Gene silencing via DNA methylation in naturally occurring Tragopogon miscellus (Asteraceae) allopolyploids

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Gene silencing via DNA methylation in naturally occurring Tragopogon miscellus (Asteraceae) allopolyploids
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Sehrish, Tina
Symonds, V Vaughan
Soltis, Douglas E.
Soltis, Pamela S.
Tate, Jennifer A.
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Background: Hybridization coupled with whole-genome duplication (allopolyploidy) leads to a variety of genetic and epigenetic modifications in the resultant merged genomes. In particular, gene loss and gene silencing are commonly observed post-polyploidization. Here, we investigated DNA methylation as a potential mechanism for gene silencing in Tragopogon miscellus (Asteraceae), a recent and recurrently formed allopolyploid. This species, which also exhibits extensive gene loss, was formed from the diploids T. dubius and T. pratensis. Results: Comparative bisulfite sequencing revealed CG methylation of parental homeologs for three loci (S2, S18 and TDF-44) that were previously identified as silenced in T. miscellus individuals relative to the diploid progenitors. One other locus (S3) examined did not show methylation, indicating that other transcriptional and post-transcriptional mechanisms are likely responsible for silencing that homeologous locus. Conclusions: These results indicate that Tragopogon miscellus allopolyploids employ diverse mechanisms, including DNA methylation, to respond to the potential shock of genome merger and doubling. Keywords: Allopolyploidy, DNA methylation, Gene silencing, Tragopogon, Whole-genome duplication.
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Sehrish et al. BMC Genomics 2014, 15:701 http://www.biomedcentral.com/1471-2164/15/701; Pages 1-7
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doi:10.1186/1471-2164-15-701 Cite this article as: Sehrish et al.: Gene silencing via DNA methylation in naturally occurring Tragopogon miscellus (Asteraceae) allopolyploids. BMC Genomics 2014 15:701.

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© 2014 Sehrish et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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RESEARCHARTICLEOpenAccessGenesilencingviaDNAmethylationinnaturally occurring Tragopogonmiscellus (Asteraceae) allopolyploidsTinaSehrish1,VVaughanSymonds1,DouglasESoltis2,3,PamelaSSoltis3andJenniferATate1*AbstractBackground: Hybridizationcoupledwithwhole-genomeduplication(allopolyploidy)leadstoavarietyofgenetic andepigeneticmodificationsintheresultantmergedgenomes.Inparticular,genelossandgenesilencingare commonlyobservedpost-polyploidization.Here,weinvestigatedDNAmethylationasapotentialmechanismfor genesilencingin Tragopogonmiscellus (Asteraceae),arecentandrecurrentlyformedallopolyploid.Thisspecies, whichalsoexhibitsextensivegeneloss,wasformedfromthediploids T.dubius and T.pratensis Results: ComparativebisulfitesequencingrevealedCGmethylationofparentalhomeologsforthreeloci(S2,S18 andTDF-44)thatwerepreviouslyidentifiedassilencedin T.miscellus individualsrelativetothediploidprogenitors. Oneotherlocus(S3)examineddidnotshowmethylation,indicatingthatothertranscriptionalandpost-transcriptional mechanismsarelikelyresponsibleforsilencingthathomeologouslocus. Conclusions: Theseresultsindicatethat Tragopogonmiscellus allopolyploidsemploydiversemechanisms,including DNAmethylation,torespondtothepotentialshockofgenomemergeranddoubling. Keywords: Allopolyploidy,DNAmethylation,Genesilencing, Tragopogon ,Whole-genomeduplicationBackgroundWhole-genomeduplication(polyploidy)hasplayeda