Cordyceps militaris, a model species in the genus Cordyceps, is widely distributed globally and is known for its significant medicinal value. It has been traditionally used in Chinese medicine to enhance immunity, alleviate fatigue, and treat tumors, among other therapeutic purposes. Here, we successfully assembled a telomere-to-telomere (T2T) level genome of C. militaris CH1 using PacBio HiFi and Hi-C technologies. The assembled genome is 32.67 Mb in size, with an N50 of 4.70 Mb. Gene prediction revealed a total of 10,749 predicted genes in the C. militaris CH1 genome, with a gene completeness of 99.20%. Phylogenetic analysis showed the evolutionary relationship between C. militaris CH1 and other Cordyceps species, suggesting that the divergence between this strain and C. militaris ATCC 34164 occurred approximately 1.36 Mya. Combined transcriptomic and metabolomic analyses identified 842 differentially expressed genes and 2052 metabolites that were significantly altered under light stress, primarily involving key pathways related to amino acid metabolism, purine metabolism, and secondary metabolite biosynthesis. Joint analysis of genes and metabolites revealed 79 genes coding for enzymes associated with the synthesis of adenine and adenosine, with the expression of 52 genes being upregulated, consistent with the accumulation trends of adenine and adenosine. Four gene clusters related to the synthesis of cordycepin were identified, with a significant upregulation of cns3 (FUN_003263), suggesting that light stress may promote cordycepin biosynthesis. This comprehensive analysis not only provides new insights into the genomics, metabolomics, and functional gene research of C. militaris CH1 but also offers a potential biological foundation for understanding the synthesis mechanisms of cordycepin and its efficient production.

Morchella is an edible and medicinal fungus valued for its distinctive flavor and nutritional properties. However, unstable yield and quality remain major constraints on its commercial cultivation. Although soybean meal is used as an organic nutrient supplement in edible mushroom production, its effects on protein accumulation and transcriptional regulation in Morchella remain unclear. This study combined fruiting-body protein measurements with transcriptome analysis to compare soybean meal supplied through soil application or exogenous nutrient bags and to characterize tissue-specific responses in the cap and stipe. Soybean meal increased protein content in an application method-, concentration-, and tissue-dependent manner. Soil application was more effective than nutrient-bag supplementation, while stipes exhibited a stronger transcriptional response than caps. RNA sequencing detected 11,982 expressed genes and revealed both shared and tissue-specific responses to the two supplementation methods. Gene Ontology analysis showed broadly similar functional categories among comparison groups, whereas KEGG enrichment analysis revealed distinct metabolic responses. In caps, soil application was associated with glutathione metabolism and amino sugar and nucleotide sugar metabolism, while nutrient-bag supplementation was associated with the pentose phosphate pathway, glyoxylate and dicarboxylate metabolism, and pentose and glucuronate interconversions. In stipes, the genes involved in ribosome and aminoacyl-tRNA biosynthesis were downregulated under soil application and nutrient-bag supplementation, respectively. Weighted gene co-expression network analysis identified three modules associated with protein content and highlighted ribosome, aminoacyl-tRNA biosynthesis, glycerophospholipid metabolism, and arginine biosynthesis as candidate pathways. The most highly connected genes in each key module were identified as hub genes, and selected expression patterns were supported by qRT-PCR. These findings provide a transcriptomic framework for understanding how soybean meal supplementation is associated with protein accumulation and tissue-specific physiological responses in Morchella.