Production of the antidepressant orcinol glucoside in Yarrowia lipolytica with yields over 6,400-fold higher than plant extraction.

Orcinol glucoside (OG), mainly found in the rhizome of the traditional Chinese herb Curculigo orchioides Gaertn, is noted for its antidepressant effects. In this study, an efficient screening pipeline was established for identifying the highly active orcinol synthase (ORS) and UDP-dependent glycosyltransferase (UGT) involved in the biosynthesis of OG by combining transcriptome analysis, structure-based virtual screening, and in vitro enzyme activity assays. By enhancing the downstream pathway, metabolic engineering and fermentation optimization, the OG production in Yarrowia lipolytica was improved 100-fold, resulting in a final yield of 43.46 g/L (0.84 g/g DCW), which is almost 6,400-fold higher than the extraction yield from C. orchioides roots. This study provides a reference for rapid identification of functional genes and high-yield production of natural products.

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.

Integrated metabolome and transcriptome analyses reveal the molecular mechanism underlying dynamic metabolic processes during taproot development of Panax notoginseng.

BACKGROUND: Panax notoginseng (Burk) F. H. Chen is one of the most famous Chinese traditional medicinal plants. The taproot is the main organ producing triterpenoid saponins, and its development is directly linked to the quality and yield of the harvested P. notoginseng. However, the mechanisms underlying the dynamic metabolic changes occurring during taproot development of P. notoginseng are unknown. RESULTS: We carried out metabolomic and transcriptomic analyses to investigate metabolites and gene expression during the development of P. notoginseng taproots. The differentially accumulated metabolites included amino acids and derivatives, nucleotides and derivatives, and lipids in 1-year-old taproots, flavonoids and terpenoids in 2- and 3-year-old taproots, and phenolic acids in 3-year-old taproots. The differentially expressed genes (DEGs) are related to phenylpropanoid biosynthesis, metabolic pathway and biosynthesis of secondary metabolites at all three developmental stages. Integrative analysis revealed that the phenylpropanoid biosynthesis pathway was involved in not only the development of but also metabolic changes in P. notoginseng taproots. Moreover, significant accumulation of triterpenoid saponins in 2- and 3-year-old taproots was highly correlated with the up-regulated expression of cytochrome P450s and uridine diphosphate-dependent glycosyltransferases genes. Additionally, a gene encoding RNase-like major storage protein was identified to play a dual role in the development of P. notoginseng taproots and their triterpenoid saponins synthesis. CONCLUSIONS: These results elucidate the molecular mechanism underlying the accumulation of and change relationship between primary and secondary metabolites in P. notoginseng taproots, and provide a basis for the quality control and genetic improvement of P. notoginseng.

Development of a stable genetic transformation system in Erigeron breviscapus: a case study with EbYUC2 in relation to leaf number and flowering time.

MAIN CONCLUSION: A stable genetic transformation system for Erigeron breviscapus was developed. We cloned the EbYUC2 gene and genetically transformed it into Arabidopsis thaliana and E. breviscapus. The leaf number, YUC2 gene expression, and the endogenous auxin content in transgenic plants were significantly increased. Erigeron breviscapus is a prescription drug for the clinical treatment of cardiovascular and cerebrovascular diseases. The rosette leaves have the highest content of the major active compound scutellarin and are an important component in the yield of E. breviscapus. However, little is known about the genes related to the leaf number and flowering time of E. breviscapus. In our previous study, we identified three candidate genes related to the leaf number and flowering of E. breviscapus by combining resequencing data and genome-wide association study (GWAS). However, their specific functions remain to be characterized. In this study, we cloned and transformed the previously identified full-length EbYUC2 gene into Arabidopsis thaliana, developed the first stable genetic transformation system for E. breviscapus, and obtained the transgenic plants overexpressing EbYUC2. Compared with wild-type plants, the transgenic plants showed a significant increase in the number of leaves, which was correlated with the increased expression of EbYUC2. Consistently, the endogenous auxin content, particularly indole-3-acetic acid, in transgenic plants was also significantly increased. These results suggest that EbYUC2 may control the leaf number by regulating auxin biosynthesis, thereby laying a foundation for revealing the molecular mechanism governing the leaf number and flowering time of E. breviscapus.

