Murata Hitoshi, Babasaki Katsuhiko, Yamada Akiyoshi
Department of Applied Microbiology and Mushroom Sciences, Forestry and Forest Products Research Institute, Matsunosato 1, 305-8687 Tsukuba, Japan.
Mycorrhiza. 2005 May;15(3):179-86. doi: 10.1007/s00572-004-0319-0. Epub 2004 Sep 16.
The ectomycorrhizal basidiomycete Tricholoma matsutake produces commercially valuable fruit bodies--matsutake--in Pinus sp. forest. Here we report that PCR with outward facing primers designed based on sequences comprising sigma(marY1), the long terminal repeat of the gypsy-type retroelement marY1, specifies strains of T. matsutake. PCR with a primer based on the 22-bp sequence conserved at the 5'-end of sigma(marY1) conferred 73 reliable bands overall whose profiles depend upon strains of T. matsutake and T. magnivelare, the latter known as 'American matsutake'. This PCR system gave no detectable band in any other species of Tricholoma tested, including T. bakamatsutake and T. fulvocastaneum, symbionts closely related to T. matsutake, as well as a host plant, Pinus densiflora. Similarly, PCR with a set of primers based on 26-bp and 28-bp sequences at bp 48-73 and bp 281-308 of sigma(marY1), internal regions that are mutated in a variant of marY1, conferred 90 reliable bands only in strains of T. matsutake. Theoretically, PCR with the 22-bp primer would allow generation of 2(73), or 9.4x10(21), types of polymorphism, and PCR with a combination of 26- and 28-bp primers, 2(90), or 1.2x10(27) types. The probability of falsely specifying two different isolates as the same strain is <1/10(21). While polymorphisms conferred by the primer based on the 5' end of sigma(marY1) rather exhibit genetic conservation of a group of T. matsutake, those resulting from primers based on the internal sequences more clearly demonstrate intra-specific diversification. Both systems revealed that T. matsutake is divergent within the species. Ectomycorrhizas formed between P. densiflora and T. matsutake were identified by the PCR systems developed in the present study. This method, using sigma(marY1) as a genetic marker, is useful in analyzing the diversity of T. matsutake, monitoring the behavior of individual mycorrhizas, and specifying the ecological background of fruit bodies traded in markets.
外生菌根担子菌松茸在松属植物林中产生具有商业价值的子实体——松茸。在此，我们报告基于包含sigma(marY1)（吉普赛型反转录元件marY1的长末端重复序列）的序列设计的外向引物进行PCR可鉴定松茸菌株。使用基于sigma(marY1) 5'端保守的22 bp序列的引物进行PCR总共产生了73条可靠条带，其条带图谱取决于松茸菌株和大孢口蘑菌株，后者被称为“美国松茸”。该PCR系统在测试的任何其他口蘑物种中均未检测到条带，包括与松茸密切相关的共生菌巴氏口蘑和黄褐口蘑，以及寄主植物赤松。同样，使用基于sigma(marY1) 48 - 73 bp处的26 bp序列和281 - 308 bp处的28 bp序列（marY1变体中发生突变的内部区域）的一组引物进行PCR，仅在松茸菌株中产生了90条可靠条带。理论上，使用22 bp引物进行PCR可产生2(73)，即9.4×10(21)种多态性类型，而使用26 bp和28 bp引物组合进行PCR可产生2(90)，即1.2×10(27)种类型。将两个不同分离株错误鉴定为同一菌株的概率<1/10(21)。虽然基于sigma(marY1) 5'端的引物赋予的多态性更体现了一组松茸的遗传保守性，但基于内部序列的引物产生的多态性更清楚地证明了种内多样性。两种系统均表明松茸在物种内存在差异。本研究开发的PCR系统可鉴定赤松与松茸形成的外生菌根。这种以sigma(marY1)作为遗传标记的方法，对于分析松茸的多样性、监测单个菌根的行为以及确定市场上交易的子实体的生态背景很有用。
