Online First Article Characterization of β-casein Gene Sequence Variants in Cholistani Cattle Ruqayya Bint Khalid 1 , Asif Nadeem 1,2 *, Maryam Javed 1 , Muhammad Zubair Shabbir 3 and Masroor Ellahi Babar 4 1 Institute of Biochemistry and Biotechnology, University of Veterinary and Animal Sciences, Lahore, Pakistan. 2 Department of Biotechnology, Virtual University of Pakistan, Lahore, Pakistan 3 Department of Microbiology, University of Veterinary and Animal Sciences, Lahore, Pakistan; 4 The University of Agriculture, Dera Ismail Khan, Khyber Pakhtunkhwa, Pakistan Article Information Received 10 June 2020 Revised 03 July 2020 Accepted 07 July 2020 Available online 06 November 2020 Authors’ Contribution AN and MJ conceived the idea and executed the project. RBK carried out the genomic work. MZS and MEB assisted in data analysis and interpretation. RBK and MJ assisted in manuscript write up and editing. Key words β-casein, Cholistani cattle, Variants β-casein is second most abundant protein of cow’s milk. β-casein gene is highly polymorphic. A1 and A2 are the frequently occurred variants. A1 is recognized as potential cause of several human diseases. It is important to evaluate the A1/A2 β-casein status in milk. Current study was conducted to molecular characterize the exonic regions of β-casein gene and to explore the status of A1/A2 β-casein type in Cholistani cattle breed of Pakistan. Blood samples of Cholistani Cattle were collected from Government Livestock Farm, Jugait Peer, Bahawalpur. Genomic DNA was extracted from whole blood by organic method. PCR primers were designed and optimized according to respective melting temperatures. PRALINE tool, MEGA 6.0 and POPGENE tool were utilized for phylogenetic tree construction and statistical analysis, respectively. Characterization of physical and chemical properties of β-casein protein was performed by ProtParam and SWISS MODEL was utilized for protein model prediction. Sequencing results of amplifed PCR products revealed total 9 SNPs including 1 in exonic and 8 in intronic regions of β-casein gene. Results represent the presence of A2 allele in milk of Cholistani Cattle of Pakistan. Multiple sequence alignment represented the presence of MKVLILACLVALALARE and QRAVPVQALLLNQE as highly conserved regions. Phylogenetic analysis revealed the evolutionary relationship among Cholistani cattle of Pakistan, Bos indicus and Bos taurus. β-casein gene is highly polymorphic and A2 allele is present in Cholistani Cattle of Pakistan. INTRODUCTION A griculture is considered as an important sector of Pakistan’s economy. Livestock species play a vital role in country’s economic, social and cultural values. It contributes about 58.3% to the agriculture sector and 11.4% to the overall GDP of Pakistan. Within livestock sector, milk is the major commodity. Annual milk production of Pakistan is over 50 billion liters (GOP, 2018). Milk is considered as a complete diet as it is composed of essential micro and macronutrients. Milk is an essential source of nutrients like proteins, carbohydrates and particular micronutrients. It is rich in vitamin B12, vitamin A, ribofavin, folate, and calcium but iron occurs in less amount (Bermudez et al., 2010). Proteins are one of the most important component of milk, out of which caseins have gained most of the attention due to their recognized association with health related * Corresponding author: asif.nadeem@vu.edu.pk 0030-9923/2021/0001-0001 $ 9.00/0 Copyright 2021 Zoological Society of Pakistan properties. Bovine milk contains four casein components including αS1 (39-46%), αS2 (8-11%), β-casein (25-35%), к-casein (8-15%) respectively (Roginski et al., 2003). β-casein is the 2 nd most abundant protein with all essential 209 amino acids. β-casein is highly polymorphic milk protein gene with 12 known variants including A1, A2, A3, B, C, D, E, F, H1, H2, I and G. However, A1 and A2 are the most common occurring variants (Farrell Jr et al., 2004; Kamiński et al., 2007). Bovine β-casein gene is located at chromosome number 6, with total length of 8,695 bp and 8 number of exons (NC_037333.1). A1 and A2 allelic variance occur at exon VII and exon VII is responsible for encoding most of the mature protein parts (Ganguly et al., 2013). A1 differs from A2 variant in such a way that A1 milk contain histidine (CAT) amino acid at position 67 while A2 milk has proline (CCT) at similar position. This polymorphism leads to conformational changes in the secondary structure of protein and affect casein micelles physical properties. After substitution of histidine, A1 variant becomes vulnerable to gastrointestinal proteolysis digestion causing the release of a 7 amino acid bioactive ABSTRACT Pakistan J. Zool., pp 1-9, 2021. DOI: https://dx.doi.org/10.17582/journal.pjz/20200610120622