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.     

Headspace Solid-Phase Micro-Extraction Gas Chromatography/Mass Spectrometry (HS-SPME-GC/MS)-Based Untargeted Metabolomics Analysis for Comparing the Volatile Components from 12 <i>Panax</i> Herbal Medicines

Quality control of ginseng currently is mainly based on ginsenoside analysis, but rarely focuses on the volatile organic components. In the current work, an untargeted metabolomics approach, by headspace solid-phase micro-extraction gas chromatography/mass spectrometry (HS-SPME-GC/MS), was elaborated and further employed to holistically compare the compositional difference of the volatile components simultaneously from 12 Panax herbal medicines, which included P. ginseng (PG), P. quinquefolius (PQ), P. notoginseng (PN), red ginseng (PGR), P. ginseng leaf (PGL), P. quinquefolius leaf (PQL), P. notoginseng leaf (PNL), P. ginseng flower (PGF), P. quinquefolius flower (PQF), P. notoginseng flower (PNF), P. japonicus (PJ), and P. japonicus var. major (PJvm). Chromatographic separation was performed on an HP-5MS elastic quartz capillary column using helium as the carrier gas, enabling good resolution within 1 h. We were able to characterize totally 259 volatile compounds, including 82 terpenes (T), 46 alcohols (Alc), 29 ketones (K), 25 aldehydes (Ald), 21 esters (E), and the others. By analyzing 90 batches of ginseng samples based on the untargeted metabolomics workflows, 236 differential ions were unveiled, and accordingly 36 differential volatile components were discovered. It is the first report that simultaneously compares the compositional difference of volatile components among 12 Panax herbal medicines, and useful information is provided for the quality control of ginseng aside from the well-known ginsenosides.     

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.     

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 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.     

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.     

Regulation and differential expression of protopanaxadiol synthase in Asian and American ginseng ginsenoside biosynthesis by RNA interferences

Asian ginseng (Panax ginseng) and American ginseng (Panax quinquefolium), are thought to be representative plant of Panax species, have important commercial value and are used in worldwide. Panax species produces triterpene saponins called ginsenosides, which are classified into two groups by the skeleton of aglycones, namely dammarane-type and oleanane-type. Dammarane-type ginsenosides dominate over oleanane-type not only in amount but also in structural varieties. Researches shows that the saponins content in American ginseng is higher than that in Asian ginseng, the higher part of ginsenosides is from dammarane-type biosynthesis. It has been proposed that protopanaxadiol derived from dammarenediol-II, is a key hydroxylation by cytochrome P450 for the biosynthesis of ginsenosides, and the gene number of protopanaxadiol synthase has been published independent in Asian ginseng (PgCYP716A47). However, little is known about genes involved in hydroxylation and glycosylation in American ginseng ginsenoside biosynthesis. Here, we first cloned and identified a P450 gene named PqD12H encoding enzymes catalyzed dammarenediol-II to protopanaxadiol by RT-PCR using degenerate primers designed based on sequence homology. In vitro, the ectopic expression of PqD12H in recombinant WAT21 yeast resulted in protopanaxadiol production after dammarenediol-II was added to the culture medium. In vivo, we established both PgCYP716A47 and PqD12H RNAi transgenic. The RT-PCR and HPLC analysis of the final products of protopanaxadiol and protopanaxatriol showed a result that declined level of protopanaxadiol-type and protopanaxatriol-type ginsenosides. It suggested that the P450 synthase content or expression in American ginseng exceed than in Asian ginseng. The result elucidated the evolution relationship of P450s and the reason of different saponins content among Panax species.     

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.     