majorroleineukaryoticevolution[1-6].Inparticular, floweringplantshaveexperiencedrepeatedepisodesof polyploidysincetheysharedacommonancestorwith thegymnospermssome300millionyearsago[7,8].Understandingthegenomicconsequencesofpolyploidization,particularlywhenaccompaniedbyhybridization (allopolyploidy),allowsinsightintothepotentialforspeciationandadaptationofthesenovelentities[9,10].In particular,themergeranddoublingoftwodivergentgenomescaninducedifferentgeneticandepigenetic changesintheresultingpolyploid[11-16].Geneticmodificationscanincludegeneloss,genomedown-sizing,variablemutationratesoftheduplicatedgenes(homeologs), chromosomalrearrangementsandregulatoryincompatibilitiesresultingfrompost-transcriptionalmodifications inthemergedgenomes[16-24].Epigeneticmodifications involveheritablechangesingeneexpressionwithout changesinthenucleotidesequence[25-27]andcanincludehistonemodification,DNAmethylation,chromatin remodeling,ormicroRNAorprionactivity[28-30].DNA methylation,theadditionofamethylgroupatposition5 ofthepyrimidineringofcytosine,isacommonmechanismassociatedwithgenesilencinginpolyploids[31-33]. Ingeneral,cytosinemethylationisimportantformaintaininggenomicstabilityandisinvolvedingenomicimprinting,transposonsilencingandepigeneticregulationof genetranscription[30,34-36]. Here,weinvestigatedgenesilencingviamethylationin theallotetraploidplant Tragopogonmiscellus .Thisspeciesformedrepeatedlyduringtheearly1900sinthe westernUnitedStates,followingtheintroductionofthe diploidprogenitors, T.dubius and T.pratensis ,from Europe[37-40].Previousstudiesidentifiedextensive homeologloss[21,41-43]andchromosomalvariation [17]innaturallyoccurring T.miscellus populations.Two studies[42,43]alsoidentifiedhomeologousgenesilencinginsomeindividualsof T.miscellus ,butthemechanismforsilencingwasnotknown.InTateetal.[43], *Correspondence: j.tate@massey.ac.nz1InstituteofFundamentalSciences,MasseyUniversity,PalmerstonNorth, NewZealand Fulllistofauthorinformationisavailableattheendofthearticle 2014Sehrishetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative CommonsAttributionLicense(http://creativecommons.org/licenses/by/4.0),whichpermitsunrestricteduse,distribution,and reproductioninanymedium,providedtheoriginalworkisproperlycredited.TheCreativeCommonsPublicDomain Dedicationwaiver(http://creativecommons.org/publicdomain/zero/1.0/)appliestothedatamadeavailableinthisarticle, unlessotherwisestated.Sehrish etal.BMCGenomics 2014, 15 :701 http://www.biomedcentral.com/1471-2164/15/701

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the T.dubius copyofonelocus(TDF-44)wassilenced inmultipleindividualsfromPullman,Washington,and Moscow,Idaho.InBuggsetal.[42],sixlocishowedvariablesilencingof T.dubius or T.pratensis homeologsina fewindividualsfromfivedifferentpopulations(Oakesdale, Pullman,andSpangle,Washington;MoscowandGarfield, Idaho).Inthepresentstudy,weusedcomparativebisulfite sequencingtodetermineiftheselociweresilencedby methylation.ResultsanddiscussionCGmethylationregulatesduplicategeneexpressionGenomicandbisulfite-convertedsequenceswereacquiredforfourloci[TDF-44(putativeleucine-richrepeattransmembraneproteinkinase)[43],S2(putative RNAbindingprotein),S3(putativeNADP/FADoxidoreductase),andS18(putativeporphyrin-oxidoreductase) [42]]fromallopolyploid Tragopogonmiscellus andthe diploidparents T.dubius and T.pratensis (Table1).Afifth locus(S8,putativeacetyltransferase)identifiedassilenced inBuggsetal.