Discrimination of Anemonefish Species by PCR-RFLP Analysis of Mitochondrial Gene Fragments
Chuta Boonphakdee* and Pichan Sawangwong
Graduate School of Environmental Science, Burapha University, Chonburi 20131, Thailand
Received 23 November 2007; Accepted 11 January 2008
Abstract
A means of discriminating among species of clown anemonefishes, based on restriction enzyme analysis of partial mitochondrial DNA sequences, was investigated. Mitochondrial 16S rRNA and cytochrome b genes from 6 species (7 strains) of anemonefish (Premnas biculeatus, Amphiprion polymnus, A. sandaracinos, A. perideraion, A. ocellaris, A. ocellaris var. and A. percula) were PCR-amplified. A 623-bp portion of 16S rRNA gene was obtained from different fishes using the same pair of primers. Further investigation of this 16S rRNA fragment, by restriction endonuclease digestion with BfuCI and RsaI, was not able to distinguish all fishes studied, but did yield 3 different digestion patterns. The first was specific to P. biculaetus, the sole member of the genus Premnas, while the remaining two separated the Amphiprion species into 2 groups: 1) A. polymnas, A. sandaracinos and A. perideraion, and 2) A. ocellaris, A. ocellaris var. and A. percula. In contrast to this, restriction endonuclease digestion of a 786-bp fragment of the cytochrome b gene with HinfI and RsaI, was able to differentiate different 7 anemonefishes. This utility marker is valuable for unambiguous species/strain identification of juvenile anemonefishes.
Keywords: anemonefish; species identification; 16S rRNA, cytochrome b; PCR-RFLP
* corresponding author : chuta@buu.ac.th