FISH and GISH analysis of the genomic relationships amongPanax species

Ginseng is a well-known medicinal plant that has been used as an anti-aging agent for many years in East Asia. In the genusPanax, only three species,P. ginseng (Oriental ginseng),P. quinquefolius (American ginseng) andP. notoginseng (Chinese ginseng), are currently considered to be important medicinal herbs. Despite the increase in their breeding value, molecular cytogenetic information on the species is not available. To analyze the genomic relationships among thePanax species, FISH (fluorescencein situ hybridization) and GISH (genomicin situ hybridization) techniques were applied. FISH analysis revealed that the 45S and 5S rRNA genes ofP. notoginseng (2n=2x=24) andP. ginseng (2n=4x=48) cluster on a single locus on different chromosomes, whileP. quinquefolius (2n=4x=48),P. japonicus (2n=4x=48), and Korean wild ginseng (2n =4x= 48) had one locus of the 45S rRNA gene and two loci of the 5S rRNA gene, respectively. GISH analysis using genomic DNA as a probe detected strong cross-hybridization of genomes betweenP. ginseng andP. quinquefolius. GISH analysis of other species showed weak or no distinct signals on the chromosomes. Our data revealed thatP. ginseng andP. quinquefolius showed the highest degree of homology, indicating that these species diverged in most recent years.     

CAPS markers using mitochondrial consensus primers for molecular identification of Panax species and Korean ginseng cultivars (Panax ginseng C. A. Meyer)

Cleaved amplified polymorphic sequence (CAPS) marker system using mitochondrial consensus primers was applied for molecular identification of Korean ginseng cultivars (Panax ginseng). Initially, a total of 34 primers were tested to six Korean ginseng cultivars and two foreign Panax species, P. quinquefolius and P. notoginseng. In the polymerase chain reaction (PCR) amplification results, four primers (mt7, mt11, mt13, and mt18) generated co-dominant polymorphic banding patterns discriminating the Korean ginseng cultivars from P. quinquefolius and P. notoginseng. In the CAPS analysis results, the majority of the cleaved PCR products also yielded additional latent polymorphisms between the Korean ginseng cultivars and two foreign Panax species. Specific latent CAPS polymorphisms for cultivar Gopoong and Chunpoong were detected from internal region amplified with mt9 primer by treating HinfI and Tsp509I endonucleases, respectively. Sequencing analysis revealed that the length of amplified region of Korean ginseng cultivars was 2,179 bp, and those of P. quinquefolius and P. notoginseng were 2,178 and 2,185 bp, respectively. Blast search revealed that the amplified region was a mitochondrial cytochrome oxidase subunit 2 (cox2) gene intron II region. Nineteen single nucleotide polymorphisms (SNP) including each specific SNP for Gopoong and Chunpoong, and three insertion and deletion (InDel) polymorphisms were detected by sequence alignment. The CAPS markers developed in this study, which are specific to Gopoong and Chunpoong, and between the Korean ginseng cultivars and two foreign Panax species, will serve as a practical and reliable tool for their identification, purity maintenance, and selection of candidate lines and cultivars.     

A phylogenetic analysis ofPanax (Araliaceae): Integrating cpDNA restriction site and nuclear rDNA ITS sequence data

A phylogenetic analysis ofPanax was performed using restriction site variations of eight PCR-amplified chloroplast regions. Twenty populations were examined, representing 13 of the 14 species ofPanax. Aralia cordata was used as the outgroup. The 11 restriction endonucleases produced a total of 105 restriction sites and length variations from the large single-copy region of cpDNA. Forty restriction variations are polymorphic. The cpDNA tree is largely congruent with the nuclear ribosomal ITS phylogeny. Similar to the ITS tree, the cpDNA dataset suggests the following relationships: (1)P. trifolius from eastern North America is sister to the clade consisting of all otherPanax species; (2)P. ginseng andP. japonicus from eastern Asia form a clade withP. quinquefolius from eastern North America; (3) the HimalayanP. pseudoginseng is most closely related toP. stipuleanatus of southwestern China; and (4) the medicinally importantP. notoginseng forms a clade with the closely relatedP. bipinnatifidus, P. ginseng, P. japonicus, P. major, P. quinquefolius, P. sinensis, P. wangianus, andP. zingiberensis. Two biogeographic disjunctions are detectable withinPanax. One is the connection of the eastern North AmericanP. trifolius with the rest ofPanax species. The other is the more recent disjunction between the North AmericanP. quinquefolius and the eastern AsianP. ginseng andP. japonicus. The active orogenies caused by the collision of the Indian Plate with Asia may have facilitated the diversification ofPanax taxa in Asia in the late Tertiary.     

