Approaches to Whole-Genome Methylome Analysis in Plants

  • Xiaodong Yang
  • Sally A. MackenzieEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2093)


Cytosine methylation as a reversible chromatin mark has been investigated extensively for its influence on gene silencing and the regulation of its dynamic association–disassociation at specific sites within a eukaryotic genome. With the remarkable reductions in cost and time associated with whole-genome DNA sequence analysis, coupled with the high fidelity of bisulfite-treated DNA sequencing, single nucleotide resolution of cytosine methylation repatterning within even very large genomes is increasingly achievable. What remains a challenge is the analysis of genome-wide methylome datasets and, consequently, a clear understanding of the overall influence of methylation repatterning on gene expression or vice versa. Reported data have sometimes been subject to stringent data filtering methods that can serve to skew downstream biological interpretation. These complications derive from methylome analysis procedures that vary widely in method and parameter setting. DNA methylation as a chromatin feature that influences DNA stability can be dynamic and rapidly responsive to environmental change. Consequently, methods to discriminate background “noise” of the system from biological signal in response to specific perturbation is essential in some types of experiments. We describe numerous aspects of whole-genome bisulfite sequence data that must be contemplated as well as the various steps of methylome data analysis which impact the biological interpretation of the final output.

Key words

DNA methylation Methylome Bisulfite-seq DMR 


  1. 1.
    Holliday R, Pugh J (1975) DNA modification mechanisms and gene activity during development. Science 187(4173):226–232. Scholar
  2. 2.
    Riggs AD (1975) X inactivation, differentiation, and DNA methylation. Cytogenet Genome Res 14:9–25. Scholar
  3. 3.
    Zhang X, Yazaki J, Sundaresan A et al (2006) Genome-wide high-resolution mapping and functional analysis of DNA methylation in Arabidopsis. Cell 126:1189–1201. Scholar
  4. 4.
    Cokus SJ, Feng S, Zhang X et al (2008) Shotgun bisulphite sequencing of the Arabidopsis genome reveals DNA methylation patterning. Nature 452:215–219. Scholar
  5. 5.
    Lister R, O’Malley RC, Tonti-Filippini J et al (2008) Highly integrated single-base resolution maps of the epigenome in Arabidopsis. Cell 133:523–536. Scholar
  6. 6.
    Jones PA (2012) Functions of DNA methylation: islands, start sites, gene bodies and beyond. Nat Rev Genet 13:484–492CrossRefGoogle Scholar
  7. 7.
    Law JA, Jacobsen SE (2010) Establishing, maintaining and modifying DNA methylation patterns in plants and animals. Nat Rev Genet 11:204–220CrossRefGoogle Scholar
  8. 8.
    Zhang H, Lang Z, Zhu JK (2018) Dynamics and function of DNA methylation in plants. Nat Rev Mol Cell Biol 19:489–506CrossRefGoogle Scholar
  9. 9.
    Dowen RH, Pelizzola M, Schmitz RJ et al (2012) Widespread dynamic DNA methylation in response to biotic stress. Proc Natl Acad Sci 109:E2183–E2191. Scholar
  10. 10.
    Crisp PA, Ganguly D, Eichten SR et al (2016) Reconsidering plant memory: intersections between stress recovery, RNA turnover, and epigenetics. Sci Adv 2:e1501340. Scholar
  11. 11.
    Bej S, Basak J (2017) Abiotic stress induced epigenetic modifications in plants: how much do we know? In: Plant epigenetics, pp 493–512CrossRefGoogle Scholar
  12. 12.
    He XJ, Chen T, Zhu JK (2011) Regulation and function of DNA methylation in plants and animals. Cell Res 21:442–465. Scholar
  13. 13.
    Du J, Johnson LM, Jacobsen SE, Patel DJ (2015) DNA methylation pathways and their crosstalk with histone methylation. Nat Rev Mol Cell Biol 16:519–532. Scholar
  14. 14.
    Chatterjee A, Rodger EJ, Morison IM, et al (2017) Tools and strategies for analysis of genome-wide and gene-specific DNA methylation patterns. In: Methods in molecular biology. Humana Press, New York, pp 249–277Google Scholar
  15. 15.
    Laird PW (2010) Principles and challenges of genome-wide DNA methylation analysis. Nat Rev Genet 11:191–203CrossRefGoogle Scholar
  16. 16.
