DNA fingerprinting of Pakistani buffalo breeds (Nili-Ravi, Kundi) using microsatellite and cytochrome b gene markers Rashid Saif • Masroor Ellahi Babar • Ali Raza Awan • Asif Nadeem • Abu-Saeed Hashmi • Tanveer Hussain Received: 6 August 2010 / Accepted: 30 April 2011 / Published online: 13 May 2011 Ó Springer Science+Business Media B.V. 2011 Abstract Cytochrome b gene markers have been proved as an efficient and powerful tool for breed characterization and species identification of buffaloes. This study repre- sents the substantial analysis of mitochondrial DNA vari- ation in Pakistani buffalo breeds and provides information about their genetic diversity. In this study partial amplifi- cation of cytochrome b gene of 1,061 bp was done and sequencing results showed ten haplotypes. Comparing all fifty samples from two buffalo breeds of Pakistan, fifteen polymorphic sites were observed out of which, twelve codons 42, 71, 118, 120, 199, 235, 269, 297, 318, 327, 350, 355 of mitochondrial cytochrome b gene are monomorphic which translate same amino acids as in the reference pro- tein sequence due to silent mutation while different in DNA sequence. Similarly three codons 163, 246, 337 of mitochondrial cytochrome b are polymorphic and different from the reference sequence with respect to DNA as well as protein sequence. For the further confirmation a panel of nine microsatellite markers was used with high polymor- phism information content (PIC). The frequency distribu- tion of these alleles varies from three to eight allele at locus CSSM66 and ILST029 respectively. The results obtained from this study may contribute to the establishment of routine genotyping service of buffalo breeds for buffalo farmers for animal forensic application in case of any dispute. Additionally this study may help for breed char- acterization and phylogeny of aforementioned breeds of buffalo. Keywords DNA fingerprinting Á Buffalo breeds Á Microsatellite markers Á Cytochrome b gene Á Genetic diversity Introduction The domestic water buffalo is a major source of meat, draught, hide, and employment to the marginal farmers and landless laborers in many Asian countries. Broadly, water buffalo (Bubalus bubalis) is of two types i.e. River buffalo distributed in the Indian sub-continent, Middle East and Eastern Europe. Swamp buffalo distributed in north-eastern part of India, Bangladesh, china and South East Asian countries [1]. In Pakistan, there is a vast population of buffalo 29.9 million. Major breeds present in Pakistan are Nile, Ravi, Nili-Ravi, Kundi and Aza-kheli. Most of the buffalo popu- lation in Pakistan is not pure bred and carrying genetic makeup from different sources and having a great threat regarding the conservation. Two riverine type buffalo breeds of Pakistan are very important i.e., Nili-Ravi of Punjab and Kundi of Sind province. Both breeds are huge animals, native to Pakistan and long lived producing good yield of milk in their life. Kundi breed is mainly of milch type while Nili-Ravi is popular not only milch animal, but also for its meat and draught capabilities. Although both breeds are among the best dairy breeds of buffalo in the world, their milk producing ability is still sub-optimal and there is a considerable scope for their production improvement [2]. It is already mentioned that the indigenous breeds Nili- Ravi and Kundi are not producing in accordance to their real potential therefore, there is a need to utilize the tech- niques available to get maximum production per animal. Buffalo breeds differences are still disputed and need clear- R. Saif Á M. E. Babar (&) Á A. R. Awan Á A. Nadeem Á A.-S. Hashmi Á T. Hussain Institute of Biochemistry and Biotechnology, University of Veterinary and Animal Sciences, Lahore, Pakistan e-mail: drbabar@hotmail.com 123 Mol Biol Rep (2012) 39:851–856 DOI 10.1007/s11033-011-0808-0