Current Genetics

, Volume 51, Issue 3, pp 209–219 | Cite as

Multiple displacement amplification, a powerful tool for molecular genetic analysis of powdery mildew fungi

  • Dolores Fernández-Ortuño
  • Juan A. Torés
  • Antonio de Vicente
  • Alejandro Pérez-García
Technical Note

Abstract

Powdery mildew fungi (Erysiphales) are probably the largest group of plant pathogens that remain uncharacterized from genetic and molecular points of view, with the only exception of the powdery mildew of cereals, Blumeria graminis. Their nature as obligate biotrophic parasites and consequent inability to grow on culture media has significantly hampered research. A common bottleneck to the molecular genetic analysis of powdery mildew fungi is the availability of genomic DNA of suitable quality and in sufficient quantity. The so-called whole genome amplification technology has the potential to overcome this limitation. Here we present the application of phi29 DNA polymerase-mediated multiple displacement amplification (MDA) to amplify the whole genome of Podosphaera fusca, the main causal agent of powdery mildew in cucurbits, to address this problem. The genome coverage and fidelity of the MDA process was evaluated by PCR amplification and sequencing of two genetics markers: the nuclear rDNA internal transcribed spacer (ITS) regions and the mitochondrial cytochrome b gene (CYTB). Our results show that MDA is a valuable tool for molecular genetic analysis of powdery mildew fungi that can be used for a number of downstream applications in different fields, such as epidemiology and population genetics or systematics.

Keywords

Cytochrome b ITS regions phi29 DNA polymerase Podosphaera fusca Whole genome amplification 

