**4. Mitochondrial investigation in sheep**

### **4.1 Whole mitogenome**

Over the half (58%) of the mitogenome was completely (included CR and CYTB, ND2, ND3, ND4L, COX3, and 12 tRNA genes, and the origin of L strand replication), and partially (12S and 16S rRNA and an additional six protein coding and six tRNA genes) analyzed by Hiendleder [44]. In that research, the CRs and the coding regions shown 4.34% and 0.44% divergence in the comparison of sheep haplogroup A and B, respectively.

**Table 1** represents the complete mtDNA molecular sequencing of the domestic sheep (*Ovis aries*) achieved by Hiendleder et al. [45]. The length of the complete ovine mtDNA presented is 16,616 nucleotides (nt), which length is variable, due to heteroplasmy caused by the occurrence of different numbers of a 75-nt-long tandem repeat in the CR. The majority of domestic sheep contained four copies of that 75 bp repeat unit in work of Meadows et al. [46] resulting in a mitogenome of 16,616 or 16,620 bps.

Using 14 restriction enzymes Hiendleder et al. [47] evaluated haplotypes of restriction fragment length polymorphisms (RFLP) based on pairwise nucleotide sequence divergence between haplotypes, and proved that the domestic sheep come exclusively from *Ovis orientalis*.

Sanna et al. [48] reported the first complete mitogenome of the (*Ovis gmelini ophion*), and compared to the known five mitochondrial haplogroups. They suggest that the Cyprus Mouflon, a feral variant of domestic sheep diverged from urial (*O. vignei*) and argali (*O. ammon*) about 0.89 and 1.11 million years ago.

Lv et al. [49] performed a meta-analysis using complete and partial mitogenomic sequences. They suggest sheep individuals migrating east from Fertile Crescent on the Mongolian Plateau region may have formed another centre for the further spread of domestic sheep 3–5 thousand year B.C., at the same time extending the haplogroup C.

The complete mitochondrial genome provides complex information for knowledge of phylogeography and population genetics in sheep. The mitochondrial genomes of several breeds of domestic sheep (*Ovis aries*) have now been mapped by Davenport et al. [33].

#### **4.2 Examination of fossils**

The sheep (*Ovies aries*), together with the dog are the earliest domesticated animal species, and had remarkable role in the life of ancient societies. Sheep were domesticated around 7–9 thousand years B.C. in the area of the Fertile Crescent. Demirci et al. [50] found a time-dependent change in the incidence and proportions of haplotypes in Anatolia. Ancient samples showed the presence of haplogroup E (3%) in the Bronze Age and the presence of haplogroup C (6%) in the Hellenistic age, while haplogroups A and B were continuously present (with nearly 50–50 percent).

**185**

*Reality of Mitogenome Investigation in Preservation of Native Domestic Sheep Breeds*

NADH1 2,745 3,700 956 ATG TAa

NADH2 3,910 4,951 1,042 ATA Taa

5,163 5,194 32

COI 5,332 6,876 1,545 ATG TAA

ATPase8 7,775 7,975 201 ATG TAA ATPase6 7,936 8,615 680 ATG TAa COIII 8,616 9,399 784 ATG Taa

NADH3 9,469 9,815 347 ATA TAa

NADH4L 9,885 10,181 297 ATG TAA NADH4 10,175 11,552 1,378 ATG Taa

NADH5 11,754 13,574 1,821 ATA TAA NADH6 (L) 13,558 14,085 528 ATG TAA

tRNA-Ser (AGY) 11,622 11,681 60 A

tRNA-Glu (L) 14,086 14,154 69 ACTA Cyt b 14,159 15,298 1,140 ATG AGA CAA

tRNA-Leu (UUR) 2,668 2,742 75 AA

tRNA-Gln (L) 3,767 3,838 72 AT

tRNA-Trp 4,952 5,018 67 A tRNA-Ala (L) 5,020 5,088 69 A

tRNA-Tyr (L) 5,263 5,330 68 C

tRNA-Ser (UCN) (L) 6,874 6,944 71 TAAAC tRNA-Asp 6,950 7,017 68 T COII 7,019 7,702 684 ATG TAA AAT tRNA-Lys 7,706 7,773 68 T

**codon**

**Stop codonb** **3′ spacer**

**Feature From To Size Start** 

*DOI: http://dx.doi.org/10.5772/intechopen.95768*

tRNA-Phe 1 68 68 12S rRNA 69 1,026 958 tRNA-Val 1,027 1,093 67 16S rRNA 1,094 2,667 1,574

tRNA-Ile 3,701 3,769 69

tRNA-Met 3,841 3,909 69

tRNA-Asn (L) 5,090 5,162 73

tRNA-Cys (L) 5,195 5,262 68

tRNA-Gly 9,400 9,468 69

tRNA-Arg 9,816 9,884 69

tRNA-His 11,553 11,621 69

tRNA-Leu (CUN) 11,683 11,753 71

tRNA-Thr 15,302 15,371 70

Origin of L-strand

repl.


