Comparative Genomics of the Ectomycorrhizal Sister Species <i>Rhizopogon vinicolor</i> and <i>Rhizopogon vesiculosus</i> (Basidiomycota: Boletales) Reveals a Divergence of the Mating Type <i>B</i> Locus

Divergence of breeding system plays an important role in fungal speciation. Ectomycorrhizal fungi, however, pose a challenge for the study of reproductive biology because most cannot be mated under laboratory conditions. To overcome this barrier, we sequenced the draft genomes of the ectomycorrhizal sister species <i>Rhizopogon vinicolor</i> Smith and Zeller and <i>R. vesiculosus</i> Smith and Zeller (Basidiomycota, Boletales)-the first genomes available for Basidiomycota truffles-and characterized gene content and organization surrounding their mating type loci. Both species possess a pair of homeodomain transcription factor homologs at the mating type <i>A</i>-locus as well as pheromone receptor and pheromone precursor homologs at the mating type <i>B</i>-locus. Comparison of <i>Rhizopogon</i> genomes with genomes from Boletales, Agaricales, and Polyporales revealed synteny of the <i>A</i>-locus region within Boletales, but several genomic rearrangements across orders. Our findings suggest correlation between gene content at the <i>B</i>-locus region and breeding system in Boletales with tetrapolar species possessing more diverse gene content than bipolar species. <i>Rhizopogon vinicolor</i> possesses a greater number of <i>B</i>-locus pheromone receptor and precursor genes than <i>R. vesiculosus</i>, as well as a pair of isoprenyl cysteine methyltransferase genes flanking the <i>B</i>-locus compared to a single copy in <i>R. vesiculosus</i> Examination of dikaryotic single nucleotide polymorphisms within genomes revealed greater heterozygosity in <i>R. vinicolor</i>, consistent with increased rates of outcrossing. Both species possess the components of a heterothallic breeding system with <i>R. vinicolor</i> possessing a <i>B</i>-locus region structure consistent with tetrapolar Boletales and <i>R. vesiculosus</i> possessing a <i>B</i>-locus region structure intermediate between bipolar and tetrapolar Boletales.