Journal of Radiation Industry 4 (2) : 171~177 (2010) 스프레이국화 Argus 와화색돌연변이체간의유전적다형성비교분석 성상엽 1,2 정성진 1 김상훈 1 김욱진 1 이지연 1 김동섭 1 김진백 1 김홍기 2 강시용 1, * 1 한국원자력연구원정읍방사선과학연구소, 2 충남대학교응용생물학전공 Analysis of Genetic Diversity among Spray-Type Chrysanthemum (Dendranthema grandiflorum) Argus and its Flower-Colored Mutants Sang Yeop Sung 1,2, Sung Jin Chung 1, Sang Hoon Kim 1, Wook-Jin Kim 1, Ji Yeon Lee 1, Dong Sub Kim 1, Jin-Baek Kim 1, Hong Gi Kim 2 and Si-Yong Kang 1, * 1 Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup 580-185, Korea 2 Department of Applied Biology, Chungnam National University, Daejeon 305-764, Korea Abstract - The original variety, chrysanthemum cv. Argus and 4 mutant lines, AM1 to AM4 derived from that were used to analyze the relationship between morphological characters and genetic diversity. Morphological characteristics were investigated by using 29 parameters including flower, leaf, stem types, and so on. The genetic relationship was analyzed among the five lines using the AFLP fingerprinting method. A total of 28 EcoRI and MseI primer combinations were used to analyze the genetic relationship to among the five lines using the ABI3130 capillary electrophoresis system. In the morphological evaluation, euclidean distance values ranged from 1.20 to 1.52. In the AFLP analysis, a total of 2085 bands were detected by the 28 primer sets, and 956(45.8%) of them were confirmed as polymorphic bands. Similarity coefficient values ranged between 0.40 and 0.72. The distribution by Euclidean distance and a similarity coefficient between the results of morphological characters and AFLP analysis showed a difference. Key words : Chrysanthemum, AFLP, UPGMA, Similarity coefficient, Euclidean distance, Mutation 서 론 국화는꽃의은은한향과아름다운모양으로세계 3 * Corresponding authors: Si-Yong Kang, Tel. +82-63-570-3310, Fax. +82-63-570-3319, E-mail. sykang@kaeri.re.kr 대절화류로서많은사람들의사랑을받고있다. 국내에서도 2008년기준으로연간 1,030억원의생산액으로장미다음의판매우위를점하고있다. 하지만우리나라는네덜란드등의다국적기업에서만들어낸외국품종을도입하여재배하기때문에종묘비도매년계속해서증가하고있어경영에압박을받는농가가늘고있다. 따라서 171
