Purification of High Molecular Weight Genomic DNA from Powdery Mildew for Long-Read Sequencing

• Published in: Journal of visualized experiments no. 121
• Main Authors: Feehan, Joanna M, Scheibel, Katherine E, Bourras, Salim, Underwood, William, Keller, Beat, Somerville, Shauna C
• Format: Journal Article
• Language: English
• Published: United States MyJove Corporation 31-03-2017
• Subjects: Ascomycota - genetics; DNA, Fungal - chemistry; DNA, Fungal - genetics; DNA, Fungal - isolation & purification; Genetics; Genome, Fungal - genetics; Molecular Weight; Plant Diseases - microbiology; Sequence Analysis, DNA; Spores, Fungal - genetics
• DOI: 10.3791/55463
• EISSN: 1940-087X

The powdery mildew fungi are a group of economically important fungal plant pathogens. Relatively little is known about the molecular biology and genetics of these pathogens, in part due to a lack of well-developed genetic and genomic resources. These organisms have large, repetitive genomes, which have made genome sequencing and assembly prohibitively difficult. Here, we describe methods for the collection, extraction, purification and quality control assessment of high molecular weight genomic DNA from one powdery mildew species, Golovinomyces cichoracearum. The protocol described includes mechanical disruption of spores followed by an optimized phenol/chloroform genomic DNA extraction. A typical yield was 7 µg DNA per 150 mg conidia. The genomic DNA that is isolated using this procedure is suitable for long-read sequencing (i.e., > 48.5 kbp). Quality control measures to ensure the size, yield, and purity of the genomic DNA are also described in this method. Sequencing of the genomic DNA of the quality described here will allow for the assembly and comparison of multiple powdery mildew genomes, which in turn will lead to a better understanding and improved control of this agricultural pathogen.