First Report of <i>Fusarium striatum</i> Causing Root Rot Disease of <i>Panax notoginseng</i> in Yunnan,China

Panax notoginseng is a traditional Chinese medicinal plant. Root rot of P. notoginseng is one of the most serious diseases affecting P. notoginseng growth and causes wilted leaves, fewer lateral roots and rotten roots. Root rot is a soil-borne disease, and mainly occurs from June to August in Yunnan Province when the temperatures are high and the air is humid. In this study, the endophytic fungal genus Fusarium isolate E-2018.1.22-#3.2 was obtained from a P. notoginseng embryo. Fusarium isolate E-2018.1.22-#3.2 was identified as Fusarium striatum based on morphological characteristics and molecular analysis. The fungus was found to have conidiophores and macroconidia, and its ITS, LSU and TEF-1α genes shared 100%, 99.2% and 99% identities with the homologous genes of Fusarium striatum, respectively. Isolate F. striatum E-2018.1.22-#3.2 can cause root rot symptoms, including black, soft roots, fewer lateral roots and leaf wilt, in 93% of the experimental P. notoginseng plants, and could be re-isolated, fulfilling Koch’s postulates. When the P. notoginseng plants were treated with the fungicide pyraclostrobin, isolate F. striatum E-2018.1.22-#3.2 was unable to cause root rot. We have therefore demonstrated that F. striatum E-2018.1.22-#3.2 is able to cause root rot disease in P. notoginseng. This is the first report of root rot disease caused by F. striatum on P. notoginseng in China.     

Application of ultra-performance LC-TOF MS metabolite profiling techniques to the analysis of medicinal <Emphasis Type="Italic">Panax</Emphasis> herbs

The morphological appearance and some ingredients of Panax ginseng, Panax notoginseng and Panax japonicus of the Panax genus are similar. However, their pharmacological activities are obviously different due to the significant differences in the types and quantity of saponins in each herb. In the present study, ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOFMS) was used to profile the abundances of metabolites in the three medicinal Panax herbs. Multivariate statistical analysis technique, that is, principle component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) were used to discriminate between the Panax samples. PCA of the analytical data showed a clear separation of compositions among the three medicinal herbs. The critical markers such as chikusetsusaponin IVa, ginsenoside R0, ginsenoside Rc, ginsenoside Rb1, ginsenoside Rb2 and ginsenoside Rg2 accountable for such variations were identified through the corresponding loading weights, and the tentative identification of biomarkers is completed by the accurate mass of TOFMS and high resolution and high retention time reproducibility performed by UPLC. The proposed analytical method coupled with multivariate statistical analysis is reliable to analyze a group of metabolites present in the herbal extracts and other natural products. This method can be further utilized to evaluate chemical components obtained from different plants and/or the plants of different geographical locations, thereby classifying the medicinal plant resources and potentially elucidating the mechanism of inherent phytochemical diversity.     

Development of peptide nucleic acid (PNA) microarray for identification of Panax species based on the nuclear ribosomal internal transcribed spacer (ITS) and 5.8S rDNA regions

This study describes the identification of Panax species using a peptide nucleic acid (PNA) microarray. P. ginseng, P. quienquefolius, and P. japonicus were distinguished from each other using 5 PNA probes designed based on three single nucleotide polymorphisms (SNPs) detected in internal transcribed spacer (ITS) and 5.8S rDNA regions. Signal intensity comparison between PNA and DNA microarrays revealed that the PNA microarray provides a significantly more stable and specific fluorescent signal intensity than the DNA microarray. Three Panax species identified by the PNA microarray were denoted as barcode numbers depending on their fluorescent signal patterns of each species using 5 PNA probes (PG-ITS-116, PG-ITS-414-1, PG-ITS-414-2, PG-ITS-425-1, and PG-ITS-425-2). P. ginseng, P. quinquefolius, and P. japonicus were denoted as ‘11010’, ‘00202’ and ‘00000’, respectively. The PNA microarray developed in this study will be useful for legitimizing the distribution of ginseng in domestic and foreign ginseng markets.     

