Korean Journal of Ichthyology, Vol. 29, No. 2, 139-145, June 2017 ISSN: 1225-8598 (Print), 2288-3371 (Online) Received: June 8, 2017 Revised: June 22, 2017 Accepted: June 28, 2017 우리나라미꾸리속 (genus Misgurnus) 알비노개체의미토콘드리아및핵유전자염기서열분석에의한유전적동정 송하윤 문신주 1 김근식 2 방인철 1, * 국립수산과학원중앙내수면연구소, 1 순천향대학교생명시스템학과, 2 한국해양과학기술원동해연구소 Genetic Species Identification by Sequencing Analysis of Nuclear and Mitochondrial Genes for Albino Misgurnus Species from Korea by Ha-Youn Song, Shin-Joo Moon 1, Keun-Sik Kim 2 and In-Chul Bang 1, * (Inland Fisheries Research Institute, National Institute of Fisheries Science, Gapyeong 12453, Republic of Korea; 1 Life Science and Biotechnology, Soonchunhyang University, Asan 31538, Republic of Korea; 2 East Sea Research Institute, Korea Institute of Ocean Science and Technology, Uljin 36315, Republic of Korea) ABSTRACT The spontaneous color mutant, albino individuals of genus Misgurnus, are rarely discovered in Korea and there are difficult to identify morphological species due to lack melanin pigmentation. In this study, we developed a genetic identification method for the species of albino Misgurnus individuals based on phylogenetic analysis by using recombination activating gene 1 (rag1) and cytochrome b (cytb) region of mitochondrial DNA. As a result of molecular phylogenetic analysis, three clades were identified as Misgurnus mizolepis, and M. mohoity. The homology of the cytb sequences of M. mohoity was best match to that of M. mohoity sequences in GenBank database. As a result of species identification of 25 albino Misgurnus individuals based on the phylogenetic tree, the red-eye type was identified as 16 and one. The remaining three individuals were identified as one, and two M. mohoity, respectively. In addition, the five black-eye type individuals were identified as one, three and one M. mohoity. Therefore, this genetic identification method will be an useful techniques for species or hybrid identification in genus Misgurnus. Key words: Loach, albino, Misgurnus, hybrid, molecular markers 서 알비노현상은일반적으로자연적인색소결핍에의해발생 하며 (Lin and Fisher, 2007), 우리나라및해외의다양한어류에 서보고되고있다 (Vielkind et al., 1971; Koga and Hori, 1997; Yoo et al., 2003; Kang et al., 2007; Oh et al., 2010). 이중에서 도최근우리나라에서황금미꾸라지 ( 또는황금미꾸리 ) 로불리 는미꾸리속 (Misgurnus) 알비노개체들이매우낮은빈도로 출현하고있다. 우리나라에서발견되는미꾸리속알비노개체 론 * Corresponding author: In-Chul Bang Tel: 82-41-530-1286, Fax: 82-41-530-1256, E-mail: incbang@sch.ac.kr 들은안구의색을기준으로빨간색과검은색 2가지타입으로분류할수있으며, 이중에서도빨간색안구를가진타입은완벽한백색증 (albinism) 의특징을가지고있어관상어류로가치가높다. 현재까지우리나라에보고된잉어목 (Cypriniformes) 미꾸리과 (Cobitidae) 에속하는미꾸리속어류는미꾸라지 (Misgurnus mizolepis) 와미꾸리 () 2종으로우리나라전국하천의중 하류에진흙바닥이나농수로에서주로서식한다 (Kim et al., 2005). 두종은염색체수 (Kim et al., 1995a), 입수염길이, 체색, 반점의유무등을근거로분류할수있는것으로보고되어있다 (Kim et al., 2005). 하지만외부형태의경우서식하는환경에따라크기와특징이달라질수있으며 139 http://www.fishkorea.or.kr
