Genomic analysis of Tulasnella spp. associated with Cypripedium macranthos var. rebunense

Orchid mycorrhizal (OM) fungi are essential for seed germination and seedling development in orchids. Although research on the molecular mechanisms underlying orchids-OM fungi interactions is progressing, detailed analyses, including fungal genomics, are limited. In this study, we performed short-read genome sequencing of two Tulasnella isolates obtained from the terrestrial orchid Cypripedium macranthos var. rebunense-both capable of inducing seed germination. Using sequencing data, we assembled genomes and predicted gene sets, each demonstrating over 95% completeness based on Benchmarking Universal Single-Copy Orthologs analysis, indicating high-quality assemblies and annotations. Phylogenetic analysis incorporating existing Tulasnella genome data revealed that these isolates belonged to Group IV, an early-diverging lineage distant from the other Tulasnella groups. Comparative genomic analyses demonstrated that Group IV Tulasnella possessed a distinct repertoire of genes encoding carbohydrate-active enzymes and lineage-specific small secreted proteins. Our results indicate that these genes are possibly involved in establishing symbiosis with Cypripedium species. These findings offer novel insights into the molecular basis of orchid-mycorrhizal interactions.