Report of Systematic Zoology Lab Practicum, August, 2011

28S rRNA partial sequence of an unidentified species of anthuridean (Crustacea: Isopoda)

Ruriko Mitani

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

Material and Methods
Material and Methods A harpacticoid specimen was obtained subtidally at Oshoro Bay, Hokkaido, Japan, about 43°12′N, 140°51′E, on 30 May 2011 by Ruriko Mitani, photographed by Hiroshi Kajihara and fixed in 99% EtOH. DNA was extracted from the entire 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.
      Amplification of mitochondrial cytochrome c oxidase subunit I gene (COI) using LCO1490 (5′-GGTCAACAAATCATAAAGATATTGG-3′) and HCO2198 (5′-TAAACTTCAGGGTGACCAAAAAATCA-3′) (Folmer et al. 1994) was unsuccessful.
      An about 1.2 K-bp fragment of mitochondrial 28S rRNA gene was amplified by polymerase chain reaction (PCR) using LSU5 (5′-ACCCGCTGAAYTTAAGCA-3′) and LSU3 (5′-TCCTGAGGGAAACTTCGG-3′) (Littlewood 1994). A hot start PCR was performed by a thermal cycler, iCycler (Bio-Rad), in a 20-μl reaction volume containing 1 µn;l of template total DNA (approximately 10?100 ng) and 19 µn;l of premix made with 632-µn;l deionized water, 80-µn;l Ex Taq Buffer (TaKara Bio), 64-µn;l dNTP (each 25 mM), 8-µn;l each primer (each 10 µn;M), and 0.1-µn;l TaKara Ex Taq (5 U/µn;l,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 BigDyeR 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 469 bp of 28S rRNA sequence was determined from ICHU22090100 (see Appendix).

Taxonomy
Order Isopoda
Anthuridea sp. (Figs 1, 2)



Fig. 1. Anthuridea sp.(ICHU22090100), lateral view.


Fig. 2. Anthuridea sp.(ICHU22090100), dorsal 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.