Background: It is generally accepted that nuclear genes in eukaryotes are located independently on chromosomes and expressed in a monocistronic manner. However, accumulating evidence suggests a more complex landscape of gene structure and transcription. Ganoderma lingzhi, a model medicinal fungus, currently lacks high-quality genome annotation, hindering genetic studies. Results: Here, we reported a golden annotation of G. lingzhi, featuring 14,147 high-confidence genes derived from extensive manual corrections. Novel characteristics of gene structure and transcription were identified accordingly. Notably, non-canonical splicing sites accounted for 1.99% of the whole genome, with the predominant types being GC-AG (1.85%), GT-AC (0.05%), and GT-GG (0.04%). 1165 pairs of genes were found to have overlapped transcribed regions, and 92.19% of which showed opposite directions of gene transcription. A total of 5,412,158 genetic variations were identified among 13 G. lingzhi strains, and the manually corrected gene sets resulted in enhanced functional annotation of these variations. More than 60% of G. lingzhi genes were alternatively spliced. In addition, we found that two or more protein-coding genes (PCGs) can be transcribed into a single RNA molecule, referred to as polycistronic genes. In total, 1272 polycistronic genes associated with 2815 PCGs were identified. Conclusions: The widespread presence of polycistronic genes in G. lingzhi strongly complements the theory that polycistron is also present in eukaryotic genomes. The extraordinary gene structure and transcriptional activity uncovered through this golden annotation provide implications for the study of genes, genomes, and related studies in G. lingzhi and other eukaryotes.

Fungi polysaccharides are nutraceutical-rich compounds with bioactive properties, offering promising applications in food formulation. This study examined the non-covalent complexation of commercial polysaccharides derived from the fruiting bodies of Auricularia auricula-judae (AA) and Ganoderma lucidum (GL) and soy protein isolate to enhance emulsifying properties. Complexes were examined across protein-to-polysaccharide ratios (0:1 to 1:0), pH levels (3 to 7), and heat treatment conditions. Results indicated a maximum insoluble association at pH 4 for both SPI-AAP and SPI-GLP complexes, with SPI-AAP complexes remaining soluble at pH 3, while SPI-GLP complexes exhibited insolubility. Heat treatment had a limited effect on electrostatically driven complexation but resulted in larger particles through a protein-denaturation-induced increase of hydrophobic interactions. In terms of emulsifying properties, individual GLPs demonstrated superior performance compared to individual AAPs. The GLPs engaged in competitive adsorption at the oil–water interface alongside SPI, resulting in larger emulsion droplet sizes compared to either component alone. The association of either AAPs or GLPs with SPI enhanced the emulsion stability against coalescence and Ostwald ripening. Commercial fungal polysaccharides demonstrate substantial potential for incorporation into manufactured food products, particularly in colloidal formulations.

Ganoderma lucidum has a wide carbon spectrum, while the expression profile of key genes relevant to carbon metabolism on different carbon sources has been seldom studied. Here, the transcriptomes of G. lucidum mycelia cultured on each of 19 carbon sources were conducted. In comparison with glucose, 16 to 1,006 genes were upregulated and 7 to 1,865 genes were downregulated. Significant gene expression dynamics and induced activity were observed in laccase genes when using agricultural and forestry residues (AFRs) as solo carbon sources. Furthermore, study of laccase gene family in two haploids of G. lucidum GL0102 was conducted. Totally, 15 and 16 laccase genes were identified in GL0102_53 and GL0102_8, respectively, among which 15 pairs were allelic genes. Gene structures were conserved between allelic laccase genes, while sequence variations (most were SNPs) existed. Nine laccase genes rarely expressed on all the tested carbon sources, while the other seven genes showed high expression level on AFRs, especially Gllac2 and Gllac7, which showed 5- to 1,149-fold and 4- to 94-fold upregulation in mycelia cultured for 5 days, respectively. The expression of H53lac7 was consistently higher than that of H8lac7_1 on all the carbon sources except XM, exhibiting a case of allelic expression bias. A total of 47 SNPs and 3 insertions/deletions were observed between promoters of H53lac7 and H8lac7_1, which lead to differences in predicted binding sites of zinc fingers. These results provide scientific data for understanding the gene expression profile and regulatory role on different carbon sources and may support further functional research of laccase.

The two haploids, Ct001_29 and Ct001_31, derived from the thermotolerant strain Coriolopsis trogii Ct001, differ in mating type and exhibit distinct thermotolerance capacities. To investigate the underlying molecular mechanisms, their phenotypic responses to different temperatures were characterized, and comparative RNA-sequencing (RNA-Seq) analyses were performed using an upgraded, manually curated genome annotation. Phenotypic observations revealed that Ct001_31 displayed more robust hyphal growth at 35 °C than Ct001_29, including denser mycelia, larger hyphal diameter, smoother hyphal surfaces, and the ability to sustain growth at 40 °C. Through manual correction, a high-quality genome annotation for Ct001_31 was generated, comprising 12,953 high-confidence genes. Comparative RNA-Seq analysis identified 108 up-regulated and 121 down-regulated genes commonly shared between the two haploids at 35 °C. The up-regulated genes in Ct001_29 were significantly enriched in functions related to protein folding and heat response, whereas no significant functional enrichment was observed among the differentially expressed genes (DEGs) in Ct001_31 at 35 °C. Notably, CtDnaJ.16 showed increased expression in Ct001_31 at 35 °C, while no significant change was observed in Ct001_29. Functional investigation revealed that Ct31DnaJ.16 was specifically involved in thermotolerance in Ct001_31, with transformants overexpressing this gene capable of germinating at 43 °C. In contrast, neither overexpression nor RNA interference of Ct29DnaJ.16 in Ct001_29 led to detectable phenotypic alterations. These findings demonstrate that CtDnaJ.16 plays a strikingly haploid-specific role in thermotolerance, deepening our understanding of functional differentiation between allelic genes.