DNA Barcode Development of Two Catfishes Heteropneustes fossilis and Mystus bleekeri from Upper Lake Bhopal, Madhya Pradesh, India

Aims: To develop cox1 based DNA barcodes for Heteropneustes fossilis and Mystus bleekeri, from Upper Lake, Bhopal, to support accurate species identification and molecular taxonomy. Study Design: The study involved the collection and morphological identification of samples followed by DNA isolation, PCR amplification, Sanger sequencing, and barcode generation. Place and Duration of Study: Laboratory of molecular Biology and Genomics, Department of Bioscience, Barkatullah University, Bhopal (M.P.), India, between March 2024 to July 2025. Methodology: The genomic DNA was extracted using Phenol:Chloroform:Isoamyl (25:24:1) method. The quality of extracted DNA was determined on a 1% agarose gel. The cox1 gene fragments were amplified using universal primer (FishF1 and FishR1). Sequenced through Sanger sequencing and analysed using BLAST, MEGA11, and BOLD systems for species identification, phylogenetic assessment and barcode development. Results: In this study, two samples were analysed to generate DNA barcodes. A 615 bases long fragment of the cox1 gene was sequenced, resulting sequences (658 bp for H. fossilis and 609 bp for M. bleekeri) were submitted to GenBank (accession number PP754237 and PX116877). Sequence composition analysis revealed a moderate AT-bias (A+T = 56.35%) with the highest GC content at the first codon position. BLAST analysis confirmed 99-100% similarity with reference sequences, validating species level identification. Phylogenetic analyses using Neighbor-Joining (NJ) trees highlighted contrasting patterns: H. fossilis sequences clustered into a single cohesive clade with low divergence across South Asia, while M. bleekeri grouped within a diverse genus level dataset showing clear interspecific separation but some overlap with congeners. Conclusion: These findings confirm the reliability of cox1 as a DNA barcode for both species. Overall, this study contributes validates barcodes for two freshwater catfishes of Upper Lake and emphasizes the value of DNA barcoding in fish taxonomy, biodiversity.