**3. Non-coding and coding region on mtDNA**

#### **3.1 Control region**

On the circle of the mtDNA, there is a specialized sequence, called control region (CR), and also called D (displacement)-loop because of its peculiar protrusion. The CR is made up of a triplex DNA structure at the site of origin of the heavy strand. This region is critical for the initiation of transcription and translation [7].

Zardoya et al. [26] determined the nucleotide sequence of the sheep mitochondrial DNA CR and its flanking tRNA genes. They found that several conserved motifs characteristic mammals have been identified along the 1189-bp sequence of the sheep control region: ten termination-associated sequences (TASs) and one conserved sequence block (CSB-1). CSB-2 and CSB-3, which are frequently determined in most species, are not present in the sheep CR, which shows instead a short direct repeat at their usual localization.

The CR contains hypervariable sites (mutational hotspots). This unstable segment gives the basis for dating estimation in many mammalian species. However, according to some authors (e.g. [27]) the high occurrence of recurrent mutations may bias dating estimates. Also within the sheep species, according to Pedrosa et al. [28], the time of separation of haplogroups is significantly earlier if hypervariable CR is taken into account. Researches on human mtDNA raise the concerns that focusing exclusively on CR can be inadequate [29].

#### **3.2 Genes**

A total of 37 genes of mtDNA are coding 2 ribosomal RNAs, 22 transfer RNAs, and 13 mitochondrial proteins as well in mammals. The latter, without exception, direct cells to produce protein subunits for enzyme complexes of the oxidative phosphorylation system. Mitogenome has a very similar conservative nucleotide sequence in all organisms.

A meta-analysis study [30] of over 1500 animal species revealed that the average within-species level of mtDNA diversity *per se* is remarkably similar across animal phyla. Reason for that is a recurrent selective sweeps which would affect mtDNA evolution in species causing frequent drops in diversity at the whole genome level. Based on that hypothesis and being high conservative gene dense, according to the report of Galtier et al. [31] mtDNA is by and large not the satisfactory marker of molecular diversity and representation of population history. After that, it may come as a surprise the most popular marker of molecular diversity in animals, a mitochondrial fragment, COX1, was recently elected as the standardized tool for molecular taxonomy and identification [32].

Some genes even overlap. In the mitochondrial genome, some triplet codons may be the final stage of one gene but also (in a functional overlapping) the initial stage of the next gene. Another feature is that the mtDNA is transcribed from several structural genes to the messenger RNA at the same time. Thus, large mitochondrial mRNAs contain instructions for the synthesis of different proteins. The inheritance of the extranuclear genes are independent of nuclear genes, but, they interact with each other in function. The mitochondrial genome is not able to produce all the proteins required for phosphorylation on its own, so mitochondria are highly demanding of gene products produced by the nuclear genome.

Investigation of Rocky Mountain bighorn sheep (*O. canadenesis,* [33]) revealed 16,466 bps, with about 40% GC content. Further on, it confirmed also the bighorn sheep mitochondrial genome has 22 tRNA genes, 2 rRNA genes (12S and 16S), and

**183**

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

of the bighorn 3 million years ago from the sheep living at that time.

13 respiratory genes (ATP6, ATP8, CYTB, COX1, COX2, COX3, ND1, ND2, ND3, ND4, ND4L, ND5, and ND6). Comparison of the genome of bighorn sheep with the genome sequence of other sheep showed 99.6% identity, indicating the separation

Since mitochondria acts as the powerhouses of cells, tissues that have high energy demands (brain, retina, skeletal muscle, and cardiac muscle) are particularly vulnerable to the harmful consequences of mutation. As an inherent part of energy production, mitochondria create reactive oxygen species (ROS) as well which is seen to cause further mitochondrial mutations. Elevated levels of ROS negatively affect cellular metabolism, thereby accelerating the cell ageing process and increasing the likelihood of cell death. Symptoms of mitochondrial diseases in humans can usually include: poor growth; muscle- weakness, pain, low tone, exercise intolerance, and movement disorders; vision and/or hearing problems; learning disabilities and mental retardation; autism and autism-like features; heart-, liver- or kidney diseases; gastrointestinal disorders, swallowing difficulties, diarrhea or constipation, unexplained vomiting, and cramping, reflux; diabetes; increased risk of infection; neurological problems, seizures, migraines; strokes; thyroid problems; respiratory (breathing) problems; lactic acidosis; dementia [35]. Fortunately, nextgeneration sequencing techniques have substantially improved genetic diagnosis. Individuals resulting from cloning procedures (*nuclear transplantation* or *somatic* 