    Jacinto FV, Ballestar E, Esteller M (2008) Methyl-DNA immunoprecipitation (MeDIP): hunting down the DNA methylome. BioTechniques 44:35–43. Scholar
  17. 17.
    Martin M (2011) Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J 17:10. Scholar
  18. 18.
    Krueger F (2016) Trim Galore. In: Babraham Bioinforma.
  19. 19.
    Langmead B (2010) Aligning short sequencing reads with bowtie. Curr Protoc Bioinformatics.
  20. 20.
    Krueger F, Andrews SR (2011) Bismark: a flexible aligner and methylation caller for bisulfite-Seq applications. Bioinformatics 27:1571–1572. Scholar
  21. 21.
    Huang KYY, Huang YJ, Chen PY (2018) BS-Seeker3: ultrafast pipeline for bisulfite sequencing. BMC Bioinformatics 19:111. Scholar
  22. 22.
    Pedersen B, Hsieh TF, Ibarra C, Fischer RL (2011) MethylCoder: software pipeline for bisulte-treated sequences. Bioinformatics 27:2435–2436. Scholar
  23. 23.
    Feng H, Conneely KN, Wu H (2014) A Bayesian hierarchical model to detect differentially methylated loci from single nucleotide resolution sequencing data. Nucleic Acids Res 42(8):e69. Scholar
  24. 24.
    Schultz MD, He Y, Whitaker JW et al (2015) Human body epigenome maps reveal noncanonical DNA methylation variation. Nature 523:212–216. Scholar
  25. 25.
    Hansen KD, Langmead B, Irizarry RA (2012) BSmooth: from whole genome bisulfite sequencing reads to differentially methylated regions. Genome Biol 13:R83. Scholar
  26. 26.
    Akalin A, Kormaksson M, Li S et al (2012) MethylKit: a comprehensive R package for the analysis of genome-wide DNA methylation profiles. Genome Biol 13:R87. Scholar
  27. 27.
    Dolzhenko E, Smith AD (2014) Using beta-binomial regression for high-precision differential methylation analysis in multifactor whole-genome bisulfite sequencing experiments. BMC Bioinformatics 15:215. Scholar
  28. 28.
    Lea AJ, Tung J, Zhou X (2015) A flexible, efficient binomial mixed model for identifying differential DNA methylation in bisulfite sequencing data. PLoS Genet 11:e1005650. Scholar
  29. 29.
    Yelagandula R, Stroud H, Holec S et al (2014) The histone variant H2A.W defines heterochromatin and promotes chromatin condensation in Arabidopsis. Cell 158:98–109. Scholar
  30. 30.
    Gouil Q, Baulcombe DC (2016) DNA methylation signatures of the plant chromomethyltransferases. PLoS Genet 12:e1006526. Scholar
  31. 31.
    Stroud H, Greenberg MVC, Feng S et al (2013) Comprehensive analysis of silencing mutants reveals complex regulation of the Arabidopsis methylome. Cell 152:352–364. Scholar
  32. 32.
    Mlura A, Yonebayashi S, Watanabe K et al (2001) Mobilization of transposons by a mutation abolishing full DNA methylation in Arabidopsis. Nature 411:212–214. Scholar
  33. 33.
    Stuart T, Eichten SR, Cahn J et al (2016) Population scale mapping of transposable element diversity reveals links to gene regulation and epigenomic variation. elife 5.
  34. 34.
    Fultz D, Slotkin RK (2017) Exogenous transposable elements circumvent identity-based silencing, permitting the dissection of expression-dependent silencing. Plant Cell 29:360–376. Scholar
  35. 35.
    Yu A, Lepere G, Jay F et al (2013) Dynamics and biological relevance of DNA demethylation in Arabidopsis antibacterial defense. Proc Natl Acad Sci 110:2389–2394. Scholar
  36. 36.
    Schmitz RJ, Schultz MD, Urich MA et al (2013) Patterns of population epigenomic diversity. Nature 495:193–198. Scholar
  37. 37.
    Dubin MJ, Zhang P, Meng D et al (2015) DNA methylation in Arabidopsis has a genetic basis and shows evidence of local adaptation. elife 4:e05255. Scholar
  38. 38.
    Kawakatsu T, Huang S shan C, Jupe F et al (2016) Epigenomic diversity in a global collection of Arabidopsis thaliana accessions. Cell 166:492–506. Scholar
  39. 39.
    Ganguly DR, Crisp PA, Eichten SR, Pogson BJ (2017) The Arabidopsis DNA methylome is stable under transgenerational drought stress. Plant Physiol 175:1893–1912. Scholar
  40. 40.
