Detection of Genetic Homogeneity of Panax notoginseng Cultivars by Sequencing Nuclear 18S rRNA and Plastid matK Genes

• Abstract
• Materials and Methods
• Acknowledgements
• References

Abstract

The nuclear 18S rRNA and chloroplast matK genes of 18 samples of Panax notoginseng and its processed material Sanqi (Radix Notoginseng) were analyzed. The two genes, regardless of cultivar origin, were found to be identical to genotype R₁ and M₁, respectively, of the published sequences (GenBank accession no. D85171 and AB027526). This phenomenon implies that the species is highly conserved, which is probably caused by the use of the same strain in cultivation and the lack of active mutation in these two genes.

Supporting information available online at http://www.thieme-connect.de.accesdistant.sorbonne-universite.fr/ejournals/toc/plantamedica

Panax notoginseng (Burk.) F. H. Chen, belongs to the genus Panax (Araliaceae). Its dry root, Sanqi (Radix Notoginseng), is one of the most famous Chinese medicines used to activate blood circulation, eliminate stasis and stop bleeding. The efficacy of Sanqi for the cadiovascular system and its antioxidant activities have been proven experimentally [1], [2]. Nowadays, Panax notoginseng is cultivated in the Yunnan Province, China. The plant is also cultivated in some areas of the Guangxi Zhuang Autonomous Region of China. The wild plant of Panax notoginseng has not been found so far.

In previous studies, phylogenetic analysis of the genus Panax has been conducted using the internal transcribed spacers (ITS) sequence of ribosomal DNA [3], together with the chloroplast trnK gene and the nuclear 18S rRNA gene in Panax notoginseng [4]. The sequence identification of Panax notoginseng and its adulterants has also been reported [5]. Molecular divergence of plant materials from different regions or localities has been attracting attention [6]. Although genetic heterogeneity of the ribosomal RNA gene and matK gene in the genus Panax was reported, the existence of genetic heterogeneity in Panax was only shown in Panax notoginseng [7]. The genotypes R₁, R₂, R₃ and R₄ for the 18S rRNA gene (GenBank accession no. D85171, AB027524, AB027525 and AF412275) and genotypes M₁, M₂ for the matK gene (GenBank accession no. AB027526 and AB027527) have been reported [7], [8], [9]. Genotypes R₁, R₂ and R₃ of the 18S rRNA gene are all 1809 bp in length, and the genotypes M₁ and M₂ of the matK gene are 1259 bp in length. There are five base substitutions between R₁ and R₂, and five base substitutions between M₁ and M₂. Sixty-one base substitutions are found between R₁ and R₃, and sixty-two between R₂ and R₃. The sequence of genotype R₄ is 991 bp long [8] and is also observed between bases 43 and 1040 of genotype R₁. Genotype R₄ is identical in sequence to genotype R₁ except for one base deletion. The phenomenon of genetic heterogeneity, on the other hand, was not found in Panax ginseng and Panax quinquefolium [7], therefore, there remains confusion on its genetic heterogeneity because of the limited samples. In the present study, PCR direct sequencing was applied to analyze Panax notoginseng cultivars, for characterizing the sequences of the 18S ribosomal RNA gene and the matK gene.

The results indicated that the nucleotide sequences of the 18S rRNA and matK genes from 14 cultivars and 4 commercial samples are 1809 bp and 1259 bp in length (Table [1]), which are identical to genotypes R₁ and M₁ (GenBank accession no. D85171 and AB027526), respectively. Our previous results showed that the 18S rRNA and matK genes were useful markers for identifying Panax notoginseng and its adulterants [5]. Our current results further confirm that these genes are highly conserved.

The lack of variation of the two genes is probably caused by use of the same strain for cultivation and the lack of active mutation in these genes. The comparison among the sequences in the genus Panax showed that genotypes R₂ and M₂ of Panax notoginseng were identical to the corresponding sequences of Panax ginseng (GenBank accession no. D83274 and D89057), respectively [7]. For a long period, Panax notoginseng has been widely cultivated in the southwestern part of China, while Panax ginseng is mainly distributed in the northeastern part of China and Korea. Although some morphological similarities exist, the two species possess different biogeographical patterns. The strict consensus tree constructed by ITS sequence analysis indicated that Panax notoginseng was at a basal position compared with Panax ginseng, and the two species did not form a monophyletic group [3]. Zhu et al. verified this hypothesis by means of chloroplast trnK gene and nuclear 18S rRNA gene sequencing analysis [4]. Therefore, in order to determine whether the genetic heterogeneity of ribosomal RNA gene and matK gene exists in Panax notoginseng species, a large number of samples distributing in diverse regions are needed to be further studied.