*cell fusion,* [36]) are heteroplasmic. The initial heteroplasmic stage of chimeric offspring cell turns usually into homoplasmy with prevailing mitochondria of the host oocyte [37]. It is likely that heteroplasmy of mitochondrial genomes will be terminated by selective elimination of donor or recipient mitochondria by chemical or other means. Establishing biotechnical approaches allows women with mitochondrial diseases to have reproductive options. Recent advances in these including *in vitro* fertilization techniques with mitochondrial donation, will serve as a solution

Mutation in mtDNA-coded ribosomal RNA, called RNR1 indicates the presence of an environmental effect. That mutation causes deafness in children, but the clinical symptoms of the deafness are related to the administration of certain

The percentual manifestation (penetrance) of a mitochondrial disease in males and females differing from the expectations points to the likely involvement of other (nuclear) genes and environmental factors. *Leber hereditary optic neuropathy* (LHON) is an example for mitochondria-associated disorders which is manifested

First reported Pal et al. [41] the association of cytochrome b (Cyt b, CYTB) gene with disease traits in sheep. Mutations of Cyt b gene (non-synonymous substitutions: F33L and D171N) interferes with the site of heme binding domain and calcium binding essential for electron transport chain causing anemia, malfunctioning of most of the vital organs. This discovery raises the possibility that the sheep may

Results of Reicher et al. [42] revealed ovine mitogenome genetic variation in protein- and tRNA coding genes (26 and 8 mutant sites, respectively) and empha-

Yüncü et al. [43] tested the restriction fragment length polymorphism (RFLP)

method (applied to CR) and the single strand conformational polymorphism

size that sequence variation is associated with ewe prolificacy.

Higher mutation rate of mtDNA will cause increased rate of genetic diseases of mitochondrial origin [34]. Therefore, such diseases are also all of maternal origin. Males can be carriers of a given genetic defect and can be affected by its manifestation, however, they are not responsible for transmitting that disease into their

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

progenies.

in the future [38].

antibiotic type [39].

in loss of vision [40].

come into play as a model of man.

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

13 respiratory genes (ATP6, ATP8, CYTB, COX1, COX2, COX3, ND1, ND2, ND3, ND4, ND4L, ND5, and ND6). Comparison of the genome of bighorn sheep with the genome sequence of other sheep showed 99.6% identity, indicating the separation of the bighorn 3 million years ago from the sheep living at that time.

Higher mutation rate of mtDNA will cause increased rate of genetic diseases of mitochondrial origin [34]. Therefore, such diseases are also all of maternal origin. Males can be carriers of a given genetic defect and can be affected by its manifestation, however, they are not responsible for transmitting that disease into their progenies.

Since mitochondria acts as the powerhouses of cells, tissues that have high energy demands (brain, retina, skeletal muscle, and cardiac muscle) are particularly vulnerable to the harmful consequences of mutation. As an inherent part of energy production, mitochondria create reactive oxygen species (ROS) as well which is seen to cause further mitochondrial mutations. Elevated levels of ROS negatively affect cellular metabolism, thereby accelerating the cell ageing process and increasing the likelihood of cell death. Symptoms of mitochondrial diseases in humans can usually include: poor growth; muscle- weakness, pain, low tone, exercise intolerance, and movement disorders; vision and/or hearing problems; learning disabilities and mental retardation; autism and autism-like features; heart-, liver- or kidney diseases; gastrointestinal disorders, swallowing difficulties, diarrhea or constipation, unexplained vomiting, and cramping, reflux; diabetes; increased risk of infection; neurological problems, seizures, migraines; strokes; thyroid problems; respiratory (breathing) problems; lactic acidosis; dementia [35]. Fortunately, nextgeneration sequencing techniques have substantially improved genetic diagnosis.

Individuals resulting from cloning procedures (*nuclear transplantation* or *somatic cell fusion,* [36]) are heteroplasmic. The initial heteroplasmic stage of chimeric offspring cell turns usually into homoplasmy with prevailing mitochondria of the host oocyte [37]. It is likely that heteroplasmy of mitochondrial genomes will be terminated by selective elimination of donor or recipient mitochondria by chemical or other means. Establishing biotechnical approaches allows women with mitochondrial diseases to have reproductive options. Recent advances in these including *in vitro* fertilization techniques with mitochondrial donation, will serve as a solution in the future [38].

