Marsdenia tenacissima genome reveals calcium adaptation and tenacissoside biosynthesis.

Marsdenia tenacissima is a medicinal plant widely distributed in the calcium-rich karst regions of southwest China. However, the lack of a reference genome has hampered the implementation of molecular techniques in its breeding, pharmacology and domestication. We generated the chromosome-level genome assembly in Apocynaceae using combined SMRT sequencing and Hi-C. The genome length was 381.76 Mb, with 98.9% of it found on 11 chromosomes. The genome contained 222.63 Mb of repetitive sequences and 21 899 predicted gene models, with a contig N50 of 6.57 Mb. Phylogenetic analysis revealed that M. tenacissima diverged from Calotropis gigantea at least 13.43 million years ago. Comparative genomics showed that M. tenacissima underwent ancient shared whole-genome duplication. This event, together with tandem duplication, contributed to 70.71% of gene-family expansion. Both pseudogene analysis and selective pressure calculations suggested calcium-related adaptive evolution in the M. tenacissima genome. Calcium-induced differentially expressed genes (DEGs) were mainly enriched in cell-wall-related processes. Domains (e.g. Fasciclin and Amb_all) and cis-elements (e.g. MYB and MYC) frequently occurred in the coding and promoter regions of cell-wall DEGs, respectively, and the expression levels of these genes correlated significantly with those of calcium-signal-related transcription factors. Moreover, calcium addition increased tenacissoside I, G and H contents. The availability of this high-quality genome provides valuable genomic information for genetic breeding and molecular design, and lends insights into the calcium adaptation of M. tenacissima in karst areas.

Unigene-based RNA-seq provides insights on drought stress responses in Marsdenia tenacissima.

Marsdenia tenacissima is a well-known anti-cancer medicinal plant used in traditional Chinese medicine, which often grows on the karst landform and the water conservation capacity of land is very poorly and drought occurrences frequently. We found M. tenacissima has strong drought resistance because of continuousdrought16 d, the leaves of M. tenacissima were fully curly and dying. But the leaves were fully almost recovering after re-watering 24h. The activity of SOD and POD were almost doubled under drought stress. The content of osmotic regulating substance proline and soluble sugar were three times than control group. But after re-watering, these indexes were declined rapidly. Three cDNA libraries of control, drought stress, and re-watering treatments were constructed. There were 43,129,228, 47,116,844, and 42,815,454 clean reads with Q20 values of 98.06, 98.04, and 97.88respectively.SRA accession number of raw data was PRJNA498187 on NCBI. A total of 8672, 6043, and 6537 differentially expressed genes (DEGs) were identified in control vs drought stress, control vs re-watering, and drought stress vs re-watering, respectively. In addition, 1039, 1016, and 980 transcription factors (TFs) were identified, respectively. Among them, 363, 267, and 299 TFs were identified as DEGs in drought stress, re-watering, and drought stress and re-watering, respectively. These differentially expressed TFs mainly belonged to the bHLH, bZIP, C2H2, ERF, MYB, MYB-related, and NAC families. A comparative analysis found that 1174 genes were up-regulated and 2344 were down-regulated under drought stress and this pattern was the opposite to that found after re-watering. Among the up-regulated genes, 64 genes were homologous to known functional genes that directly protect plants against drought stress. Furthermore, 44 protein kinases and 38 TFs with opposite expression patterns under drought stress and re-watering were identified, which are possibly candidate regulators for drought stress resistance in M. tenacissima. Our study is the first to characterize the M. tenacissima transcriptome in response to drought stress, and will serve as a useful resource for future studies on the functions of candidate protein kinases and TFs involved in M. tenacissima drought stress resistance.

Integrated approach for identifying and evaluating the quality of Marsdenia tenacissima in the medicine market.

The accurate identification and quality evaluation of herbal medical plants is highly necessary to ensure their safety and efficacy. In present study, a new strategy combining DNA barcoding techniques with thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) was proposed to facilitate the identification and quality control of M. tenacissima. In present work, the internal transcribed spacer 2 (ITS2) barcode was successfully used to identify 58 M. tenacissima samples and its adulterants. TLC successfully identified the other three M. tenacissima samples that failed to produce ITS2 regions. An adulterant was found in all the 62 samples. Moreover, the content of active medicinal ingredients is important for herbal plants quality. The content of tenacissoside H (TS-H) of M. tenacissima samples was determined by HPLC to range from 0.39% to 1.09%, which meets the criterion of the Chinese Pharmacopoeia. Thus, DNA barcoding coupled with TLC and HPLC is very promising to identify and evaluate the quality of M. tenacissima in the medicine market.

Analysis of the transcriptome of Marsdenia tenacissima discovers putative polyoxypregnane glycoside biosynthetic genes and genetic markers.

Marsdenia tenacissima is a well-known anti-cancer medicinal plant used in traditional Chinese medicine due to bioactive constituents of polyoxypregnane glycosides, such as tenacissosides, marsdenosides and tenacigenosides. Genomic information regarding this plant is very limited, and rare information is available about the biosynthesis of polyoxypregnane glycosides. To facilitate the basic understanding about the polyoxypregnane glycoside biosynthetic pathways, de novo assembling was performed to generate a total of 73,336 contigs and 65,796 unigenes, which represent the first transcriptome of this species. These included 27 unigenes that were involved in steroid biosynthesis and could be related to pregnane backbone biosynthesis. The expression patterns of six unigenes involved in polyoxypregnane biosynthesis were analyzed in leaf and stem tissues by quantitative real time PCR (qRT-PCR) to explore their putative function. Furthermore, a total of 15,295 simple sequence repeats (SSRs) were identified from 11,911 unigenes, of which di-nucleotide motifs were the most abundant.

[Genetic relationship on several medicinal plants in Marsdenia from Yunnan in ISSR marker].

OBJECTIVE: To analyze the genetic relationship of 9 Marsdenia species from Yunnan, especially the traditional Dai medicine "Dai Bai Jie" (M. auricularis). METHOD: Applying the inter-simple sequence repeats (ISSR) markers technology. RESULT: Twenty-five primers were screened out of 60 ISSR random primers and produced 391 bands totally, every primer produce 8-21 bands and the mean number was 15.6. The range of the GS (genetic similarity) value was 0.6675-0.8210. In 9 Marsdenia species, M. auricularis is a relative of M. tenacissima. M. balansae and M. officinalis have the closest genetic relationship. CONCLUSION: It is supported by ISSR that the M. auricularis which is sib species of M. tenacissima, and the folk medicine of Marsdenia are worthy deep investigation and study.