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The sclerotia of  Pachyma hoelen  are one of the traditional Chinese medicines and foods that are widely used in East Asian countries. The strains used for cultivation showed bad performance in recent years, and breeding of superior strains has become increasingly important for this fungus. Nevertheless, the mating system and life cycle of  P. hoelen  were still ambiguous. In this study, the methods for distinguishing between homokaryotic offspring with different mating types were established, as well as confirmation of strain hybridization based on allelic polymorphism at a locus of the  rpb2  gene. The bipolar mating system was confirmed according to the mating results of homokaryotic SSIs. The fact that heterokaryotic parents produce homokaryotic meiospores proves that the life cycle is heterothallic. Combining scanning electron microscope observation and DAPI (4′,6-diamidino-2-phenylindole) fluorescent staining of hymenium and basidiospores  in situ  and  ex situ , nuclear migration pattern from basidia to spores was revealed. The heterothallic life cycle was verified, revised, and supplemented step by step. This is the first report of systematic research on the mating system, life cycle, and outcrossing of homokaryotic offspring in  P. hoelen . It will be helpful for the biological research, strain improvement, and development of the  P. hoelen  industry.

Bipolar system of sexual incompatibility and heterothallic life cycle in the basidiomycetes Pachyma hoelen Fr. (Fuling)