Highly polymorphic DNA markers to specify strains of the ectomycorrhizal basidiomycete Tricholoma matsutake based on sigmamarY1, the long terminal repeat of gypsy-type retroelement marY1.

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.