAC4,AC6andAC7belongingtoManipurwerefoundtobetriploids  Table 1  Acorus calamus  accessions collected from different geographical locations in India with their ploidy level and  b -asarone content (%).Sr. no. Accession code no. Place of collection Geograpical locations Ploidy level  b -Asarone content (%)1 AC1 Palampur (HP) 32 8 N 76 8 E Tetraploid 88.02 AC2 Mandi (HP) 31 8 N 76 8 E Tetraploid 87.03 AC3 Palampur (HP) 32 8 N 76 8 E Tetraploid 80.04 AC4 Manipur (MN) 25 8 N 94 8 E Triploid 7.05 AC5 Shamshi (HP) 31 8 N 77 8 E Tetraploid 78.06 AC6 Manipur (MN) 25 8 N 94 8 E Triploid 7.347 AC7 Manipur (MN) 25 8 N 94 8 E Triploid 7.858 AC8 Narkara, Kashmir (J&K) 34 8 N 74 8 E Triploid 8.09 AC9 NGC Kullu (HP) 31 8 N 77 8 E Tetraploid 76.010 AC10 Kakpura, Kashmir (J&K) 34 8 N 74 8 E Triploid 6.9211 AC11 TP Batote, Jammu (J&K) 32 8 N 74 8 E Tetraploid 76.012 AC12 Rani Sidhpur, Jammu (J&K) 32 8 N 74 8 E Tetraploid 82.013 AC13 Hirabagh, Jammu (J&K) 32 8 N 74 8 E Tetraploid 78.014 AC14 Chimbelhar (HP) 32 8 N 76 8 E Tetraploid 82.415 AC15 Tikaphata (HP) 31 8 N 77 8 E Tetraploid 84.016 AC16 Tipovan (HP) 34 8 N 77 8 E Tetraploid 87.217 AC17 Manali (HP) 32 8 N 77 8 E Tetraploid 76.018 AC18 Manali (HP) 32 8 N 77 8 E Tetraploid 73.019 AC19 Manali (HP) 32 8 N 77 8 E Tetraploid 81.020 AC20 Manali (HP) 32 8 N 77 8 E Tetraploid 84.021 AC21 Manali (HP) 32 8 N 77 8 E Tetraploid 80.022 AC22 Barotiwala (HP) 31 8 N 77 8 E Tetraploid 86.023 AC23 Dehradun (UK) 30 8 N 78 8 E Tetraploid 86.0HP: Himachal Pradesh; UK: Uttarakhand; MN: Manipur; J&K: Jammu & Kashmir.  A. Ahlawat et al./Scientia Horticulturae 124 (2010) 294–297   295  Author's personal copy with 6.92–8.0%  b -asarone, whereas rest of accessions collectedfromHimachalPradesh,UttarakhandandJammu(J&K)werefoundtobetetraploidwithhigh b -asaronecontent(73–88%).Ploidyleveland  b -asarone content of all the accessions except Manipur andKashmir accessions are in accordance with the previous reports(Ref. cited in Mcgaw et al., 2002; Bertea et al., 2005) where in they reported that Indian accessions were tetraploid with high  b -asarone content. Kashmir accessions were triploid with less  b -asarone content similar to that reported by Rost and Bos (1979cited by Mcgaw et al., 2002). Contrarily this study observed thataccessions belonging to Manipur are also triploid and have less b -asarone content (6.92–8.0%).  3.2. RAPD analysis Twenty-three accessions of   A. calamus  L. were analyzed usingforty random decamer primers. Out of which 34 primersgenerated amplification products with a very low number of bands and the remaining 6 primers produced polymorphic andrepeatable amplification products (Fig. 1). They produced 5–11amplification fragments with an average of 8 bands per primer.Out of these six primers, four primers with G + C content of 60%resulted in better polymorphism (62.5–100%) (Table 2) (Smel- cerovic et al., 2006; Ahmad et al., 2006). A total of 48 RAPDmarkers were detected from  Acorus  accessions with 6 primersand out of which 36 bands showed polymorphism. The averageproportion of polymorphic markers across primers was 75%,ranging between 20% (OPK-04) to 100% (OPK-13) (Table 2). Thesize of the amplification products varied from 0.1 to 2 kb. Thelarge number of exclusive markers account for a substantialportion of the genetic