[42]wasnotamenableforstudybecauseno singlenucleotidepolymorphisms(SNPs)betweenthediploidsweremaintainedfollowingbisulfiteconversionto distinguishtheparentalcopiesintheallopolyploid (Figure1a).Forthefourlociexamined,wetookadvantage ofSNPsbetweenthediploidstodetermineifaparental homeologwassilencedbymethylationinthe T.miscellus individuals.Inadditiontothepartialgenesequencesretrievedinthetwopreviousstudies[42,43],5 ’ genome walkingwasundertakentodeterminemethylationstatus ofthepromoterregions.ThenewsequencesweredepositedinGenBank(KM260156-KM260165). InspectionofthepromoterandcodingregionsidentifiedCGsites,whicharecommonmethylationsitesin plants[44,45].Theintegrityofbisulfiteconversionwas determinedfromtheconversionofalltheCsnotadjacent Table1ResultsofmethylationanalysisPopulationSpeciesLineageLocussilencedMethylated?aPullman T.dubius 2613-1NA T.dubius 2613-11NA T.miscellus 2605-4TDF44dYes-A/S T.miscellus 2605-7TDF44dYes-A/S T.miscellus 2605-13TDF44dYes-A/S S18dYes-A T.miscellus 2605-24TDF44dYes-A/S T.miscellus 2605-28TDF44dYes-A/S T.miscellus 2605-46TDF44dYes-A/S Moscow T.pratensis 2608-31NA T.pratensis 2608-35xNA T.miscellus 2604-4TDF44dYes-A/S T.miscellus 2604-11TDF44dYes-A/S T.miscellus 2604-15TDF44dYes-A/S T.miscellus 2604-22* T.pratensis genomiccopylost T.miscellus 2604-24TDF44dYes-A/S T.miscellus 2604-35TDF44dYes-A/S Spangle T.miscellus 2693-7S3pNo T.miscellus 2693-8S3pNo T.miscellus 2693-14S3pNo S18dNo Garfield T.dubius 2687-11NA T.pratensis 2689-17NA T.miscellus 2688-3S2dYes-A/S S3dNo S18dNo Oakesdale T.miscellus 2671-11S2dYes-A/SNA=Notapplicable;subscriptdorpindicatesthehomeologsilenced;aA=antisensestrand,S=sensestrand. Individualplantsusedinthestudyandtheirmethylationstatusforthegenesstudied;silencingdatafrom[ 42 43 ].Sehrish etal.BMCGenomics 2014, 15 :701 Page2of7 http://www.biomedcentral.com/1471-2164/15/701

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toaGintoTs.Thelocistudiedhereallshowedcomplete bisulfiteconversioninthegenicregions,whileincomplete conversionatafewsiteswasdetectedinthepromoterregionofTDF-44forthreepolyploidindividuals(2604-4, 2604-35and2605-14).Giventhatmostofthepromoter andgenicregionswereproperlyconverted,theincomplete conversionforTDF-44doesnotinfluencetheoverall interpretationoftheresults.Suchlowfrequencyof partialbisulfiteconversioniscommonlyduetoreaction temperature[46,47].Alternatively,thesesitescouldrepresentvaryinglevelsofCHH(H=A,C,orT)orCHG methylation[48]. CGmethylationofbothsenseandantisensestrands wasdetectedinthegenicandpromoterregionsofS2 (putativeRNAbindingprotein)andTDF-44(putative leucine-richrepeattransmembraneproteinkinase). TDF-44includedsevenCGsitesinthepromoterregion andfouringenicregions;the T.dubius homeologwas methylatedin11of12 T.miscellus individualsfrom PullmanandMoscow(Figure1b,Table1),whichreveals themechanismofsilencingobservedinTateetal.[43]. Theexceptionwasindividual2604-22,whichretained onlythe T.dubius genomichomeologandtherefore expressedthatcopy[43].Similarly,wefoundmethylation Figure1 Methylationofhomeologouslociin Tragopogonmiscellus. Sequencepolymorphismsbetweenthediploidparents( Tragopogon dubius and T.pratensis )wereusedtodeterminehomeolog-specificsilencingin T.miscellus allopolyploids. (a) Diagrammaticillustrationofthe expectedchromatogrampeaksforgenomicandbisulfite-convertedsequenceswhenun-methylatedormethylatedinallopolyploid T.miscellus Thisexampleshowssilencingofthe T.dubius homeolog. (b) ChromatogramsofTDF-44indicatingthepositionofamethylatedCGadjacenttoa polymorphicsite(redbox)in T.miscellus comparedtothediploids. (c) ChromatogramsofS18showinganun-methylatedCGsitein T.miscellus (blackbox)andthelocationofapolymorphicsitebetweenparentalcopies(redbox).Red,blue,greenandyellowcolorsofthechromatogram correspondtoA,C,TandG,respectively.IUPACambiguitycodes:W=A/T,Y=C/T,R=A/G.BS-converted=bisulfite-converted. Sehrish etal.BMCGenomics 2014, 15 :701 Page3of7 http://www.biomedcentral.com/1471-2164/15/701

