The T2T genome of Butyriboletus roseoflavus confirms genomic adaptations to ectomycorrhizal symbiosis

Butyriboletus roseoflavus is an edible ectomycorrhizal bolete that is currently only available from wild collection. To investigate genomic features associated with the ectomycorrhizal nutritional mode, we generated gap-free, haplotype-resolved telomere-to-telomere (T2T) genome assemblies of B. roseoflavus specimen HN25B01 using PacBio HiFi sequencing. The two haplotype assemblies, Hap1 and Hap2, were 31.49 Mb and 32.26 Mb in length, respectively. Phylogenomic analysis placed B. roseoflavus close to Butyriboletus appendiculatus and Butyriboletus subappendiculatus , with their common ancestor estimated to have existed approximately 67 million years ago. Using Hap1 as the representative genome, we compared carbohydrate-active enzymes (CAZymes), secreted proteases, small secreted proteins (SSPs), and secondary metabolite biosynthetic gene clusters across fungi representing ectomycorrhizal, dual symbiotrophic/saprotrophic, and saprotrophic nutritional modes. Seven of the 15 functional categories (CE, GH, PL, AP, GP, SP, and T1PKS) showed significant differences in copy numbers among nutritional modes (Kruskal–Wallis test, p < 0.05). Saprotrophic fungi generally exhibited higher CE, GH, PL, SP, and T1PKS copy numbers, whereas ectomycorrhizal fungi generally exhibited higher AP and GP copy numbers. The B. roseoflavus Hap1 genome contained nine CE, 100 GH, two PL, and 21 SP genes, as well as 108 genes encoding SSPs. Metagenomic analysis revealed differences in microbial community composition between soil associated with the stipe base (SBS) of B. roseoflavus fruiting bodies and the surrounding soil (SS). Nitrogen-fixing and phosphate-solubilizing taxa, including Bradyrhizobium and Paraburkholderia , were more abundant in the SBS samples than in the SS samples. Together, these findings offer a high-quality genomic resource and confirm genomic adaptations to ectomycorrhizal symbiosis, which may facilitate future investigations into the biology, nutritional ecology, and cultivation of B. roseoflavus .