한수지 49(6), 867-874, 2016 Note Korean J Fish Aquat Sci 49(6),867-874,2016 마루자주새우 [Crangon hakodatei (Rathbun, 1902)] 의전장미토콘드리아유전체에대한분석연구 김경률 1 김현우 1,2 * 1 부경대학교의생명기계전기융합공학협동과정, 2 부경대학교자원생물학과 Complete Mitochondrial Genome of Crangon hakodatei (Rathbun, 1902) (Crustacea: Decapoda: Crangonidae) Gyungryul Kim 1 and Hyun-Woo Kim 1,2 * 1 Interdisciplinary Program of Biomedical, Mechanical and Electrical Engineering, Pukyong National University, Busan 48513, Korea 2 Department of Marine Biology, Pukyong National University, Busan 48513, Korea Although shrimps belonging to family Crangonidae are known to be genetically divergent and ecologically important among the various benthos, any of their mitochondrial genome has not been reported yet. We here determined the complete mitochondrial genome sequence of Crangon hakodatei (Rathbun, 1902), which was collected from East China Sea (124 E and 34.5 N). Total mitochondrial genome length of C. hakodatei was 16,060 bp, in which 13 proteins, 2 ribosomal RNAs, 22 transfer RNAs and a putative control region were encoded. Secondary structure prediction analysis showed that twenty trna genes exhibit the conserved structure but two genes, trna Cys and trna Ser (AGN), lack T and D arm, respectively. Based on the sequence similarity of the COI region from the currently reported five species belonging to genus Crangonidae, C. hakodatei was most closely related to Crangon crangon. Phylogenetic analysis of full COXI genes belonging to infraorder Caridea showed that only crangonid shrimps were clustered together with those of Dendrobranchiata. Gene order were well conserved from Penaeoidea to Caridea but trna Pro and trna Thr in Palaemonid shrimp were flipped each other by the recombination. Further study about mitochondrial genome sequences of shrimps belonging to Crangonidae should be made to know better about their evolutional relationships with other those in infraorder Caridea. Key words: Crangonidae, Decapod crustacean, Barcode, Mitochondrial genome 서론 (Fabricius, 1798) 24 (Hayashi and Kim, 1999). (gastric spine), 3 (maxilliped) (arthrobranch), (pereopod) (merus) 1 (ventral spine) (Hayashi and Kim, 1999). [Crangon hakodatei (Rathbun, 1902)],,, 200 m (Cha et al., 2001; Han and Li, 2015; Hayashi and Kim, 1999). 3 5 (abdominal somites) (dorsomedial carina) (Han and Li, 2015; Maher et al., 2013). C. hakodatei (Han and Li, 2015). http://dx.doi.org/10.5657/kfas.2016.0867 Korean J Fish Aquat Sci 49(6) 867-874, December 2016 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Licens (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Received 8 December 2016; Accepted 19 December 2016 *Corresponding author: Tel: +82. 51. 629. 5926 Fax: +82. 51. 629. 5930 E-mail address: kimhw@pknu.ac.kr Copyright 2016 The Korean Society of Fisheries and Aquatic Science 867 pissn:0374-8111, eissn:2287-8815
