Nuclear Functional Divergence and Genetic Basis of Cap Colour Variation Revealed by T2T Genomes of Two Compatible Monokaryons in Pholiota adiposa

Basidiomycete fungi maintain a dikaryotic state throughout their life cycle, in which two genetically distinct haploid nuclei coexist within the same cytoplasm. However, the extent and functional significance of inter-nuclear genomic divergence remain poorly understood. Here, we present the first telomere-to-telomere assemblies of two compatible monokaryons (D1 and D11) from a wild isolate of Pholiota adiposa, achieving 13 complete chromosomes with zero gaps and Benchmarking Universal Single-Copy Orthologs completeness of 98.6% and 98.3%, substantially outperforming the previous fragmented assembly. The two nuclei exhibit only 75% collinear genes, with 570 (D1) and 668 (D11) strain-specific genes. D1-specific genes are enriched in energy metabolism, consistent with faster mycelial growth, while D11-specific genes are enriched in genetic information processing and mitogen-activated protein kinase signalling, revealing functional complementarity between the two haploid nuclei. Resequencing of 18 wild accessions identified two candidate regions associated with red pileus colour on chromosomes 6 and 12, the former as a major-effect locus and the latter harbouring multiple pigment-associated genes. Kyoto Encyclopedia of Genes and Genomes enrichment implicated phenylalanine metabolism, tyrosine metabolism, and terpenoid biosynthesis in colour formation, suggesting coordinated regulation across metabolic networks. This framework facilitates dissection of nuclear functional divergence and provides candidate targets for understanding fungal phenotypic variation.