Complete Mitochondrial DNA Sequence of the Japanese Sardine Sardinops Melanostictus†

FISHERIES SCIENCE 2000; 66: 924–932

Original Article

Complete mitochondrial DNA sequence of the Japanese sardine Sardinops melanostictus†

Jun G INOUE,1,* Masaki MIYA,2 Katsumi TSUKAMOTO1 AND Mutsumi NISHIDA1

1Ocean Research Institute, University of Tokyo, Nakano-ku, Tokyo 164-8639 and 2Department of Zoology, Natural History Museum & Institute, Chiba, Chuo, Chiba 260-8682, 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 [16 881 base pairs (bp)] contained the same 37 mitochondrial structural genes (two ribosomal RNA, 22 transfer RNA, and 13 protein- coding genes) as found in other vertebrates, with the gene order identical to that in typical vertebrates. A major non-coding region between the tRNAPro and tRNAPhe 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 In such studies, a short segment of the mtDNA [notably those from the control (D-loop) region of Animal mitochondrial DNA (mtDNA) is a closed 300–1000 base pairs (bp)] were commonly amplified circular molecule, typically 16–20 kilobases (kb) in using the polymerase chain reactions (PCR), the length, comprising 37 genes encoding 22 transfer RNA products subsequently digested by various restriction (tRNA), two ribosomal RNA (rRNA), and 13 proteins, endonucleases [restriction fragment length polymor- plus a control region that initiates replication and tran- phism (RFLP) analyses] or directly sequenced. In some scription.1–3 Because of its compactness, maternal in- cases, however, analyses of genetic variations have heritance, fast evolutionary rate compared to that of the revealed ambiguous geographic structures of the local nuclear DNA, and the resulting short coalescence time, populations, partly because the segment was too short to mtDNA is a useful marker for population genetic stu- contain sufficient genetic variations or the evolutionary dies, such as analyses of gene flow, hybridization, and rate of the segment was not suitable for a specific purpose introgression.4,5 Consequently, its usefulness as a genetic of the study. Unfortunately, our knowledge on the com- marker has received much attention from applied bio- plete mitochondrial genomes in fisheries resources is logical sciences, such as fisheries biology.6 Numerous restricted to several fishes, such as loach,16 carp,17 trout,18 studies have employed mtDNA as a genetic marker cod,19 ginbuna,20 deep-sea fish Gonostoma gracile,21 and to investigate the geographic population structures of Atlantic salmon.22 Also the genetic analyses have fisheries resources in Japanese waters, including ayu,7,8 heavily depended upon the early availability of ‘univer- Japanese flounder,9–11 red sea bream,12,13 diamond-shaped sal’ PCR primers for several short, partial segments of the squid Thysanoteuthis rhombus,14 and abalone Haliotis mitochondrial genome, such as those of the control diversicolor.15 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 *Corresponding author: Tel: 81-3-5351-6520. Fax: 81-3-5351-6514. Email: [email protected] from various fisheries resources. With limited time and †Mitogenomics of the commercially important fishes in Japan—I. resources, however, it has been technically difficult to Received 13 December 1999. Accepted 22 May 2000. obtain such longer mtDNA sequences from a wide variety Japanese sardine mitochondrial genome FISHERIES SCIENCE 925

