Location: Sunflower and Plant Biology ResearchTitle: Purification of high molecular weight genomic DNA from powdery mildew for long-read sequencing Author
Submitted to: Journal of Visualized Experiments
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/17/2016
Publication Date: 3/31/2017
Citation: Feehan, J.M., Schneibel, K.E., Bouras, S., Underwood, W., Keller, B., Somerville, S.C. 2017. Purification of high molecular weight genomic DNA from powdery mildew for long-read sequencing. Journal of Visualized Experiments. doi:10.3791/55463. Interpretive Summary: Recent advances in DNA sequencing technology now allow for whole genome sequencing of phytopathogens. Data obtained from genome sequences facilitate comparative studies and novel approaches to address plant disease problems. Powdery mildew fungi infect a wide array of agriculturally and horticulturally important plant species and cause significant economic losses. Sequencing of powdery mildew genomes has been limited by the large size and repetitive nature of these genomes, but new sequencing technologies capable of longer read-lengths provide an opportunity to overcome these barriers. In this article we describe a procedure for obtaining clean, high molecular weight genomic DNA suitable for long-read sequencing from powdery mildew fungi. Sequencing and analysis of genomes from multiple powdery mildew species will improve our understanding of these significant agricultural pathogens.
Technical Abstract: 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 for most of the over 900 powdery mildew species. 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. The genomic DNA that is isolated using this procedure is suitable for long-read (>20kb) sequencing. 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 better control of this agricultural pathogen.