بررسي ناحيه HVR-III ژنوم ميتوكندري گوسفندان ايراني با روش توالي‌يابي

Sequencing of HVR-III region of mtDNA in Iranian sheep breeds

كشور ايران جزء يكي از محدود مناطقي است كه كشاورزي و اهلي شدن حيوانات توسط انسان ­هاي اوليه در آن اتفاق افتاده است و در نتيجه باعث شده است كه ايران داراي ذخاير ژنتيكي ارزشمندي در حيوانات اهلي باشد. از طرفي ايران با داشتن بيش از 27 نژاد گوسفند اهلي به همراه 5 زيرگونه گوسفند وحشي، داراي ذخيره ژنتيكي با تنوع كم نظيري در دنيا مي باشد. هدف: هدف از انجام اين تحقيق توالي­يابي ناحيه HVR-III از ژنوم ميتوكندري گوسفندان اهلي نژاد مغاني، لري­بختياري، سنگسري، بلوچي، كرماني، شال، قره­گل و كردي به منظور بررسي ارتباط فيلوژنيك بين نژادهاي مذكور و همچنين مقايسه با هاپلوتايپ­هاي شاخص شناسايي شده گوسفند اهلي مي ­باشد. روش كار: براي اين منظور تعداد 80 نمونه خون جمع‌آوري و پس از استخراج DNA، ناحيه موردنظر توسط پرايمرهاي اختصاصي به روش PCR تكثير و سپس توالي­يابي شدند. نتايج: با تجزيه و تحليل داده­هاي حاصل از تعيين توالي و مقايسه توالي HVR-III گوسفندان ايراني با يكديگر و با توالي ناحيه مشابه از ژنوم گوسفندان ساير نژاد­ها، مشخص شد كه عليرغم تنوع فنوتيپي قابل توجه در بين نژاد­هاي مورد مطالعه، اين نژاد­ها همگي متعلق به گروه هاپلوتايپي A مي­ باشد. اين امر ممكن است به دليل نزديكي جغرافيايي مكان زيستي آن ها و يا به دليل روش خاص زندگي عشاير باشد كه انتظار مي رود نژاد­هاي متنوع امروزي ايران از توده هاي محدودي با تنوع نسبتا كم مشتق شده باشند. نتيجه گيري نهايي: نتيجه گيري كامل تر نيازمند مطالعه بخش­ هاي بزرگتري از DNA ميتوگندري و همچنين بر روي تعداد بيشتر نمونه از نژادهاي اهلي به همراه مطالعه تنوع نژادهاي وحشي گوسفند ايران است.

Introduction: Sheep are a highly adaptable domestic species, which has made them an important resource for human around the world. Archaeozoological evidence indicates that sheep were first domesticated in the Fertile Crescent (Meadows et al. 2007). Iran is located in the area of agriculture initiation and animal domestication by early humans. As a result Iran has a unique gene pool and genetic diversity in farm animals. There are more than 32 distinctive domestic sheep breeds in addition to wild sheep populations in Iran. The complete mitochondrial DNA (mtDNA) molecule of the domestic sheep, Ovis aries, was sequenced by Hiendleder et al (1998). Sequence variation within the mitochondrial genome has proven highly informative for investigation of the origin of domestic animal species due to the extremely low rate of recombination of mtDNA, its maternal lineage heredity and its relatively faster substitution rate than nuclear DNA. Phylogenetic studies showed that there are five clades in sheep populations around the world. Clade B is dominated by animals localized to Europe, clade C by sheep from the Middle East and Asia, and clade A to be a mixture from the Middle East, Asia, and Europe (Pereira et al. 2006). A comprehensive study on a large group of Iranian sheep breeds to study the genetic relationship wthin these animals is yet missing. The present study aimed to determine the sequence of the HVR-III region from mtDNA in Iranian Moghani, Lori-Bakhtiari, Sangsari, Baluchi, Kermani, Shall, Karakul and Kordi sheep breeds and determining phylogenetic relationships between these breeds and known five haplotypes that have been identified in domestic sheep worldwide. Material and methods: In total, blood samples from 80 sheep were collected. Genomic DNA was extracted from whole blood using guanidium thiocyanate-silicagel method (Diatom DNA Prep. 100, Isogene, Russia) following the manufacturer’s instruction. The integrity of the extracted DNA was analyzed by electrophoresis on a 0.8% agarose gel and the purity of the obtained DNA was verified by Nano Drop spectrophotometer (Thermo, USA). After DNA extraction, a 600bp from HVR-III region of mtDNA was amplified by a standard PCR reaction with specific primers. Sanger sequencing was done after purification of PCR products. Sequencing data was assessed and analyzed by CLC Main workbench 7.6.4 and BLAST applications. Bayesian method was employed to analyze the phylogenetic tree. Mr.Bayse version 3.1 was used with General Time Reversible (GTR) model of nucleotide substitution and Markov chain Monte Carlo (MCMC) strands, 1,000,000 generations, with trees sampled every 100 generations. Results and discussion: PCR products were assessed by electrophoresis on 1% agarose gel and visualized under UV light after staining with GelRedTM and results showed that specific fragment of mtDNA amplified successfully (Figure 2). A Bayesian method was used to generate a phylogenic tree based on the HVRIII sequencing. This method has a stronger branch support in compare to other methods such as maximum parsimony or minimum evolution methods, therefore the results of this study on Iranian indigenous sheep breeds have higher reliability than older studies. The results showed that all of studied breeds belonged to the clade A (Figure 4), despite of the notable phenotypic variation between them. This resemblance might be due to geographical vicinity (Meadows et al. 2005) and the particular way of nomad life style that suggests today's Iranian sheep breeds may have been derived from a limited number of founder population. The results of this research was in accordance with Mohammadhashemi et al (2011) and Shafagh Motlagh (2007) who used D-loop region of mtDNA. Also Meadows et al (2005) sequenced a large fragment of mtDNA including cytb and control region reported that clade A were found mostly in breeds from Asia (Such as India, Indonesia, Mongolia, and Tibet), while type haplotype B individuals were observed at the highest frequency in breeds originated from Europe (Such as Austria, Finland, Spain, and northwestern Russia). In 1996, Wood and Phua identified two domestic ovine lineages in sheep from New Zealand, and in 1998, Hiendleder et al. characterized these as of Asian (clade A) or European (clade B) origin after comparing the distribution of haplotypes within multiple breeds sampled from Russia, Germany and Kazakhstan. Meadows et al (2011) demonstrated that the control region of mtDNA had the highest rate of mutation and it can be used as a better region to estimate diversity in populations. Although conserved regions would be better choice to use in divergence time estimation between different populations or species. Conclusion: We concluded that clade A is the main haplotype in Iranian domestic sheep however, this claim needs to be verified based on experiments with more animals in addition to whole mitogenome sequencing.