of animals. Miya and Nishida21 have overcome this diffi- Long PCR was done in a Model 9700 thermal cycler culty by developing a PCR-based approach for sequenc- (Perkin-Elmer, Foster City, CA, USA), and reactions ing the complete mitochondrial genome of fishes that were carried out with 30 cycles of a 25 mL reaction employs a long PCR technique and a number of fish- volume containing 15.25 mL of sterile distilled H2O, versatile primers. In this approach, the entire mitochon- 2.5 mL of 10 ¥ LA PCR buffer II (TaKaRa, Otsu, Shiga, drial genome was purified by gene amplification using Japan), 4.0 mL dNTP (4 mM), 1.0 mL each primer long PCR and the products were subsequently used as (5 mM), 0.25 mL of 1.25 unit LA Taq (TaKaRa), and 1.0 templates for PCR with a number of newly designed, fish- mL of template containing approximately 5 ng DNA. versatile primers that amplify contiguous overlapping The thermal cycle profile was that of ‘shuttle PCR’: segments of the entire genome.21 The complete mtDNA denaturation at 98°C for 10 s, and annealing and exten- sequence is obtained by the direct sequencing of these sion combined at the same temperature (68°C) for 16 contiguous PCR products and preliminary experiments min. Long-PCR products were electrophoresed on a have revealed that this PCR-based approach for sequenc- 1.0% L 03 agarose (TaKaRa) gel and later stained with ing the complete mitochondrial genome is applicable to ethidium bromide for band characterization via ultravi- a wide variety of fishes (Miya M & Nishida M, unpubl. olet transillumination. The long-PCR products were data).21 It should be noted that this approach not only diluted with TE buffer (1:20) for subsequent use as PCR greatly reduces the possibility of amplification of mito- templates. chondrial pseudogenes in the nuclear genome, but also allows an accurate determination of the complete PCR and sequencing mtDNA sequences that is faster than sequencing cloned mtDNA.21,24–26 Also, it should be useful for small, rare, or We used 30 sets of primers that amplify contiguous, over- endangered species.21 lapping segments of the entire genome. These primers This paper, the first in a series of papers entitled include 57 fish-versatile primers that were designed with ‘Mitogenomics of the commercially important fishes in reference to the aligned, complete nucleotide sequences Japan,’ describes the mitochondrial genome and its gene from the mitochondrial genome of six species of bony organization of a clupeid, Sardinops melanostictus, one of fishes (loach,16 carp,17 trout,18 cod,19 bichir,31 and lung- the five Sardinops species that have been recognized on fish32). Three species-specific primers were supplemented the basis of geographic separation of the populations.27–29 in regions where no appropriate fish-versatile primers were available. MATERIALS AND METHODS The PCR was done in a Model 9700 thermal cycler (Perkin-Elmer), and reactions were carried out with Fish sample and DNA extraction 30 cycles of a 25 mL reaction volume containing 14.4 mL of sterile, distilled H2O, 2.5 mL of 10 ¥ PCR buffer II A Japanese sardine specimen was obtained from a com- (Perkin-Elmer), 2.0 mL of dNTP (4 mM), 2.5 mL of each mercial source and tissues for DNA extraction were primer (5 mM), 0.1 mL of 0.5 unit Ex Taq (TaKaRa), and immediately preserved in 99.5% ethanol. Total genomic 1.0 mL of template. The thermal cycle profile was as DNA was extracted from the muscle tissue using a follows: denaturation at 94°C for 15 s, annealing at 50°C QIAamp tissue kit (QIAGEN Hilden, Germany) fol- for 15 s, and extension at 72°C for 30 s. The PCR prod- lowing the manufacturer’s protocol. A voucher specimen ucts were electrophoresed on a 1.0% L 03 agarose gel and was deposited in the Fish Collection, National Science stained with ethidium bromide for band characterization Museum, Tokyo (NSMT-P 58444). via ultraviolet transillumination. Double-stranded PCR products were purified by fil- tration through a Microcon-100 (Amicon Inc., Bedford, Mitochondrial DNA purification by long PCR MA, USA), which were subsequently used for direct cycle sequencing with dye-labeled terminators (Perkin- We previously determined partial sequences for the 16S Elmer). Primers used were the same as those for PCR. All rRNA and cyt b genes from the S. melanostictus specimen sequencing reactions were performed according to the (Inoue JG, Miya M, Tsukamoto K, Nishida M, unpubl. manufacturer’s instructions. Labeled fragments were ana- data) using primer pairs 4 (L2510-16S H3058-16S) and + lyzed on a Model 310 DNA sequencer (Perkin-Elmer). 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 Sequence analyses designed so as to amplify the 16S–cyt b region (Fig. 1). The cyt b–16S region, a remaining portion of the whole The DNA sequences were analyzed using the computer mitochondrial genome, was amplified using another set software package program DNASIS version 3.2 (Hitachi of fish-versatile primers (L15285-CYB + H2582-16S; Software Engineering Co. Ltd, Yokohama, Japan). The Table 1). location of the 13 protein-coding genes was determined 926 FISHERIES SCIENCE JG Inoue et al.

