Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing, 100081, China.
J Microbiol Methods. 2013 Mar;92(3):316-22. doi: 10.1016/j.mimet.2012.11.021. Epub 2013 Jan 10.
Genotyping is fundamental to population analysis. To accommodate fast, accurate and cost-effective genotyping, a one-step multiplex PCR method employing twelve simple sequence repeat (SSR) markers was developed for high-throughput screening of Phytophthora infestans populations worldwide. The SSR markers reported for this species were evaluated and the twelve most informative and easily scored were selected. To accomplish a single step genotyping procedure, we optimized primers, fluorescent labels and PCR conditions to genotype using a capillary electrophoresis system with four fluorescent labels (FAM, NED, PET and VIC) and a labeled LIZ standard for sizing of the SSR fragments. The results obtained using commercially available multiplex PCR kits on a set of reference isolates were in agreement with that obtained using primer pairs in simplex reactions. In testing on many thousands of isolates, we have found the markers appropriate for resolving distinct multilocus genotypes (MLGs) of isolates of European and wider populations. Here we demonstrate the utility of the assay on a set of 19 reference isolates plus 77 others sampled from The Netherlands and Great Britain. In most isolates one to two alleles were observed at each locus but the presence of three alleles at a single locus in some isolates was consistent with increased ploidy. Methods are presented that are appropriate for the analysis of datasets comprising isolates of mixed ploidy levels. We also report on the direct P. infestans genotyping from infected field material to collect, store and extract pathogen DNA. A critical step in this multiplex method was the standardization of the protocol between two laboratories in The Netherlands and Great Britain. Reference isolates were exchanged and an allele nomenclature and scoring system agreed. Such co-operation is facilitating the genotyping of international collections of P. infestans isolates in wider networks of laboratories and providing the data required to expand an existing international database of pathogen diversity.
基因分型对于种群分析至关重要。为了适应快速、准确和具有成本效益的基因分型，我们开发了一种一步多重 PCR 方法，该方法使用 12 个简单重复序列（SSR）标记，用于高通量筛选全球马铃薯晚疫病菌种群。对该物种报告的 SSR 标记进行了评估，并选择了 12 个最具信息量和易于评分的标记。为了完成一步基因分型程序，我们优化了引物、荧光标记物和 PCR 条件，以使用带有 4 种荧光标记物（FAM、NED、PET 和 VIC）和一个标记的 LIZ 标准的毛细管电泳系统进行基因分型，用于 SSR 片段的大小测定。使用商业可得的多重 PCR 试剂盒在一组参考分离物上获得的结果与使用单重反应中的引物对获得的结果一致。在对数千个分离物进行测试时，我们发现这些标记物适合于区分欧洲和更广泛种群的分离物的不同多位点基因型（MLGs）。在这里，我们在一组 19 个参考分离物以及从荷兰和英国采集的另外 77 个分离物上展示了该测定的实用性。在大多数分离物中，每个位点观察到一个到两个等位基因，但在一些分离物中一个位点存在三个等位基因与倍性增加一致。我们提出了适用于分析包含混合倍性水平分离物的数据集的方法。我们还报告了直接从受感染的田间材料中进行马铃薯晚疫病菌的基因分型，以收集、存储和提取病原体 DNA。该多重方法的一个关键步骤是在荷兰和英国的两个实验室之间标准化方案。参考分离物进行了交换，并达成了等位基因命名法和评分系统。这种合作正在促进国际马铃薯晚疫病菌分离物的基因分型，在更广泛的实验室网络中提供所需的数据，并扩展现有的病原体多样性国际数据库。
