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DOI: 10.1055/s-2004-832651
© Georg Thieme Verlag KG Stuttgart · New York
Phylogenetic Analysis of the DNA Sequence of the Non-Coding Region of Nuclear Ribosomal DNA and Chloroplast of Ephedra Plants in China
Dr. N. Kakiuchi
Faculty of Pharmaceutical Sciences
Kanazawa University
13-1 Takaramachi
Kanazawa 920-0934
Japan
Fax: +81-76-234-4490
Email: kakiuchi@p.kanazawa-u.ac.jp
Publication History
Received: February 6, 2004
Accepted: March 14, 2004
Publication Date:
18 November 2004 (online)
- Abstract
- Abbreviations
- Introduction
- Materials and Methods
- Results
- Discussion
- Acknowledgements
- References
Abstract
Twenty-four Ephedra plants belonging to 8 species grown in the northern and western parts of China were phylogenetically analyzed for their non-coding DNA sequences, internal transcribed spacers (ITSs) of nuclear ribosomal DNA as well as trnL intron and intergenic spacers between trnL and trnF (trnL/trnF) of the chloroplast. Based on the ITS sequences, the 8 species could be divided into 3 groups: Group 1 (Ephedra intermedia, E. sinica, E. przewalskii), Group 2 (E. equisetina, E. monosperma, E. gerardiana), and Group 3 (E. likiangensis, E. minuta). The species classified into Group 1 grow mainly in the north, Group 3 in the south and Group 2 in the center, suggesting their genetic and geographic relationships. A specific primer set was designed to classify the 3 groups by routine PCR. Combined analysis of ITS and trnL/trnF differentiated the 8 Ephedra species.
#Abbreviations
ITS1:internal transcribed spacer 1
ITS2:internal transcribed spacer 2
trnL/trnF:intron of trnL and intergenic spacer between trnL and trnF
EI:Ephedra intermedia
ES:Ephedra sinica
EP:Ephedra przewalskii
EE:Ephedra equisetina
EM:Ephedra monosperma
EG:Ephedra gerardiana
EL:Ephedra likiangensis
EMu:Ephedra minuta
Key words
Ephedra - Ephedraceae - nuclear ribosomal DNA - internal transcribed spacer (ITS) - chloroplast DNA - intron of trnL and intergenic spacer between trnL and trnF (trnL/trnF)
Introduction
Mahuang is a crude drug that has been utilized for perspiratory, antitussive, antipyretic and anti-inflammatory purposes in traditional Chinese medicine for centuries. The herbal origin of Mahuang is confined to the aerial part of Ephedra sinica, E. intermedia and E. equisetina in the Pharmacopoeia of China [1]. The habitat of these 3 species is mainly the northern and northwestern parts of China, such as Inner Mongolia, Gansu and Qinghai Provinces. Although other Ephedra species such as E. gerardiana, E. likiangensis, E. przewalskii, E. minuta, are also used as Mahuang [2], their reputation is not as good as the Ephedra species listed in the Pharmacopoeia of China, and their ephedrine content is reportedly lower. It is therefore inappropriate to use these species because their therapy effect has not been confirmed scientifically. However, it is difficult to exclude these species from clinical use because of the morphological resemblance among Ephedra species when they do not bear flowers or seeds. Anatomic research has been conducted to differentiate them [3], [4], [5], but its accuracy was discounted due to geographic variation. DNA analysis is a powerful tool for the identification of Ephedra plants: the RAPD method was used by Takeuchi et al., but the results were unclear because the plants in the study were not differentiated [6]. In the course of research into medicinal plants in 2001 and 2002, we surveyed Ephedra plants in northern and western China during flowering and collected 8 Ephedra species [7]. Since non-coding regions tend to evolve faster than the coding region, nuclear ribosomal internal transcribed spacers (ITSs) were often used to identify the botanical origin of the herbal drugs as well as to resolve the phylogenetic relationship [8], [9], [10], [11]. The ITS of Ephedra plants, a member of the gymnosperm family whose ITS are longer than in angiosperm plants, can provide further information for identification [12], [13]. The trnL/trnF of chloroplast DNA is another molecular marker effective in differentiating the herbal origin [14]. The plastids are inherited maternally in Ephedra, and nuclear DNA is inherited biparentally [15]. The combined data derived from rDNA and chloroplast DNA provide a more precise resolution in phylogeny [16].
