Specificity, sensitivity and discrimination of primers for PCR–RFLP of larger basidiomycetes and their applicability to identification of ectomycorrhizal fungi in Eucalyptus forests and plantations
Specificity, sensitivity and discrimination of primers for PCR–RFLP of larger basidiomycetes and their applicability to identification of ectomycorrhizal fungi in Eucalyptus forests and plantations
复制标题
大型担子菌 PCR-RFLP 引物的特异性、敏感性和辨别力及其在桉树林和人工林外生菌根真菌鉴定中的适用性
DOI：
10.1017/s0953756200003361
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发表时间：
2001
期刊：
影响因子：
2.5
通讯作者：
Philip A. O'Brien
中科院分区：
文献类型：
--
作者：
M. Glen;I. Tommerup;N. Bougher;Philip A. O'Brien
快速识别外生菌根群落的担子菌真菌伙伴的技术将是对种植园、森林、荒地和其他本地或自然植被中表层和地下形式的生态、真菌种群动态和生活史研究的主要优势。 DNA区域的PCR-RFLP（聚合酶链式反应-限制性片段长度多态性）鉴定是一种可用的技术；然而，需要很有可能仅扩增担子菌 DNA 的引物。在这里，我们评估了六种不同引物对（三个靶向核区域和三个线粒体区域）的特异性、敏感性和辨别力，用于通过将 PCR-RFLP 模式与形态学定义的物种相匹配来鉴定来自桉树森林的澳大利亚担子菌。两套引物，一套是新设计的，针对核糖体DNA内转录间隔区（ITS），另一套是扩增线粒体大亚基核糖体DNA片段，满足高特异性和灵敏度的要求，扩增来自多种较大担子菌的DNA，不扩增植物、细菌或子囊菌DNA。将ITS引物对的特异性与ITS1-F/ITS4-B进行比较。两个地区的 PCR-RFLP 在物种水平上区分了 28 科 91 种真菌。因此，这两个 DNA 区域和特异性引物是一种潜在的实用 PCR-RFLP 工具，用于从田间样品中识别与植物相关的担子菌。
Techniques to rapidly identify the basidiomycete fungal partner of ectomycorrhizal associations would be a major advantage for ecological, fungal population dynamics and life history studies of epigeous and hypogeous forms in plantations, forests, wild lands and other native or natural vegetation. PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) identification of DNA regions is an available technique; however, primers which have a high probability of amplifying only the basidiomycete DNA are needed. Here we have assessed the specificity, sensitivity and discrimination of six different primer pairs, three targeting nuclear and three mitochondrial regions, for use in identification of Australian basidiomycete fungi from Eucalyptus forests by matching PCR-RFLP patterns to morphologically defined species. Two sets of primers, one newly designed and targeting the nuclear ribosomal DNA internal transcribed spacers (ITS) and the other amplifying a fragment of mitochondrial large subunit ribosomal DNA met the requirements of high specificity and sensitivity, amplifying DNA from a broad range of larger basidiomycetes, with no amplification of plant, bacterial or ascomycete DNA. The specificity of the ITS primer pair was compared with that of ITS1-F/ITS4-B. PCR-RFLP of the two regions discriminated fungi to species level for 91 fungal species from 28 families. Hence these two DNA regions and the specific primers are a potential practical PCR-RFLP tool for identifying basidiomycetes associated with plants from field samples.