140 송하윤 문신주 김근식 방인철 (West-Eberhard, 2005; Kerschbaumer et al., 2011), 이로인해종동정이어려운경우가있다. 특히색소결핍에의해발생하는알비노개체는기존의분류방법인체색차이와반점유무를기준으로구분하기에는어려움이있다. 최근외부환경에의해변이가심한외부형질에의존하는전통적인분류학적연구방법의한계를극복및보완하고자다양한분자생물학적기법을사용하고있다 (Kim et al., 2003). 특히유전자의염기서열을확보하여비교분석하는분자계통학적기법이보편적으로이용되고있다 (Slechtova et al., 2007; Sonnenberg et al., 2007; Cebrat et al., 2008; Lee et al., 2009; Yun et al., 2009; Kim et al., 2015). 분자계통학적기법을이용하여동일속내에종을분류하기위해서는적절한분자마커의선택이필수적이다. 핵유전자중하나인 recombination activating gene 1 (rag1) 영역은면역글로빈과 T-세포수용체유전자의재결합에관련된유전자로서염기서열이잘보존되어있고, 진화를잘반영하는것으로알려져있다 (Kim and Bang, 2010). 이와더불어미토콘드리아유전자는일반적으로모계유전을하는특징을가지며, 유전자재조합이일어나지않아점진적인변화를관찰할수있는장점이있다 (Hauswith and Clayton, 1985; Irwin et al., 1991; Howard and Berlocher, 1998). 따라서다양한어류에서핵유전자와미토콘드리아유전자를동시에이용하여분자계통학적분석을수행한바있으며 (Mayden et al., 2008; Lee et al., 2009; Song and Bang 2015), 유전적변이분석 (Song and Park, 2006), 자연잡종의동정 (Yun et al., 2009; Lee et al., 2009; Kim et al., 2015) 등에널리이용되고있다. 따라서본연구는핵유전자인 rag1 영역과미토콘드리아유전자인 cytochrome b (cytb) 영역의염기서열을비교분석함으로써국내하천에분포하는미꾸리속알비노개체의유전적동정을수행하고자하였다. Table 1. Sequence information of primers used for genetic identification in this study Primer name Sequences (5 3 ) Annealing temp. ( ) Recombination activating gene 1 (rag1) rag1 1492F AGY CAR TAY CAY AAR ATG TA 54 rag1 2476R TCC TGR AAG ATY TTG TAR AA 54 Cytochrome b (cytb) cytb 14513F TGR CTT GAA RAA CCA YCG TTG T 55 cytb 15868R GTG GGA GTT ARA ATC TC 55 *Y=C+T, R =A+G genomic DNA를추출하였으며 (Asahida et al., 1996), 추출한 genomic DNA는 NanoDrop ND-1000 (Thermo Scientific Inc., Germany) 을이용하여양을측정하고, 0.8% 아가로즈젤에전기영동후질을확인하여실험에이용하였다. 핵유전자 rag1 영역과미토콘드리아유전자 cytb 영역을 PCR 증폭할수있는프라이머를디자인하기위해 GenBank 와 Ensembl에서이용가능한어류종들의염기서열을확보하였다. 이염기서열들은 BioEdit 프로그램 (ver. 7.1.11; www. mbio.ncsu.edu/bioedit/bioedit.html) 의 ClustalW (Thompson et al., 1997) 를이용하여다중서열정렬을수행한후, 보존성이높은부분의염기서열정보를바탕으로프라이머를제작하였다 (Table 1). 중합효소연쇄반응 (polymerase chain reaction, PCR) 은 genomic DNA 100 ng과각프라이머 5 μm을사용하여 AccuPower PCR Premix (Bioneer Inc., Korea) 로반응하였다. PCR 조건은최초 94 에서 4분간초기열변성반응이후, 94 에서 1분, 55 annealing 온도에서 1분, 72 에서 1 분반응을 35회실시하였으며, 최종 72 에서 5분간반응하였다. 증폭된산물은 1.5% 아가로즈젤상에서전기영동한후에 EcoDye TM solution (BioFact, Korea) 으로염색하여증폭된 PCR 산물의유무를확인하였다. 재료및방법 1. 실험어수집본연구에사용한미꾸리속알비노개체는전라북도완주군봉동읍만경강일대에서채집한 25개체를이용하였다. 실험어눈의색차이를기준으로빨간색눈과검은색눈두가지타입으로구분하였다. 빨간눈알비노개체는 RGL (Red eyed, golden color, loach) 로표기하였으며, 검은눈알비노개체는 BGL (Black eyed, golden color, loach) 로표기하였다. 