The Neurospora crassa Pangenome: A Robust Framework for Population-Scale Analysis and Structural Variant Discovery.

Neurospora crassa is a widely distributed ascomycete with high genetic diversity, yet reliance on limited reference genomes has hindered a comprehensive understanding of its genetic landscape. To address this limitation, we integrated the functional annotation of the FGSC2225 genome with a comprehensive comparative genomic analysis of N. crassa strains. FGSC2225 gene and transposable element (TE) proportions mirrored those of FGSC2489, though TE levels were significantly higher than those in sister species Sordaria macrospora. Phylogenetic analysis resolved the N. crassa population into two primary lineages: Clade A (including FGSC2489 and FGSC2225) and Clade B (including FGSC4830), with the former exhibiting larger genome sizes. Leveraging de novo assemblies of 72 high-quality draft genomes, we constructed a comprehensive pangenome to investigate the molecular evolution of various gene families. For example, systematic phylogenetic analysis of the HET-domain-containing gene family and three stress-related families-heat shock transcription factor, basic leucine zipper, and Cytochrome P450-demonstrated varying degrees of conservation and presence/absence variation across the lineages. Addressing the limitations of current genomic resources, this work provides a pangenomic framework to detect rapid adaptive evolution in filamentous fungi. This methodology serves as a robust template for identifying transcription factors, effectors, and structural variations critical to stress response and virulence in diverse fungi.