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Accurate CpG and non-CpG cytosine methylation analysis by high-throughput locus-specific pyrosequencing in plants

hal.structure.identifierFondation Jean Dausset - Centre d’Étude du Polymorphisme Humain
dc.contributor.authorHOW-KIT, Alexandre
hal.structure.identifierFondation Jean Dausset - Centre d’Étude du Polymorphisme Humain
dc.contributor.authorDAUNAY, Antoine
hal.structure.identifierCommissariat à l'énergie atomique et aux énergies alternatives [CEA]
dc.contributor.authorMAZALEYRAT, Nicolas
hal.structure.identifierCommissariat à l'énergie atomique et aux énergies alternatives [CEA]
dc.contributor.authorBUSATO, Florence
hal.structure.identifierCommissariat à l'énergie atomique et aux énergies alternatives [CEA]
dc.contributor.authorDAVIAUD, Christian
hal.structure.identifierBiologie du fruit et pathologie [BFP]
dc.contributor.authorTEYSSIER, Emeline
hal.structure.identifierFondation Jean Dausset - Centre d’Étude du Polymorphisme Humain
hal.structure.identifierCommissariat à l'énergie atomique et aux énergies alternatives [CEA]
dc.contributor.authorDELEUZE, Jean-Francois
hal.structure.identifierBiologie du fruit et pathologie [BFP]
dc.contributor.authorGALLUSCI, Philippe
hal.structure.identifierCommissariat à l'énergie atomique et aux énergies alternatives [CEA]
dc.contributor.authorTOST, Jörg
dc.date.issued2015
dc.identifier.issn0167-4412
dc.description.abstractEnPyrosequencing permits accurate quantification of DNA methylation of specific regions where the proportions of the C/T polymorphism induced by sodium bisulfite treatment of DNA reflects the DNA methylation level. The commercially available high-throughput locus-specific pyrosequencing instruments allow for the simultaneous analysis of 96 samples, but restrict the DNA methylation analysis to CpG dinucleotide sites, which can be limiting in many biological systems. In contrast to mammals where DNA methylation occurs nearly exclusively on CpG dinucleotides, plants genomes harbor DNA methylation also in other sequence contexts including CHG and CHH motives, which cannot be evaluated by these pyrosequencing instruments due to software limitations. Here, we present a complete pipeline for accurate CpG and non-CpG cytosine methylation analysis at single base-resolution using high-throughput locus-specific pyrosequencing. The devised approach includes the design and validation of PCR amplification on bisulfite-treated DNA and pyrosequencing assays as well as the quantification of the methylation level at every cytosine from the raw peak intensities of the Pyrograms by two newly developed Visual Basic Applications. Our method presents accurate and reproducible results as exemplified by the cytosine methylation analysis of the promoter regions of two Tomato genes (NOR and CNR) encoding transcription regulators of fruit ripening during different stages of fruit development. Our results confirmed a significant and temporally coordinated loss of DNA methylation on specific cytosines during the early stages of fruit development in both promoters as previously shown by WGBS. The manuscript describes thus the first high-throughput locus-specific DNA methylation analysis in plants using pyrosequencing.
dc.language.isoen
dc.publisherSpringer Verlag (Germany)
dc.subjectplant
dc.subject.enpyrosequencing
dc.subject.encpg and non cpg methylation
dc.subject.enmethylation analysis
dc.subject.entomato
dc.title.enAccurate CpG and non-CpG cytosine methylation analysis by high-throughput locus-specific pyrosequencing in plants
dc.typeArticle de revue
dc.identifier.doi10.1007/s11103-015-0336-8
dc.subject.halSciences du Vivant [q-bio]
dc.subject.halSciences du Vivant [q-bio]/Biologie végétale
bordeaux.journalPlant Molecular Biology
bordeaux.page471-485
bordeaux.volume88
bordeaux.issue4
bordeaux.peerReviewedoui
hal.identifierhal-02634424
hal.version1
hal.popularnon
hal.audienceInternationale
hal.origin.linkhttps://hal.archives-ouvertes.fr//hal-02634424v1
bordeaux.COinSctx_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.jtitle=Plant%20Molecular%20Biology&rft.date=2015&rft.volume=88&rft.issue=4&rft.spage=471-485&rft.epage=471-485&rft.eissn=0167-4412&rft.issn=0167-4412&rft.au=HOW-KIT,%20Alexandre&DAUNAY,%20Antoine&MAZALEYRAT,%20Nicolas&BUSATO,%20Florence&DAVIAUD,%20Christian&rft.genre=article


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