Skip to main content
SpringerLink
Log in

Analysis of DNA Cytosine Methylation Patterns Using Methylation-Sensitive Amplification Polymorphism (MSAP)

  • Protocol
  • First Online:
Plant Epigenetics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1456))

Abstract

Different molecular techniques have been developed to study either the global level of methylated cytosines or methylation at specific gene sequences. One of them is the methylation-sensitive amplified polymorphism technique (MSAP) which is a modification of amplified fragment length polymorphism (AFLP). It has been used to study methylation of anonymous CCGG sequences in different fungi, plants, and animal species. The main variation of this technique resides on the use of isoschizomers with different methylation sensitivity (such as HpaII and MspI) as a frequent-cutter restriction enzyme. For each sample, MSAP analysis is performed using both EcoRI/HpaII- and EcoRI/MspI-digested samples. A comparative analysis between EcoRI/HpaII and EcoRI/MspI fragment patterns allows the identification of two types of polymorphisms: (1) methylation-insensitive polymorphisms that show common EcoRI/HpaII and EcoRI/MspI patterns but are detected as polymorphic amplified fragments among samples and (2) methylation-sensitive polymorphisms which are associated with the amplified fragments that differ in their presence or absence or in their intensity between EcoRI/HpaII and EcoRI/MspI patterns. This chapter describes a detailed protocol of this technique and discusses the modifications that can be applied to adjust the technology to different species of interest.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 159.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Vos P, Hogers R, Bleeker M, Reijans M, van de Lee T, Hornes M, Frijters A, Pot J, Peleman J, Kuiper M, Zabeau M (1995) AFLP: a new technique for DNA fingerprinting. Nucleic Acids Res 23:4407–4414

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Laird PW (2010) Principles and challenges of genome wide DNA methylation analysis. Nat Rev Genet 11:191–203

    Article  CAS  PubMed  Google Scholar 

  3. Reyna-López GE, Simpson J, Ruiz-Herrera J (1997) Differences in DNA methylation patterns are detectable during the dimorphic transition of fungi by amplification of restriction polymorphisms. Mol Gen Genet 253:703–710

    Article  PubMed  Google Scholar 

  4. Li Y, Shan X, Liu X, Hu L, Guo W, Liu B (2008) Utility of the methylation-sensitive amplified polymorphism (MSAP) marker for detection of DNA methylation polymorphism and epigenetic population structure in a wild barley species (Hordeum brevisubulatum). Ecol Res 23:927–930

    Article  CAS  Google Scholar 

  5. Herrera CM, Bazaga P (2010) Epigenetic differentiation and relationship to adaptive genetic divergence in discrete populations of the violet Viola cazorlensis. New Phytol 187:867–876

    Article  CAS  PubMed  Google Scholar 

  6. Herrera CM, Bazaga P (2011) Untangling individual variation in natural populations: ecological, genetic and epigenetic correlates of long-term inequality in herbivory. Mol Ecol 20:1675–1688

    Article  CAS  PubMed  Google Scholar 

  7. Ocaña J, Walter B, Schellenbaum P (2013) Stable MSAP markers for the distinction of Vitis vinifera cv Pinot noir clones. Mol Biotechnol 55:236–248

    Article  PubMed  Google Scholar 

  8. Sáez-Laguna E, Guevara MA, Díaz LM, Sánchez-Gómez D, Collada C, Aranda I, Cervera MT (2014) Epigenetic variability in the genetically uniform forest tree species Pinus pinea L. PLoS One 9:e103145

    Article  PubMed  PubMed Central  Google Scholar 

  9. Long Y, Xia W, Li R, Wang J, Shao M, Feng J, King GJ, Meng J (2011) Epigenetic QTL mapping in Brassica napus. Genetics 189:1093–1102

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Lira-Medeiros CF, Parisod C, Fernandes RA, Mata CS, Cardoso MA, Ferreira PCG (2010) Epigenetic variation in mangrove plants occurring in contrasting natural environment. PLoS One 5(4):e10326

    Article  PubMed  PubMed Central  Google Scholar 

  11. Wang WS, Pan YJ, Zhao XQ, Dwivedi D, Zhu LH, Ali J, Fu BY, Li ZK (2011) Drought-induced site-specific DNA methylation and its association with drought tolerance in rice (Oryza sativa L.). J Exp Bot 62:1951–1960

