Plant Molecular Biology Reporter

, Volume 18, Issue 4, pp 361–368 | Cite as

AFLP-Based detection of DNA methylation

  • Mingliang Xu
  • Xiangqian Li
  • Schuyler S. Korban


By using the isoschizomersHpa II andMsp I which display differential sensitivity to cytosine methylation, a modified amplified fragment length polymorphism (AFLP) technique has been developed to investigate DNA methylation profiles in eukaryotic organims. Genomic DNA was digested with a mixture ofEcoR I and one of the isoschizomers, and ligated to oligonucleotide adapters. After two rounds of selective PCR amplification, followed by DNA separation on a Long Ranger gel electrophoresis, a subset of restriction fragments can be displayed on an X-ray film. Comparison of AFLP banding patterns betweenHpa II andMsp I revealed the extent of DNA methylation. The technique has been successfully applied in this study to investigate DNA methylation profiles of apple (Malus domestica cv. Gala) genomic DNA extracted from leaves of field-grown adult trees andin vitro-grown shoot cultures. The results showed that up to 25 percent of AFLP bands were derived from methylated sequences, and among those, a few bands unique to either adult trees orin vitro shoots were observed. These results demonstrated that this protocol is effective in identifying methylated DNA profiles.

Key words

amplified fragment length polymorphism (AFLP) apple isoschizomer methylation 


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  1. Ballvora A, Hesselbach J, Niewhner J, Dario Leister, Salmini F and Gebhardt C (1995) Marker enrichment and high-resolution map of the segment of potato chromosome VII harbouring the nematode resistance geneGrol. Mol Gen Genet 249: 82–90.PubMedCrossRefGoogle Scholar
  2. Doerfler W (1983) DNA methylation and gene activity. Annu Rev Biochem 52: 93–124.PubMedCrossRefGoogle Scholar
  3. Gonzalgo ML and Jones PA (1997a) Mutagenic and epigenetic effects of DNA methylation. Mutat Res 386: 107–118.PubMedCrossRefGoogle Scholar
  4. Gonzalgo ML and Jones PA (1997b) Rapid quantitation of methylation differences at specific sites using methylation-sensitive single nucleotide primer extension (Ms-SNuPE). Nucleic Acids Res 25: 2529–2531.PubMedCrossRefGoogle Scholar
  5. Magill JM and Magill CW (1989) DNA methylation in fungi. Dev Genet 10: 63–99.PubMedCrossRefGoogle Scholar
  6. McClelland M, Nelson M and Raschke E (1994) Effect of site-specific modification on restriction edonucleases and DNA modification methyltransferases. Nucleic Acids Res 22: 3640–3659.PubMedCrossRefGoogle Scholar
  7. Meyer P, Niedenhof I and ten Lohuis M (1994) Evidence for cytosine methylation of non-symmetrical sequences in transgenicPetunia hybrida. EMBO J 13: 2084–2088.PubMedGoogle Scholar
  8. Money T, Reader S, Qu LJ, Dunford RP and Moore G (1996) AFLP-based mRNA fingerprinting. Nucleic Acids Res 24: 2616.PubMedCrossRefGoogle Scholar
  9. Reyna-L’opez GE, Simpson J and 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.CrossRefGoogle Scholar
  10. Rossi V, Motto M and Pelligrini L (1997) Analysis of the methylation pattern of the maizeOpaque-2 (O2) promoter and in vitro binding studies indicate that the O2 B-Zip protein and other endosperm factors can bind to methylated target sequences. J Biol Chem 272: 13758–13765.PubMedCrossRefGoogle Scholar
  11. Thomas CM, Vos P, Zabeau M, Jones DA, Norcott KA, Chadwick BP and Jones JDG (1995) Identification of amplified restriction fragment polymorphism (AFLP) markers tightly linked to the tomatoCf-9 gene for resistance toCladosporium fulvum. Plant J 8: 785–794.PubMedCrossRefGoogle Scholar
  12. Ulian EC, Magill JM, Magill CW and Smith RH (1996) DNA methylation and expression of NPT II in transgenic petunias and progeny. Theor Appl Genet 92: 976–981.CrossRefGoogle Scholar
  13. Ushijima T, Morimura K, Hosoya Y, Okonogi H, Tatematsu M, Sugimura T and Nagao M (1997) Establishment of methylation-sensitive-representational difference analysis and isolation of hypo- and hypermethylated genomic fragments in mouse liver tumors. Proc Natl Acad Sci USA 94: 2284–2289.PubMedCrossRefGoogle Scholar
  14. Vos P, Hogers R, Bleeker M, Reijans M, Lee TV, Hornes M, Frijters A, Pot J, Peleman J, Kuiper M and Zabeau M (1995) AFLP: a new technique for DNA fingerprinting. Nucleic Acids Res 23: 4407–4414.PubMedCrossRefGoogle Scholar
  15. Xiong LZ, Xu CG, Saghai Maroof MA and Zhang QF (1999) Patterns of cytosine methylation in an elite rice hybrid and its parental lines by a methylation-sensitive amplification polymorphism technique. Mol Gen Genet 261: 439–446.PubMedCrossRefGoogle Scholar
  16. Xu ML, Melchinger AE, Xia XC and Luebberstedt T (1999) High-resolution mapping of loci conferring resistance to a sugarcane mosaic virus in maize using RFLP, SSR, and AFLP markers. Mol Gen Genet 261: 574–581.PubMedCrossRefGoogle Scholar
  17. Xu ML and Korban SS (2000) Saturation mapping of the apple scab resistance geneVf using AFLP markers. Theor Appl Genet 101: 844–851.CrossRefGoogle Scholar

Copyright information

© Springer 2000

Authors and Affiliations

  • Mingliang Xu
    • 1
  • Xiangqian Li
    • 1
  • Schuyler S. Korban
    • 1
  1. 1.Department of Natural Resources and Environmental SciencesUniversity of IllinoisUrbana

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