SEQUENCING REPORT OF COX1 GENE-BASED AMPLICONS IN TEN ISOLATES OF GRASS CARP

One specific PCR fragment partially covering the coding regions of the cytochrome c oxidase subunit I encoding gene was selected in this study. The amplified fragments were directly exposed to Sanger sequencing experiments to assess the pattern of genetic polymorphism in the collected fish samples. Then, a specific comprehensive tree was generated to assess the accurate identification of the observed variants and their phylogenetic distribution. Sequencing reactions showed the accurate identity of the investigated samples due to the presence of one species, as it was confirmed that S1-S10 belonged to grass carp (Ctenopharyngodon idella). Further details were also observed from the investigated sequencing reactions as a total of five nucleic acid substitutions were observed in some of the analyzed fishes. It was found that the most variable type was grass carp due to the presence of five nucleic acid variants compared with its referring sequences (GenBank acc. no. MG827396.1). The identified five nucleic acid variations (324T>C, 506T>A, 609T>A, 473T>C, and 578T>G) were found in some of the grass carp with three silent (p.135N=, p.196L=, and p.230L=) and two missense (p.185V>A and p.220F>C) effects respectively. The generated phylogenetic tree was constructed to incorporate five phylogenetic clades with distinct phylogenetic distances. It was inferred from the tree that the grass carp group was suited in the vicinity to the clade of Prussian carp. This positioning referred to the fact that both species shared the highest homology than the other incorporated outgroup species. The detected nucleic acid substitutions showed a slight effect of the observed variations on the evolutionary positioning of grass carp samples in comparison with the other investigated referring sequences. This was due to the positioning of the variant samples (S1, S2, and S6) slightly away from the other wild-type seven samples of the grass carp group.