Identification of six grouper species under the genus Epinephelus (Bloch, 1793) from Indian waters using PCR-RFLP of cytochrome c oxidase I (COI) gene fragment

Abstract

Groupers are biologically diverse, commercially important and highly regarded of all fish species associated with reef fisheries globally. Given the huge demand and high value in live fish trade, restaurants and supermarkets around the world, the accurate identification of the closely related grouper species is crucial to prevent any adulteration. Partial nucleotide sequence data of the mitochondrial cytochrome c oxidase I (COI) gene, which is globally accepted as fish DNA barcoding gene, was generated for six commercially important grouper species belonging to the genus, Epinephelus viz. E. fuscoguttatus, E. coioides, E. logispinis, E. malabaricus, E. bleekeri and E. tauvina collected from Indian waters. Based on the analysis of 704 bp COI gene sequences, a PCR-RFLP method was developed to identify all the six species accurately. The PCR products of COI gene fragment was digested with selected restriction enzymes viz. HinfI, AseI, HindIII and BsaJI. Restriction endonuclease HinfI was enabled to produce species-specific fragments for E. coioides, E. malabaricus, E. logispinis and E. bleekeri. The result shows that AseI restriction enzyme could be used to differentiate E. fuscoguttatus from other five species. The expected and actual molecular weight of the digested products was similar indicating the authenticity of the methods followed. This is the first report on differentiating closely related grouper species using above mentioned restriction endonucleases and COI gene fragment. PCR-RFLP profiles generated could be useful for detecting seafood fraud by identifying the grouper species under the genus Epinephelus using samples from fresh, frozen, cooked and even processed products.