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1 ª Ÿ (Korean J. Medicinal Crop Sci.) 18(6) : (2010) w p (Adenophora racemosa) mw e *Á *Á *Á *Á ¼** Á½y * *w w w, ** w Molecular Phylogenetic Position of Adenophora racemosa, an Endemic Species in Korea Yunui Ji*, Byeong Cheol Moon*, A Yeong Lee*, Jin Mi Chun*, Byung Kil Choo** and Ho Kyoung Kim* *Korea Institute of Oriental Medicine, Daejeon , Korea. **Chonbuk National University Jeonju , Korea ABSTRACT : Adenophora racemosa is recently reported as a new Korean endemic plant species. However, the phylogenetic relationship of this genus has been controversial due to the morphological similarity and frequent morphological change of aerial parts. To verify the phylogenetic position of Adenophora racemosa and phylogenetic relationship of genus Adenophora, we analyzed the internal transcribed spacer (ITS) sequence of nuclear ribosomal DNA (nrdna) and random amplified polymorphic DNA (RAPD) using 21 individual of 6 Adenophora species, A. verticillata, A. divaricata, A. racemosa, A. remotiflora, A. stricata and A. tetraphylla. In comparative analysis of the nrdna-its sequences, we could not found not only any species specific nucleotide sequence but also could not estimated their inter or intra species. In the phylogenic analysis based on the RAPD derived DNA polymorphism, Adenophora species were classified into four groups by clustering analysis of the UPGMA. These results suggest that the DNA fingerprinting based on RAPD is more suitable than nrdna-its sequence for the phylogenetic analysis of Adenophora species Key Words : Adenophora racemosa, RAPD, nrdna-its, Genetic Relationship. (Adenophora ) (Campanulaceae) w (, Adenophora Radix) w (Cooperation teaching materials compilation committee of oriental medicine college in Korea, 2004) w w. sw, ù d d (A. verticillata Fisch.), (A. divaricata Franch. & Sav.), (A. stricta Miq.), (A. remotiflora Miq.) 29 wš (Lee, 2006). ü w w p (Endemic plant species)» (Kim, 2004) (A. racemosa, 1990) xk w w, y ¼ s š, y y ¼ s, y š š,» l, w l š, y x š w š (Lee and Lee, 1990). ù ƒ š z w p ƒ w w w ƒ š. w» w w d ƒ š (Lee et al., 2010; Eu et al., 2009), m w p w» ù w (DNA fingerprinting) š (Kress et al., 2005; Weising, 2005). p» nuclear ribosomal DNA (nrdna) ITS (internal transcribed spacer) ƒ Corresponding author: (Phone) ( ) hkkim@kiom.re.kr, (Phone) ( ) bkchoo@jbnu.ac.kr Received 2010 September 20 / 1st Revised 2010 October 15 / 2nd Revised 2010 November 4 / Accepted 2010 November 9 379

2 Á Á Á Á ¼Á½y š (Sucher and Carles, 2008; Xie et al., 2009; Kim et al., 2007; Xu et al., 2006; Lin et al., 2008; Li and Jin, 2006), (cpdna) matk, rbcl, atpa, trnl-f š (Yoo et al., 2007; Yang et al., 2004). ITS» w Tricholoma matsutake Rhizina undulata» w (Lee et al., 2004; Lee et al., 2007), š w (Jigden et al., 2009)ƒ. w, 1980 z l PCR (Polymerase Chian Reaction)» x (Polymorphism) w RAPD (Randon Primer Polymorphic DNA, Williams et al., 1990), AFLP (Amplified Fragment Length Polymorphism, Zabeau and Vos, 1993; Vos et al., 1995), ISSR (Inter-Simple Sequence Repeat, Zietkiewicz et al., 1994) w w w šw (Ghada et al., 2009; Han et al., 2007; Kim et al., 2007). p, RAPD x y ƒ w w ù w w w f (Weising et al, 2005). RAPD mw m (Artemisia spp.) (Park et al., 2005) ù ³ (Lee et al., 2006). w RAPD w wš w ww» w (Yoo et al., 1996). ù w, š š jš ƒ w w w ù p dd,,,, nrdna ITS» RAPD w ³ wš w. 1. œ w w ƒƒ ü w (Table 1), w w, w, w ƒ Á z y. ƒ» t w w w w t w, 70æ þ š w DNA wš nrdna- ITS s RAPD w DNA fingerprinting w. Table 1. List and information of Adenophora species used in this study. Science name Sample No. Location Line No. 1-1 Jangsu-gun, Jeollabuk-do, Korea 1 Adenophora verticillata Hoengseong-gun, Gangwon-do, Korea Adenophora divaricata 2-2 Hoengseong-gun, Gangwon-do, Korea Inje-gun, Gangwon-do, Korea 8 Adenophora racemosa Pyeongchang-gun Gangwon-do, Korea Inje-gun, Gangwon-do, Korea 13 Adenophora remotiflora 4-2 Hwajeong, Gillim-seong, China Hamyang, Gyeongsangnam-do, Korea Muhan, Hebei, China 16 Adenophora stricta Ryongnam, Gansusheng, China Adenophora tetraphylla 6-2 Hwajeong, Jilin, China

