1Chapter1AnalysisofDNAMethylationinPlantsbyBisulfiteSequencingAndreaM.FoersterandOrtrunMittelstenScheidAbstractMethylationofcytosinesisaveryimportantepigeneticmodificationofgenomicDNAinmanydifferenteukaryotes,anditisfrequentlyinvolvedintranscriptionalregulationofgenes.Inplants,DNAmethyla-tionisregulatedbyacomplexinterplaybetweenseveralmethylatinganddemethylatingenzymes.Analysisoftheresultingcytosinemethylationpatternswiththehighestresolutionisachievedaftersodiumbisulfitetreatment,deaminatingnonmethylatedcytosinestouracil.SubsequentPCRandsequenceanalysisofindividualampliconsdisplaysthedegree,position,andsequencecontextofmethylationofeverycytosineresidueinindividualgenomicsequences.WedescribetheapplicationofbisulfitesequencingfortheanalysisofDNAmethylationatdefinedindividualsequencesofplantgenomicDNA.Keywords:DNAmethylation,5-methylcytosine(5mC),Bisulfitesequencing,Bisulfiteprimerdesign,BisulfiteconversioncontrolMethylationatposition5ofcytosinesisamajorepigeneticmodifi-cationineukaryotesandtheonlyknowncovalentchangeofplantgenomicDNAitself.AlterationsinDNAmethylationarefre-quentlyinvolvedintranscriptionalgeneregulation(1,2).Therefore,thereisagreatinterestinanalyzingcytosinemethylationlevelsanddistributionwithinthegenome.Methylatedandunmethylatedcytosinescanbedistinguishedbybisulfitegenomicsequencingatsingle-baseresolution.TreatinggenomicDNAwithsodiumbisulfiteconvertsunmethylatedcytosinetouracil,while5-methylcytosineremainsunchanged(Fig.1).Amplificationbythepolymerasechainreaction(PCR)ofconvertedDNAfollowedbysequencingrevealspositionsof5-methylcytosineinthesequenceofinterest.1.IntroductionIgorKovaIchukandFranzZemp(eds.),PlantEpigenetics:MethodsandProtocols,MethodsinMolecularBiology,vol.631,DOI10.1007/978-1-60761-646-7_1,©SpringerScience+BusinessMedia,LLC20102FoersterandMittelstenScheidThisprinciple,firstdescribedbyFrommeretal.(3)andClarketal.(4),hassinceundergoneseveralexperimentalsimplificationsandrefinementsandiswidelyappliedtoDNAfrommanydifferentorganisms.Itisals...
