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Mitochondrial DNA
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Complete mitochondrial genome of the rabbitfish Siganus fuscescens (Perciformes, Siganidae)

Dae-Ju Oh a; Ji-Young Kim a; Jung-A Lee a; Weon-Jong Yoon a; Soo-Yeong Park a; Yong-Hwan Jung a a Jeju Biodiversity Research Institute, Jeju Hi-Tech Industry Development Institute, Jeju, South Korea First Published:2007

To cite this Article Oh, Dae-Ju, Kim, Ji-Young, Lee, Jung-A, Yoon, Weon-Jong, Park, Soo-Yeong and Jung, Yong-

Hwan(2007)'Complete mitochondrial genome of the rabbitfish Siganus fuscescens (Perciformes, Siganidae)',Mitochondrial DNA,18:4,295 301
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DNA Sequence, August 2007; 18(4): 295301

FULL LENGTH RESEARCH PAPER

Complete mitochondrial genome of the rabbitsh Siganus fuscescens (Perciformes, Siganidae)

DAE-JU OH, JI-YOUNG KIM, JUNG-A LEE, WEON-JONG YOON, SOO-YEONG PARK, & YONG-HWAN JUNG
Jeju Biodiversity Research Institute, Jeju Hi-Tech Industry Development Institute, Jeju 690-121, South Korea
(Received 5 October 2006)
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Abstract We determined the complete nucleotide sequence of the mitochondrial genome for the rabbitsh Siganus fuscescens (Perciformes, Siganidae). This mitochondrial genome, consisting of 16,491 base pairs (bp), included 13 protein-coding genes, 2 ribosomal RNAs, 22 transfer RNAs, and a noncoding control region similar those found in other vertebrates; the gene order was identical to that of typical vertebrates. Most of the genes of S. fuscescens were encoded on the H-strand, while the ND6 and eight tRNA (Gln, Ala, Asn, Cys, Tyr, Ser [UCN], Glu, and Pro) genes were encoded on the L-strand. The reading frames of ATPase 8 and 6 and those of ND4L and ND4 overlapped by ten and seven nucleotides, respectively. All mitochondrial protein-coding genes began with an ATG start codon, except for CO1, which started with GTG. Open reading frames of S. fuscescens ended with TAA (ND1, CO1, ATPase 8, ND4L, ND5 and ND6), and the remainder had incomplete stop codons, either TA (ATPase 6 and CO3) or T (ND2, CO2, ND3, ND4, and Cytb). The origin of L-strand replication in S. fuscescens was located in a cluster of ve tRNA genes (WANCY) and was 34 nucleotides in length. A major noncoding region between the tRNA-Pro and tRNA-Phe genes (828 bp) was considered to be the control region (D-loop). Within this sequence, we identied a conserved sequence block characteristic of this region. The rabbitsh was grouped with Siganus canaliculatus in most parsimony analyses, which showed 100% bootstrap support for their divergence. These ndings are useful for inferring phylogenetic relationships and identication within the suborder Acanthuroidei.

Keywords: Mitochondrial genome, rabbitsh, Siganus fuscescens, WANCY, conserved sequence block, parsimony analyses Database accession number: EF025185

Introduction The rabbitsh Siganus fuscescens belongs to the family Siganidae, which includes the single genus Siganus. This sh is one of important marine food sh species in Jeju Island, Korea. The body coloration of rabbitsh is brown or olive green, with a silvery belly and small spots. When frightened, adults become mottled. Some confusion exists in the taxonomy of S. fuscescens, which has often been misidentied as a small spotted form of Siganus canaliculatus (Lam 1974; de la Paz and Aragones 1990). In addition, S. fuscescens differs from S. argenteus in details of coloration and in having a less deeply forked tail. These shes are distributed in tropic and subtropical

regions of the western Pacic, including southern Korea, Japan, Southeast Asia, and Australia. Juveniles feed on lamentous algae, and adults feed on leafy algae and seagrasses. From March to May, the sh aggregate to spawn (Woodland 1977). Structurally, most animal mitochondrial genomes contain the same 37 genes encoding 22 transfer RNA (tRNA), 2 ribosomal RNA (rRNA), and 13 proteins; the gene order is highly conserved among vertebrates (Brown 1985; Boore 1999). Vertebrate mitochondrial genomes normally consist of 16 18 kilobases (kb) and are extremely compact, with no introns and few if any intergenic spacers. The only major noncoding sequence is the control region, which is involved in