    Kawakatsu T, Nery JR, Castanon R, Ecker JR (2017) Dynamic DNA methylation reconfiguration during seed development and germination. Genome Biol 18:171. Scholar
  41. 41.
    Kaplowitz PB, Jennings SS (1987) Effect of growth hormone therapy on caloric intake in children with growth hormone deficiency. Nutr Res 7:901–906. Scholar
  42. 42.
    Mirouze M, Vitte C (2014) Transposable elements, a treasure trove to decipher epigenetic variation: insights from Arabidopsis and crop epigenomes. J Exp Bot 65:2801–2812CrossRefGoogle Scholar
  43. 43.
    Moarefi AH, Chédin F (2011) ICF syndrome mutations cause a broad spectrum of biochemical defects in DNMT3B-mediated de novo DNA methylation. J Mol Biol 409:758–772. Scholar
  44. 44.
    Bewick AJ, Schmitz RJ (2017) Gene body DNA methylation in plants. Curr Opin Plant Biol 36:103–110CrossRefGoogle Scholar
  45. 45.
    Yang H, Chang F, You C et al (2015) Whole-genome DNA methylation patterns and complex associations with gene structure and expression during flower development in Arabidopsis. Plant J 81:268–281. Scholar
  46. 46.
    Schmid MW, Heichinger C, Coman Schmid D et al (2018) Contribution of epigenetic variation to adaptation in Arabidopsis. Nat Commun 9:4446. Scholar
  47. 47.
    Walker J, Gao H, Zhang J et al (2018) Sexual-lineage-specific DNA methylation regulates meiosis in Arabidopsis. Nat Genet 50:130–137. Scholar
  48. 48.
    Derreumaux S, Chaoui M, Tevanian G, Fermandjian S (2001) Impact of CpG methylation on structure, dynamics and solvation of cAMP DNA responsive element. Nucleic Acids Res 29:2314–2326. Scholar
  49. 49.
    Severin PMD, Zou X, Gaub HE, Schulten K (2011) Cytosine methylation alters DNA mechanical properties. Nucleic Acids Res 39:8740–8751. Scholar
  50. 50.
    Pérez A, Castellazzi CL, Battistini F et al (2012) Impact of methylation on the physical properties of DNA. Biophys J 102:2140–2148. Scholar
  51. 51.
    Ngo TTM, Yoo J, Dai Q et al (2016) Effects of cytosine modifications on DNA flexibility and nucleosome mechanical stability. Nat Commun 7.
  52. 52.
    Roeler K, Takuno S, Gaut BS (2016) CG methylation covaries with differential gene expression between leaf and floral bud tissues of Brachypodium distachyon. PLoS One 11:e0150002. Scholar
  53. 53.
    Takuno S, Gaut BS (2012) Body-methylated genes in Arabidopsis thaliana are functionally important and evolve slowly. Mol Biol Evol 29:219–227. Scholar
  54. 54.
    Bewick AJ, Ji L, Niederhuth CE et al (2016) On the origin and evolutionary consequences of gene body DNA methylation. Proc Natl Acad Sci 113:9111–9116. Scholar
  55. 55.
    Wang X, Zhang Z, Fu T et al (2017) Gene-body CG methylation and divergent expression of duplicate genes in rice. Sci Rep 7:2675. Scholar
  56. 56.
    Zilberman D (2017) An evolutionary case for functional gene body methylation in plants and animals. Genome Biol 18:87. Scholar
  57. 57.
    Xing M-Q, Zhang Y-J, Zhou S-R et al (2015) Global analysis reveals the crucial roles of DNA methylation during Rice seed development. Plant Physiol 168:1417–1432. Scholar
  58. 58.
    Zhong S, Fei Z, Chen YR et al (2013) Single-base resolution methylomes of tomato fruit development reveal epigenome modifications associated with ripening. Nat Biotechnol 31:154–159. Scholar
  59. 59.
    Lang Z, Wang Y, Tang K et al (2017) Critical roles of DNA demethylation in the activation of ripening-induced genes and inhibition of ripening-repressed genes in tomato fruit. Proc Natl Acad Sci 114:E4511–E4519. Scholar
  60. 60.
    Candaele J, Demuynck K, Mosoti D et al (2014) Differential methylation during maize leaf growth targets developmentally regulated genes. Plant Physiol 164:1350–1364. Scholar
  61. 61.