Functional characterization of genes related to triterpene and flavonoid biosynthesis in Cyclocarya paliurus.

MAIN CONCLUSION: The oxidosqualene cyclases (OSCs) generating triterpenoid skeletons in Cyclocarya paliurus were identified for the first time, and two uridine diphosphate (UDP)-glycosyltransferases (UGTs) catalyzing the glycosylation of flavonoids were characterized. Cyclocarya paliurus, a native rare dicotyledonous plant in China, contains an abundance of triterpenoid saponins and flavonoid glycosides that exhibit valuable pharmaceutical effects in preventing hypertension, hyperlipidemia, and diabetes. However, the molecular mechanism explaining the biosynthesis of triterpenoid saponin and flavonoid glycoside in C. paliurus remains unclear. In this study, the triterpene content in different tissues and the expression pattern of genes encoding the key enzymes associated with triterpenoid saponin and flavonoid glycoside biosynthesis were studied using transcriptome and metabolome analysis. The eight upstream oxidosqualene cyclases (OSCs) involved in triterpenoid saponin biosynthesis were functionally characterized, among them CpalOSC6 catalyzed 2,3;22,23-dioxidosqualene to form 3-epicabraleadiol; CpalOSC8 cyclized 2,3-oxidosqualene to generate dammarenediol-II; CpalOSC2 and CpalOSC3 produced ß-amyrin and CpalOSC4 produced cycloartenol, while CpalOSC2-CpalOSC5, CpalOSC7, and CpalOSC8 all produced lanosterol. However, no catalytic product was detected for CpalOSC1. Moreover, two downstream flavonoid uridine diphosphate (UDP)-glycosyltransferases (UGTs) (CpalUGT015 and CpalUGT100) that catalyze the last step of flavonoid glycoside biosynthesis were functionally elucidated. These results uncovered the key genes involved in the biosynthesis of triterpenoid saponins and flavonoid glycosides in C. paliurus that could be applied to produce flavonoid glycosides and key triterpenoid saponins in the future via a synthetic strategy.

Identification of two key UDP-glycosyltransferases responsible for the ocotillol-type ginsenoside majonside-R2 biosynthesis in Panax vietnamensis var. fuscidiscus.

MAIN CONCLUSION: Two UDP-glycosyltransferases from Panax vienamensis var. fuscidiscus involved in ocotillol-type ginsenoside MR2 (majonside-R2) biosynthesis were identified. PvfUGT1 and PvfUGT2 sequentially catalyzes 20S,24S-Protopanxatriol Oxide II and 20S,24R-Protopanxatriol Oxide I to pseudoginsenoside RT4/RT5 and RT4/RT5 to 20S, 24S-MR2/20S, 24S-MR2. Ocotilol type saponin MR2 (majonside-R2) is the main active component of Panax vietnamensis var. fuscidiscus (commonly known as 'jinping ginseng') and is well known for its diverse pharmacological activities. The use of MR2 in the pharmaceutical industry currently depends on its extraction from Panax species. Metabolic engineering provides an opportunity to produce high-value MR2 by expressing it in heterologous hosts. However, the metabolic pathways of MR2 remain enigmatic, and the two-step glycosylation involved in MR2 biosynthesis has not been reported. In this study, we used quantitative real-time PCR to investigate the regulation of the entire ginsenoside pathway by MeJA (methyl jasmonate), which facilitated our pathway elucidation. We found six candidate glycosyltransferases by comparing transcriptome analysis and network co-expression analysis. In addition, we identified two UGTs (PvfUGT1 and PvfUGT2) through in vitro enzymatic reactions involved in the biosynthesis of MR2 which were not reported in previous studies. Our results show that PvfUGT1 can transfer UDP-glucose to the C6-OH of 20S, 24S-protopanaxatriol oxide II and 20S, 24R-protopanaxatriol oxide I to form pseudoginsenoside RT4 and pseudoginsenoside RT5, respectively. PvfUGT2 can transfer UDP-xylose to pseudoginsenoside RT4 and pseudoginsenoside RT5 to form 20S, 24S-MR2 and 20S, 24S-MR2. Our study paves the way for elucidating the biosynthesis of MR2 and producing MR2 by synthetic biological methods.