2. Genomic DNA의분리및 PCR 실험어의지느러미의일부를절단하여페놀추출법으로 3. 염기서열분석및계통도작성증폭된 PCR 산물은 HiGene TM Gel & PCR purification system Kit (BioFact, Korea) 를이용하여정제하였으며, 이후염기서열분석기 (ABI 3730xl DNA Analyzer) 를이용하여염기서열을결정하였다. 확보된염기서열을대상으로 BioEdit 프로그램 (ver. 7.1.11) 을이용하여각각의유전자염기서열을다중배열하여정리하였다 (Thompson et al., 1997). 계통수작성은 MEGA (ver. 6.06) 프로그램을이용하여 Kimura 2-parameter model로 Neighbour joining (NJ) 방법을이용하였고 (Tamura et al., 2013), 비교군으로 GenBank 에등록되어있는국내산미꾸리 () 및미꾸라지 () 염기서열, 일본 (AB473348) 및중국 (EU 131139, JN858850) 산미꾸리속어류의염기서열을비교군으
미꾸리속알비노개체의유전적동정 141 로이용하였고, 외그룹으로새코미꾸리 (Koreocobitis rotundicaudata) 의염기서열을이용하였다 (Table 2). 분자계통도내분지의지지도를통계적으로계산하기위해 1,000회 bootstrap 분석을수행하였다 (Felsenstein, 1981). 분석결과나타난계통별염기서열을 National Center for Biotechnology Information (NCBI) 의 Basic Local Alignment Search Tool (BLAST, http://www.ncbi.nlm.nit.gov/blast) 를이용하여유전자은행에확보되어있는염기서열중가장높은유사성을보이는염기서열을확인하였다. Table 2. Accession number of reference sequences in Genbank for this study Species rag1 GenBank accession No. cytb Misgurnus mizolepis EU670843 EU670767, KF732663 Misgurnus anguillicaudatus EF508651 EU131139, AB473348 Misgurnus mohoity JN858810 JN858850 Koreocobitis rotundicaudata EU670841 EU670765 결과및고찰 본연구에서제작한프라이머를이용하여 PCR을수행한결과확보된 rag1 영역은 828 bp의염기서열을확보하였으며, cytb 영역은 1,121 bp의염기서열을확보하였다. 확보된염기서열을이용하여미꾸리속알비노개체들의 rag1 영역과 cytb 영역의분자계통도를작성한결과두유전자모두 3개의단계통 Fig. 1. Neighbour joining phylogenetic tree by the Kimura 2-parameter model showing phylogenetic relationship among genus Misgurnus species based on recombination activating gene 1 (rag1) sequence data.
142 송하윤 문신주 김근식 방인철 Fig. 2. Neighbour joining phylogenetic tree by the Kimura 2-parameter model showing phylogenetic relationship among genus Misgurnus species based on cytochrome b (cytb) sequence data. 군이형성되었으며 (Figs. 1, 2), Clade Ⅰ은미꾸리 () 계통군으로분리되었고 Clade Ⅲ은미꾸라지 (M. mizolepis) 계통군으로분리되었다. 따라서미꾸라지속알비노개체는단계통군으로뚜렷하게구분이되어 Kim (2009) 의분류체계와동일하게종구분이가능하였다. 하지만 Clade Ⅱ 는국내에보고된미꾸리속어류계통군에포함되지않는별도의계통군을형성하였고, cytb 영역의분자계통도에서더욱뚜렷하게나타남으로써현재국내에분포하는미꾸리와미꾸라지외에미기록종인 M. mohoity가서식하고있는것으로추정되었다. Clade Ⅱ에해당하는개체의염기서열을이용해 Genbank에등록된염기서열과의 BLAST 분석결과미꾸리속어류인 M. mohoity의염기서열과가장높은유사성을나타내었다 (Table 3). 분자계통도를기준으로종동정을수행한결과는 Table 3에 제시하였으며, RGL 20개체중 16개체는미꾸리로판별하였고 1개체는미꾸라지로판별하였다. 나머지 3개체중 RGL 수컷 2번과 3번은 rag1 분자계통도에서미꾸리계통군 (Clade Ⅰ) 에포함되고, cytb 분자계통도는 M. mohoity 계통군 (Clade Ⅱ) 에포함됨에따라 M. mohoity 미꾸리 의교잡종으로판별하였다. 또한 RGL 수컷 4번은 rag1 분자계통도에서미꾸리계통군 (Clade Ⅰ) 에포함되고, cytb 분자계통도는미꾸라지계통군 (Clade Ⅲ) 에포함됨에따라미꾸라지 미꾸리 교잡종으로판별하였다. 한편 BGL 5개체는미꾸리 1개체와미꾸라지 3 개체로판별하였으며, 나머지 BGL 암컷 3번개체는 M. mohoity 로판별하였다. 이상과같이 RGL 개체 20마리중 3마리가잡종으로판별되어 15% 의빈도를보였으나, BGL 개체 5마리중잡종개체는전혀확인되지않았다. 추후샘플수를늘려분석한다면 BGL 잡종개체의확인이가능할것으로판단된다.
