Molecular Effects of Polymorphism in the 3’UTR of Osteopontin Gene in Riverine Buffalo

Sidra Manzoor1, Asif Nadeem1,*, Masroor Ellahi Babar2, Wasim Shehzad1, Abu Saeed Hashmi1, Muhammad Imran1, Tanveer Hussain2, Abdul Wajid1 and Maryam Javed1 1Institute of Biochemistry and Biotechnology, University of Veterinary and Animal Sciences, Lahore, Pakistan 2Virtual University of Pakistan, Lahore, Pakistan Article Information Received 07 January 2017 Revised 25 March 2017 Accepted 19 April 2017 Available online 26 June 2017

Nucleotide sequencing of bovine osteopontin gene showed a novel DNA polymorphism 37421762 (C>A) in the 3'-untranslated region (3'-UTR) of the gene. Association test was performed to analyze its relation with milk composition but it was found non-significant in all analyzed traits. So, it was inferred that it might be due to environmental or epigenetic effect, sampling number or a breed difference. To know the functional consequences of the identified polymorphism, RNA secondary structure analysis was performed and SNP was found changing the termination loop shape in mRNA. However, to increase the selection efficacy of dairy animals, the effects of candidate polymorphism should further be screened in different environments and genetic backgrounds.
O steopontin (SPP1, OPN) is a multifunctional protein involved in a number of biological processes; chemotaxis, bone remodeling, cell adhesion, inhibition of ectopic calcification, fetal growth and development, cancer metastasis and immune modulatory functions (Schack et al., 2009). Its role in initiating and maintaining pregnancy, mammary gland development and differentiation has made it a potential biomolecule for genetic studies (Nemir et al., 2000). A large number of polymorphisms located in noncoding regions, including promoters, 5' and 3' untranslated gene regions (UTR), introns, and intergenic portion, associated with milk production traits have been identified so far (Yudin and Voevoda, 2015). Osteopontin gene has been reported for 8514C>T (intron IV) and other novel polymorphisms in coding and non-coding regions that were associated with milk production traits and body weight in various breeds (Khatib et al., 2007;Pareek et al., 2008;Mello et al., 2012;Kowalewska-Luczak and Kulig, 2013;Nani et al., 2015;Pasandideh et al., 2015). This work was planned to genetically characterize the osteopontin gene in riverine buffalo and to identify the single nucleotide polymorphism (SNP) as genetic markers.

Materials and methods
In order to analyze the Osteopontin polymorphism, fifty blood samples of Nili Ravi (Riverine buffalo) were collected from different livestock farms and processed for genomic DNA extraction (Maryam et al., 2012).
Sequences were aligned by using ClustalW and analyzed for genetic variation. p.37421762 (C/A) was found in 3 ' UTR of Osteopontin gene. Polymorphism was further analyzed for Chi 2 testing, Hardy Weinberg Equilibrium (HWE) and association analysis by One Way ANOVA (Mean±SE) (Tables 1, II). SNP was found obeying HWE. Further p.37421762 was analyzed for RNA secondary structure (Supplementary Fig. 1) and there was
The SPP1 gene was selected for association test with milk composition traits as several reported markers has showed its effects on milk production (Leonard et al., 2005). Alligned sequence showed an SNP in 3' UTR of SPP1 gene in riverine buffalo (Table I). DNA mutations in 3' UTR and intronic portion of the genes may alter the mRNA stability and splice sites of pre-mRNA, consequently leading to the assemblage of truncated proteins (Yudin and Voevoda, 2015). The identified SNP 37421762 (C>A) in the 3′UTR of the gene was found nonsignificant in all analyzed milk production traits. The SPP1 gene has been reported for g.8514C>T polymorphism associated with milk composition traits (Leonard et al., 2005;Khatib et al., 2007). However, the current study was found contradictory to them and no SNP was identified in this region of the gene. Cohen et al. (2004) reported that SPP1 is a candidate gene affecting milk protein percentage, but the identified SNP in 3′UTR did not show any significant relationship with milk protein content. It may be due to environmental or epigenetic effect, sampling number or a breed difference. However, the effects of genes used in marker-assisted selection (MAS) program should be known and consistent in different environments and genetic backgrounds to increase the selection efficacy of dairy animals.

Statement of conflict of interest
Authors have declared no conflict of interest.