EbbHLH80 Enhances Salt Responses by Up-Regulating Flavonoid Accumulation and Modulating ROS Levels.

bHLH transcription factors are involved in multiple aspects of plant biology, such as the response to abiotic stress. Erigeron breviscapus is a composite plant, and its rich flavonoids have strong preventive and therapeutic effects on cardio cerebral vascular disease. EbbHLH80, a gene from E. breviscapus that positively regulates flavonoid synthesis, was previously characterized. However, it is unclear whether EbbHLH80 increases flavonoid accumulation, which affects salt tolerance. The function of EbbHLH80 in transgenic tobacco seeds was identified by phylogenetic analysis and metabolome-transcriptome analysis. We investigated the role of EbbHLH80 in salt stress response. Our results showed that the expression of EbbHLH80 increased following salt treatment. Integrating the metabolome and transcriptome analysis of EbbHLH80-OE and Yunyan 87 (WT) seeds, we identified several genes and metabolites related to flavonoid biosynthesis and salt stress. Moreover, EbbHLH80-OE plants displayed higher salt tolerance than wild-type plants during seed germination and seedling growth. After salt treatment, transgenic tobacco had significantly lower levels of reactive oxygen species (ROS) than WT, with enhanced levels of antioxidant enzyme expression. Altogether, our results demonstrated that EbbHLH80 might be a positive regulator, promoting salt tolerance by modulating ROS scavenging and increasing stress-responsive genes.

MtGSTF7, a TT19-like GST gene, is essential for accumulation of anthocyanins, but not proanthocyanins in Medicago truncatula.

Anthocyanins and proanthocyanins (PAs) are two end products of the flavonoid biosynthesis pathway. They are believed to be synthesized in the endoplasmic reticulum and then sequestered into the vacuole. In Arabidopsis thaliana, TRANSPARENT TESTA 19 (TT19) is necessary for both anthocyanin and PA accumulation. Here, we found that MtGSTF7, a homolog of AtTT19, is essential for anthocyanin accumulation but not required for PA accumulation in Medicago truncatula. MtGSTF7 was induced by the anthocyanin regulator LEGUME ANTHOCYANIN PRODUCTION 1 (LAP1), and its tissue expression pattern correlated with anthocyanin deposition in M. truncatula. Tnt1-insertional mutants of MtGSTF7 lost anthocyanin accumulation in vegetative organs, and introducing a genomic fragment of MtGSTF7 could complement the mutant phenotypes. Additionally, the accumulation of anthocyanins induced by LAP1 was significantly reduced in mtgstf7 mutants. Yeast-one-hybridization and dual-luciferase reporter assays revealed that LAP1 could bind to the MtGSTF7 promoter to activate its expression. Ectopic expression of MtGSTF7 in tt19 mutants could rescue their anthocyanin deficiency, but not their PA defect. Furthermore, PA accumulation was not affected in the mtgstf7 mutants. Taken together, our results show that the mechanism of anthocyanin and PA accumulation in M. truncatula is different from that in A. thaliana, and provide a new target gene for engineering anthocyanins in plants.

Identification of two UDP-glycosyltransferases involved in the main oleanane-type ginsenosides in Panax japonicus var. major.

MAIN CONCLUSION: Two UDP-glycosyltransferases from Panax japonicus var. major were identified, and the biosynthetic pathways of three oleanane-type ginsenosides (chikusetsusaponin IVa, ginsenoside Ro, zingibroside R1) were elucidated. Chikusetsusaponin IVa and ginsenoside Ro are primary active components formed by stepwise glycosylation of oleanolic acid in five medicinal plants of the genus Panax. However, the key UDP-glycosyltransferases (UGTs) in the biosynthetic pathway of chikusetsusaponin IVa and ginsenoside Ro are still unclear. In this study, two UGTs (PjmUGT1 and PjmUGT2) from Panax japonicus var. major involved in the biosynthesis of chikusetsusaponin IVa and ginsenoside Ro were identified based on bioinformatics analysis, heterologous expression and enzyme assays. The results show that PjmUGT1 can transfer a glucose moiety to the C-28 carboxyl groups of oleanolic acid 3-O-ß-D-glucuronide and zingibroside R1 to form chikusetsusaponin IVa and ginsenoside Ro, respectively. Meanwhile, PjmUGT2 can transfer a glucose moiety to oleanolic acid 3-O-ß-D-glucuronide and chikusetsusaponin IVa to form zingibroside R1 and ginsenoside Ro. This work uncovered the biosynthetic mechanism of chikusetsusaponin IVa and ginsenoside Ro, providing the rational production of valuable saponins through synthetic biology strategy.