172 성상엽 정성진 김상훈 김욱진 이지연 김동섭 김진백 김홍기 강시용 다양하고우수한신품종육성을통해국제적경쟁으로부터국내화훼산업을보전해야한다 (Yun et al. 2002). 과거새로운품종의육성은주로교배육종에이루어져왔으며, 최근에는생명공학기법을이용한유전자변형육종방법이시도되고있다. 또한기존품종의형질개량, 중간모본용육종소재확대및유전자풀 (Gene pool) 의확대를위해다양한방법이시도되고있으며, 이중한방법이돌연변이육종법이다. 연구결과에따르면생물의종류나유전자좌에따라다소차이를보이지만보통자연돌연변이의경우유전자좌당 10-5 ~10-7 정도의아주낮은빈도로나타난다 (Kimura and Ohta 1971). 인위적으로변이발생빈도를높이기위하여 EMS (ethyl methanesulfonate) 등의화학돌연변이유기원을처리하기도하지만쉽게분해되며처리과정에서인체에질병및암을유발하는문제점이제기되고있다. 한편지구상에는우주및지각으로부터나오는자연방사선이존재하며자연방사선에의한돌연변이빈도는극히낮다. 이에반해감마선, 이온빔, 전자빔등방사능잔류에대한안전성이검증된다양한방사선을이용한방사선돌연변이육종법은다양한식물종에적정선량을조사함으로써더안전하고단기간에돌연변이품종육성을할수있게하였다 (Sidrak and Suess 1973; Nagatomi et al. 1991; Brenda et al. 1992; Nagatomi et al. 1993, 1997; Mandel et al. 2000a; Misra et al. 2003; Naito et al. 2005; Hiroyasu et al. 2008, 2009; Jia and Li 2008). FAO- IAEA 돌연변이품종데이터베이스 (http://www.mvgs.iaea. org/mvd/default.htm) 에의하면돌연변이육종에의해등록된화훼류는약 600여품종으로, 그중254개가국화품종이며 χ선과 γ선을이용하여 50% 이상이네덜란드와인도에서개발되었다. 최근일본에서도 Imajin, Arajin, Emi-akari, Princess kagawa, Dreaming, Ray Sunrise 등 ion beam과 χ선조사를통한품종개발이보고된바있으며국내에서도조직배양기술과감마선돌연변이육종기술을접목하여국화신품종개발을위한육종연구가보고되고있다 (Park 2005; Park et al. 2007; Lee et al. 2008). 한편돌연변이체간의유연관계를밝히고다양한정보를축척하여돌연변이유전자원의지적재산권을확보하기위해서는변이체간의명확한구분및유연관계분석이나유전표식을확인하는작업이필요하다. 현재까지연구되고있는유전표식으로 RFLP (Restriction Fragment Length Polymorphism), RAPD (Random Amplified Polymorphic DNA), AFLP (Amplified Fragment Length Polymorphism), SNP (Single Nucleotide Polymorphism) 및 EST (Expressed Sequence Tags) 등이개발되어이용되고있으 며, 이러한유전표식은다양한분석을통해그응용의폭이매우광범위하게활용되고있다 (Ulrich et al. 1999; Gagnaire et al. 2009). 또한분석대상종에따라각유전표식이나타내는유전적변이성의정도및응용성은차이를나타내고있기때문에분석목적에따라적합한유전표식을사용할필요가있다. DNA를이용한유전표식에있어AFLP분석은 PCR 기반의 DNA 다형성분석법으로서적용이쉽고, 결과가안정적이며단시간내에전체유전체를대상으로다양한유전자좌를포함하는다량의 polymorphism을획득할수있는장점이있어널리이용되고있다 (Vos et al. 1995). 본연구에서는감마선조사에유도된 4개의선발화색돌연변이체와원품종간의형태적특성조사와아울러 AFLP 분석을통해유전적차이및유연관계를명확히파악하여유전자원의기초자료로활용함과동시에방사선조사에의한신품종육성의유용성을확인하는것을목적으로하였다. 재료및방법 1. 실험재료 Dendranthema grandiforum Argus 를 2005년에줄기의기내배양을통해대량증식하였다. 증식된유식물체에한국원자력연구원저준위감마선조사시설 ( 60 Co) 에서 40 Gy를조사하여 3주간의발근후방사선육종시험장온실에정식하였다. 2006년에서 2007년사이에삽목증식을통해 MV3세대를육성하였다 ( 박 2007). 이중유전적으로고정된 4계통의화색변이계통을선발하여원품종과함께실험재료로사용하였다. 2. 형태적분석안정적인특성평가를위해각계통별총 40주로부터특성조사를실시하였으며, 정상적인생육이이루어지고완전히개화가되어진식물체를이용하였다. 형태적특성조사는생장유형, 꽃의형태, 화반의형태, 설상화및관상화의주된색등을세분화하여조사를진행하였다. 형태적분석데이터를통계적으로수치화하기위하여각조사특성에따른차이를 5단계로나누어원품종 Argus 의특성을 3 으로기준하고각계통의수치에따라 1~5 단위로표시하여 matrix code를작성하였다. NTSYS 프로그램 (Rohlf 1992) 을사용하여각개체간의 Euclidean distance를구하고 UPGMA (Unweighted Pair- Group Method using Arithmetic) 로연관관계를분석하였