3 w p (Adenophora racemosa) mw e 2. Genomic DNA Á w 70æ» þ g w kƒ w z, Plant genomic DNA Prep Kit (Solgent, Korea) DNeasy Plant Mini Kit (QIAGEN, Germany) w ƒ œw protocol Á w. DNA y w» w 1.5% agarose gel w» z, EtBr (Ethidium Bromide) w UV light DNA band y w, UV spectrophotometer w Ÿ d w w. 3. nrdna-its RAPD PCR s 5~20 ng genomic DNA ƒ 20 pmole ITS1 (5'- TCC GTA GGT GAA CCT GCG G-3') ITS4 (5'-TCC TCC GCT GAT TGA TAT GC-3 ) primer (White et al., 1990), š 2unit DNA polymerase 50 1 (75 mm Tris-HCL (ph 8.8), 20 mm (NH 4 ) 2 SO 4, 0.1% (v/v) Tween 20, 20 µm dntp, 200 µm MgCl 2 ) ƒƒ ƒw DNA Engine Dyad Thermal Cycler (MJ Research, USA) w sw. 95æ 5 predenaturationw z 95æ 30 denaturation, 51æ 40 annealing, 72æ 1 extension 35z wwš 72æ 10 extension g sw. RAPD w PCR s Williams (1990) w Operon 10-mer RAPD Kits RAPD kit A, B, C, D w (Seoul, Korea) 20-mer URP random primer kit w ww (Table 2). 5~20 ng total DNA 30 pmole RAPD primer 2 unit DNA polymerase 30 1 (75 mm Tris-HCL (ph 8.8), 20 mm (NH 4 ) 2 SO 4, 0.1% (v/v) Tween 20, 20 µm dntp, 200 µm MgCl 2 ) ƒƒ ƒw DNA Engine Dyad Thermal Cycler (MJ Research, USA) w s w. 95æ 5 predenaturationw z 95 æ 30 denaturation, 46æ 30 annealing, 72æ 45 extension 35z wwš 72æ 5 extension g. 4. nrdna-its s» nrdna-its s DNA» w total s 1.5% agarose gel 100 bp DNA ladder (Solgent, Korea) wì» wš EtBr w w z, yw s s agarose gel l z w Agarose Gel Extraction Kit (SolGent, Korea) w w, pgem-teasy Vector Systems (Promega, USA) w. s XL1-Blue MRF' Table 2. List of oligonucleotide primer sequences and application for RAPD analysis. No. Primer SequenceG(5' 3') No. Primer SequenceG(5' 3') No. Primer SequenceG(5' 3') 1 OPA1 CAG GCC CTT C 22 OPB2 TGA TCC CTG G 43 OPC11 AAA GCT GCG G 2 OPA2 TGC CGA GCT G 23 OPB3 CAT CCC CCT G 44 OPC12 TGT CAT CCC C 3 OPA3 AGT CAG CCA C 24 OPB4 GGA CTG GAG T 45 OPC13 AAG CCT CGT C 4 OPA4 AAT CGG GCT G 25 OPB5 TGC GCC CTT C 46 OPC14 TGC GTG CTT G 5 OPA5 AGG GGT CTT G 26 OPB6 TGC TCT GCC C 47 OPC15 GAC GGA TCA G 6 OPA6 GGT CCC TGA C 27 OPB7 GGT GAC GCA G 48 OPC16 CAC ACT CCA G 7 OPA7 GAA ACG GGT G 28 OPB8 GTC CAC ACG G 49 OPC17 TTC CCC CCA G 8 OPA8 GTG ACG TAG G 29 OPB9 TGG GGG ACT C 50 OPC19 GTT GCC AGC C 9 OPA9 GGG TAA CGC C 30 OPB10 CTG CTG GGA C 51 OPD1 ACC GCG AAG G 10 OPA10 GTG ATC GCA G 31 OPB11 GTA GAC CCG T 52 OPD2 GGA CCC AAC C 11 OPA11 CAA TCG CCG T 32 OPB12 CCT TGA CGC A 53 OPD3 GTC GCC GTC A 12 OPA12 TCG GCG ATA G 33 OPC1 TTC GAG CCA G 54 OPD4 TCT GGT CAG G 13 OPA13 CAG CAC CCA C 34 OPC2 GTG AGG CGT C 55 OPD5 TGA GCG GAC A 14 OPA14 TCT GTG CTG G 35 OPC3 GGG GGT CTT T 56 OPD6 ACC TGA ACG G 15 OPA15 TTC CGA ACC C 36 OPC4 CCG CAT CTA C 57 OPD7 TTG GCA CGG G 16 OPA16 AGC CAG CGA A 37 OPC5 GAT GAC CGC C 58 OPD8 GTG TGC CCC A 17 OPA17 GAC CGC TTG T 38 OPC6 GAA CGG ACT C 59 OPD9 CTC TGG AGA C 18 OPA18 AGG TGA CCG T 39 OPC7 GTC CCG ACG A 60 OPD10 GGT CTA CAC C 19 OPA19 CAA ACG TCG G 40 OPC8 TGG ACC GGT G 61 OPD11 AGC GCC ATT G 20 OPA20 GTT GCG ATC C 41 OPC9 CTC ACC GTC C 62 OPD12 CAC CGT ATC C 21 OPB1 GTT TCG CTC C 42 OPC10 TGG ACC GGT G 381

