疫霉属（Phytophthora）DNA条形码的选择与评价

DNA barcoding is a technique for identifying and characterizing species of organisms using a short DNA region. It is becoming more and more attractive to biologists as a new method to aid in species identification. DNA barcoding has been successfully employed for species identification in diverse groups of organisms such as insects, fishes, birds and prokaryotes. Phytophthora is a genus of plant pathogens responsible for severe damage to crops, natural vegetation and forestry worldwide. However, a universal DNA barcode for genus Phytophthora not been formally established. Our knowledge on genus Phytophthora DNA barcoding is still limited compared with that on animals and plants. . The aim of the project is to determine the candidate sequences which can be used as DNA barcode to identify species in genus Phytophthora by screening out from eleven different DNA fragments sequences of COI, Ypt1, (mtSSU)rDNA, TEF1-α, ITS, β-tubulin, rbcL, matK, LSU, and ITS2 genes, and the PCR amplification, sequencing efficiency, differential intra- and inter-specific divergences, DNA barcoding gap and identification efficiency were evaluated.. It is expected that this project will absolutely solve the problem of species identification of genus Phytophthora. which will benefits relative studies in ecological investigation, disease management and molecular system phylogenetic of fungi or oomycetes..

DNA条形码是通过一个标准DNA片段对物种进行快速准确鉴定的分子生物学技术，该技术在部分物种鉴别中已得到成功的应用。疫霉属卵菌是农林业中的植物重要病原菌，与动物、植物相比，针对疫霉菌开展的DNA 条形码研究十分有限，目前尚没有统一的疫霉菌DNA条形码。本项目拟以疫霉属中概念清晰的种为材料，通过比较COI、Ypt1、（mtSSU）rDNA、TEF1-α、ITS、β-tubulin、rbcL、matK、LSU和ITS2等9个基因的PCR 扩增成功率、测序效率、种内和种间变异、barcoding gap 和鉴定成功率等指标，从中筛选出可用于疫霉属卵菌鉴定的DNA条形码通用序列。研究结果将有助于疫霉属卵菌的简便、准确、快速识别，对制定植物卵菌病害防制对策具有重要意义，还可为其它真菌类群的分类与系统发育研究提供参考。

由于疫霉属种间形态学特征差异小，相似性高，其分类一直难以确定。为探讨DNA条形码技术对疫霉属种类进行分类鉴定的可行性，本项目以29种疫霉菌147菌株的总DNA为模板，利用PCR技术扩增了6个DNA条形码片段（ITS、TEF-1α、Ypt1、β-tubulin、COΙ和COⅡ），并进行测序，比较6个基因序列的扩增率及测序成功率，应用MEGA 7.0 软件对序列进行比对及分析其种内与种间遗传距离，采用Neighbor-joining (NJ)法构建系统发育树。结果表明，Ypt1和β-tubulin基因PCR扩增与测序成功率高，且种内、种间遗传距离频率分布不存在重叠，TEF-1α基因可以恰当区分种内和种间差异，但测序成功率相对较低，ITS、COΙ和COⅡ基因虽然具有较高的PCR扩增与测序成功率，但种内、种间距离频率分布存在重叠。运用NJ法构建的疫霉菌系统进化树中，基于Ypt1和β-tubulin基因序列所构建的物种系统进化树要优于基于TEF-1α、ITS、COΙ和COⅡ序列所构建的系统进化树，基于Ypt1和β-tubulin基因序列构建的系统发育树的聚类分析与形态学鉴定结果基本一致。综合上述指标可看出，Ypt1和β-tubulin基因是疫霉属理想的条形码基因，可成功应用于疫霉种鉴定，TEF-1α效果次之，ITS、COΙ和COⅡ不宜作为疫霉属卵菌DNA条形码。基于DNA条形码基因Ypt1和β-tubulin开展了疫霉菌部分重要物种的快速分子检测和鉴定技术。以DNA条形码基因Ypt1和β-tubulin为PCR和LAMP分子检测靶标，设计出了番茄晚疫病菌、豇豆疫霉菌、芋疫霉菌、樟疫霉菌、恶疫霉菌、苜蓿疫霉菌和棕榈疫霉菌等物种的特异性引物，开展了PCR和LAMP可视化快速检测技术研究。基于DNA条形码基因开发的PCR和LAMP方法，可应用于疫霉菌的快速、特异性检测和鉴定，为疫霉菌所致病害的及时有效防治提供了技术手段。本项目发表学术论文4篇，会议论文1篇，投稿论文3篇，申请国家发明专利6项，培养硕士研究生1名。