국화화색돌연변이체간의다형성분석 173 으며, 이를토대로덴드로그램을작성하여형태적유사도를확인하였다. 3. Genomic DNA의분리및AFLP 분석실험에이용되어진 Genomic DNA는 Doyle and Doyle (1990) 의방법에따라 10 g의어린식물체의잎과줄기로부터분리되었다. 분리되어진 Genomic DNA는흡광광도계측정 (Jenway 6505, Essex, U.K.) 과 0.7% agarose gel 전기영동을통해서 DNA의양과질을확인한후실험에이용하였다. AFLP 분석은 Vos et al. (1995) 와 Meudt and Clarke (2007) 의방법을일부변형하여사용하였다. Genomic DNA 250 ng을 EcoRI/MseI (Promega,USA) 제한효소를이용하여절단시켰다. 절단산물에 EcoRI/MseI adaptor 를첨가하여 ligation 반응을실시하고 preamplification 반응을수행하였다. 반응액을 TE buffer를이용하여 50 배희석하여 5가지의 fluorescent dyes (FAM, HEX, NED, ROX and LIZ) 로라벨되어진 EcoRI/MseI selective primer 를이용하여 selective amplification을수행하였다. 최종 PCR product를 ABI 3130xl genetic analyzer (Applied Biosystems, CA, USA) 를이용하여 capillary electrophoresis 를통해 AFLP분석을수행하였다 (Applied Biosystems, CA, USA). PCR 증폭을포함한모든반응조건은 Analysis system I (Invitrogen, Carlsbad, California) 을참조하여실행하였다. 유전적다형성분석은 100~500 bp에서의 polymorphic peak 유무에따라 0 또는 1로표시하여 matrix code를작성한후, NTSYS 프로그램을사용하여각개체간의유사도 matrix를 UPGMA (Unweighted Pair- Group Method using Arithmetic) 로분석하였고이를토대로덴드로그램을작성하고유전적유사도를확인하였다. 결과및논의 1. 형태적특성조사본연구에서의국화원품종 Argus 는스프레이타입아네모네형으로연분홍빛을띄는흰색계열의설상화와자주색을띄는통상화로크게 2가지색상을띈다 (Table 1). Argus 와변이체의줄기와잎의특성을조사한결과화서의형태는산방화서 (corymbiform) 로감마선에의한변화가없었으며, 잎의형태 (rounded) 나기부의주된모양 (rounded), 잎자루의형태 (moderately upwards), 잎의색깔또한조사결과방사선에의한큰변화가없었다. 줄기의색깔에서는 Argus 와변이체 AM3, AM5 에서진한녹색을띠었으며, AM1 은청녹색, AM2 는연한녹색으로약간의차이가나타났다 (Table 2). 잎의가장자리톱니수 (indentations) 에서 Argus 의경우 48개로 AM2 45개와비슷하였으나 AM1, AM3, AM5 는 72~74개로상당한수의차이를나타냈다. 잎의넓이는 Table 1. Morphological characteristics of stem and leaf parts among Argus and its derived 4 mutant lines Lines Plant type Inflorescence form Leaf Petiole Stem Color of Number of Predominant Shape upper side indentations shape of base Attitude Wild-type Argus Non bushy Corymbiform Green 137A Rounded 48 Rounded Moderately upwards Green N199B AM1 Non bushy Corymbiform Green 137C Rounded 72 Rounded Moderately upwards Green 165C AM2 Non bushy Corymbiform Green 137A Rounded 45 Rounded Moderately upwards Green 137D AM3 Non bushy Corymbiform Green 137A Rounded 72 Rounded Moderately upwards Green 199A AM5 Non bushy Corymbiform Green 137B Rounded 74 Rounded Moderately upwards Green 199A Royal Horticultural Society color chart (RHS chart) Color Table 2. Characteristics of floral parts in Argus and its derived 4 mutant lines Lines Flower color Flower bud Color before Color at Natural flowering period Color of outer side anther anther Flower head Ray floret Tubular floret just before opening dehiscence dehiscence type Wild-type Argus Oct E-Nov M Light pink 69C Ruby red 