AssessmentofMagnaporthegriseamatingtypebysporePCR

IsolationofDNAfromfilamentousfungiforPCRanalysisisusuallytimeconsumingandinvolvesuseoftoxicchemicalssuchasphenol/chloroform.InSaccharomycescerevisiae,PCRassaycanbeperformedwithyeastcolonies(Huxleyetal.TIG19906:236).HerewedescribeaPCRprotocolwhichusesM.griseaconidiadirectlyforPCRanalysiswithoutextractionofDNA.

ThePCRassayisperformedwithconidiaproducedonanoatmealculture(Crawfordetal.1986Genetics,114:1111-1129)thathasbeengrownatroomtemperatureforoneweek.Tocollectconidia,5mlsterilewaterisaddedtoeach0.5mlmicrotube.Aflat-endedtoothpickiswettedinsterilewaterandusedtogentlyscrapeconidiafromthesurfaceoftheculturewithbothsidesofthetoothpick.CareshouldbeexercisedtoavoidtransferringmediumsinceagarmayinhibitthePCRreaction.Conidiacanbereleasedbyrotatingthetoothpickbrieflyinthewaterinthemicrotube.Thesamplesarefrozeninliquidnitrogenorina-80Cfreezerfor10min.

ForM.grisea,10(4)-10(6)sporesperreactiongivethebestresult.Toomanyspores(over10(6))willinhibitthePCRreaction.PCRiscarriedoutbyaddingtoeachtubeofconidia45mlof10mMTris-HCl(pH9.0at25C)containing50mMKCl,0.1%TritonX-100,2.5mMMgCl2,0.2mMeachdNTP,1.0mMeachprimer,1.25unitsofTaqDNApolymerase(Promega).Samplesareoverlaidwith50ulmineraloilandsubjectedtothefollowingPCRcondition:95Cfor5min;30cyclesof95Cfor1min/52Cfor2min/72Cfor2min;followedby72Cfor5minutes.

PCRprimersusedforM.griseaareA1(AGCCTCATCAACGGCAA)andA5(GTAGCGTACAAGCACGG)forMat1-1,B15(CTCAATCTCCGTAGTAG)andB16(CATCCGATATGACGACA)forMat1-2.TheexpectedPCRproductsare372bpforMat1-1and376bpforMat1-2(CourtesyofDr.SeochangKang).Forotherfungi,the18SrDNAprimersNS1andNS2(Whiteetal.,1989.PCRprotocols,AcademicPress)wereusedinsporePCRtests.

ThissporePCRprotocolallowstherapiddeterminationofthematingtype(Mat1-1andMat1-2)ofM.grisea.Itisparticularlyusefulforscoringmatingtypewhenlowfertilityorsterilityisaproblemincrossingoranalyzingfieldcollections.ThematingtypeallelesdeterminedbysporePCRareconsistentwithmatingtestson58Progenyofacrossbetweenstrains4375-R-26X4136-4-3.FungalmyceliumcanbeusedinsteadofsporesforPCR,butitmaybemoredifficulttocollectmyceliumthansporesfrompetridishculturewithouttouchingtheagar.

ThesameprotocolhasbeenusedsuccessfullywithNeurosporacrassa,FusariummoniliformeandAspergillusnidulanswith18SrDNAprimersNS1andNS2.ThissporePCRprotocolcanbeapplicabletootherfungiandmaybeusefulforrapidPCRassaywithoutDNApreparationinanalyzingtransformantsandfieldcollections.