    Song Q, Zhang T, Stelly DM, Chen ZJ (2017) Epigenomic and functional analyses reveal roles of epialleles in the loss of photoperiod sensitivity during domestication of allotetraploid cottons. Genome Biol 18:99. Scholar
  62. 62.
    Holoch D, Moazed D (2015) RNA-mediated epigenetic regulation of gene expression. Nat Rev Genet 16:71–84CrossRefGoogle Scholar
  63. 63.
    Matzke MA, Kanno T, Matzke AJM (2015) RNA-directed DNA methylation: the evolution of a complex epigenetic pathway in flowering plants. Annu Rev Plant Biol 66:243–267. Scholar
  64. 64.
    Hossain MS, Kawakatsu T, Do KK et al (2017) Divergent cytosine DNA methylation patterns in single-cell, soybean root hairs. New Phytol 214:808–819. Scholar
  65. 65.
    Lauria M, Echegoyen-Nava RA, Rodríguez-Ríos D et al (2017) Inter-individual variation in DNA methylation is largely restricted to tissue-specific differentially methylated regions in maize. BMC Plant Biol 17:52. Scholar
  66. 66.
    Turco GM, Kajala K, Kunde-Ramamoorthy G et al (2017) DNA methylation and gene expression regulation associated with vascularization in Sorghum bicolor. New Phytol 214:1213. Scholar
  67. 67.
    Johannes F, Porcher E, Teixeira FK et al (2009) Assessing the impact of transgenerational epigenetic variation on complex traits. PLoS Genet 5(6):e1000530. Scholar
  68. 68.
    Yang DL, Zhang G, Tang K et al (2016) Dicer-independent RNA-directed DNA methylation in Arabidopsis. Cell Res 26:66–82. Scholar
  69. 69.
    Scheid OM, Probst AV, Afsar K, Paszkowski J (2002) Two regulatory levels of transcriptional gene silencing in Arabidopsis. Proc Natl Acad Sci 99:13659–13662. Scholar
  70. 70.
    Li D, Palanca AMS, Won SY et al (2017) The MBD7 complex promotes expression of methylated transgenes without significantly altering their methylation status. elife 6:e19893. Scholar
  71. 71.
    Williams BP, Pignatta D, Henikoff S, Gehring M (2015) Methylation-sensitive expression of a DNA demethylase gene serves as an epigenetic rheostat. PLoS Genet 11:e1005142. Scholar
  72. 72.
    Wibowo A, Becker C, Marconi G et al (2016) Hyperosmotic stress memory in Arabidopsis is mediated by distinct epigenetically labile sites in the genome and is restricted in the male germline by DNA glycosylase activity. elife 5:e13546. Scholar
  73. 73.
    Zhu H, Wang G, Qian J (2016) Transcription factors as readers and effectors of DNA methylation. Nat Rev Genet 17:551–565. Scholar
  74. 74.
    Neri F, Rapelli S, Krepelova A et al (2017) Intragenic DNA methylation prevents spurious transcription initiation. Nature 543:72–77. Scholar
  75. 75.
    Wang X, Hu L, Wang X et al (2016) DNA methylation affects gene alternative splicing in plants: an example from rice. Mol Plant 9:305–307CrossRefGoogle Scholar
  76. 76.
    Jackson JP, Lindroth AM, Cao X, Jacobsen SE (2002) Control of CpNpG DNA methylation by the KRYPTONITE histone H3 methyltransferase. Nature 416:556–560. Scholar
  77. 77.
    Wollmann H, Stroud H, Yelagandula R et al (2017) The histone H3 variant H3.3 regulates gene body DNA methylation in Arabidopsis thaliana. Genome Biol 18:94. Scholar
  78. 78.
    Luo M, Yu CW, Chen FF et al (2012) Histone deacetylase HDA6 is functionally associated with AS1 in repression of KNOX genes in Arabidopsis. PLoS Genet 8:e1003114. Scholar
  79. 79.
    Kim JM, To TK, Seki M (2012) An epigenetic integrator: new insights into genome regulation, environmental stress responses and developmental controls by histone deacetylase 6. Plant Cell Physiol 53:794–800CrossRefGoogle Scholar
  80. 80.
    Iwasaki M, Takahashi H, Iwakawa H et al (2013) Dual regulation of ETTIN (ARF3) gene expression by AS1-AS2, which maintains the DNA methylation level, is involved in stabilization of leaf adaxial-abaxial partitioning in Arabidopsis. Development 140:1958–1969. Scholar
  81. 81.
