Introduction

The Animal domestication played an important role in human civilization and changing of man’s lifestyle from hunter-gatherer to agricultural one.¹ The horse (Equus ferus caballus L.) was firstly domesticated in the Eurasian region.² More than 300 horse breeds – or actually types because sometimes the use of breed term for horses is not right – are present all over the world and employed in different activities.³

The Arabian horse is a breed originated on the Arabian Peninsula with a distinctive head shape and high tail carriage. Arabian horse is one of the most easily recognizable horse breeds in the world and also it is one of the oldest breeds with archaeological evidence of horses in the Middle East.⁴ Arabian horses have spread around the world by both war and trade, used to improve other breeds by adding speed, refinement, endurance, and strong bone.⁵ Today, Arabian bloodlines are found in almost every modern breed of riding. The Arabian developed in a desert climate and was prized by the nomadic Bedouin people. It is a versatile breed; Arabian horses dominate the discipline of endurance riding and compete today in many other fields of equestrian sport.⁶ They are one of the top ten most popular horse breeds in the world. They are now found worldwide, including the United States and Canada, United Kingdom, Australia, continental Europe, South America (especially Brazil) in addition to their land of origin, the Middle East horse.⁷

The Thoroughbred is a horse breed known for its use in horse racing. It is developed in at 17^thcentury in England, when native mares were crossbred with Oriental stallions of Arabian, Barb and Turkoman breeding (www.britannica.com/animal/Thoroughbred). During the 18^thand 19^thcenturies, the Thoroughbred breed spread throughout the world; they were imported into North America, Australia, Europe, Japan and South America (www.ansi.okstate.edu/breeds/horses/thoroughbred). Thoroughbreds are used mainly for racing, but are also bred for other riding disciplines such as show jumping, combined training, dressage, polo and fox hunting. They are also commonly crossbred to create new breeds or to improve existing ones, and have been influential in the creation of the Quarter Horse, Standardbred, Anglo-Arabianand other various horse breeds (www.tbheritage.com).

The race efficiency characteristics of horses are inherited mainly through the maternal genetics with highly significant effect.⁸ MtDNA genes plays an important role in various characteristic associated with speed and force which have effect on race performance efficiency.⁹ The variations in mtDNA genes influence the potential and stamina of different horse breeds.^10,3 This work aimed to identify genetic variations and SNPs in two effective mtDNA genes; ATP6 and ND2; among Arabian and Thoroughbred horses.

Material and Methods

Blood Samples and Genomic DNA Extraction

Blood samples were collected from 24 horses belonging to two breeds; Arabian and Thoroughbred horses. Genomic DNA was extracted from the whole blood according to the method described by¹¹ with minor modifications. Briefly, Blood samples were mixed with cold 2x sucrose-triton and centrifuged at 5000 rpm for 15 min at 4°C. The nuclear pellet was suspended in lysis buffer, sodium dodecyl sulfate and proteinase K and incubated overnight in a shaking water bath at 37°C. Nucleic acids were extracted with saturated NaCl solution. The DNA was picked up and washed in 70% ethanol. The DNA was dissolved in 1xTE buffer. The DNA concentration was determined, using Nano Drop1000 thermo scientific spectrophotometer and then diluted to the working concentration of 50 ng/μl.

Polymerase Chain Reaction (PCR)

The PCR amplifications were conducted in a 50 μL volume containing 5 μL of 10x reaction buffer, 1.5 mM MgCl₂, 0.2 mM dNTPs, 0.2 μM each primer (Table 1), 1.5U Taq DNA polymerase (Fermentas, Germany) and approximately 100 ng genomic DNA. The reaction was cycled for 1 min at 94°C, 1 min at an optimized annealing temperature that was determined for each primer (Table 1) and 2 min at 72°C for 35 cycles. The PCR products were electrophoresed on 2% agarose gel stained with ethidium bromide to test the amplification success. The PCR products were purified and sequenced by Macrogen Incorporation (Seoul, Korea).

