| Summary: | <i>Hericium rajendrae</i> is an emerging species in the genus <i>Hericium</i> with few members. Despite being highly regarded due to its rarity, knowledge about <i>H. rajendrae</i> remains limited. In this study, we sequenced, de novo assembled, and annotated the complete genome of <i>H. rajendrae</i> NPCB A08, isolated from the Qinling Mountains in Shaanxi, China, using the Illumina NovaSeq and Nanopore PromethION technologies. Comparative genomic analysis revealed similarities and differences among the genomes of <i>H. rajendrae</i>, <i>H. erinaceus</i>, and <i>H. coralloides</i>. Phylogenomic analysis revealed the divergence time of the <i>Hericium</i> genus, while transposon analysis revealed evolutionary characteristics of the genus. Gene family variation reflected the expansion and contraction of orthologous genes among <i>Hericium</i> species. Based on genomic bioinformation, we identified the candidate genes associated with the mating system, carbohydrate-active enzymes, and secondary metabolite biosynthesis. Furthermore, metabolite profiling and comparative gene clusters analysis provided strong evidence for the biosynthetic pathway of erinacines in <i>H. rajendrae</i>. This work provides the genome of <i>H. rajendrae</i> for the first time, and enriches the genomic content of the genus <i>Hericium</i>. These findings also facilitate the application of <i>H. rajendrae</i> in complementary drug research and functional food manufacturing, advancing the field of pharmaceutical and functional food production involving <i>H. rajendrae</i>.
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