ORIGINAL SCIENTIFIC ARTICLE / IZVORNI ZNANSTVENI ČLANAK Molecular Phylogeny and Genetic Diversity of Domestic Yaks (Bos grunniens) in Pakistan based on Mitochondrial and Microsatellite Markers T. Hussain γ, A. Wajidγ, M. Soail, A. Ali, K. Abbas, F. M. M. T. Marikar*, M. M. Musthafa and M. E. Babar Abstract The complete Cytchrome b gene and partial cluster into two highly divergent maternal mtDNA control region were sequenced for the lineages (lineages I and II), while three Pakistani domestic yak (Bos grunniens) within haplogroups A, C, and D were identified the Bovidae family. A total of 300 samples were of the six previously known haplogroups. genotyped using 27 bovine microsatellite Haplogroups A and C were dominant and markers from the Gilgit-Baltistan and Skardu widely distributed among all investigated yak regions of Pakistan. We identified a total of 35 samples. All microsatellites were polymorphic mutations and 9 haplotypes based on D-loop and a total of 138 alleles were observed, with sequences, with a haplotype and nucleotide average polymorphic information content diversity of 0.9640±0.051 and 0.02172±0.00224, (PIC) of 0.56 indicating their effectiveness. respectively. For the Cyt b gene, a total of 23 The average heterozygosity was observed at variable sites and six different haplotypes 0.6071 with allele diversity of 5.1111 and gene were observed with 0.885±0.067 haplotype diversity of 0.4830. The implications of these and 0.00989±0.003 nucleotide diversity. findings can be applied for yak conservation. Phylogenetic analysis of D-loop and Cyt b Key words: domestic yak; mtDNA D-loop; gene suggested that domestic yak sequences Cyt b gene; microsatellites; phylogeny; Pakistan Tanveer HUSSAIN, Abdul WAJID, Akhtar ALI, Kamran ABBAS, Ellahi BABAR, The University of Agriculture, Dera Ismail Khan, Khyber, Pakhtunkhwa, Pakistan; Mudassir SOAIL, Department of Livestock and Dairy Development, Gilgit Baltistan, Pakistan; Faiz M. M. T. MARIKAR*, DVM, PhD, (Corresponding author, e-mail: [email protected]), General Sir John Kotelawala Defense University, Ratmalana, Sri Lanka, *The Open University of Sri Lanka, Faculty of Health Sciences, Nawala Sri Lanka*; Muneeb M. MUSTHAFA, Department of Biosystems Technology, Faculty of Technology, South Eastern University of Sri Lanka, University Park, Oluvil, Sri Lanka γ First two authors contributed equally VETERINARSKA STANICA 52 (6), 2021. | https://doi.org/10.46419/vs.52.6.5 671 T. HUSSAIN, A. WAJID, M. SOAIL, A. ALI, K. ABBAS, F. M. M. T. MARIKAR, M. M. MUSTHAFA and M. E. BABAR Introduction Yak (Bos grunniens) is a member of molecular markers such as mitochondrial the Artiodactyla, family Bovidae and displacement loop, Cytochrome b gene genus Bos. Yak can be considered to and bovine microsatellite markers. be one of the world’s most significant In Pakistan, yak pastoralism is domestic animals, as it can survive in restrained to the Northern highlands extreme environments and ensures a i.e. Skardu district, Astore in district livelihood for the local people. Domestic Diamer and Ganche districts in Baltistan yak is distributed at high altitudes and Chitral regions, lying at altitude between 2000 to 5000 m, in areas of the of 3100 m. Yaks are endemic to the vast Hindu Kush and Karakoram in Pakistan mountainous regions characterized by and Afghanistan, Himalayas in India, cold and high altitude environments of Nepal, Tibetan plateau and Tien Shan Hindu Kush and Karakoram highlands mountains of northwestern China and of Pakistan and Afghanistan, bordered western and northern Mongolia (Miller on Xinjiang and Qinghai-Tibetan Plateau and Steane, 1997). Yaks are multipurpose (QTP) and their distribution extends into animals providing products such as milk, central Asia to Afghanistan, Tajikistan meat, leather, hair and manure for fuel and to the Hangai, Hovsgol, Altai and for people living at high altitudes, and Hentii mountains of Mongolia and are useful as riding and pack animals Altai, East and West mountains of (Nguyen et al., 2005). The domestic Sayan (Buryatia and Republics of Tuva, yak is one of the most significant respectively) Russia and some of the domestic animal genetic resources in central independent states of the former the corresponding region and plays a USSR. vital role in the life of pastoralists and Mitochondrial DNA is a useful genetic agro-pastoralists (Wiener et al., 2003). marker for both intra and interspecies Indispensable knowledge of the genetic studies (Loftus et al., 1994; Kikkawa et variability of the yak population is useful al., 1995). Mitochondrial DNA (mtDNA) for conservation efforts to protect yak contains highly informative polymorphic genetic resources. The principal objective sites and in many organisms its of breeding yaks and other livestock simple maternal inheritance without is to improve productivity and fitness, recombination makes it useful for and to influence desirable changes in the population studies (Luikart et al., 2001). animal’s genome (Nguyen et al., 2005). The mitochondrial genome (mtDNA) of Unfortunately, quantitative genetic vertebrates has become a common tool