Biosci. Biotechnol. Biochem., 69 (9), 1771–1773, 2005
Note
Genetic Identification of Panax ginseng and Panax quinquefolius by Pyrosequencing Methods Kanghyun L EEM,1 Sang Chan K IM,2 Chae Ha Y ANG,2 and Jungchul S EO2; y 1 2
Department of Herbal Pharmacology, College of Oriental Medicine, Semyung University, Jecheon 390-711, Korea Je-Han Oriental Medical Academy, College of Oriental Medicine, Daegu Haany University, Daegu 706-828, Korea
Received February 2, 2005; Accepted June 3, 2005
This study was performed to determine whether two ginseng species (Panax ginseng and Panax quinquefolius) can be identified by genetic analysis and to verify pyrosequencing analysis, which was used to assess genetic variation. The pyrosequencing results constituted clear data. Panax quinquefolius showed a very different pattern than Panax ginseng. Pyrosequencing analysis might be able to identify the Panax species. Key words:
Panax ginseng; Panax quinquefolius; pyrosequencing
The ginseng root has been used in traditional Korean and Chinese medicine for a long time.1) The herbal supplement market has been growing rapidly in recent decades.2,3) Panax is one of the most medicinally important genera in Oriental medicine. It is one of the approximately 120 genera of plants with an eastern Asian and eastern North American disjunct distribution.4) Two of the species (Panax ginseng, known as Korean ginseng, and Panax quinquefolius, American ginseng) are regarded as important medicines in Korea and China.3,4) Of the two species, Panax ginseng is thought to tonify the Qi (energy) more effectively against aging, weakness, and stress than Panax quinquefolius. In the Korean market, the price of Panax ginseng is much higher than that of Panax quinquefolius. A few dealers have practiced the deception of disguising Panax quinquefolius as Panax ginseng, because the general consumer can not distinguish between the two. Other ginseng species, including Panax notoginseng and Panax japonica, can easily be identified. But, many commercial ginseng products are extremely difficult to identify in the form of slice, powder, or extract.5) Identification via analysis of chemical profiles is also very difficult due to many variables, such as the soil condition, climate, and nutritional factors.6) Because the internal transcribed spacer region (ITS) of nuclear ribosomal DNA is known to be a good molecular site for analysis of species in plants,7,8) we studied the genetic variance between the
two species using pyrosequencing analysis of ITS. Panax ginseng samples were purchased from Korean drug stores in Jinan, Kumsan, Youngju, Kanghwa, and Punggi, Korea as standardized packages inspected by national agricultural cooperative federations. Panax quinquefolius samples were purchased from Liaoning, China, and New York, U.S.A. Three g of ginseng plant material in the form of whole root was first minced with a sterile scalpel and pulverized to a powder using a sterilized mortar and pestle. A DNA isolation kit (DNeasy, no. 69104, Qiagen, Valencia, CA) was used according to the manufacturer’s instructions with slight modifications. Three hundred milligrams of the powdered ginseng sample was used in the purification procedure. Before sample elution, the columns were dried at 37 C for 5 min to evaporate residual ethanol. Samples were eluted in a total volume of 200 ml of TE buffer (10 mM Tris– HCl, 1 mM EDTA, pH 8.0). The extracted DNA was amplified by polymerase chain reaction (PCR). The ITS of each ginseng sample was amplified using 25 ng of DNA, and 5 pmol of each primer; the forward was 50 -CCAAGGAAATCAAACTGAAC-30 and the reverse was 50 -TCTGCAATTCACACCAAGTA-30 . PCR amplification was performed using 0.5 unit Taq polymerase (HT Biotechnology, Cambridge, UK). The 30 ml of PCR reaction mixtures were composed of 10 mM Tris–HCl, pH 9.0, 1.5 mM magnesium chloride, 50 mM potassium chloride, 0.1% Triton-X 100, 0.01% (v/v) stabilizer, 0.25 mM of each deoxynucleotide triphosphate (dNTP), and 0.1 M of each oligonucleotide primer. The PCR steps were denaturation of 5 min at 95 C, 30 cycles of 30 seconds at 95 C, 30 seconds at 60 C, and 30 seconds at 72 C with a PCR System (Astec, Fukuoka, Japan). The reverse primer was biotinylated to allow the preparation of single-stranded DNA. The quality of PCR products was controlled by 1.5% of agarose gel electrophoresis. DNA preparation for pyrosequencing was performed according to the manufacturer’s standard protocol (Pyrosequencing AB, Uppsala, Sweden).7) Streptavidin
y To whom correspondence should be addressed. Tel: +82-54-452-2200; Fax: +82-505-245-9279; E-mail:
[email protected] Abbreviations: PCR, polymerase chain reaction; ITS, internal transcribed spacer regions; dNTP, deoxynucleotide triphosphate
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Fig. 1. Typical Pyrosequencing Profile for Panax quinquefolius Cultivated in Liaoning, China, and New York, U.S.A. The boxed regions identify sequence disparities between Panax quinquefolius and Panax ginseng. The peak was absent in the first T nucleotide base and the peak of the next C nucleotide base was higher than that of the next G nucleotide base. The sequence depicted for Panax quinquefolius is GCCGAGA.
