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首页> 美国卫生研究院文献>Applied and Environmental Microbiology >The Nuclear Ribosomal DNA Intergenic Spacer as a Target Sequence To Study Intraspecific Diversity of the Ectomycorrhizal Basidiomycete Hebeloma cylindrosporum Directly on Pinus Root Systems
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The Nuclear Ribosomal DNA Intergenic Spacer as a Target Sequence To Study Intraspecific Diversity of the Ectomycorrhizal Basidiomycete Hebeloma cylindrosporum Directly on Pinus Root Systems

机译：核糖体DNA基因间隔子作为目标序列直接研究松树根系上的菌根担子菌Helicoma cylindrosporum的种内多样性

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Polymorphism of the nuclear ribosomal DNA intergenic spacer (IGS) of the ectomycorrhizal basidiomycete Hebeloma cylindrosporum was studied to evaluate whether this sequence could be used in field studies to estimate the diversity of strains forming mycorrhizas on individual Pinus pinaster root systems. This sequence was amplified by PCR from 125 haploid homokaryotic strains collected in 14 P. pinaster stands along the Atlantic coast of France by using conserved oligonucleotide primers. Restriction enzyme digestion of the amplified 3.4-kbp-long IGS allowed us to characterize 24 alleles whose frequencies differed. Nine of these alleles were found only once, whereas about 60% of the strains contained four of the alleles. Local populations could be almost as diverse as the entire population along a 150-km stretch of coastline that was examined; for example, 13 alleles were found in a single forest stand. The IGS from one strain was partially sequenced, and the sequence data were used to design oligonucleotides which allowed separate PCR amplification of three different segments of the IGS. Most polymorphisms observed among the full-length IGS regions resulted from polymorphisms in an internal ca. 1,500-bp-long sequence characterized by length variations that may have resulted from variable numbers of a T2AG3 motif. This internal polymorphic sequence could not be amplified from the genomes of nine other Hebeloma species. Analysis of this internal sequence amplified from the haploid progenies of 10 fruiting bodies collected in a 70-m2 area resulted in identification of six allelic forms and seven distinct diplotypes out of the 21 possible different combinations. Moreover, optimization of the PCR conditions resulted in amplification of this sequence from more than 80% of the DNA samples extracted from individual H. cylindrosporum infected P. pinaster mycorrhizal root tips, thus demonstrating the usefulness of this sequence for studying the below-ground diversity of mycorrhizas formed by genets belonging to the same fungal species.
机译：研究了外生菌根的担子菌Helicoma cylindrosporum的核糖体DNA基因间隔子（IGS）的多态性，以评估该序列是否可用于田间研究，以估计在单个松属松树根系上形成菌根的菌株的多样性。通过使用保守的寡核苷酸引物，通过PCR从在法国大西洋沿岸的14个P. pinaster林分中收集的125个单倍体同核生物菌株中扩增出该序列。扩增的3.4kbp长的IGS的限制性酶消化使我们能够表征频率不同的24个等位基因。这些等位基因中的九个仅被发现一次，而约60％的菌株包含四个等位基因。在经过检查的150公里长的海岸线上，当地居民的多样性几乎与整个人口一样多；例如，在一个林分中发现了13个等位基因。对来自一个菌株的IGS进行部分测序，并将序列数据用于设计寡核苷酸，从而允许对三个不同IGS片段进行单独的PCR扩增。在全长IGS区域中观察到的大多数多态性是由内部ca中的多态性引起的。 1,500 bp长的序列，其特征在于可能由可变数目的T2AG3基序导致的长度变化。无法从其他9个Hebeloma物种的基因组中扩增出此内部多态序列。通过从70-m 2 区域中收集的10个子实体的单倍体后代扩增的内部序列的分析，在21种可能的不同组合中鉴定出6个等位基因形式和7个不同的双倍型。而且，PCR条件的优化导致该序列从80％的DNA样品中扩增而来，该DNA样品是从单个被H.cylindrosporum感染的P. pinaster菌根根尖中提取的，因此证明了该序列对研究地下多样性的有用性属于相同真菌物种的种系形成的菌根。

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