*Reality of Mitogenome Investigation in Preservation of Native Domestic Sheep Breeds DOI: http://dx.doi.org/10.5772/intechopen.95768*

*Landraces - Traditional Variety and Natural Breed*

**4. Mitochondrial investigation in sheep**

available.

16,620 bps.

**4.1 Whole mitogenome**

haplogroup A and B, respectively.

exclusively from *Ovis orientalis*.

ing the haplogroup C.

Davenport et al. [33].

**4.2 Examination of fossils**

(SSCP) method (applied to NADH dehydrogenase subunit 2 and 4) for reliability in haplogroup classification. Among these the SSCP analysis of NADH dehydrogenase subunit 2 exhibited the highest discrimination power among these. Starting with that, authors advice a stepwise screening, when whole sequencing is not easy

Over the half (58%) of the mitogenome was completely (included CR and CYTB, ND2, ND3, ND4L, COX3, and 12 tRNA genes, and the origin of L strand replication), and partially (12S and 16S rRNA and an additional six protein coding and six tRNA genes) analyzed by Hiendleder [44]. In that research, the CRs and the coding regions shown 4.34% and 0.44% divergence in the comparison of sheep

**Table 1** represents the complete mtDNA molecular sequencing of the domestic sheep (*Ovis aries*) achieved by Hiendleder et al. [45]. The length of the complete ovine mtDNA presented is 16,616 nucleotides (nt), which length is variable, due to heteroplasmy caused by the occurrence of different numbers of a 75-nt-long tandem repeat in the CR. The majority of domestic sheep contained four copies of that 75 bp repeat unit in work of Meadows et al. [46] resulting in a mitogenome of 16,616 or

Using 14 restriction enzymes Hiendleder et al. [47] evaluated haplotypes of restriction fragment length polymorphisms (RFLP) based on pairwise nucleotide sequence divergence between haplotypes, and proved that the domestic sheep come

Sanna et al. [48] reported the first complete mitogenome of the (*Ovis gmelini ophion*), and compared to the known five mitochondrial haplogroups. They suggest that the Cyprus Mouflon, a feral variant of domestic sheep diverged from urial

Lv et al. [49] performed a meta-analysis using complete and partial mitogenomic sequences. They suggest sheep individuals migrating east from Fertile Crescent on the Mongolian Plateau region may have formed another centre for the further spread of domestic sheep 3–5 thousand year B.C., at the same time extend-

The complete mitochondrial genome provides complex information for knowl-

edge of phylogeography and population genetics in sheep. The mitochondrial genomes of several breeds of domestic sheep (*Ovis aries*) have now been mapped by

The sheep (*Ovies aries*), together with the dog are the earliest domesticated animal species, and had remarkable role in the life of ancient societies. Sheep were domesticated around 7–9 thousand years B.C. in the area of the Fertile Crescent. Demirci et al. [50] found a time-dependent change in the incidence and proportions of haplotypes in Anatolia. Ancient samples showed the presence of haplogroup E (3%) in the Bronze Age and the presence of haplogroup C (6%) in the Hellenistic age, while haplogroups A and B were continuously present (with nearly 50–50

(*O. vignei*) and argali (*O. ammon*) about 0.89 and 1.11 million years ago.

**184**

percent).


*a Nucleotide number 1 is the 5′ end of the tRNA-Phe-specifying gene. Anticodons for the two tRNA-Leu and the two tRNA-Ser are given in parentheses. (L) denotes light-strand sense. Positions include the 58 and 38 nt of each feature. ATPase6 and ATPase8, genes encoding subunits 6 and 8 of ATPase; COI-III, genes encoding subunits I–III of cytochrome c oxidase; Cyt b, gene encoding cytochrome b; NADH1–6, genes encoding subunits 1–6 of nicotinamide adenine dinucleotide dehydrogenase.*

*b Incomplete stop signals are denoted by lowercase letters.*

#### **Table 1.**

*Features of the* Ovis aries *mitochondrial genomea [45].*

Dymova et al. [51] carried out archeological mitochondrial DNA D-loop fragment analysis based on about 4,000–1,000 years old sheep bone remains in Altai. They found all the previously determined haplogroups (A, B, C, D and E lineages). That richness of diversity led them to conclude that the Altai region had been a migratory area for many sheep and peoples in the past.

Study of Horsburgh and Rhines [52] evaluating sheep finds excavated in a South African Neolithic Age cave shown their assignation to haplogroup B.

In comparative study of samples dated primarily by archeological context and ranged from Late Bronze Age, through Iron Age to post-medieval period Rannamäe et al. [53] identified four novel ancient haplotypes specific to Estonia (H3, H4, H5 and H9), and haplogroups A and B in a ratio of one to two.

#### **4.3 Displacement-loop**

Mitochondrial displacement-loop (D-loop), called also as control region (CD) is a frequently investigated sequence in researches, also in sheep.

