Genome Research And Platform Construction Of Model Medicinal Mushroom Ganoderma Lucidum

Ganoderma lucidum, a white rot basidiomycete belonging to the order Polyporales, is one of the most well-known medicinal mushrooms in the world. G. lucidum has been used as a remedy for thousands years in East Asia. This species is documented as LingZhi in Chinese Pharmacopoeia and is included in the American Herbal Pharmacopoeia and Therapeutic Compendium. G. lucidum is known as a "cellular factory for biologically useful compounds," and more than400bioactive compounds have been identified in G. lucidum, including polysaccharides, triterpenoids, fatty acids and alkaloids. Modern studies have reported that G. lucidum possesses multiple pharmacological activities, including antitumor. anti-hypertensive, anti-inflammatory and immunomodulatory activities. This species is amenable to genetic transformation and artificial cultivation, making it an ideal system for traditional medicine research.Here, we sequenced and assembled its genome using next-generation sequencing and optical mapping approaches. The43.3-Mb genome of G. lucidum contained13chromosomes and was assembled into82scaffolds. The gene prediction and annotation was using MAKER pipeline, combining the ab initio method and comparative ways, constructed gene models were further manual curated using Apollo programm. Gene expression profile was detected using454transcriptome technology and RNA-seq technology. Results show that G.lucidum’s genome encoded16,113predicted genes, some genes’expression level changed dramatically during different development stages. More importantly, the analysis revealed an impressive array of genes encoding cytochrome P450s (CYPs), transporters, and regulatory proteins which cooperate in secondary metabolism. The genome also encodes one of the richest sets of wood degradation enzymes among all sequenced basidiomycetes. Twenty-four physical CYP gene clusters were identified. Moreover,78CYP genes were co-expressed with lanosterol synthase, and16of these show high similarity to fungal CYPs that specifically hydroxylate testosterone, suggesting their possible roles in triterpenoid biosynthesis. The elucidation of the relationship between G.lucidum secondary metabolites and development based on omic scale analysis enables this organism to be a potential model system for the study of secondary metabolic pathways and their regulation in fungi.The G. lucidum genome and transcriptome have wild applications in this specie’s breeding, cultivation, production and research. In this paper, we introduced two examples. First, mating-type loci were identified based on genomic data. A90kb region in scaffold1was deemed as mating type A(matA) locus, two genes (GL30604and GL30607) in this region were inferred to encoding the HDl and HD2proteins. There are also29other genes in this region. Thirteen sequences were identified as mating type B (matB) locus genes. Six pheromone encoding genes were clustered with seven STE3-like pheromone receptor genes in a region of approximately100kb on scaffold14. The results suggested that mating-type locus in G. lucidum were conserved with other fungi which have been sequenced. The RNA-seq analysis showed that the expression level of different pheromone coding genes and pheromone receptor coding genes were not the same. This result indicated that different pheromones and their receptors may play diverse roles in variety conditions. The phylogenetic analysis of MIP coding genes from14fungi indicated a conserved region in Ganodermae. Based on the consensus sequences of mip in Ganodermae, a pair of primer was designed for the identification of the ploidy and the mating type of fungi strains from Ganodermae. Second, reference genes for qRT-PCR were selected from predicted gene models. Quantitative real-time reverse transcription PCR (qRT-PCR) is a rapid, sensitive and reliable technique for gene expression studies. The accuracy and reliability of qRT-PCR results depend on the stability of the reference genes used for gene normalization. Therefore, a systematic process of reference gene evaluation is needed. Ganoderma lucidum is a famous medicinal mushroom in East Asia. In the current study,10potential reference genes were selected from the G. lucidum genomic data. The sequences of these genes were manually curated, and primers were designed following strict criteria. The experiment was conducted using qRT-PCR, and the stability of each candidate gene was assessed using4commonly used statistical programs-geNorm, NormFinder, BestKeeper and RefFinder. According to our results, PP2A was expressed at the most stable levels under different fermentation conditions, and RPL4was the most stably expressed gene in different tissues. RPL4, PP2A and β-tubulin are the most commonly recommended reference genes for normalizing gene expression in the entire sample set.