Correspondence: Y.-H. Jung, Jeju Biodiversity Research Institute, Jeju HiDI, 4-8 Ara-1, Jeju, Jeju 690-121, South Korea. Tel: 82 64 720 2321. Fax: 82 64 720 2301. E-mail: yhjung@jejuhidi.or.kr
ISSN 1042-5179 print/ISSN 1029-2365 online q 2007 Informa UK Ltd. DOI: 10.1080/10425170701248525

296 D. -J. Oh et al. regulating transcription and replication (Clayton 1982; Shadel and Clayton 1997) and is usually , 5% of the total genome size. Because of several special features (compactness, maternal inheritance, fast evolutionary rate compared to nuclear DNA, and the resulting short coalescence time), mitochondrial DNA is a useful marker for population genetic studies, such as analyses of gene ow, genetic variation, hybridization, and introgression (Moore 1995; Jiggins et al. 1997; Chubb et al. 1998; McLean and Taylor 2001; Jung et al. 2002; Miya et al. 2003). The complete mitochondrial DNA sequences from approximately 300 species of teleostei have been reported since Tzeng et al. (1992) rst sequenced the mitochondrial genome from the freshwater loach (Crossostoma lacustre). To further understanding of mitochondrial genome evolution in lower vertebrates and invertebrates, the Miya group has reported complete mitochondrial genomes in sh and in other taxa, such as the copepod Tigriopus japonicus (Machida et al. 2002). Recently, Miya et al. (2003) reported major patterns of higher teleostean phylogenies based on 100 complete mitochondrial DNA sequences. In particular, they reported the complete mitochondrial genome sequence in nine families of Perciformes, Carangidae Emmelichthyidae, Lutjanidae, Zoarcidae, Pholidae, Blennidae, Gobiesocidae, Rhyacichthyidae, and Eleotridae, to infer phylogenies among the higher teleostean (Miya et al. 2003). Broughton and Reneau (2006), also analyzed the complete mitochondrial genome of the Percidae, Centrarchidae, and Latidae, and Kim et al. (2004) reported the complete mitochondrial genome of the javeline goby Acanthogobius hasta with phylogenetic considerations. However, no one has yet analyzed the complete mitochondrial genome of the Siganidae species of Perciformes. In this paper, we present the rst complete nucleotide sequence for the mitochondrial genome of the rabbitsh S. fuscescens and show its mitochondrial genomic structure. We also report on the organization, gene arrangement, and codon usage of S. fuscescens mitochondrial DNA and compare it to the mitochondrial DNAs of other Perciformes. Based on a phylogenetic analysis using CLUSTRAL X (Thompson et al. 1997) to compare the 16S ribosomal RNA partial sequences of the 13 previously reported shes in the suborder Acanthuroidei, we were able to place S. fuscescens within the molecular phylogeny of the Acanthuroidei. individual of S. fuscescens using a Wizard Genomic DNA Purication kit (Promega, USA) following the manufacturers protocol.