Á Á Á Á ¼Á½y š Fig. 1. Amplification of nrdna-its region from Adenophora species using ITS1 and ITS4 primer. competent cell (Stratagene, USA) x yw ampicillin X-gal/IPTGƒ ƒ LB agar w 20 w.

4 Á Á Á Á ¼Á½y š Fig. 1. Amplification of nrdna-its region from Adenophora species using ITS1 and ITS4 primer. competent cell (Stratagene, USA) x yw ampicillin X-gal/IPTGƒ ƒ LB agar w 20 w. ƒ 3 white colony wš Plasmid mini preparation kit (SolGent, Korea) w plasmid DNA w z, T7 SP6 primer w ABI3730 automatic DNA sequencer (Applied Biosystems, USA)» wš ƒ rdna-its» y w. ƒ l nrdna-its» ITS1, 5.8S ITS2 GenBank» w w, 21 nrdna-its» BioEdit program (Version 7.0.9,) w multiple align wwš ƒ,» e» (insertion), (deletion) ey (substitution) w. w, 21 DNAstar program (Version 5.0) MegAlign w ClustalW phylogenetic tree w w. 5. RAPD s RAPD w 62 Operon 10- mer primer w (Table 2) sw 1.5% agarose gel 1kb plus DNA ladder (Invitrogen, USA) wì» z EtBr mw polymophic band image y w (Syngene, UK). y image GeneTools program (Syngene, UK) w ƒ primer w s s ql w, primer GeneDirctory program (Syngene, UK) w Á x library wš ƒ library UPGMA mw similarity matrix phylogenetic tree wš w. 1. nrdna-its» nrdna-its» w w l w genomic DNA x w ITS1 ITS4 primer 800 bp s š (Fig. 1), s 21 nrdna-its» w, nrdna sww s 793~795 bp» (Fig. 2). w nrdna-its s j» š kù 633~654 bp (Son et al., 2007), ë 646~652 bp (Park et al., 2006) w ¼ r l y w nrdna-its» w, ü» identity 98.5~100%,» identity 98.2~99.9%,, œm indels ( / ) ù nucleotide substitution y w, p indel»ey y š, w» ƒ w. w xkƒ. w, 6 21 nrdna-its» w ü w, xkw š w w group x w w (Fig. 3).» w š w x k ƒ w (Lee, 1989; Yoo, 1995). ITS»» ü w xk w xk w w pw p ƒ. w ywš yw y w xk š x ƒ w q. 2. RAPD mw nrdna-its» mw Adenophora ù m w yw. genome fingerprinting mw y w RAPD ww. 62 Operon 10-mer primer w RAPD 382

한국특산종 외대잔대 Adenophora racemosa 의 분자계통학적 위치 ( Fig. 2. ) Alignment of nucleotide sequences of nrdna-its region for the comparition of Adenophora species.