미꾸리속알비노개체의유전적동정 143 Table 3. Result of genetic identification of albinism genus Misgurnus species Type RGL BGL No. of fish Species categorizations Species Best-matched database sequence (GenBank accession No.) Sequence homology (%) rag1 cytb rag1 cytb rag1 cytb 16 AA AA 2 AA MMO Hybrid (OA) 1 AA MM Hybrid (MA) 1 MM MM 1 AA AA 1 MMO MMO M. mohoity 3 MM MM (EU670843) M. mohoity (JN858810) (EU670843) (EU131139, AB473348) M. mohoity (JN858850) (EU670767, KF732663) (EU670767, KF732663) (EU131139, AB473348) M. mohoity (JN858850) (EU670677, KF732663) E-value 98 99 0.0 *RGL: Red eyed golden loach, BGL: Black eyed golden loach, AA: Misgurnus anguillicaudatus, MM:, MMO: M. mohoity, MA: M. anguillicaudatus hybrid, OA: M. mohoity hybrid. A B Fig. 3. Photographs of albinism (A) and leucism (B) fishes from genus Misgurnus species from Korea. 일반적으로백색증현상은멜라닌색소결핍에의해두가지형태로발생하는것으로알려져있으며 (Kinnear et al., 1985), 미꾸리속어류의경우어체에발현되는표현형에따라다음과같이두가지표현형으로구분할수있다. 첫째, RGL 개체는유전자돌연변이에의해표피와망막의멜라닌색소포 (melanophore) 의결핍으로인해완벽한백색증으로표현되는알비노와둘째, BGL 개체와같이발생생리학적문제로인해색소침착이줄어들거나없지만망막은색소침착이이루어진 부분적백색증 (partial albinism) 또는루시즘 (leucism) 으로구분하고있다 (Kinnear et al., 1985). 이와같이비정상적인피부색소침착과함께안구에정상적인색소침착이나타나는경골어류를루시즘 (leucism and leucistic phenotype) 으로일반적인알비노개체들과구분하고있다 (Clark, 2002; Veena et al., 2011; Muto et al., 2013). 따라서 BGL 개체는눈을제외하고전체신체표면에색소침착이없는루시즘표현형이발현되는개체로서일반적인백색증을가지는 RGL 개체와구분되었
144 송하윤 문신주 김근식 방인철 다 (Fig. 3). 루시즘은유전변이, 서식지오염및내교배 (inbreeding) 등이발생원인으로알려져있으나 (Veena et al., 2014), 포획한후실내에서사육시일정시간이경과되면피부에색소침착이이루어진것으로보아유전적돌연변이인백색증현상과는뚜렷하게구분되었다. 한편종간잡종화는인위적인방류, 산란시기의일치, 산란장소의부족현상등의다양한이유로발생하고있다 (Jansson et al., 1991; Kuriiwa et al., 2007). 이중미꾸리과어류는자연상태에서도종간잡종이일어난사례가보고되어있으며 (Lee et al., 2009; Kwon et al., 2014), 미꾸리속어류인미꾸리와미꾸라지의경우자원조성, 방생등의이유로무분별하게국내하천생태계에이입되고있는실정이다 (Kim et al., 1995b; Park et al., 1997). 특히최근중국에서생산또는포획한미꾸리속어류를국내에지속적으로수입하고있어국내하천생태계에이입될확률또한높다. 이로인해현재까지기록되지않은 M. mohoity가출현한것으로추정된다. 따라서추후 M. mohoity에대한표본의추가확보및원기재논문검토등을통한정확한종동정과유입경로파악등에대한후속연구가필요할것으로판단된다. 일반적으로미꾸라지와미꾸리는입수염길이에서종간차이를보이며, 미꾸라지는가장긴입수염이눈지름의약 4 배정도로긴반면에미꾸리는 2.0~2.5배정도로미꾸라지에비해상대적으로짧아두종간구분이가능하다 (Kim et al., 2005). 그러나두종간에유도된잡종의경우입수염길이의차이가명확하게구분이되지않는한계점이존재한다 (Park et al., 1997). 미꾸리속알비노개체의경우에는체색과반문이발현되지않을뿐만아니라잡종개체도나타남으로써외부형질에기반을두는종동정방법과함께본연구에서이용한분자마커및분자계통학적분석을추가할시보다정확하게종동정을할수있을것으로판단된다. 요약 최근우리나라에서자연발생적인미꾸리속알비노개체들이낮은빈도로출현하고있으나, 색소결핍으로인해형태적종동정이어렵다. 따라서본연구에서는핵유전자인 recombination activating gene 1 (rag1) 영역및미토콘드리아유전자 cytochrome b (cytb) 영역을이용한분자계통학적분석을이용해미꾸리속알비노개체의분자동정을수행하였다. 그결과 rag1과 cytb의분자계통도에서미꾸라지, 미꾸리, 그리고 M. mohoity로 3개의 clade가확인되었다. 