Polymerase chain reaction (PCR) We used 14 sets of primers that amplify contiguous, overlapping segments of the complete mitochondrial genome of S. fuscescens as shown in Table I. We could not nd any sequence information of the complete mitochondrial genome for the Acanthuroidei. Therefore, the primers used to amplify the mitochondrial genome of the rabbitsh were designed based on the complete mitochondrial genome sequences for Emmelichthys struhsakeri (Perciformes, Emmelichthyidae) (GenBank Accession No. AP004446; Miya et al. 2003). The PCR mixture consisted of 8 ml sterile distilled water, 1.5 ml 10 reaction buffer, 1.2 ml dNTPs (4 mM), 1.5 ml of each primer (5 mM), 1.5 units Taq DNA polymerase (TaKaRa, Japan), and 1 ml template. Amplication was performed in a GeneAmp PCR system 9700 thermal cycler (Applied Biosystems, USA) under the following cycle conditions: an initial denaturation step (2 min at 948C) followed by 30 cycles of denaturation (15 s at 948C), annealing (15 s at 53 558C), and extension (1 min at 728C), then a nal primer extension step (7 min at
Table I. PCR primers in the analysis of the S. fuscescens mitochondrial genome. Primer 1F 1R 2F 2R 3F 3R 4F 4R 5F 5R 6F 6R 7F 7R 8F 8R 9F 9R 10F 10R 11F 11R 12F 12R 13F 13R 14F 14R Sequence (50 ! 30 ) GCA TAA CAC TGA AGA TGT TAA G CAT GAT GCA AAA GGT ACG AG CTC GTA CCT TTT GCA TCA TG CCA ACA TCG AGG TCG TAA AC GTT TAC GAC CTC GAT GTT GG ACC TTA TCA AAG TGG CCC T AGG GCC ACT TTG ATA GAG T GCG CTT AGC TGT TAA CTA AG AGA CCA AGG GCC TTC AAA GC TTA CCA GAG TAG TAG GCT AC GTA GCC TAC TAC TCT GGT AA GCT TCA GTA TCA TTG GTG GCC GGC CAC CAA TGA TAC TGA AGC GCT GGG GTC AAC TAT GTG GTA TAC CAC ATA GTT GAC CCC AGC CCG GGT GAT TGG AAG TCA CT AGT GAC TTC CAA TCA CCC GG TTG ATC CGG CTA CTG GGG CCT AGG CCC CAG TAG CCG GAT CAA TGC TAT TGC GAA GAT CAG GC CCT AAT CCC CCT GCT CCT AC CAG GCG TTT AGG TGG GAT GTG CAC ATC CCA CCT AAA CGC CTG CAA CGG TGG TTT TTC AAG CC GGC TTG AAA AAC CAC CGT TG AGA ATC CTA GCT TTG GGA G CTC CCA AAG CTA GGA TTC T CTT AAC ATC TTC AGT GTT ATG C

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Materials and methods Fish sample and DNA extraction S. fuscescens blood was provided by Marine & Environmental Research Institute, Cheju National University, Jeju, Korea. Total genomic DNA was extracted from the blood lymphocytes of a single

Complete mitochondrial genome of the rabbitsh Siganus fuscescens (Perciformes, Siganidae) 728C). The PCR products were electrophoresed on a 1.0% agarose gel and stained with ethidium bromide for band characterization via ultraviolet transillumination. The products were recovered and concentrated using the GeneClean II system (Bio 101, USA) according to the manufacturers protocol Phylogenetic analysis

297

Cloning and sequencing the PCR products The PCR products were cloned using the TOPO TA Cloning Kit (Invitrogen, USA) according to the manufacturers instructions. Each cloned DNA fragment was sequenced according to the manufacturers protocol with TOPO vector inner primers (Invitrogen, USA): T7 and M13R. Labeled fragments were analyzed on an ABI Prism 3100 DNA sequencer (Applied Biosystems, USA).

To determine the phylogenetic position of S. fuscescens within the suborder Acanthuroidei, we aligned the 16S rRNA gene sequences of the S. fuscescens mitochondrial genome with those of the Acanthuroidei available in DDBJ, EMBL, and GenBank using CLUSTAL X (Thompson et al. 1997). Emmelichthys struhsakeri (Perciformes, Emmelichthyidae) was chosen as the outgroup. Maximum parsimony (MP) analysis was performed with a heuristic search using PAUP 4.0b8 (Swofford 1999). Strict consensus trees were constructed from all most-parsimonious trees. Bootstrap analyses were carried out with 1000 replicates using TBR branch-swapping of the heuristic search with random taxon addition (1000 replicates; Felsenstein 1985).