diversity. Genetic diversity betweenprimers as illustrated by Shannon index per primer was rangedfrom 0.121532 (OPK-04) to 1.790443 (OPK-11) with a totaldiversity ( H  ) of 7.301575 and an average diversity of 1.2169(Table 2).Using 6 RAPD primers on the set of 23 accessions, 41 RAPDpatterns were obtained. The number of banding patterns ( T  p )ranged from 2 in primer-OPK-4 to 11 in Primer-OPK-14 with anaverage of 7 banding patterns. The discriminating power rangedfrom 0.23 in primer-OPK-4 to 0.88 in primer-OPK-13 suggestingthat OPK-13 primer is best for discriminating 23 accessions of   Acorus . Similarly Tessier et al. (1999) evaluated the efficiency of aprimer for the purpose of identification of varieties usingdiscrimination power.Cluster analysis of RAPD data based on similarity matrixgenerated a dendrogram (Fig. 2) with two clusters at  > 80%similarity level (Dice coefficient 0.815). Cluster II included theplants AC4, AC6, AC7, AC8 and AC10 whereas rests of the plantswere grouped into Cluster I. Bootstrap analyses based on 1000pseudo-samples also validated the dendrogram; classifying the 23accessions in two major clusters.Cluster analysis revealed a strong distinctness of the profilesfrom different geographical regions: accessions from Manipur(MN) and Kashmir (J&K) were grouped in Cluster II whereasaccessions belonging to Himachal Pradesh, Jammu and Uttarak-hand were grouped in Cluster I. But within a cluster, all theaccessions of particular geographical region were not clustered indistinct groups reflecting a high genetic differentiation amonggenotypes from same locations per se. In accessions of Cluster II,there is no chance of immigration of germplasm from onegeographicalregiontootherbecauseoftheirgeographicalpositioni.e.oneatthenorthendandotherontheeasternend.Thecasemaybe vice versa for Cluster I.All accessions which were triploid with trace amount of   b -asarone (6.92–8.0%) were clustered in Cluster II whereas thoseaccessions which were tetraploid with high  b -asarone content(73–88%) were grouped in Cluster I. Similar to our studies, in lastdecade on the basis of 700 bp sequence of 5S-rRNA gene spacerregion, three chemotypes of   A. calamus  (chemotype A-predomi-nant Z-asarone, chemotype B-predominant sesquiterpenoids,chemotype M-various ratio of Z-asarone and sesquiterpenoids)wereclusteredseparately(Sugimotoetal.,1999).Recentlybecauseof the presence/absence of EcoRI site in these sequences, Berteaet al. (2005) identified the  b -asarone free diploid  A. calamus caryotypes from triploid plants. Similar to our study, there was astrong correlation for secondary metabolite contents with RAPDdata than with SSR data among the six  Hypericum  species fromSerbia (Smelcerovic et al., 2006). Fig. 1.  RAPD profile of 23accessions of   Acorus calamus  using primer OPK-14. Lanes 1–23accessions of   A. calamus  (AC 1 –AC 23 ).Lane M—Gene Ruler Lamda DNA/HindIII Digest(Bangalore Genei, Bangalore).  