    Article  CAS  PubMed  Google Scholar 

  12. Karan R, DeLeon T, Biradar H, Subudhi PK (2012) Salt stress induced variation in DNA methylation pattern and its influence on gene expression in contrasting rice genotypes. PLoS One 7:e40203

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Herrera CM, Bazaga P (2013) Epigenetic correlates of plant phenotypic plasticity: DNA methylation differs between prickly and nonprickly leaves in heterophyllous Ilex aquifolium (Aquifoliaceae) trees. Bot J Linn Soc 171:441–452

    Article  Google Scholar 

  14. Ruiz-García L, Cervera MT, Martinez-Zapater JM (2005) DNA methylation increases throughout Arabidopsis development. Planta 222:301–306

    Article  PubMed  Google Scholar 

  15. Meng FR, Li YC, Yin J et al (2012) Analysis of DNA methylation during the germination of wheat seeds. Biol Plantarum 56:269–275

    Article  CAS  Google Scholar 

  16. Osabe K, Clement JD, Bedon F, Pettolino FA, Ziolkowski L, Llewellyn DJ, Finnegan EJ, Wilson IW (2014) Genetic and DNA methylation changes in cotton (Gossypium) genotypes and tissues. PLoS One 9:e86049

    Article  PubMed  PubMed Central  Google Scholar 

  17. Salmon A, Ainouche ML, Wendel JF (2005) Genetic and epigenetic consequences of recent hybridization and polyploidy in Spartina (Poaceae). Mol Ecol 14:1163–1175

    Article  CAS  PubMed  Google Scholar 

  18. Zhao Y, Yu S, Xing C, Fan S, Song M (2008) Analysis of DNA methylation in cotton hybrids and their parents. Mol Biol 42:169–178

    Article  CAS  Google Scholar 

  19. Hegarty MJ, Batstone T, Barker GL, Edwards KJ, Abbott RJ, Hiscock SJ (2011) Nonadditive changes to cytosine methylation as a consequence of hybridization and genome duplication in Senecio (Asteraceae). Mol Ecol 20:105–113

    Article  CAS  PubMed  Google Scholar 

  20. Li A, Hu BQ, Xue ZY, Chen L, Wang WX, Song WQ, Chen CB, Wang CG (2011) DNA methylation in genomes of several annual herbaceous and woody perennial plants of varying ploidy as detected by MSAP. Plant Mol Biol Report 29:784–793

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Rodriguez MP, Cervigni GDL, Quarin CL, Ortiz JPA (2012) Frequencies and variation in cytosine methylation patterns in diploid and tetraploid cytotypes of Paspalum notatum. Biol Plantarum 56:276–282

    Article  CAS  Google Scholar 

  22. Hanai LR, Floh EIS, Fungaro MHP, Anta-Catarina C, de Paula FM, Viana AM, Vieira MLC (2010) Methylation patterns revealed by MSAP profiling in genetically stable somatic embryogenic cultures of Ocotea catharinensis (Lauraceae). In Vitro Cell Dev Biol Plant 46:368–377

    Article  CAS  Google Scholar 

  23. Bobadilla Landey R, Cenci A, Georget F, Bertrand B, Camayo G, Dechamp E, Herrera JC, Santoni S, Lashermes S, Simpson J, Etienne H (2013) High genetic and epigenetic stability in Coffea arabica plants derived from embryogenic suspensions and secondary embryogenesis as revealed by AFLP, MSAP and the phenotypic variation rate. PLoS One: e56372.

