Development of SCAR Marker for Discrimination of Artemisia Princeps and A

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 purposes 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 characterized 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 characterized 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

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 (5→3) Sequence(5→3) 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 performed 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 products 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 amplification 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. iwayomogi. was used in this work. As a result of RAPD, using 31 non- Sequencing of PCR Products from A. princeps and A. specific UBC primers (Table 2), 11 primers including 354 argyi Among the non-specific primers that showed poly- and 357 showed polymorphism among Artemisia herbs (Fig. morphism between the Artemisia herbs, primer 329, in par- 1). Based on the RAPD results, we then analyzed the phylo- ticular, generated strong amplified products of 800—850 bp genetic similarity of Artemisia herbs and constructed a in size, specifically to A. princeps and A. argyi (Fig. 3). phenogram with UPGMA (unweighted pair-group method Based on the nucleotide sequences of the amplified products, with arithmetic average) using the NTSYS (numerical taxon- we attempted to design primers that could not only efficiently omy and multi analysis system) program (Fig. 2).15) The discriminate A. princeps and A. argyi from other Artemisia phenogram consists of three groups: A. princeps (lanes 1—3) herbs, but that would also be available for PCR amplification April 2006 631

Fig. 2. Phenogram of 17 Artemisia Plants Listed in Table 1 Constructed from RAPD Results Deﬁned by 11 Non-speciﬁc Primers Showing Polymorphism

DISCUSSION

Although studies of the efficacy of A. princeps and A. argyi as medicinal herbs have been conducted,16,17) there has yet to be any reported study on the classification of any Artemisia herbs used as medicinal materials in Korea, China, and Japan. In this study, using genetic markers, we attempted to discriminate objectively both A. princeps and A. argyi from other Artemisia herbs, particularly, A. capillaris and A. iwayomogi, as discrimination is otherwise subjective through a Fig. 3. Polymorphism among Artemisia Plants Detected by Non-specific morphological approach. Since Artemisia herbs are used in Primer 329 the form of dried leaves as medicinal material, species-spe- Lanes 1—17, Artemisia plants listed in Table 1. M, 100-bp ladder. cific primers are needed, which is a single reaction step. As shown in Fig. 1, polymorphisms between Artemisia using fragmented genomic DNA prepared from samples in a herbs were detected efficiently by RAPD analysis. The main dried state. Thus, after sub-cloning the amplified products of causal factor for these polymorphisms could be the base sub- primer 329, the nucleotide sequencing of the amplified prod- stitution of the non-specific primer’s binding sequences that ucts was determined (Fig. 4). We registered the determined exists in the genomic DNA of each Artemisia herbs sam- nucleotide sequences of the amplified products of 838 bp to ple.13) However, the observed polymorphism may take place NCBI (AY485210 and AY485209) and confirmed a 97% as a result of technical factors, such as differences in the con- similarity between the sequences of the primer 329 amplified centrations of template genomic DNA and primers used for products of A. princeps and A. argyi. PCR. Also, we cannot exclude the possibility that several SCAR Marker Development of A. princeps and A. argyi non-specific amplified products were generated by a rela- from Other Artemisia Herbs Based on the sequences of tively shorter 10-m primer used in the RAPD experiment.14) the amplified products of primer 329 from A. princeps and A. Due to these causal factors, different patterns in RAPD could argyi, we devised a primer set that generated a SCAR marker be detected between different samples of the same species of of 254 bp. Fb (5-CAT CAA CCA TGG CTT ATC CT-3) Artemisia plants. However, despite polymorphism between was devised as a forward primer and R7 (5-GCG AAC CTC different specimens of the same species of Artemisia herbs, CCC ATT CCA-3) was devised as a reverse primer. Using A. princeps and A. argyi were clearly discriminated from the Fb/R7 primer pair, the expected amplified products, hav- other Artemisia herbs in the phenogram (Fig. 2). This ing a size of 254 bp, were generated from A. princeps and A. phenogram, constructed from 11 RAPD analysis, and the argyi. Meanwhile, no amplified products were generated RAPD marker were utilized to design a SCAR marker for the from A. capillaris or A. iwayomogi (Fig. 5). Therefore, we discrimination of A. princeps and A. argyi from other confirmed that the primer set can be utilized to amplify the Artemisia herbs. In particular, primer 329 generated strong SCAR marker for the discrimination of A. princeps and A. amplified products with a size of 838 bp from only A. prin- argyi from other Artemisia herbs. We subsequently tested ceps and A. argyi (Fig. 3). To confirm whether the nucleotide whether the Fb/R7 primer set could efficiently amplify the sequences of the amplified products of A. princeps and A. SCAR marker in Aeyup handled in a dried state from a argyi are homologous, and whether 838 bp products could be herbal market (Fig. 6). From this result, no amplified prod- available for the development of the SCAR marker, we con- ucts were generated in the specimens of Injinho and Haninjin ducted nucleotide sequencing (Fig. 4). As a result of compar- but amplified products with a size of 254 bp were generated ing the sequences of 838 bp products from A. princeps and in specimens of Aeyup. A. argyi, it was found that one bp gap (56th and 501st) ex- isted in A. princeps and A. argyi, respectively, and among the total 838 bp compared, 23 bp showed differences. Thus, we 632 Vol. 29, No. 4

Fig. 4. Comparison of the Nucleotide Sequences of PCR Products of A. princeps and A. argyi Ampliﬁed by UBC Primer 329 Dots represent identical nucleotides. Bold blocks of nucleotides indicate the designed primer pair.

