Complete mitochondrial DNA sequence of the Japanese sardine Sardinops melanostictus

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1 FISHERIES SCIENCE 2000; 66: Original Article Complete mitochondrial DNA sequence of the Japanese sardine Sardinops melanostictus Jun G INOUE, 1, * Masaki MIYA, 2 Katsumi TSUKAMOTO 1 AND Mutsumi NISHIDA 1 1 Ocean Research Institute, University of Tokyo, Nakano-ku, Tokyo and 2 Department of Zoology, Natural History Museum & Institute, Chiba, Chuo, Chiba , Japan SUMMARY: We determined the complete nucleotide sequence of the mitochondrial genome for a Japanese sardine, Sardinops melanostictus (Teleostei: Clupeiformes). The entire genome was purified by gene amplification using long polymerase chain reactions (PCR), and the products were subsequently used as templates for PCR with 30 sets of fish-versatile primers (including three species-specific primers) that amplify contiguous, overlapping segments of the entire genome. Direct sequencing of the PCR products demonstrated that the genome [ base pairs (bp)] contained the same 37 mitochondrial structural genes (two ribosomal RNA, 22 transfer RNA, and 13 proteincoding genes) as found in other vertebrates, with the gene order identical to that in typical vertebrates. A major non-coding region between the trna Pro and trna Phe genes (1200 bp) was considered as the control (D-loop) region, as it has several conservative blocks characteristic to this region. KEY WORDS: complete mitochondrial DNA sequence, Japanese sardine, long-pcr, mitogenomics, Sardinops melanostictus. INTRODUCTION Animal mitochondrial DNA (mtdna) is a closed circular molecule, typically kilobases (kb) in length, comprising 37 genes encoding 22 transfer RNA (trna), two ribosomal RNA (rrna), and 13 proteins, plus a control region that initiates replication and transcription. 1 3 Because of its compactness, maternal inheritance, fast evolutionary rate compared to that of the nuclear DNA, and the resulting short coalescence time, mtdna is a useful marker for population genetic studies, such as analyses of gene flow, hybridization, and introgression. 4,5 Consequently, its usefulness as a genetic marker has received much attention from applied biological sciences, such as fisheries biology. 6 Numerous studies have employed mtdna as a genetic marker to investigate the geographic population structures of fisheries resources in Japanese waters, including ayu, 7,8 Japanese flounder, 9 11 red sea bream, 12,13 diamond-shaped squid Thysanoteuthis rhombus, 14 and abalone Haliotis diversicolor. 15 *Corresponding author: Tel: Fax: jinoue@ori.u-tokyo.ac.jp Mitogenomics of the commercially important fishes in Japan I. Received 13 December Accepted 22 May In such studies, a short segment of the mtdna [notably those from the control (D-loop) region of base pairs (bp)] were commonly amplified using the polymerase chain reactions (PCR), the products subsequently digested by various restriction endonucleases [restriction fragment length polymorphism (RFLP) analyses] or directly sequenced. In some cases, however, analyses of genetic variations have revealed ambiguous geographic structures of the local populations, partly because the segment was too short to contain sufficient genetic variations or the evolutionary rate of the segment was not suitable for a specific purpose of the study. Unfortunately, our knowledge on the complete mitochondrial genomes in fisheries resources is restricted to several fishes, such as loach, 16 carp, 17 trout, 18 cod, 19 ginbuna, 20 deep-sea fish Gonostoma gracile, 21 and Atlantic salmon. 22 Also the genetic analyses have heavily depended upon the early availability of universal PCR primers for several short, partial segments of the mitochondrial genome, such as those of the control region, two rrna, and cytochrome b genes. 23 For more effective uses of the mtdna as a genetic marker in fisheries biology, it appears that more mitochondrial genomic (mitogenomic) information is needed from various fisheries resources. With limited time and resources, however, it has been technically difficult to obtain such longer mtdna sequences from a wide variety

