Species Identification of 3 Hypsibarbus spp. (Pisces: Cyprinidae) Using PCR-RFLP of Cytochrome b Gene

Polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) was used to identify 3 closely related Hypsibarbus spp: Hypsibarbus wetmorei, H. vernayi, and H. malcolmi. Mitochondrial cytochrome b gene (993 bp) in 3 Hypsibarbus spp. showed single PCR– product. The sequencing results of PCR–products in 3 Hypsibarbus spp. showed very low interspecific variation. However it could be used to discriminate these species by RFLP analysis. The combination of 2 restriction enzymes; Bsp143I and BcuI were used to identify 3 Hypsibarbus spp. Bsp143I could discriminate H. vernayi from H. wetmorei and H. malcolmi, by generating 3 fragments (535 bp, 234 bp and 224 bp) in H. vernayi whereas 2 fragments of 769 bp and 224 bp in H. wetmorei and H. malcolmi. Thereafter, BcuI was effectively discriminated H. wetmorei from H. malcolmi by generating 3 fragments (591 bp, 288 bp and 114 bp) in H. malcolmi and uncut fragment in H. wetmorei. There were intraspecific restriction polymorphism in H. vernayi using BcuI which generated 2 patterns; an uncut fragment and 2 fragment of approximately 700 bp and 300 bp. Thus, PCR–RFLP technique could be used to identify 3 closely related Hypsibarbus spp.