G.2072Page1GlobalResearchjournalofNaturalScience&Technology(GRJNST)Volume:04-Issue2(2026),2072ISSNP:2790-7643ISSNE:2790-7651www.grjnst.nethttps://doi.org/10.53762/grjnst.04.02.23GeneticDiversityAnalysisofIndigenousFloraUsingMolecularMarkersReceived:27March2025.Accepted:3April2026.Published:27April2026InstituteofplantSciencesUniversityofSindhJamshoroSohailRajajatoisohail.raja333@gmail.comSobiaSattarInstituteofplantSciencesUniversityofSindhJamshorosobiasattararain@gmail.comKosarparveenInstituteofplantSciencesUniversityofSindhJamshorokosarparveen422@yahoo.comImranAliBhurtInstituteofplantSciencesUniversityofSindhJamshoroemranali007007@gmail.comSadiaTalpurInstituteofplantSciencesUniversityofSindhsadiatalpur.ss@gmail.comGRJNST,Volume:04-Issue2(2026)/ISSNP:2790-7643ArticleID:2072https://doi.org/10.53762/grjnst.04.02.23Copyright©2026GRJNST.ThisarticleispublishedunderanOpenAccessmodel.ItismadeavailabletothepublicunderthetermsoftheCreativeCommonsAttribution4.0International(CCBY4.0)license,whichpermitsunrestricteduseanddistributionG.2072Page2Abstract:Theadaptability,survivalandsustainableuseofindigenousplantsdependuponitsgeneticrichness.ThisresearchfocusedonthediverseneedfortheassessmentofgeneticdiversityoflocalfloraofSindhthroughtheaidofmoleculartechnique.Thegeneticstructureandconservationofvariousindigenousplantspeciesfromdifferentecologicalzoneswerestudied.Thisstudyexploresgeneticdiversitypatternsinferredfrommolecularmarkertechniquestoilluminatepopulationdifferentiationandecologicaladaptation.Italsodrawsattentiontotheneedforincorporatingnovelgenetictoolsforbiodiversityassessmentinlessdevelopedareas.TheobjectiveofthisstudywastoevaluateandcomparethegeneticdiversityofindigenousplantpopulationsfromvariousecologicalzonesofSindhusingRAPDandSSRmarkers.Thesamplesofleavesweretakenfromdifferentecozonescoastal,desertandirrigatedzoneofSindh.GenomicDNAwasextractedusingtheCTAB-modifiedmethodandPCRamplificationwascarriedoutusingRAPDandSSRprimers.Agarosegelelectrophoresiswasperformedonamplifiedproductsandbinary-codedgeneticdatawerescoredforstatisticaltheuseofPOPGENE,NTSYS,andPASTsoftware.Highanalysiswithpolymorphism(78.17%RAPD;81.25%SSR)indicatedlargegeneticdiversityamongpopulationsofthetwospecies,asshownfromtheresults.ThehighestgeneticdiversitywasnotedindesertregionssuchasTharparkar,andcomparativelylowerwasobservedinirrigatedregions.ClusterandPCoAanalysesfurtherindicatedthatpopulationsclusteredaccordingtoecologicalaffinities.TheauthorsconcludethatthegeneticdiversitywithinspeciesofthenativefloraofSindhisconsiderablebutnotevenlydistributed.Itsuggeststheprioritizationofgeneticallyrichpopulationsforin-situconservationandadvisesembeddingmoleculardataongeneticdiversityintonationalbiodiversitymanagementprogramsforeffectiveconservationplanning.Keywords:Plants,Genetic,Molecular,Flora,EcozonesIntroductionResearchonplantgeneticdiversityhasgainedrenewedattentionforitspotentialtoprovideinsightintoevolutionaryhistory,ecologicaladaptationandconservationofbiologicalresourcesovertime.Indigenousplantsformanimportantgeneticresourceforecosystemfunctioningaswellasasourceofrawmaterialforagriculture,medicineandindustrialpurposes(Zhaoetal.,2023).Theuseofmolecularmarkersisamethodthathasrevolutionizedgeneticvariationassessment,quantifyinglevelsattheDNAlevelwithhighaccuracyandprecision.MolecularapproachesarelessenvironmentalcontextdependentandGRJNST,Volume:04-Issue2(2026)/ISSNP:2790-7643ArticleID:2072https://doi.org/10.53762/grjnst.04.02.23G.2072Page3yieldmorereproducibledatasetsthanclassicalmorphologicalandbiochemicalmethods(Nybom,2004).Together,theseadvanceshaveallowedresearcherstoundertakemoreaccurategeneticstructure,populationdynamicsandphylogeneticinvestigations.Withglobalanthropogenicdeclineofbiodiversity,measuringthegeneticcompositionofaplantspeciesondemandhasneverbeenmoreneeded.Geneticdiversityissignificantforitsdirectpurpose.Thishasimplicationsforconservationbiology,andsustainabledevelopment(Borahetal.,2021).GradientanalysisofgeneticSo,variabilitywithinandbetweenplantpopulationsisalsousefultoidentifyuniquegenotypeswhichareunderthreatandneedimmediateprotection.Ithelpstodevisenewstrategiesforconservationtoo:eachliferequiresbothinsituandexsituconservation(X.Zhangetal.,2024).Atalargerscale,itisallaboutgeneticdiversitycriticalintheenhancedcapacityofplantstorespondtoevolvingenvironmentalchanges,climateincluded.change,pestsanddiseases.Plantbreedingprogramsalsousemolecularmarkertechniques.toidentifythosecropsthathavethepotentialtoberesilientfornegativebioticorabioticconditions(Mondinietal.,2009).Asthattheuseofmoleculartoolstostudybiodiversitygivesnotonlytheoreticalknowledge,butalsothepotentialtoapplythatknowledgeinagricultureandenvironmentalmanagement.Pakistanhasbeenbestowedwithdiverseecologicalzonesfromcoastaltohillyareasofavarietyofnaturalnativeplantspecies.Assuch,thisdiversityisanirreplaceablenationalblessingthathasecological,economicandculturalvalue.Highgrowingpopulation&urbanization,deforestation&climatevariability.Theexpansionofhabitatfragmentationandtheunsustainableuseofspecieshaveincreasedmanyplantspeciesatriskofgeneticerosion.Though,studiesongeneticdiversityusingextensivemoleculartechniquesarenon-existentinPakistan.Aretrospectivehassignaledforthefuture–onthekindsofconversationalstrategieslargelyclassicalwithmostfailingincorporateconsiderthatgeneticdiversity-isandtheexistinplantpopulations.Themagnitudeoftheseshortcomingsunderscoresthecompellingneedfornewmoleculartoolstoadvanceconservationofnativeplantsacrossthenation.Althoughmolecularmarkertechnologieshavebeenusedworldwide,fewstudiesconcerningthegeneticsofnativefloraareavailable,especiallyindevelopingcountrieslikePakistan.ItrestrictsthecapacityofGRJNST,Volume:04-Issue2(2026)/ISSNP:2790-7643ArticleID:2072https://doi.org/10.53762/grjnst.04.02.23G.2072Page4formulatingeffectiveconservationandmanagementmeasuresdrivenbyspecificempiricaldata.Thisstudyaimedtoaddressthelackofadoptionofmolecularmarkertechniquesinstudyinggeneticdiversityforindigenousplantspeciesduetolimitedunderstandingneededforconservationplanning.Developanaccurategenotypingdatasetforregionalflorausingmodernmolecularmarkersystems.Suchanambitionistodevelopageneticarchivethatwillinformconservationandbreedinginitiatives.Besides,thenationalbiodiversitypoliciesmustincorporatetheseresearchresultstoguaranteesustainableuseandconservationofplantgeneticresources.LiteratureReviewGeneticdiversityisfundamentaltotheabilityofnativeplantspeciestoadapttoenvironmentalchangeandsurviveextinction.Thismoleculardiversitycanbeaccuratelymeasuredbymolecularmarkers,anditwouldaidtheconservationintreesandsustainableuseoflocalplants(Hasnain&Mehvish,2020).TheliteraturedisplaysaquickevolutionfrombasicPCR-basedmarkerstogenome