Table1 PCR and sequencing primers in the analysis of Japanese sardine mitochondrial genome Primers Sequence (5¢Æ3¢) Long PCR primers Same-16S-L1 CAA CCA CGA AAA GCG GCC CTA ATT GGA GCC Same-CYB-H1 GGC AGA TAG GAG GTT AGT AAT GAC AGT GGC L15285-CYB CCC TAA CCC GVT TCT TYG C H2582-16S ATT GCG CTA CCT TTG CAC GGT PCR and sequencing primers 1. L709-12S TAC ACA TGC AAG TCT CCG CA H1552-12S ACT TAC CGT GTT ACG ACT TGC CTC 2. L1340-12S ACG TCA GGT CGA GGT GTA GC H1999-16S GCA ACC AGC TAT AAC TAG GCT CGG T 3. L1854-16S AAA CCT CGT ACC TTT TGC AT H2590-16S2 ACA AGT GAT TGC GCT ACC TT 4. L2510-16S2 CGC CTG TTT AAC AAA GAC AT H3058-16S TCC GGT CTG AAC TCA GAT CAC GTA 5. L2949-16S2 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-ND12 CGA TAT GAT CAA CTM ATK CA H4866-ND22 AAK GGK GCK AGT TTT TGT CA 8. L4633-ND22 CAC CAC CCW CGA GCA GTT GA H5334-ND22 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-Ala2 GCA AMT CAG ACA CTT TAA TTA A H6371-CO12 TTG ATT GCC CCK AGG ATW GA 11. L6199-CO12 GCC TTC CCW CGA ATA AAT AA H6855-CO12 AGT CAG CTG AAK ACT TTT AC 12. L6730-CO12 TAT ATA GGA ATR GTM TGA GC H7480-Ser2 ATG TGG YTG GCT TGA AA 13. L7255-CO12 GAT GCC TAC ACM CTG TGA AA H8168-CO22 CCG CAG ATT TCW GAG CAT TG 14. L7863-CO22 ATA GAC GAA ATT AAT GAC CC H8589-ATP2 AAG CTT AKT GTC ATG GTC AGT 15. L8329-Lys2 AGC GTT GGC CTT TTA AGC H9076-ATP2 GGG CGG ATA AAK AGG CTA AT 16. L8894-ATP2 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-Arg2 AAC CAT GGW TTT TTG AGC CGA AAT 19. L10056-Gly CTT GGT TAA AKT CCA AGG AAA G H10970-ND42 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-ND42 TGA CTT CCW AAA GCC CAT GTA GA H12632-ND52 GAT CAG GTT ACG TAK AGK GC 22. L12191-His TTG TGA TTC TAA AAA TAG GGG TTA AA H13069-ND52 GTG CTG GAG TGK AGT AGG GC 23. L12936-ND5 AAC TCM TGG GAG ATT CAA CAA H13727-ND52 GCG ATK ATG CTT CCT CAG GC 24. L13562-ND52 CTW AAC GCC TGA GCC CT H14718-Gln TTT TTG TAG TTG AAT WAC AAC GGT 25. Same-ND5-L1 GCA CAA CTT CTC AAA TAT ACT TGG Same-ND6-H1 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-L1 CCC GGT AAA TCG ATT AAA CCC C H1065-12S 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 genome30 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 Japanese sardine mitochondrial genome FISHERIES SCIENCE 927

