Comparison of three molecular methods for species identification of the family Cichlidae in Kwan Phayao, Thailand

Abstract

The species diversity of cichlids was investigated in Kwan Phayao from August 2016 to May 2017. Four cichlid species were found, including Oreochromis niloticus, Oreochromis mossambicus, Coptodon rendalli and Coptodon zillii. Due to similar characterizations, it is very difficult to identify each species. Three molecular methods were used to distinguish these four species. DNA barcodes or partial cytochrome c oxidase I (COI) gene sequences were amplified by PCR and sequenced. In Oreochromis sp. and Coptodon sp., 707- and 704-bp fragments were amplified, respectively. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis showed clear differences between the four cichlid species after digestion with three restriction enzymes, ScaI, HindIII and PdiI. ScaI and HindIII separated Oreochromis sp. from Coptodon sp. due to different fragment sizes. PdiI distinguished each cichlid species in the same genus. Finally, high resolution melting (HRM) analysis showed the sensitivity of the primers for discriminating these species with small amplicons and melting curves. From the comparison, HRM analysis was the most efficient method because the primer was shown to be sensitive for discriminating the four cichlids. In addition, it was inexpensive and required a short time to detect large samples. However, direct sequencing or DNA barcodes were still necessary in the case of the COI sequences of organisms of interest, which have not been reported in any databases. These four cichlids are alien species in Thailand; thus, species identification is very important for fishery management.