Aijia Ji

Analysis of the Genome Sequence of the Medicinal Plant Salvia miltiorrhiza

Document S1. Supplemental Methods and Supplemental ReferencesxDownload (.04 MB ) Document S1. Supplemental Methods and Supplemental ReferencesSupplemental Figure 1. Flow Cytometry of S. miltiorrhiza for Genome Size EstimationxDownload (.08 MB ) Supplemental Figure 1. Flow Cytometry of S. miltiorrhiza for Genome Size EstimationSupplemental Figure 2. Evolutionary Relationship of the Putative SmCPS1- and SmCPS2- Gene ClustersxDownload (.04 MB ) Supplemental Figure 2. Evolutionary Relationship of the Putative SmCPS1- and SmCPS2- Gene ClustersSupplemental Figure 3. Identification of SNPs and INDELs between Purple- and White-Flower S. miltiorrhiza VarietiesxDownload (.07 MB ) Supplemental Figure 3. Identification of SNPs and INDELs between Purple- and White-Flower S. miltiorrhiza VarietiesSupplemental Table 1. Statistics of Raw Sequencing Data for S. miltiorrhiza Genome AssemblyxDownload (.03 MB ) Supplemental Table 1. Statistics of Raw Sequencing Data for S. miltiorrhiza Genome AssemblySupplemental Table 2. Summary Statistics of De Novo Genome Assembly from Different DatasetsxDownload (.03 MB ) Supplemental Table 2. Summary Statistics of De Novo Genome Assembly from Different DatasetsSupplemental Table 3. Statistics of SNPs in the S. miltiorrhiza GenomexDownload (.01 MB ) Supplemental Table 3. Statistics of SNPs in the S. miltiorrhiza GenomeSupplemental Table 4. Summary of Gene and Repeat Annotation of the S. miltiorrhiza GenomexDownload (.03 MB ) Supplemental Table 4. Summary of Gene and Repeat Annotation of the S. miltiorrhiza GenomeSupplemental Table 5. List of 1620 Transcription Factors Identified in the S. miltiorrhiza GenomexDownload (.05 MB ) Supplemental Table 5. List of 1620 Transcription Factors Identified in the S. miltiorrhiza GenomeSupplemental Table 6. List of 82 TPS Genes in the S. miltiorrhiza GenomexDownload (.02 MB ) Supplemental Table 6. List of 82 TPS Genes in the S. miltiorrhiza GenomeSupplemental Table 7. List of 437 Putative CYPs in the S. miltiorrhiza GenomexDownload (.03 MB ) Supplemental Table 7. List of 437 Putative CYPs in the S. miltiorrhiza GenomeSupplemental Table 8. Co-Expression Analysis of CYPs with SmCPS1xDownload (.01 MB ) Supplemental Table 8. Co-Expression Analysis of CYPs with SmCPS1Supplemental Table 9. Putative Genes Encoding Tanshinone Biosynthetic Enzymes in S. miltiorrhizaxDownload (.01 MB ) Supplemental Table 9. Putative Genes Encoding Tanshinone Biosynthetic Enzymes in S. miltiorrhiza

Genome-wide characterisation and analysis of bHLH transcription factors related to tanshinone biosynthesis in Salvia miltiorrhiza

Salvia miltiorrhiza Bunge (Labiatae) is an emerging model plant for traditional medicine, and tanshinones are among the pharmacologically active constituents of this plant. Although extensive chemical and pharmaceutical studies of these compounds have been performed, studies on the basic helix-loop-helix (bHLH) transcription factors that regulate tanshinone biosynthesis are limited. In our study, 127 bHLH transcription factor genes were identified in the genome of S. miltiorrhiza, and phylogenetic analysis indicated that these SmbHLHs could be classified into 25 subfamilies. A total of 19 sequencing libraries were constructed for expression pattern analyses using RNA-Seq. Based on gene-specific expression patterns and up-regulated expression patterns in response to MeJA treatment, 7 bHLH genes were revealed as potentially involved in the regulation of tanshinone biosynthesis. Among them, the gene expression of SmbHLH37, SmbHLH74 and SmbHLH92 perfectly matches the accumulation pattern of tanshinone biosynthesis in S. miltiorrhiza. Our results provide a foundation for understanding the molecular basis and regulatory mechanisms of bHLH transcription factors in S. miltiorrhiza.

Identification and evaluation of reference genes for qRT-PCR normalization in Ganoderma lucidum.

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, 10 potential 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 using four commonly 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 RPL4 was 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. The current study provides a foundation for the further use of qRT-PCR in G. lucidum gene analysis.