1 2 3 4 56789 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 2627 29 30 FV LIMWDK GR HSL T

Q ANCY S EP 30 1 2 3 4 567 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 2524 26 27 28 29

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 single- letter 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 + H2582-16S) 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 the previously determined sequences. Consequently, the bony fish mitochondrial genomes. The 22 tRNA genes mitochondrial genome of Japanese sardine was purified were identified by their proposed cloverleaf secondary by gene amplification, providing templates for subse- structures and anticodon sequences.33 The two rRNA quent amplifications and direct sequencing of contigu- genes were identified by sequence homology and pro- ous, overlapping segments of the entire genome using the posed secondary structure.34 Sequence data are available 30 sets of primers (Fig. 1; Table 1).24 from DDBJ/EMBL/GenBank under accession number AB032554. Genome content and base composition

RESULTS AND DISCUSSION The total length of the Japanese sardine genome was 16 881 bp. The complete L-strand nucleotide sequence of Long PCR and sequencing strategy Japanese sardine, is shown in Fig. 2. The genome content of Japanese sardine included two rRNA, 22 tRNA, 13 Recent development of a long-PCR technique enabled protein-coding genes, and a control region, as found in us to amplify up to a 35 kb target sequence with high other vertebrates (Figs 1,2; Table 2). As in other verte- fidelity.35,36 Although Cheng et al.37 successfully ampli- brates, most genes were encoded on the H-strand, except fied 16.3 kb of the 16.6 kb human mitochondrial genome for the ND6 and eight tRNA genes, and all genes were in a single long PCR, our preliminary experiments similar in length to those in other bony fishes. The gene demonstrated that such a single long PCR was not fea- order is identical to those so far obtained in other sible for the Japanese sardine mitochondrial genome, vertebrates. probably because of GC-rich regions in the putative The base composition of Japanese sardine was ana- control region. Strings of C and G bases often inhibit lyzed separately for rRNA, tRNA, and protein-coding PCR reactions. genes (Table 3). In the protein-coding gene, anti-G bias Alternatively, we decided to divide the circular mito- was observed in the third codon positions (18.0%). chondrial genome into two segments (Fig. 1): one long Pyrimidines were overrepresented in the second codon segment was expected to cover all protein-coding and positions (66.5%), as has been noted for other vertebrate most tRNA genes, spanning from the 16S rRNA to the mitochondrial genomes, owing to the hydrophobic cyt b genes, and another short segment was expected to character of the proteins.38 Japanese sardine tRNA genes cover the two rRNA genes and the entire putative were slightly A + T rich (52.0%), as in other vertebrates, control region, spanning from the cyt b to the 16S rRNA while rRNA genes have a high adenine content genes. Since we had already determined two partial (30.9%), as in other bony fishes.39 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 Protein-coding genes long segment. The short segment, on the other hand, was amplified using two fish-versatile primer (L15285-CYB + Among the 13 protein-coding genes of Japanese sardine, H2582-16S), because of their nearly perfect matching to there were two reading-frame overlaps on the same strand 928 FISHERIES SCIENCE JG Inoue et al.

