Abstract

Casein proteins of milk are the key factors in defining the nutritional quality and processing characteristics of milk and the genetic variation in casein genes is known to affect such characteristics in dairy species. Nevertheless, there is a lack of complete data on the diversity of indigenous Pakistani buffalo breeds in terms of casein gene. The objectives of the current study were to describe genetic diversity in the key casein genes of Nili-Ravi, Kundi, and Azakheli buffalo and evaluate the possible structural and functional consequences of the identified variants. Blood samples were used to extract genomic DNA, amplified using conventional PCR and sequenced using Sanger sequencing of regions of interest. The bioinformatic pipelines were used to analyze sequence data to determine genetic variants, conduct phylogenetic analysis, and predict the structural effects of missense mutations. The number of variants that have been identified in the three casein genes (CSN2, CSN3 and CSN1S1) was 2254, and CSN1S1 had the most polymorphic variants. The breed-specific analysis also indicated a significant genetic differentiation, with the Kundi buffalo having the highest count of variants. There was a specific nonsynonymous mutation in CSN3 (c.467C>T) in Nili-Ravi buffalo that caused a p.T156I amino acid substitution, which was a predicted localized structural and physicochemical change of κ-casein. Phylogeny also favored breed-level separation. The results indicate a significant amount of diversity of casein genes in Pakistani buffalo which could serve as a source of important molecular evidence in further association studies, breed improvement efforts, and conservation of native buffalo genetic resources.