5 한국특산종 외대잔대 Adenophora racemosa 의 분자계통학적 위치 ( Fig. 2. ) Alignment of nucleotide sequences of nrdna-its region for the comparition of Adenophora species. Bars ( ) indicate gaps required for alignment and dots ( ) indicate sequences identical with the first sequences. 1:A. verticillata, 2:A. divericata, 3:A. racemosa, 4:A. remotiflora, 5:A. stricta, 6:A. tetraphylla. 383

6 지윤의 문병철 이아영 천진미 추병길 김호경 Fig. 2. Continued. 384

w p (Adenophora racemosa) mw e Fig. 3. Phylogenetic relationship of the nrdna-its sequence among 21 samples of 6 Adenophora species. 1-1~1-3:A. verticillata, 2-1~2-3:A. divericata, 3-1~3-6:A.

7 w p (Adenophora racemosa) mw e Fig. 3. Phylogenetic relationship of the nrdna-its sequence among 21 samples of 6 Adenophora species. 1-1~1-3:A. verticillata, 2-1~2-3:A. divericata, 3-1~3-6:A. racemosa, 4-1~4-3:A. remotiflora, 5-1~5-3:A. stricta, 6-1~6-3:A. tetraphylla. Fig. 4. RAPD profile of 6 Adenophora species using Operon 10-mer primer kit., 45 primer l ƒ š s w j» DNA r s y w. s PCR j» 150 bp~4.5 kb w ù kû, œm s wr ü p s. 3,403 RAPD s band y w š primer DNA s 2~8 s³ 3.6 bandƒ ùkù p s w (Fig. 4). primer w ù 17 primer DNA r s ƒ (data not shown). DNA band wš 6 21 w (Table 3), ü 80~98%, 44~63%, RAPD w UPGMA phylogenetic tree w, nrdna-its» (Fig. 3) w group x w y w (Fig. 5). w dd ƒ 95% w š, dd w û ùkþ. 385

8 Á Á Á Á ¼Á½y Table 3. Divergence matrix for Adenophora species. Species A. tetraphylla A. tetraphylla A. tetraphylla A. divaricata A. divaricata A. divaricata A. racemosa A. racemosa A. racemosa A. racemosa A. racemosa A. racemosa A. stricta A. stricta A. stricta A. remotiflora A. remotiflora A. remotiflora A. verticillata A. verticillata A. verticillata Fig. 5. Phylogenetic relationship of 6 Adenophora species and 21 samples from RAPD-based UPGMA analysis. ƒ¾ 80% (Table 3). y w, Calamun thwaitesii ƒ 64~96% (Sreekumar and Renuka, 2006), Isoetes coreana (Kim et al., 2008) Stipa grandis (Zhao, 2006) 80% ùkü š, ù ù (Abies koreana) (Kim and Hyun, 2000) 92~96% ùkü. RAPD l w 50~60% ùkþ (Table 3), Ligustrum ƒ 34~46% (Zheng et al., 2009), Vanilla ƒ 64% w w, 386

