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Weed Turf. Sci. 4(3):225~229 http://dx.doi.org/10.5660/wts.2015.4.3.225 Print ISSN 2287-7924, Online ISSN 2288-3312 Research Article Weed & Turfgrass Science Weed & Turfgrass Science was renamed from both formerly Korean Journal of Weed Science from Volume 32 (3), 2012, and formerly Korean Journal of Turfgrass Science from Volume 25 (1), 2011 and Asian Journal of Turfgrass Science from Volume 26 (2), 2012 which were launched by The Korean Society of Weed Science and The Turfgrass Society of Korea founded in 1981 and 1987, respectively. 국내에유입되는열대피 (Echinochloa colona) 동정 : DNA 바코드중심 이정란 * 김창석 이인용국립농업과학원 Discrimination of Echinochloa colona (L.) Link from other Echinochloa Species using DNA Barcode Jeongran Lee*, Chang-Seok Kim, and In-Yong Lee National Academy of Agricultural Science, Iseo 565-852, Korea ABSTRACT. Echinochloa colona is one of the most problematic weeds in the paddy fields of the world. In recent years, this species is likely to be introduced in Korea due to global warming, the expansion of international trade including agricultural products, and increasing tourists. We tried to identify the species from Korean Echinochloa crus-galli and E. oryzicola in order to establish the control measures in case of the initial influx. For this study, Echinochloa colona collected from the National Plant Germplasm System, USA were examined and E. crus-galli and E. oryzicola were collected in Korea. It is, however, very difficult to identify for Echinochloa species using morphological characters because of numerous interspecific and intraspecific types found in nature. Thus, we barcoded the species using rbcl, matk, and ITS. All three markers identified E. colona very well from the others. ITS alone may be enough as a DNA barcode for E. colona identification, when considering cost and effectiveness. The barcode sequences were deposited to the National Center for Biotechnology Information database for public use. Key words: DNA barcode, Echinochloa colona, E. crus-galli, E. oryzicola, Identification Received on August 07, 2015; Revised on September 08, 2015; Accepted on September 10, 2015 *Corresponding author: Phone) +82-63-238-3322, Fax) +82-63-238-3838; E-mail) kongsarang@korea.kr 2015 The Korean Society of Weed Science and The Turfgrass Society of Korea This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 서론 외부형태적으로뚜렷한형질이없어동정하기매우어려운돌피속 (Echinochloa (L.) P. Beauv.) 식물은전세계적으로약 30~40여종이온대, 아열대, 열대에골고루분포하는것으로알려져있다 (Clayton and Renvoize, 1999). 돌피속은소수의형태와서식지등의특징으로주로동정해왔으나이런형태적특징은종간, 종내변이가매우심하여많은나라에서본속의종동정에각기다른동정체계를따르고있다 (Mennan and Kaya-Altop, 2012). 따라서돌피속의일부종들은여전히학명까지혼동되어사용되고있다. 그중돌피 (E. crus-galli (L.) P. Beauv), 열대피 (E. colona (L.) Link), 논피 (E. oryzicola (Vasinger) Vasinger) 등은특히논에서악성잡초로알려져있다. 