Sequence Variations of Mitochondrial DNA Displacement-Loop in Iranian Indigenous Sheep Breeds

Document Type: Research Articles


1 Department of Animal Science, Ashkezar Branch, Islamic Azad University, Ashkezar, Iran

2 Gilan Branch, Agricultural Biotechnology Research Institute of Iran/Agricultural Research, Education and Extension Organization (AREO), Rasht, Iran


Mitochondrial DNA (mtDNA) has been used extensively to study population genetics because it has the unique features of maternal inheritance, a relatively fast rate of evolution and lack of recombination. A total of 82 unrelated sheep from 10 Iranian indigenous sheep breeds were investigated to determinate the maternal genetic diversity using a sequence of a 685 bp segment of the displacement loop (D-loop) of mtDNA. Analysis of this region revealed 74 haplotypes and 123 polymorphic sites. Haplotype diversity, nucleotide diversity and the average number of nucleotide differences were estimated to be 0.996 ± 0.003, 0.0372 ± 0.0001 and 25.23, respectively. The sequence analysis also revealed high level of genetic diversity among the native Iranian breeds. Analysis of molecular variance revealed that 3.43 percent of the variation is found among populations compared with 96.57 percent variation found within populations. The Neighbor-Joining (NJ) tree indicated four (A, B, C and E) of the five haplogroups described so far are present in Iranian sheep breeds. The phylogenetic tree did not show any distinct genetic structure among the studied populations, which suggested that there existed strong gene flow and intermixing among sheep populations probably caused by extensive transportation of sheep in history and similar maternal lineages among the regions.



Sheep domestication occurred about 10000 to 11000 years ago in an area extending from northern Zagros Mountains of Iran to South East Anatolia, Turkey. Asia Minor and the Middle East regions established an important geographical connection between Asia and Europe continents. For this reason, species in this area reflect a high-level of gene flow, mixing and differentiation from the time of the domestication event (Meadows et al. 2011). Sheep are one of the most economically important domestic animals in Iran, as well as in other parts of the world (Guo et al. 2005). The contribution of sheep in Iran can be categorized as a source of meat, wool, skin and milk. Genetic characterization of different sheep breeds is a first step to collect basic information for any breeding and conservation plan in order to maintain and improve production efficiency in this industry. Mitochondrial DNA (DNA) has shown good potential to study population genetics and evolution. MtDNA is the only extra-nuclear genome in the cytoplasm, and exists as multiple copies. It has a high mutation rate and the rate of mtDNA evolution is about 5 to 10 times faster than nuclear DNA (Xingbo et al. 2000; Reicher et al. 2012). MtDNA is inherited maternally, apparently lacks recombination and is highly variable within a species because of its elevated mutation rate that may be the result of lack of repair mechanisms and/or the presence of free radicals formed during the phosphorylation process. As a result, mtDNA is an important material for phylogenetic inference and for analyzing genetic diversity (Witas and Zawicki, 2004; Wang et al. 2007; Galtier et al. 2009). Studies performed on the control region fragment and / or the cytochrome b (cytB) gene of mtDNA of modern sheep from a wide geographical range divided domestic sheep's genetic diversity into five different haplogroups: A, B, C, D and E (Wood and Phua, 1996; Hiendleder et al. 1998; Hiendleder et al. 2002; Guo et al. 2005; Pedrosa et al. 2005; Meadows et al. 2005; Meadows et al. 2007). The aim of present research was to study the genetic diversity within and between Iranian sheep populations. In this study, we investigated the mtDNA control region polymorphisms in 10 Iranian sheep breeds to obtain deeper insight into sequence variation, type of haplogroups and their diversity. The information for main use and population size of studied breed has been indicated in Table 1.


