Plant-RRBS: DNA Methylome Profiling Adjusted to Plant Genomes, Utilizing Efficient Endonuclease Combinations, for Multi-Sample Studies

  • Martin Schmidt
  • Magdalena Woloszynska
  • Michiel Van Bel
  • Frederik Coppens
  • Mieke Van LijsebettensEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2093)


In plants, methylation at cytosines often leads to changes in gene expression and inactivation of transposable elements. Changes in cytosine methylation (epimutations) might produce epialleles with distinct phenotypes. We present a genome-wide cytosine methylation profiling method based on bisulfite conversion and next-generation sequencing, which is applicable for plant species with available reference genomes. This so-called plant-RRBS profiling method reproducibly covers specific genomic regions and enriches for coverage of cytosine positions that are suitable for comparative analyses in multi-sample studies in basic biology and breeding studies. The plant-RRBS workflow consists of genomic DNA digestion with coverage-efficient restriction endonuclease combinations followed by a performant library generation and next-generation sequencing and a straightforward, publically available methylation data processing pipeline. Plant-RRBS has a twofold higher ratio of cytosine coverage per covered genome as compared to whole-genome bisulfite sequencing, covering tens of millions of cytosine positions, and allows detection of differential cytosine methylation, which was evaluated using rice epilines.

Key words

Cytosine methylation DpnII MspI and ApeKI endonucleases Reduced representation bisulfite sequencing (RRBS) Epiline Breeding Oryza sativa 



We thank Annick Bleys and Martine De Cock for precious help in preparing the manuscript. This research was funded by the European Union Seventh Framework Programme through the Marie Curie Intra-European program “Lighter” to M.W. and the Research Training Network “Chromatin in Plants–European Training and Mobility” to M.V.L. and fellow M.S. (CHIP-ET, FP7-PEOPLE-2013-ITN607880).


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Martin Schmidt
    • 1
    • 2
  • Magdalena Woloszynska
    • 1
    • 2
    • 3
  • Michiel Van Bel
    • 1
    • 2
  • Frederik Coppens
    • 1
    • 2
  • Mieke Van Lijsebettens
    • 1
    • 2
    Email author
  1. 1.Department of Plant Biotechnology and BioinformaticsGhent UniversityGhentBelgium
  2. 2.Center for Plant Systems Biology, VIBGhentBelgium
  3. 3.Department of GeneticsWrocław University of Environmental and Life SciencesWrocławPoland

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