Genetic Diversity and Phylogenetic Analysis of D-loop Region of mtDNA in Baluchi Sheep Breed

Native animals are part of the national capital and strategic reserves of any country which their diversity is very important. Mitochondrial genome (mtDNA) in sheep is 16.58 Kbp. MtDNA has a region named D-loop or control region with no coding gene. Rate of nucleotide mutation in this region is 10 times the nucleus DNA. D-loop has promotors to regulate mtDNA transcription. This region is consisted of HVR1 and HVR2 sites. As the mtDNA is haploid and no meiosis occurs in it, so D-loop region of the mitochondrial genome is a powerful and applicable tool to determine the level of genetic diversity, to study the phylogenetic relationship between the populations and species as well as study the origin and dispersion of animal species. Baluchi sheep breed is one of the important Iranian sheep breeds which has a major role in production of red meat. Due to high strength and resistance to water scarcity it has been able to adapt with hot and dry weather conditions in East and South East of Iran. Due to the high diversity of species and subspecies, the importance of maintaining the purity of native breeds and incomplete information on sheep domestication in Iran, this study was performed to investigate the variation in Baluchi sheep breed and phylogenetic analyzes of mitochondrial D-loop region.
Material and Blood samples collection was done randomly from 27 non relative sheep which were kept in Animal Breeding center of Northeast of Iran (Abbasabad breeding station). DNA extraction was done using Diatom DNA Prep kit. DNA quality and quantity were checked using 8% agarose gel and spectrophotometer Nano drop ND-200, respectively. Primers were designed using Primer Premier5 software to amplify 1180 bps fragment of D-loop region of mitochondrial DNA. Primers specificity was checked in BLASTPrimer of NCBI. To Sequence the amplified region, samples were send to Bioneer Company. To enhance the accuracy of sequencing, each sample was sequenced from both sides. Nucleotide sequences were edited with Chromas Lite 2.01 software. After proofing the quality of sequences they were reformatted from ab1 to FASTA. Then homology of the sequences with registered sequences of the same gene in NCBI and with the sequences themselves was determined using BLASTN in NCBI database and CLC Main workbench 5.5 software, respectively. To draw the phylogenetic tree of Baluchi sheep breed, 15 sequences from each 4 haplogroups were identified and along with consensus sequence from samples were used. To draw the phylogenetic tree with 1000 iterations, the Neighbor-Joining method of MEGA5 software was used and to determine the genetic distance the Create Pairwise Comparison procedure of CLC Main workbench 5.5 software was used.
Eight haplogroups were observed. The frequencies of haplogroups were 7.40, 7.40, 14.81, 11.11, 18.51, 14.81, 11.11 and 14.81. Haplogroups 2 and 7 had the highest difference between the nucleotides (10 nucleotides) with 99.15 percent genetic similarity, and haplogroups 1 and 4 had the highest genetic similarity (99.83 percent) with 2 nucleotide difference. The present genetic diversity among the 27 samples was estimated 0.0131±0.005. This level of diversity is in the average range of nucleotide diversity which is reported in eukaryotes. The Phylogenetic analysis in this study showed that Baluchi sheep breed is located in the A haplotype.
Since the origin of haplotype A is from Asia and Middle East, and according to the results of earlier studies on native Iranian sheep breeds shuch as Moghani, Shal, Sangsari and Afshari it can be concluded that the Baluchi sheep was in the mentioned haplotype therefore the placement of this breed in this haplotype is justifiable.