Report of Systematic Zoology Lab Practicum, Volume 4: e11; August, 2013


Cytochrome c oxidase subunit I partial sequence of the chiton Lepidozona coreanica (Mollusca: Polyplacophora: Neoloricata) from Oshoro, Hokkaido, northern Japan


Satoko Mori and Kusuko Obara

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


Material and Methods
A chiton was obtained in Oshoro Bay, Hokkaido, Japan, about 43°12′N, 140°51′E on 12 June 2013 by Satoko Mori and Kusuko Obara. It was photographed, fixed in 99% EtOH, and identified by Hiroshi Kajihara as Lepidozona coreanica by a reference to Saito (2000). DNA was extravted from the caudal portion 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 ICHU2110816 (contact: Dr. Hiroshi Kajihara, kazi@mail.sci.hokudai.ac.jp).
      An about 700-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 (ration at Applied Biosystems) in a 20-µl reaction volume containing 1µl 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 MEGA v5 software (Tamura et al. 2011).


Results
After eliminating the primer sites, a total of 676 bp of COI sequence was determined from Lepidozona coreanica (see Appendix).


Taxonomy
Phylum Mollusca
Class Polyplacophora
Order Neuloricata
Family Ischnochitonidae Dall, 1899
Genus Lepidozona Pilsby, 1892
Lepidozona coreanica (Reeve, 1847)
[Japanese name: yasuri-hizaragai]
(Figs 1, 2)


Fig. 1. Lepidozona coreanica (ICHU2110816) from Oshoro, Hokkaido, Japan, dorsal view.



Fig. 2. Lepidozona coreanica (ICHU2110816) from Oshoro, Hokkaido, Japan, ventral veiw.




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.

Saito, H. 2000. Ischnochitonidae. Pp. 9–15. In: Okutani, T. Marine Molluscks in Japan. Tokai University Press, Tokyo.

Tamura, K.,Peterson, D., Peterson, N., Stecher, G., Nei, M. and Kumar, S. 2011. MEGA5: molecullar evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution 28: 2731–2739.




Appendix
Partical COI sequence (676 bp) from ICHU2110816 identidied as Lepidozona coreanica collected in Oshoro Bay, Hokkaido, Japan.

TGGTCAACAAAATCATAAAACTCTTTATATTTTATTTGGAATTTGAGCAGGGTTAGTTGGGACGGCTTTAAGATTATTAATTCGAGCTGAATTAGGCCAACCTGGGGCTTTATTAGGGGATGATCAATTATATAATGTTATTGTAACTGCTCATGCTTTTGTTATAATTTTTTTTTTAGTTATGCCTATAATAATTGGAGGATTCGGAAACTGGTTAGTTCCTTTAATATTAGGGGCTCCAGATATAGCGTTTCCTCGTTTAAACAATATAAGATTTTGATTACTTCCACCTGCTTTATGTTTATTACTAGGATCTGCAGCAGTTGAAAGAGGAGCTGGGACAGGATGAACTGTTTACCCTCCTTTAGCTAGTAATATCGCGCATGCTGGCGGGTCAGTAGATTTAGCAATTTTTTCATTACACTTAGCAGGGGTTTCTTCTATTTTAGGTGCAGTAAATTTTATTACTACGGTGTTTAATATACGTTGAAAGGGAATACAAATAGAACGGTTACCCTTATTTGTTTGATCTGTAAAAATTACCGCTGTACTATTACTTTTATCTTTACCAGTTTTAGCTGGAGGGATTACTATATTATTAACAGATCGAAATTTTAATACCACATTTTTTGATCCTGCAGGAGGAGGAGACCCTATTTTATATCAGCATTTATTT