Global Identification of the Full-Length Transcripts and Alternative Splicing Related to Phenolic Acid Biosynthetic Genes in Salvia miltiorrhiza.

Salvianolic acids are among the main bioactive components in Salvia miltiorrhiza, and their biosynthesis has attracted widespread interest. However, previous studies on the biosynthesis of phenolic acids using next-generation sequencing platforms are limited with regard to the assembly of full-length transcripts. Based on hybrid-seq (next-generation and single molecular real-time sequencing) of the S. miltiorrhiza root transcriptome, we experimentally identified 15 full-length transcripts and four alternative splicing events of enzyme-coding genes involved in the biosynthesis of rosmarinic acid. Moreover, we herein demonstrate that lithospermic acid B accumulates in the phloem and xylem of roots, in agreement with the expression patterns of the identified key genes related to rosmarinic acid biosynthesis. According to co-expression patterns, we predicted that six candidate cytochrome P450s and five candidate laccases participate in the salvianolic acid pathway. Our results provide a valuable resource for further investigation into the synthetic biology of phenolic acids in S. miltiorrhiza.

Chromosome-level genome map provides insights into diverse defense mechanisms in the medicinal fungus Ganoderma sinense.

Fungi have evolved powerful genomic and chemical defense systems to protect themselves against genetic destabilization and other organisms. However, the precise molecular basis involved in fungal defense remain largely unknown in Basidiomycetes. Here the complete genome sequence, as well as DNA methylation patterns and small RNA transcriptomes, was analyzed to provide a holistic overview of secondary metabolism and defense processes in the model medicinal fungus, Ganoderma sinense. We reported the 48.96 Mb genome sequence of G. sinense, consisting of 12 chromosomes and encoding 15,688 genes. More than thirty gene clusters involved in the biosynthesis of secondary metabolites, as well as a large array of genes responsible for their transport and regulation were highlighted. In addition, components of genome defense mechanisms, namely repeat-induced point mutation (RIP), DNA methylation and small RNA-mediated gene silencing, were revealed in G. sinense. Systematic bioinformatic investigation of the genome and methylome suggested that RIP and DNA methylation combinatorially maintain G. sinense genome stability by inactivating invasive genetic material and transposable elements. The elucidation of the G. sinense genome and epigenome provides an unparalleled opportunity to advance our understanding of secondary metabolism and fungal defense mechanisms.

Abundant and Selective RNA Editing Events in the Medicinal Mushroom Ganoderma lucidum

RNA editing is a widespread, post-transcriptional molecular phenomenon that diversifies hereditary information across various organisms. However, little is known about genome-scale RNA editing in fungi. In this study, we screened for fungal RNA editing sites at the genomic level in Ganoderma lucidum, a valuable medicinal fungus. On the basis of our pipeline that predicted the editing sites from genomic and transcriptomic data, a total of 8906 possible RNA-editing sites were identified within the G. lucidum genome, including the exon and intron sequences and the 5′-/3′-untranslated regions of 2991 genes and the intergenic regions. The major editing types included C-to-U, A-to-G, G-to-A, and U-to-C conversions. Four putative RNA-editing enzymes were identified, including three adenosine deaminases acting on transfer RNA and a deoxycytidylate deaminase. The genes containing RNA-editing sites were functionally classified by the Kyoto Encyclopedia of Genes and Genomes enrichment and gene ontology analysis. The key functional groupings enriched for RNA-editing sites included laccase genes involved in lignin degradation, key enzymes involved in triterpenoid biosynthesis, and transcription factors. A total of 97 putative editing sites were randomly selected and validated by using PCR and Sanger sequencing. We presented an accurate and large-scale identification of RNA-editing events in G. lucidum, providing global and quantitative cataloging of RNA editing in the fungal genome. This study will shed light on the role of transcriptional plasticity in the growth and development of G. lucidum, as well as its adaptation to the environment and the regulation of valuable secondary metabolite pathways.

Genome-Wide Identification of the AP2/ERF Gene Family Involved in Active Constituent Biosynthesis in