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Results and discussion Sequence analysis We cloned and sequenced three clones from each PCR fragment. Sequences that were identical in at least two of the three clones were selected and used in the molecular genetic analysis. DNA sequences of the complete mitochondrial genome of S. fuscescens were determined by comparison with published sequences for various bony shes (Tang et al. 1999; Clements et al. 2003; Kim et al. 2004; Klanten et al. 2004; Westneat and Alfaro 2005; Smith and Wheeler 2006). The locations of the 13 protein-coding genes were determined by comparisons of nucleotide or amino acid sequences of bony sh mitochondrial genomes. The 22 transfer RNA genes were identied by their proposed cloverleaf secondary structures and anticodon sequences (Kumazawa et al. 1998). The two ribosomal RNA genes and the second control region were identied by sequence homology and proposed secondary structures (Gutell et al. 1993). Genome organization We amplied the mitochondrial genome of S. fuscescens using 14 regenerated primer sets (Table I; Figure 1). The total length of the S. fuscescens mitochondrial genome was 16,491 bp, with overall base compositions as follows: A, 28.05% (4626/ 16491); C, 29.79% (4912/16491); G, 16.76% (2764/16491); and T, 25.40% (4189/16491). The mitochondrial genome of S. fuscescens included 13 protein-coding genes, 22 transfer RNAs (tRNAs), two ribosomal RNAs (rRNAs), and a control region, as found in other vertebrates (Figure 1; Table II). The newly determined sequence has been deposited in GenBank databases (Accession No. EF025185). As seen in other vertebrates, most of the genes of S. fuscescens were encoded on the H-strand, with only the NADH dehydrogenase subunits (ND) 6 and 8 tRNAs (Gln, Ala, Asn, Cys, Tyr, Ser [UCN], Glu, and Pro) genes encoded on the L-strand, and all genes were

Figure 1. Gene organization and sequencing strategy for the rabbitsh mitochondrial genome. All protein-coding genes are encoded by the H strand with the exception of ND6, which is coded by the L strand. Transfer RNA genes are designated by single letter amino acid codes, and those encoded by the H strand and L strand are shown above and below the gene map, respectively. The small arrows indicate the positions and orientations of the primers used. Primer sequences are given in Table I. *12S and 16S, 12S and 16S rRNA; ND1-6, NADH dehydrogenase subunits 16; CO1-3, cytochrome c oxidase subunits 1 3; ATPase 8 and 6, ATPase subunit 8 and 6; Cytb, cytochrome b.

298 D. -J. Oh et al.

Table II. Location of features in the mitochondrial genomes of S. fuscescens. Size (bp) 68 947 72 1691 74 975 70 71(L) 69 1045 72 69(L) 73(L) 66(L) 71(L) 1551 71(L) 72 691 75 168 683 785 73 349 69 297 1381 69 68 73 1839 522(L) 69(L) 1141 72 70(L) 828

Gene* tRNAPhe 12S rRNA tRNAVal 16S rRNA tRNALeu (UUR) ND1 tRNAIle tRNAGln tRNAMet ND2 tRNATrp tRNAAla tRNAAsn tRNACys tRNATyr CO1 tRNASer (UCN) tRNAAsp CO2 TRNA Lys ATPase 8 ATPase 6 CO3 tRNAGly ND3 tRNAArg ND4L ND4 tRNAHis tRNASer (AGY tRNALeu (CUN) ND5 ND6 tRNAGlu CytB tRNAThr tRNAPro Control region

Location 1 . . . 68 69 . . . 1015 1016 . . . 1087 1088 . . . 2778 2779 . . . 2852 2853 . . . 3827 3832 . . . 3901 3901 . . . 3971 3971 . . . 4039 4040 . . . 5084 5085 . . . 5156 5157 . . . 5225 5228 . . . 5300 5335 . . . 5400 5401 . . . 5471 5473 . . . 7023 7026 . . . 7096 7100 . . . 7171 7180 . . . 7870 7871 . . . 7945 7947 . . . 8114 8105 . . . 8787 8788 . . . 9572 9573 . . . 9645 9646 . . . 9994 9995 . . . 10063 10064 . . . 10360 10354 . . . 11734 11735 . . . 11803 11804 . . . 11871 11879 . . . 11951 11952 . . . 13786 13783 . . . 14308 14309 . . . 14377 14382 . . . 15522 15523 . . . 15594 15594 . . . 15663 15664 . . . 16491

Start

Stop

ATG

TAA

ATG

oxidase subunit 1 (COI), which used GTG (Table II). Open reading frames of S. fuscescens ended with TAA (ND1, CO1, ATPase 8, ND4L, ND5 and ND6), or incomplete stop codons, either TA (ATPase 6 and CO3) or T (ND2, CO2, ND3, ND4 and CytB, Table II). Variance in termination codons seems to be a common tendency in sh mitochondrial genomes (Tzeng et al. 1992; Miya and Nishida 1999; Kim et al. 2004). This condition is not uncommon among vertebrate mitochondrial genes, and it appears that TAA stop codons are created via posttranscriptional polyadenylation (Ojala et al. 1981). Incomplete stop codons have been previously reported in various bony shes (Tzeng et al. 1992; Inoue et al. 2000; Broughton et al. 2001; Ishiguro et al. 2001). Transfer RNA genes

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GTG

TAA

ATG ATG ATG ATG ATG ATG ATG

T TAA TA TA T TAA T

ATG ATG ATG

TAA TAA T

* ND1-6, NADH dehydrogenase subunits 16; CO1-3, cytochrome c oxidase subunits 13; ATPase 8 and 6, ATPase subunit 8 and 6; CytB, cytochrome b; (L) indicates a gene encoded on the L-strand.

similar in length to those of other bony shes (Tzeng et al. 1992; Miya et al. 2003). The gene order was identical to those obtained for other vertebrates (Figure 1; Table II).

Protein-coding genes Among the 13 protein-coding genes of S. fuscescens, two reading-frame overlaps were found on the same strand: ATPase subunit (ATPase) 8 and 6 overlapped by ten nucleotides, and ND4L and ND4 overlapped by seven nucleotides (Table II). This is a common vertebrate feature and has been found in other bony shes (Tzeng et al. 1992; Inoue et al. 2000; Kawaguchi et al. 2001; Miya et al. 2003; Kim and Lee 2004). All protein encoding genes in the rabbitsh mitochondrial genome use the initiation codon ATG except for cytochrome c

The S. fuscescens mitochondrial genome contains 22 tRNA genes, which are interspersed between the rRNA and protein-coding genes. Twenty-two tRNAs sequences and their inferred structures were searched by Web-based tRNAscan-SE software (http://lowelab. ucsc.edu/tRNAscan-SE). Of these tRNAs, we identied two forms of tRNALeu (UUR and CUN) and tRNA Ser (UCN and AGY, Figure 1 and Table II). The three tRNA clusters, IQM (isoleucine [I], glutamime [Q], and methionine [M]), WANCY (tryptophan [W], alanine [A], asparagine [N], cysteine [C], and tyrosine [Y]), and HSL (histidine [H], serine [S], and leucine [L]), were well conserved in S. fuscescens, as in typical vertebrate mitochondrial genomes. The tRNA genes ranged in size from 66 (tRNA Cys) to 75 (tRNA Lys) nucleotides (Table II), large enough that the encoded tRNAs can fold into the cloverleaf secondary structure characteristic of tRNAs (data not shown). However, G U wobbles and other atypical pairings were identied in the stem regions, as has been reported in previous studies (Noack et al. 1996; Broughton et al. 2001; Ishiguro et al. 2001). These mutations appear to accumulate in mitochondrial genes, in part because mitochondrial DNA is not subject to the process of recombination, which may facilitate the elimination of deleterious mutations (Lynch 1997). The postulated tRNA cloverleaf structures generally contained 7 bp in the aminoacyl stem, 5 bp in the TcC stem and the anticodon stem, and 4 bp in the DHU stem. The tRNA Ser (AGY) found in the S. fuscescens mitochondrial genome had no discernible DHU stem, similar to that shown in the lamprey (Lee and Kocher 1995), bichir (Noack et al. 1996), and ayu (Ishiguro et al. 2001). Ribosomal RNA genes The 12S and 16S rRNA genes of S. fuscescens were 947 and 1,691 nucleotides long, respectively (Table II). As in other vertebrates, these genes were located