Cytogenetic analysis of Lycopersicon esculentum (+) Solanum etuberosum somatic hybrids and their androgenetic regenerants

The aim of the study was to characterize genomic relationships among cultivated tomato (Lycopersicon esculentum Mill.) (2n=2x=24) and diploid (2n=2x=24) non-tuberous wild Solanum species (S. etuberosum Lindl.). Using genomic in situ hybridization (GISH) of mitotic and meiotic chromosomes, we analyzed intergeneric somatic hybrids between tomato and S. etuberosum. Of the five somatic hybrids, two plants were amphidiploids (2n=4x=48) mostly forming intragenomic bivalents in their microsporocytes, with a very low frequency of multivalents involving the chromosomes of tomato and S. etuberosum (less than 0.2 per meiocyte). Tomato chromosomes showed preferential elimination during subsequent meiotic divisions of the amphidiploids. Transmission of the parental chromosomes into microspores was also evaluated by GISH analysis of androgenic plants produced by direct embryogenesis from the amphidiploid somatic hybrids. Of the four androgenic regenerants, three were diploids (2n=2x=24 or 2n=2x+1=25) derived from reduced male gametes of the somatic hybrids, and one plant was a hypertetraploid (2n=4x+4=52). GISH revealed that each anther-derived plant had a unique chromosome composition. The prospects for introgression of desirable traits from S. etuberosum into the gene pool of cultivated tomato are discussed. Received: 2 August 2000 / Accepted: 4 December 2000     

Karyotype Analysis of Gossypium arboreum × G. bickii by Genome in situ Hybridization

By using genome in situ hybridization (GISH) on root somatic chromosomes of allotetraploid derived from the cross Gossypium arboreum × G. bickii with genomic DNA (gDNA) of G. bickii as a probe, two sets of chromosomes, consisting of 26 chromosomes each, were easily distinguished from each other by their distinctive hybridization signals. GISH analysis directly proved that the hybrid GarboreumxG. bickii is an allotetraploid amphiploid. The karyotype formula of the species was 2n = 4x = 52 = 46m (4sat) + 6sm (4sat). We identified four pairs of satellites with two pairs in each sub-genome. FISH analysis using 45S rDNA as a probe showed that the cross G. arboreumxG. bickii contained 14 NORs. At least five pairs of chromosomes in the G sub-genome showed double hybridization (red and blue) in their long arms, which indicates that chromatin introgression from the A sub-genome had occurred.     

Peg-mediated fusion of Rubus idaeus (raspberry) and R. fruticosus (blackberry) protoplasts,selection and characterisation of callus lines

ABSTRACT

Cell suspension-derived protoplasts of two cultivated Rubus species, Rubus idaeus-raspberry (subgenus Idaeobatus 2n=2x=14) and R. fruticosus-blackberry (a complex species aggregate within the subgenus Eubatus, 2n=4x=28) were fused using different polyethylene glycol (PEG) fusion treatments. Duration of PEG treatment and choice of culture media influenced the rate of cell divisions and plating efficiency. Colony formation was initiated on solid media for the production of several callus lines. Cytological analyses were performed on selected callus lines with hexaploid chromosome number. Two hexaploid fusion callus lines, selected for their homogeneity in growth and ploidy level, were examined by molecular cytogenetic techniques of fluorescent in situ hybridisation (FISH) and genomic in situ hybridisation (GISH). GISH revealed the presence of the heterokaryon within the fusion callus lines. FISH probed with ribosomal DNA (rDNA) showed variable numbers and sizes of loci. Aberrant distribution and condensation of rDNA were common in interphase cells. FISH results suggest that large karyotype rearrangements occurred, including variation in chromosome number and rDNA loci translocations. Attempts to regenerate plants from the hexaploid callus lines following several applications of plant growth regulator combinations were unsuccessful. This may be attributed to the genomic reorganisation and instability of these long-term fusion callus cultures.     