In this paper, we report the results of the combined analysis of rDNA ITS and trnL/trnF to differentiate 8 Ephedra species grown in China.
#Materials and Methods
#Plant materials
All 24 Ephedra plant specimens belonging to 8 species (1 - 5 plant specimens per species) were collected in the Inner Mongolia Autonomous Region and the Sichuan, Hebei, Shanxi, Gansu and Qinghai Provinces, as shown in Table [1]. Gnetum leptostachyum was a generous gift from Dr. M. Tamura, identified by M. Tamura and J. F. Maxwell. The plants were deposited in the Herbarium of the Medicinal Plants of the Faculty of Pharmaceutical Sciences in Kanazawa University.
Species | Voucher No. | Locality of voucher | Date of collection |
GenBank Accession No. | ||
ITS1 | ITS2 | trnL/trnF | ||||
E. intermedia (EI) | KANP02309 | Guinan, Qinghai Prov., China | 2 002.7.31 | |||
KANP02341 | Akese, Gansu Prov., China | 2 002.8.7 | ||||
KANP02363 | Yuzhong, Gansu Prov., China | 2 002.8.13 | AY394070 | AY394062 | AY423430 | |
KANP02364 | Qingshui Xiang, Yuzhong, Gansu Prov., China |
2 002.8.13 | ||||
KANP02369 | Longxi, Gansu Prov., China | 2 002.8.14 | ||||
E. sinica (ES) | KANP02109 | E-er-duo-si, Neimenggu, China | 2 002.6.5 | |||
KANP02133 | Datong, Shanxi Prov., China | 2 002.6.8 | ||||
KANP02139 | Chengde, Hebei Prov,, China | 2 002.6.10 | AY394071 | AY394063 | AY423431 | |
KANP02143 | Chifeng, Neimenggu, China | 2 002.6.11 | ||||
KANP02145 | Tongliao, Neimenggu, China | 2 002.6.11 | ||||
E. przewalskii (EP) | KANP02321 | Dulan, Qinghai Prov., China | 2 002.8.4 | |||
KANP02326 | Ge-er-mu, Qinghai Prov., China | 2 002.8.4 | AY394072 | AY394064 | AY423432 | |
KANP02340 | Xi-meng-gu-zu-zi-zhi-zhou,, Qinghai Prov., China |
2 002.8.7 | ||||
KANP02351 | Jiuquan, Gansu Prov., China | 2 002.8.10 | ||||
E. equisetina (EE) | KANP02303 | Xunhua, Qinghai Prov., China | 2 002.7.30 | |||
KANP02314 | Xining, Qinghai Prov., China | 2 002.8.1 | AY394073 | AY394065 | AY423433 | |
KANP02356 | Shandan, Gansu Prov., China | 2 002.8.11 | ||||
KANP02136 | Zhangjiakou, Hebei Prov., China | 2 002.6.8 | AY423434 | |||
E. monosperma (EM) | KANP02378 | Qilianshan, Qinghai Prov., China | 2 002.8.1 | AY394077 | AY394066 | AY423435 |
E. gerardiana (EG) | KANP0101117 | Shiqu Xian, Sichuan Prov., China | 2 001.8.6 | AY394074 | AY394067 | AY423436 |
E. likiangensis (EL) | KANP0101001 | Kangding Xian,, Sichuan Prov., China | 2 001.7.27 | AY394075 | AY394068 | AY423437 |
KANP0101126 | Xinlong Xian, Sichuan Prov., China | 2 001.8.8 | ||||
E. minuta (EMu) | KANP0101136 | Litang Xian, Sichuan Prov., China | 2 001.8.11 | AY394076 | AY394069 | AY423438 |
KANP0101039 | Kangding Xian, Sichuan Prov., China | 2 001.7.28 | AY423439 | |||
G. leptostachyum | Doi Suthep, Thailand | 2 002.3 | AY445622 | AY445623 | AY445621 |
DNA extraction and PCR amplification
The plant stem was cut into 2-mm pieces, frozen in liquid nitrogen and ground into powder. Using the DNeasy Plant Mini Kit (QIAGEN, Germany), the DNA was extracted according to the manufacturer’s protocol. Total DNA was used as a template for amplifying the ITS1 and ITS2 by PCR. The primers were designed based on 18S, 5.8S and 26S nuclear ribosomal DNA sequences from the Genbank. The primers, Eph-F (GAC GTC GCG AGA AGT TCA TT) and 5.8S-R (CGG GAT TCT GCA ATT CAC AC), were used to amplify ITS1 and primers 5.8-F (GAA CGT AGC GAA ATG CGA TA) and Eph-R (GTA AGT TTC TCT TCC TCC GC) were used for ITS2. The primers C (CGA AAR CGG