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Deep RNA sequencing experiment was employed to detect putative single nucleotide polymorphisms (SNP) in mammary epithelial cells between two diverse cattle breeds (Jersey and Kashmiri) to understand the variations in the coding regions that reflect differences in milk production traits. The low milk-producing Kashmiri cattle are being replaced by crossbreeding practices with Jersey cattle with the aim of improving milk production. However, crossbred animals are prone to infections and various other diseases resulting in unsustainable milk production. In this study, we tend to identify high-impact SNPs from Jersey and Kashmiri cows (utilizing RNA-Seq data) to delineate key pathways mediating milk production traits in both breeds. A total of 607 (442 SNPs and 169 INDELs) and 684 (464 SNPs and 220 INDELs) high-impact variants were found specific to Jersey and Kashmir cattle, respectively. Based on our results, we conclude that in Jersey cattle, genes with high-impact SNPs were enriched in nucleotide excision repair pathway, ABC transporter, and metabolic pathways like glycerolipid metabolism, pyrimidine metabolism, and amino acid synthesis (glycine, serine, and threonine). Whereas, in Kashmiri cattle, the most enriched pathways include endocytosis pathway, innate immunity pathway, antigen processing pathway, insulin resistance pathway, and signaling pathways like TGF beta and AMPK which could be a possible defense mechanism against mammary gland infections. A varied set of SNPs in both breeds, suggests a clear differentiation at the genomic level; further analysis of high-impact SNPs are required to delineate their …