Report of Systematic Zoology Lab Practicum, Volume 4: e09; August, 2013


Cytochrome c oxidase submit I partial sequence of Patiria pectinifera (Echinodermata: Asterinidae)


Daiki Wakita and Takashige Funatsu

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


Material and Methods
A starfish was obtained subtidally at Oshoro Bay, Hokkaido, Japan, about 43°12′N, 140°51′E, on 27 May 2013 by Daiki Wakita, Takashige Funatsu, photographed and fixed in 99% EtOH by Hiroshi Kajihara. The specimen was identified as Asterina pectinifera by a reference to e.g. Oguro (1995), but the generic affiliation of the species herein adopted follows a molecular phylogenetic analysis by O'Loughlin and Waters (2004). DNA was extracted from the tubefoot of the animal 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 ICHU2110673 (contact: Hiroshi Kajihara, kazi@mail.sci.hokudai.ac.jp).
      An about 600-bp fragment of mitochondrial cytochrome c oxidase submit I gene (COI) was amplified by polymerase chain reaction (PCR) using LCO_As (5′-CCTTTTAGGCACTTCTATAAGAC-3′) and HCO_As(5′-CAAAACAAATGCTGGTAGAGAACG-3′) (unpublished). A hot start PCR was performed by a thermal cycler, 2720 Thermal Cycler (Applied Biosystems), 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 40°C for 30 sec, and elongation at 72°C for 45 sec 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. 2011).


Results
A total of 619 bp of COI sequence was determined from Patiria pectinifera (see Appendix).


Taxonomy
Phylum Echinodermata
Class Asteroidea
Order Valvatida
Family Asterinidae
Genus Patiria
Patiria pectinifera (Müller and Troschel, 1842)
[Japanese name: itomaki-hitode]
(Figs 1, 2)


Fig. 1. Patiria pectinifera (Müller and Troschel, 1842), ICHU2110673, aboral view.


Fig. 2. Patiria pectinifera (Müller and Troschel, 1842), ICHU2110673, oral 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.

O'Loughlin, P. M. and Waters, J. M. 2004. A molecular and morphological revision of genera of Asterinidae (Echinodermata: Asteroidea). Memoirs of Museum Victoria 61(1): 1–40.

Oguro, C. 1995. Asteroidea. Pp. 513–529. In: Nishimura, S. (Ed.) Guide to Seashore Animals of Japan with Color Pictures and Keys, Vol. II. Hoikusha, Osaka.

Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., and Kumar, S. 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution 28: 2731–2739.




Appendix

COI sequence from ICHU2110673 identified as Patiria pectinifera (Müller and Troschel, 1842).

GTGGTATTGACTTTTCGGTCTGTAAGAAGCATCGTAATTGCACCTGCAAGTACTGGTAGGGATAGAAGTAGGAGGAAAGCTGTTACAAATACTGATCAGACAAATAGGGGAAGTCGGTCAAATGAAATTCCAGGGGTTCGCATATTTATAACAGTTGTTATAAATTTTATTGAAGCAAGAATTGAGGAAGCTCCCGCCAGGTGAAGTGAGAATATCGCCAAGTCTACTGATCCGCCAGCATGCGCTAATCCCCTAGACAAAGGAGGGTAGATCGTTCATCCTGTTCCAGCACCTCTTTCTACACCTGCAGAAGCTAATAGGAGGAGAAAGGAAGGGGGGATTAGTCAGAATCTCATATTGTTCATTCGGGGGAAGGCCATGTCTGGAGCTCCGATCATTAGAGGTATTAGTCAGTTCCCGAATCCTCCAATCATGATTGGCATCACCATAAAGAAGATCATGACTAGGGCGTGTGCAGTAACTATTACTTTGTATATTTGGTCGTCTTGCAGTAAGGATCCAGGTTGCGCCAGTTCTGTTCGTATTATTACGCTCATTGCTGTTCCGGCCATTCCGGCTCAAGCCCCAAAGATAAGATAAAGGGTTCCAATGTCCTTGT