2 Japanese sardine mitochondrial genome FISHERIES SCIENCE 925 of animals. Miya and Nishida 21 have overcome this difficulty by developing a PCR-based approach for sequencing the complete mitochondrial genome of fishes that employs a long PCR technique and a number of fishversatile primers. In this approach, the entire mitochondrial genome was purified by gene amplification using long PCR and the products were subsequently used as templates for PCR with a number of newly designed, fishversatile primers that amplify contiguous overlapping segments of the entire genome. 21 The complete mtdna sequence is obtained by the direct sequencing of these contiguous PCR products and preliminary experiments have revealed that this PCR-based approach for sequencing the complete mitochondrial genome is applicable to a wide variety of fishes (Miya M & Nishida M, unpubl. data). 21 It should be noted that this approach not only greatly reduces the possibility of amplification of mitochondrial pseudogenes in the nuclear genome, but also allows an accurate determination of the complete mtdna sequences that is faster than sequencing cloned mtdna. 21,24 26 Also, it should be useful for small, rare, or endangered species. 21 This paper, the first in a series of papers entitled Mitogenomics of the commercially important fishes in Japan, describes the mitochondrial genome and its gene organization of a clupeid, Sardinops melanostictus, one of the five Sardinops species that have been recognized on the basis of geographic separation of the populations MATERIALS AND METHODS Fish sample and DNA extraction A Japanese sardine specimen was obtained from a commercial source and tissues for DNA extraction were immediately preserved in 99.5% ethanol. Total genomic DNA was extracted from the muscle tissue using a QIAamp tissue kit (QIAGEN Hilden, Germany) following the manufacturer s protocol. A voucher specimen was deposited in the Fish Collection, National Science Museum, Tokyo (NSMT-P 58444). Mitochondrial DNA purification by long PCR We previously determined partial sequences for the 16S rrna and cyt b genes from the S. melanostictus specimen (Inoue JG, Miya M, Tsukamoto K, Nishida M, unpubl. data) using primer pairs 4 (L S + H S) and 27 (L14734-Glu + H15557-CYB) designated in Table 1. On the basis of these two sequences, a set of species-specific primers (Same-16S-L + Same-CYB-H; Table 1) were designed so as to amplify the 16S cyt b region (Fig. 1). The cyt b 16S region, a remaining portion of the whole mitochondrial genome, was amplified using another set of fish-versatile primers (L15285-CYB + H S; Table 1). Long PCR was done in a Model 9700 thermal cycler (Perkin-Elmer, Foster City, CA, USA), and reactions were carried out with 30 cycles of a 25 ml reaction volume containing ml of sterile distilled H 2 O, 2.5 ml of 10 LA PCR buffer II (TaKaRa, Otsu, Shiga, Japan), 4.0 ml dntp (4 mm), 1.0 ml each primer (5 mm), 0.25 ml of 1.25 unit LA Taq (TaKaRa), and 1.0 ml of template containing approximately 5 ng DNA. The thermal cycle profile was that of shuttle PCR : denaturation at 98 C for 10 s, and annealing and extension combined at the same temperature (68 C) for 16 min. Long-PCR products were electrophoresed on a 1.0% L 03 agarose (TaKaRa) gel and later stained with ethidium bromide for band characterization via ultraviolet transillumination. The long-pcr products were diluted with TE buffer (1:20) for subsequent use as PCR templates. PCR and sequencing We used 30 sets of primers that amplify contiguous, overlapping segments of the entire genome. These primers include 57 fish-versatile primers that were designed with reference to the aligned, complete nucleotide sequences from the mitochondrial genome of six species of bony fishes (loach, 16 carp, 17 trout, 18 cod, 19 bichir, 31 and lungfish 32 ). Three species-specific primers were supplemented in regions where no appropriate fish-versatile primers were available. The PCR was done in a Model 9700 thermal cycler (Perkin-Elmer), and reactions were carried out with 30 cycles of a 25 ml reaction volume containing 14.4 ml of sterile, distilled H 2 O, 2.5 ml of 10 PCR buffer II (Perkin-Elmer), 2.0 ml of dntp (4 mm), 2.5 ml of each primer (5 mm), 0.1 ml of 0.5 unit Ex Taq (TaKaRa), and 