characterization has been largely absent for resolving phylogenetic relationships for the yak because of the unavailability at different evolutionary depths due to of pedigree data and performance its distinctive properties (Carmela et al., records in yak populations. Other reasons 2000). The current study was planned to may be due to the remoteness and harsh use the mitochondrial Cytochrome b and conditions of yak territories, and the care D-loop region for the genetic diversity of the yak. To design typical breeding and phylogenetic analysis of Pakistani strategies for the optimum utilization yak populations. The most recent and conservation of genetic variability studies on the domestication patterns of Pakistani yak population, it is crucial and phylogenetic histories of ungulate to understand their genetic architecture domestic animals have been based on and variation. To obtain knowledge the mtDNA D-loop region (Lau et al., of their genetic structure, we utilized 1998; Luikart et al., 2001; Troy et al., 2001; 672 VETERINARSKA STANICA 52 (6), 671-684, 2021. Molecular Phylogeny and Genetic Diversity of Domestic Yaks (Bos grunniens) in Pakistan based on Mitochondrial and Microsatellite Markers Molekularna filogenija i genetička raznolikost domaćih jakova (Bos grunniens) u Pakistanu na temelju mitohondrijskih i mikrosatelitskih markera Jansen et al., 2002; Savolainen et al., 2002; DNA was brought to 50 ng/uL and stored Kierstein et al., 2004; Larson et al., 2005; at -80oC, before further use. Approval Guo et al., 2006; Wang et al., 2010; Babar from the Institutional Animal Care and et al., 2011). This mitochondrial region Use Committee (IACUC) from Virtual is highly variable and informative in University of Pakistan was obtained elucidating the origin of domestication, before the experiment. diversification and phylogenetic analysis of animals. Mitochondrial genome analysis For microsatellites (or short tandem To amplify the complete mitochon- repeats), some authors have claimed drial Cyt b gene (1609 bp), three pairs of that single nucleotide polymorphisms primers were designated from Bos indicus may more be well-known and are (NCBI accession number AF492350) using stable, polymorphic, easily analysed software Primer3 (Steve and Skaletsky, and occurs throughout an animal 2000). Primer set 1: MtCCF1 (5’-CGAAA- genome, making them especially GTCCCACCCACTAAT-3’) and MtCCR1 suitable for genetic analysis (Boyce et (5’-TCCTCATGGTAGGACGT ATCC-3’); al., 1996). Microsatellites are used as primer set 2: MtCCF2 (5’-GCACGTAG- valuable genetic markers for genetic and GACGAGGCTT AT 3’) and MtCCR2 population studies, assessment of the (5’-AAGAGGGCCCCTAAGATGTC-3’); population relationship, identification primer set 3: MtCCF3 (5’-AAATCCCAT- of animals, parentage determination, TCCACCCTAC-3’) and MtCCR3 determination of genetic variation within (5’-GTGCCGGCTGTTGGTATTAG-3’). and among breeds, reconstruction of The partial mtD-loop region of 695 bp phylogenetic relationships among was amplified using two sets of oligos, populations, disease diagnostics, forensic set 1: MtCDF1 (5’-TCCATAAATACAAA- analysis, development of genetic maps, GAGCCTTATCAGT-3’) and MtCDR1 and in marker-assisted breeding. (5’-GCGGCAT GGTAATTAAGCTC-3’), In the present study, we performed and set 2: MtCDF2 (5’-TTATATAT- a preliminary analysis based on TCCCTACCATTAGATCACG-3’) and mitochondrial DNA (mtDNA) and MtCDR2 (5’- GGGAAAAGTCTGT- microsatellite markers to characterize the TAAAA GTGGTG-3’), designated from genetic diversity of domestic yaks reared Bos indicus: accession number AF492350. across the Northern region of Pakistan. PCR was performed in a 25 µL reaction This study will aid future conservation mixture containing 1 µL template (the efforts and breeding programmes of genomic DNA of each sample was used domestic yak to improve meat quality as a template for PCR), 1 µL each prim- and milk yields. er (10 pmoL/µL), 12.5 µL of 2 × Taq PCR MasterMix and 9.5 µL ddH2O. Negative controls were always included in PCR re- Materials and Methods actions to assess possible contamination. Blood collection, DNA extraction and The standard PCR conditions for Cyt Ethics clearance b were followed: initial denaturation Blood samples (10 mL) were collected temperature at 95 °C for 4 min, 35 from 300 yaks from Gilgit-Baltistan n=18 cycles of denaturation at 94 °C for 30 s, and Sakardu n=7 in ethylenediamine annealing at 57.2 oC, 59 oC, 61 oC for 30 tetra-acetic acid (EDTA) added tubes. s for primer sets 1, 2 and 3, respectively, The inorganic method (Sambrook and and extension at 72 °C for 45 s followed Russell, 2001) was used for genomic DNA by final extension at 72 °C for 10 min. PCR extraction. The final concentration of was performed in 25 µL reaction mixture VETERINARSKA STANICA 52 (6), 671-684, 2021. 673 T. HUSSAIN, A. WAJID, M. SOAIL, A. ALI, K. ABBAS, F. M. M. T. MARIKAR, M. M. MUSTHAFA and M. E. BABAR using about 50 ng DNA as a template (American bison) accession numbers with 2 units Taq DNA polymerase JN632601, GU947005; Bison bonasus (Fermentas, Thermo Fisher Scientific Inc. (European bison) accession number USA).