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Three rapid methods for direct PCR of powdery mildew samples for later sequencing

Irum Mukhtar, Arend F. van Peer, Gerardo Vázquez–Marrufo and Ma. Soledad Vázquez-Garcidueñas, Baogui Xi

Sydowia 70: 155-160

Published online on August 30th, 2018

Powdery mildews are an important obligate parasitic fungal group reported in nearly all regions of the world. DNA isolation for molecular identification of powdery mildew samples is now a standard practice and is becoming more affordable but remains a lengthy process. In this study, three direct DNA isolation methods have been developed for rDNA internal transcribed spacer (ITS) amplification of different powdery mildew species. This reduces time taken for traditional DNA extraction from hours to minutes and rapid PCR means large quantities of DNA are available for sequencing in an hour. Methods proposed in this paper were found rapid and efficient for sequencing. ITS sequences obtained, were neat, clear and comparable to sequences obtained by conventional DNA extraction methods. However, ITS remains insufficient for separation of certain closely related species. In conclusion, our methods are simple, cost-effective and produced enough genomic DNA for quick and high quality sequences. Methods should be trialed further for preparation of DNA libraries of further genomic regions for rapid and accurate powdery mildew identification.

Keywords: ITS, powdery mildews, DNA isolation.

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