Molecular and bioinformatic approaches of BAC analyses in Clarias magur for gene mining, marker development, improving genome assembly and understanding genome organization

Bacterial artificial chromosome (BAC) is a library of clones, each containing a large DNA insert, which are reported to be valuable resource for physical chromosome mapping, gene mining, positional cloning and other genomic studies. Application of BAC library could be maximized when both the ends’ sequences of the clones' insert DNA are known and the genome information of the species is deciphered and available in the public domain. The purpose of this study was to highlight the utility of BAC resources. Thus, we describe here an inclusive approach using BAC end sequences and whole genome information to reveal the complete sequence of the clones, mine genes located on the clone and physically map the genes on the chromosome(s). We utilized the library of the BAC clones of Clarias magur, developed and maintained at our laboratory. BAC ends of the clones were sequenced on a capillary sequencer using plasmid purified DNA as well as T7 and pbRP1 primers. Both end sequences were then aligned onto the C. magur genome scaffolds and gene as well as repeat sequences were mined using bioinformatic tools. DNA probe was constructed and anchoring of genes on the chromosomes was achieved using FISH tool. Aligning of 3691 BAC end sequences on the genome scaffolds of C. magur followed by gene annotation of the aligned region of scaffolds resulted in unfolding of complete sequences of the clones and identification of 3734 annotated genes in the clones. The GC content and repetitive DNA elements of the aligned sequences of clones were 39.24% and 36.17%, respectively. Gene ontology assignment revealed genes’ involvement in the regulation of translation, signal transduction and many other functions. This approach is useful in retrieving full sequence of the clone, mining of genes from the clone, anchoring genes on the chromosomes, developing markers for chromosome cataloguing, understanding genome organization, improving genome assembly to chromosome level, targeted markers development etc. Further, the identified BAC clones containing unique and important genes could separately be maintained and may serve as an important genomic resource for downstream studies.