Effect of growth regulators on ginsenoside production in the cell culture of two ginseng species

Plants belonging to the genus Panax produce ginsenosides that possess pharmacological properties. The ability to synthesize these compounds is preserved in some cultured cells of ginseng. In this work, we used suspension cell cultures of two species of ginseng: Panax japonicus var. repens C. A. Mey and P. ginseng C. A. Mey. The first culture was grown on MS medium supplemented with α-NAA. After one subculturing, cell biomass increased 5–6 times with the level of ginsenosides being equal to 2.5–3.0% of dry weight. The second culture was grown on the same medium supplemented with 2,4-D. In this case, biomass increment was 3–5-fold, and ginsenosides were produced in trace amounts. Substitution of 2,4-D for NAA in suspension cell culture of P. japonicus brought about deterioration of growth characteristics, but the content and composition of ginsenosides was not changed. In the suspension cell culture of P. ginseng, substitution of NAA for 2,4-D decreased the rate of biomass accumulation and increased the extent of cell aggregation, with total content of ginsenosides increasing 25 times and their assortment being complete.     

Cell culture system versus adventitious root culture system in Asian and American ginseng: a collation

Panax ginseng and Panax quinquefolius of Panax genus are valuable as health foods as well as pharmaceuticals for the treatment of cancer, diabetes and ageing as these plants possess saponins. In the current study, Cell and adventitious root cultures of P. ginseng and P. quinquefolius were investigated for the biomass, cell division, saponin content and ginsenosides profile from four lines namely P. quinquefolius (AM), P. ginseng mountain (Mt.) Baekdu line, P. ginseng Cheong-sol line (CS) and P. ginseng CBN line (CBN) with the objective of comparing cell and adventitious root systems to check their efficacy for the production of ginseng saponins. Additionally, genes related to ginsenoside biosynthesis were also analyzed concerning to cell and adventitious root lines. The results indicated that various cell lines were better in multiplication and growth compared to adventitious root lines. However, adventitious root lines showed higher accumulation of dry biomass (1.5–2 fold) than that of cell lines. CS adventitious root line showed higher saponin content and ginsenoside productivity (10.48 mg·g^?1 DW, 12.88 mg·L^?1, respectively) than that of CS cell line (9.50 mg·g^?1 DW, 2.39 mg·L^?1, respectively). Especially, Rd ginsenoside productivity of CS adventitious root line recorded fourfold higher than CS cell line. Genes which are related to ginsenoside biosynthesis such as P. ginseng squalene synthase (PgSS2), P. ginseng squalene epoxidase (PgSE2), P. ginseng protopanaxadial synthase (PgPPDS) and P. ginseng protopanaxatriol synthase (PgPPTS) were analyzed by real time quantitative polymerase chain reaction to support ginsenoside production. The adventitious root culture system described in this study is useful system for biomass and ginsenoside production.     

Development of interspecies hybrids to increase ginseng biomass and ginsenoside yield

Key message

Interspecific hybrids between Panax ginseng and P. quinquefolius results in hybrid vigor and higher ginsenoside contents.

Abstract

Ginseng is one of the most important herbs with valued pharmaceutical effects contributing mainly by the presence of bioactive ginsenosides in the roots. However, ginseng industry is impeded largely by its biological properties, because ginseng plants are slow-growing perennial herbs with lower yield. To increase the ginseng yield and amounts of ginsenosides, we developed an effective ginseng production system using the F₁ progenies obtained from the interspecific reciprocal cross between two Panax species: P. ginseng and P. quinquefolius. Although hybrid plants show reduced male fertility, F₁ hybrids with the maternal origin either from P. ginseng or P. quinquefolius displayed heterosis; they had larger roots and higher contents of ginsenosides as compared with non-hybrid parental lines. Remarkably, the F₁ hybrids with the maternal origin of P. quinquefolius had much higher ginsenoside contents, especially ginsenoside Re and Rb1, than those with the maternal origin of P. ginseng. Additionally, non-targeted metabolomic profiling revealed a clear increase of a large number of primary and secondary metabolites including fatty acids, amino acids and ginsenosides in hybrid plants. To effectively identify the F₁ hybrids for the large-scale cultivation, we successfully developed a molecular marker detection system for discriminating F₁ reciprocal hybrids. In summary, this work provided a practical system for reciprocal hybrid ginseng production, which would facilitate the ginseng production in the future.