Table2 Location of features in the mitochondrial genome of Table3 Base composition of the mitochondrial genome of Japanese sardine Japanese sardine Gene Position number Size (bp) Codon ACG T From To Start Stop Proteins tRNAPhe 16969 1st 25.8 27.1 26.8 20.3 12S rRNA 70 1028 959 2nd 19.3 27.8 14.2 38.7 tRNAVal 1029 1100 72 3rd 24.5 34.3 18.0 23.2 16S rRNA 1101 2783 1683 Total 23.2 29.7 19.7 27.4 tRNALeu(UUR) 2784 2858 75 tRNA 28.5 26.3 21.7 23.5 ND1 2859 3833 975 ATG TAG rRNA 30.9 26.2 22.7 20.2 tRNAIle 3843 3914 72 tRNAGln 3914 3984 71 (L) tRNAMet 3984 4052 69 ND2 4053 5098 1046 ATG TA– tRNATr p 5099 5170 72 tRNAAla 5174 5242 69 (L) tRNAAsn 5243 5315 73 (L) protein-coding genes (Figs 1,2). The tRNA genes range tRNACys 5349 5414 66 (L) in size from 66 to 76 nucleotides (Table 2), large enough tRNATy r 5417 5487 71 (L) so that the encoded tRNA can fold into the cloverleaf COI 5489 7039 1551 GTG TAA secondary structure characteristic of tRNA (data not tRNASer(UCN) 7040 7110 71 (L) shown). This is possible provided that formation of the tRNAAsp 7115 7183 69 G–U wobble and other atypical pairings were allowed in COII 7197 7887 691 ATG T— the stem regions. All postulated cloverleaf structures Lys tRNA 7888 7961 74 contained 7 bp in the amino acid stem, 5 bp in the TYC ATPase 8 7963 8130 168 ATG TAA stem, 5 bp in the anticodon stem, and 4 bp in the DHU ATPase 6 8121 8803 683 ATG TA– stem (3 bp in tRNASer(AGY)). COIII 8804 9588 785 ATG TA– tRNAGly 9589 9660 72 ND3 9661 10 009 349 ATG T— tRNAArg 10 010 10 079 70 Ribosomal RNA genes ND4L 10 080 10 376 297 ATG TAA ND4 10 370 11 750 1381 ATG T— The 12S and 16S rRNA genes of Japanese sardine were tRNAHis 11 751 11 820 70 959 and 1683 nucleotides long, respectively (Table 2). tRNASer(AGY) 11 821 11 887 67 They were located, as in other vertebrates, between Leu(CUN) tRNA 11 888 11 959 72 genes of the tRNAPhe and tRNALeu, being separated by ND5 11 960 13 795 1836 ATG TAA the tRNAVal gene (Figs 1,2). Preliminary assessment of ND6 13 792 14 310 519 (L) ATG TAG their secondary structure indicated that the present tRNAGlu 14 329 14 397 69 (L) Cyt b 14 402 15 542 1141 ATG T— sequences could be reasonably superimposed on the pro- tRNAThr 15 543 15 612 70 posed secondary structure of carp 12S rRNA and loach 34 tRNAPro 15 612 15 681 70 (L) 16S rRNA, respectively. Control region 15 682 16 881 1200

(ATPases 8 and 6 shared 10 nucleotides; ND4L and ND4 shared seven nucleotides) (Fig. 2). As in other bony Fig. 2 The complete L-strand nucleotide sequence of the fishes, all the mitochondrial protein-coding genes began Japanese sardine mitochondrial genome. Position 1 corre- with an ATG start codon, except for COI, which starts sponds to the first nucleotide of the tRNAPhe gene. Direction with GTG (Table 2). Open-reading frames of Japanese of transcription for each gene is shown by arrows. Beginning sardine ended with TAA (COI, ATPase 8, ND4L, and and end of each gene are indicated by vertical bars ( | ). Trans- ND5), TAG (ND1 and ND6), and the remainder had fer RNA genes are boxed; corresponding anticodons are indi- incomplete stop codons, either TA (ND2, ATPase 6, and cated in black boxes. Amino acid sequences presented below COIII) or T (COII, ND3, ND4, and cyt b) (Table 2). 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 Transfer RNA genes underlined with dots. TAS, putative termination-associated sequence; CSB 2, 3, and D, conserved sequence blocks. The Japanese sardine mitochondrial genome contained Sequence data are available from DDBJ/EMBL/GenBank with 22 tRNA genes interspersed between the rRNA and accession number AB032554. Japanese sardine mitochondrial genome FISHERIES SCIENCE 929 930 FISHERIES SCIENCE JG Inoue et al. Japanese sardine mitochondrial genome FISHERIES SCIENCE 931

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