Feehan Joanna M, Scheibel Katherine E, Bourras Salim, Underwood William, Keller Beat, Somerville Shauna C
Department of Plant and Microbial Biology, University of California Berkeley; John Innes Centre, Norwich Research Park.
Department of Plant and Microbial Biology, University of California Berkeley.
J Vis Exp. 2017 Mar 31(121):55463. doi: 10.3791/55463.
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.
白粉菌是一类具有重要经济意义的真菌植物病原体。人们对这些病原体的分子生物学和遗传学了解相对较少，部分原因是缺乏完善的遗传和基因组资源。这些生物体具有庞大的重复基因组，这使得基因组测序和组装极其困难。在此，我们描述了从一种白粉菌——菊科白粉菌（Golovinomyces cichoracearum）中收集、提取、纯化和进行高分子量基因组DNA质量控制评估的方法。所描述的方案包括对孢子进行机械破碎，然后进行优化的酚/氯仿基因组DNA提取。典型产量为每150毫克分生孢子产生7微克DNA。使用此程序分离的基因组DNA适用于长读长测序（即>48.5千碱基对）。该方法还描述了确保基因组DNA大小、产量和纯度的质量控制措施。对这里所述质量的基因组DNA进行测序将有助于多个白粉菌基因组的组装和比较，进而更好地了解并改进对这种农业病原体的控制。
