As a valuable medicinal plant, the yield of Panax ginseng is seriously affected by autotoxicity, which is a common phenomenon due to continuous cropping. However, the mechanism of autotoxicity in P. ginseng is still unknown. In total, high throughput sequencing of 18 RNA-Seq libraries produced 996,000 000 100-nt reads that were assembled into 72,732 contigs. Compared with control, 3697 and 2828 genes were significantly up- and down-regulated across different tissues and time points, respectively. Gene Ontology enrichment analysis showed that ‘enzyme inhibitor activity’, ‘carboxylesterase activity’, ‘pectinesterase activity’, ‘centrosome cycle and duplication’ and ‘mitotic spindle elongation’ were enriched for the up-regulated genes. Transcription factors including AP2s/ERFs, MYBs, and WRKYs were up-regulated in roots after benzoic acid treatment. Moreover, reactive oxygen species, peroxidases and superoxide dismutase contigs were up-regulated in roots after benzoic acid treatment. Physiological and biochemical indexes showed that the proline and malondialdehyde content were restored to lower levels at a later stage after benzoic acid treatment. Benzoic acid inhibited the root hair development in a dose-dependent manner, and several differential expressed genes potentially involved in hair development were identified. Several key contigs in the flavonoid and ginsenoside biosynthesis pathways were repressed. Finally, 58,518 alternative splicing (AS) events from 12,950 genes were found after benzoic acid treatment. Interestingly, contigs in the ginsenoside biosynthetic pathway underwent AS, providing useful information about post-transcriptional regulation in P. ginseng. This study revealed the stress-response molecular mechanisms in P. ginseng induced by benzoic acid.

Background: Digitalis purpureais an important ornamental and medicinal plant. There is considerable interest in exploring its transcriptome.Results: Through high-throughput 454 sequencing and subsequent assembly, we obtained 23532 genes, of which 15626 encode conserved proteins. We determined 140 unigenes to be candidates involved in cardiac glycoside biosynthesis. It could be grouped into 30 families, of which 29 were identified for the first time inD. purpurea. We identified 2660 mRNA-like npcRNA (mlncRNA) candidates, an emerging class of regulators, using a computational mlncRNA identification pipeline and 13 microRNA-producing unigenes based on sequence conservation and hairpin structure-forming capability. Twenty five protein-coding unigenes were predicted to be targets of these microRNAs. Among the mlncRNA candidates, only 320 could be grouped into 140 families with at least two members in a family. The majority ofD. purpureamlncRNAs were species-specific and many of them showed tissue-specific expression and responded to cold and dehydration stresses. We identified 417 protein-coding genes with regions significantly homologous or complementary to 375 mlncRNAs. It includes five genes involved in secondary metabolism. A positive correlation was found in gene expression between protein-coding genes and the homologous mlncRNAs in response to cold and dehydration stresses, while the correlation was negative when protein-coding genes and mlncRNAs were complementary to each other.Conclusions: Through comprehensive transcriptome analysis, we not only identified 29 novel gene families potentially involved in the biosynthesis of cardiac glycosides but also characterized a large number of mlncRNAs. Our results suggest the importance of mlncRNAs in secondary metabolism and stress response inD. purpurea.

Pueraria thomsonii Benth is an important medicinal plant. Transcriptome sequencing, unigene assembly, the annotation of transcripts and the study of gene expression profiles play vital roles in gene function research. However, the full-length transcriptome of P. thomsonii remains unknown. Here, we obtained 44,339 nonredundant transcripts of P. thomsonii by using the PacBio RS II Isoform and Illumina sequencing platforms, of which 43,195 were annotated genes. Compared with the expression levels in the plant roots, those of transcripts with a |fold change| ≥ 4 and FDR < 0.01 in the leaves or stems were assigned as differentially expressed transcripts (DETs). In total, we found 9,225 DETs, 32 of which came from structural genes that were potentially involved in isoflavone biosynthesis. The expression profiles of 8 structural genes from the RNA-Seq data were validated by qRT-PCR. We identified 437 transcription factors (TFs) that were positively or negatively correlated with at least 1 of the structural genes involved in isoflavone biosynthesis using Pearson correlation coefficients (r) (r > 0.8 or r < -0.8). We also identified a total of 32 microRNAs (miRNAs), which targeted 805 transcripts. These miRNAs caused enriched function in ‘ATP binding’, ‘defense response’, ‘ADP binding’, and ‘signal transduction’. Interestingly, MIR156a potentially promoted isoflavone biosynthesis by repressing SBP, and MIR319 promoted isoflavone biosynthesis by repressing TCP and HB-HD-ZIP. Finally, we identified 2,690 alternative splicing events, including that of the structural genes of trans-cinnamate 4-monooxygenase and pullulanase, which are potentially involved in the biosynthesis of isoflavone and starch, respectively, and of three TFs potentially involved in isoflavone biosynthesis. Together, these results provide us with comprehensive insight into the gene expression and regulation of P. thomsonii.

