Single nucleotide polymorphisms in the ectomycorrhizal mushroom Tricholoma matsutake Jianping Xu, 1,2 Hong Guo 2 and Zhu-Liang Yang 1 Correspondence Jianping Xu jpxu@mcmaster.ca fungi@mail.kib.ac.cn 1 Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, Yunnan, People’s Republic of China 2 Department of Biology, McMaster University, Hamilton, ON, Canada Received 28 December 2006 Revised 8 March 2007 Accepted 28 March 2007 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 SNP 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 SNP 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. INTRODUCTION Molecular markers are essential tools to study a variety of biological properties and processes, such as recombination, population structure, and genetic relatedness among individuals. Among these markers, single nucleotide polymorphisms (SNPs) are among the fastest-developing categories in biomedical and biological research. This is mainly because SNPs are the most frequently observed differences between DNA sequences obtained from differ- ent individuals, or between alleles from within an in- dividual in diploid or higher ploidy organisms. In addition, SNPs have several properties, such as a relatively low mutation rate and the ease of scoring and data sharing, that make them highly desirable for a variety of biological analyses (Brumfield et al., 2003; Xu, 2006b). So far, most such studies have focused on model organisms, and relatively little is known about the patterns and distribu- tions of SNPs in non-model organisms, including most fungi. Our objective in this study was to develop and analyse a set of SNP markers for the economically and ecologically important fungus Tricholoma matsutake. T. matsutake is an ectomycorrhizal basidiomycete that produces the economically important edible mushrooms commonly known as ‘matsutake’. It has a monokaryon– dikaryon alternating life cycle typical of most basidiomy- cete mushrooms. Specifically, haploid monokaryotic basidiospores are released from mature fruiting bodies and germinate to form monokaryotic mycelia. When monokaryotic mycelia with different mating types meet, they can mate and form dikaryotic mycelia. Under appropriate conditions, the dikaryotic mycelia can form mature mushrooms and release basidiospores to complete the life cycle (Tominaga, 1978). Abbreviations: IGS, intergenic spacer; ITS, internal transcribed spacer; SNP, single nucleotide polymorphism; SSR, simple sequence repeat. Microbiology (2007), 153, 2002–2012 DOI 10.1099/mic.0.2006/005686-0 2002 2006/005686 G 2007 SGM Printed in Great Britain