www.IndianJournals.com Members Copy, Not for Commercial Sale

Downloaded From IP - 61.247.228.217 on dated 27-Jun-2017 Indian J. Genet J. Indian Badrinath, Ranikhet andKilbury, Nainital), Badrinath, Ranikhet Himalayan the geneticrelationshipamongmainspeciesof Asian pinesarestillnotwellsettled[1-4]. species aswellrelationshipsamongandbetween The positionsofseveralendemic the watersheds. significantly totheeconomyofcountryandprotect *Corresponding author’se-mail: [email protected];[email protected] P.kesiya The genus (Received: December2008;Revised:January2009;Accepted:2009) Kaulagarh Road,Dehradun248195 Division ofGeneticsandTreePropagation,ForestResearchInstitute,POIPE, H. S. Ginwal, Pallavi Bisht, PritiChauhan, ShaliniSinghMaurya andSantanBarthwal occurrence Genetic relationshipbetweenthreeHimalayan pinesof Indian Communucation Short (CTAB) methodofStange usingthecetyltrimethylammoniumbromide Genomic DNAwasisolatedfromneedlesofindividual the study.Ineachpopulationfivetreeswereincluded. Gangolihat andThalisian,)weretakenfor representing thepopulation wasmade.Testswere was bulkeduniformlyand acompositesample The isolatedDNAfromfive treesofeachpopulation template DNAforPolymeraseChainReaction(PCR). to auniformconcentrationof50ng/µlbeusedas was quantifiedandallthesampleswerebroughtdown Assam andMeghalaya) . Howeveronlyfourspecies studies relatedtonaturalpopulation[5]. shown thatISSRmarkershavegreatpotentialforsuch technique wasemployed.Thepublishedinformationhas seven speciesofpines genera ofconifersintheNorthernHemisphere.InIndia P. wallichiana, P. gerardiana gerardiana P. wallichiana, P. wallichiana, P. armandi, P. gerardiana, P. kesiya, P. kesiya, P. gerardiana, P. armandi, P. wallichiana, bhutanica bhutanica In ordertoestablishabetterunderstandingabout Two populationseachof and and Pinus P. gerardiana P.gerardiana ., 69(1):81-83(2009) P. merkusii merkusii P. isoneofthemostwidelydistributed viz ., P. roxburghii, P. wallichiana wallichiana P. roxburghii, P. a hyper-variableISSRmarker et al. al. et viz are knowntooccurinthe and ., [6]. TheisolatedDNA Pinus roxburghii, P. roxburghii, Pinus P. wallichiana wallichiana P. P. roxburgii roxburgii P. P. kesiya kesiya P. viz ., P. kesiya kesiya P. P . roxburghii, contribute (from (from (from and average of6bandsperprimer. Incaseof ranged fromaslow2to a maximumof11,withan Table 1.Thenumberofamplification productsproduced nucleotide sequenceoftheISSRprimersisgivenin for scoringandgeneticanalysis.Thedetails reproducible andconsistentamplificationwereused Software wasobtained. genetic distancematrix[7]valueusingGeneProfiler with ArithmeticAverages(UPGMA)andNEILi dendrogram basedonUnweightedPairGroupMethod order toanalyzetherelatednessamongspecies,a profiles wereanalyzedusingthesoftwareNTSYS.In absent foreachDNAsampleandfurthertheISSR Documentation system. staining andphotographedunderUVlightusingGel hours. Gelswerevisualizedbyethidiumbromide agarose gels(Fig.1)with1xTBEbufferatpH8.0for3 amplification productswereelectrophoresedusing1.5% (GibcoBRL) inatotalvolumeof50ml.Subsequently of theprimerpair,and1.5units GibcoBRL, LifeTechnologies,USA),0.5mmolofeach 200 mmolofeachdeoxyribonucleotide(dNTP, sec, andterminationby5minat72°C. temperatures giveninTable1,extensionat72°Cfor60 at 94°Cfor30sec,primerannealingsec initial denaturation,followedby35cyclesofdenaturation PCR amplificationwascarriedoutat94°Cfor5min performed forstandardizingPCRconditionsandfinally Out oftwentyISSRprimersscreened,sixgave All theamplifiedbandswerescoredaspresentor The PCRmixturecontained10ngDNAtemplate, Taq Taq DNA polymerase P . gerardiana www.IndianJournals.com Members Copy, Not for Commercial Sale