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oftheS2locus,whichwasshownbyBuggsetal.[42]to besilencedintwo T.miscellus individuals(oneeachfrom GarfieldandOakesdale).AllCGsitesinthepromoter (four)andgenic(six)regionsofS2weremethylatedin bothindividuals.However,bothparentalhomeologs showedCGmethylationandsequencingthecloned bisulfite-convertedsequencesrevealedtwiceasmany methylated T.dubius clonedcopiesas T.pratensis copies. Thisresultsuggeststhatmethylationcanquantitatively regulatethelevelofexpressionofparentalcopiesrather thancompletelysilencingonehomeolog.ForlocusS18 (putativeporphyrin-oxidoreductase),whichhad11CG sitesinthepromoterand11inthegenicregions,theonly methylationdetectedwasoneindividualthatshowed hemimethylationoftheantisensestrand(Table1).Interestingly,thisindividualonlyshowedmethylationatfiveof the11CGsitesinthegenicregions. Analysisofthepromoterandgenicregionsoftheother locus(S3-putativeNADP/FADoxidoreductase)didnot showmethylationofanyoftheCGsites(Figure1c, Table1;S3includedsixCGsitesinthepromoterand threeCGsitesinthegenicregions).Thus,theremaybe mechanismsotherthanDNAmethylationthatareresponsibleforhomeolog-specificsilencing.Forexample, histonedeacetylation(causingchromatincondensation)is thoughttoberesponsiblefortranscriptionalrepression [49-51].RNAinterference(RNAi)isalsowidelyassociated withpost-transcriptionalsilencingviaanumberofdifferentmechanisms,includingmRNAdegradation,translationalinhibitionandtherepressionoftranscription elongation[52-55]. Naturalvariationinepigeneticpatterningisnotwell understood,butcanbeanimportantdriverofecological speciation,ashasbeenfoundin Viola [56]and Dactylorhiza [57,58].Herewefinddifferencesinthemethylation statusandsilencingmechanismsinallopolyploidindividualsfromdifferentpopulations(Table1).Forthe17 Tragopogonmiscellus polyploidsstudiedhere,most showedsilencingofonlyonelocusinthepreviousstudiesofTateetal.[43]andBuggsetal.[42],butthreeindividualsshowedsilencingoftwoormoreloci.Someof theselociaresilencedbymethylation,butothersare not,suggestingdiversemechanismsexistwithinanallopolyploidindividualtoregulateduplicategeneexpression.Forthelocithatweremethylated,twoshowed 100%CGmethylationingenicandpromoterregions (TDF-44andS2),whilethethirdwasmethylatedat50% ofthegenicCGsites.Asmethylationofgeneregionsis notusuallyassociatedwithgenesilencinginplants[48], howthispatternofmethylationcontributestosilencing thisgene,ifatall,isnotunderstood.Comparisonofthe methylationstatusofsilencedvs.unsilencedlocicould lendfurtherinsightintotheroleofgenebodymethylationin Tragopogon Hence,asinotherpolyploidspecies[ Spartinaanglica [11], Brassica ,[59],wheat,[60],rice,[61], Arabidopsis suecica ,[62]],genomeevolutionin Tragopogonmiscellus includesDNAmethylationasamechanismtoregulate duplicategeneexpression,whichwedemonstratehere forthefirsttime.Previousstudiesin Tragopogon showed homeologloss[41-43]andchromosomalrepatterning [63,64]followingallopolyploidformation.Theselatter phenomenaseemtobemorecommonmechanismsin T.miscellus populationsthanexpressionchangesfor dealingwiththe ‘ genomeshock ’ thataccompanies hybridizationandwholegenomeduplication[65].The locisilencedviamethylationhadthe T.dubius copysilenced,which,althoughasmallnumber,mayindicatea ‘ preference ’ forsilencinglociofoneprogenitor ’ sgenome.Thisresultistrueofthe T.miscellus polyploids formedwitheither T.dubius (Pullman)or T.pratensis (Garfield,Moscow,Oakesdale,Spangle)asthematernal parent,sotheredoesnotseemtobeamaternal ‘ imprinting ’ influenceforthelocistudiedhere.Thisinterpretationisinlinewithpreviousstudiesthathave