DNA barcoding and inferred genetic diversity of epinephelinae groupers from Peninsular Malaysia using mitochondrial cytochrome c oxidase I gene and microsatellites

This study was conducted with the purpose of validating the species identity and genetic diversity of groupers from Peninsular Malaysia. All samples were collected from across the Peninsular Malaysian waters and stored in ethanol (95%) for short term storage before being placed into -20°C freezer. All steps were done in a sterile environment to avoid any contamination of samples. A total of 131 individuals comprising of 12 species from three genera were collected from coastal areas of Johor (N=4), Kedah (N=12), Kelantan (N=4), Malacca (N=4), Negeri Sembilan (N=2), Pahang (N=14), Perak (N=15), Selangor (N=22) and Terengganu (N=54). All 12 species were identified as Cephalopholis boenak, C. formosa, C. miniata, Epinephelus areolatus, E. bleekeri, E. coioides, E. corallicola, E. erythrurus, E. fuscoguttatus, E. poecilonotus, Plectropomus leopardus and P. maculatus. The first part of the study was on DNA barcoding analysis using Cytochrome c Oxidase I gene on all 131 collected individuals. Deoxyribonucleic acid extraction and Polymerase Chain Reaction were done to all collected samples before being sent for sequencing. The successful sequences were validated through National Center for Biotechnology Information database (using BLAST: Basic Local Alignment Search Tool) and most of the sequences were correctly matched with their respective grouper species (identity index ≥95%), except for eight individuals which showed discrepancy in taxonomic identification. A barcode gap was present and the lowest genetic pairwise distance was between P. leopardus and P. maculatus (4.5%), while the highest distance was between C. miniata and P. leopardus (23.8%). The phylogenetic trees showed that there were two main clades present which separated the genera Cephalopholis and Epinephelus with genus Plectropomus. Sister clades were found between P. leopardus and P. maculatus with strong bootstrapping confidence level. This concludes that species with the same genus tend to be closely related. In the second part of the study, five sets of microsatellite markers which were used for genetic diversity analysis showed polymorphism in the three selected species (Epinephelus areolatus, E. bleekeri and E. coioides: N=64) from two populations (East Peninsular Malaysia and West Peninsular Malaysia). PCR annealing temperatures are different for all species. The markers were substituted into labeled primers, and all successful PCR products of the polymorphic markers were sent for fragment analysis (genotyping). The highest number of alleles per locus can be found in E. areolatus populations ranging from seven to 32 alleles, followed by E. bleekeri populations ranging from four to 23 alleles, and lastly E. coioides populations which ranged from five to 15 alleles. An estimation of Fixation index (FST) value for all microsatellite loci showed a small data. The highest value of percentage variation was in E. areolatus (79.74%), followed by E. coioides (67.88%) and E. bleekeri (67.47%). In addition, the population structuring for all three species showed a similar pattern. Hence, the two populations for all species did not show significant differences which indicated that all individuals were in the same gene pool. In conclusion, DNA barcoding approach is suitable for species validation and phylogenetic study among groupers and polymorphic microsatellites is proven to be suitable for determining the genetic diversity of a species.

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