Henrion B, Tacon F LE, Martin F
Laboratoire de Microbiologie Forestière, Centre de Recherches Forestières de Nancy, Institut National de la Recherche Agronomique, Champenoux 54280, France.
New Phytol. 1992 Oct;122(2):289-298. doi: 10.1111/j.1469-8137.1992.tb04233.x.
There is a clear requirement to develop sensitive methods for detecting denned isolates of ectomycorrhizal fungi within the complex microbial communities of natural ecosystems and reforestation sites. We present a method that permits the rapid identification of an ectomycorrhizal isolate using enzymatic amplification (polymerase chain reaction) of DNA extracted either from pure cultures or ectomycorrhizas. A set of oligonucleotide primers capable of amplifying full-length nuclear 17S and 25S ribosomal RNA genes, together with the ribosomal internal transcribed spacer and intergenic spacer, have been designed and could be used for amplifying target sequences from a wide range of ectomycorrhizal genera. Length polymorphism in the amplified rDNA and restriction endonuclease analysis of nearly 6-0 kbp of amplified rDNA provided useful criteria for the rapid typing of isolates from different genera and species. Restriction endonuclease analysis of amplified DNA from 26 isolates representing four species of Laccaria (L. bicolor, L. laccata, L. proxima, L. tortilis) yielded up to 20 scored RFLPs and revealed interspecific and intraspecific polymorphism. Most of the polymorphisms were located within the regions corresponding to the internal transcribed spacer and intergenic spacer. The degree of variation observed was sufficient to discriminate several isolates from the same species. Genetic variation was correlated to some extent with geographical origin of the isolates. However, RFLPs of the rRNA genes cannot unambiguously discriminate all selected isolates within Laccaria species, requiring the development of additional DNA probes. Alone, or in combination with other DNA probes, the amplified rDNA genes may serve in the determination of pure fungal cultures and in the characterization of genetic variation of field ectomycorrhizal populations.
在自然生态系统和重新造林地点的复杂微生物群落中，开发用于检测外生菌根真菌特定分离株的灵敏方法有着明确需求。我们提出了一种方法，该方法能够利用从纯培养物或外生菌根中提取的DNA进行酶促扩增（聚合酶链反应），从而快速鉴定外生菌根分离株。已经设计了一组能够扩增全长核17S和25S核糖体RNA基因以及核糖体内部转录间隔区和基因间隔区的寡核苷酸引物，这些引物可用于从多种外生菌根属中扩增靶序列。扩增的rDNA中的长度多态性以及对近6.0 kbp扩增rDNA的限制性内切酶分析为不同属和种的分离株快速分型提供了有用标准。对代表四种蜡蘑属（双色蜡蘑、松塔牛肝菌、近蜡蘑、扭柄蜡蘑）的26个分离株的扩增DNA进行限制性内切酶分析，产生了多达20个可计分的限制性片段长度多态性，并揭示了种间和种内多态性。大多数多态性位于对应于内部转录间隔区和基因间隔区的区域内。观察到的变异程度足以区分同一物种的多个分离株。遗传变异在一定程度上与分离株的地理来源相关。然而，rRNA基因的限制性片段长度多态性不能明确区分蜡蘑属内所有选定的分离株，需要开发额外的DNA探针。扩增的rDNA基因单独或与其他DNA探针结合，可用于确定纯真菌培养物以及表征田间外生菌根群体的遗传变异。