Table 1 Main use and population size per each breed in the distributed areas



Sample collection and DNA extraction

Blood samples were collected from 82 unrelated sheep from 10 Iranian indigenous sheep breeds including Balouchi, GharehGol, Kalakui, Lori-Bakhtiari, Moghani, Makui, Naini, Shal, Taleshi and Zel. The number of individuals per each breed is indicated in Table 2. Geographic distribution of the sampling sites of the 10 Iranian sheep breeds is shown in Figure 1. Genomic DNA was extracted by standard salting-out method (Mburu and Hanotte, 2005). Purity and concentration of DNA samples measured using NanoDrop and electrophoresis on 1% agar gel.


Polymerase chain reaction (PCR)

A 685 bp fragment from 15448 to 16127 bp of the mtDNA control region was amplified by using forward primer (15388F): 5’ GCC CCA CTA TCA ACA CCC AAA G 3’ and reverse primer (CD744R): 5’ AAT GGG CGA TTT TAG ATG AGA TGG C 3’ (Hiendleder, 1998). The PCR amplification reaction system consisted of genomic DNA 40 ng, dNTPs 0.08 mM, primers 0.2 M, MgCl2 1.25 mM, Taq DNA polymerase 0.05 U/µL. The PCR conditions were initial denaturation step at 95 ˚C for 5 min, 30 cycles of denaturation at 94 ˚C for 30 s, annealing at 59 ˚C for 60 s and extension at 72 ˚C for 60 s and a final extension step at 72 ˚C for 10 min. The PCR products were electrophoresed through 2.0% (wt/vol) agarose gel which was stained with ethidium bromide solution.


Sequencing analysis of mtDNA

The forward primer was used for sequencing PCR product using Genetic analyzer 3130X. All the sequences were edited and aligned using the BioEdit software (Hall, 1999). The polymorphism of the haplotypes was analyzed with the DnaSP 5.10.00 software (Librado and Rozas, 2009). The Neighbor-Joining phylogenetic tree based on pairwise distance between pairs of individual was constructed using the MEGA ν.5.0program (Tamura et al. 2011). Analysis of molecular variance (AMOVA) was performed with ARLEQUIN version 3.11 to infer the amount of diversity between and within populations. The levels of statistical significance were tested by performing 1000 permutations (Excoffier et al. 2005). The representative sequences of each haplogroups A (HM236174, HM236175), B (HM236176, HM236177), C (HM236178, HM236179), D (HM236180, HM236181) and E (HM236182, HM236183) was retrieved from NCBI and added to the primary data set for performing phylogenic analysis (Neighbor-Joining tree) to identify type of haplogroups of Iranian sheep breeds (Meadows et al. 2011).



In the present study, we identified 74 haplotypes in the analyzed 82 animals with 123 polymorphic sites. Estimates of genetic diversity within the sheep populations have been indicated in Table 2. The average of haplotype diversity, nucleotide diversity and the average number of nucleotide differences were estimated to be 0.996 ± 0.003, 0.0372 ± 0.0001 and 25.23, respectively. Breed-specific estimates of genetic diversity indicated that Kalakui contained the highest (0.04762) and Zel the lowest (0.00872) nucleotide diversity (π) within the Iranian populations (Table 2). AMOVA allocates only a small portion of the total diversity to the between-breeds component. It reveals that mitochondrial diversity is mainly distributed within breeds (96.57 %) and only in part among regions (3.43 %) (Table 3). To determine the clade membership of each haplotype, a Neighbor-Joining tree was constructed using the 92 haplotypes (Figure 2). The tree contained haplogroups of A, B, C and E (but not D). Sequence analysis of the mtDNA from Iranian sheep showed high genetic diversity within the breeds.


Table 2 Estimates of genetic diversity within the sheep populations

ID: identity document; N: number of individual sampled; Hn: observed haplotypes; π: population nucleotide diversity and Hd: haplotype diversity.