이런잡초들은벼와닮은형태, 매우빠른발아력, 신속한성장력, 풍부한종자생산력과같은광범위한생태적내성에의해과거 60여년이상악성잡초로문제시되어왔다 (Barrett, 1983). 국내에는그동안문헌기록에의하면열대피와대만피 (E. glabrescens Munro ex Hook. f.) 를포함하는 8분류군의돌피속식물이분포하는것으로알려져왔으나 (Lee et al., 2013) 최근에한국, 중국, 인도, 필립핀, 미국등의돌피속식물을핵 DNA internal transcribed spacer (ITS), External transcribed spacer (ETS) 와엽록체 DNA trnl intron, trnl- F intergenic spacer, matk의염기서열을이용한연구결과와 225

226 이정란 김창석 이인용 simple sequence repeat을이용한분자계통학적분류결과, 국내에분포하는돌피속식물은돌피, 피 (E. esculenta (A.Braun) H.Scholz), 논피, 나도논피 (E. oryzoides (Ard.) Fritsch) 4종인것으로보고되었다 (Lee et al., 2014b; Lee et al., 2014c). 최근에는기후변화및국제무역의확대, 특히농산물교역량의증가와여행자의증가등의영향으로의도하지않은병, 해충, 잡초등이국내로유입되어국내에새로운문제병, 해충, 잡초가되는경향이있다. 특히잡초의경우, 곡물의수입에의해잡초종자가혼입되어국내에유입되어항만, 사일로, 곡물수송도로부근과축산농가주변에서발견되는경우가있다 (Oh et al., 2002). 최근에는국제결혼과외국관광객의증가에의해새로운수요가되고있는아열대채소의재배농가주변에서도비의도적도입에의해발견될수있을것으로추정된다. 열대아시아원산의열대피는세계 3대악성잡초중의하나로돌피속잡초중에국내에유입될경우지구온난화로더욱따뜻해진국내기후에적응이잘될가능성이매우높다 (Holm et al., 1977). 열대피는일단정착이되면돌피, 논피와더불어국내논에서악성잡초가될가능성이높을뿐만아니라, 제초제에저항성을갖게될가능성역시매우높기때문에국내에도입되기전에종을쉽게동정할수있는방법을제시하여도입시초기방제에활용할수있어야한다. 최근에 Hebert et al. (2003) 에의해제안되어생물다양성협약 (Convention on Biodiversity) GTI 9조및 13조에서권고하는표준종동정방법인 DNA 바코드는돌피속과같이형태적으로분류가어려운종을비교적짧은시간에객 관적으로동정할수있는강력한대안으로이용되고있다. 외국원산열대피와, 국내논피, 돌피를 DNA 바코드하고그정보를데이터베이스화하여열대피가국내에도입될경우빠르게동정에활용할수있도록본연구를수행하였다. 재료및방법 식물재료, DNA 추출, PCR 증폭과염기서열분석본연구에이용된열대피는미국, 인도, 이스라엘, 에디오피아, 남아프리공화국원산의 6종이었고, 국내에서채집한논피 2점, 외부형태가다양한돌피 4점이포함되었다. 열대피는미국 National Plant Germplasm System (NPGS) 에서종자를분양받아국립농업과학원온실에서파종하여식물체를얻었으며, 돌피와논피는국내에서직접수집하였다 (Table 1). 본연구에이용한식물재료의확증표본은국립농업과학원식물균류표본보존센터 (HCCN) 에보관하였다. 전체게놈 DNA는종자를파종후 4~5엽기에생체를채집하여 Genomic DNA isolation kit (NucleoGen, Germany) 를이용하여동봉되어있는프로토콜에따라추출하였으며, 추출한 DNA는 Nanodrop ND-1000을이용하여 DNA의농도와순도를측정한후 25 ng μl -1 로맞추어 PCR 증폭에이용하였다. PCR 증폭반응과증폭온도는 Lee et al. (2014a), 바코드 primer는 Lee et al. (2015) 에따랐다. 바코드부위의염기서열분석은 GenoTech (Dajeon, Korea) 에서수행하였다. 바코드구간염기서열분석및계통수작성분석된염기서열은 Lasergen 8.1.5 (CodonCode Corp, Table 1. The plant list and NCBI GenBank numbers deposited in this study. Species Passport Origin NCBI GenBank No. ITS matk rbcl E. colona PI292598 Israel KR706351 KT365299 KT365292 E. colona PI331385 Ethiopia KR706352 KT365300 KT365293 E. colona PI364794 South Africa KR706353 KT365301 KT365294 E. colona PI463640 India KR706354 KT365302 KT365295 E. colona PI463829 India KR706355 KT365303 KT365296 E. colona PI491424 USA KR706356 KT365304 KT365297 E. oryzicola E46 Korea KC164280 KT365309 KT365290 E. oryzicola E55 Korea KF010187 KT365310 KT365291 E. crus-galli var. echinata PB-159 Korea KF163624 KF163726 KF163507 E. crus-galli var. crus-galli PB-170 Korea KF163621 KF163722 KF163504 E. crus-galli var. crus-galli PB-187 Korea KF163622 KF163723 KF163505 E. crus-galli var. echinata PB-240 Korea KF163625 KF163724 KF163506