sepharose beads (Streptavidin Sepharose HP, Amersham Pharmacia Biotech, Uppsala, Sweden) were immobilized to PCR products. The sequencing primer of the ginseng was 50 -GCCGAGATATCCGTT-30 , designed so that the terminal residue hybridized to the base immediately adjacent to the T/C mutation from Pyrosequencing AB (http://www.pyrosequencing.com). By incubation at room temperature for 10 min, 20 ml of biotinylated PCR products were immobilized onto streptavidin-coated sepharose beads, and the immobilized PCR products were transferred to a Millipore 96well filter plate (Millipore, Bedford, MA). A vacuum was used to eliminate the various solutions and reagents to obtain pure, single-stranded DNA while the beads remained in the wells.9) In 55 ml of 4 M acetic acid containing 0.35 uM of sequencing primer, the beads with the immobilized template were resuspended. Then the 45 ml of resuspension was transferred to a PSQ 96 plate (Pyrosequencing AB).10) Using a PSQ 96 Sample Prep Thermoplate (Pyrosequencing AB) the PSQ 96 plate containing the samples was heated at 90 C for 5 min for sequencing primer annealing, and left at room temperature for 10 min. Then the PSQ 96 plate was placed into the process chamber of the PSQ 96 instrument (Pyrosequencing AB).11) The enzymes, substrates, and nucleotides were dispensed from a reagent cassette into the wells using a PSQ 96 SNP Reagent Kit (Pyrosequencing AB), During the process a light was generated when a nucleotide was incorporated into a growing DNA strain.12) By this process the polymorphism of the ginseng was genotyped automatically. There are several types of DNA sequence variation, including insertions and deletions, differences in the copy number of repeated sequences, and single base-pair differences. The latter is the most frequent. Characterization and scoring of genetic variations is increasingly important to correlate phenotype and genotype differences. We have investigated the possibility of typing single-base variation Panax species in DNA using a
recently developed sequencing technique called pyrosequencing. To determine whether two ginseng species can be identified by pyrosequencing method, we performed pyrosequencing analyses of Panax ginseng cultivated in Korea and of Panax quinquefolius cultivated in China or U.S.A. The pyrosequencing results constituted clear data. Panax quinquefolius showed a very different pattern than Panax ginseng. The peak of Panax quinquefolius was absent in the first T nucleotide base and the peak of the next C nucleotide base was higher than that of the next G nucleotide base (Fig. 1). But in Panax ginseng the peak of the first T nucleotide base was same height as that of the next C nucleotide base (Fig. 2). We designed ginseng species-specific sequencing primer to identify Panax ginseng and Panax quinquefolius. In sequence, Panax ginseng has a T nucleotide base, but Panax quinquefolius has a C nucleotide base. By these results, we verified that our ginseng species-specific sequencing primer was welldesigned. At present, pyrosequencing is performed in an automated microtiter-based pyrosequencer instrument, which allows simultaneous analysis of samples within 15 min. Each round of nucleotide dispensing takes approximately 1 min and thus offers a more rapid and convenient way to determine the exact sequence of the genetic variations than do other methods (RAPD, RFLP, etc.). Ginseng is cultivated world-wide and the origins of the product and the quality differ. So a method for identifying the origin is very important. Our results suggest that the pyrosequencing method is suitable for authentification of Panax species. This study shows that typing of genetic variations can be performed efficiently by pyrosequencing using an automated system for pattern recognition software. In conclusion, pyrosequencing analysis might be able to provide identification of Panax species.
Genetic Identification of Panax Species by Pyrosequencing
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Fig. 2. Typical Pyrosequencing Profile for Panax ginseng Cultivated in Jinan, Kumsan, Youngju, Kanghwa, and Punggi, Korea. The boxed regions identify sequence disparities between Panax quinquefolius and Panax ginseng. The peak of the first T nucleotide base was same height as that of the next C nucleotide base. The sequence depicted for Panax ginseng is GTCGAGA.
Acknowledgments This work was supported by grant no. R12-2003-00200006-0 from the Basic Research Program of the Korea Science and Engineering Foundation and and Korea Institute of Oriental Medicine.
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