Figure 1 Geographic distribution of the sampling sites of the 10 Iranian sheep breeds

For breed abbreviations, see Table 2


The haplotype diversity in the analysed breeds was higher than that in other European breeds (Pariset et al. 2011). The values of haplotype and nucleotide diversities observed in this study was higher than haplotype diversity of 0.792 ± 0.37 and nucleotide diversity of 0.00392 ± 0.00046 obtained from analysis of mtDNA variation and matrilineal structure in blue sheep populations of Helan Mountain, China (Wang et al. 2006). Pariset et al. (2011) assessed genetic diversity of sheep breeds from Albania, Greece and Italy by mtDNA and nuclear polymorphisms (SNPs) and observed increased nucleotide diversity from the South to the East, which was consistent with the approach to the sheep domestication center. Lancioni et al. (2013) also assessed the genetic diversity of three Italian Merino-derived (IMD) breeds by mtDNA and observed three distinct subhaplogroups within B haplogroup.


Table 3 Analysis of molecular variance for 10 Iranian sheep breeds based on displacement-loop mitochondrial DNA sequence data

** (P<0.01).

NS: non significant.


Comparing the number of individuals sampled and the number of haplotypes (Table 2) indicates that each breed has its own haplotype that can be due to high diversity in Iranian sheep breeds and / or sampling strategy (collecting unrelated individuals). Sequencing more individual may endorse this claim in future studies. In the present study, the analysis of molecular variance attributes most genetic variation to the within-population component (97%). These results were similar to the findings from an AMOVA analysis on Turkish sheep that showed the main genetic variation distributed within populations (95.61%) and the rest between populations and different geographical regions (Oner et al. 2013). Genetic diversity analysis of the mitochondrial D-loop of Nigerian indigenous sheep also showed 99.77% of genetic variation within populations and 0.32 % of variation between populations (Agaviezor et al. 2012). Higher level of genetic diversity within rather than between Iranian sheep populations demonstrates that there is no clear structure between populations and also indicates a high level of gene flow through the maternal lineage between the populations (Tserenbataa et al. 2004). Phylogenic analysis did not yield distinct clusters representing the populations (Figure 2). Low differentiation between populations can be attributed to limited pastures, random mating, lacking of appropriate breeding plans or management policies. Moreover, migration leading to natural gene flow between populations can also be important factors in reducing the differentiation between populations (Kantanen et al. 1995). However, Gizaw et al. (2011) declared that an important feature which should be noted about genetic diversity study of domesticated animal species is that genetic variation within populations is much higher that genetic variation between populations (Gizaw et al. 2011). In this study phylogenetic analysis demonstrated four major haplotypes (A, B, C and E) in 10 Iranian native breeds. Haplogroup A and haplogroup B are the most frequent and have been found in every geographic region where the domestic sheep have been sampled. Haplogroup A is mainly represented in Asian breeds, while haplogroup B is found in high frequency in breeds sampled in Europe (Wood and Phua, 1996; Hiendleder et al. 1998). Haplogroup C is less frequent and only few samples have been isolated in the Fertile Crescent (Asia), and the Caucasus and the Iberian Peninsula (Europe) (Guo et al. 2005; Pedrosa et al. 2005; Meadows et al. 2005; Tapio et al. 2006; Pereira et al. 2006).


Figure 2 Phylogenetic relationship between maternal haplotype identified in the sheep populations (individual level)

For breed abbreviations, see Table 2


Haplogroup D and haplogroup E are the rarest and have only been found in the Caucasus and Turkey so far (Meadows et al. 2005). A recent study on the complete mitochondrial genome of ten domestic sheep and six wild sheep examined the relationship between domestic and wild sheep. The phylogenetic analysis confirms the division of domestic sheep into the five (A, B, C, D, E) haplogroups (Meadows et al. 2011). The only investigation on mtDNA sequence variation in Iranian sheep breeds was performed by Mohammadhashemi et al. (2012) on Moghani sheep breed. They obtained five haplotypes that were distributed within the 10 individuals analyzed. All the haplotypes of that study were subdivided into the haplogroup A.



High diversity, high gene flow, and low levels of breed differentiation are the characteristic of Iranian native sheep populations. There could be several reasons for this, including implementation of inappropriate breeding programs, random mating, and exchange of populations between different geographical areas. Alternatively, Iran’s location close to or inside the domestication center has likely led to preservation of a large part of wild diversity in populations of this region and surrounding area (Benjelloun et al. 2011). Another explanation for the findings is Iran’s location along the Silk Route that has been traversed by commercial caravans connecting China, India, Iran, Turkey and other locations for millennia. This route was important in trade of a variety of crops and animals and probably contributed to random mixing between different populations. So, the weak genetic structure of Iranian native sheep populations can be attributed to long-term severe genetic flow between Iranian native sheep, induced by the historical movement of humans.