국내에유입되는열대피 (Echinochloa colona) 동정 : DNA 바코드중심 227 Dedham, MA, USA) 를이용하여 contig를작성하고, contig 들은 Clustal W를이용하여정렬한후육안편집을거쳤다. 종간염기서열차이는육안으로확인하였으며 MEGA 6.06 프로그램으로 Kimura-2-parameter distance model을적용하여 Neighbor-joining 계통수를작성하여종을구별하였다 (Saitou and Nei, 1987; Tamura et al., 2013). 바코드염기서열은미국의국립생물공학정보센터 (National Center for Biotechnology Information) 에제출하여 GenBank 번호를부여받았다. 결과및고찰 열대피의형태적특징문헌에의하면열대피는총상화서 (raceme) 에달린소수가가지런하게 4줄로배열되어있으며소화경의길이가 0.7-2(4) cm로 2차소화경은없고, 소수의길이는 2-3 mm로돌피속의돌피, 논피, 피등에비하여작은것으로기록되어있다 (Chen and Peterson, 2006; Michael, 2007). 그러나이러한형태적특징은종간, 종내잡종에의해중간타입이많이관찰되어외부형태적형질에기초하여종을동정하는데는어려움이많기때문에객관적인동정법이필요한현실이다. 실제로인천항, 전남곡성의열대작물재배농가주변에서외부형태적특징이모호하여열대피로의심되는종들을채집하였으나 DNA 바코드결과돌피와같은종으로동정되었다 (data not shown, provided upon request). DNA 염기서열분석과변이각마커별바코드염기서열의 NCBI GenBank에제출하여부여받은번호는 Table 1과같으며 GenBank 번호로분석된염기서열은검색가능하다. 전분류군의분석된구간의길이는전종에서 rbcl과 matk는염기서열이각각 1096 bp와 1020 bp로같은길이로분석되었으나, ITS는열대피는 585 bp로, 돌피와논피는 588 bp로분석되었다. 고등식물의표준바코드구간인엽록체 DNA의 rbcl 유전자로분석한결과열대피와국내돌피, 논피와는분석된염기서열 1096 bp 중에 744번째염기서열 A가 T로, 774번 째염기서열 T가 C로, 858번째염기서열 A가 T로, 984번째염기서열 T가 C로의치환이발견되어 0.36% 의치환이있었다. 또한, 137번째염기서열 A가 T로치환된논피특이염기서열이발견되었다 (Table 2). rbcl과마찬가지로표준바코드구간이며모계유전하는유전자부위인 matk에서는 33번째염기서열 C가 T로, 348 번째염기서열 A가 T로, 362번째염기서열 T가 C로치환되어열대피특이치환은 0.29% 로나타났다 (Table 2). 특히 matk에서논피는 435번째염기서열 A가 G로, 774번째염기서열 A가 T로, 1012번째염기서열 C가 T로치환되어논피를돌피로부터구분해낼수있었으며, 253번째염기서열 T에서 G로의치환은돌피에서만관찰되어돌피특이염기서열로구분되었다. 이는 rbcl과 matk 조합은바코드갭이매우낮아적절하게종을동정하기어려운문제점이있다는 Lahaye et al. (2008) 의보고와달리돌피속은고등식물의표준마커만으로도충분히종을동정할수있어서, matk는분류군에따라바코드에이용하는것을고려해야한다는 Kress and Erickson (2007) 과 Lee et al. (2015) 등의보고와같은결과를얻었다. 모계에서유래한 cpdna의 rbcl과 matk 유전자의염기서열을이용하여분석하였을때돌피와논피가구분되었던특이점은아마도잡종의경우모계종으로동정이될수있다는보고와일치하는것으로생각된다 (Chase and Fay, 2009). 국내의돌피, 논피와달리열대피는종내변이도 0.06% 발견되었다. 양친유래마커즉, 핵 DNA 유전자의경우다수의카피로존재하거나종종클로닝을해야하는번거로움이있기때문에표준마커로서이용이어려운단점이있음에도불구하고, rbcl+matk가분류군에따라종식별력이떨어질수있기때문에핵 DNA의 ITS 구간은표준바코드부위와함께바코드분석에이용되면보완이될것이라는많은주장이있다 (Chase and Fay, 2009; Chen et al., 2010; China Plant BOL Group, 2011; Kress and Erickson, 2007). 5.8S를포함하는 ITS는전체길이가 rbcl이나 matk에비하여매우짧기때문에단한번의 PCR로전체길이를증폭할수있는장점이있어바코드의효율성을고려하기위하여본분석에포함시켰다. 본분석에이용된 ITS 구간은 2개의 Table 2. Characters identifying Echinochloa colona from E. crus-galii and E. oryzicola. rbcl (0.36%)* matk (0.29%) ITS (3.2%) 744** 774 858 984 33 348 362 27 45 63 82 85 87 88 91 92 173 181 375 396 401 402 403 436 481 521 539 542 576 E. colona T C C C T T C T C C - A G A A A T T A A T - - C C C A T T E. crus-galli A T G A C A T C T T T C A G G G C G C C G T A T A T T A C E. oryzicola A T G A C A T C T T T C A G G G C G C C G T A T A T T A C *percentage of E. colona-specific nucleotide substitution. **indicate the nucleotide numbers sequenced using each barcode marker.

