Bipolar, not tetrapolar: mating system determination in Inonotus hispidus through genomic and phenotypic analysis.

Inonotus hispidus is a traditional medicinal mushroom in China with significant potential for development of health products and future foods, owing to its diverse functional components and pharmacological activities. Recent advancements in cultivation techniques, coupled with growing market demand, have expanded the production scale of I. hispidus. Breeding superior strains is essential for industry progress, but the absence of clamp connections in I. hispidus complicates mating system studies, making accurate identification of homokaryotic strains a critical step. In this study, we first confirmed the multinucleate nature of both heterokaryotic and homokaryotic mycelia, revising the traditional concepts of monokaryotic and dikaryotic mycelia in this species. Additionally, the mating type loci of I. hispidus were identified through genome sequencing and homologous gene BLAST analysis. Homokaryotic and heterokaryotic strains were distinguished based on sequence differences at the mating type loci between different mating types, which also allowed for differentiation of the mating types themselves. Furthermore, by combining traditional mating tests, we clearly elucidated the bipolar mating system of I. hispidus, refuting previous reports of a tetrapolar system. The growth rate of mycelium, its performance on a wheat grain substrate, as well as the antagonism between the homokaryotic strain and the heterokaryotic parent strain have been demonstrated to be useful for distinguishing the homokaryons. This study established a reliable method for identifying homokaryotic strains and systematically characterized the mating system of I. hispidus for the first time. These findings provide scientific foundation for uncovering the life cycle and presents methods for creating new germplasms. KEY POINTS: • First confirmation of a bipolar mating system in Inonotus hispidus • Single-spore isolates are multinucleate homokaryons • The significant growth rate differences provide method for homokaryon identification.