Report of Systematic Zoology Lab Practicum, August, 2011


Observation of an unidentified species of ascidian (Chordata: Ascidiacea)


Shohei Yamauchi

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



Material and Methods
An ascidian was obtained among laminarian holdfast at Oshoro Bay, Hokkaido, Japan, about 43°12′N, 140°51′E, on 13 June 2011 by Shohei Yamauchi, photographed by Hiroshi Kajihara, then fixed in 99% EtOH. DNA was extracted from the whole specimen, 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.
      A hot start PCR was performed using LCO1490 (5′-GGTCAACAAATCATAAAGATATTGG-3′) and HCO2198 (5′-TAAACTTCAGGGTGACCAAAAAATCA-3′) (Folmer et al. 1994), 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 &mul; 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 version 5.0 (Tamura et al. 2011).

Results
Due to the low quality of the chromatogram, the sequencing of COI region was unsuccessful.

Taxonomy
Phylum Chordata
Class Ascidiacea (Fig. 1)



Fig. 1. ICHU22090310, an unidentified species of ascidian, overall view.





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., 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 Phylogenetics and Evolution 28: 2731–2739.