1.0 ml of template. The thermal cycle profile was as follows: denaturation at 94 C for 15 s, annealing at 50 C for 15 s, and extension at 72 C for 30 s. The PCR products were electrophoresed on a 1.0% L 03 agarose gel and stained with ethidium bromide for band characterization via ultraviolet transillumination. Double-stranded PCR products were purified by filtration through a Microcon-100 (Amicon Inc., Bedford, MA, USA), which were subsequently used for direct cycle sequencing with dye-labeled terminators (Perkin- Elmer). Primers used were the same as those for PCR. All sequencing reactions were performed according to the manufacturer s instructions. Labeled fragments were analyzed on a Model 310 DNA sequencer (Perkin-Elmer). Sequence analyses The DNA sequences were analyzed using the computer software package program DNASIS version 3.2 (Hitachi Software Engineering Co. Ltd, Yokohama, Japan). The location of the 13 protein-coding genes was determined

3 926 FISHERIES SCIENCE JG Inoue et al. Table 1 Primers PCR and sequencing primers in the analysis of Japanese sardine mitochondrial genome Sequence (5 Æ3 ) Long PCR primers Same-16S-L 1 CAA CCA CGA AAA GCG GCC CTA ATT GGA GCC Same-CYB-H 1 GGC AGA TAG GAG GTT AGT AAT GAC AGT GGC L15285-CYB CCC TAA CCC GVT TCT TYG C H S ATT GCG CTA CCT TTG CAC GGT PCR and sequencing primers 1. L709-12S TAC ACA TGC AAG TCT CCG CA H S ACT TAC CGT GTT ACG ACT TGC CTC 2. L S ACG TCA GGT CGA GGT GTA GC H S GCA ACC AGC TAT AAC TAG GCT CGG T 3. L S AAA CCT CGT ACC TTT TGC AT H S 2 ACA AGT GAT TGC GCT ACC TT 4. L S 2 CGC CTG TTT AAC AAA GAC AT H S TCC GGT CTG AAC TCA GAT CAC GTA 5. L S 2 AGT TAC CCT GGG GAT AAC AGC GCA ATC H3976-ND1 ATG TTG GCG TAT TCK GCK AGG AA 6. L3686-ND1 TGA GCM TCW AAT TCM AAA TA H4432-Met TTT AAC CGW CAT GTT CGG GGT ATG 7. L4166-ND1 2 CGA TAT GAT CAA CTM ATK CA H4866-ND2 2 AAK GGK GCK AGT TTT TGT CA 8. L4633-ND2 2 CAC CAC CCW CGA GCA GTT GA H5334-ND2 2 CGK AGR TAG AAG TAK AGG CT 9. L4822-ND2 CAG TTC TGA KTG CCA GAR GT H5669-Asn AAC TGA GAG TTT GWA GGA TCG AGG CC 10. L5644-Ala 2 GCA AMT CAG ACA CTT TAA TTA A H6371-CO1 2 TTG ATT GCC CCK AGG ATW GA 11. L6199-CO1 2 GCC TTC CCW CGA ATA AAT AA H6855-CO1 2 AGT CAG CTG AAK ACT TTT AC 12. L6730-CO1 2 TAT ATA GGA ATR GTM TGA GC H7480-Ser 2 ATG TGG YTG GCT TGA AA 13. L7255-CO1 2 GAT GCC TAC ACM CTG TGA AA H8168-CO2 2 CCG CAG ATT TCW GAG CAT TG 14. L7863-CO2 2 ATA GAC GAA ATT AAT GAC CC H8589-ATP 2 AAG CTT AKT GTC ATG GTC AGT 15. L8329-Lys 2 AGC GTT GGC CTT TTA AGC H9076-ATP 2 GGG CGG ATA AAK AGG CTA AT 16. L8894-ATP 2 TTG GAC TAC TWC CST ATA C H9375-CO3 CGG ATR ATG TCT CGT CAT CA 17. L9220-CO3 AAC GTT TAA TGG CCC ACC AAG C H10035-Gly CTT TCC TTG GGK TTT AAC CAA G 18. L9655-CO3 GTA ACW TGG GCT CAT CAC AG H10433-Arg 2 AAC CAT GGW TTT TTG AGC CGA AAT 19. L10056-Gly CTT GGT TAA AKT CCA AGG AAA G H10970-ND4 2 GAT TAT WAG KGG GAG WAG TCA 20. L10440-Arg AAG ATT WTT GAT TTC GGC T H11618-ND4 TGG CTG ACK GAK GAG TAG GC 21. L11424-ND4 2 TGA CTT CCW AAA GCC CAT GTA GA H12632-ND5 2 GAT CAG GTT ACG TAK AGK GC 22. L12191-His TTG TGA TTC TAA AAA TAG GGG TTA AA H13069-ND5 2 GTG CTG GAG TGK AGT AGG GC 23. L12936-ND5 AAC TCM TGG GAG ATT CAA CAA H13727-ND5 2 GCG ATK ATG CTT CCT CAG GC 24. L13562-ND5 2 CTW AAC GCC TGA GCC CT H14718-Gln TTT TTG TAG TTG AAT WAC AAC GGT 25. Same-ND5-L 1 GCA CAA CTT CTC AAA TAT ACT TGG Same-ND6-H 1 TTC TTT TCT TAA TCT ATT TGG GTG G 26. L14504-ND6 GCC AAW GCT GCW GAA TAM GCA AA H15149-CYB GGT GGC KCC TCA GAA GGA CAT TTG KCC TCA 27. L14734-Glu AAC CAC CGT TGT TAT TCA ACT H15557-CYB GGC AAA TAG GAA RTA TCA YTC 28. L15285-CYB CCC TAA CCC GCT TAT TYG C H15990-Pro AGT TTA ATT TAG AAT CYT GGC TTT GG 29. L15774-CYB ACA TGA ATT GGA GGA ATA CCA GT H16500-CR GCC CTG AAA TAG GAA CCA GA 30. Same-CR-L 1 CCC GGT AAA TCG ATT AAA CCC C H S GGC ATA GTG GGG TAT CTA ATC CCA GTT TGT Primers are designated by their 3 ends, which correspond to the position of the human mitochondrial genome 30 by convention. L and H denote heavy strand and light strand, respectively. For relative positions of primers in the mitochondrial genome, see Fig. 1. Positions with mixed bases are labeled with their IUB codes: R indicates A or G; Y, C or T; K, G or T; M, A or C; S, G or C; W, A or T. 1 Japanese sardine specific primers. 2 After Miya and Nishida. 21

