TheDNAfragmentsabout1600bpwereamplifiedusingrandomamplifiedpolymorphismDNA(RAPD)primerOPA12withthetemplatesofmitochondrialDNAofZhenshan97AandZhenshan97B,andweresequenced.ThenucleotidesequencesandlengthsofthefragmentsfromZhenshan97AandZhenshan97Bshowednodifference.Thepreciselengthofthefragmentwas1588bp.Sequencecharacterizedamplificationregion(SCAR)primerswerethendevelopedtodiscriminatethecytoplasmicmalesterile(CMS)linesandtheirmaintainerlines.Aspecific1588bpfragmentcouldbeamplifiedwithSCARprimers,CHI19F2/CHI19R2andCHI20F3/CHI23R3,inthemitochondrialDNAofZhenshan97A,butnotZhenshan97B.Furthermore,thespecificfragmentcouldbealsoamplifiedfromthetotalDNAfromgreenleaftissuesofZhenshan97AwithSCARprimers,butnotZhenshan97B.Withthecorrespondingprimers,thespecificfragmentcouldalsobeamplifiedfromthetotalDNAofgreenleavesofothertwoCMSlineswithwildabortivetypecytoplasm(CMS-WA),namelyZhenpinAandTianfengA,butnotintheirmaintainerlines.Moreover,usingtotalDNAastemplate,eachofthefourpairsofSCARprimerscouldalsobeusedtoamplifythe1588bpfragmentinCMS-ID(Indonesiapaddytype)lineⅡ-32A,butnotinII-32B,andthespecificfragmentwasamplifiedfromtheDNAofbothF1andF2seedlingsofShanyou63.Theresultsofdetectingthegeneticpurityofaman-mademixtureoftheseedsofZhenshan97AusingCHI20F3/CHI23R3werecompletelyconsistentwiththephenotypes.Takentogether,theseresultsindicatedthatthespecific1588bp-fragmentamplifiedbyCHI20F3/CHI23R3wastheuniqueamplificationproductsofCMSmitochondrialDNA,andcouldbeusedtodistinguishCMS-WAandCMS-IDlinesfromtheircorrespondingmaintainerlinesattheseedlingstage.
