Malapi-Wight Martha, Demers Jill E, Veltri Daniel, Marra Robert E, Crouch Jo Anne
United States Department of Agriculture, Agricultural Research Service (ARS), Systematic Mycology and Microbiology Laboratory, Beltsville, MD 20705, USA.
Oak Ridge Institute for Science and Education, ARS Research Participation Program, Oak Ridge, TN 37831, USA.
Sci Rep. 2016 May 20;6:26140. doi: 10.1038/srep26140.
Rapid and accurate molecular diagnostic tools are critical to efforts to minimize the impact and spread of emergent pathogens. The identification of diagnostic markers for novel pathogens presents several challenges, especially in the absence of information about population diversity and where genetic resources are limited. The objective of this study was to use comparative genomics datasets to find unique target regions suitable for the diagnosis of two fungal species causing a newly emergent blight disease of boxwood. Candidate marker regions for loop-mediated isothermal amplification (LAMP) assays were identified from draft genomes of Calonectria henricotiae and C. pseudonaviculata, as well as three related species not associated with this disease. To increase the probability of identifying unique targets, we used three approaches to mine genome datasets, based on (i) unique regions, (ii) polymorphisms, and (iii) presence/absence of regions across datasets. From a pool of candidate markers, we demonstrate LAMP assay specificity by testing related fungal species, common boxwood pathogens, and environmental samples containing 445 diverse fungal taxa. This comparative-genomics-based approach to the development of LAMP diagnostic assays is the first of its kind for fungi and could be easily applied to diagnostic marker development for other newly emergent plant pathogens.
快速准确的分子诊断工具对于尽量减少新出现病原体的影响和传播至关重要。新型病原体诊断标志物的鉴定面临诸多挑战，尤其是在缺乏种群多样性信息且遗传资源有限的情况下。本研究的目的是利用比较基因组学数据集来寻找适合诊断两种导致黄杨木新出现疫病的真菌物种的独特靶标区域。从亨氏拟茎点霉和假舟形拟茎点霉以及三种与该疾病无关的相关物种的基因组草图中鉴定出环介导等温扩增（LAMP）检测的候选标志物区域。为了提高识别独特靶标的概率，我们基于（i）独特区域、（ii）多态性和（iii）数据集中区域的存在/缺失，采用三种方法挖掘基因组数据集。从一组候选标志物中，我们通过检测相关真菌物种、常见黄杨木病原体以及包含445种不同真菌类群的环境样本，证明了LAMP检测的特异性。这种基于比较基因组学的LAMP诊断检测方法是首次用于真菌，并且可以很容易地应用于其他新出现植物病原体的诊断标志物开发。