Fig. 6. PCR Products of the Designed Primer Pair (Fb/R7) from Dried Artemisia Plants Handled in Herbal Markets in Korea Aeyup (lanes 1—5); Injinho (lanes 6—10); Haninjin (lanes 11—15).

Fig. 5. PCR Products of the Designed Primer Pair (Fb/R7) from Four primer 329 were compared with other sequences deposited in Kinds of Artemisia Plants the data bank. Part of the sequence of the amplified product from A. princeps was consistent with the sequences of a re- confirmed that 97% homology exists between the primer 329 gion of chromosome No. 4 in Arabidopsis (AL021711) and a amplified products of A. princeps and A. argyi. Based on the region of chromosome No. 11 in Oryza sativa (AC135512), similarity between the two PCR products, Fb and R7 primers the functions of which were not identified. Meanwhile, some generating a SCAR marker in A. princeps and A. argyi were of the sequence of amplified products of A. argyi were con- devised. Since we speculated that the genomic DNA ex- sistent with the sequences of the region of the PIN promoter tracted from dried samples and used for the PCR template is of Brassica juncea (AJ308229). Based on these observations, likely to be fragmented, Fb and R7 were devised to amplify it is possible that the amplified products of primer 329 are 254 bp, part of the amplified product of primer 329. not part of the ORF region. The nucleotide sequences of A. princeps and A. argyi by As shown in Fig. 5, while Fb and R7 generated 254 bp April 2006 633 bands in A. princeps and A. argyi, no amplified products 6) Gilani A. H., Janbaz K. H., Gen. Pharmacol., 26, 309—315 (1995). were generated in A. capillaris and A. iwayomogi. In the 7) White N. J., Trans. R. Soc. Trop. Med. Hyg., 88 (Suppl. 1), S3—4 (1994). samples in dried states, as handled in Korean herbal markets, 8) Xu Q., Mori H., Sakamoto O., Koda A., Nishioka I., Ogawa Y., 254 bp bands were also generated only from Aeyup (Fig. 6). Hosaka K., J. Med. Pharm. Soc. Wakan-Yaku, 6, 1—7 (1989). Therefore, we confirmed that Fb and R7 could be used for 9) “The Korean Herbal Pharmacopoeia,” Korea Food & Drug Adminis- the discrimination of Aeyup from Injinho and Hanjinho dis- tration, 2002. p. 251. tributed in herbal markets, so as to prevent misuse between 10) Zhao Q. C., Kiyohara H., Yamada H., Phytochemistry, 35, 73—77 (1994). Artemisia herbs. 11) Kim J. O., Kim Y. S., Lee J. H., Kim M. N., Rhee S. H., Moon S. H., Park K. Y., J. Korean Soc. Food Nutr., 21, 308—313 (1992). REFERENCES 12) McClelland M., Welsh J., PCR Methods Applic., 4, S59—65 (1994). 13) Williams J. G. K., Kubelik A. R., Livak K. J., Rafalski J. A., Tingey S. 1) McArthur E. D., “Sagebrush Ecosystem Symposium,” Utah State Uni- V., Nucl. Acids Res., 18, 6531—6535 (1990). versity, Logan, UT, 1979, pp. 14—22. 14) Paran I., Michelmore R. W., Theor. Appl. Genet., 85, 985—993 (1993). 2) Bremer K., Humphries C. J., Bull. Nat. Hist. Mus. Lond. (Bot.), 23, 15) Rohlf F. J., “NTSYSpc Numerical Taxonomy and Multivariate Analy- 71—177 (1993). sis System,” Ver. 2.1, New York, 2000. 3) Torrell M., Cerbah M., Siljak-Yakovlev S., Valles J., Plant Syst. Evol., 16) Tan R. X., Zheng W. F., Tang H. Q., Planta Med., 64, 295—302 239, 141—153 (2003). (1998). 4) Piezzi R. S., Guzman J. A., Guaridia T., Pestchanker M. J., Microsc. 17) Kim J. H., Kim H. K., Jeon S. B., Son K. H., Kim E. H., Kang S. K., Electron. Biol. Celular, 16, 45—55 (1992). Sung N. D., Kwon B. M., Tetrahedron Lett., 43, 6205—6208 (2002). 5) Gilani A. H., Janbaz K. H., J. Pak. Med. Assoc., 44, 65—68 (1994).