Epigenetic Approaches in Non-Model Plants

  • M. Teresa Boquete
  • Niels C. A. M. Wagemaker
  • Philippine Vergeer
  • Jeannie Mounger
  • Christina L. RichardsEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2093)


Reduced representation bisulfite sequencing is an emerging methodology for evolutionary and ecological genomics and epigenomics research because it provides a cost-effective, high-resolution tool for exploration and comparative analysis of DNA methylation and genetic variation. Here we describe how digestion of genomic plant DNA with restriction enzymes, subsequent bisulfite conversion of unmethylated cytosines, and final DNA sequencing allow for the examination of genome-wide genetic and epigenetic variation in plants without the need for a reference genome. We explain how the use of several combinations of barcoded adapters for the creation of highly multiplexed libraries allows the inclusion of up to 144 different samples/individuals in only one sequencing lane.

Key words

Epigenetics DNA methylation Bisulfite treatment epiGBS Next-generation sequencing Restriction enzymes Multiplexing Reduced representation sequencing 



This work was supported by funding from the National Science Foundation (U.S.A.) IOS-1556820 (to C.L.R.), and through the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska Curie grant agreement No. 704141 (to M.T.B.).


  1. 1.
    Robertson MH, Richards CL (2015) Opportunities and challenges of Next Generation Sequencing applications in ecological epigenetics. Mol Ecol 24:3799–3801CrossRefGoogle Scholar
  2. 2.
    Richards CL, Alonso C, Becker C, Bossdorf O, Bucher E, Colomé-Tatché M, Durka W, Engelhardt J, Gaspar B, Gogol-Döring A, Grosse I, van Gurp TP, Heer K, Kronholm I, Lampei C, Latzel V, Mirouze M, Opgenoorth L, Paun O, Prohaska SJ, Rensing SA, Stadler PF, Trucchi E, Ullrich K, Verhoeven KJF (2017) Ecological plant epigenetics: evidence from model and non-model species, and the way forward. Ecol Lett 20:1576–1590CrossRefGoogle Scholar
  3. 3.
    Paun O, Verhoeven KJF, Richards CL (2019) Opportunities and limitations of reduced representation bisulfite sequencing in ecological epigenomics. New Phytol 221:738–742CrossRefGoogle Scholar
  4. 4.
    Schrey AW, Alvarez M, Foust CM, Kilvitis HJ, Lee JD, Liebl SL, Martin LB, Richards CL, Robertson MH (2013) Ecological epigenetics: beyond MS-AFLP. Integr Comp Biol 53:340–350CrossRefGoogle Scholar
  5. 5.
    Alvarez M, Robertson MH, van Gurp TP, Wagemaker CAM, Giraurd D, Ainouche ML, Salmon A, Verhoeven KJF, Richards CL (2018) Reduced-representation bisulfite sequencing finds novel epigenome-wide response to oil pollution in the foundation plant Spartina alterniflora. BioRxiv. doi:
  6. 6.
    van Gurp TP, Wagemaker NCAM, Wouters B, Vergeer P, Ouborg JNJ, Verhoeven KJ (2016) epiGBS: reference-free reduced representation bisulfite sequencing. Nat Methods 13:322–324CrossRefGoogle Scholar
  7. 7.
    Dubin MJ, Zhang P, Meng D, Remigereau MS, Osborne EJ, Paolo Casale F, Drewe P, Kahles A, Jean G, Vilhjalmsson B et al (2015) DNA methylation in Arabidopsis has a genetic basis and shows evidence of local adaptation. Elife 4:e05255CrossRefGoogle Scholar
  8. 8.
    Niederhuth CE, Bewick AJ, Ji L, Alabday M, Kim KD, Li Q, Rohr NA, Rambani A, Burke JM, Udall JA et al (2016) Widespread natural variation of DNA methylation within angiosperms. Genome Biol 17:194CrossRefGoogle Scholar
  9. 9.
    Trucchi E, Mazzarella AB, Gilfillan GD, Lorenzo MT, Schönswetter P, Paun O (2016) BsRADseq: screening DNA methylation in natural populations of non-model species. Mol Ecol 25:1697–1713CrossRefGoogle Scholar
  10. 10.
    Niederhuth CE, Schmitz RJ (2017) Putting DNA methylation in context: from genomes to gene expression in plants. Biochim Biophys Acta 1860:149–156CrossRefGoogle Scholar
  11. 11.
    Wang X, Zhang Z, Fu T, Hu L, Xu C, Gong L, Wendel JF, Liu B (2017) Gene-body CG methylation and divergent expression of duplicate genes in rice. Sci Rep 7:2675CrossRefGoogle Scholar
  12. 12.
    van Moorsel SJ, Schmid MV, Wagemaker CAM, van Gurp T, Schmid B, Vergeer P (2019) Evidence for rapid evolution in a grassland biodiversity experiment. Molecular Ecology 28:4097–4117. doi: Scholar

Copyright information

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

Authors and Affiliations

  • M. Teresa Boquete
    • 1
    • 2
  • Niels C. A. M. Wagemaker
    • 3
  • Philippine Vergeer
    • 3
    • 4
  • Jeannie Mounger
    • 2
  • Christina L. Richards
    • 2
    Email author
  1. 1.Estación Biológica de Doñana, Consejo Superior de Investigaciones Científicas (CSIC)SevillaSpain
  2. 2.Department of Integrative BiologyUniversity of South FloridaTampaUSA
  3. 3.Institute of Water and Wetland Research, Radboud University NijmegenNijmegenThe Netherlands
  4. 4.Plant Ecology and Nature Conservation GroupWageningen University and ResearchWageningenThe Netherlands

Personalised recommendations