Complete mitochondrial genome of the rabbitsh Siganus fuscescens (Perciformes, Siganidae)

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Figure 2. Sequences and the conserved elements of the control region from the rabbitsh S. fuscescens mitochondrial genome. Primary sequence features (CSB-1, CSB-2, CSB-3 and CSB-D) are underlined. CSB, conserved sequence block.

between genes of the tRNA Phe and tRNA Leu (UUR), and were separated by the tRNA Val gene (Figure 1). Preliminary assessment of their secondary structure indicated that the sequences could be reasonably superimposed on the proposed secondary structure of carp 12S rRNA and loach 16S rRNA, respectively. The A T content of S. fuscescens rRNA genes was 53.39% (1,408/2,637), which is slightly A T-rich compare to other bony shes (Zardoya

and Meyer 1997; Kim and Lee 2004; Kim et al. 2004). Non-coding sequences A noncoding sequence associated with the putative L-strand replication origin (OL) is 34 bp long located between tRNAAsn and tRNACys (Figure 1). The conserved motif (50 -GCCGG-30 ) was found at the base

Figure 3. One of the four most parsimonious trees for phylogenetic relationships among the mitochondrial 16S rRNA genes of various Acanthuroidei shes. Bootstrap values from 1000 replicates are labeled below the relevant nodes. Emmelichthys struhsakeri, belonging to the family Emmelichthyidae, was used as the outgroup taxon. The 16S rRNA sequences of the Acanthuroidei shes used in this study were obtained from the DDBJ/EMBL/GenBank database.

300 D. -J. Oh et al. of the stem within the tRNACys gene. The control region of the S. fuscescens mitochondrial genome spans 828 bp, which is consistent with that of other vertebrates (Lee et al. 1995). Within this sequence, we identied a conserved sequence block (CSB) 1, 2, 3, and D boxes, which are characteristic of this region (Figure 2, Walberg and Clayton 1981; Doda et al. 1981). The region may be useful in analyzing interspecies variation within S. fuscescens samples collected from different habitats in the western Pacic Ocean (e.g. Korea, Japan, Southesat Asia, and Australia).
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Phylogenetic analysis To place S. fuscescens within the molecular phylogeny of the Acanthuroidei, we constructed a MP tree with 16S rRNA sequences from several acanthuroid shes. MP analysis for the unambiguously aligned 16S rRNA sequences from 14 acanthuroid shes, including outgroup taxon, produced four equally likely MP trees, when the coded gaps were excluded from the data set. One of the four MP trees is shown in Figure 3. The bootstrap values for individual clades based on 1000 replicates ranged from 75 to 100%. S. fuscescens grouped with S. canaliculatus, with 100% bootstrap support for their divergence (Figure 3). These two species belong to the family Siganidae of the suborder Acanthuroidei. In addition, S. fuscescens and S. canaliculatus were more closely related to Luvarus imperialis (Luvaridae) than to any other sh (Figure 3). These ndings are useful for inferring phylogenetic relationships and identication within the suborder Acanthuroidei.

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Acknowledgements This work was supported by grant No. RTI04-02-07 from the Regional Technology Innovation Program of the Ministry of Commerce, Industry and Energy (MOCIE).

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