-greatmethodswithseveracasestudiesinwildandendangeredplants.ThepartofGeneticDiversityinNativeandEndangeredPlantsItalsounderpinstheconservationofwildandendangeredspeciesbecausegeneticvariationdeterminesplants'abilitytorespondtoclimate,pests,andhabitatchange.Remarkably,taxathatarethreatenedorendemicoftendisplayhighlevelsofwithin-populationdiversitybutdifferentdegreesofpopulationdifferentiationwhichreflectgeographicstructure,geneflowandorexploitationMarker-baseddataonpatternsofdiversityhavebecomingacommoncomponentinprioritysettingforinsituandexsituconservationandbreeding.DifferentTypesofMarkerSystemsandWhatDefinesThemS.No.MarkerType/UseFeaturesandApplicationsRelatedStudiesSSR/microsatellitesCo‑dominant,highly(Mondinietal.,polymorphic;preferred2009;Nametal.,forpopulationstructure,2021;Rai,2023)corecollections,cultivarGRJNST,Volume:04-Issue2(2026)/ISSNP:2790-7643ArticleID:2072https://doi.org/10.53762/grjnst.04.02.23G.2072IDPage5RAPD,ISSR,AFLP,SRAP,SPARDominant,lowpriorinfo;(Goswamietal.,rapidscreeningof2021;Nametal.,wild/endemicspecies,2021;Szaboetoftencombinedforal.,2021)robustnessGene‑targeted(SCoT,CBDP,iPBS,Focusonfunctionalor(Hasnain&R‑ISSR)retrotransposonregions;Mehvish,2020;usefulforfinestructurePorth&El-andtrait‑linkedvariationKassaby,2014;Zenetal.,2004;Y.Zhang&Maginn,2014)NGS/SNP&RAD‑seqderivedHigh‑throughput,(Courregelonguegenome‑wide;usedto&Pons,2024;developdenseSSR/SNPHaque,2025;panelsinnon‑model,rareNametal.,2021;speciesZhaoetal.,2023)GeneticDiversityinIndigenousPlantPopulations:PatternsInfact,mostvariation(65–90%)isfoundwithinpopulationsformanywildandindigenousspecies.Moderatetohighlevelsofamong-populationdifferentiation(e.g.Gst/Fst≈0·16–0·40)aretypicallyassociatedwithgeographicisolation,habitatfragmentationandgeneflowrestriction(Haque,2025;Nametal.,2021).Conservationbasedonhabitat-selectionisurgentlyneededratherthanthepresumptionofseveregeneticerosionforsomeendangeredtaxawhichexhibitunexpectedlyhighdiversityandrelativelylargegeneflow.GRJNST,Volume:04-Issue2(2026)/ISSNP:2790-7643ArticleID:2072https://doi.org/10.53762/grjnst.04.02.23G.2072Page6MethodologicalConsiderationsandTrendsComparativestudiesshowthatdominantmarkers(RAPD,AFLP,ISSR)providebroadlysimilarestimatesbutlowerlevelsofdiversitycomparedwithSSRs,whichalsoindicatehigherwithin-populationheterozygosityandfinerstructure.Clusterresolutionanddiscriminatinguniquebreeding/restorationgenotypescanbeachievedbyusingmultiplemarkersystemsincombinationorintegratingmorphology/phytochemistrywithDNAdata(Huetal.,2024;Pandeyetal.,2023).MaterialsandMethodsGeneticDiversityamongselectedindigenousplantspeciescollectedfrommainecologicalzonesofSindh,Pakistan.SamplesiteswerelikeCoastalbeltsofThatta,Badin,desertareasofTharparkarandUmerkot,docileirrigatedplainsofHyderabadandSukkur.Theseregionsshowecologicalvariationacrosstheprovince.Itwascollectedduringperiodsofactivevegetativegrowthbywayoffieldsurveys,ensuringthatampleviablebiologicalmaterialwasavailable.Toaccountforintra-populationvariability,healthyyoungleaftissueswerecollectedfromseveralindividualsofeachspecies.EverysamplewasassignedtoitsownIDcode.HandheldGPS(GlobalPositioningSystem)recordsgeographiccoordinates.Werecordedhabitatvariablesincludingsoiltype,vegetationtype,andmoisturelevel.SampleswereinitiallypreservedinsilicagelforDNAintegrityduringtransport.TheexperimentalworkwasdoneinmolecularlaboratoriesatUniversityofSindh,andSindhAgricultureUniversity.LaboratorySetupCentrifugeMicropipettesetVortexmixerWaterbathLaminarflowcabinetThermalcyclerElectrophoresisunitUVtransilluminatorGeldocumentationsystemThegenomicDNAwasextractedfollowingamodifiedCTABprotocolappropriateforthepresenceofsecondarymetabolitesinplanttissues.Usingliquidnitrogen,theleafsamplesweregroundtoafinepowder.CelllysiswithCTABextractionbuffer.PurificationofDNAwasbychloroform–isoamylalcohol.ColdisopropanolwasusedforDNAprecipitation.Pelletswerewashedwithethanol,driedandresuspendedinTEbuffer.DNAqualitywasdeterminedandvisualizedon1%agarosegel[8].MeasurementofDNAconcentrationwasdoneusingUV–Visiblespectrophotometerbymeansof260/280nmratio.GRJNST,Volume:04-Issue2(2026)/ISSNP:2790-7643ArticleID:2072https://doi.org/10.53762/grjnst.04.02.23G.2072Page7SelectedfordownstreamprocessingweresamplesstillshowinghighmolecularweightDNAintactintegrityfollowinglysis.MolecularanalysisbasedonRandomAmplifiedPolymorphicDNAmarkersSimpleSequenceRepeatmarkers.ReactionmixturecontainstemplateDNA,primermix,dNTPs,MgCl₂),buffersolutionandTaqDNApolymerase.PCRamplificationwasperformedinthermalcyclerTheamplificationprogramwasasfollows:aninitialdenaturationstepat95degreesCfor5min,followedbycyclicdenaturation,primerannealingandstrandextension(25cyclesof30sat95degreesC,30satthedeterminedTmminus^C,and90sat72degreesC),togetherwithafinalextensionphaseof72°Cforanother^starttime>min.TAEBufferSystemAgaroseGelTransfectionAmplifiedfragmentsofDNADNAbandingwasdonewithsafenucleicaciddye.ThevisualizationwascarriedoutusingaUVtransilluminator.Thegelimageswerecapturedusingageldocumentationsystem.Forbandscoringbinaryapproachappliedas1forpresence,0forabsence.Genotypecallingwasperformedbasedonabinarydatamatrix.StatisticalanalyseswereperformedusingPOPGENE,version1.32.Geneticparameterswereestimatedaspercentagepolymorphism(PP),geneticdiversity(He:Nei’sgenediversity)andShannon’sinformationindex.Computinggeneticsimilaritymatrix,NTSYSversion2.1ClusteranalysisusedUnweightedPairGroupMethodwithArithmeticMeanalgorithmfollowed.Geneticrelationshipsamongpopulationsareshownbydendrogramconstruction.PASTversion4.03forPrincipalCoordinateAnalysiswasusedformultivariateanalysis.Geneticdispersionpatternswererepresentedbygraphicalplots.Allprocedureswereperformedaccordingtostandardmolecularbiologyprotocols.Experimentsweredesignedtomatchlaboratories’facilitiesavailableindifferentlocationsofSindhregion.Reproducibility,reliability,regionalapplicabilityforgeneticdiversityassessmentofindigenousflorawasensuredusingMethod.ResultsandDiscussionThe3selectedindigenousplantspeciesfromSindhweresubjectedtogeneticanalysis,whichshowedasignificantlevelofpolymorphisminmostofthepopulationssampled.Scorablebandswith20RAPDprimersaddeduptoatotalof142all,including111polymorphicband,whichgivetheaverageofpolymorphismequalto(78.17%).Asreported(Pandeyetal.,2023;Porth&El-Kassaby,2014),theSSRanalysiswitheightprimerpairsgeneratedatotalof64GRJNST,Volume:04-Issue2(2026)/ISSNP:2790-7643ArticleID:2072https://doi.org/10.53762/grjnst.04.02.23G.2072alleleswithanaverageof5.33allelesperlocus.Thecomprehensivemarkerperformanceissummarizedin(Table1),exhibitingdifferencesinallelenumberandpolymorphismamongtheloci.Thehighdegreeofpolymorphismreflectsthefairamountofgeneticvariationbetweenthepopulationsstudied,particularlythosefromenvironmentallychallengedregionslikeTharparkar.Page8Table1.SummaryofMolecularMarkerAnalysisMarkerTypeNo.ofPrimersTotalBands/AllelesPolymorphicBands%PolymorphismMeanAllelesperLocusRAPDSSR2012142641115278.17%81.25%—5.33Table2indicateslargedifferencesingeneticdiversityindicesacrosssamplingregions.MaximumNei’sgenediversity(H=0.36)wasrecordedinTharparkarpopulations,whilethelowestvalue(H=0.21)wasinvestigatedinHyderabad.Shannon'sinformationindexvariedbetween0.32to0.51,givingamoderatetohighlevelofgeneticvariation.Increaseddiversityindesertpopulationsmayrelatetoevolutionaryorecologicaladaptivenesstoharshenvironments,whilereducedbiodiversityinirrigatedsettingsmayindicateanthropogenicstressorhabitathomogeneity.Table2.RegionsofSindhGeneticDiversityParametersRegion%PolymorphismNei’sGeneDiversity(H)ShannonIndex(I)Tharparkar82.4%Umerkot79.2%0.360.330.510.47GRJNST,Volume:04-Issue2(2026)/ISSNP:2790-7643ArticleID:2072https://doi.org/10.53762/grjnst.04.02.23G.2072Region%PolymorphismNei’sGeneDiversity(H)ShannonIndex(I)Page9Thatta76.5%Badin74.8%Sukkur72.6%Hyderabad69.3%0.300.280.250.210.440.410.360.32Geneticsimilaritycoefficientswerebetween0.62and0.89,confirmingdivergenceatthesametimeasrelatednessofpopulations.UPGMAbasedclusteranalysisdrewthreedistinctclustersaccordingtoecologicalzonesinwhichthepopulationswerefound(Borahetal.,2021;Goswamietal.,2021).OneclusterwascomposedofcoastalpopulationsfromThattaandBadin,indicatingadaptationtosalineenvironments.IndividualsfromaridregionsofTharparkarandUmerkotclusteredwithoneanotherindicatinggeneticsimilarityassociatedwithdryconditions.IrrigatedpopulationsfromHyderabadandSukkurclusteredseparately,indicatingdecreasedvariabilityduetolessvariabilityassociatedwithenvironmentofuniformagriculturalenvironments.Thefirstthreeaxesofprincipalcoordinateanalysis(PCoA)explained67.4%oftotalgeneticvariation.Populationswereclearlyseparatedaccordingtotheirecologicalzonesintermsofspatialdistribution.Hyderabadpopulationsexhibitedmorecompacthyperecologicalclusters,indicatingPLINinsteadofTharparkarpopulationswhichrevealedbroaderdispersionthusrepresentinghigherintra-populationdiversitywhichisresponsibleforgeneticuniformity(Bidyanandaetal.,2024;Mondinietal.,2009;Szaboetal.,2021).ThesefindingsagreewiththeresultsofTable2,whichshowedanoveralllowestvalueofdiversityindicesforirrigatedregions.ThegeneticvariationobservedcouldbesignificantforconservationplanningofSindh.Higherdiversitypopulations,particularlyfromdesertenvironments,shouldbeprioritisedforin-situconservationbecauseoftheiradaptivepotential.Ontheotherhand,lowerdiversitypopulationsmightneedtobemaintainedexsitutoavoidfurthergeneticdepletion(Haque,2025;Hasnain&Mehvish,2020;Nametal.,2021).Theregionaldifferencesindiversitypatternshighlighttheimpactofenvironmentalstress,habitatGRJNST,Volume:04-Issue2(2026)/ISSNP:2790-7643ArticleID:2072https://doi.org/10.53762/grjnst.04.02.23G.2072fragmentationandhumanprocessesongeneticstructure.EvaluationRemarks:ThefindingsindicatethatRAPDandSSRmarkershavegoodpotentialforestimatinggeneticdiversityunderlocallabconditions.OurresultsnotonlyhighlightgeneticstructurevariationatthespecieslevelofindigenousflorainSindhbutalsoendorseincorporationofmoleculardatatoPage10informbiodiversityconservationstrategies.ConclusionandFutureRecommendationsThehighamountofgeneticdiversityfoundinnativeplantspeciesofSindhasshownbyourstudyseemstobeduetoobviouslythevariationconsideringfromoneecologicalzonetoanother.ThemolecularmarkersanalysisperformedusingRAPDandSSRtechniquesrevealedaveryhighlevelofpolymorphism,whichindicatesthatthesemethodsarehighlyusefulfortheassessmentofgeneticdiversityunderlocallaboratoryconditions.DetectionofallelicdiversityconfirmingpopulationstructureismoreaccuratelydefinedusingSSRmarkersthathavehigherresolution.TheregionalhistoricalanalysisshowsthatTharparkarandUmerkotdesertecosystemshavegreatergeneticvariability,whereasirrigatedareaslikeHyderabadandSukkurarefoundtobegeneticallylessvariable.Thegeneticstructureofcoastalbrowntroutwasinfluencedbyenvironmentalstress,habitatheterogeneityandhumanactivitieswhichshapedthispattern.Theycollectivelyprovidefurtherevidenceofgeographicandenvironmentalcontrolongeneticdifferentiationbetweenpopulations(asisevidencedbytheirecologicalgroupingshownintheclusterandPCoAanalyses).Futurestudiesshouldsamplemorebroadlytoincludeagreaterdiversityofspeciesandunder-representedhabitatswhichwouldallowforafullergeneticinventoryofnativeflora.Novelgenomictechnologies(SNPgenotyping,RAD-seqandwhole-genomesequencing)shouldallowsignificantimprovementinresolutionwithrespecttoadaptivevariationandevolution.AlthoughmolecularresearchofseveralspeciesisextensiveinPakistan,collaborationbetweenresearchinstitutions,forestrydepartmentsandpolicy-makingbodiesshouldbedesignedtoensurethatsuchgenomicinformationisconvenientlytransferredforimprovedimplementationintothesustainableconservationstrategiesacrossdifferentregionsinPakistan.GRJNST,Volume:04-Issue2(2026)/ISSNP:2790-7643ArticleID:2072https://doi.org/10.53762/grjnst.04.02.23G.2072ReferencesPage11Bidyananda,N.,Jamir,I.,Nowakowska,K.,Varte,V.,Vendrame,W.,Devi,R.S.,&Nongdam,P.(2024).PlantGeneticDiversityStudies:InsightsfromDNAMarkerAnalyses.InternationalJournalofPlantBiology.https://doi.org/10.3390/ijpb15030046Borah,R.,Bhattacharjee,A.,Rao,S.,Kumar,V.,Sharma,P.,Upadhaya,K.,&Choudhury,H.(2021).GeneticdiversityandpopulationstructureassessmentusingmolecularmarkersandSPARapproachinIlliciumgriffithii,amedicinallyimportantendangeredspeciesofNortheastIndia.JournalofGeneticEngineering&Biotechnology,19.https://doi.org/10.1186/s43141-021-00211-5Courregelongue,M.,&Pons,A.(2024).Thearomaoftoastedoakwood(Quercuspetraea):fromsensoryanalysistomolecularcharacterisation.OENOOne,58(4).Goswami,B.,Gadi,B.,&Rao,S.(2021).MorphologicalandmolecularmarkersbasedassessmentofgeneticdiversityofavaluableendemicplantLasiurussindicusHenr.inthearidregionofRajasthan,India.AridLandResearchandManagement,36,298–313.https://doi.org/10.1080/15324982.2021.2012728Haque,I.(2025).AdvancementsinDNA-basedmolecularmarkerstounravelthegeneticdiversityofendangeredplants.CurrentBotany.https://doi.org/10.25081/cb.2025.v16.9010Hasnain,H.,&Mehvish,N.(2020).Assessmentofplantgeneticvariationsusingmolecularmarkers:Areview.https://doi.org/10.7324/jabb.2020.80514Hu,L.,Wang,J.,Wang,X.,Zhang,D.,Sun,Y.,Lu,T.,&Shi,W.(2024).DevelopmentofSSRMarkersandEvaluationofGeneticDiversityofEndangeredPlantSaussureainvolucrata.Biomolecules,14.https://doi.org/10.3390/biom14081010Mondini,L.,Noorani,A.,&Pagnotta,M.(2009).AssessingPlantGeneticDiversitybyMolecularTools.Diversity,1,19–35.https://doi.org/10.3390/d1010019Nam,V.T.,Hằng,P.L.B.,Linh,N.N.,Ly,L.H.,Hue,H.,Ha,N.H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