The recently described procedure of microsatellite-enriched library pyrosequencing was used to isolate microsatellite loci in the gourmet and medicinal mushroom Agaricus subrufescens. Three hundred and five candidate loci containing at least one simple sequence repeats (SSR) locus and for which primers design was successful, were obtained. From a subset of 95 loci, 35 operational and polymorphic SSR markers were developed and characterized on a sample of 14 A. subrufescens genotypes from diverse origins. These SubSSR markers each displayed from two to 10 alleles with an average of 4.66 alleles per locus. The observed heterozygosity ranged from 0 to 0.71. Several multiplex combinations can be set up, making it possible to genotype up to six markers easily and simultaneously. Cross-amplification in some closely congeneric species was successful for a subset of loci. The 35 microsatellite markers developed here provide a highly valuable molecular tool to study genetic diversity and reproductive biology of A. subrufescens.

Comparative linkage mapping can rapidly facilitate the transfer of genetic information from model species to orphan species. This macrosynteny analysis approach has been extensively used in plant species, but few example are available in fungi, and even fewer in mushroom crop species. Among the latter, the Agaricus genus comprises the most cultivable or potentially cultivable species. Agaricus bisporus, the button mushroom, is the model for edible and cultivable mushrooms. We have developed the first genetic linkage map for the basidiomycete A. subrufescens, an emerging mushroom crop known for its therapeutic properties and potential medicinal applications. The map includes 202 markers distributed over 16 linkage groups (LG), and covers a total length of 1701 cM, with an average marker spacing of 8.2 cM. Using 96 homologous loci, we also demonstrated the high level of macrosynteny with the genome of A. bisporus. The 13 main LG of A. subrufescens were syntenic to the 13 A. bisporus chromosomes. A disrupted synteny was observed for the three remaining A. subrufescens LG. Electronic mapping of a collection of A. subrufescens expressed sequence tags on A. bisporus genome showed that the homologous loci were evenly spread, with the exception of a few local hot or cold spots of homology. Our results were discussed in the light of Agaricus species evolution process. The map provides a framework for future genetic or genomic studies of the medicinal mushroom A. subrufescens.