9B 69B 61A 65C Single AM1 Oct E-Nov M Heather violet 72C Raspberry red 77C 72C 59A 72A Single AM2 Oct E-Nov M White pink 69D Ruby red 9B 69C 59A 65D Single AM3 Oct E-Nov M White pink 69D Sulfur yellow 75B 69C 71A 7B Single AM5 Oct E-Nov M Light pink 69C Traffic red N74C 69C N79A 84C Single E, M and L: early, mid and late of a month, respectively Royal Horticultural Society color chart (RHS chart)
174 성상엽 정성진 김상훈 김욱진 이지연 김동섭 김진백 김홍기 강시용 Table 3. Measured morphological characteristics in Argus and its derived 4 mutant lines Lines Plant height (cm) Leaf Flower Inflorescence Length Tubular width at Stipule Ray floret Width Length of including size (mm) Diameter Height floret widest point peduncle (cm) petiole (cm) (cm) (mm) Length Width Length (mm) (cm) (mm) (mm) (mm) Wild-type Argus 51.0±3.62 4.6 10.5 11 7 4.2 14 20 8 14 54 AM1 49.2±3.75 4.3 7.3 10 3 3.8 14 18 7 14 40 AM2 51.0±4.23 5.1 7.3 12 4 4.8 17 22 7 17 45 AM3 46.5±6.51 4.2 11.0 10 2 3.7 15 16 8 8 53 AM5 62.3±10.75 3.5 9.1 12.5 3 3.9 13 16 7 14 54 A B C D E Fig. 1. The flower features of chrysanthemum [Dendranthema grandiforum (Ramat) Kitam] Argus and mutants. A, Argus; B, AM1; C, AM2; D, AM3; E, AM5. Argus, AM1, AM3 이 4.2~4.6 cm로유사하였으며, AM2 가 5.1로가장크고 AM5 가 3.5로가장작았다. 식물체초장의길이는 AM5 가 62.3±10.7 cm로가장컸으며, Argus, AM1, AM2 는대부분 50~51 cm로
국화화색돌연변이체간의다형성분석 175 1.52 1.44 1.36 1.28 1.20 Euclidean distance Fig. 2. Phylogenetic tree between original variety, Argus and its derived four mutants from the UPGMA clustering analysis based on the morphological data. 유사하고, AM3 은 46.5±6.4 cm로가장작게나타났다 (Tables 1, 3). 개화특성은차이가없었으며, 꽃의형태에서도큰차이가없었다. 화색에있어서는설상화 (ray floret) 와통상화 (tubular floret) 모두에서변화가있었는데 AM1 의경우설상화에서 heather violet으로, AM3 은통상화가 sulfur yellow로가장확연한차이가있었다. AM2 와 AM3 의통상화에서는분홍빛이또렷해지는것을관찰할수있었다. 그외에도전반적인화색의변화가있었다 (Table 2, Fig. 1). 형태적특성조사의결과를정도에따라 1 ~ 5 로표시하여 matrix code를작성한후, NTSYS 프로그램을사용하여각개체간의 Euclidean distance를이용하여덴드로그램을작성하고 형태적유사도를확인한결과, AM2, AM5 가 Euclidean distance에서 1.20로높았으며, AM3 은가장낮은것을확인할수있었다 (Fig. 2). 그리고 Argus 를기준으로 AM1, AM2, AM5 가한그룹을이루고, AM3 은형태적으로동떨어져있음을확인할수있었으며, Euclidean distance는 1.52에서 1.20으로분포됨을확인할수있었다 (Fig. 2). 2. AFLP 분석 AM1 AM2 AM5 WT AM3 형태적특성조사를통해서원품종 Argus 와변이체는잎, 줄기와꽃에서변이가확인되었다. 생물의형태적차이는그종이보유하고있는염색체 DNA 염기서열의차이와밀접한관련이있으며, 따라서 DNA상의차이를확인하는 AFLP분석은돌연변이체간의유연관계를밝히는데중요한수단이라할수있다 (Lee et al. 2002). AFLP 분석은총 28개의 primer 조합이사용되었다 (Table 4). 