An Integrated genetic approach for mating-type identification and nuclear analysis in the dikaryotic fungus Stropharia rugosoannulata

The prolonged dikaryotic phase is a defining feature of many basidiomycete fungi, but it complicates the study of mating compatibility, nuclear organization, and gene function. Mating tests typically require monokaryotic tester strains, which are difficult to isolate from dikaryons. In Stropharia rugosoannulata, a highly heterozygous edible mushroom, the limited availability of genetic tools has further constrained these studies. This study aimed to establish an integrated genetic approach for mating-type identification and nuclear analysis in this fungus. PacBio HiFi sequencing was used to generate haplotype-resolved genome assemblies of the dikaryotic strain SR18, enabling the identification of homeodomain and pheromone/pheromone receptor mating-type genes. Based on haplotype-specific polymorphisms, diagnostic polymerase chain reaction markers were developed for rapid and reliable identification of all four mating types without genome resequencing. An endogenous glyceraldehyde-3-phosphate dehydrogenase promoter containing its first intron was identified and shown to consistently enhance heterologous gene expression. Combined with a mycelial fragment-based Agrobacterium tumefaciens-mediated transformation method, this promoter-intron combination enabled the stable use of multiple selectable markers within the same strain background. Furthermore, histone H2B-based green fluorescent protein and mCherry reporters were introduced into compatible monokaryons. These reporters enabled the visualization of heterokaryon formation and dikaryotic nuclear organization in living hyphae. This study establishes an integrated genetic approach that combines haplotype-resolved genome analysis, mating-type identification, genetic transformation, and fluorescent nuclear labeling in S. rugosoannulata. The resulting tools provide a basis for investigating mating and nuclear organization in this dikaryotic basidiomycete. An integrated genetic approach was developed for studying mating types and nuclear organization in Stropharia rugosoannulata. PCR-based markers enable reliable identification of four mating types in a dikaryotic background. An endogenous GPD promoter with intron-mediated enhancement improves transformation efficiency. Fluorescent nuclear labeling allows visualization of heterokaryon formation in living hyphae.