Single nucleotide polymorphisms in the ectomycorrhizal mushroom Tricholoma matsutake

Single nucleotide polymorphisms (SNPs) are becoming increasingly popular markers for studying a variety of biological phenomena. This paper describes the development and analysis of a set of SNIP markers for the basidiomycete fungus Tricholoma matsutake. T. matsutake is a gourmet mycorrhizal mushroom primarily associated with pine forests. However, little is known about its genetics and genomic variation, including SNIP variation. To identify and analyse SNPs in T matsutake, a genomic library was constructed and >72 000 nt were analysed from >200 random clones. Primers from 20 sequenced fragments were then designed to amplify and sequence > 10 000 bp sequences from the original strain, from which the genomic library was constructed, as well as another strain from >350 km away; both strains were from south-western China. These two strains had similar intra-strain SNP frequencies (1.104 and 1.278 % per nucleotide, respectively). The combined analysis revealed that 14 of the 20 examined fragments contained SNPs, ranging from two to 47 per fragment, and yielding a total of 178 SNPs out of the 10 428 sequenced nucleotides (an SNP frequency of 1.707 %). Among the 178 SNPs, one site had three alternative nucleotides, while the remaining 177 had two each, with 148 transitions and 29 transversions, resulting in a combined transition to transversion ratio of 5.1. In addition, the haplotype phases of all SNPs within individual fragments for both strains were determined. Phylogenetic analyses of these haplotypes revealed three kinds of haplotype relationship, including haplotype sharing both within and between strains. Furthermore, a subset of the SNPs detectable by restriction enzyme digests was screened for its distribution among 31 additional wild strains from five distinct locations in south-western China. The implications of these SNPs and haplotypes for our understanding of the genetics, population history, ecology and evolution of this important mushroom species are discussed.