분석결과 4개의변이체와원품종 Argus 간의 28개의 Table 4. Polymorphic patterns observed from 28 fluorescent labeled AFLP primer combinations among chrysanthemum Argus and its derived 4 mutants lines Primer set No. of No. of Polymorphism total bands polymorphic (%) (EA) bands (EA) E-ACA/M-CAA 179 126 70.4 E-ACA/M-CCA 87 29 33.3 E-ACA/M-CGA 44 6 13.6 E-ACC/M-CAA 204 158 77.5 E-ACC/M-CCA 109 35 32.1 E-ACC/M-CGA 93 27 29 E-ACC/M-CTA 36 13 36.1 E-AGC/M-CAA 163 107 65.6 E-AGC/M-CCA 55 25 45.5 E-AGC/M-CGA 80 20 25 E-AAC/M-CAA 50 9 18 E-AAC/M-CAT 40 10 25 E-AAC/M-CAC 79 24 30.4 E-AAC/M-CCA 17 6 35.3 E-AAC/M-CGA 58 26 44.8 E-AAC/M-CTA 44 11 25 E-AGG/M-CAA 54 36 66.7 E-AGG/M-CAT 78 39 50 E-AGG/M-CAC 78 47 60.3 E-AGG/M-CCA 105 32 30.5 E-AGG/M-CGA 102 35 34.3 E-AGG/M-CTA 36 11 30.6 E-ACG/M-CAA 37 12 32.4 E-ACG/M-CAT 53 20 37.7 E-ACG/M-CAC 44 24 54.5 E-ACG/M-CAA 58 24 41.4 E-ACG/M-CGA 38 27 71.1 E-ACG/M-CTA 64 17 26.6 Total 2085 956 - Mean 74.5 34.1 40.8 primer 조합에서전체 2,085 개의밴드가확인되었고, primer 당평균밴드수는 74.5 개였다. 다형성밴드는총 956 개, 평균 34.1개의특이적밴드가확인되었다. Primer E- ACA/M-CAA조합에서최대 179개의전체밴드와 126 개의다형성밴드가확인되었으며, E-AAC/M-CCA조합에서 17개의전체밴드와 6개의다형성밴드가확인되어최소값을보여주었다. Polymorphism은 E-ACA/ M- CAA조합에서 70.4% 로최대값을보였으며, E-ACA/ M- CGA조합에서 13.6% 로최소값, 평균 40.8% 의 polymorphism을보였다. 그리고또한 NTSYS-pc program을이용하여 similarity coefficient를확인하였는데 AM2 와 AM3 이 0.72정도의유연계수가나타나 Argus 보다유전적으로더가까웠다. 또한전체적인유사도는 0.40~ 0.72의범위였는데, Kim et al. (2009) 의무궁화품종구분에서 0.66~0.98의유사도보고와 Lee et al. (2002) 의카네이션근연야생종에서 0.52~0.71의유사도보고가본실험의결과와유사함을확인하였다 (Fig. 3). 형태적특성조사와 AFLP분석결과를바탕으로한
176 성상엽 정성진 김상훈 김욱진 이지연 김동섭 김진백 김홍기 강시용 0.40 0.48 0.56 0.64 0.72 Similarity coefficient 덴드로그램은다른양상를보였다. 그리고 AFLP결과 0.4의유연계수를보였던 AM1 은유전적유사도가가장낮게확인되었는데이는가시적으로확인할수있었던화색의변화가가장높았던변이체로서화색의변화와유전적변이율이일치하는결과를보여주었다. 결 AFLP분석은 genomic DNA를제한효소로절단하여단편의양쪽에 primer와상보적인 adaptor배열을접합하여임의의염기배열을추가한 primer를이용하여 DNA 를증폭시켜그증폭단편의길이의다형을검출하는방법으로 RFLP분석의신뢰성및 PCR의간편성을조합한기술이다. 따라서본연구에서는돌연변이체간의형태적으로명확히구분되는 4개의돌연변이체와원품종을대상으로 AFLP분석을수행하였으며그결과유전적차이및유연관계를명확히파악할수있었다. 한편 AFLP분석에대한 similarity coefficient와형태적분석을통한 Euclidean distance 결과가일치하지않았으며, 환경에영향을많이받는표현형을기반으로한결과보다는환경에거의영향을받지않는유전형에가산한결과가더욱신뢰도가높은것으로판단된다. 향후얻어진돌연변이계통을이용한유전자마커및특이적발현유전자에대한심도깊은추가연구가필요할것으로판단된다. 사 론 사 WT AM3 AM2 AM5 AM1 Fig. 3. Phylogenetic tree between original variety, Argus and its derived four mutants induced by gamma-ray from the UPGMA clustering analysis using the AFLP data. 