reportedagreatertendencyofhomeologlossofthe T. dubiuscopycomparedto T.pratensis [21,41-43,66-68]. Curiously,inthecaseofrDNA,although T.dubius homeologsaremorefrequentlylostfromthepolyploid genomes,transcriptionratesofremaining T.dubius copiesarehigherthan T.pratensis copies[67].As T.miscellus hasshownahighfrequencyofhomeologloss,but littlegenesilencingbasedonthestudiestodate [21,41-43,69],amorecomprehensivegenome-wideanalysisofmethylationwouldhelptodeterminetheroleof thisepigeneticmechanisminshapingtheevolutionof Tragopogon allopolyploidgenomes.ConclusionsAllopolyploidscanemploydiversemechanismstocope withduplicateandredundantgenomes.Whileprevious studiesof Tragopogon allopolyploidsshowedthathomeologlossisacommonconsequenceofallopoyploidization, hereweshowthatDNAmethylationcansilenceoneprogenitorhomeologoritcanregulatethelevelofexpression ofthetwoprogenitorhomeologs.Asfurthergenomicresourcesfor Tragopogon aredeveloped,genome-wide methylationanalysesshouldbeundertakentoassesshow extensivehomeologmethylationiswithintheallopolyploidspecies.MethodsPlantmaterialDNAforthediploidparents( Tragopogondubius and T. pratensis )and Tragopogonmiscellus usedwasthesame aspreviousstudies[42,43].Briefly,DNAwasextracted byamodifiedCTABmethod[70]fromtissuepreviously flash-frozeninliquidnitrogen.LeaftissuewascollectedSehrish etal.BMCGenomics 2014, 15 :701 Page4of7 http://www.biomedcentral.com/1471-2164/15/701

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fromseedlingsgrownunderstandardizedglasshouseconditions.Intotal,17 T.miscellus individuals,eachofwhich previouslyshowedgenesilencing(TDF-44in[43];S2,S3 andS18in[42];Table1),wereexamined.Threerepresentativesofeachdiploidspecieswerealsoincluded.BisulfiteconversionPriortobisulfiteconversion,genomicDNAofthediploidandpolyploidsampleswasdigestedwith Eco RV (NewEnglandBiolabs,UK),whichdoesnotcutwithin thegenesofinterest.TwomicrogramsofgenomicDNA weredigestedinatotalvolumeof100 lwith80units of Eco RV,10Xbufferand10 gBSA.Thereactionwas incubatedat37Covernight(16-18hours)andthe digestedDNAcleanedbyethanolprecipitation.Bisulfite conversionwascarriedoutusingtheEZDNAMethylationkit(ZymoResearch,USA).Afterbisulfiteconversion,thesingle-strandedDNAwasquantifiedusing parametersforRNA-40onaNanodrop-1000(Thermo FisherScientific,USA).Amplificationandsequencingofgenomicand bisulfite-ConvertedDNAPrimersweredesignedfollowingahome-madegenome walkingkit[71].Separateprimersweredesignedto amplifysenseandantisensestrands,becauseafterbisulfiteconversionthetwostrandswerenotpreciselycomplementary,withadditionalprimersdesignedtoperform nestedPCR,usingMethylPrimerExpresssoftwarev.1.0 (AppliedBiosystems,USA).Primers26-29bpinlength weredesignedtogenerateanampliconof~300bpand withaCorTnearthe3 ’ endtoavoidnon-specificbindinginthebisulfite-convertedDNA.Theprimersusedfor amplificationofgenomicDNAandbisulfite-converted DNAarelistedinAdditionalfile1:TableS1. Amplificationofbisulfite-convertedDNAfortheprimaryPCRreactionwasconductedinatotalvolumeof 25 lwith10ngtemplateDNA,10 Mofbothgenespecificforwardandreverseprimers,10XPCRbuffer,10 mMdNTPsand1unitofTakaraExTaqTMpolymerase (TakaraBiotechnology,Japan).GenomicandbisulfiteconvertedDNAwasamplifiedusingthefollowingPCR program:95Cfor5min,95Cfor1min,53Cfor1 min,72Cfor1minforthefirst5cycles,then44cycles with95Cfor1min,48Cfor1min,72Cfor1min,and afinalextensionat72Cfor7min.Usingthenested primers,anotherPCRwasperformedusingtheprimary PCRproductastemplate.TheresultingnestedPCR productswererunona1.5%agarosegelstainedwith ethidiumbromideandexaminedusingaGelDoc2000 system(Bio-Rad,UK).Forsequencing,PCRproducts