228 이정란 김창석 이인용 primer에의해간단하게 PCR 증폭과염기서열분석이가능하였으며분석된구간은 ITS1, 5.8S ribosomal DNA와 ITS2 가포함되었다. 분석된 ITS1 구간은 207 bp 이었으며 1개의삽입또는결실부위와 10 bp의염기서열치환이조사되었다. 163 bp의 5.8S ribosomal DNA 구간은전분류군에서 100% 동일한염기서열이보존되어있었다. 염기서열변이가가장높은것으로알려진 ITS2 구간에서는 2bp의삽입또는결실부위가발견되었고, 9 bp의염기서열치환이조사되었다. 그러므로 ITS 구간전체에서열대피는국내의돌피, 논피와비교하였을때 3bp의삽입또는결실부위와 19 bp의염기서열치환이조사되어분석된 589 bp의 3.2% 의염기서열치환율을나타냈다 (Table 2). 논피또는돌피특이염기서열은발견되지않았다. Fig. 1. Neighbor-joining dendrograms derived from rbcl (A), matk (B), ITS (C), rbcl+matk (D) and rbcl+matk+its (E) sequences. Echinochloa colona species are well separated from the other species of Echinochloa in all dendrograms. Neighbor-joining 계통수에의한종동정고등식물표준바코드마커와 ITS 염기서열을이용하여종간 pairwise sequence divergence를산출하였으며이를기초로 Neighbor-joining (NJ) 계통수를작성하였다 (Fig. 1). 본연구에이용된세마커는모두열대피와국내의논피, 돌피를구분할수있는바코드갭 ( 평균 1.36%) 을갖는것으로나타났다. rbcl, matk, ITS의단일 NJ 계통수와이들조합의계통수모두열대피를국내의논피, 돌피와구분하는데성공하였다. 열대피는 DNA 바코드로빠른시간내에정확하게종동정이가능하기때문에국내에서열대피로의심되는생태형이발견될경우, 바코드를활용하면정확한종동정을할수있다. 특히비교적긴구간을분석해야하는 cpdna의 rbcl과 matk 이외에비교적짧은부위인핵 DNA의 ITS만으로도열대피의정확한동정이가능하다. 그러므로열대피로의심되는생태형을발견할경우 ITS 부위를단독으로이용하면쉽고빠르고낮은비용으로정확하게동정할수있을것으로생각한다. 농사에있어서잡초의오동정은정확한방제법을놓칠수있기때문에잡초관리에있어서정확한종동정은중요한이슈가되고있다 (Mennan and Kaya-Altop, 2012). 종에따라어떤종들은특히정확한동정이필요할수있다. 예를들면, 캐나다온타리오남서부지방에자생하는 3종의비름속 (Amaranthus L.) 잡초중 A. retroflexus L. 와 A. powell S. Wats 는 atrazine 저항성이지만 A. hybridus L. 는감수성이었다 (Warwick and Weaver, 1980). 이런경우각종에따라다른관리법이필요하므로정확한종동정은농업적으로매우중요하다. 특히피속잡초는연속변이를포함하는형태적특징들때문에정확한종동정이매우어려운실정이다. 그러므로 DNA 바코드를이용한동정은매우유용한대안이될수있다.