TAG ACG CTA CG) and F (ATT TGA ACT GGT GAC ACG AG) were used to amplify the region of trnL/trnF [17]. PCR was performed in 50 μL of reaction mixture containing 5 μL of 10 × PCR buffer for KOD-Plus, 0.2 mM each of dNTP, 1 mM MgSO4, 0.4 μM of each primer, 100 ng of the template and 1 unit of KOD-Plus DNA polymerase (TOYOBO). PCR was carried out as follows: hot start at 94 °C for 2 min, 30 cycles of denaturation at 94 °C for 15 sec, annealing at 55 °C for 30 sec and elongation at 68 °C for 45 sec, and final elongation at 68 °C for 5 min. Five μL PCR product was checked with 1.5 % agarose gel electrophoresis and the remaining product was purified using the QIA quick PCR Purification Kit (QIAGEN, Germany).
#Sequencing and phylogenetic analysis
The purified PCR product was subjected to direct sequencing using a Bigdye Terminator Cycle Sequencing Kit (Applied Biosystem) with ABI PRISM 310 (Applied Biosystem). The primers Eph-F, 5.8S-R and ITS-1A (GCG GGG ACG TGG ACG GTC TT) were used for sequencing ITS1 and primer 5.8S-F was used for ITS2. Primers C and F were used to sequence the region of trnL/trnF. The DNA sequences were aligned by ‘DNASIS’ version 3.0 (Hitachi).
Based on ITS1, ITS2 and trnL/trnF sequences, the phylogenetic trees were constructed using the program PAUP* (Version4.0b10, Sinauer Assoc. Inc., U.S.A.). Parsimony analysis was performed using the heuristic search method with TBR branch swapping with Gnetum leptostachym as an outgroup. Confidence in the tree was estimated by bootstrap analysis.
#Results
The sequence analysis of multiple specimens of EI, ES, EP and EL showed their uniformity in the sequences analyzed in this study, ITS 1 and 2 (Fig. [1]), and trnL/trnF, within the same species. This is true for EE and EMu in the ITS1 and 2. The length of ITS2 found to be conserved was 246 bp, whereas that of ITS1 ranged from 1120 to 1139, as shown in Table [2]. EI and ES, EL and EMu had identical ITS1 and ITS2 lengths and sequences. Sequence divergence of ITS1 in 8 Ephedra species ranged from 0 to 3.49 % while that of ITS2 ranged from 0 to 3.69 %. At the end of ITS1, 76 bp were repeated 3 times in EI, ES and EP, whereas each of the sub-repeat units of EE, EM, EG, EL and EMu has a 4 bp deletion (Table [3]). According to the sequences of ITS1 and ITS2, 8 Ephedra species can be divided into 3 groups: Group 1 (EI, ES and EP), Group 2 (EE, EM and EG), and Group 3 (EL and EMu).
Based on the difference in the sub-repeat unit of ITS1 (Table [3]), a specific primer to Group 1, ITS-B-R (GTG AGC GGC AAG TAA GAT CC), was designed. Differentiation of the 3 groups of Ephedra species was conducted using a primer set of ITS-B-R and ITS-1A (357 - 376). When the annealing temperature was set at 55 °C, PCR products with about 450 bp were detected in Group 1 and Group 3, but no amplified fragment was seen in Group 2 (Fig. [2] A). In addition to 450 bp, faint amplified bands of about 530 and 610 bp were also detected in Group 1. When the annealing temperature rose to 65 °C, PCR fragments were detected only in Group 1 analysis (Fig. [2] B).
The sequences of trnL/trnF were found at 465 bp in EI, EP, EE, EM, EG, EMu, EL, and 463 bp in ES (Fig. [3]). One specimen of EE, EE02136 was found to be different at the 398th position from the other 3. Similarly, 2 EMu were found to differ from each other at the 271st position. EI and EP have identical sequences. EE has an identical sequence to EM, EG, EL, but not EE02136.