weretreatedwithExonucleaseI(5units)andShrimpalkalinephosphatase(0.5unit)priortothecyclesequencingreactionusingBigDyeTerminatorv.3.1(Applied Biosystems).Thepurifiedproductsweresequencedwith bothforwardandreverseprimersonanABIDNA Analyzer3770atMasseyGenomeService(Palmerston North,NewZealand).TheresultingsequenceswereassembledandanalyzedinSequencherv.4.10.1(Gene CodesCorporation,USA). Becausebothparentalhomeologsin T.miscellus polyploidsshowedCGmethylationoftheS2locus,cloning wasundertakentodeterminethemethylationstatusof theparentalcopies.PCRproductsofBS-convertedDNA wereclonedfrom Tragopogonmiscellus individuals 2671-11and2688-3usingtheTOPOTAcloningkit (Invitrogen,CA,USA).TwelvepositiveclonespersampleweresequencedwithT3andT7primersusingthe above-mentionedprotocolsforsequencing.GenomewalkingInordertodeterminethemethylationstatusofthepromoterregion,5 ’ genomewalkingwasperformedfollowingtheGenomeWalkermanual(ClontechLaboratories, USA)[72].GenomicDNAof Tragopogondubius (adiploidparentalspecies)wasdigestedwiththreedifferent restrictionenzymes: Eco RV, Dra Iand Sca I(NewEngland Biolabs,USA)inseparatereactiontubescontaining2.5 gofgenomicDNA,80unitsofrestrictionenzymeand 10Xbuffer(NewEnglandBiolabs)inatotalvolumeof 100 l.Reactionswereincubatedat37Cfor16-18 hours.Thesereactionswereethanolprecipitatedinthe presenceof20 gglycogenand3Msodiumacetate. Adapterligationtotheprecipitated,digestedgenomic DNAwasperformedinatotalvolumeof8 lcontaining 25 Madapter,10Xligationbuffer,3unitsofT4DNA ligase(NewEnglandBiolabs)and0.5 gofpurified DNA.PrimaryPCRwasperformedin50ltotalvolume using10mMdNTPs,10XPCRbuffer(TakaraBiotechnology,Japan),10 MofadapterprimerAP1(Forward) andgene-specificprimer(Reverse)(gene-specificreverse primersforallthegenesS2,S3,S8,S18andTDF-44are listedinAdditionalfile1:TableS1)and1unitofTakara ExTaqpolymerase(TakaraBiotechnology,Japan).The PCRprofilefortheprimaryPCRwasasfollows:first7 cyclesat94Cfor25sec,72Cfor3min,thenremaining 32cyclesat94Cfor25sec,67Cfor3min,andafinal extensionat67Cfor7min.PrimaryPCRproductsfor thenestedroundwerediluted1:50inddH2O.Inthe secondaryPCR,10 MnestedadapterprimerAP2(forward)andinternalgene-specificprimers(reverse)were used(TableS1)and2 lofdilutedprimaryPCRproduct wereusedastemplate.ThesecondaryPCRprofilewas asfollows:94Cfor25sec,72Cfor3minfor5cycles and94Cfor25sec,67Cfor3minfornext20cycles, thenfinalextensionat67Cfor7min.SecondaryPCR productswereseparatedona1%agarosegelandproductsfromeachlibrarywerecloned.AtleasttenpositiveSehrish etal.BMCGenomics 2014, 15 :701 Page5of7 http://www.biomedcentral.com/1471-2164/15/701

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clonespergeneperindividualweresequenced.The resultingsequencesforeachgenewerealignedwithpreviouslyobtainedsequencesofthatgeneinSequencher. Newmethylation-specificprimersweredesignedto amplifypromoterregionsfrombisulfite-convertedDNA. Theamplifiedpromoterregionsfrombisulfite-converted DNAandgenomicDNAofallfivegenesweresequencedforthe T.miscellus polyploidsandtheprogenitors T.dubius and T.pratensis .AdditionalfileAdditionalfile1:TableS1. Listofprimersusedforamplificationof bisulfite-convertedDNAand5 ’ genomewalking. Competinginterests Theauthorsdeclarethattheyhavenocompetinginterests. Authors ’ contributions TSandJATdesignedtheexperiments.TSconductedexperiments.TS,VS, DES,PSSandJATanalyzeddataandwrotethemanuscript.Allauthorsread andapprovedthefinalmanuscript. Acknowledgements ThisworkwassupportedbyaMasseyUniversityResearchFundgranttoJAT. TSwassupportedbyaPh.D.scholarshipfromtheHigherEducation CommissionofPakistan.Twoanonymousreviewersprovidedvaluable commentsthatimprovedthemanuscript. 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