[Identification and expression profiling of R2R3-MYB transcription factors in Erigeron breviscapus].

R2 R3-MYB transcription factors are ubiquitous in plants, playing a role in the regulation of plant growth, development, and secondary metabolism. In this paper, the R2 R3-MYB transcription factors were identified by bioinformatics analysis of the genomic data of Erigeron breviscapus, and their gene sequences, structures, physical and chemical properties were analyzed. The functions of R2 R3-MYB transcription factors were predicted by cluster analysis. Meanwhile, the expression patterns of R2 R3-MYB transcription factors in response to hormone treatments were analyzed. A total of 108 R2 R3-MYB transcription factors, named EbMYB1-EbMYB108, were identified from the genome of E. breviscapus. Most of the R2 R3-MYB genes carried 2-4 exons. The phylogenetic tree of MYBs in E. breviscapus and Arabidopsis thaliala was constructed, which classified 234 MYBs into 30 subfamilies. The MYBs in the five MYB subfamilies of A.thaliala were clustered into independent clades, and those in E. breviscapus were clustered into four clades. The transcriptome data showed that MYB genes were differentially expressed in different tissues of E. breviscapus and in response to the treatments with exogenous hormones such as ABA, SA, and GA for different time. The transcription of 13 R2 R3-MYB genes did not change significantly, and the expression patterns of some genes were up-regulated or down-regulated with the extension of hormone treatment time. This study provides a theoretical basis for revealing the mechanisms of R2 R3-MYB transcription factors in regulating the growth and development, stress(hormone) response, and active ingredient accumulation in E. breviscapus.

EbARC1, an E3 Ubiquitin Ligase Gene in Erigeron breviscapus, Confers Self-Incompatibility in Transgenic Arabidopsis thaliana.

Erigeron breviscapus (Vant.) Hand.-Mazz. is a famous traditional Chinese medicine that has positive effects on the treatment of cardiovascular and cerebrovascular diseases. With the increase of market demand (RMB 500 million per year) and the sharp decrease of wild resources, it is an urgent task to cultivate high-quality and high-yield varieties of E. breviscapus. However, it is difficult to obtain homozygous lines in breeding due to the self-incompatibility (SI) of E. breviscapus. Here, we first proved that E. breviscapus has sporophyte SI (SSI) characteristics. Characterization of the ARC1 gene in E. breviscapus showed that EbARC1 is a constitutive expression gene located in the nucleus. Overexpression of EbARC1 in Arabidopsis thaliana L. (Col-0) could cause transformation of transgenic lines from self-compatibility (SC) into SI. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays indicated that EbARC1 and EbExo70A1 interact with each other in the nucleus, and the EbARC1-ubox domain and EbExo70A1-N are the key interaction regions, suggesting that EbARC1 may ubiquitinate EbExo70A to regulate SI response. This study of the SSI mechanism in E. breviscapus has laid the foundation for further understanding SSI in Asteraceae and breeding E. breviscapus varieties.

Comparative transcriptome and metabolome analyses provide new insights into the molecular mechanisms underlying taproot thickening in Panax notoginseng.

BACKGROUND: Taproot thickening is a complex biological process that is dependent on the coordinated expression of genes controlled by both environmental and developmental factors. Panax notoginseng is an important Chinese medicinal herb that is characterized by an enlarged taproot as the main organ of saponin accumulation. However, the molecular mechanisms of taproot enlargement are poorly understood. RESULTS: A total of 29,957 differentially expressed genes (DEGs) were identified during the thickening process in the taproots of P. notoginseng. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment revealed that DEGs associated with "plant hormone signal transduction," "starch and sucrose metabolism," and "phenylpropanoid biosynthesis" were predominantly enriched. Further analysis identified some critical genes (e.g., RNase-like major storage protein, DA1-related protein, and Starch branching enzyme I) and metabolites (e.g., sucrose, glucose, fructose, malate, and arginine) that potentially control taproot thickening. Several aspects including hormone crosstalk, transcriptional regulation, homeostatic regulation between sugar and starch, and cell wall metabolism, were identified as important for the thickening process in the taproot of P. notoginseng. CONCLUSION: The results provide a molecular regulatory network of taproot thickening in P. notoginseng and facilitate the further characterization of the genes responsible for taproot formation in root medicinal plants or crops.

