Molecular techniques to identify freshwater eels: RFLP analyses of PCR-amplified DNA fragments and allele-specific PCR from mitochondrial DNA

Abstract

Because of morphological ambiguity, traditional classification of freshwater eel elvers (of the genus Anguilla) has always been difficult and unreliable. This study analyzes 2 mitochondrial genes viz., cytochrome b and 12S ribosomal RNA (rRNA) genes from previous studies, to establish molecular standards for eel identification. Prediction of restriction fragment length polymorphism (RFLP) indicated that a combination of 3 enzyme results for cytochrome b revealed good resolution power. We also selected 39 specific nucleotide sites of the 2 genes at which the site is unique for individual species: thus these can potentially be used for identification by polymerase chain reaction (PCR). To verify the accuracy of our predictions, we examined 58 specimens from 4 species by DpnII and HaeIII digestions and by PCR amplification using species-specific primers. Consistent with the putative mutation rates, more than 95% and 99% of the specimens could be successfully identified by the RFLP and PCR methods, respectively. The molecular techniques developed here can be helpful in eel aquaculture and ecological studies.