Materials and Methods

Fourteen fresh plant materials of Panax notoginseng (Burk.) F. H. Chen (No. 1 - 14) and four dried crude samples of Radix Notoginseng (No. 15 - 18) were collected from different localities and markets in Yunnan, Guangxi and Guangdong, P. R. China (Table [1]), and authenticated by Prof. Hui Cao and Prof. Baozuo He. All samples were stored at -20 °C until extraction. The voucher specimens are stored in the herbarium of the Shenzhen Research Institute of City University of Hong Kong, Shenzhen, China.

Fresh and/or silica gel-dried leaves and roots were used for genomic DNA isolation. Genomic DNA was isolated using the DNeasy^TM plant kit (Qiagen; Hilden, Germany) and a modified CTAB miniprocedure [10]. The PCR primers for the 18S rRNA gene and matK gene are as follows: 18SF: 5′-CAA CCT GGT TGA TCC TGC CAG T-3′; 18SR: 5′-CTG ATC CTT CTG CAG CTT CAC CTA C-3′ and matKAF: 5′-CTA TAT CCA CTT ATC TTT CAG GAG T-3′;matK8R: 5′-AAA GTT CTA GCA CAA GAA AGT CGA-3′, respectively. The PCR reaction volumes (50 μL) contained 1.5 U of Taq polymerase (TAKARA; Tokyo, Japan), 1 × buffer (Mg²⁺ plus), 1.5 mmol · L^-1 of MgCl₂, 0.2 mmol · L^-1 of dNTPs, 0.25 μmol · L^-1 of PCR primer and 50 - 100 ng of template DNA. PCR reactions were performed in a GeneAmp 9700 system (Applied Biosystems Inc.; Foster, CA, USA). 18S rRNA PCR amplification was carried out at 94 °C for 3 min for initial denaturation, followed by 30 cycles of denaturation at 94 °C for 40 s, primer annealing at 60 °C for 1 min, an extension at 72 °C for 2 min, and a termination at 72 °C for 10 min. matK gene PCR amplification was carried out at 94 °C for 3 min, followed by 35 cycles of 94 °C for 1 min, 45 °C for 1 min, 72 °C for 2 min, and a termination at 72 °C for 10 min. The purification of the PCR products was performed using the NaAc-EtOH precipitation procedure.

Double-stranded purified PCR products were sequenced using the dideoxy chain termination method with an ABI PRISM^TM Bigdye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems Inc.). Reactions and programs were carried out according to the manufacturer’s protocol. Sequencing primers were designed and listed as follows: 18SF: 5′-CAA CCT GGT TGA TCC TGC CAG T-3′; 18S462R: 5′-CCG TGT CAG GAT TGG-3′; 18S446F: 5′-CAA TCC TGA AAT ACG-3′; 18S910R: 5′-TCA CCT CTG ACT ATG AAA TAC G-3′; 18S924F: 5′-ATG AAA GAC GAA CAA CTG C-3′; 18S1330F: 5′-AAC GAG TCA GCC TG-3′; 18SR: 5′-CTG ATC CTT CTG CAG GTT CAC C-3′ and matKAF: 5′-CTA TAT CCA CTT ATC TTT CAG GAG T-3′; matK444R: 5′-GCG AAG AGT TTG AAC CAA G-3′; matK491F: 5′-TCC ACG AGT ATT GTA ATT GG-3′; matK867F: 5′-TTA CCT GTG GTC TCA G-3′; matK8R: 5′-AAA GTT CTA GCA CAA GAA AGT CGA-3′. Samples were electrophoresed on an ABI 310 Genentic Analyzer (Applied Biosystems Inc.), and sequenced two to three times for each individual sample.

