腐霉属的DNA条形码研究

In the previous research of Pythium, it was found that systematic trees generated based on ITS, COXI and β-tubulin sequences showed corresponding relationship with morphological characters and the same isolate of some species contained two or more polymorphic ITS copies (intra-isolate variation) resulting in it impossible sequencing ITS directly. In this research, more Pythium strains in China will be collected and some Pythium strains will be bought from CBS and ＡTCC. Genomic DNA of important strains representing various morphological characteristics will be extracted and the ITS, ?-tubulin, COXI, EF-1α, Gpd and Cal genes of these strains will be amplified and sequenced. Finally, corresponding relationship between sequences and morphological characteristics of Pythium species will be evaluated and identification abilities of ITS, ?-tubulin, COXI, EF-1α, Gpd and Cal to Pythium species will be discriminated. ITS sequence will be chosen as basic DNA barcode of Pythium species and supplementary DNA barcode will be chosen from ?-tubulin, COXI, EF-1α, Gpd and Cal genes. Pythium species which could be distinguished based on ITS or supplementary DNA barcode will be preliminarily determined. The standard of molecular identification of Pythium genus will be set up.

在前期的腐霉研究中发现ITS、β-tubulin和COXI等三个片段序列构建的系统树与腐霉的形态特征存在对应关系，还发现腐霉属部分种类的ITS序列存在同一个菌株含有多态拷贝（菌株内变异）导致无法对ITS直接测序。拟在此基础上增加收集国内的腐霉菌株，从CBS和ＡTCC购买部分重要种类的菌株，选择代表性菌株进行DNA的提取，对ITS、COXI、β-tubulin、EF-1α、Gpd和Cal基因序列进行扩增、测序和聚类分析，分析各序列对腐霉种类的鉴别作用。以ITS序列为腐霉属的基本DNA条形码，从COXI、β-tubulin、EF-1α、Gpd和Cal等基因序列中选出最适合腐霉分类的补充DNA条形码。初步确定ITS能够鉴别的腐霉种类或需要补充DNA条形码才能鉴别的腐霉种类，制定腐霉属的分子鉴定标准。

从全国各地收集收集到525株腐霉菌株经鉴定为51个分类单元，其中研究期间已发表的新种5个，中国新记录种12个和待发表的疑似新种10个。从4个DNA片段即COI、COII、ITS和ß-tub中筛选腐霉的条形码，发现COI和ITS序列是腐霉属合适的DNA条形码。单基因和多基因系统发育分析结果显示，ITS、ITS与COI组合以及COI、COII、ITS和ß-tub四片段组合都能反映出腐霉属的系统演化关系，四基因结合系统树上的主要进化分枝与形态特征的对应关系最好，而传统分类检索表中作为区别腐霉属物种的有性繁殖体的形态特征与各分子系统发育树上的大进化枝没有明显的对应关系。应用DNA条形码对从广西草坪土壤中分离到的122个腐霉菌株进行鉴定得到24个腐霉物种,证明这是便捷和准确的腐霉鉴定方法。项目研究实现了中国腐霉条形码与国际腐霉条形码成果接轨。