Transcriptome analysis of Panax zingiberensis identifies genes encoding oleanolic acid glucuronosyltransferase involved in the biosynthesis of oleanane-type ginsenosides.

MAIN CONCLUSION: Oleanolic acid glucuronosyltransferase (OAGT) genes synthesizing the direct precursor of oleanane-type ginsenosides were discovered. The four recombinant proteins of OAGT were able to transfer glucuronic acid at C-3 of oleanolic acid that yields oleanolic acid 3-O-ß-glucuronide. Ginsenosides are the primary active components in the genus Panax, and great efforts have been made to elucidate the mechanisms underlying dammarane-type ginsenoside biosynthesis. However, there is limited information on oleanane-type ginsenosides. Here, high-performance liquid chromatography analysis demonstrated that oleanane-type ginsenosides (particularly ginsenoside Ro and chikusetsusaponin IV and IVa) are the abundant ginsenosides in Panax zingiberensis, an extremely endangered Panax species in southwest China. These ginsenosides are derived from oleanolic acid 3-O-ß-glucuronide, which may be formed from oleanolic acid catalyzed by an unknown oleanolic acid glucuronosyltransferase (OAGT). Transcriptomic analysis of leaves, stems, main roots, and fibrous roots of P. zingiberensis was performed, and a total of 46,098 unigenes were obtained, including all the identified homologous genes involved in ginsenoside biosynthesis. The most upstream genes were highly expressed in the leaves, and the UDP-glucosyltransferase genes were highly expressed in the roots. This finding indicated that the precursors of ginsenosides are mainly synthesized in the leaves and transported to different parts for the formation of particular ginsenosides. For the first time, enzyme activity assay characterized four genes (three from P. zingiberensis and one from P. japonicus var. major, another Panax species with oleanane-type ginsenosides) encoding OAGT, which particularly transfer glucuronic acid at C-3 of oleanolic acid to form oleanolic acid 3-O-ß-glucuronide. Taken together, our study provides valuable genetic information for P. zingiberensis and the genes responsible for synthesizing the direct precursor of oleanane-type ginsenosides.

Illumina-based transcriptomic analysis on recalcitrant seeds of Panax notoginseng for the dormancy release during the after-ripening process.

Panax notoginseng (Burk) F.H. Chen is an economically and medicinally important plant of the family Araliacease, with seed dormancy being a key factor limiting the extended cultivation of P. notoginseng. The seeds belong to the morphophysiological dormancy (MPD) group, and it has also been described as the recalcitrant seed. To date, the molecular mechanism of dormancy release in the recalcitrant seed of P. notoginseng is unknown. In the present study, the transcript profiles of seeds from different after-ripening stages (0, 20, 40 and 60 days) were investigated using Illumina Hiseq 2500 technology. 91 979 946 clean reads were generated, and 81 575 unigenes were annotated in at least one database. In addition, the differentially expressed genes (DEGs) were identified by the pairwise comparisons. We screened out 2483 DEGs by the three key groups of 20 days vs 0 d, 40 d vs 0 d and 60 d vs 0 d. The DEGs were analyzed by gene ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway annotation. Meanwhile, we obtained 78 DEGs related to seeds dormancy release at different after-ripening stages of P. notoginseng, of which 15 DEGs were associated with abscisic acid and gibberellin. 26 DEGs that encode late embryogenesis abundant protein and antioxidant enzyme were correlated with desiccation tolerance in seeds. In summary, the results obtained here showed that PECTINESTERASE-2-LIKE, GA-INSENSITIVE, ENT-KAURENE SYNTHASE, PROTEIN PHOSPHATASE 2C, GIBBERELLIN 2-BETA-DIOXYGENASE, SUPEROXIDE DISMUTASE, L-ASCORBATE PEROXIDASE, CATALASE, LATE EMBRYOGENESIS ABUNDANT PROTEIN DC3 and DEHYDRIN 9 were potentially involved in dormancy release and desiccation sensitivity of P. notoginseng seeds. The data might provide a basis for researches on MPD.

Molecular cloning and expression analysis of SRLK1 gene in self-incompatible Asteraceae species Erigeron breviscapus.

Based on the transcriptome data, using RACE techniques, we cloned the full-length EbSRLK1 gene in a medicinal, self-incompatible Asteraceae species, Erigeron breviscapus. Bioinformatics approaches were used to analyze the DNA and protein sequences, physical and chemical properties, and domains of the encoded protein. The full-length EbSRLK1 cDNA is 2891 base pairs (bp) with an open reading frame (ORF) of 2634 bp, which encodes the EbSRLK1 protein with 878 amino acids and an estimated molecular weight of 98.13 kD. The EbSRLK1 protein has the characteristic domain structure of S-locus receptor-like protein kinases, which contains one transmembrane domain but lacks the signal peptide. Quantitative real-time PCR (qRT-PCR) analysis showed that the EbSRLK1 gene is lowly expressed in roots, stems and leaves, but highly expressed in flowers, especially in flowers one day prior to opening. Western blot analysis showed that the EbSRLK1 protein is expressed in stems, leaves, and flowers, but is almost undetectable in roots. The EbSRLK1 protein expression is induced in self-pollinated but not in cross-pollinated E. breviscapus flowers. Cloning and expression analysis of EbSRLK1 lay a solid foundation for elucidating the role of EbSRLK1 in regulating self-incompatibility in E. breviscapus.

[A semimicroquality evaluation method on Panax notoginseng and its application in analysis of continuous cropping obstacles research samples].

Panax notoginseng is a commonly used traditional Chinese medicine with blood activating effect while has continuous cropping obstacle problem in planting process. In present study, a semimicroextraction method with water-saturated n-butanol on 0.1 g notoginseng sample was established with good repeatability (RSD<2.5%) and 9.6%-20.6% higher extraction efficiency of seven saponins than the conventional method. A total of 16 characteristic peaks were identified by LC-MS-IT-TOF, including eight 20(S)-protopanaxatriol (PPT) type saponins and eight 20(S)-protopanaxadiol (PPD) type saponins. The established method was utilized to evaluate the quality of notoginseng samples cultivated by manual intervened methods to overcome continuous cropping obstacles.As a result, HPLC fingerprint similarity, content of Fa and ratio of notoginsenoside K and notoginsenoside Fa (N-K/Fa) were found out to be as valuatable markers of the quality of samples in continuous cropping obstacle research, of which N-K/Fa could also be applied to the analysis of notoginseng samples with different growth years.Notoginseng samples with continuous cropping obstacle had HPLC fingerprint similarity lower than 0.87, in consistent with normal sample, and had significant lower content of notoginsenoside Fa and significant higher N-K/Fa (2.35-4.74) than normal group (0.45-1.33). All samples in the first group with manual intervention showed high similarity with normal group (>0.87), similar content of common peaks and N-K/Fa (0.42-2.06). The content of notoginsenoside K in the second group with manual intervention was higher than normal group. All samples except two displayed similarity higher than 0.87 and possessed content of 16 saponins close to normal group. The result showed that notoginseng samples with continuous cropping obstacle had lower quality than normal sample. And manual intervened methods could improve their quality in different levels.The method established in this study was simple, fast and accurate, and the markers may provide new guides for quality control in continuous cropping obstacle research of notoginseng.

Transcriptome analysis of Panax vietnamensis var. fuscidicus discovers putative ocotillol-type ginsenosides biosynthesis genes and genetic markers.

