Sequence analysis and identification of new variations in the 5 0 -flanking region of aS2-casein gene in Indian zebu cattle Amit Kishore • M. Sodhi • M. Mukesh • B. P. Mishra • R. C. Sobti Received: 13 January 2012 / Accepted: 29 April 2013 / Published online: 9 May 2013 Ó Springer Science+Business Media Dordrecht 2013 Abstract Regulatory region of milk protein alpha S2- casein (aS2-CN) gene sequence was characterized and analyzed for nucleotide variations in animals representing 13 Indian zebu cattle (Bos indicus) breeds. A total of 15 variations; 11 in promoter region (1.56 Kb): -1481 (C [ T), -1412 (C [ T), -1342 (C [ T), -1084 (G [ A), -979 (A [ G), -657 (A [ T), -508 (A [ G), -186 (T [ C), -184 (T [ C), -151 (T [ C) and -135 (C [ T); 1 in 5 0 -UTR (44 bp): 7 (C [ T) while, 3 in intron-I region (73 bp): 186 (C [ T), 194 (A [ C) and 301 (A [ T) were identified. Addi- tionally, single deletion was observed at -975 (A [ -) but not involve any known potential transcription factor bind- ing sites (TFBS). Comparison with Bos taurus sequence revealed two additional variations -1085 (T [ C) and -739 (A [ G). Out of the total 18 variations observed between indicine and taurine aS2-CN regulatory region sequence, 15 were novel to B. indicus and are reported for the first time. Among these, four variations were located within the potential TFBSs; -1342 (C [ T) within HNF-3beta, -739 (A [ G) within C/EBP-alpha while -657 (A [ T) and -508 (A [ G) were found within glucocorticoid receptor TFBSs. Variations located within or in proximity to putative TFBSs could possibly influence the binding affinity of nuclear factors towards DNA binding domains, thus affecting transcriptional rate of aS2-CN gene. Phyloge- netically, as expected, Indian zebu cattle were grouped close to B. taurus and were most distantly placed in comparison to human. The study indicated possible genetic variations in the regulatory regions of aS2-CN gene within Indian native cattle (B. indicus) and also its comparison with evolutionary different B. taurus breeds. Keywords Alpha S2-casein Á Promoter sequence Á Genetic variation Á Phylogenetic relationship Á Indian native cattle Introduction Bovine milk has been regarded as nature’s perfect food, providing an important source of nutrients including protein, carbohydrates and essential minerals and vitamins. It gen- erally contains about 3.5 % protein, of which *80 % are caseins and 20 % are whey proteins. More than 95 % of the cow milk proteins are constituted by caseins (aS1-, aS2-, b-, k-CN) and whey (a-LA and b-LG) protein genes [1]. Among the different caseins, Alpha S2-casein (aS2-CN) holds importance. It comprises *10 % of the casein fraction of the cow’s milk [2] and is phosphorylated at multiple sites. It plays an important role in calcium phosphate transport. Further, aS2-CN is predicted to yield peptides with defensin- like activity and aid the immune system with its anti-bacte- rial properties [3]. Preparations or proteolytic digests enri- ched with aS2-CN may have use in value-added or health- promoting dairy products [4]. Variations within the coding regions of milk protein genes; either alone or in combina- tions (haplotypes), might alter protein structure and its function and thus influence milk protein content and its properties [5–12]. Till date, four such protein variants (A, B, Electronic supplementary material The online version of this article (doi:10.1007/s11033-013-2539-x) contains supplementary material, which is available to authorized users. A. Kishore Á M. Sodhi (&) Á M. Mukesh Á B. P. Mishra Cattle Genomics Lab, National Bureau of Animal Genetics Resources, P.O. Box 129, Karnal 132 001, Haryana, India e-mail: monikasodhi@yahoo.com A. Kishore Á R. C. Sobti Department of Biotechnology, Panjab University, Chandigarh 160 014, India 123 Mol Biol Rep (2013) 40:4473–4481 DOI 10.1007/s11033-013-2539-x