Genetic characterization of freshwater fishes using DNA barcoding with 12S rRNA gene from rivers of Malakand division, Pakistan

Identification and classification at the species level are central to fisheries research and planning conservation strategies. The current study aimed to identify, classify and characterize selected fish species at the molecular level using the 12S rRNA gene in the freshwater of Malakand division, Pakistan. A total of nine samples representing six species were sampled and identified morphologically. Muscle tissue was employed to extract DNA, conduct PCR and sequence using specific markers. All sequences were edited and trimmed using BioEdit, followed by submission to GenBank for accession numbers. MEGA12 was used to calculate genetic distances and the construction of phylogenetic trees through ML, NJ, ME, MP and UPGMA models. The DnaSP software package was used to calculate the genetic diversity indices. The number of nucleotide character sites was 509 singleton, 99 parsimony informative, 609 variable and 320 conserved. The average numbers of T, C, A and G were 167, 210, 269 and 183, respectively. GC% content was lower (47.36%) than AT% content (52.64%), with an average base-pair length of 845 nucleotides. The range of within-species K2P distances was between 0.000 and 0.00329. Similarly, the mean K2P within the genus distance for Carassius was 0.0067, Tor 0.012, Channa 0.105 and Garra 0.038. The number of haplotype sites for Carassius auratus, Tor putitora, Tariqilabeo latius, Channa punctata and Mastacembelus armatus were 2, 3, 3, 1 and 1, respectively, while the nucleotide diversity π was 0.429, 1.000, 0.800, 0.000 and 0.000, respectively. All phylogenetic trees were congruent. The current study favors the use of 12S rRNA as a marker for the identification and characterization of fish species and urges its extension to other species globally.