Report of Systematic Zoology Lab Practicum, Volume 5: e17; August, 2014


28S rRNA partial sequence of cf. Epiactis japonica (Cnidaria: Anthozoa) from Oshoro Bay, Hokkaido, Japan


Ryotaro Nouda an Nodoka Satoh

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


Material and Methods
A sea anemone was obtained under rock at Oshoro Bay, Hokkaido, Japan, about 43°12′N, 140°51′E, on 9 June 2014 by Ryotaro Nouda; it was tentatively identified by Hiroshi Kajihara as Epiactis japonica (Verrill, 1869), and photographed and fixed in 99% EtOH by Takumi Onishi. Total DNA was extracted from a piece of body wall 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 2122044 (contact: Dr. Hiroshi Kajihara, kazi@mail.sci.hokudai.ac.jp).
      Hot start PCRs were 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). Amplification of the mitochondrial cytochrome c oxidase subunit I (COI) gene using LCO1490 (5′-GGTCAACAAATCATAAAGATATTGG-3′) and HCO2198 (5′-TAAACTTCAGGGTGACCAAAAAATCA-3′) (Folmer et al. 1994) was unsuccessful. An about 1.2K-bp fragment of the 28S rRNA gene was amplified by using the primer pair LSU5 (ACCCGCTGAAYTTAAGCA) and LSU3 (TCCTGAGGGAAACTTCGG) (Littlewood. 1994). The 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 sec (COI) or 3 min (28S); 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. 2007).


Results
Phylum Cnidaria
Class Anthozoa
Order Actiniaria
Family Actiniidae Rafinesque, 1815
Genus Epiactis Verrill, 1869
Epiactis japonica (Verrill, 1869)
[Japanese name: komochi-isoginchaku]

(Fig. 1)

Because the chromatogram corresponding to the reverse primer was largely distrubed, only 227 bp near the forward primer of the 28S rRNA gene was reliably determined (see Appendix).



Fig. 1. Epiactis japonica (Verrill, 1869), ICHU2122044, photograph taken in life.




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.

Littlewood, D. T. 1994. Molecular phylogenetics of cupped oysters based on partial 28S rRNA gene sequences. Molecular Phylogenetics and Evolution 3: 221–229.

Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., and Kumar, S. 2011. MEGA5: Molecullar Evolutionary Genetics Analysis (MEGA) software version 5.0. Molecular Phylogenetics and Evolution 28: 2731–2739.

Appendix
28S rRNA sequence from ICHU2122044 identidied as Epiactis japonica (Verrill, 1869).

CCACCAGAGTTTCCTCTGGCTTCACCCTATTCAGGCATAGTTCACCATCTTTCGGGTCCCAACAGATGCGCTCTTACTCAAACCTTCCTAAGAGTAGAATAGGTCGGTCGATGATGCGCCCCCCGACGAGGGGGGGTTCTCACCTCAGCGGCCGAGGCCGCCTTCACTTTCATTCCGCCTGGGAGTTTCGATCACTCGAAGACTCGCGCACATGTTAGACTCCTTGG