BACKGROUND: P. vietnamensis var. fuscidiscus, called "Yesanqi" in Chinese, is a new variety of P. vietnamensis, which was first found in Jinping County, the southern part of Yunnan Province, China. Compared with other Panax plants, this species contains higher content of ocotillol-type saponin, majonoside R2. Despite the pharmacological importance of ocotillol-type saponins, little is known about their biosynthesis in plants. Hence, P. vietnamensis var. fuscidiscus is a suitable medicinal herbal plant species to study biosynthesis of ocotillol-type saponins. In addition, the available genomic information of this important herbal plant is lacking. RESULTS: To investigate the P. vietnamensis var. fuscidiscus transcriptome, Illumina HiSeq™ 2000 sequencing platform was employed. We produced 114,703,210 clean reads, assembled into 126,758 unigenes, with an average length of 1,304 bp and N50 of 2,108 bp. Among these 126,758 unigenes, 85,214 unigenes (67.23%) were annotated based on the information available from the public databases. The transcripts encoding the known enzymes involved in triterpenoid saponins biosynthesis were identified in our Illumina dataset. A full-length cDNA of three Squalene epoxidase (SE) genes were obtained using reverse transcription PCR (RT-PCR) and the expression patterns of ten unigenes were analyzed by reverse transcription quantitative real-time PCR (RT-qPCR). Furthermore, 15 candidate cytochrome P450 genes and 17 candidate UDP-glycosyltransferase genes most likely to involve in triterpenoid saponins biosynthesis pathway were discovered from transcriptome sequencing of P. vietnamensis var. fuscidiscus. We further analyzed the data and found 21,320 simple sequence repeats (SSRs), 30 primer pairs for SSRs were randomly selected for validation of the amplification and polymorphism in 13 P. vietnamensis var. fuscidiscus accessions. Meanwhile, five major triterpene saponins in roots of P. vietnamensis var. fuscidicus were determined using high performance liquid chromatography (HPLC) and evaporative light scattering detector (ELSD). CONCLUSIONS: The genomic resources generated from P. vietnamensis var. fuscidiscus provide new insights into the identification of putative genes involved in triterpenoid saponins biosynthesis pathway. This will facilitate our understanding of the biosynthesis of triterpenoid saponins at molecular level. The SSR markers identified and developed in this study show genetic diversity for this important crop and will contribute to marker-assisted breeding for P. vietnamensis var. fuscidiscus.

Transcriptomic comparison of the self-pollinated and cross-pollinated flowers of Erigeron breviscapus to analyze candidate self-incompatibility-associated genes.

BACKGROUND: Self-incompatibility (SI) is a widespread and important mating system that promotes outcrossing in plants. Erigeron breviscapus, a medicinal herb used widely in traditional Chinese medicine, is a self-incompatible species of Asteraceae. However, the genetic characteristics of SI responses in E. breviscapus remain largely unknown. To understand the possible mechanisms of E. breviscapus in response to SI, we performed a comparative transcriptomic analysis with capitulum of E. breviscapus after self- and cross-pollination, which may provide valuable information for analyzing the candidate SI-associated genes of E. breviscapus. METHODS: Using a high-throughput next-generation sequencing (Illumina) approach, the transcriptionexpression profiling of the different genes of E. breviscapus were obtained, some results were verified by quantitative real time PCR (qRT-PCR). RESULTS: After assembly, 63,485 gene models were obtained (average gene size 882 bp; N50 = 1485 bp), among which 38,540 unigenes (60.70% of total gene models) were annotated by comparisons with four public databases (Nr, Swiss-Prot, KEGG and COG): 38,338 unigenes (60.38% of total gene models) showed high homology with sequences in the Nr database. Differentially expressed genes were identified among the three cDNA libraries (non-, self- and cross-pollinated capitulum of E. breviscapus), and approximately 230 genes might be associated with SI responses. Several these genes were upregulated in self-pollinated capitulum but downregulated in cross-pollinated capitulum, such as SRLK (SRK-like) and its downstream signal factor, MLPK. qRT-PCR confirmed that the expression patterns of EbSRLK1 and EbSRLK3 genes were not closely related to SI of E. breviscapus. CONCLUSIONS: This work represents the first large-scale analysis of gene expression in the self-pollinated and cross-pollinated flowers of E. breviscapus. A larger number of notable genes potentially involved in SI responses showed differential expression, including genes playing crucial roles in cell-cell communication, signal transduction and the pollination process. We thus hypothesized that those genes showing differential expression and encoding critical regulators of SI responses, such as MLPK, ARC1, CaM, Exo70A1, MAP, SF21 and Nod, might affect SI responses in E. breviscapus. Taken together, our study provides a pool of SI-related genes in E. breviscapus and offers a valuable resource for elucidating the mechanisms of SI in Asteraceae.