The phylogenetic tree based on the combined sequences of ITS1, ITS2 and trnl/trnF is shown in Fig. [4]. The results suggest that 8 Ephedra species were divided into two clusters, i. e., 1 (EI, ES and EP) and 2 (EE, EM, EG, EL and EMu) with high bootstrap probabilities. Cluster 2 can be divided into two sub-clusters, i. e., EE, EM and EG, and EL and EMu.
EI | ES | EP | EE | EM | EG | EL | EMu | |
ITS1 | 1 139 | 1 139 | 1 139 | 1 120 | 1 120 | 1 120 | 1 124 | 1 124 |
ITS2 | 246 | 246 | 246 | 246 | 246 | 246 | 246 | 246 |
791 - 810 | 866 - 885 | 945 - 964 | |
EI, ES | GGATCTTACTTGCCGCTCAC | GGATCTTACTTGCCGCTCAA | GGATCTTACTTGCCGCTCAC |
EP | GGATCTTACTTGCCGCTCAC | GGATCTTACTTGCCGCTCAC | GGATCTTACTTGCCGCTCAC |
EE, EM | GGATCTCAC- - - -CGCTCAA | GGATCTCAC- - - -CGCTCAA | GGATCTCAC- - - -CGCTCAC |
EG | GGATCTCAC- - - -CGCTCAA | GGATCTCAC- - - -CGCTCAA | GGATTTCAC- - - -CGCTCAC |
EL, EMu | GGATCTTAC - - - -CGCTCAC | GGATCTCAC- - - -CGTTCAA | GGATATCAC- - - -CGCTCAC |

Fig. 1 Organization of plant ITS1 and ITS2. Arrows indicate orientation and approximate position of the primers.

Fig. 2 Specific amplification using primers ITS-1A and ITS-B-R. A: Annealing temperature at 55 °C. B: Annealing temperature at 65 °C. Lane 1 : 100-bp marker. Lane 2: E. intermedia. Lane 3: E. sinica. Lane 4: E. przewalskii. Lane 5: E. equisetina. Lane 6: E. monosperma. Lane 7: E. gerardiana. Lane 8: E. likiangensis. Lane 9: E. minuta.

Fig. 3 Comparison of the region of trnL intron and trnL-trnF intergenic spacer sequence. Arrows indicate orientation and approximate position of the primers. The numbers above sequences are the aligned nucleotide positions. Asterisks indicate the identical nucleotides with E. intermedia in the first line, and hyphens represent gaps.

Fig. 4 Phylogenetic relationship of 8 Ephedra species based on DNA sequences of ITSs and trnL/trnF. The 50 % majority rule consensus tree built on the basis of maximum parsimonious analysis of combined ITS1, ITS2 and trnL/trnF. Tree length = 609, CI = 0.9803, RI = 0.9226, RC = 0.9044. Number above line is branch length, and number below line is the bootstrap value with 1000 replicates.
Discussion
Ephedra and other Gnetalean genera are thought to share evolutional positions between the angiosperm and gymnosperm. Out of 50 - 60 Ephedra species distributed worldwide, 12 species and 4 varieties are reported to grow in China [18], [19]. The length of ITS1 varies greatly within the same genus in many gymnosperm plants. On the other hand, variations in the ITS1 length of the 8 Ephedra species were limited in range from 1120 bp to 1139 bp. The region of trnL/trnF was about 460bp in the Ephedra plants, much shorter than that in the angiosperm. Nuclear ribosomal DNA of plants is often reported to be heterogeneous due to DNA recombination. However, we observed no ambiguous sequences in the ITS2 of Ephedra plants determined by direct sequencing, except for E. equisetina whose 16th and 17th positions of ITS2 were C or T, suggesting that sequence heterogeneity is limited. As for ITS1, although E. intermedia, E. sinica and E. przewalskii were determined without ambiguity, E. likiangensis and E. minuta that were classified into Group 3, were not clear at the 497th, 595th and 731st positions, supposedly due to heterogeneity in the nucleotide sequence of these points. Furthermore, Ephedra species classified into Group 2 showed heterogeneity, C or T, at the 33rd position. Those nucleotide sequences were excluded from the sequence analysis. As reported previously on E. fragilis, ITS1 of the 8 Ephedra species had three tandem repeats of about 70 bp, the sequences of which were unique to each group and can be used to discriminate the groups. Although E. intermedia and E. sinica as well as E. likiangensis and E. minuta are identical in ITS1 and ITS2, they differ in the region of trnL/trnF. The information deduced from chloroplast DNA sequence analysis is different to that from ribosomal DNA ITS. Furthermore, one E. equisetina that was collected in Hebei Province, northeast China, had a different trnL/trnF sequence from the other 3 samples collected in the northwest. Two E. minuta that were collected in Sichuan Province, also showed differences in the trnL/trnF region. Chloroplast non-coding DNA mutates faster than nuclear DNA.