Divergence times estimated for types B and A (which was about 1.5–0.45 Mya) can be overestimated when it is based solely on hypervariable sequence of CR [54]. In regard of calibrating a molecular clock, the consideration of the codifying cytochrome b gene seems to be more accurate. To eliminate the distortive effect (known heteroplasmic behavior) of CR the repeat unit located within the CR region was removed before phylogenetic inference made by Meadows et al. [46].

The purpose of sequencing is, in addition to the genetic characterization of a given breed (population), to compare its genetic material to genetic material of other already sequenced breeds (GenBank sequences). Thus, we try to get an answer to the origin of the given breed and its genetic relatives. This is important when studied in the former natural range of the sheep species (primarily Asia, then Europe and Africa), but it is also important on the continents (America and Australia) where sheep individuals later entered with migrants.

For example, based on the CR, Annus et al. [55] confirmed the common origin of the Hungarian Tsigai with European sheep after finding that they are belonging to the haplogroup B (with the exception of 6% to the haplogroup A). An example of the latter case is the evaluation of the Mexican Creole sheep carried out by Alonso et al. [56], which revealed a narrow Iberian maternal origin.

Lancioni et al. [57] discovered relationships of the three Italian Merino-derived sheep breeds, and obtained that these are representatives of the predominant haplogroup B (99%). Since almost all the animals are carrying an own individual haplotype these are characterized with a diverse genetic background. On the other hand, this processing gives an example of how, despite upgrading (with Merino), the maternal background (Appeninica) is clearly discernible.

**187**

level.

**4.6 Haplogroup dispersion**

*Reality of Mitogenome Investigation in Preservation of Native Domestic Sheep Breeds*

It was observed cytochrome b (Cyt b) gene is also quite mutable than other mtDNA coding regions. For this reason, phylogenetic studies have used that marker too to investigate the genetic relationships among breeds. By use of cytochrome b gene and displacement-loop together or individually, like before, five haplogroups

In the phylogenetic sequence analysis based on cytochrome b gene Bunch et al. [58] revealed an about 3.12 million yearlong evolution of true sheep (*Ovis*). On the course of its evolutionary history there have been three major genetic groups developed. Foremost, the Argaliforms (*Ovis ammon*, 2n = 56) and Moufloniforms (*Ovis musimon* or *O. orientalis*, 2n = 54, and Urial/*Ovis vignei* 2n = 58) diverged from the initial ancestral stock 2.3 million years ago and spread on Eurasian continent. The domestic sheep (*Ovis aries*, 2n = 54) descend solely from *O. orientalis*. Second, the snow sheep group (*Ovis nivicola*, 2n = 52) as a variant of Pachyceriforms took their own shape in Eastern Asia from about 1.96 million years ago, then the other variants of Pachyceriforms (*O. canadiensis*, 2n = 54; *O. dalli*, 2n = 54) separated from them at about 1.41 million years ago, and evolved further in North America. Argaliforms are represented by only one species, *O. ammon*. The ancestral karyotype had 2n = 60 chromosomes. During the evolutionary development of variants of the species, also acrocentric fusions

According to the initial studies (e.g. [59, 60]) haplogroup A predominates in Asian sheep, while haplogroup B predominates in European sheep. Nevertheless, haplogroup C seems to have a wide geographical distribution [61]. Pedrosa et al. [28] and Chen et al. [61] suggested the divergence time of haplogroup C from haplogroup A and B to be approximately 0.42–0.76 million year ago and approximately 0.45–0.75 Mya from the analysis of control region and Cyt b gene sequences, respectively. However, a study of Meadows et al. [46] using 12 protein-coding genes of mtDNA puts the separation between the haplogroups less early (0.59–1.17 Mya

Tserenbataa et al. [62] collected 71 argali sheep (*O. ammon*) samples from three main geographical regions of Mongolia, and additionally from Kazakhstan and Kyrgyzstan. Based on the sequenced 556 bp of the mitochondrial ND5 gene. They differentiated 17 haplotypes which were differed far from each other by transitional substitutions in most of the cases. Nucleotide diversity was low within the three regions from Mongolia (π = 0.0029) compared to Kazakhstan and Kyrgyzstan. While the variance occurred within populations was as much as 85.76%. Finally, the use of mitochondrial ND5 gene provided an opportunity to detect divergence between the Altai and Gobi, and Altai and Khangai populations at a significant

For phylogenetic relationship between mitochondrial haplogroups of domestic sheep Meadows et al. [46] observed the greatest distance between B and C (nucleotide difference, D = 163.5), closely followed by B–E and C–D (D = 162.0, identically). The lowest number of nucleotide differences was 93.0 and 58.5 between A and B, and C and E, respectively. **Table 2** reveals the genetic distance between

*DOI: http://dx.doi.org/10.5772/intechopen.95768*

of sheep can be distinguished from each other [46].

between A and B, and 0.26–0.09 Mya between C and E.

domestic sheep haplogroups in addition to Urial, Argali to us.

**4.4 Cytochrome b gene**

of chromosomes are observed.

**4.5 ND5 gene**