Chromosomal localization of two novel repetitive sequences isolated from the Chenopodium quinoa Willd. genome

The chromosomal organization of two novel repetitive DNA sequences isolated from the Chenopodium quinoa Willd. genome was analyzed across the genomes of selected Chenopodium species. Fluorescence in situ hybridization (FISH) analysis with the repetitive DNA clone 18-24J in the closely related allotetraploids C. quinoa and Chenopodium berlandieri Moq. (2n = 4x = 36) evidenced hybridization signals that were mainly present on 18 chromosomes; however, in the allohexaploid Chenopodium album L. (2n = 6x = 54), cross-hybridization was observed on all of the chromosomes. In situ hybridization with rRNA gene probes indicated that during the evolution of polyploidy, the chenopods lost some of their rDNA loci. Reprobing with rDNA indicated that in the subgenome labeled with 18-24J, one 35S rRNA locus and at least half of the 5S rDNA loci were present. A second analyzed sequence, 12-13P, localized exclusively in pericentromeric regions of each chromosome of C. quinoa and related species. The intensity of the FISH signals differed considerably among chromosomes. The pattern observed on C. quinoa chromosomes after FISH with 12-13P was very similar to GISH results, suggesting that the 12-13P sequence constitutes a major part of the repetitive DNA of C. quinoa.     

Inter-genomic relationships among three medicinal herbs: Cnidium officinale, Ligusticum chuanxiong and Angelica polymorpha

Cnidium officinale, Ligusticum chuanxiong and Angelica polymorpha have long been used as Chinese medicinal herbs to treat the same conditions. To elucidate the inter-specific relationships among these species, molecular and molecular cytogenetic analyses were conducted. Cytogenetically, C. officinale and A. polymorpha were found to be diploid with 2n=2x=22, but L. chuanxiong had a triploid chromosome number (2n=3x=33). FISH analysis revealed that each two pairs of the 45S- and 5S rRNA genes were detected on the different chromosomes of C. officinale, and that triplet of the rRNA genes were localized on the same loci of the homologue in L. chuanxiong. GISH conducted using a genomic DNA probe detected strong cross-hybridization of genomes between C. officinale and L. chuanxiong, while no distinct GISH signal between C. officinale and A. polymorpha was observed. ITS and NTS data also revealed a very high sequence homology (95–96%) between C. officinale and L. chuanxiong. The RFLP profile of the rDNA-ITS enabled us to confirm the genetic distance among species. An effective species-specific SCAR marker for the identification of the wild plant, A. polymorpha, was also developed. Taken together, the results of this study suggest two possibilities; (1) L. chuanxiong originated from C. officinale, or (2) L. Chuanxiong and C. officinale diverged from the same parental plant.     

Physical Mapping of rRNA Gene Loci and Inter-specific Relationships in Wild <Emphasis Type="Italic">Lilium</Emphasis> Distributed in Korea

Molecular cytogenetic analyses using fluorescence in situ hybridization (FISH) and genomic in situ hybridization (GISH) were carried out to elucidate inter-specific relationships among wild Lilium species distributed in Korea. FISH revealed four to eight 45S rRNA gene loci, which are located on chromosomes 1–7, 10, and 11 among the different species. In contrast, the 5S rRNA gene locus was conserved on the long arm of chromosome 3, occasionally with two adjacent sites on the same chromosome arm in a few species. The 5S rDNA site was located adjacent to the 45S rDNA site in only three species, Lilium distichum, Lilium hansonii, and Lilium tsingtauense. GISH analysis using genomic DNA probes detected strong hybridization of genomes between diploid and triploid Lilium lancifolium species, demonstrating that triploid plants were derived from diploid L. lancifolium and not from Lilium maximowiczii. Phylogenetic analysis of the ITS and NTS sequences supported the cytogenetic data as well as Comber’s classification of the genus Lilium.     

Relationships of the woody Medicago species (section Dendrotelis) assessed by molecular cytogenetic analyses

Background and Aims

The organization of rDNA genes in the woody medic species from the agronomically important Medicago section Dendrotelis was analysed to gain insight into their taxonomic relationships, to assess the levels of infraspecific variation concerning ribosomal loci in a restricted and fragmented insular species (M. citrina) and to assess the nature of its polyploidy.