In the traditional classification of Ephedra species, according to the morphological difference in seed cones, Ephedra species in China were divided into 2 sections: Sect. Alatae and Sect. Ephedra. The seed cones in Sect. Alatae are membranous at maturity, whereas the seed cones in Sect. Ephedra are fleshy at maturity. E. przewalskii belongs to Sect. Alatae, and the other 7 Ephedra species belong to Sect. Ephedra [19]. However, analysis of ITS and the region of trnL/trnF shows that E. przewalskii has a genetically close relationship with E. intermedia and E. sinica. In addition, E. likiangensis and E. minuta that inhabit southern China differ greatly in size of plant, but are genetically close according to the ITS variation. The results from ITS and trnL/trnF did not correlate well with the morphology.
Karyomorphological studies showed that Ephedra species are diploid with 2n = 14 (E. przewalskii , E. equisetina and E. minuta), or tetraploid with 2n = 28 (E. sinica, E. likiangensis), as well as a mixture of those two 2n = 14, 28 (E. intermedia, E. gerardiana) [20]. No polyploid relationship of either species distribution or DNA classification was observed. On the other hand, in our survey, the species classified into Group 1 grew mainly in northern China; Inner Mongolia, Hebei, Shanxi, Gansu, and Qinghai. Group 3 grew in the south, Sichuan, and Group 2 grew in the center, suggesting genetic and geographic relationships. The species of Group 1 are thought to better tolerate harsh climates such as water-deficiency and low temperatures. The narrow DNA divergence within the species may result from selection under such climate stress. Considering their adaptive character, they may be suitable for cultivation in the dry land of northern China where progressive desert conditions have accelerated in the last 10 years.
#Acknowledgements
This work was supported by Grant-in-Aids from the Ministry of Education, Science, Sports, and Culture, Japan.
#References
- 1 Chinese Pharmacopoeia Editorial C ommittee. Chinese Pharmacopoeia. 2000 edition, Volume 1 Beijing; Chemical Technology Press 2000: p 262
- 2 Zhonghua Bencao (Chinese Herb) Editorial C ommittee. Zhonghua Bencao (Chinese Herb). Volume 2 Shanghai; Shanghai Science & Technology Press 1999: pp 349-357
- 3 Zhang J S, Li S H, Lou Z C. Morphological and histological studies of Chinese Ephedra mahuang I. Seven species produced in north China. Acta Pharmaceutica Sinica. 1989; 24 937-48
- 4 Zhang J S, Li S H, Lou Z C. Morphological and histological studies of Chinese Ephedra mahuang II. Species produced in southwestern China and other species. Acta Pharmaceutica Sinica. 1990; 25 54-65
- 5 Mikage M, Kondo N. Medico-botanical studies of Ephedra plants from the Himalayan region, part I. Anatomical studies of the herbal stems and botanical origin of Tibetan crude drugs ”TSHE” and ”BALU”. J Jpn Bot. 1996; 71 323-32
- 6 Takeuchi M, Nakashima A, Mizukami H, Hiraoka N, Kohda H. RAPD analysis of Ephedra plants. Natural Medicines. 2003; 57 50-4
- 7 Mikage M, Takahashi A, Chen H B, Li Q S. Studies of Ephedra plants in Asia. Part 1. On the resources of Ephedra plants in China. Natural Medicines. 2003; 57 202-8
- 8 Lau D TW, Shaw P C, Wang J, But P PH. Authentication of medicinal Dendrobium species by the internal transcribed spacer of ribosomal DNA. Planta Medica. 2001; 67 456-60