본연구는 2009 년도농촌진흥청 (RDA) 바이오그린 21 사 업 (Code 20070301034033) 및한국원자력연구원 (KAERI) 주요사업을통해수행되었으며, 그지원에감사드립니다. 참고문헌 Doyle JJ and Doyle JL. 1990. Isolation of plant DNA from fresh tissue. Focus. 12:13-15. Gagnaire PA, Albert V, Jonsson B and Bernatchez L. 2009. Natural selection influences AFLP intraspecific genetic variability and introgression patterns in Atlantic eels. Molecular Ecology 18:1678-1691. Hiroyasu Y, Akemi S, Konosuke D and Toshikazu M. 2008. Effects of dose and dose rate of gamma ray irradiation on mutation induction and nuclear DNA content in chrysanthemum. Breeding Sci. 58:331-335. Hiroyasu Y, Akemi S, Yoshihiro H, Konosuke D, Atsushi T and Toshikazu M. 2009. Mutation induction with ion beam irradiation of lateral buds of chrysanthemum and analysis of chimeric structure of induced mutants. Euphytica 165: 97-103. Jia CF and Li AL. 2008. Effect of gamma radiation on mutant induction of Fagopyrum dibotrys Hara. Photosynthetica 46(3): 363-369 Kim SH, Kim WJ, Lee WJ, Song HS, Kim DS, Kim JB and Kang SY. 2009. Genetic relationship of Hibiscus syriacus L. clarified by AFLP & morphological evaluation. J. Kor. Soc. Hort. Sci. 50(6):555-565. Kimura M and Ohta T. 1971. Protein polymorphism as a phase of molecular Evolution. Nature. 229:467-469. Lee GJ, Chung SJ, Park IS, Lee JS, Kim JB, Kim DS and Kang SY. 2008. Variation in the phenotypic features and transcripts of color mutants of chrysanthemum (Dendranthema grandiflorum) Derived from Gamma ray Mutagenesis. J Plant Biol. 51(6):418-423. Lee SY, Kim KS and Joung HY. 2002. Genetic similarity and cross compatibility of interspecific hybridization in wild species of carnation. J. Kor. Soc. Hort. Sci. 43:355-358. Mandal AKA, Chakrabarty D and Datta SK. 2000a. In vitro isolation of solid novel flower colour mutants from induced chimeric ray florets of chrysanthemum. Euphytica 114:9-12. Meudt HM and Clarke AC. 2007. Almost forgotten or latest practice? AFLP applications, analyses and advances. Trends Plant Sci. 12:106-117. Misra P, Datta SK and Chakrabarty D. 2003. Mutation in flower colour and shape of chrysanthemum morifolium induced by γ-radiation. Biol. Plantarum. 47:153-156. Mueller UG and LaReesa Wolfenbarger L. 1999. AFLP genotyping and fingerprinting. TREE 14:389-394. Nagatomi S. 1991. Flower color mutants derived from floral
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