References

  1. Agrios GN (2005) Plant pathology, 5th edn. Elsevier, AmsterdamGoogle Scholar
  2. Álvarez B, Torés JA (1997) Cultivo in vitro de Sphaerotheca fuliginea (Schlecht. ex. Fr.), efecto de diferentes fuentes de carbono sobre su desarrollo. Bol San Veg Plagas 23:283–288Google Scholar
  3. Banke S, Peschon A, McDonal BA (2004) Phylogenetic analysis of globally distributed Mycosphaerella graminicola populations based on three DNA sequence loci. Fungal Genet Biol 41:226–238PubMedCrossRefGoogle Scholar
  4. Bardin M, Nicot PC, Normand P, Lemaire JM (1997) Virulence variation and DNA polymorphism in Sphaerotheca fuliginea, causal agent of powdery mildew in cucurbits. Eur J Plant Pathol 103:545–554CrossRefGoogle Scholar
  5. Bergen AW, Qi Y, Haque KA, Welch RA, Chanock SJ (2005) Effects of DNA mass on multiple displacement whole genome amplification and genotyping performance. BMC Biotechnol 5:24–35PubMedCrossRefGoogle Scholar
  6. Biswas SK, Yokoyama K, Nishimura K, Miyaji M (2001) Molecular phylogenetics of the genus Rhodotorula and related basidiomycetous yeast inferred from the mitochondrial cytochrome b gene. Int J Syst Evol Microbiol 51:1191–1199PubMedGoogle Scholar
  7. Dean FB, Nelson JR, Giesler TL, Lasken RS (2001) Rapid amplification of plasmid and phage DNA using phi29 DNA polymerase and multiply-primed rolling circle amplification. Genome Res 11:1095–1099PubMedCrossRefGoogle Scholar
  8. Dean FB, Honoso S, Fang LH, Wu XH, Faruqi AF, Bray-Ward P, Sun ZY, Zong QL, Du YF, Du J, Driscoll M, Song WM, Kingsmore SF, Egholm M, Lasken RS (2002) Comprehensive human genome amplification using multiple displacement amplification. Proc Natl Acad Sci USA 99:5261–5266PubMedCrossRefGoogle Scholar
  9. Esteban JA, Salas M, Blanco L (1993) Fidelity of the phi29 DNA polymerase. Comparison between protein-primed initiation and DNA polymerization. J Biol Chem 268:2719–2726PubMedGoogle Scholar
  10. Fernández-Ortuño D, Pérez-García A, López-Ruiz F, Romero D, de Vicente A, Torés JA (2006) Occurrence and distribution of resistance to QoI fungicides in populations of Podosphaera fusca in south central Spain. Eur J Plant Pathol 115:215–222CrossRefGoogle Scholar
  11. Foster SJ, Monahan BJ (2005) Whole genome amplification from filamentous fungi using phi29-mediated multiple displacement amplification. Fungal Genet Biol 42:367–375PubMedCrossRefGoogle Scholar
  12. Fraaije BA, Butters JA, Coelho JM, Jones DR, Hollomon DW (2002) Following the dynamics of strobilurin resistance in Blumeria graminis f. sp. tritici using quantitative allele-specific real-time PCR measurements with the fluorescent dye SYBR Green I. Plant Pathol 51:45–54CrossRefGoogle Scholar
  13. Gadkar V, Rillig MC (2005a) Application of phi29 DNA polymerase mediated whole genome amplification on single spores of arbuscular mycorrhizal (AM) fungi. FEMS Microbiol Lett 242:65–71CrossRefGoogle Scholar
  14. Gadkar V, Rillig MC (2005b) Suitability of genomic DNA synthesized by strand displacement amplification (SDA) for AFLP analysis: genotyping single spores of arbuscular mycorrhizal (AM) fungi. J Microbiol Methods 63:157–164CrossRefGoogle Scholar
  15. Gisi U, Sierotzki H, Cook A, McCaffery A (2002) Mechanisms influencing the evolution of resistance to Qo inhibitor fungicides. Pest Manag Sci 58:859–867PubMedCrossRefGoogle Scholar
  16. Green JR, Carver TLW, Gurr SJ (2002) The formation and function of infection feeding structures. In: Bélanger RR, Bushnell WR, Dik AJ, Carver TLW (eds) The powdery mildews. A comprehensive treatise. APS Press, St. Paul, pp 66–82Google Scholar
  17. Hawkins TL, Detter JC, Richardson PM (2002) Whole genome amplification—applications and advances. Curr Opin Biotechnol 13:65–67PubMedCrossRefGoogle Scholar
  18. Hewitt HG (1998) Fungicides in crop protection. CABI Publishing, WallingfordGoogle Scholar
  19. Hirata T, Cunnington JH, Paksiri U, Limkaisang S, Shishkoff N, Grigaliunaite B, Sato Y, Takamatsu S (2000) Evolutionary analysis of subsection Magnicellulatae of Podosphaera section Spaherotheca (Erysiphales) based on the rDNA internal transcribed spacer sequences with special reference to host plants. Can J Bot 78:1521–1530CrossRefGoogle Scholar
  20. Ishii H, Fraaije BA, Sugiyama T, Noguchi K, Nishimura K, Takeda T, Amano T, Hollomon DW (2001) Occurrence and molecular characterization of strobilurin resistance in cucumber powdery mildew and downy mildew. Phytopathology 91:1166–1171Google Scholar
  21. Kim YS, Dixon P, Vincelli P, Farman ML (2003) Field resistance to strobilurin (QoI) fungicides in Pyricularia grisea caused by mutations in the mitochondrial cytochrome b gene. Phytopathology 93:891–900Google Scholar
  22. Lasken RS, Egholm M (2003) Whole genome amplification: abundant supplies of DNA of precious samples or clinical specimens. Trends Biotechnol 21:531–535PubMedCrossRefGoogle Scholar
  23. McDonal BA (1997) The population genetics of fungi: tools and techniques. Phytopathology 87:448–452Google Scholar
  24. Paez JG, Lin M, Beroukhim R, Lee JC, Zhao X, Richter DJ, Gabriel S, Herman P, Sasaki H, Altshuler D, Li C, Meyerson M, Sellers WR (2004) Genome coverage and sequence fidelity of phi29 polymerase-based multiple strand displacement whole genome amplification. Nucleic Acids Res 18:e71CrossRefGoogle Scholar
  25. Pedersen C, Wu B, Giese H (2002) A Blumeria graminis f. sp. hordei BAC library—contig building and microsynteny studies. Curr Genet 42:103–113PubMedCrossRefGoogle Scholar
  26. Pérez-García A, Mingorance E, Rivera ME, del Pino D, Romero D, Torés JA, de Vicente A (2006) Long-term preservation of Podosphaera fusca using silica gel. J Phytopathol 154:190–192CrossRefGoogle Scholar
  27. Raghunathan A, Ferguson Jr HR, Bornarth CJ, Song W, Driscoll M, Lasken RJ (2005) Genomic DNA amplification from a single bacterium. Appl Environ Microbiol 71:3342–3347PubMedCrossRefGoogle Scholar
  28. Robbertse B, Reeves JB, Schoch CL, Spatafora JW (2006) A phylogenomic analysis of the Ascomycota. Fungal Genet Biol 43:715–725PubMedCrossRefGoogle Scholar
  29. Saenz GS, Taylor JW (1999) Phylogeny of the Erysiphales (powdery mildews) inferred from internal transcribed spacer (ITS) ribosomal DNA sequences. Can J Bot 77:150–169CrossRefGoogle Scholar
  30. Takamatsu S (2004) Phylogeny and evolution of the powdery mildew fungi (Erysiphales, Ascomycota) inferred from nuclear ribosomal DNA sequences. Mycoscience 45:147–157CrossRefGoogle Scholar
  31. Taylor JW, Geiser DM, Burt A, Koufopanou V (1999) The evolutionary biology and population genetics underlying fungal strain typing. Clin Microbiol Rev 12:126–146PubMedGoogle Scholar
  32. Telenius H, Carter NP, Bebb CE, Nordenskjold M, Ponder BAJ, Tunnacliffe A (1992) Degenerate oligonucleotide-primed PCR—general amplification of target DNA by a single degenerate primer. Genomics 13:718–725PubMedCrossRefGoogle Scholar
  33. Wang L, Yokoyama K, Miyaji M, Nishimura K (1998) The identification and phylogenetic relationship of pathogenic species of Aspergillus based on the mitochondrial cytochrome b gene. Med Mycol 36:153–164PubMedCrossRefGoogle Scholar
  34. Zhang L, Cui XF, Schmitt K, Hubert R, Navidi W, Arnheim N (1992) Whole genome amplification from a single cell–implications for genetic analysis. Proc Natl Acad Sci USA 89:5847–5851PubMedCrossRefGoogle Scholar
  35. Zhang Z, Henderson C, Perfect E, Carver TLW, Thomas BJ, Skamnioti P, Gurr SJ (2005) Of gene and genomes, needles and haystacks: Blumeria graminis and functionality. Mol Plant Pathol 6:561–575CrossRefGoogle Scholar
  36. Zitter TA, Hopkins DL, Thomas CE (1996) Compendium of cucurbits diseases. APS Press, St. PaulGoogle Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Dolores Fernández-Ortuño
    • 1
  • Juan A. Torés
    • 1
  • Antonio de Vicente
    • 2
  • Alejandro Pérez-García
    • 2
  1. 1.Estación Experimental “La Mayora” (CSIC)MálagaSpain
  2. 2.Grupo de Microbiología y Patología Vegetal-Unidad Asociada a CSIC, Departamento de Microbiología, Facultad de CienciasUniversidad de MálagaMálagaSpain

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