DNA sequences were analyzed using Chromas (Ver. 2.0) and software of Contig (Sangon Co.; Shanghai, China), and aligned using Blast and Clustalw (www.). Manual adjustment was performed where necessary to reduce gap value.

Supporting Information: A comparison of the variable sites in the 18S rRNA sequences is available as Supporting Information.

Acknowledgements

This work was partly supported by State Hi-Tech Development Program of China (863 Program) (No. 2003AA2Z2052), and Key Scientific and Technological Program of Zhuhai Bureau of Science and Technology (No. 2002 - 2-14).

The authors are grateful to Prof. Bao-Zuo He, Guangxi College of Traditional Chinese Medicine, Dr. Li Zhang, South China Botanical Garden, Chinese Academy of Science, Mr. Lin Zhou, Kunming Institute of Botany, Chinese Academy of Science, Mr. Yan-Xin Chen, Wenshan Qi Hua Company Ltd., and Mr. Yu-Qi Yu, Yunnan Miaoxiang Notoginseng Industrial Corporation for the collection of plant material.

References

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Prof. Hui Cao

National Engineering Research Center for Modernization of Traditional Chinese Medicine

Zhuhai

People’s Republic of China

Phone: +86-756-813-5676

Fax: +86-756-828-9500

Email: kovhuicao @yahoo.com.cn

References

• 1 Ng T B, Liu F, Wang H X. The antioxidant effects of aqueous and organic extracts of Panax quinquefolium, Panax notoginseng, Codonopsis pilosula, Pseudostellaria heterophylla and Glehnia littoralis .  J Ethnopharmacol. 2004;  93 285-8
  CrossrefPubMedSearch in Google Scholar
• 2 Liu J C, Cheng T H, Lee H M, Lee W S, Shih N L, Chen Y L. Inhibitory effect of trilinolein on angiotensin II-induced cardiomyocyte hypertrophy.  Eur J Pharmacol. 2004;  484 1-8
  CrossrefPubMedSearch in Google Scholar
• 3 Wen J, Elizabeth A Z. 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
  CrossrefPubMedSearch in Google Scholar
• 4 Zhu S, Fushimi H, Cai S Q, Komatsu K. Phylogenetic relationship in the genus Panax: inferred from chloroplast trnK gene and nuclear 18S rRNA gene sequences.  Planta Med. 2003;  69 647-53
  Thieme ConnectPubMedSearch in Google Scholar
• 5 Cao H, Liu Y P, Fushimi H, Komatsu K. Sequencing identification of Panax notoginseng and its adulterants.  J Chin Med Mat. 2001;  24 398-401
  PubMedSearch in Google Scholar
• 6 Kondo K, Terabayashi S, Higuchi M. Discrimination between ”Banxia” and ”Tiannanxing” based on rbcL sequences.  Natural Med. 1998;  52 253-8
  PubMedSearch in Google Scholar
• 7 Fushimi H, Komatsu K, Namba T, Isobe M. Genetic heterogeneity of ribosomal RNA gene and matK gene in Panax notoginseng .  Planta Med. 2000;  66 659-61
  Thieme ConnectPubMedSearch in Google Scholar
• 8 Fushimi H, Komatsu K, Isobe M, Namba T. 18S ribosomal RNA gene sequences of three Panax species and the corresponding Ginseng drugs.  Biol Pharm Bull,. 1996;  19 350-2
  PubMedSearch in Google Scholar
• 9 Wu Y S, Steven S. Sequencing of ribosomal 18S rRNA gene from Panax pseudoginseng var. notoginseng .  Chin Trad Herb Drugs. 2001;  32 1116-9
  PubMedSearch in Google Scholar
• 10 Doyle J J, Doyle J L. A rapid DNA isolation procedure for small quantities of fresh leaf tissue.  Phytochem Bull. 1987;  19 11-5
  PubMedSearch in Google Scholar

Prof. Hui Cao

National Engineering Research Center for Modernization of Traditional Chinese Medicine

Zhuhai

People’s Republic of China

Phone: +86-756-813-5676

Fax: +86-756-828-9500

Email: kovhuicao @yahoo.com.cn

• Supplementary Material