Methods

Fluorescence in situ hybridization (FISH) was used for physical mapping of 5S and 45S ribosomal DNA genes in the three species of section Dendrotelis (M. arborea, M. citrina, M. strasseri) and the related M. marina from section Medicago. Genomic in situ hybridization (GISH) was used to assess the genomic relationships of the polyploid M. citrina with the putatively related species from section Dendrotelis.

Key Results

The diploid (2n = 16) M. marina has a single 45S and two 5S rDNA loci, a pattern usually detected in previous studies of Medicago diploid species. However, polyploid species from section Dendrotelis depart from expectations. The tetraploid species (2n = 32) M. arborea and M. strasseri have one 45S rDNA locus and two 5S rDNA loci, whereas in the hexaploid (2n = 48) M. citrina four 45S rDNA and five 5S rDNA loci have been detected. No single chromosome of M. citrina was uniformly labelled after using genomic probes from M. arborea and M. strasseri. Instead, cross-hybridization signals in M. citrina were restricted to terminal chromosome arms and NOR regions.

Conclusions

FISH results support the close taxonomic interrelationship between M. arborea and M. strasseri. In these tetraploid species, NOR loci have experienced a diploidization event through physical loss of sequences, a cytogenetic feature so far not reported in other species of the genus. The high number of rDNA loci and GISH results support the specific status for the hexaploid M. citrina, and it is suggested that this species is not an autopolyploid derivative of M. arborea or M. strasseri. Further, molecular cytogenetic data do not suggest the hypothesis that M. arborea and M. strasseri were involved in the origin of M. citrina. FISH mapping can be used as an efficient tool to determine the genomic contribution of M. citrina in somatic hybrids with other medic species.Key words: Medicago arborea, M. citrina, M. strasseri, rRNA genes, 18S-5·8S-25-S, 5S, FISH mapping, GISH, polyploidy     

Molecular cytogenetic analysis of the Vigna species distributed in Korea

Vigna plants distributed in Korea were analyzed by molecular cytogenetic fluorescence in situ hybridization (FISH), genomic in situ hybridization (GISH) and rDNA ITS/NTS sequences. FISH revealed that variable 45S rRNA gene loci (one to four) were localized on the terminal regions of chromosomes, while two conserved 5S rRNA gene loci from all species examined, except for rice bean (single locus), were detected. FISH and GISH showed the characteristic organization of rRNA gene loci and genomic homology on the chromosomes, indicating their cytogenetic relatationships. ITS sequence revealed that there was considerable variation in length (190–207 bp in ITS1, 205–221 bp in ITS2) and nucleotide composition (7–67 bp). The 5S rRNA gene unit comprised coding region (118 bp) and extensive sequence heterogeneity (97–221 bp). Phylogenetic analysis of the ITS and NTS sequences demonstrated that the Vigna species are divided into two groups: angularis (V. angularis, V. umbellata, V. nakashimae and V. nipponensis) and unguiculata (V. unguiculata, V. sesquipedalis and V. vexillata). Sequence data also showed that mung bean was closer to the angularis group.     

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.     

Characterization of polymorphic microsatellite loci in cultivated and wildPanax ginseng

Ginseng (Panax ginseng) is one of the most important herbal remedies used in East Asia. The present study investigated six polymorphic microsatellite markers (PG29, PG281, PG287, PG668, PG1319, and PG1481) in samples of cultivated and wildP. ginseng collected in Korea. Total allelic number observed in this study was 27 (average allelic numbers per locus: 4.5). All examined loci exhibited deviation from the Hardy-Weinberg equilibrium and deficiency of heterozygosity in both cultivated and wild groups. Although the wild ginseng group exhibited slightly more polymorphic behavior (mean PIC=0.392, GD=0.454 and H_obs=0.129), compared with the cultivated group (mean PIC=0.383, GD=0.438 and H_obs=0.105), no significant differences of allele frequencies and genotype distributions were revealed. By combined analysis of six loci in 100 cultivated ginsengs, 71 different types were observed. The analyzed microsatellite loci in this study will be helpful for understanding genetic variation, QTL mapping and phylogenic studies inPanax species.