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ª Ÿ (Korean J. Medicinal Crop Sci.) 18(6) : 379 388 (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 305-811, Korea. **Chonbuk National University Jeonju 561-756, 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) +82-42-868-9502 (E-mail) hkkim@kiom.re.kr, (Phone) +82-63-270-2526 (E-mail) bkchoo@jbnu.ac.kr Received 2010 September 20 / 1st Revised 2010 October 15 / 2nd Revised 2010 November 4 / Accepted 2010 November 9 379

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 1-2 2 Hoengseong-gun, Gangwon-do, Korea 1-3 3 2-1 4 Adenophora divaricata 2-2 Hoengseong-gun, Gangwon-do, Korea 5 2-3 6 3-1 7 3-2 Inje-gun, Gangwon-do, Korea 8 Adenophora racemosa 3-3 9 3-4 10 3-5 Pyeongchang-gun Gangwon-do, Korea 11 3-6 12 4-1 Inje-gun, Gangwon-do, Korea 13 Adenophora remotiflora 4-2 Hwajeong, Gillim-seong, China 14 4-3 Hamyang, Gyeongsangnam-do, Korea 15 5-1 Muhan, Hebei, China 16 Adenophora stricta 5-2 17 Ryongnam, Gansusheng, China 5-3 18 6-1 19 Adenophora tetraphylla 6-2 Hwajeong, Jilin, China 20 6-3 21 380

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 260 280 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. 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. 6 21 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. 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

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. 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

Á Á Á Á ¼Á½y Table 3. Divergence matrix for Adenophora species. Species 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 1. A. tetraphylla 6-1 1.00 2. A. tetraphylla 6-2 0.98 1.00 3. A. tetraphylla 6-3 0.95 0.97 1.00 4. A. divaricata 2-3 0.51 0.54 0.54 1.00 5. A. divaricata 2-1 0.51 0.53 0.53 0.85 1.00 6. A. divaricata 2-2 0.52 0.55 0.55 0.86 0.92 1.00 7. A. racemosa 3-2 0.55 0.55 0.54 0.63 0.58 0.57 1.00 8. A. racemosa 3-1 0.52 0.53 0.51 0.63 0.62 0.59 0.92 1.00 9. A. racemosa 3-3 0.55 0.55 0.54 0.62 0.62 0.60 0.91 0.92 1.00 10. A. racemosa 3-6 0.54 0.55 0.54 0.61 0.61 0.59 0.80 0.80 0.80 1.00 11. A. racemosa 3-4 0.54 0.53 0.53 0.62 0.60 0.60 0.83 0.83 0.87 0.86 1.00 12. A. racemosa 3-5 0.55 0.56 0.55 0.62 0.59 0.59 0.83 0.83 0.86 0.87 0.97 1.00 13. A. stricta 5-3 0.56 0.57 0.57 0.51 0.52 0.51 0.57 0.56 0.56 0.53 0.54 0.53 1.00 14. A. stricta 5-1 0.56 0.56 0.56 0.50 0.52 0.50 0.56 0.55 0.56 0.53 0.54 0.53 0.89 1.00 15. A. stricta 5-2 0.57 0.58 0.57 0.51 0.52 0.51 0.56 0.54 0.57 0.54 0.54 0.53 0.92 0.94 1.00 16. A. remotiflora 4-3 0.50 0.51 0.51 0.54 0.53 0.52 0.56 0.56 0.57 0.59 0.58 0.60 0.56 0.55 0.54 1.00 17. A. remotiflora 4-2 0.50 0.51 0.51 0.54 0.53 0.51 0.55 0.56 0.57 0.58 0.58 0.60 0.59 0.58 0.58 0.92 1.00 18. A. remotiflora 4-1 0.51 0.51 0.50 0.53 0.52 0.51 0.55 0.56 0.57 0.58 0.59 0.60 0.59 0.58 0.58 0.88 0.94 1.00 19. A. verticillata 1-1 0.52 0.53 0.53 0.58 0.61 0.59 0.54 0.52 0.54 0.51 0.52 0.50 0.48 0.46 0.46 0.48 0.50 0.50 1.00 20. A. verticillata 1-2 0.45 0.45 0.44 0.54 0.56 0.56 0.51 0.52 0.54 0.48 0.49 0.49 0.47 0.45 0.45 0.48 0.48 0.49 0.81 1.00 21. A. verticillata 1-3 0.46 0.47 0.47 0.52 0.57 0.57 0.49 0.49 0.52 0.45 0.48 0.46 0.46 0.44 0.45 0.44 0.45 0.45 0.80 0.89 1.00 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

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