국내에유입되는열대피 (Echinochloa colona) 동정 : DNA 바코드중심 229 요약 열대피와같은논농사지역의악성잡초는국내에유입될경우문제잡초로정착될가능성이높기때문에유입되는초기에방제가될수있도록정확한동정이되어야한다. 그러나피속잡초는형태적으로연속변이가많이존재하여종간구별이매우어려운잡초이다. 본연구는미국 NPGS 에서분양받은열대피와국내에서채집한돌피와논피를고등식물표준바코드마커 rbcl과 matk를이용하여바코드하고, 추가적으로핵 DNA ITS 부위를바코드하여표준바코드구간과 ITS 구간의열대피를동정할수있는능력과바코드활용성을비교하였다. 바코드결과, rbcl은 0.36%, matk는 0.29%, ITS는 3.2% 의열대피특이염기서열이조사되었고 Neighbor-joining 계통수에서종별유집이뚜렷하게나타나표준바코드마커와 ITS 모두쉽고간편하게열대피를국내의돌피, 논피와동정할수있었다. 특히 ITS는분석구간은짧지만열대피를국내의논피, 돌피와정확하게구분해낼수있어서 ITS 단독으로국내에유입되는열대피동정에활용할수있을것으로생각한다. 주요어 : 열대피, 논피, 돌피, DNA 바코드, 동정 Acknowledgment This research was supported by a project from the Cooperative Research Program for Agricultural Science & Technology Development (Project No. PJ009962) of the RDA. References Barrett, S.C.H. 1983. Crop mimicry in weeds. Econ. Bot. 37:255-282. Chase, M.W. and Fay, M.F. 2009. Barcoding of plants and fungi. Sci. 325:682-683. Chen, S. and Peterson, P.M. 2006. Poaceae (Gramineae). Vol. 22, Poaceae (Graminae), pp. 515-518. In: Wu et al. (Eds.). Flora of China. Science Press, Beijing, China and Missouri Botanical Garden Press St. Louis, USA. Chen, S., Yao, H., Han, J., Liu, C., Song, J., et al. 2010. Validation of the ITS2 region as a novel DNA barcode for identifying medicinal plant species. PLoS ONE 5:e8613(8611-8618). China Plant BOL Group. 2011. Comparative analysis of a large dataset indicates that internal transcribed spacer (ITS) should be incorporated into the core barcode for seed plants. P. Natl. Acad. Sci. USA. 108:19641-19646. Clayton, W.D. and Renvoize, S.A. 1999. Genera graminum, grasses of the world. London: Royal Botanic Gardens, Kew. pp. 280-281. Hebert, P.D.N., Cywinska, A., Ball, S.L. and Waard, J.R.d. 2003. Biological identifications through DNA barcodes. P. R. Soc. London, Series B. 270:313-321. Holm, L.G., Plucknett, D.L., Pancho, J.V. and Herberger, J.P. 1977. The world s worst weeds: Distribution and biology. The University Press of Hawaii, Honolulu, USA. pp. 32-46. Kress, W.J. and Erickson, D.L. 2007. A two-locus global DNA barcode for land plants: the coding rbcl gene complements the non-coding trnh-psba spacer region. PLoS ONE 2:e508. Lahaye, R., Bank, M.v.d., Bogarin, D., Warner, J., Pupulin, F., et al. 2008. DNA barcoding the floras of biodiversity hotspots. P. Natl. Acad. Sci. USA. 105:2923-2928. Lee, J., Kim, C.S. and Lee, I.Y. 2015. Molecular identification of Pooideae, Poaceae in Korea. Weed Turf. Sci. 4:18-25. (In Korean) Lee, J., Kim, C.S. and Lee, I.Y. 2014a. Evaluating the discriminatory power of DNA barcodes in Panicoideae, Poaceae. J. Agric. Sci. Technol. B 4:533-544. Lee, J., Kim, C.S. and Lee, I.Y. 2014b. Taxonomic review of the genus Echinochloa in Korea (I): inferred from sequences of cpdna and nrdna. Weed Turf. Sci. 3:183-189. Lee, J., Kim, C.S. and Lee, I.Y. 2014c. Taxonomic review of the genus Echinochloa in Korea (II): inferred from simple sequence repeats. Weed Turf. Sci. 3:190-195. Lee, J., Kim, C.S. and Lee, I.Y. 2013. Identification of Echinochloa oryzicola (Vasinger) Vasinger and E. oryzoides (Ard.) Fritsch in Korea. Kor. J. Pla. Tax. 43:56-62. (In Korean) Mennan, H. and Kaya-Altop, E. 2012. Molecular techniques for discrimination of late watergrass (Echinochloa oryzicola) and early watergrass (Echinochloa oryzoides) species in Turkish rice production. Weed Sci. 60:525-530. Michael, P.W. 2007. Echinochloa P. Beauv. Vol. 25, Magnoliophyta: Commelinidae (in part): Poaceae, part 2. pp. 390-403. In: Barkworth et al. (Eds.). Flora of North America, North of Mexico. Oxford University Press, New York, USA. Oh, S.M., Kim, C.S., Moon, B.C. and Lee, I.Y. 2002. Inflow information and habitat current status of exotic weeds in Korea. Kor. J. Weed Sci. 22:280-295. (In Korean) Saitou, N. and Nei, M. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4:406-425. Tamura, K., Stecher, G., Peterson, D., Filipski, A. and Kumar, S. 2013. MEGA6: Molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30:2725-2729. Warwick, S.I. and Weaver, S.E. 1980. Atrazine resistance in Amaranthus retroflexus (redroot pigweed) and Amaranthus powellii (green pigweed) from southern Ontario. Can. J. Plant Sci. 60:1485-1488.