4 Japanese sardine mitochondrial genome FISHERIES SCIENCE F V L I M W D K G R HSL T Q ANCY S E P Fig. 1 Gene organization and sequencing strategy for the Japanese sardine 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 singleletter amino acid codes, those encoded by the H strand and L strand are shown above and below the gene map, respectively. Two pairs of long-pcr primers (Same-16S-L + Same-CYB-H and L15285-CYB + H S) amplify two segments covering the entire mitochondrial genome except two previously determined partial sequences from 16S rrna and cyt b genes. Relative positions of other primers are shown by small arrows with numerals designated in Table 1. 12S and 16S indicate genes of the 12S and 16S rrna; ND1 6, and 4L, NADH dehydrogenase subunits 1 6 and 4L; COI III, cytochrome c oxidase subunits I III; ATPase 6 and 8, ATPase subunits 6 and 8; cyt b, cytochrome b; CR, control region. by comparisons of nucleotide or amino acid sequences of bony fish mitochondrial genomes. The 22 trna genes were identified by their proposed cloverleaf secondary structures and anticodon sequences. 33 The two rrna genes were identified by sequence homology and proposed secondary structure. 34 Sequence data are available from DDBJ/EMBL/GenBank under accession number AB RESULTS AND DISCUSSION Long PCR and sequencing strategy Recent development of a long-pcr technique enabled us to amplify up to a 35 kb target sequence with high fidelity. 35,36 Although Cheng et al. 37 successfully amplified 16.3 kb of the 16.6 kb human mitochondrial genome in a single long PCR, our preliminary experiments demonstrated that such a single long PCR was not feasible for the Japanese sardine mitochondrial genome, probably because of GC-rich regions in the putative control region. Strings of C and G bases often inhibit PCR reactions. Alternatively, we decided to divide the circular mitochondrial genome into two segments (Fig. 1): one long segment was expected to cover all protein-coding and most trna genes, spanning from the 16S rrna to the cyt b genes, and another short segment was expected to cover the two rrna genes and the entire putative control region, spanning from the cyt b to the 16S rrna genes. Since we had already determined two partial sequences from the 16S rrna and cyt b genes for the Japanese sardine, two species-specific primers were designed on the basis of their sequences to amplify the long segment. The short segment, on the other hand, was amplified using two fish-versatile primer (L15285-CYB + H S), because of their nearly perfect matching to the previously determined sequences. Consequently, the mitochondrial genome of Japanese sardine was purified by gene amplification, providing templates for subsequent amplifications and direct sequencing of contiguous, overlapping segments of the entire genome using the 30 sets of primers (Fig. 1; Table 1). 24 Genome content and base composition The total length of the Japanese sardine genome was bp. The complete L-strand nucleotide sequence of Japanese sardine, is shown in Fig. 2. The genome content of Japanese sardine included two rrna, 22 trna, 13 protein-coding genes, and a control region, as found in other vertebrates (Figs 1,2; Table 2). As in other vertebrates, most genes were encoded on the H-strand, except for the ND6 and eight trna genes, and all genes were similar in length to those in other bony fishes. The gene order is identical to those so far obtained in other vertebrates. The base composition of Japanese sardine was analyzed separately for rrna, trna, and protein-coding genes (Table 3). In the protein-coding gene, anti-g bias was observed in the third codon positions (18.0%). Pyrimidines were overrepresented in the second codon positions (66.5%), as has been noted for other vertebrate mitochondrial genomes, owing to the hydrophobic character of the proteins. 38 Japanese sardine trna genes were slightly A + T rich (52.0%), as in other vertebrates, while rrna genes have a high adenine content (30.9%), as in other bony fishes. 39 Protein-coding genes Among the 13 protein-coding genes of Japanese sardine, there were two reading-frame overlaps on the same strand