9 w p (Adenophora racemosa) mw e ƒ ƒ¾ q. ƒ 60% ù w ƒ ƒà ùkûš, 57% ƒ¾ group (Table 3, Fig. 5), xk w wš, y j» w» š (Lee, 1989) w yƒ xk» wš y w. w nrdna-its» RAPD mw ƒ š ùkû. w» w y» (L08020) w w, x w œ œ. LITERATURE CITED Cooperation teaching materials compilation committee of oriental medicine college in Korea. (2005). Herbal Medicine. Younglimsa Press. Seoul, Korea. p Eu GS, Park MR and Yun SJ. (2009). Internal transcribed spacer (ITS) regions reveals phylogenic relationships of Rubus species cultivated in Korea. Korean Journal of Medicinal Crop Science. 17: Ghada B, Khaled Ci, Olfa S, Messaoud M, Mohamed M, Mokhtar T and Amel SH. (2009). Genetic analysis of tunisian fig (Ficus carica L.) cultivars using amplified fragment length polymorphism (AFLP) markers. Scientia Horticulturae. 120: Han JP, Zhang WS, Cao HB, Chen SI and Wang YY. (2007). Genetic diversity and biogeography of the traditional chinese medicine, Gardenia jasminoides, based on AFLP markers. Biochemical Systematics and Ecology. 35: Jigden B, Kim MK, Noh JH, Sun H and Yang DC. (2009). Phylogenetic analysis of schizonepeta spike on the basis of DNA sequences. Korean Journal of Medicinal Crop Science. 17: Kress WJ, Wurdack KJ, Zimmer EA, Weigt LA and Janzen DH. (2005). Use of DNA barcodes to identify flowering plants. Proceedings of the National Academy of Sciences. 102: Kim CK, Na HR and Choi HK. (2008). Genetic diversity and population structure of endangered Isoetes coreana in south Korea based on RAPD analysis. Aquatic Botany. 89: Kim IS and Hyun JO. (2000). Genetic diversity of Abies koreana Wilson based on RAPD analysis. Korean Journal of Breeding. 32: Kim MY. (2004). Korean Endemic Plant. Solkwahak. Seoul, Korea. p Kim OT, Bang KH, In DS, Lee JW, Kim YC, Shin YS, Hyun DY, Lee SS, Cha SW and Seong NS. (2007). Molecular authentication of ginseng cultivars by comparison of internal transcribed spacer and 5.8S rdna sequences. Plant Biotechnol Report. 1: Kim YD, Kim KJ, Kim SH and Kim HT. (2007). Genetic diversity in three populations of Hibiscus hamabo(malvaceae) in Jeju Island, Korea. Korean Journal of Plant Taxonomy. 37: Lee JH, Jo IH, Lee JW, Park CG, Bang KH, Kim HS and Park CB. (2010). Molecular authentication of Scrophularia herbs by PCR-RFLP based on rpl-5 region of mitochondrial DNA. Korean Journal of Medicinal Crop Science. 18: Lee JK. (1989). Taxonomy of the genus Adenophora (Adenophorae) in Korea. Sungkyunkwan University. Seoul, Korea. p. 75. Lee JK and Lee ST. (1990). Adenophora racemosa (Campanulaceae), a new species from Korea. Journal of Korean Plant Taxonomy. 20: Lee JS, Noh EW, Choi YI, Lee WY and Kah KH. (2004). Development of nuclear ribosomal ITS DNA marker for Tricholoma matsutake. Journal of Korean Forest Society. 93: Lee TB. (2006). Coloured Flora of Korea (V. 2). Hyangmunsa Press. Seoul, Korea. p Lee SK, Lee JK, Kim KH, Lee SK and Lee SY. (2007). PCR detection and sequence analysis of the rdna ITS regions of Rhizina undulata. Journal of Korean Forestry Society. 96: Lee SY, Lee SG, Lee JG, Kim GH and Lee SG. (2006). Cultural characteristics and genetic diversity of Rhizina undulata isolates by Randim Amplified Polymorphic DNA (RAPD). Journal of Korean Forestry Society. 95: Lin WY, Chen LR and Lin TY. (2008). Rapid authentication of Bupleurum species using an array of immobilized sequencespecific oligonucleotide probes. Planta Medica. 74: Li UM and Jin ZX. (2006). High genetic differentiation revealed by RAPD analysis of narrowly endemic Sinocalycanthus chinensis Cheng et S.Y. Chang, an endangered species of China. Biochemical Systematics and Ecology. 34: Park JU, Kim MH, Gwak MH and Won HS. (2006). Variation of nuclear ribosomal ITS sequences of polygonum section Persicaria (Polygonaceae) in Korea. Korean Journal of Plant Taxonomy. 36: Park SK, Chung BH, Kim HS and Cho YG. (2005). Classification of Artemisia spp. collections based on morphological characters and RAPD analysis. Korean Journal of Medicinal Crop Science. 13: Son SW, Kim JH, Kim YS and Park SJ. (2007). ITS sequence variations in populations of Ilex cornuta (Aquifoliaceae). Korean Journal of Plant Taxonomy. 37: Sreekumar VB and Renuka C. (2006). Assessment of genetic diversity in Calamus thwaitesii BECC. (Arecaceae) using RAPD markers. Biochemical Systematics and Ecology. 34: Sucher NJ and Carles MC. (2008). Genome-based approaches to the authentication of medicinal plants. Planta Medica. 74:

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Æ¯ÇãÃ» '' - 1 - - 2 - - 3 - - 4 - - 5 - ' - 6 - - 7 - - 8 - - 9 - - 10 - - 11 - Super-Bio Co., Ltd. KWON, Suk-Tae Thermostable DNA Polymerase-encoding Gene from Thermus sp. X-1 an d Amino Acid Sequence