Siraitia grosvenorii fruit, known as Luo-Han-Guo, has been used as a traditional Chinese medicine for many years, and mogrosides are its primary active ingredients. Unfortunately, Siraitia siamensis, its wild relative, might be misused due to its indistinguishable appearance, not only threatening the reliability of the medication but also partly exacerbating wild resource scarcity. Therefore, high-resolution genetic markers must be developed to discriminate between these species. Here, the complete chloroplast genomes of S. grosvenorii and S. siamensis were assembled and analyzed for the first time; they were 158,757 and 159,190 bp in length, respectively, and possessed conserved quadripartite circular structures. Both contained 134 annotated genes, including 8 rRNA, 37 tRNA and 89 protein-coding genes. Twenty divergences (Pi > 0.03) were found in the intergenic regions. Nine protein-coding genes, accD, atpA, atpE, atpF, clpP, ndhF, psbH, rbcL, and rpoC2, underwent selection within Cucurbitaceae. Phylogenetic relationship analysis indicated that these two species originated from the same ancestor. Finally, four pairs of molecular markers were developed to distinguish the two species. The results of this study will be beneficial for taxonomic research, identification and conservation of Siraitia Merrill wild resources in the future.

Circular RNAs (circRNAs) play important roles in animals, plants, and fungi. However, no circRNAs have been reported in Ganoderma lucidum. Here, we carried out a genome-wide identification of the circRNAs in G.lucidum using RNA-Seq data, and analyzed their features. In total, 250 and 2193 circRNAs were identified from strand-specific RNA-seq data generated from the polyA(−) and polyA(−)/RNase R-treated libraries, respectively. Six of 131 (4.58%) predicted circRNAs were experimentally confirmed. Across three developmental stages, 731 exonic circRNAs (back spliced read counts ≥ 5) and their parent genes were further analyzed. CircRNAs were preferred originating from exons with flanking introns, and the lengths of the flanking intron were longer than those of the control introns. A total of 200 circRNAs were differentially expressed across the three developmental stages of G. lucidum. The expression profiles of 119 (16.3%) exonic circRNAs and their parent genes showed significant positive correlations (r ≥ 0.9, q < 0.01), whereas 226 (30.9%) exonic circRNAs and their parent genes exhibited significant negative correlations (r ≤ −0.9, q < 0.01), in which 53 parent genes are potentially involved in the transcriptional regulation, polysaccharide biosynthesis etc. Our results indicated that circRNAs are present in G. lucidum, with potentially important regulatory roles.

Licorice (Glycyrrhiza) is a staple Chinese herbal medicine in which the primary bioactive compound is glycyrrhizic acid (GA), which has important pharmacological functions. To date, the structural genes involved in GA biosynthesis have been identified. However, the regulation of these genes in G. uralensis has not been elucidated. In this study, we performed a comprehensive analysis based on the transcriptome and small RNAome by high-throughput sequencing. In total, we identified 18 structural GA genes and 3924 transporter genes. We identified genes encoding 2374 transporters, 1040 transcription factors (TFs), 262 transcriptional regulators (TRs) and 689 protein kinases (PKs), which were coexpressed with at least one structural gene. We also identified 50,970 alternative splicing (AS) events, in which 17 structural genes exhibited AS. Finally, we also determined that miRNAs potentially targeted 4 structural genes, and 318, 8, and 218 miRNAs potentially regulated 150 TFs, 34 TRs, and 88 PKs, respectively, related to GA. Overall, the results of this study helped to elucidate the gene expression and regulation of GA biosynthesis in G. uralensis, provided a theoretical basis for the synthesis of GA via synthetic biology, and laid a foundation for the cultivation of new varieties of licorice with high GA content.