Report of Systematic Zoology Lab Practicum, August, 2010

Cytochrome c oxidase subunit I partial sequence of Coryphella athadona (Nudibranchia: Coryphellidae: Coryphella)

Yuka Mori

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

Material and Methods
A sea slug specimen was obtained subtidally at Oshoro Bay, Hokkaido, Japan, about 43°12′N, 140°51′E, on 24 May 2010 by Yuka Mori, photographed and identified by Hiroshi Kajihara as Coryphella athadona (Bergh, 1875) based on Hamatani (2000: 803, pl. 399, fig. 1), Anterior half of the body wasfixed in 10% formalin as a morphological voucher and posterior half in 99% EtOH. DNA was extracted 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 ICHU22080101 (contact: Dr. Hiroshi Kajihara, kazi@mail.sci.hokudai.ac.jp).
      An about 600-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, iCycler (Bio-Rad), 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 MEGA v4 software (Tamura et al. 2007).

Results
Due to the poor chromatogram data, only a total of 214 bp of COI sequence was determined from the material (see Appendix).

Taxonomy
Order Nudibranchia
Family Coryphellidae
Genus Coryphella
Coryphella athadona (Bergh, 1875)




Fig. 1. Coryphella athadona (Bergh, 1875), ICHU22080101, dorsal view.




Fig. 2. Coryphella athadona (Bergh, 1875), ICHU22080101, head.



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 Microbiology28: 495–503.