Long-read sequencing technologies are having a major impact on our approaches to studying non-model organisms and microbial communities. By significantly reducing the cost and facilitating the genome assembly pipelines, any laboratory can now develop its own genomics program regardless of the complexity of the genome studied. The most crucial current challenge is to develop efficient protocols for extracting genomic DNA (gDNA) with high quality and integrity adapted to the organism of interest. This can be particularly complex for obligate pathogens that must maintain intimate interactions inside infected host tissues. Here we propose a simple and cost-effective method for high molecular weight gDNA extraction from spores of Plasmopara halstedii, an obligate biotroph oomycete pathogen responsible for downy mildew in sunflower. We optimized the yield, the quality and the integrity of the extracted gDNA by fine-tuning three critical parameters, the grinding, the lysis temperature and the lysis duration. We obtained gDNA with a fragment size distribution reaching a peak ranging from 79 to 145 kb. More than half of the extracted gDNA consisted of DNA fragments larger than 42 kb, with 23% of fragments larger than 100 kb. We then demonstrated the relevance of this protocol for long-read sequencing using PacBio RSII technology. With this protocol, we were able to obtain a mean read length of 9.3 kb, a max read length of 71 kb and an N50 of 13.3 kb. The development of such DNA extraction protocols is an essential prerequisite for fully exploiting technologies requiring high molecular weight gDNA (e.g. long-read sequencing or optical mapping). These technological advances will help generate data to answer questions such as the role of newly duplicated gene clusters, repeated regions, genomic structural variations or to define number of chromosomes that still remains undefined in many species of pathogenic fungi and oomycetes.

长读长测序技术对我们研究非模式生物和微生物群落的方法产生了重大影响。通过显着降低成本和促进基因组组装流程，任何实验室现在都可以开发自己的基因组学计划，而不管研究的基因组的复杂性如何。当前最关键的挑战是开发适用于感兴趣的生物体的高质量和完整性提取基因组 DNA (gDNA) 的有效方案。对于必须在受感染的宿主组织内保持密切相互作用的专性病原体来说，这可能特别复杂。在这里，我们提出了一种从Plasmopara halstedii孢子中提取高分子量 gDNA 的简单且经济高效的方法，一种专性生物营养卵菌病原体，负责向日葵霜霉病。我们通过微调研磨、裂解温度和裂解持续时间这三个关键参数来优化提取的 gDNA 的产量、质量和完整性。我们获得了片段大小分布达到峰值范围为 79 到 145 kb 的 gDNA。超过一半的提取 gDNA 由大于 42 kb 的 DNA 片段组成，其中 23% 的片段大于 100 kb。然后，我们使用 PacBio RSII 技术证明了该协议与长读长测序的相关性。使用此协议，我们能够获得 9.3 kb 的平均读取长度、71 kb 的最大读取长度和 13.3 kb 的 N50。例如长读长测序或光学作图）。这些技术进步将有助于生成数据来回答诸如新复制基因簇的作用、重复区域、基因组结构变异等问题，或者确定许多病原真菌和卵菌物种中仍未确定的染色体数量。