Downloaded From IP - 61.247.228.217 on dated 27-Jun-2017 82 was observedbetween P Results indicatedthat and variation betweenpopulationof from eachother.Thedendrogramrevealedthatthe differentiate only threepinespecieswereusedforcomparison. no amplificationwithISSRprimerswasachieved,hence related to similarity coefficientwasfoundbetween respectively. Acrossthespecieshighestvalueof value ofsimilaritycoefficient0.900,0.875and0.612 variation betweenthespecies,asindicatedbyhigher cluster analysisdifferentiated reproducible geneticrelationshipwasobtained(Fig.2). SR1 CCCCCCCCG53 51 54 ACACACACACACACACYG 44 ISSR-10 ACACACACACACACACYT 43 ISSR-8 GTGTGTGTGTGTGTGTYG ISSR-5 GAGAGAGAGAGAGAGAA ISSR-4 GAGAGAGAGAGAGAGAC ISSR-3 SR 1TTTTTTTTR 54 TGTGTGTGTGTGTGTGRT ISSR- 11 primer Basesequence(5'-3') Name of Table 1. wallichiana wallichiana . Fig. 1a&b. DNAbandsasamplifiedby - primers wallichiana wallichiana P The UPGMAbaseddendrogram(Fig.2)clearly . kesiya. kesiya. study List ofISSRprimerssequencesusedforthe P and . P kesiya kesiya . roxburghii, P. kesiya kesiya P. roxburghii, clearly fromeachotherandahighly The lowestsimilaritycoefficientvalue ISSR-8 (1a)andISSR-11(1b) P . kesiya kesiya than P P . P is lessincomparisontothe . roxburghii roxburghii . wallichiana wallichiana P wallichiana. wallichiana. . roxburghii, P. kesiya P.kesiya roxburghii, and P . is moreclosely temperature ( roxburghii, P. roxburghii, and P The UPGMA Annealing P. roxburghii P. . wallichiana P. kesiya. P. H. S. Ginwal Ginwal S. H. and o C) et al et of Wang 3]. Thepresentrelationshipisinsupporttothefindings suggested highlevelsofdivergenceamongthem[11, (cp) DNArestrictionsitedataandITSsequenceshave Islands [9,10].Howeverrecentanalysisofchloroplast Tertiary andledtotheriseof Himalayan regionintheUpperCretaceous-Lower followed theTethyscoasttoeastandreached .- .50045 .35050 .001 0.9000 0.5500 1 0.4375 0.4250 0.5500 0.4500 1 0.5125 0.4250 P.r-2 0.4500 0.6125 0.5750 1 P.r-1 0.4750 0.2875 P.k-2 1 0.2875 P.k-1 0.8750 1 P.w-2 P.w-1 from Mediterraneanancestorsof intron sequences. chloroplast relationships ofthirty-twoEurasianpinesbasedon Table 2. [8]. considerable divergencefromalltheotherhardpines character of considered ascloserelativesof Fig. 2.Dendrogram showing divergence among3 . P Klaus [10]suggestedthat The Himalayan . roxburghii roxburghii .- .- .- .- .- P.r-2 P.r-1 P.k-2 P.k-1 P.w-2 P.w-1 of threedifferentspeciesPinus Similarity indexforsimplematchingcoefficient et al. al. et rbcl, math, rpl20-rps18 rpl20-rps18 math, rbcl, P . roxburghii roxburghii [8] whoestablishedphylogenetic and species of P . roxburghii P as revealedbyourpresent . P wallichiana have have wallichiana . roxburghii. roxburghii. P P . . canariensis canariensis Pinus roxburghii roxburghii P is . spacer, and

canariensis canariensis known toshow [Vol. 69,No.1 The divergent originated of Canary been trnv that www.IndianJournals.com Members Copy, Not for Commercial Sale

Downloaded From IP - 61.247.228.217 on dated 27-Jun-2017 3. 4. 2. 5. 1. kesiya. of and other[3]molecularevidencesuggestsanearlysplit February, 2009] References ancestral stocktotheAsianhardpines[8]. P . Buylla E. R. Listom A., Robinson W. A., Pinero D. andAlvarez- 498. restriction siteanalysis.AmericanJ.Botany, Pinus Pinus C. Schirone B., Piovesan G., Bellarosa R. andPelosi and Evolution, spacer regionsequences.MolecularPhylogenetics based onnuclearribosomalDNAinternaltranscribed subgenus Phylogenetic analysisofthehardpines( Krupkin A.B.,Liston,I.andStrausstS.H. Ten., 11-18.MinistryofForestry,Ankara. Assessing hybridizationinnaturalpopulationsof Wolfe A. D., XiangQ. Y. S. R. andKephart Evolution, Frankis M. P. brutia. In In brutia. roxburghii roxburghii Alternatively, 1991. Ataxonomicanalysisofseedproteinsin spp. ().PlantSystematicsand International Symposiumon 178 Pinus, Pinus, 1999. lineage from 1993. Morphologyandaffinitiesof : 43-53. 11 P : 95-109. Pinaceae) fromchloroplastDNA

Phylogenetics of . roxburghii roxburghii Genetic relationship in Himalayan pines of Indian occurrence Indian of pines Himalayan in relationship Genetic P . wallichiana wallichiana might representan Pinus Pinus Pinus (Pinaceae) 83 than 1998b. Pinus : 489- Pinus 1996 P . 8. 7. 11. 10. 6. 9. of Eurasianpines( K. andSzmidtA.E. Y.,R., Tsumura Nagasaka X. H., YoshimaruWang 5273. endonucleases. Proc.Nat.Acad.Sci.,USA, studying geneticvariationintermsofrestriction Nei M. and 1-8. based phylogenyofAsian Wang X. R. andSzmidtA. E. 163. history. PlantSystematicsandEvolution, Klaust W. Company, NewYork,NY. RAPD analysis.PlantMolecularBiologyReporter, Isolation ofPinusradiatagenomicDNAsuitablefor C., PrehnD.Stange andJohnsonP. A. Ecology, inter simplesequencerepeatmarkers.Molecular chloroplast Penstemon Penstemon SystematicsandEvolution, Mirov N. intron sequences.Amer.J.Hot,

7 T. T. : 1107-1125. rbcL, matK, rpl20-rps18 rpl20-rps18 matK, rbcL,

1989. Mediterraneanpinesandtheir (Scrophulariaceae) usinghypervariable Li W. H. 1967. Thegenus 1999. Phylogeneticrelationships Pinus, Pinaceae Pinus, 1979. Mathematicalmodelfor Pinus Pinus 1993. ChloroplastDNA- Pinus. Pinus. species ( 86 188 : 1742-1753. spacer, andtrnV : 197-211. Ronald Press ) basedon Pinaceae 162 76 : 5269- : 133- 1998. 16 83 ). :