확보된 M. mohoity 의염기서열을유전자은행의 BLAST를이용해유사성을검색한결과 M. mohoity와가장유사하였다. 분자계통도를기준 으로 25마리의알비노미꾸리속개체의종동정을수행한결과빨간눈타입은미꾸리 16마리, 미꾸라지 1마리로판별되었고, 나머지 3개체는미꾸라지 미꾸리 잡종 1마리와 M. mohoity 미꾸리 잡종이 2마리판별되었다. 또한검은눈타입 5마리는미꾸리 1마리와미꾸라지 3마리및 M. mohoity 1마리로판별되었다. 따라서본연구에이용한분자마커를활용함으로써미꾸리속어류의정확한종또는잡종을동정하기위한유용한방법으로이용될수있을것이다. References Asahida, T., T. Kobayashi, K. Saitoh and I. Nakayama. 1996. Tissue preservation and total DNA extraction from fish stored at ambient temperature using buffers containing high concentration of urea. Fish. Sci., 62: 727-730. Cebrat, A., A. Cebula, A. Laszkiewicz, M. Kasztura, A. Miazek and P. Kisielow. 2008. Mechanism of lymphocyte-specific inactivation of RAG-2 intragenic promoter of NWC: Implications for epigenetic control of RAG locus. Mol. Immunol., 45: 2297-2306. Clark, S. 2002. First report of albinism in the white spotted bamboo shark, Chiloscyllium plagiosum (Orectolobiformes: Hemiscyllidae), with a review of reported color aberrations in elasmobranchs. Zoo. Biol., 21: 519-524. Felsenstein, J. 1981. Evolutionary trees from DNA sequences: a maximum likelihood approach. J. Mol. Evol., 17: 368-376. Hauswith, W.W. and D.A. Clayton. 1985. Length heterogeneity of a conserved displacement-loop sequence in human mitochondrial DNA. Nucl. Acids. Res., 13: 8093-8104. Howard, D.J. and S.H. Berlocher. 1998. Endless forms: species and speciation. Oxford Univ. Press., 470pp. Irwin, D.M., T.D. Kocher and A.C. Wilson. 1991. Evolution of cytochrome b gene of mammals. J. Mol. Evol., 32: 128-144. Jansson, H., I. Holmgren, K. Wedin and T. Andersson. 1991. High frequency of natural hybrids between Atlantic salmon, Salmo salat L., and brown trout S. trutta L., in a Swedish river. J. Fish Biol., 39: 343-348. Kang, D.Y., H.C. Kim, J.H. Kim, K.G. Kim and J.I. Myeong. 2007. Effects of environment factors on the occurrence of pseudo-albinism in cultured flounder, Paralichthys olivaceus. Korean J. Ichthyol., 40: 234-242. (in Korean) Kerschbaumer, M., L. Postl, M. Koch, T. Wiedl and C. Sturmbauer. 2011. Morphological distinctness despite large-scale phenotypic plasticity-analysis of wild and pond-bred juveniles of allopatric populations of Tropheus moorii. Naturwissenschaften, 98: 125-134. Kim, D.S., Y.K. Nam and I.S. Park. 1995a. Survival and karyological analysis of reciprocal diploid and triploid hybrids between mud loach (Misgurnus mizolepis) and cyprinid loach (Misgurnus anguillicaudatus). Aquaculture, 135: 257-265. Kim, D.S., Y.K. Nam and I.S. Park. 1995b. Performance of diploid
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