     

A fast SNP identification and analysis of intraspecific variation in the medicinal Panax species based on DNA barcoding

Medicinal plants of the Panax genus belonging to Araliaceae family are well-known, rare plants used as tonics in traditional Chinese medicine and have been described in the Chinese Pharmacopoeia. Because of the high price and the huge human demand, these commercial products often contain adulterants. In this study, 377 sequences from four species were analyzed. Single nucleotide polymorphisms (SNPs) were detected and patterns of intragenomic variation in internal transcribed spacer 2 (ITS2) from the four Panax species were studied. Intraspecific variations were analyzed based on three typical DNA barcodings (ITS2, matK and psbA-trnH). Results from this study revealed that intraspecific genetic distances in Panax ginseng and Panax quinquefolius were quite low (0–0.002) and the multi-copy ITS2 could be considered a single locus in the genomes of these two species. Five stable SNPs were detected in ITS2 region to identify the Panax medicinal species. Considering the mixed powder of P. ginseng and P. quinquefolius, double peaks could be clearly examined at SNP positions and the height of the peaks could indicate the mixed ratio roughly. Our findings indicate that SNP-based molecular barcodes could be developed as a routine method for the identification of the Panax genus with closely related species and the mixed powder P. ginseng and P. quinquefolius.     

Development and characterization of new microsatellite markers in <Emphasis Type="Italic">Panax</Emphasis><Emphasis Type="Italic">ginseng</Emphasis> (C.A. Meyer) from BAC end sequences

Panax ginseng, commonly known as Korean ginseng, is a valued source of herbal medicine in Korea and China. We have developed and characterized 35 microsatellite markers in P. ginseng from available BAC end sequences. Characterization of these 35 SSR primer pairs in 14 cultivars of P. ginseng showed 12 primer pairs to be polymorphic and 19 primer pairs to be monomorphic, while the remaining four primer pairs did not produce any product. The number of alleles amplified ranged from 4 to 8 per primer pair, with an average of six alleles per locus. The expected and observed heterozygosities ranged from 0.7500 to 0.9678 and 0.5645 to 0.7109, respectively. None of these loci deviated from Hardy–Weinberg equilibrium. All of the functional primer pairs of P. ginseng showed cross-species transferability with Panax quinquefolium. The cross-species transferable markers could be used for ginseng cultivar identification, for genomic studies, including mapping of specific trait QTL/genes, and for measuring conservation of ginseng.     

Chloroplast DNA variation of Panax (Araliaceae) in Nepal and its taxonomic implications

The restriction site and size variation of five PCR amplified fragments of noncoding chloroplast DNA (cpDNA) was examined in material from 13 populations ofPanax from Nepal and China. Fourteen restriction endonucleases produced 81 restriction site and length variations from the large single-copy region of cpDNA, 27 of which are polymorphic. The cpDNA dataset suggests two distinct groups ofPanax from Nepal (clades I and II). Clade I consists of two populations ofP. pseudoginseng subsp.pseudoginseng, and clade II is composed of material referable toP. pseudogingeng subsp.himalaicus (vars.himalaicus, angustifolius, andbipinnatifidus). The three accessions ofP. pseudoginseng subsp.japonicus andP. ginseng studied from China had cpDNA characters that differed from the HimalayanPanax. The highly distinctive cpDNA profile and morphology ofP. pseudoginseng subsp.pseudoginseng sensu Hara (1970) from central Nepal support its status as a separate species, which has an extremely restricted distribution.     

