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Phytoplasma pp 405-416 | Cite as

Mapping the Phytoplasma Chromosome

  • Sylvie Malembic-MaherEmail author
  • Patricia Carle
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 938)

Abstract

Physical and genetic mapping of the phytoplasma chromosome can be a useful tool in a genome sequencing project in order to assemble the in silico-predicted contigs robustly. Mapping consists of four distinct steps: preparation of phytoplasma chromosomes from infected plants, single- and double-digestion of chromosomes with rare-cutting restriction enzymes, separation of large DNA fragments by pulsed-field gel electrophoresis, and hybridization with various genetic markers. Materials and methods needed for each step are described and the technique is illustrated using the flavescence dorée phytoplasma genome map as an example.

Key words

Flavescence dorée phytoplasma Genetic map Mollicutes Physical map Pulsed-field gel electrophoresis Restriction endonucleases Southern blot 

Notes

Acknowledgements

This research was funded by the French National Institute of Agronomical Research (INRA) and by the Interprofessional Council of Bordeaux Wine (CIVB). We thank our colleagues Pascal Salar and Géraldine Gourgues for elaborating the protocols for phytoplasma chromosome isolation and for PFGE.

References

  1. 1.
    Hogenhout SA, Šeruga MM (2010) Phytoplasma genomics, from sequencing to comparative and functional genomics—what have we learnt? In: Weintraub PG, Jones P (eds) Phytoplasmas: genomes, plant hosts and vectors. CABI Publishing, Oxfordshire, pp 19–36Google Scholar
  2. 2.
    Sugio A et al (2011) Diverse targets of phytoplasma effectors: from plant development to defense against insects. Annu Rev Phytopathol 49:175–195PubMedCrossRefGoogle Scholar
  3. 3.
    Firrao G, Smart CD, Kirkpatrick BC (1996) Physical map of the Western X-disease phytoplasma chromosome. J Bacteriol 178:3985–3988PubMedGoogle Scholar
  4. 4.
    Padovan AC et al (2000) Chromosome mapping of the sweet potato little leaf phytoplasma reveals genome heterogeneity within the phytoplasmas. Microbiology 146:893–902PubMedGoogle Scholar
  5. 5.
    Lauer U, Seemüller E (2000) Physical map of the chromosome of the apple proliferation phytoplasma. J Bacteriol 182:1415–1418PubMedCrossRefGoogle Scholar
  6. 6.
    Marcone C, Seemüller E (2001) A chromosome map of the European stone fruit yellows phytoplasma. Microbiology 147:1213–1221PubMedGoogle Scholar
  7. 7.
    Malembic-Maher S et al (2008) A chromosome map of the Flavescence dorée phytoplasma. Microbiology 154:1454–1463PubMedCrossRefGoogle Scholar
  8. 8.
    Bautsch W (1988) Rapid physical mapping of the Mycoplasma mobile genome by two-dimensional field inversion gel electrophoresis techniques. Nucl Acids Res 16:11461–11467PubMedCrossRefGoogle Scholar
  9. 9.
    Herschleb J, Ananiev G, Schwartz DC (2007) Pulsed-field gel electrophoresis. Nat Protoc 2:677–684PubMedCrossRefGoogle Scholar
  10. 10.
    Schwartz DC, Cantor CR (1984) Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophoresis. Cell 37:67–75PubMedCrossRefGoogle Scholar
  11. 11.
    Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
  12. 12.
    Schwartz DC et al (1993) Ordered restriction maps of Saccharomyces cerevisiae chromosomes constructed by optical mapping. Science 262:110–114PubMedCrossRefGoogle Scholar
  13. 13.
    Latreille P et al (2007) Optical mapping as a routine tool for bacterial genome sequence finishing. BMC Genomics 8:321PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  1. 1.UMR1332 Fruit Biology and PathologyINRA, University of BordeauxVillenave d’OrnonFrance

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