868 김경률ㆍ김현우. cytochrome oxidase subunit I (COXI) COI DNA (Folmer et al., 1994), (Lang and Burger, 2007; Ma et al., 2015; Yamauchi et al., 2002). Sanger (Dideoxy termination reaction) (~15 kb), (NGS) (Williams et al., 2014; Zhou et al., 2013)., 2015 5,319 GenBank (http://www.ncbi.nlm.nih.gov). (Matzen da Silva et al., 2011),. PCR C. hakodatei. 샘플링및 DNA 추출 재료및방법 (124 DNA lva. 99% (SK, ) -20%. genomic DNA Tissue Lyser II (QIAGEN). genomic DNA AccuPrep Genomic DNA Extraction Kit (Bioneer, ). DNA -70. MiSeq 시스템을이용한전장미토콘드리아게놈분석 PCR, COXI b (cyt b) PCR. COXI LCO1490 HCO2198 (Folmer et al., 1994) cytb Crus-mt-F10 Crus-mt-R10 (Table 1). IDT SciTools Table 1. The primer sequences used in the study Name Crus-mt-F10 Crus-mt-R10 Crh-F1 Crh-R1 Crh-F2 Crh-R2 Crh-F3 Crh-R3 Crh-F41 Crh-R4 Crh-F5 Crh-R5 Crh-F6 Crh-F7 Crh-R7 M13-F M13-R Sequences (5' 3') GCWATACAYTAYACDGC TTRTTDGGRATDGAHCG CGCCCGCTAAACTAGAAGCAGAG CTCTGCTTCTAGTTTAGCGGGCG CCTCCTCTCTCAGCAGGAATTGCTCAC TAAAGAAGAATGAAGCGCCGTTGGC CTTAGAAATGAGAGCGACGGGCG AGTATGTACACATCGCCCGTCGC CGAACTATTCATGCCAACGGCGC AGCTCCTAGAATGGAAGATACACCAGC GTTAGTATAACCGCGGATGCTGGCAC CCTGCATTATGTTTTGGTGTAAAGGCACG GCTTCATGGTTTACGGCTTGACTTGG GCGACGGGCGATGTGTACATAC CAGGCCGACTACCATGTTCCATCTC GTAAAACGACGGCCAGT GCGGATAACAATTTCACACAGG (http://eu.idtdna.com/analyzer/applications/oligoanalyzer/) (Macrogen, Korea). PCR Gel Extraction Kit (Bioneer, Daejeon, Korea) pgem-t Easy vector (Promega, Madison, WI). DH5d 37. M13 PCR PCR (Table 1). DNA. PCR (Yamauchi et al., 2002) Crh-F2 Crh-R2 Crh-F4 Crh-R4 PCR (Table 1). 2 PCR Covaris M220 (Covaris Inc, Woburn, MA, USA) 600 bp. TruSeq Sample Preparation V2, (Illumina, San Diego, USA). PCR, 2%. 500 bp 700 bp MiniElute PCR (Qiagen, Hilden, Germany). MiSeq System 300 2. 염기서열조합및생물정보학적분석 MiSeq sequencer CLC Ge-
마루자주새우의전장미토콘드리아게놈 869 nomic Workbench v.8.0 (CLC Bio Aarhus, ) QV 20 2. Mothur (v.1.36.1) 7 bp. Geneious R8 (Biomatters, Auckland, New Zealand) 20 bp 100%. C. hakodatei PCR (Table 1). 13 2 rrna mtdna. 22 trna trnascan-se (Lowe and Ward, 1997) ARWEN (Laslett and Canbäck, 2008). C. hakodatei Organellar Genome DRAW (Laslett and Canbäck, 2008). Evolutionary Genetics Analysis (MEGA6, http://www.megasoftware.net) Minimum Evolution. 결과및고찰 Crangon hakodatei 의전장미토콘드리아게놈 Crangon hakodatei. C. hakodatei 16,060 bps (GenBank Accession Number, KU641481) 13, 22 trna 2 rrna (Fig. 1, Table 2). 9 (atp6, atp8, cox1, cox2, cox3, cob, nad2, nad3 nad6) H 4 (nad1, nad4, nad4l nad5) L (Table 2). ACG COI ATN Nad4 Cytb (Table 2). COXI COXII 11 7 bp (Cook, 2005; Yamauchi et al., 2003). C. hakodatei LrRNA SrRNA 1297 bp 853 bp. LrRNA trna Leu trna- Val, SrRNA trna Val (control region). C. hakodatei A/T (85.1 %) Table 2. Location of genes in the mitochondrial genome of the Crangon hakodatei Featrures (anticodon) Start Stop Str. L nt In./term. L aa COI 1 1535 + 1535 ACG/TA- 511 trnl1 (taa) 1536 1599 + 64 CO2 1602 2288 + 687 ATG/TAA 228 trnk (ttt) 2292 2359 + 68 trnd (gtc) 2360 2423 + 64 Atp8 2424 2582 + 159 ATC/TAG 52 Atp6 2576 3253 + 678 ATG/TAA 