The authors are thankful to the anonymous reviewers and the shepherds for their contributions on sheep identification and sampling. This research was supported by grant from Agricultural Biotechnology Research Institute of Iran, North branch.

Agaviezor B.O., Adefenwa M.A., Peters S.O., Yakubu A., Adebambo A.O., Ozoje M.O., Ikeobi C.O.N., Ilori B.M., Wheto M., Okpeku M., De Donato M. and Imumorin I.G. (2012). Mitochondrial D-loop genetic diversity of Nigerian indigenous sheep. Anim. Genet. 50, 13-20.

Benjelloun B., Pompanon F., Ben Bati M., Chentouf M., Ibnelbachyr M., El Amiri B., Rioux D., Boulanouar B. and Taberlet P. (2011). Mitochondrial DNA polymorphism in Morrocan goats. Small Rumin. Res. 98(1), 201-205.

Excoffier L., Laval G. and Schneider S. (2005). Arlequin ver. 3.0: an integrated software package for population genetics data analysis. Evol. Bioinform. 1, 47-50.

Galtier N., Nabholz B., Glemin S. and Hurst G.D.D. (2009). Mitochondrial DNA as a marker of molecular diversity. Mol. Ecol. 18, 4541-4550.

Gizaw S., Komen H., Hanote O., Van Arendonk J.A.M., Kemp S., Haile A., Mwai O. and Dessie T. (2011). Characterization and conservation of indigenous sheep genetic resources: a practical framework for developing countries. Pp. in 41-45 in Proc. ILRI Res. Report. Nairobi, Kenya.

Guo J., DuL X., Ma Y.H., Guan W.J., Li H.B., Zhao Q.J., Li X. and Rao S.Q. (2005). A novel maternal lineage revealed in sheep (Ovis aries). Anim. Genet. 36, 331-336.

Hall T.A. (1999). BioEdit: a user-friendly biological sequence alignment editor and analysis program for windows 95/98/NT. Nucl. Acids. Symp. Ser. 41, 95-98.

Hiendleder S. (1998). A low rate of replacement substitutions in two major Ovis aries mitochondrial genomes. Anim. Genet. 29(2), 116-122.

Hiendleder S., Kaupe B., Wassmuth R. and Janke A. (2002). Molecular analysis of wild and domestic sheep questions current nomenclature and provides evidence for domestication from two different subspecies. Proc. Biol. Sci. 269, 893-904.

Hiendleder S., Mainz K., Plante Y. and Lewalski H. (1998). Analysis of mitochondrial DNA indicates that domestic sheep are derived from two different ancestral maternal sources: no evidence for contributions from Urial and Argali sheep. J. Hered. 89, 113-120.

Kantanen J., Vilkki J., Elo K. and Maki-Tanila A. (1995). Random amplified polymorphic DNA in cattle and sheep: application for detecting genetic variation. Anim. Genet. 26, 315-320.

Lancioni H., Di Lorenzo P., Ceccobelli S., Perego U.A., Miglio A., Landi V., Antognoni M.T., Sarti F.M., Lasagna E. and Achilli A. (2013). Phylogenetic relationships of three Italian Merino-derived sheep breeds evaluated through a complete mitogenome analysis. PLoS. One. 8(9), 1-15.

Librado P. and Rozas J. (2009). DnaSP vae5: a software for comprehensive analysis of DNA polymorphism data. Bioinform. 25, 1451-1452.

Mburu D. and Hanotte O. (2005). A practical approach to microsatellite genotyping with special reference to livestock population genetics. Pp. 21-27 in Proc. ILRI Biodiver. Project. Nairobi, Kenya.

