Report of Systematic Zoology Lab Practicum 1: e1 (August 2010)

28S rDNA partial sequence of Anthopleura pacifica Actinia equina (Cnidaria: Anthozoa: Actiniaria: Actiniidae)

Marina Takahashi

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

Material and Methods
A sea anemone speciman was obtained subtidally at Oshoro Bay, Hokkaido, Japan, about 43°12′N, 140°51′E, on 23 May 2010 by Marina Takahashi, photographed and identified by Hiroshi Kajihara as Actinia equina based on Uchida (1992: 136, pl. 31-1), and fixed 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 ICHU22080080 (contact: Dr. Hiroshi Kajihara, kazi@mail.sci.hokudai.ac.jp).
      An about 1.2K-bp fragment of 28S rRNA gene was amplified by polymerase chain reaction (PCR) using LSU5 (5′-ACCCGCTGAAYTTAAGCA-3′) and LSU3 (5′-TCCTGAGGGAAACTTCGG-3′) (Littlewoodet 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
A total of 1074 bases of 28S rDNA sequence was determined from Actinia equina (see Appendix).

Taxonomy
Class Anthozoa
Order Actiniaria
Family Actiniidae Rafinesque, 1815
Genus Actinia Linnaeus, 1767
Actinia equina (Linnaeus, 1758)
(Figs 1–3)



Fig. 1. Anthopleura pacifica Actinia equina (ICHU22080080), showing the mouth and tentacles.


Fig. 2. Anthopleura pacifica Actinia equina (ICHU22080080), oblique view of the body.


Fig. 3. Anthopleura pacifica Actinia equina (ICHU22080080), close-up of the mouth and tentacles.




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.