INTERNATIONAL JOURNAL OF AGRICULTURE & BIOLOGY ISSN Print: 1560–8530; ISSN Online: 1814–9596 15–662/2016/18–5–903–905 DOI: 10.17957/IJAB/15.0149 http://www.fspublishers.org Short Communication To cite this paper: Khan, W.A., T. Hussain, M.E. Babar, A. Nadeem, A.R. Awan, A. Fatima and R. Saif, 2016. Polymorphic status of PRKAA2 gene in Pakistani buffaloes. Int. J. Agric. Biol. 18: 903‒905 Polymorphic Status of PRKAA2 Gene in Pakistani Buffaloes Waqas Ahmad Khan 1 , Tanveer Hussain 2* , Masroor Ellahi Babar 2 , Asif Nadeem 1 , Ali Raza Awan 1 , Ambrin Fatima 3 and Rashid Saif 2 1 Institute of Biochemistry and Biotechnology, University of Veterinary and Animal Sciences, Lahore, 54000, Pakistan; Department of Biotechnology, University of Sargodha, Sargodha, Pakistan 2 Department of Molecular Biology, Virtual University of Pakistan, Lahore, Pakistan 3 Health Biotechnology Division, National Institute of Biotechnology and Genetic Engineering, Faisalabad, Pakistan *For correspondence: tanveer.hussain@vu.edu.pk Abstract This study was designed to find single nucleotide polymorphism (SNPs) in coding and non-coding region of PRKAA2 gene in Nili-Ravi and Kundi buffaloes. The PRKAA2 gene (AMPKα2) of 100 animals from both buffalo breeds were sequenced for SNPs identification. A total number of 43 inter and intra-generic SNPs were found. Out of which 17 SNPs were detected among buffalo breeds (intra-generic). All SNPs were intronic and may have a role in gene regulation and splicing patterns. These SNPs might be associated with commercially important production traits. This is the first study to identify novel SNPs that are linked to energy metabolism and production traits of buffalo. © 2016 Friends Science Publishers Keywords: AMPK; PRKAA2; Buffalo; SNP; Pakistan Introduction AMPK has a vital role in maintaining energy balance in all eukaryotes and regulates different aspects of cellular function (Hardie, 2011). Indirectly, AMPK also promotes feeding and feeding behavior of the cell (Hardie et al., 2012). AMPK α2 or PRKAA2 is activated by leptin kindles during the fatty acid oxidation in muscle. Activation of PRKAA2 by drugs like 5-aminoamidazole-4-carboxamide 1-β-D-ribofuranoside restrains protein synthesis in rat skeletal muscle (Bolster, 2002). Variations in hypothalamic PRKAA2 regulate food intake as well as body weight gain (Minokoshi, 2004). The variants in PRKAA2 are significantly associated with serum lipoproteins in normal Caucasian females and showed significant associations between variants of AMPK genes subunit and weight gain activated by clozapine and olanzapine (Souza, 2012). Involvement of this gene was also reported in the regulation of lipids, glucose metabolism and protein synthesis (Zhang, 2011). Importance of this energy-status sensor has driven scientists to work on the structure-function relationship studies of its isoforms, especially the constitution of γ regulatory subunit. Similar roles of subject gene are anticipated in mammalians species based on high conservation status of this gene in eukaryotes. In current study, sequencing and SNPs analysis of PRKAA2 gene in two Pakistani buffalo breeds, Nili-Ravi and Kundi was carried out to find out novel SNPs in the coding and non- coding region of PRKAA2 gene. Materials and Methods Blood samples from 100 pure Nili-Ravi and Kundi buffaloes were collected in tubes containing EDTA and DNA was extracted by the standard organic method (Maryam et al., 2012). The extracted DNA was quantified by agarose gel electrophoresis and spectrophotometry. The genomic DNA was amplified by PCR (polymerase chain reaction) using primers designed by EPIC approach (exon primed intron crossing) from cattle PRKAA2 gene sequence available at GenBank accession number AC_000160 (Table 1). Both sense and antisense strands of the amplified DNA was sequenced by using Big Dye terminator cycle sequencing chemistry v 3.1 (Applied Biosystems, USA) and electrophoresis was done by an automated DNA sequencer (ABI Prism 3130xL Genetic Analyzer, Applied Biosystems, USA). Sequences were analyzed manually through ChromasLite software 1.45 (http://www.technelysium.com.au/chromas.html). The sequences were BLAST against reference GenBank sequences using Blast2Sequences bioinformatics tool (Tatusova and Madden, 1999) followed by the identification of novel SNPs detection. Results In the current study, a total of 43 inter and intra-generic SNPs were detected in PRKAA2 gene after alignment of