Multiple displacement amplification, a powerful tool for molecular genetic analysis of powdery mildew fungi
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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.
KeywordsCytochrome b ITS regions phi29 DNA polymerase Podosphaera fusca Whole genome amplification
This study was funded by a grant from Plan Nacional de Recursos y Tecnologías Agroalimentarias of Ministerio de Educación y Ciencia, Spain (AGL2004–06056). The authors thank Yun-Sik Kim (University of Kentucky, Lexington, USA) for providing us with the fragment of the CYTB gene of M. grisea that was used as a probe.
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