High-level sustainable production of the characteristic protopanaxatriol-type saponins from Panax species in engineered Saccharomyces cerevisiae

The Chinese medicinal plant Panax notoginseng has been traditionally used to activate blood flow and circulation, and to prevent blood stasis. P. notoginseng contains protopanaxatriol (PPT)-type saponins as its main active compounds, thus distinguishing it from the other two famous Panax species, P. ginseng and P. quinquefolius. Ginsenoside Rg1 (Rg1), notoginsenoside R1 (NgR1), and notoginsenoside R2 (NgR2) are three major PPT-type saponins in P. notoginseng and possess potential cardiovascular protection activities. However, their use in medical applications has long been hampered by the lack of sustainable and low-cost industrial-scale preparation methods. In this study, a PPT-producing yeast chassis strain was designed and constructed based on a previously constructed and optimized protopanaxadiol (PPD)-producing Saccharomyces cerevisiae strain, and further optimized by systemically engineering and optimizing the expression level of its key P450 biopart. Rg1-producing yeast strains were constructed by introducing PgUGT71A53 and PgUGT71A54 into the PPT chassis strain. The fermentation titer of Rg1 reached 1.95 g/L. A group of UDP-glycosyltransferases (UGT) from P. notoginseng and P. ginseng were characterized, and were found to generate NgR1 and NgR2 by catalyzing the C₆–O-Glc xylosylation of Rg1 and Rh1, respectively. Using one of these UGTs, PgUGT94Q13, and the previously identified PgUGT71A53 and PgUGT71A54, the biosynthetic pathway to produce saponins NgR1 and NgR2 from PPT could be available. The NgR1 cell factory was further developed by introducing PgUGT94Q13 and a heterologous UDP-xylose biosynthetic pathway from Arabidopsis thaliana into the highest Rg1-producing cell factory. The NgR2-producing cell factory was constructed by introducing PgUGT71A54, PgUGT94Q13, and the UDP-xylose biosynthetic pathway into the PPT chassis. De novo production of NgR1 and NgR2 reached 1.62 g/L and 1.25 g/L, respectively. Beyond the realization of artificial production of the three valuable saponins Rg1, NgR1, and NgR2 from glucose, our work provides a green and sustainable platform for the efficient production of other PPT-type saponins in engineered yeast strains, and promotes the industrial application of PPT-type saponins as medicine and functional foods.     

RAPD and Allozyme Analysis of Genetic Diversity in Panax ginseng C.A. Meyer and P. quinquefolius L.

Inter- and intraspecific variation of two ginseng species Panax ginseng and P. quinquefolius was estimated by studying 159 RAPD and 39 allozyme loci. Parameters of polymorphism and genetic diversity were determined and a tree was constructed to characterize the differences between individual plants, samples, and species. Genetic variation in P. ginseng proved to be lower than in P. quinquefolius. Gene diversity in the total P. ginseng sample was comparable with the mean expected heterozygosity of herbaceous plants. This suggests that wild P. ginseng plants in various areas of the currently fragmented natural habitat and cultivated plants of different origin have retained a significant proportion of their gene pool. The mean heterozygosity calculated per polymorphic locus for the RAPD phenotypes is similar to that of the allozyme loci and may be helpful in estimating gene diversity in populations of rare and endangered plant species.     

秦巴山区三种人参属药用植物叶绿体基因组特征分析

为明确珠子参、羽叶三七和秀丽假人参3种药用植物叶绿体基因组特征与系统发育关系,该文以秦巴山区3种人参属药用植物为研究对象,运用生物信息学技术,分析其叶绿体基因组特征及密码子使用偏好性,并探讨三者之间的亲缘关系。结果表明:(1)3种人参属药用植物的叶绿体基因组为典型的四分体结构,序列全长为 156 071～156 104 bp,总 GC 含量为 38.10%,基因组大小相似度较高。(2)均注释到 133 个基因,包括 88 个蛋白编码基因、37 个tRNA基因和 8 个 rRNA 基因。(3)3种人参属药用植物叶绿体密码子使用偏好性相似,密码子第 3 位碱基以 A/U 结尾为主,密码子使用模式在受到突变影响的同时,主要受到自然选择的影响。(4)系统发育结果显示,3种人参属药用植物的亲缘关系较近,并且秀丽假人参同羽叶三七亲缘关系更近。综上认为,秀丽假人参与珠子参基源植物之间存在近缘关系,这项发现对于珠子参中药材的资源开发利用和分子鉴定,以及进一步研究人参属物种的分类、系统发育和进化机制提供了重要依据。     