Mutation in mtDNA-coded ribosomal RNA, called RNR1 indicates the presence of an environmental effect. That mutation causes deafness in children, but the clinical symptoms of the deafness are related to the administration of certain antibiotic type [39].

The percentual manifestation (penetrance) of a mitochondrial disease in males and females differing from the expectations points to the likely involvement of other (nuclear) genes and environmental factors. *Leber hereditary optic neuropathy* (LHON) is an example for mitochondria-associated disorders which is manifested in loss of vision [40].

First reported Pal et al. [41] the association of cytochrome b (Cyt b, CYTB) gene with disease traits in sheep. Mutations of Cyt b gene (non-synonymous substitutions: F33L and D171N) interferes with the site of heme binding domain and calcium binding essential for electron transport chain causing anemia, malfunctioning of most of the vital organs. This discovery raises the possibility that the sheep may come into play as a model of man.

Results of Reicher et al. [42] revealed ovine mitogenome genetic variation in protein- and tRNA coding genes (26 and 8 mutant sites, respectively) and emphasize that sequence variation is associated with ewe prolificacy.

Yüncü et al. [43] tested the restriction fragment length polymorphism (RFLP) method (applied to CR) and the single strand conformational polymorphism

*Landraces - Traditional Variety and Natural Breed*

**3.1 Control region**

translation [7].

**3.2 Genes**

sequence in all organisms.

repeat at their usual localization.

**3. Non-coding and coding region on mtDNA**

focusing exclusively on CR can be inadequate [29].

molecular taxonomy and identification [32].

demanding of gene products produced by the nuclear genome.

On the circle of the mtDNA, there is a specialized sequence, called control region (CR), and also called D (displacement)-loop because of its peculiar protrusion. The CR is made up of a triplex DNA structure at the site of origin of the heavy strand. This region is critical for the initiation of transcription and

Zardoya et al. [26] determined the nucleotide sequence of the sheep mitochondrial DNA CR and its flanking tRNA genes. They found that several conserved motifs characteristic mammals have been identified along the 1189-bp sequence of the sheep control region: ten termination-associated sequences (TASs) and one conserved sequence block (CSB-1). CSB-2 and CSB-3, which are frequently determined in most species, are not present in the sheep CR, which shows instead a short direct

The CR contains hypervariable sites (mutational hotspots). This unstable segment gives the basis for dating estimation in many mammalian species. However, according to some authors (e.g. [27]) the high occurrence of recurrent mutations may bias dating estimates. Also within the sheep species, according to Pedrosa et al. [28], the time of separation of haplogroups is significantly earlier if hypervariable CR is taken into account. Researches on human mtDNA raise the concerns that

A total of 37 genes of mtDNA are coding 2 ribosomal RNAs, 22 transfer RNAs, and 13 mitochondrial proteins as well in mammals. The latter, without exception, direct cells to produce protein subunits for enzyme complexes of the oxidative phosphorylation system. Mitogenome has a very similar conservative nucleotide

A meta-analysis study [30] of over 1500 animal species revealed that the average within-species level of mtDNA diversity *per se* is remarkably similar across animal phyla. Reason for that is a recurrent selective sweeps which would affect mtDNA evolution in species causing frequent drops in diversity at the whole genome level. Based on that hypothesis and being high conservative gene dense, according to the report of Galtier et al. [31] mtDNA is by and large not the satisfactory marker of molecular diversity and representation of population history. After that, it may come as a surprise the most popular marker of molecular diversity in animals, a mitochondrial fragment, COX1, was recently elected as the standardized tool for

Some genes even overlap. In the mitochondrial genome, some triplet codons may be the final stage of one gene but also (in a functional overlapping) the initial stage of the next gene. Another feature is that the mtDNA is transcribed from several structural genes to the messenger RNA at the same time. Thus, large mitochondrial mRNAs contain instructions for the synthesis of different proteins. The inheritance of the extranuclear genes are independent of nuclear genes, but, they interact with each other in function. The mitochondrial genome is not able to produce all the proteins required for phosphorylation on its own, so mitochondria are highly

Investigation of Rocky Mountain bighorn sheep (*O. canadenesis,* [33]) revealed 16,466 bps, with about 40% GC content. Further on, it confirmed also the bighorn sheep mitochondrial genome has 22 tRNA genes, 2 rRNA genes (12S and 16S), and

**182**

(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 available.