Tanshinones and phenolic acids are the major bioactive constituents in the traditional medicinal crop Salvia miltiorrhiza; however, transcription factors (TFs) are seldom investigated with regard to their regulation of the biosynthesis of these compounds. Here a complete overview of the APETALA2/ethylene‐responsive factor (AP2/ERF) transcription factor family in S. miltiorrhiza is provided, including phylogeny, gene structure, conserved motifs, and gene expression profiles of different organs (root, stem, leaf, flower) and root tissues (periderm, phloem, xylem). In total, 170 AP2/ERF genes were identified and divided into five relatively conserved subfamilies, including AP2 (25 genes), DREB (61 genes), ethylene responsive factor (ERF; 79 genes), RAV (4 genes), and Soloist (1 gene). According to the distribution of bioactive constituents and the expression patterns of AP2/ERF genes in different organs and root tissues, the genes related to the biosynthesis of bioactive constituents were selected. On the basis of quantitative real‐time polymerase chain reaction (qRT‐PCR) analysis, coexpression analysis, and the prediction of cis‐regulatory elements in the promoters, we propose that two genes (Sm128 and Sm152) regulate tanshinone biosynthesis and two genes (Sm008 and Sm166) participate in controlling phenolic acid biosynthesis. The genes related to tanshinone biosynthesis belong to the ERF‐B3 subgroup. In contrast, the genes predicted to regulate phenolic acid biosynthesis belong to the ERF‐B1 and ERF‐B4 subgroups. These results provide a foundation for future functional characterization of AP2/ERF genes to enhance the biosynthesis of the bioactive compounds of S. miltiorrhiza.

Biosynthesis and Regulation of Active Compounds in Medicinal Model Plant Salvia miltiorrhiza

Abstract Natural products from plant secondary metabolits are a major source of clinical drugs and industrial chemicals. Salvia miltiorrhiza is one of the most important plants in traditional Chinese medicine. Its dried roots and rhizomes are highly valued for use in the treatment of vascular diseases and for their anti-oxidative activities. Furthermore, S. miltiorrhiza is described as a medicinal model plant mainly due to its biosynthesis of active compounds. Here, we reviewed the research on S. miltiorrhiza in genomics, transcriptomics, biosynthesis of tanshinones and phenolic acids, biotic and abiotic elicitors, and regulation of transcription factors. This will provide a solid foundation for new breeding and synthetic biology approaches to produce and study natural products.

Genome-wide analysis of auxin response factor gene family members in medicinal model plant Salvia miltiorrhiza

ABSTRACT Auxin response factors (ARFs) can function as transcriptional activators or repressors to regulate the expression of auxin response genes by specifically binding to auxin response elements (AuxREs) during plant development. Based on a genome-wide strategy using the medicinal model plant Salvia miltiorrhiza, 25 S. miltiorrhiza ARF (SmARF) gene family members in four classes (class Ia, IIa, IIb and III) were comprehensively analyzed to identify characteristics including gene structures, conserved domains, phylogenetic relationships and expression patterns. In a hybrid analysis of the phylogenetic tree, microRNA targets, and expression patterns of SmARFs in different organs, root tissues, and methyl jasmonate or indole-3-acetic acid treatment conditions, we screened for candidate SmARFs involved in various developmental processes of S. miltiorrhiza. Based on this analysis, we predicted that SmARF25, SmARF7, SmARF16 and SmARF20 are involved in flower, leaf, stem and root development, respectively. With the further insight into the targets of miR160 and miR167, specific SmARF genes in S. miltiorrhiza might encode products that participate in biological processes as described for ARF genes in Arabidopsis. Our results provide a foundation for understanding the molecular basis and regulatory mechanisms of SmARFs in S. miltiorrhiza. Summary: Genome-wide analysis identified 25 ARF gene members (seven transcriptional activators and 18 repressors) in S. miltiorrhiza. The gene structures, functional domains, miRNA targets and expression patterns were analyzed in detail.

Genome-wide selection of superior reference genes for expression studies in Ganoderma lucidum.

Quantitative real-time polymerase chain reaction (qRT-PCR) is widely used for the accurate analysis of gene expression. However, high homology among gene families might result in unsuitability of reference genes, which leads to the inaccuracy of qRT-PCR analysis. The release of the Ganoderma lucidum genome has triggered numerous studies to be done on the homology among gene families with the purpose of selecting reliable reference genes. Based on the G. lucdum genome and transcriptome database, 38 candidate reference genes including 28 novel genes were systematically selected and evaluated for qRT-PCR normalization. The result indicated that commonly used polyubiquitin (PUB), beta-actin (BAT), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were unsuitable reference genes because of the high sequence similarity and low primer specificity. According to the evaluation of RefFinder, cyclophilin 5 (CYP5) was ranked as the most stable reference gene for 27 tested samples under all experimental conditions and eighteen mycelial samples. Based on sequence analysis and expression analysis, our study suggested that gene characteristic, primer specificity of high homologous genes, allele-specificity expression of candidate genes and under-evaluation of reference genes influenced the accuracy and sensitivity of qRT-PCR analysis. This investigation not only revealed potential factors influencing the unsuitability of reference genes but also selected the superior reference genes from more candidate genes and testing samples than those used in the previous study. Furthermore, our study established a model for reference gene analysis by using the genomic sequence.