- 9 Ding X Y, Xu L S, Xu H, Wang Z T, Zhou K Y. Morphological and DNA molecular evidence for authentication of Dendrobium flexicaule from ITS allied species of Dendrobium . Acta Pharmaceutica Sinica. 2001; 36 868-73
- 10 Chen Y Q, Wang N, Zhou H, Qu L H. Differentiation of medicinal Cordyceps species by rDNA ITS sequence analysis. Planta Medica. 2002; 68 635-9
- 11 Wen J, Zimmer E A. Phylogeny and biogeography of Panax L. (the Ginseng genus, Araliaceae): inferences from ITS sequences of nuclear ribosomal DNA. Mol Phylogenet Evol. 1996; 6 167-77
- 12 Liston A, Robinson W A, Oliphant J M. Length variation in the nuclear ribosomal DNA internal transcribed spacer region of non-flowering seed plants. Syst Botany. 1996; 21 109-20
- 13 Maggini F, Marrocco R, Gelati M T, Dominicis R ID. Lengths and nucleotide sequences of the internal spacers of nuclear ribosomal DNA in gymnosperms and pteridophytes. Pl Syst Evol. 1998; 213 199-205
- 14 Yang M H, Zhang D M, Liu J Q, Zheng J H. A molecular marker that is specific to medicinal rhubarb based on chloroplast trnL/trnF sequences. Planta Medica. 2001; 67 784-6
- 15 Mogensen H L. The hows and whys of cytoplasmic inheritance in seed plants. Am J Bot. 1996; 83 383-404
- 16 Zhu S, Fushimi H, Cai S Q, Komatsu K. Phylogenetic relationship in the Genus Panax: inferred from choroplast trnK gene and nuclear 18S rRNA gene sequences. Planta Medica. 2003; 69 647-653
- 17 Taberlet P, Gielly L, Pautou G, Bouvet J. Universal primers for amplification of three non-coding regions of chloroplast DNA. Plant Mol Biol. 1991; 17 1105-9
- 18 Price R A. Systematics of the Gnetales: a review of morphological and molecular evidence. Int J Plant Sci. 1996; 157 40-9
- 19 Agenda academiae sinicae e dita. Flora Reipublicae Popularis Sinicae. Volume 7 Beijing; Science Press 1978: pp 468-89
- 20 Chouhdry A S. Karyomorphological and cytogenetical studies in Ephedra . J Sci Hiroshima Univ Ser B, Div 2. 1984; 19 57-109
Dr. N. Kakiuchi
Faculty of Pharmaceutical Sciences
Kanazawa University
13-1 Takaramachi
Kanazawa 920-0934
Japan
Fax: +81-76-234-4490
Email: kakiuchi@p.kanazawa-u.ac.jp
References
- 1 Chinese Pharmacopoeia Editorial C ommittee. Chinese Pharmacopoeia. 2000 edition, Volume 1 Beijing; Chemical Technology Press 2000: p 262
- 2 Zhonghua Bencao (Chinese Herb) Editorial C ommittee. Zhonghua Bencao (Chinese Herb). Volume 2 Shanghai; Shanghai Science & Technology Press 1999: pp 349-357
- 3 Zhang J S, Li S H, Lou Z C. Morphological and histological studies of Chinese Ephedra mahuang I. Seven species produced in north China. Acta Pharmaceutica Sinica. 1989; 24 937-48
- 4 Zhang J S, Li S H, Lou Z C. Morphological and histological studies of Chinese Ephedra mahuang II. Species produced in southwestern China and other species. Acta Pharmaceutica Sinica. 1990; 25 54-65
- 5 Mikage M, Kondo N. Medico-botanical studies of Ephedra plants from the Himalayan region, part I. Anatomical studies of the herbal stems and botanical origin of Tibetan crude drugs ”TSHE” and ”BALU”. J Jpn Bot. 1996; 71 323-32
- 6 Takeuchi M, Nakashima A, Mizukami H, Hiraoka N, Kohda H. RAPD analysis of Ephedra plants. Natural Medicines. 2003; 57 50-4
- 7 Mikage M, Takahashi A, Chen H B, Li Q S. Studies of Ephedra plants in Asia. Part 1. On the resources of Ephedra plants in China. Natural Medicines. 2003; 57 202-8