5 928 FISHERIES SCIENCE JG Inoue et al. Table 2 Location of features in the mitochondrial genome of Japanese sardine Gene Position number Size (bp) Codon From To Start Stop trna Phe S rrna trna Val S rrna trna Leu(UUR) ND ATG TAG trna Ile trna Gln (L) trna Met ND ATG TA trna Trp trna Ala (L) trna Asn (L) trna Cys (L) trna Tyr (L) COI GTG TAA trna Ser(UCN) (L) trna Asp COII ATG T trna Lys ATPase ATG TAA ATPase ATG TA COIII ATG TA trna Gly ND ATG T trna Arg ND4L ATG TAA ND ATG T trna His trna Ser(AGY) trna Leu(CUN) ND ATG TAA ND (L) ATG TAG trna Glu (L) Cyt b ATG T trna Thr trna Pro (L) Control region Table 3 Base composition of the mitochondrial genome of Japanese sardine protein-coding genes (Figs 1,2). The trna genes range in size from 66 to 76 nucleotides (Table 2), large enough so that the encoded trna can fold into the cloverleaf secondary structure characteristic of trna (data not shown). This is possible provided that formation of the G U wobble and other atypical pairings were allowed in the stem regions. All postulated cloverleaf structures contained 7 bp in the amino acid stem, 5 bp in the TYC stem, 5 bp in the anticodon stem, and 4 bp in the DHU stem (3 bp in trna Ser(AGY) ). Ribosomal RNA genes A C G T Proteins 1st nd rd Total trna rrna The 12S and 16S rrna genes of Japanese sardine were 959 and 1683 nucleotides long, respectively (Table 2). They were located, as in other vertebrates, between genes of the trna Phe and trna Leu, being separated by the trna Val gene (Figs 1,2). Preliminary assessment of their secondary structure indicated that the present sequences could be reasonably superimposed on the proposed secondary structure of carp 12S rrna and loach 16S rrna, respectively. 34 (ATPases 8 and 6 shared 10 nucleotides; ND4L and ND4 shared seven nucleotides) (Fig. 2). As in other bony fishes, all the mitochondrial protein-coding genes began with an ATG start codon, except for COI, which starts with GTG (Table 2). Open-reading frames of Japanese sardine ended with TAA (COI, ATPase 8, ND4L, and ND5), TAG (ND1 and ND6), and the remainder had incomplete stop codons, either TA (ND2, ATPase 6, and COIII) or T (COII, ND3, ND4, and cyt b) (Table 2). Transfer RNA genes The Japanese sardine mitochondrial genome contained 22 trna genes interspersed between the rrna and Fig. 2 The complete L-strand nucleotide sequence of the Japanese sardine mitochondrial genome. Position 1 corresponds to the first nucleotide of the trna Phe gene. Direction of transcription for each gene is shown by arrows. Beginning and end of each gene are indicated by vertical bars ( ). Transfer RNA genes are boxed; corresponding anticodons are indicated in black boxes. Amino acid sequences presented below the nucleotide sequence were derived using mammalian mitochondrial genetic code (one-letter amino acid abbreviations placed below first nucleotide of each codon). Stop codons are overlined and indicated by asterisks. Non-coding sequence are underlined with dots. TAS, putative termination-associated sequence; CSB 2, 3, and D, conserved sequence blocks. Sequence data are available from DDBJ/EMBL/GenBank with accession number AB

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8 Japanese sardine mitochondrial genome FISHERIES SCIENCE 931 Non-coding sequences As in most vertebrates, the origin of light strand replication (O L ) in Japanese sardine was in a cluster of five trna genes (WANCY region, Fig. 2) and comprised 48 nucleotides in length. This region has the potential to fold into a stable stem-loop secondary structure with 12 bp in the stem, and 12 nucleotides in the loop. The conserved motif 5 -GCCGG-3 was found at the base of the stem within the trna Cys gene. The major non-coding region found in the Japanese sardine mtdna was located between the trna Pro and trna Phe genes. This non-coding sequence (1200 bp) appears to correspond to the control region, because it has a conserved sequence block (CSB) and terminationassociated sequence (TAS) (Fig. 1) that are characteristic to this region. 40,41 The control region of the Japanese sardine was longer than that of other bony fishes ( bp). 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