April 2006 Biol. Pharm. Bull. 29(4) 629—633 (2006) 629 Development of SCAR Marker for Discrimination of Artemisia princeps and A. argyi from Other Artemisia Herbs a b b a b Mi Young LEE, Eui Jeong DOH, Chae Haeng PARK, Young Hwa KIM, Eung Soo KIM, a ,b Byong Seob KO, and Seung-Eun OH* a Korea Insititute of Oriental Medicine; Daejon 305–811, Korea: and b Department of Biological Sciences, Konkuk University; Seoul 143–701, Korea. Received September 21, 2005; accepted January 11, 2006 Some Artemisia herbs are used for medicinal purposes. In particular, A. princeps and A. argyi are classified as ‘Aeyup’ and are used as important medicinal material in traditional Korean medicine. On the other hand, A. capillaris and A. iwayomogi, which are classified as ‘Injinho’ and ‘Haninjin’, respectively, are used for other pur- poses distinct from those of ‘Aeyup’. However, sometimes ‘Aeyup’ is not clearly discriminated from ‘Injinho’ and/or ‘Haninjin’. Furthermore, Artemisia capillaris and/or A. iwayomogi have been used in place of A. princeps and A. argyi. In this study, we developed an efficient method to discriminate A. argyi and A. princeps from other Artemisia plants. The RAPD (random amplified polymorphic DNA) method efficiently discriminated various Artemisia herbs. In particular, non-specific primer 329 (5؅-GCG AAC CTC C-3؅), which shows polymorphism among Artemisia herbs, amplified 838 bp products, which are specific to A. princeps and A. argyi only. Based on (nucleotide sequence of the primer 329 product, we designed a Fb (5؅-CAT CAA CCA TGG CTT ATC CT-3؅ -and R7 (5؅-GCG AAC CTC CCC ATT CCA-3؅) primer-set to amplify a 254 bp sized SCAR (sequence character ized amplified regions) marker, through which A. princeps and A. argyi can be efficiently discriminated from other Artemisia herbs, particularly, A. capillaris and A. iwayomogi. Key words Artemisia princeps; Artemisia argyi; Artemisia herb; random amplified polymorphic DNA (RAPD); sequence char- acterized amplified regions (SCAR) marker About 500 plants belong to the genus Artemisia.1,2) Most fied polymorphic DNA (RAPD) analysis have been used for Artemisia herbs are perennials, growing in the northern this purpose.12) The advantages of the RAPD technique are hemisphere3) and are used for various purposes, such as med- that it is simple to carry out and can be completed within a icine, food, spices, food and ornamentation. The medicinal short time.13) However, because the RAPD technique is sen- effects of Artemisia herbs are extremely diverse and include sitive to PCR conditions, reproducibility of RAPD results is the following: cell protection from peptic ulcers,4) liver pro- low. To overcome this problem, Paran and Michelmore14) tection,5,6) anti-malarial7) and anti-tumor effects.8) Among suggested a sequence characterized amplified region (SCAR) Artemisia herbs, A. princeps, A. argyi, A. capillaris, and A. marker. iwayomogi are important medicinal materials in traditional In this work, we developed a reproducible SCAR marker, medicine in Asia. based on the PCR product amplified by a non-specific primer In Korea, A. princeps and A. argyi are classified as the for RAPD, to efficiently discriminate A. princeps and A. same Artemisia herb, called ‘Aeyup’.9) Without discrimina- argyi from other Artemisia herbs, particularly from A. capil- tion between A. princeps and A. argyi, both plants have been laris and A. iwayomogi. used in traditional Korean medicine for the treatment of colic pain, vomiting and diarrhea, and irregular bleeding from the MATERIALS AND METHODS uterus.10) On the other hand, A. capillaris and A. iwayomogi are classified as ‘Injinho’ and ‘Haninjin’, respectively, and Plant Materials The following 17 fresh leaf samples, have been used in China and Japan for anti-inflammation and presented in Table 1, were used: three samples of A. diuresis.11) princeps, two samples of A. argyi, five samples of A. capil- For discrimination of medicinal plants, subjective identifi- laris, five samples of A. iwayomogi and a sample each of A. cation methods, based on the plants morphological features, japonica and A. keiskeana. are generally used. With these methods, however, it is very Fresh leaves of Artemisia herbs acquired in Korea were difficult to discriminate between medicinal plants. For in- purchased from the Rural Development Administration stance, young leaves of A. capillaris, harvested particularly (RDA) of Korea. One sample of A. argyi was purchased in in early spring and classified as ‘Myun-injin’,9) are not easily Guangxi province in China. Authenticity of samples was distinguished from those of ‘Aeyup’. Therefore, a part of ‘In- conducted by the National Institute of Crop Science in RDA jinho’ is distributed as ‘Aeyup’ in the market of traditional of Korea and samples were deposited in the Korea Institute Korean medicine. Also, some ‘Haninjin’ is distributed as of Oriental Medicine (KIOM). In addition, to determine ‘Aeyup’. Furthermore, instead of ‘Aeyup’, ‘Haninjin’ and whether the designed primers amplified the expected SCAR ‘Injinho’ are sometimes used for medicinal prescriptions. To marker, dried herbal market samples deposited in KIOM solve this problem, an efficient discrimination method, which were used. can be utilized to identify Artemisia herbs, is needed. Preparation of Genomic DNA The genomic DNA of Various molecular biological techniques, utilizing the dif- each sample was extracted according to the manual for the ferent genetic information of organisms, are employed for PureGene DNA separation kit (Gentra, U.S.A.). To improve species discrimination of plants. In particular, random ampli- DNA quality, phenolic compounds and polysaccharides were ∗ To whom correspondence should be addressed. e-mail: [email protected] © 2006 Pharmaceutical Society of Japan 630 Vol. 29, No. 4 Table1. List of Artemisia Plants Used in This Study Table2. List of RAPD Non-specific Primers Used in This Study Medicine Date of Primer GC content Primer GC content Species Locality Lane Sequence (5Ј→3Ј) Sequence(5Ј→3Ј) name collection no. (%) no. (%) Aeyup Artemisia princeps Bonghwa, Korea 2002. 9 1 301 CGG TGG CGA A 70 327 ATA CGG CGT C 60 Jeonju, Korea 2002. 5 2 302 CGG CCC ACG T 80 328 ATG GCC TTA C 50 Uiseong, Korea 2002. 9 3 308 AGC GGC TAG G 70 329 GCG AAC CTC C 70 A. argyi Suwon, Korea 2002. 6 4 309 ACA TCC TGC G 60 328 ATG GCC TTA C 50 Guangxi, China 2002. 7 5 310 GAG CCA GAA G 60 330 GGT GGT TTC C 60 Injinho A. capillaris Jeonju, Korea 2002. 5 6 311 GGT AAC CGT A 50 352 CAC AAC GGG T 60 Jinan, Korea 2002. 5 7 312 ACG GCG TCA C 70 353 TGG GCT CGC T 70 Jinan, Korea 2002. 5 8 313 ACG GCA GTG G 70 354 CTA GAG GCC G 90 Ullungdo, Korea 2002. 7 9 314 ACT TCC TCC A 50 355 GTA TGG GGC T 60 Bonghwa, Korea 2002. 9 10 315 GGT CTC CTA G 60 356 GCG GCC CTC T 80 Haninjin A. iwayomogi Jecheon, Korea 2002. 7 11 317 CTA GGG GCT G 70 357 AGG CCA AAT G 50 Uiseong, Korea 2002. 9 12 319 GTG GCC GCG C 90 360 CTC TCC AGG C 70 Jinan, Korea 2002. 5 13 321 ATC TAG GGA C 50 391 GCG AAC CTC G 70 Jinan, Korea 2002. 5 14 324 ACA GGG AAC G 60 392 CCT GGT GGT T 60 Bonghwa, Korea 2002. 9 15 Others A. japonica Suwon, Korea 2002. 6 16 A. keiskeana Uiseong, Korea 2002. 9 17 removed using 10% cetyltrimethyl ammonium bromide (CTAB) and 0.7 M NaCl. PCR Amplification for Analysis of RAPD The PCR was carried out according to the method of Williams et al.,13) using a T-personal cycler (Biometra, Germany) via mixing 600 nM UBC primer (University of British Columbia, Canada), presented in Table 2, and 1 U Taq polymerase (ABgene, U.S.A.) and 50 ng of genomic DNA extracted from each samples. In the PCR process, pre-denaturation was conducted at 94 °C for 5 min and denaturation was per- formed at 94 °C for 30 s. The annealing process was carried out at 37 °C for 30 s and the extension was performed at 72 °C for 1 min and, finally, the reaction was carried out at 72 °C for 10 min. After the separation of the amplified prod- ucts on 1.5% agarose gel, the gel was stained with EtBr (Sigma, U.S.A.). The amplified products were analyzed using the MyImager (Seoulin Biotechnology, Korea). Nucleotide Sequencing of PCR Products A PCR am- plification product separated from agarose gel was cloned using the pGEM T-easy vector I (Promega, U.S.A.) or the p- Drive cloning kit (Qiagen, Germany). The nucleotide se- Fig. 1. Polymorphism among Artemisia Plants Detected by Non-specific quences of the subcloned PCR products were determined by Primers Company Bionex (Korea). Numbers on upper left side are UBC primer numbers. Lanes 1—17, Artemisia plants listed in Table 1. M, 100-bp ladder. RESULTS and A. argyi (lanes 4, 5) belong to group I, A. capillaris Discrimination of Artemisia Herbs by RAPD Analysis (lanes 6—10) and A. japonica (lane 16) belong to group II, First, we tested whether Artemisia herbs grown in Korea, in- and A. iwayomogi (lanes 11—15) and A. keiskeana (lane 17) cluding A. princeps, A. argyi, A. capillaris, and A. iway- belong to group III. The results of the phenogram suggest omogi, could be efficiently discriminated through RAPD that A. princeps and A. argyi can be efficiently discriminated analysis using non-specific primers. Since many A. argyi from other Artemisia herbs, particularly from A. capillaris samples are not collected in Korea, one sample from China and A.