225 CO3 3257 4045 + 789 ATG/TAA 262 trng (tcc) 4045 4107 + 63 Nad3 4108 4461 + 354 ATG/TAG 117 trna (tgc) 4460 4524 + 65 trnr (tcg) 4526 4590 + 65 trnn (gtt) 4594 4658 + 65 trns1 (tct) 4658 4726 + 69 trne (ttc) 4726 4791 + 66 trnf (gaa) 4790 4853-64 Nad5 4853 6577-1725 ATG/TAA 574 trnh (gtg) 6578 6641-64 Nad4 6643 7971-1329 ATG/--- 343 Nad4L 7965 8264-300 ATG/TAA 99 trnt (tgt) 8267 8329 + 63 trnp (tgg) 8329 8396-68 Nad6 8398 8913 + 516 ATC/TAA 171 Cyt b 8913 10049 + 1137 ATG/--- 378 trns2 (tga) 10048 10117 + 70 Nad1 10117 11076-960 ATA/TAA 319 trnl2 (tag) 11088 11152-65 rrnl 11153 12449-1297 trnv (tac) 12450 12515-66 rrns 12516 13368-853 CR(non-coding) 13369 14506 + 1138 trni (gat) 14507 14573 + 67 trnq (ttg) 14683 14753-70 trnm (cat) 14761 14828 + 68 Nad2 14829 15830 + 1002 ATT/TAA 333 trnw (tca) 15828 15896 + 69 trnc (gca) 15909 15975-67 trny (gta) 15989 16051-63 1,138 bp. trna 63 70
870 김경률ㆍ김현우 Fig. 1. The Map of Crangon hakodatei mitochondrial genome. Genes encoded by the heavy strand were shown outside the circle, and encoded by the light strand were shown inside the circle respectively. The gray arrow indicate the direction of transcription for the two DNA strands. The inner circle denotes the GC content of this genome. The map of complete mitochondria genome was drawn using OGDRAW (Lohse et al., 2013). (Table 2). 20 trna trnascan-se (Lowe and Eddy, 1997) ARWEN ARWEN (Laslett and Canback, 2008) 2. trna Ser (AGN) D arm (Wolstenholme, 1992) T arm trna Cys 2 C. hakodatei (Fig. 2). (Malacostraca) (Abascal et al., 2006). (Abascal et al., 2006), C. hakodatei (Fig. 3). World Class of Marine Species (WoRMS; http://www.marinespecies.org) 15 19
마루자주새우의전장미토콘드리아게놈 871 Fig. 2. Predicted secondary structure of 22 trnas of the Crangon hakodatei mitochondrial genome. The trnas are labeled with the three letters of abbreviations for each corresponding amino acids. GenBank. (Fig. 3)., trna Pro trna Thr (Fig. 3). 미토콘드리아게놈을통한 Crangon hakodatei 의분자계통학적분석 C. hakodatei 9 70-73%, (superfamily Bresilioidea) Opaepele loihi.. COI (Folmer et al., 1994), C.hakodatei Crangon crangon 94% (Fig. 4A). COXI, C. hakodatei
872 김경률ㆍ김현우 Fig. 3. Gene orders of mitochondrial genomes from Decapoda. All genes are transcribed from left to right except for those underlined to indicate opposite orientation. trna genes are designed by the corresponding single-letter amino acid code. Red colars indicate genes that are located differently from their positions in Crangon hakodatei mitochondrial genome. (A) (B) Fig. 4. Phylogenetic trees of mitochondrial COXI genes (A) and COI regions (B) from shrimps belonging to Caridea with other decapod crustaceans. Molecular Evolutionary Genetics Analysis ver. 6.0 (MEGA6) program was used for constructing phylogenetic tree with the minimum evolution algorithm. The evolutionary distances were computed using the Kimura 2-parameter method
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