Pollination Biology of the Endangered Herbal Medicines <i>Dendrobium chrysotoxum</i> (Orchidaceae)

Pollination biology studies of the endangered herbal medicines Dendrobium chrysotoxum were conducted in natural pollination conditions using flower observation, pollinator observation and artificial pollination experiments. Populations of D. chrysotoxum with fragrance and nectar were pollinated by Ctenoplectra davidi Valhalla (Hymenoptera: Apidae) species. The floral structure of D. chrysotoxum adapted precisely to its pollinators. Flowers had a low capsule setting (0.17%) under natural conditions. However, compared to open pollination, artificial pollination experiments showed a significant increase in capsule setting, and D. chrysotoxum was cross-compatible and self-compatible, but there was pollinator limitation also. This study will provide important information for the preservation of this endangered species.     

AM Fungi and <i>Piriformospora indica</i> Improve Plant Growth of <i>Pinus elliottii</i> Seedlings

Pinus elliottii is an exotic afforestation pine extensively distributed in southern parts of China. In order to understand whether endophytic fungi can affect seedling growth of P. elliottii, Piriformospora indica (Pi), Funnelifcrmis mosseae (Fm), and Diversispora tortuosa (Dt) were inoculated respectively, and the non-inoculated group was set as control. The growth indexes, the contents of soluble sugar and soluble protein, and plant endogenous hormone levels in the leaves of P. elliottii, were analyzed. The results showed that Fm, Dt and Pi colonized the P. elliottii roots to form mycorrhizal structure and chlamydospores arranged in beads respectively. Three fungal inoculants exhibited the stimulated growth responses, whilst Dt illustrated the most positive effect on plant height, single fresh weight, trunk diameter and root system structure, compared with the control. On the other hand, the soluble sugar and soluble protein contents were increased distinctively in mycorrhizal plants. The endogenous IAA, GA3, ZR contents were increased, while the ABA contents were reduced in mycorrhizal plants versus non-mycorrhizal plants. The fungi-induced endogenous hormone changes triggered plant growth improvement of P. elliottii seedlings. This research unraveled the positive effect of AM fungi and P. indica on growth of pine seedlings, while, more application of endophytic fungi to fields needs to be explored.     

Structure and expression profiling of a novel calcium-dependent protein kinase gene <Emphasis Type="Italic">PgCDPK1a</Emphasis> in roots,leaves, and cell cultures of <Emphasis Type="Italic">Panax ginseng</Emphasis>

Calcium-dependent protein kinases (CDPKs) are proposed to play an essential role in plant defense responses. In this study, we aimed to define the full sequence of a CDPK gene of Panax ginseng and analyze its expression in roots, leaves, and cell cultures of P. ginseng, one of the most valuable Chinese traditional medicinal herbs. We isolated the full-length cDNA of a P. ginseng CDPK gene, which was designated PgCDPK1a. PgCDPK1a shares high sequence identity at the amino acidic level with previously reported CDPK sequences for other plant species. We analyzed PgCDPK1a expression in the leaves of wild-growing P. ginseng plants, and in the roots and leaves of cultivated P. ginseng plants growing in an open experimental nursery at a natural ginseng habitat. PgCDPK1a was more actively expressed in the young leaves of cultivated P. ginseng plants than in that of wild-growing ones. Finally, we analyzed the expression of the gene in control GV and five rolC and rolB transgenic callus cultures of P. ginseng with different levels of fresh biomass accumulation, pathogen-related gene expression, and ginsenoside production. We observed a strong positive correlation between fresh biomass accumulation of P. ginseng cell cultures and expression of the PgCDPK1a gene. There was a less clear negative correlation between the expression of pathogen-related genes and the content of ginsenosides with the PgCDPK1a expression in cell cultures of P. ginseng. Perhaps, PgCDPK1a is involved in ginseng growth, as a positive regulator.