G.2050Page1GlobalResearchjournalofNaturalScience&Technology(GRJNST)Volume:04-Issue2(2026),2050ISSNP:2790-7643ISSNE:2790-7651www.grjnst.nethttps://doi.org/10.53762/grjnst.04.02.02Cross-SpeciesPCRAmplificationPerformanceofWheatStripeRustResistanceGene-SpecificPrimersinCommonBean(PhaseolusVulgarisL.)Received:31December2025.Accepted:30January2025.Published:31March2026MuhammadMehrajRiasat(CorrespondingAuthor)DepartmentofAgriculture,HazaraUniversity,Mansehra21300,PakistanEmail:mehrajhum30278@gmail.comMuhammadWaleedDepartmentofAgriculture,HazaraUniversity,Mansehra21300,PakistanUzairAhmadDepartmentofAgriculture,HazaraUniversity,Mansehra21300,PakistanFarhanTahirDepartmentofAgriculture,HazaraUniversity,Mansehra21300,PakistanShafaqMunirDepartmentofAgriculture,HazaraUniversity,Mansehra21300,PakistanAtikaLiaqatDepartmentofAgriculture,HazaraUniversity,Mansehra21300,PakistanSaqibWaqarAhmedDepartmentofAgriculture,HazaraUniversity,Mansehra21300,PakistaGRJNST,Volume:04-Issue2(2026)/ISSNP:2790-7643ArticleID:2050https://doi.org/10.53762/grjnst.04.02.02Copyright©2026GRJNST.ThisarticleispublishedunderanOpenAccessmodel.ItismadeavailabletothepublicunderthetermsoftheCreativeCommonsAttribution4.0International(CCBY4.0)license,whichpermitsunrestricteduseanddistributionG.2050Page2Abstract:PCR-basedgene-specificprimersareessentialincropgenetics,buttheircross-genomeeffectivenessisunclear.Thisstudytestedwhetherwheat(TriticumaestivumL.)striperustresistancemarkersYr15andYr30canbeamplifiedincommonbean(PhaseolusvulgarisL.).GenomicDNAfrom30beangenotypeswasamplifiedbyPCR,withwheatDNAandnuclease-freewaterservingaspositiveandnegativecontrols.ThePCRprotocolusedaninitialdenaturationat94°Cfor5minutes,40cycles(94°Cfor60seconds,55.8°CforYr15or52.7°CforYr30for60seconds,72°Cfor45seconds),andafinalextensionat72°Cfor10minutes.PCRproductswereseparatedviaagarosegelandvisualized.Yr15wasnotdetectedinanybeangenotype,whileYr30showedtheexpected120bpbandinsomegenotypeswithadditionalnon-specificbands.Thesefindingshighlightthatgene-specificprimersmayfailoryieldambiguousresultsinunrelatedspecies,emphasizingtheneedforrigorousmarkervalidationacrossspecies.Keywords:PCR,Gene,Yr15,Yr30,Amplification,TriticumaestivumL.,PhaseolusVulgarisL.IntroductionPolymerasechainreaction(PCR)isacommonlabmethodincropbreedingandgeneticsthathelpsfind,copy,andstudyspecificpiecesofDNA(Mullis&Faloona,1987;Saikietal.,1988).PCRmethodsareimportantforselectingplantswithdesiredtraits,figuringoutwheregenesare,andstudyingusefultraitsincrops(Collard&Mackill,2008;Varshneyetal.,2005).ThesuccessofPCRdependsmostlyonhowwellthestartingpiecesofDNA,calledprimers,matchtheDNA,howtheyattach,andtheconditionsused,sinceprimersdecidewhichpartsofDNAarecopiedinplants(Dieffenbachetal.,1993;Kwoketal.,1990;Thornton&Basu,2011).Gene-specificprimersamplifyuniqueDNAregionslinkedtodistinctgenesorloci.Theseprimersareusuallyhighlyspecificwithintheirtargetspecies(Dieffenbachetal.,1993;Thornton&Basu,2011).However,sequencesimilarity,conservedmotifs,primer–templatemismatches,andexperimentalconditionscandecreasespecificity.Theseissuesmaycausenon-specificorcross-speciesamplification(Kwoketal.,1990;Lefeveretal.,2013).Undetectedunintendedamplificationcanleadtoinaccurateorincorrectexperimentalinterpretations,especiallywhenprimerperformanceisnotthoroughlyassessedorwhenprimersareusedoutsidetheiroriginalgeneticcontext(Thornton&Basu,2011;Bustinetal.,2009).Striperust(yellowrust),causedbyPucciniastriiformisf.sp.tritici,isamongthemostdestructivefungaldiseasesaffectingwheat(TriticumaestivumL.)globally(Chen,2005;Wellings,2011).Toaddressthischallenge,numerousstriperustresistance(Yr)geneshavebeenidentifiedandincorporatedintobreedingprograms(McIntoshetal.,2010).Amongthese,Yr15confersbroad-spectrumresistance,whileYr30providesadultplantresistance.Accordingly,gene-specificorcloselylinkedprimersfortheselociarewidelyutilizedformolecularscreeningandgermplasmevaluationinwheat(Klymiuketal.,2018;Kokhmetovaetal.,2021).GRJNST,Volume:04-Issue2(2026)/ISSNP:2790-7643ArticleID:2050https://doi.org/10.53762/grjnst.04.02.02G.2050Page3PrimersdesignedforwheatYrgenesaregenerallyvalidatedexclusivelywithinwheatgeneticbackgrounds,andthereislimitedinformationregardingtheiramplificationperformanceinnon-targetplantspecies.Therefore,assessingprimerspecificitybeyondthetargetgenomeiscrucialforidentifyingthelimitationsofPCR-basedassaysandforpreventingmisinterpretationduetonon-targetamplification(Bustinetal.,2009;Lefeveretal.,2013).TheobjectiveofthisstudywastoevaluatethePCRamplificationbehaviorofwheatstriperustresistancegene-specificprimers,Yr15andYr30,bothrecognizedfortheirrolesinconferringresistancetowheatstriperustwhenappliedtocommonbean(PhaseolusvulgarisL.)genomicDNA.Toclarifythescope,thisresearchfocusessolelyonamplificationpatternsandprimerspecificity,anddoesnotaddressgenefunction,phenotypictraits,ordiseaseresistance.MaterialandMethodsThirty(30)commonbeangenotypeswerecollectedfromdiverseagroecologicallocationsintheMansehraregionofPakistan.GenomicDNAfrom30beangenotypeswasamplifiedbyPCR,withwheatDNAandnuclease-freewaterservingaspositiveandnegativecontrols,respectively.Astandardbuffer-basedprotocolwasfollowed,includingpurificationandprecipitation.DNAintegrityandqualitywereverifiedbyagarosegelelectrophoresispriortoPCRamplification.Gene-specificprimerslinkedtowheatstriperustresistancegenes,Yr15andYr30,wereusedtoexaminecross-speciesamplificationbehavior.PCRreactionswereperformedina10µLvolumeusingacommercialmastermix,genomicDNAtemplate,andprimerpairs.Amplificationwasconductedunderoptimizedcyclingconditions.Theseincludeddenaturationat94°Cfor5minutes;40cyclesof94°Cfor60seconds;annealingat55.8°CforYr15and52.7°CforYr30for60seconds;72°Cfor45seconds;andfinalextensionat72°Cfor10minutes.PCRproductswereresolvedon1.5%agarosegelsalongsideaDNAladderforfragmentsizeestimation.Gelelectrophoresiswasperformedunderstandardconditions.Bandingpatternswerevisualizedunderultravioletilluminationanddocumentedusingagelimagingsystem.Interpretationwasbasedondirectvisualizationofamplificationprofilesinthegelimages.ForYr15,theexpecteddiagnosticfragmentsizewas390bp.ForYr30,theexpectedfragmentsizewas120bp.Onlybandscorrespondingtotheseexpectedsizeswereconsideredrelevantforinterpretation.Non-specificfragments,includinglowermolecularweightbandsandprimerdimers,weredisregarded.Theresultsarepresentedasgelimages,whichallowclearvisualassessmentofamplificationpatternsacrossgenotypes.ResultsandDiscussionYr15amplificationprofilePCRamplificationwiththeYr15gene-specificprimerfailedtogeneratetheexpected390bpdiagnosticfragmentinanyofthetestedcommonbeangenotypes(Figure1).Nobandscorrespondingtothetargetsizeweredetectedinanysample.ThislackofamplificationalignswiththeestablishedspeciesspecificityofYr15,whichencodesthetandemkinaseproteinWTK1andhasbeenfunctionallycharacterizedinwheatandcloselyrelatedspecies(Klymiuketal.,2018).Theabsenceofcross-amplificationincommonbean,aphylogeneticallydistantlegume,likelyresultsfromthelackofGRJNST,Volume:04-Issue2(2026)/ISSNP:2790-7643ArticleID:2050https://doi.org/10.53762/grjnst.04.02.02G.2050conservedprimer-bindingregionsnecessaryforspecificamplification.Comparablefindingshavebeenreportedforwheatrustresistancemarkers,whichgenerallydonotamplifyinnon-cerealspeciesduetogenomicdivergence(Lagudahetal.,2009;McIntoshetal.,2010).Theconsistentabsenceofthe390bpfragment,therefore,constitutesabiologicallymeaningfulresult,underscoringthehighspecificityoftheYr15markerwithinitsnativegenomiccontext.Page4Figure1.PCRamplificationprofileofYr15incommonbeangenotypes.Yr30amplificationprofilePCRamplificationwiththeYr30gene-specificprimerproducedheterogeneousbandingpatternsamongtheanalysedcommonbeangenotypes(Figure2).Somesampleshadfragmentsclosetotheexpectedsizeof120bp,whileothersshowedbandswithslightlylowermolecularweight(about90–110bp)ornoamplificationatall.Toensureconsistency,onlyclearlyresolvedbandsnear120bpwereinterpreted,withlowermolecularweightfragmentsexcludedfromanalysis.Thisobservedvariabilityinamplificationprofileslikelyresultsfrompartialornon-specificprimerbinding,aphenomenoncommonwhengene-specificmarkersareusedoutsidetheirnativegenomiccontext.Suchsizedifferenceshavebeenwelldocumentedandareattributedtosequencedivergenceandprimertemplatemismatchesinnon-targetspecies(Kwoketal.,1990;Dieffenbachetal.,1993;Smithetal.,2007).Importantly,thepresenceof120bpfragmentsinsomegenotypesdoesnotconfirmthattheYr30resistancegeneispresent,butratherreflectsamplificationbehaviorundercross-speciesconditions.TheabsenceofamplificationforYr15contrastswiththeseresults,furtherhighlightingdifferencesinprimerspecificityandbindingefficiency.GRJNST,Volume:04-Issue2(2026)/ISSNP:2790-7643ArticleID:2050https://doi.org/10.53762/grjnst.04.02.02G.2050Page5Figure2.PCRamplificationprofileofYr30incommonbeangenotypes.ConclusionThisstudyshowsthatwheatgene-specificprimersvaryinamplificationwithnon-targetspecies.TheYr15primerfailedtoamplifytarget-sizefragmentsinanycommonbeangenotype,whiletheYr30primershowedvariableresults,sometimesproducingexpected-sizefragments.Theseresultssuggestprimerspecificity,notbiologicalresistance,causedthedifferences.Thishighlightsthelimitationofusingcrop-specificmolecularmarkersindifferentplantgenomesandstressestheneedforcarefulvalidationbeforeapplyingPCR-basedprimersinbroadermolecularstudies.AuthorContributionMuhammadMehrajRiasat:Supervisetheresearch,MuhammadWaleedandUzairAhmad:Labwork,FarhanTahir:laboratoryassistance,ShafaqMunirandAtikaLiaqat:Writeoriginalmanuscript,SaqibWaqarAhmed:Samplecollection.Allauthorscontributedtothereviewandeditingofthemanuscript.DeclarationofFundingThisresearchwassupportedbytheDepartmentofAgriculture,HazaraUniversity,Mansehra,Pakistan.GRJNST,Volume:04-Issue2(2026)/ISSNP:2790-7643ArticleID:2050https://doi.org/10.53762/grjnst.04.02.02G.2050ReferencesPage6Bustin,S.A.,Benes,V.,Garson,J.A.,Hellemans,J.,Huggett,J.,Kubista,M.,&Wittwer,C.T.(2009).TheMIQEGuidelines:MinimumInformationforPublicationofQuantitativeReal-TimePCRExperiments.Collard,B.C.,&Mackill,D.J.(2008).Marker-assistedselection:anapproachforprec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