Meadows J.R., CemalI., Karaca O., Gootwine E. and Kijas J.W. (2007). Five ovine mitochondrial lineages identified from sheep breeds of the Near East. Genetics. 175, 1371-1379.

Meadows J.R., Li K., Kantanen J., Tapio M., Sipos W., Pardeshi V., Gupta V., Calvo J.H., Whan V., Norris B. and Kijas J.W. (2005). Mitochondrial sequence reveals high levels of gene flow between breeds of domestic sheep from Asia and Europe. J. Hered. 96, 494-501.

Meadows J.R.S., Hiendleder S. and Kijas J.W. (2011). Haplogroup relationships between domestic and wild sheep resolved using a mitogenome panel. Heredity. 106, 700-706.

Mohammadhashemi A., Pirany N., Nassiri M.R., Abbassidaloii T. and BaghbanKkohnegroz B. (2012). Studying the partially sequenced mtDNA hypervariable region1 (HVR1) of Iranian Moghani sheep. Ann. Biol. Res. 3(6), 2906-2910.

Oner Y., Calvo J.H. and Elmaci C. (2013). Investigation of the genetic diversity among native Turkish sheep breeds using mtDNA polymorphisms. Trop. Anim. Health Prod. 45, 947- 951.

Pariset L., Mariotti M., Gargani M., Joost S., Negrini R., Perez T., Bruford M., Marsan P.A. and Valentini A. (2011). Genetic diversity of sheep breeds from Albania, Greece and Italy assessed by mitochondrial DNA and nuclear polymorphisms (SNPs). Sci. World. J. 11, 1641-1659.

Pedrosa S., Uzun M., Arranz J.J., Gutie´rrez-Gil B., San Primitivo F. and Bayon Y. (2005). Evidence of three maternal lineages in Near Eastern sheep supporting multiple domestication events. Proc. Biol. Sci. 272, 2211-2217.

Pereira F., Davis S., Pereira L., McEvoy B., Bradley D.G. and Amorim A. (2006). Genetic signatures of a Mediterranean influence in Iberian Peninsula sheep husbandry. Mol. Biol. Evol. 23, 1420-1426.

Reicher S., Seroussi E., Weller J.I., Rosov A. and Gootwine A. (2012). Ovine mitochondrial DNA sequence variation and its association with production and reproduction traits within an Afec-Assaf flock. J. Anim. Sci. 90, 2084-2091.

Tamura K., Peterson D., Peterson N., Stecher G., Nei M. and Kumar S. (2011). MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28, 2731-2739.

Tapio M., Marzanov N., Ozerov M., Cinkulov M., Gonzarenko G., Kiselyova T., Murawski M., Viinalas H. and Kantenen J. (2006). Sheep mitochondrial DNA variation in European, Caucasian and Central Asian areas. Mol. Biol. Evol. 23, 1776-1783.

Tserenbataa T., Ramey R.R., Ryder O.A., Quinn T.W. and Reading R.P. (2004). A population genetic comparison of argali sheep (Ovis ammon) in Mongolia using the ND5 gene of mitochondrial DNA: implications for conservation. Mol. Ecol. 13, 1333-1339.

Wang X., Cao L., Liu Z. and Fang S. (2006). Mitochondrial DNA variation and matrilineal structure in blue sheep populations of Helan Mountain, China. Canadian J. Zool. 84, 1431-1439.

Wang X., Chen H. and Lei C.Z. (2007). Genetic diversity and phylogenetic analysis of the mtDNA D-loop region in Tibetan sheep. Asian-Australas J. Anim. Sci. 3, 313-315.

Witas H.W. and Zawicki P. (2004). Mitochondrial DNA and human evolution. Prz. Antropol.-Anthropol. Rev. 67, 97-110.

WoodN.J. and Phua S.H. (1996). Variation in the control region sequence of the sheep mitochondrial genome. Anim. Genet. 27, 25-33.

Xingbo Z., Mingxing C., Ning L. and Changxin W. (2000). Paternal inheritance of mitochondrial DNA in the sheep (Ovine aries). Sci. China. 44(3), 321-326.