Table 2 Details of primers used for RAPD analysis and comparison of genetic diversity among 23 accessions of   Acorus calamus  by RAPDs.Sr. no. Primer no. Sequence PercentageGC contentTotalbandsNo. of polymorphicbandsPercentagepolymorphismGenetic diversity interms of Shannon indexDiscriminatingpower1 OPK-3 CCAGCTTAGG 60 8 5 62.5 1.009468 0.7312252 OPK-4 CCGCCCAAAC 70 5 1 20 0.121532 0.2371543 OPK-8 GAACACTGGG 60 8 7 87.5 1.567377 0.7667984 OPK-11 AATGCCCCAG 60 11 9 81.81 1.790443 0.6363645 OPK-13 GGTTGTACCC 60 7 7 100 1.291497 0.8814236 OPK-14 CCCGCTACAC 70 9 7 77.7 1.521258 0.80632448 7.301575  A. Ahlawat et al./Scientia Horticulturae 124 (2010) 294–297  296  Author's personal copy 4. Conclusion In conclusion, thisstudy improvedthe knowledge of  b -asaronecontentinIndian  A.calamus accessions.  A.calamus accessionsfromManipur were found triploid with 6.92–8.0%  b -asarone content.RAPD analysis clustered separately the triploid  A. calamus accessions belonging to Manipur and Kashmir with 6.92–8.0% b -asarone content and the tetraploid  A. calamus  accessionsbelonging to Jammu, Himachal Pradesh and Uttarakhand with73–88% b -asarone content. This will provide proper identificationof plant material for drug purposes and ensure the reproducibilityandtheefficacyofcommercialherbalproductswhere  A.calamus L.accessions are used.  Acknowledgements The authors are grateful to Department of Biotechnology,Government of India for the financial support and the Director of the Indian Institute of Integrated Medicine, Jammu for providingthe facilities. References Abel, G., 1987. Chromosome damaging effect on human lymphocytes by beta-asarone. 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Mutagenicity testing of   b -asarone andcommercial calamus drugs with  Salmonella typhimurium . Mutat. Res. 121,191–194.Keil, M., Griffin, A.R., 1994. Use of Random Amplified Polymorphic DNA (RAPD)markersinthediscriminationandverificationofgenotypes.Theor.Appl.Genet.89, 442–450.Khan, S.A., Hussain, D., Askari, E., Stewart, J., Mc, D., Malik, K.A., Zafar, Y., 2000.Molecular phylogeny of   Gosspium  species by DNA fingerprinting. Theor. Appl.Genet. 101, 931–938.Lewontin,R.C.,1972.Theapportionmentofhumandiversity.Evol.Biol.6,381–398.Mcgaw, L.J., Jager, A.K., Staden, J., 2002. Isolation of  b -asarone, an antibacterial andanthelmintic compound, from  Acorus calamus  in South Africa. South African J.Bot. 68, 31–35.Mehrotra, S., Mishra, K.P., Maurya, R., Srimal, R.C., Yadav, V.S., Pandey, R., et al.,2003. Anticellular and immunosuppressive properties of ethanolic extract of   Acorus calamus  rhizome. Int. Immunopharmacol. 3, 53–61.Motley, T.J., 1994. The ethnobotany of sweet flag,  Acorus calamus  L. Econ. Bot. 48,397–412.Raina, V.K., Srivastava, S.K., Syamasunder, K.V., 2003. Essential oil composition of   Acoruscalamus L.fromthelowerregionoftheHimalayas.FlavourFrag.J.18,18–20.Rohlf, F.J., 1993. Exter Software. NTSYS-pc. Version 1.80. Applied Biostatistics Inc.,Setauket, NY.Rost, L.C.M., Bos, R., 1979. Biosystematic investigations with  Acorus  L. 3. Communi-cation. Constitutions of essential oils. Planta Med. 36, 350–361.Smelcerovic, A., Verma, V., Spiteller, M., Ahmad, S.M., Puri, S.C., Qazi, G.N., 2006.Phytochemical analysis and genetic characterization of six  Hypericum  speciesfrom Serbia. Phytochemistry 67, 171–177.Sugimoto, N., Kiuchi, F., Mikage, M., Mori, M., Mizukami, H., Tsuda, Y., 1999. DNAprofiling of   Acorus calamus  chemotypes differing in essential oil composition.Biol. Pharm. Bull. 22, 481–485.Tessier, C., David, J., This, P., Boursiquot, J.M., Charrier, A., 1999. 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