Report of Systematic Zoology Lab Practicum, Volume 4: e01; August, 2013


Cytochrome c oxidase subunit I partial sequence of Crassostrea gigas (Mollusca: Bivalvia: Ostreidae)


Yuuki Kawagishi and Mio Sato

Division of Biology, Department of Biological Sciences, School of Science, Hokkaido University, Sapporo 060-0810, Japan


Material and Methods
A pacific oyster was obtained in Oshoro Bay, Hokkaido, Japan, about 43°12′N, 140°51′E, on 20 May 2013 by Yuuki Kawagishi and Mio Sato, photographed and identified by Hiroshi Kajihara as Crassostrea gigas (Thunberg, 1793), and fixed in 99% EtOH. DNA was extracted from a piece of the body using the silica method (Boom et al. 1990) with some modifications. Extracted DNA was dissolved in 30 µl of deionized water and has been preserved at –20°C. Remaining morphological voucher specimen has been deposited at the Hokkaido University Museum under the catalogue number ICHU2110040 (contact: Dr. Hiroshi Kajihara, kazi@mail.sci.hokudai.ac.jp).
      An about 500-bp fragment of mitochondrial cytochrome c oxidase subunit I gene (COI) was amplified by polymerase chain reaction (PCR) using LCO1490 (5′-GGTCAACAAATCATAAAGATATTGG-3′) and HCO2198 (5′-TAAACTTCAGGGTGACCAAAAAATCA-3′) (Folmer et al. 1994). A hot start PCR was performed by a thermal cycler, 2720 Thermal Cycler (Applied Biosystems), in a 20-µl reaction volume containing 1 µl of template total DNA (approximately 10–100 ng) and 19 µl of premix made with 632-µl deionized water, 80-µl Ex Taq Buffer (TaKara Bio), 64-µl dNTP (each 25 mM), 8-µl each primer (each 10 µM), and 0.1-µl TaKara Ex Taq (5 U/µl,TaKara Bio). Thermal cycling condition comprised an initial denaturation at 95°C for 30 sec; 30 cycles of denaturation at 95°C for 30 sec, annealing at 45°C for 30 sec, and elongation at 72°C for 45°C and a final elongation at 72°C for 7 min.
      The PCR product was purified with the silica method (Boom et al. 1990). Both strands were sequenced with a BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) following the manufacturer's protocol, using the same primer set as the initial PCR amplification. Sequencing was performed with ABI Prism 3730 DNA Analyzer (Applied Biosystems). Chromatogram and sequence data were operated with MEGA5.05 software (Tamura et al. 2011).

Results
A total of 624 bp of COI sequence was determined from Crassostrea gigas (Thunberg, 1793) (see Appendix).


Taxonomy
Phylum Mollusca
Class Bivalvia Linnaeus, 1758
Order Ostreoida
Family Ostreidae Rafinesque, 1815
Genus Crassostrea Sacco, 1897
Crassostrea gigas (Thunberg, 1793)
[Japanese name: magaki]
(Figs 1, 2)



Fig. 1. Crassostrea gigas (Thunberg, 1793), ICHU2110040, entire specimen.


Fig. 2. Crassostrea gigas (Thunberg, 1793), ICHU2110040, inside of the specimen.




References

Boom, R., Sol, C. J. A., Salimans, M. M. M., Jansen, C. L., Wertheim-van Dillen, P. M. E., and van der Noordaa, J. 1990. Rapid and simple method for purification of nucleic acids. Journal of Clinical Microbiology 28: 495–503.

Folmer, O., Black, M., Hoeh, W., Lutz, R. and Vrijenhoek, R. 1994. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology 3: 294–299.

Tamura, K.,D.,Peterson,N.,stecher,G., Nei, M. and Kumar, S. 2011. MEGA5.05: Molecullar Evolutionary Genetics Analysis (MEGA) software version 5.05 Molecular Phylogenetics and Evolution 28: 2731–2739.




Appendix

COI sequence (624 bases) from ICHU2110040, identified as Crassostrea gigas (Thunberg, 1793)

GCTGAAATAAGAAGGGTCCCCCCCTCCGACAGGGTCAAAAAAAGAGGTATTAAAATGACGATCAGTCAAAAGTATAGTAAGACCTCCAGCTAACACTGGGAGAGTAGTCAAAAGCAAGAATGAAGTAACCTTAATAGATCAAGGGAATAGTGCTAGTAAATGGCCCCCAACAGATCGCATATTTCTAATCGTTACTATGAAATTAATTGACCTGAAAATAGAGCTAATACCAGCAAGGTGAAGGCTTAGAATTGCAAGGTCTATACAAACTCCATGATAAGAGTAAGTTGATAAAGGAGGGTAAATTGTTCACCCTGCCCCAACTCCGTTTTCTACAATGTTAGACATAAGCATAAGATAAAGAGACCCTGGCAAAACTCAAAATCTAAATGCATTTAATCGAGGAAATTGCATGTCTGCTACTAGAAGCATCAAAGGGATAAGCCAGTTACCAAACCCCCCAATTATTACAGGTATAACAAAGAAAAAAATCATAACCAACGCATGCCTAGTTACAACTGCATTATAAGTCACGGGGTCTAAAAACTTAGCTCCAGGGTTATAAAGTCTCCAACGAATAAGAGACCTAAACCTAGTTCCCGCAAGAACAGCTCAAAATCCAAA