- 8 Lau D TW, Shaw P C, Wang J, But P PH. Authentication of medicinal Dendrobium species by the internal transcribed spacer of ribosomal DNA. Planta Medica. 2001; 67 456-60
- 9 Ding X Y, Xu L S, Xu H, Wang Z T, Zhou K Y. Morphological and DNA molecular evidence for authentication of Dendrobium flexicaule from ITS allied species of Dendrobium . Acta Pharmaceutica Sinica. 2001; 36 868-73
- 10 Chen Y Q, Wang N, Zhou H, Qu L H. Differentiation of medicinal Cordyceps species by rDNA ITS sequence analysis. Planta Medica. 2002; 68 635-9
- 11 Wen J, Zimmer E A. Phylogeny and biogeography of Panax L. (the Ginseng genus, Araliaceae): inferences from ITS sequences of nuclear ribosomal DNA. Mol Phylogenet Evol. 1996; 6 167-77
- 12 Liston A, Robinson W A, Oliphant J M. Length variation in the nuclear ribosomal DNA internal transcribed spacer region of non-flowering seed plants. Syst Botany. 1996; 21 109-20
- 13 Maggini F, Marrocco R, Gelati M T, Dominicis R ID. Lengths and nucleotide sequences of the internal spacers of nuclear ribosomal DNA in gymnosperms and pteridophytes. Pl Syst Evol. 1998; 213 199-205
- 14 Yang M H, Zhang D M, Liu J Q, Zheng J H. A molecular marker that is specific to medicinal rhubarb based on chloroplast trnL/trnF sequences. Planta Medica. 2001; 67 784-6
- 15 Mogensen H L. The hows and whys of cytoplasmic inheritance in seed plants. Am J Bot. 1996; 83 383-404
- 16 Zhu S, Fushimi H, Cai S Q, Komatsu K. Phylogenetic relationship in the Genus Panax: inferred from choroplast trnK gene and nuclear 18S rRNA gene sequences. Planta Medica. 2003; 69 647-653
- 17 Taberlet P, Gielly L, Pautou G, Bouvet J. Universal primers for amplification of three non-coding regions of chloroplast DNA. Plant Mol Biol. 1991; 17 1105-9
- 18 Price R A. Systematics of the Gnetales: a review of morphological and molecular evidence. Int J Plant Sci. 1996; 157 40-9
- 19 Agenda academiae sinicae e dita. Flora Reipublicae Popularis Sinicae. Volume 7 Beijing; Science Press 1978: pp 468-89
- 20 Chouhdry A S. Karyomorphological and cytogenetical studies in Ephedra . J Sci Hiroshima Univ Ser B, Div 2. 1984; 19 57-109
Dr. N. Kakiuchi
Faculty of Pharmaceutical Sciences
Kanazawa University
13-1 Takaramachi
Kanazawa 920-0934
Japan
Fax: +81-76-234-4490
Email: kakiuchi@p.kanazawa-u.ac.jp

Fig. 1 Organization of plant ITS1 and ITS2. Arrows indicate orientation and approximate position of the primers.

Fig. 2 Specific amplification using primers ITS-1A and ITS-B-R. A: Annealing temperature at 55 °C. B: Annealing temperature at 65 °C. Lane 1 : 100-bp marker. Lane 2: E. intermedia. Lane 3: E. sinica. Lane 4: E. przewalskii. Lane 5: E. equisetina. Lane 6: E. monosperma. Lane 7: E. gerardiana. Lane 8: E. likiangensis. Lane 9: E. minuta.

Fig. 3 Comparison of the region of trnL intron and trnL-trnF intergenic spacer sequence. Arrows indicate orientation and approximate position of the primers. The numbers above sequences are the aligned nucleotide positions. Asterisks indicate the identical nucleotides with E. intermedia in the first line, and hyphens represent gaps.

Fig. 4 Phylogenetic relationship of 8 Ephedra species based on DNA sequences of ITSs and trnL/trnF. The 50 % majority rule consensus tree built on the basis of maximum parsimonious analysis of combined ITS1, ITS2 and trnL/trnF. Tree length = 609, CI = 0.9803, RI = 0.9226, RC = 0.9044. Number above line is branch length, and number below line is the bootstrap value with 1000 replicates.