    Google Scholar 

  24. Tiwari JK, Chandel P, Gupta S, Gopal J, Singh BP, Bhardwaj V (2013) Analysis of genetic stability of in vitro propagated potato microtubers using DNA markers. Physiol Mol Biol Plants 19:587–595

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Rathore MS, Mastan SG, Agarwal PK (2015) Evaluation of DNA methylation using methylation-sensitive amplification polymorphism in plant tissues grown in vivo and in vitro. Plant Growth Regul 75:11–19

    Article  CAS  Google Scholar 

  26. Dellaporta SL, Wood J, Hicks JB (1985) Maize DNA miniprep. In: Malberg R, Messing J, Sussex I (eds) Molecular biology of plants. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp 36–37

    Google Scholar 

  27. Meudt HM, Clarke AC (2007) Almost forgotten or latest practice? AFLP applications, analyses and advances. Trends Plant Sci 12:106–117

    Article  CAS  PubMed  Google Scholar 

  28. Schulz B, Eckstein RL, Durka W (2013) Scoring and analysis of methylation-sensitive amplification polymorphisms for epigenetic population studies. Mol Ecol Resour 13:642–653

    Article  CAS  PubMed  Google Scholar 

  29. Salmon A, Clotault J, Jenczewski E, Chable V, Manzanares-Dauleux MJ (2008) Brassica oleracea displays a high level of DNA methylation polymorphism. Plant Sci 174:61–70

    Article  CAS  Google Scholar 

  30. Vergeer P, Wagemaker N, Ouborg NJ (2012) Evidence for an epigenetic role in inbreeding depression. Biol Lett 8:798–801

    Article  PubMed  PubMed Central  Google Scholar 

  31. Paun O, Bateman RM, Fay MF et al (2010) Stable epigenetic effects impact adaptation in allopolyploid orchids (Dactylorhiza: Orchidaceae). Mol Biol Evol 27:2465–2473

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Cervera MT, Ruiz-García L, Martínez-Zapater JM (2002) Analysis of DNA methylation-sensitive AFLP markers. Mol Genet Genomics 268:543–552

    Article  CAS  PubMed  Google Scholar 

  33. Xiong LZ, Xu CG, Saghai Maroof MA, Zhang Q (1999) Patterns of cytosine methylation in an elite rice hybrid and its parental lines, detected by a methylation-sensitive amplification polymorphism technique. Mol Gen Genet 261:439–446

    Article  CAS  PubMed  Google Scholar 

  34. Peraza-Echeverria S, Herrera-Valencia VA, Kay A (2001) Detection of DNA methylation changes in micropropagated banana plants using methylation-sensitive amplification polymorphism (MSAP). Plant Sci 161:359–367

    Article  CAS  PubMed  Google Scholar 

  35. Candaele J, Demuynck K, Mosoti D, Beemster GTS, Inzé D, Nelissen H (2014) Differential methylation during maize leaf growth targets developmentally regulated genes. Plant Physiol 164:1350–1364

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Cervera MT, Remington D, Frigerio JM, Storme V, Ivens B, Boerjan W, Plomion C (2000) Improved AFLP analysis of tree species. Can J For Res 30:1608–1616

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Forest Ecology and Genetic, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria - Centro de InvestigaciónForestal (INIA-CIFOR), Ctra. de La Coruña Km 7,5, Madrid, 28040, Spain

    María Ángeles Guevara, Nuria de María, Enrique Sáez-Laguna, María Dolores Vélez, María Teresa Cervera & José Antonio Cabezas

Authors
  1. María Ángeles Guevara
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Nuria de María
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Enrique Sáez-Laguna
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. María Dolores Vélez
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. María Teresa Cervera
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. José Antonio Cabezas
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to María Teresa Cervera or José Antonio Cabezas .

Editor information

Editors and Affiliations

  1. Department of Biological Sciences, University of Lethbridge, Lethbridge, Alberta, Canada

    Igor Kovalchuk

Rights and permissions

Reprints and permissions

Copyright information

© 2017 Springer Science+Business Media New York

About this protocol

Cite this protocol

Guevara, M.Á., de María, N., Sáez-Laguna, E., Vélez, M.D., Cervera, M.T., Cabezas, J.A. (2017). Analysis of DNA Cytosine Methylation Patterns Using Methylation-Sensitive Amplification Polymorphism (MSAP). In: Kovalchuk, I. (eds) Plant Epigenetics. Methods in Molecular Biology, vol 1456. Humana Press, Boston, MA. https://doi.org/10.1007/978-1-4899-7708-3_9

Download citation

  • DOI: https://doi.org/10.1007/978-1-4899-7708-3_9

  • Published:

  • Publisher Name: Humana Press, Boston, MA

  • Print ISBN: 978-1-4899-7706-9

  • Online ISBN: 978-1-4899-7708-3

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation