PCR amplification, sequencing, and in silico analysis of holandric genes TSPYand SRY in Nili Ravi buffalo

The present work has been designed to study the evolutionary perspectives of two important holandric genes, namely, testis-specific protein, Y-encoded (TSPY), and sex determining region, Y-encoded (SRY) in Nili Ravi buffalo. These Y-chromosomal genes are responsible for male sex development. The evolutionary aspects of these genes have not been studied in the Nili Ravi buffaloes from India. A study was carried out on the peripheral blood samples of three adult Nili Ravi bulls belonging to university farm. DNA was extracted using the phenol-chloroform isoamyl alcohol (PCI) technique and reported primer sequences were used to amplify the genes (partially) using a thermocycler. Three biological samples were analyzed using Sanger sequencing and were aligned using Clustal Omega. Separate phylogenetic trees for the genes were constructed using MEGA7 software. The selection pressure analysis and evolutionary divergence were individually studied for each of the genes. The current study reports the partial TSPY and SRY gene sequences of Indian Nili Ravi buffalo. Besides, the molecular evolution analysis indicates that the genes have experienced some purifying selection during the evolutionary process. Molecular characterization of these holandric genes (TSPY and SRY) in Nili Ravi will find applications in sex determination in Nili Ravi and other buffaloes.