Combining nested PCR and restriction digest of the internal transcribed spacer region to characterize arbuscular mycorrhizal fungi on roots from the field
Combining nested PCR and restriction digest of the internal transcribed spacer region to characterize arbuscular mycorrhizal fungi on roots from the field
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DOI：
10.1007/s00572-002-0214-5
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发表时间：
2003-08-01
期刊：
影响因子：
3.9
通讯作者：
Buscot, F
中科院分区：
文献类型：
--
作者：
Renker, C;Heinrichs, J;Buscot, F
用形态学方法鉴定根上的丛枝菌根真菌（AMF）几乎是不可能的，而且由于存在污染真菌，用分子生物学技术也很困难。为了在野外广泛研究AMF的种群结构，我们建立了一种新的方法，用独特的引物选择性扩增大多数AMF的内部转录间隔区（ITS）区域。在现有rDNA序列的基础上，设计了AMF和其他真菌类群的特异性引物对，并与已建立的引物对ITS5/ITS4进行巢式PCR组合。在巢式PCR的第一次反应后，通过AluI限制减少了来自污染生物体的扩增。该方法在代表不同生境类型的五个不同野外站点进行了评估。在不同的地点发现了所有主要类群的小球菌科成员（除了太古孢子科）。利用培养菌株的方法也可以检测到Gigasporaceae。
Identification of arbuscular mycorrhizal fungi (AMF) on roots is almost impossible with morphological methods and, due to the presence of contaminating fungi, it is also difficult with molecular biological techniques. To allow broad investigation of the population structure of AMF in the field, we have established a new method to selectively amplify the internal transcribed spacer (ITS) region of most AMF with a unique primer set. Based on available sequences of the rDNA, one primer pair specific for AMF and a few other fungal groups was designed and combined in a nested PCR with the already established primer pair ITS5/ITS4. Amplification from contaminating organisms was reduced by an AluI restriction after the first reaction of the nested PCR. The method was assessed at five different field sites representing different types of habitats. Members of all major groups within the Glomeromycota (except Archaeosporaceae) were detected at the different sites. Gigasporaceae also proved detectable with the method based on cultivated strains.