    Blevins T, Pontvianne F, Cocklin R et al (2014) A two-step process for epigenetic inheritance in Arabidopsis. Mol Cell 54:30–42. Scholar
  82. 82.
    Xu Y-Z, de la Rosa Santamaria R, Virdi KS et al (2012) The chloroplast triggers developmental reprogramming when MUTS HOMOLOG1 is suppressed in plants. Plant Physiol 159:710–720. Scholar
  83. 83.
    Virdi KS, Laurie JD, Xu YZ et al (2015) Arabidopsis MSH1 mutation alters the epigenome and produces heritable changes in plant growth. Nat Commun 6:6386. Scholar
  84. 84.
    Shao MR, Kumar Kenchanmane Raju S, Laurie JD et al (2017) Stress-responsive pathways and small RNA changes distinguish variable developmental phenotypes caused by MSH1 loss. BMC Plant Biol 17:47. Scholar
  85. 85.
    Jenkinson G, Pujadas E, Goutsias J, Feinberg AP (2017) Potential energy landscapes identify the information-theoretic nature of the epigenome. Nat Genet 49:719–729. Scholar
  86. 86.
    Jenkinson G, Abante J, Feinberg AP, Goutsias J (2018) An information-theoretic approach to the modeling and analysis of whole-genome bisulfite sequencing data. BMC Bioinformatics 19:87. Scholar
  87. 87.
    Hofmeister BT, Lee K, Rohr NA et al (2017) Stable inheritance of DNA methylation allows creation of epigenotype maps and the study of epiallele inheritance patterns in the absence of genetic variation. Genome Biol 18:155. Scholar
  88. 88.
    Taudt A, Roquis D, Vidalis A et al (2018) METHimpute: imputation-guided construction of complete methylomes from WGBS data. BMC Genomics 19:444. Scholar
  89. 89.
    Tran H, Zhu H, Wu X et al (2018) Identification of differentially methylated sites with weak methylation effects. Genes (Basel) 9. Scholar
  90. 90.
    Srivastava A, Karpievitch YV, Eichten SR et al (2019) HOME: a histogram based machine learning approach for effective identification of differentially methylated regions 2. 20(1):253.
  91. 91.
    Sanchez R, Yang X, Kundariya H et al (2018) Enhancing resolution of natural methylome reprogramming behavior in plants. bioRxiv:252106.
  92. 92.
    Becker C, Hagmann J, Müller J et al (2011) Spontaneous epigenetic variation in the Arabidopsis thaliana methylome. Nature 480:245–249. Scholar
  93. 93.
    Schmitz RJ, Schultz MD, Lewsey MG et al (2011) Transgenerational epigenetic instability is a source of novel methylation variants. Science 334:369–373. Scholar
  94. 94.
    Sanchez R, Mackenzie SA (2016) Genome-wide discriminatory information patterns of cytosine DNA methylation. Int J Mol Sci 17(6):938. Scholar
  95. 95.
    Sanchez R, Mackenzie SA (2016) Information thermodynamics of cytosine DNA methylation. PLoS One 11:e0150427. Scholar
  96. 96.
    Yang X, Kundariya H, Xu Y-Z et al (2015) MutS HOMOLOG1-derived epigenetic breeding potential in tomato. Plant Physiol 168:222–232. Scholar
  97. 97.
    Raju SKK, Shao MR, Sanchez R et al (2018) An epigenetic breeding system in soybean for increased yield and stability. Plant Biotechnol J 16:1836–1847. Scholar
  98. 98.
    Reinders J, Wulff BBH, Mirouze M et al (2009) Compromised stability of DNA methylation and transposon immobilization in mosaic Arabidopsis epigenomes. Genes Dev 23:939–950. Scholar
  99. 99.
    Lang-Mladek C, Popova O, Kiok K et al (2010) Transgenerational inheritance and resetting of stress-induced loss of epigenetic gene silencing in Arabidopsis. Mol Plant 3:594–602. Scholar
  100. 100.
    Uller T, English S, Pen I (2015) When is incomplete epigenetic resetting in germ cells favoured by natural selection? Proc R Soc B Biol Sci 282:1–8. Scholar
  101. 101.
    Quadrana L, Colot V (2016) Plant transgenerational epigenetics. Annu Rev Genet 50:467–491. Scholar

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Authors and Affiliations

  1. 1.Departments of Biology and Plant ScienceThe Pennsylvania State UniversityUniversity ParkUSA

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