Report of Systematic Zoology Lab Practicum, Volume 5: e10; August, 2014

An attempt to sequence the mitochondrial cytochrome c oxidase subunit I gene of an unidentified gammaridean amphipod (Arthropoda: Malacostraca: Amphipoda) from Oshoro Bay, Hokkaido, Japan

Yuka Ogata and Konomi Toyooka

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

Material and Methods
A gammaridean amphpod was obtained in subtidal area at Oshoro Bay, Hokkaido, Japan, about 43°12′N, 140°51′E, on 2 June 2014 by Yuka Ogata and Konomi Toyooka, then photographed and fixed in 99% EtOH by Takumi Onishi. DNA was extracted from the posterior half of the EtOH-fixed 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 ICHU 2120798 (contact: Dr. Hiroshi Kajihara, kazi@mail.sci.hokudai.ac.jp).
      Amplification of the mitochondrial cytochrome c oxidase subunit I gene (COI) using LCO1490 (5′-GGTCAACAAATCATAAAGATATTGG-3′) and HCO2198 (5′-TAAACTTCAGGGTGACCAAAAAATCA-3′) (Folmer et al. 1994) was performed by a hot start PCR 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 min 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 ver. 5 (Tamura et al. 2011).

Results
Unfortunately, the chromatogram data generated by the sequencer were so dirty that no reliable sequence was determined from our material.

Taxonomy
Phylum Arthropoda
Class Crustacea
Order Amphipoda
Gammaridea sp.
(Fig. 1)



Fig. 1. Gammaridea sp. (ICHU22090030) from Oshoro Bay, Hokkaido, northern Japan.




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.