Abstract
Floccularia luteovirens is a valuable medicinal and edible ectomycorrhizal fungus that is endemic to alpine meadows on the Qinghai–Tibet Plateau. It is of significant ecological and pharmacological importance. To overcome the genomic limitations of previous fragmented assemblies, we present the first haplotype-phased, chromosome-scale genome of the Qinghai-derived QHU-1 strain using an integrated approach of PacBio HiFi, Hi-C, and Illumina sequencing. The high-contiguity assembly spans 13 chromosomes with 97.6% BUSCO completeness. Phylogenomic analysis of 31 basidiomycetes clarified a historical misclassification by placing F. luteovirens closest to Mycocalia denudata/Crucibulum laeve, thus confirming its distinct lineage from Armillaria spp. through low synteny and divergent gene family dynamics. Analyses of adaptive evolution revealed strong purifying selection and stable transposable elements, suggesting genomic adaptations to extreme UV/cold stress. AntiSMASH identified 15 biosynthetic gene clusters (BGCs), which encode diverse terpenoids (7), NRPS-like enzymes (4), PKSs (2), and a hybrid synthase with unique KS-AT-PT-A domains, which have the potential to generate novel metabolites. This chromosome-level resource sheds light on the genetic basis of F. luteovirens’ taxonomy, alpine survival, and symbiotic functions while also unlocking its potential for bioprospecting bioactive compounds.