Table 1: Sequences of primers used in this study and amplification conditions             

Data Analysis

The sequences of amplified fragments of each tested genes were aligned using the BioEdit software.¹² The DnaSP 5.00 software¹³ was used to identify the polymorphic sites and SNPs between different animals. Neighbor-joining tree was computed with Mega version 5.0.¹⁴

Results and Discussion

The mitochondrial genome is a principal component in the metabolism process of living organism. The mitochondrion is responsible for the producing of about 95% energy for eukaryotic cells and it encodes 13 proteins through four protein complexes. These protein groups are cytochrome bcl, cytochrome oxidase, nicotinamide adenine dinucleotide (NADH) dehydrogenase and adenosine triphosphate (ATP) synthase. These proteins are involved in the oxidative phosphorylation process which has an essential role in energy metabolism.¹⁵ Also in the mitochondrion, there are seventy-six nuclear genes encoding proteins which are functional in oxidative phosphorylation system.¹⁶ All these proteins can modify the metabolic fitness by functional interactions and have an important role in race efficiency characteristics of horses like force and speed.^17-19

Many studies focused on mtDNA genes and their effective roles in racing and sporting efficiency of horses as well as fitness and performance phenotypes.^8,20 Other studies discussed the effect of the combination and interaction between mitochondrial genes on the efficiency of race horses. So, the genetic characterization and phylogeny of mitochondrial genes will be of great interest in horses due to their important effects on horse race performances. This study aimed to identify the genetic variations and SNPs of two effective mtDNA genes; ATP6 and ND2; in Arabian and Thoroughbred horses. This work also aimed to clarify the phylogeny relationship between these two breeds.

MT-ATP6 gene

ATP6 is a mitochondrial gene encoding the MT-ATP6 protein which forms one part of a large enzyme called ATP synthase. This enzyme is responsible for the final step of oxidative phosphorylation process. ATP has an important role in the energy transferring within the cell, diffusing from the place in which it is produced to the place in which it is utilized. Thirty percent of total ATP consumption is utilized in ion transport processes.²¹ Due to the important role of ATP6 in energy process which has great effect on the race efficiency of horses, its genetic diversity in Arabian and Thoroughbred horses was included in this work.

The primer used in this study amplified 340-bp fragments from ATP6 gene in Arabian and Thoroughbred horses. These amplified fragments were sequenced and the nucleotide sequences of 24 samples belonging to these two horse breeds were aligned using BioEdit software. DnaSP 5.00 software was used to identify the sequence variation and polymorphic sites in the aligned sequences.

The results showed the presence of 6 polymorphic sites leading to the construction of 7 different haplotypes (Fig. 1). The sequences of all detected haplotypes of ATP6 were submitted to GenBank database with the accession numbers KX377925-KX377931. The 14 Arabian samples showed 6 haplotypes with 6 polymorphic sites whereas the 10 Thoroughbred horses showed 4 haplotypes with 4 polymorphic sites. Haplotype no. 6 was the most detected oneand found in 6 samples; 3 horses samples from each breed followed by haplotype no. 5 which was found in 5 samples; 4 from Thoroughbred and one from Arabian horses. Haplotypes nos. 2 an6 6 are specific for Arabian breed which were present in 3 and 4 samples, respectively whereas haplotype no. 7 is specific for Thoroughbred and present in 2 samples. Fig. 2 declared the presence of 7 clusters; 3 specific for Arabian horses, 1 specific for Thoroughbred and 3 mixing for both.

Figure 1: The sequences of seven detected haplotypes of ATP6 gene with 6 polymorphic sites in red   Click here to View figure

Figure 2: Phylogeny tree of 24 tested samples for ATP6 gene showed their segregation in 7 clusters.   Click here to View figure

ARA: Arabian in red,

TH: Thoroughbred in blue

The haplotype diversities (HD) in Arabian and Thoroughbred horses were 0.857 and 0.778 and the average nos. of nucleotide diversities (K) were 2.363 and 2.067, respectively. The HD and K in all 24 tested samples were 0.866 and 2.326, respectively. The nucleotide diversity was 0.00695 for Arabian, 0.00608 for Thoroughbred and 0.00684 for all tested horses (Table 2).The statistical analysis of haplotypes diversity of Arabian and Thoroughbred horses showed that the average number of nucleotide differences between two populations was 2.386, the average number of nucleotide substitutions per site between populations (Dxy) was 0.00702 and the number of net nucleotide substitutions per site between populations (Da) was 0.00050.

Table 2: The genetic diversity data of ATP6 gene

Neighbor-joining (Phylogeny) tree was constructed using the Mega 5.0 software. The sequences of the 24 tested samples were aligned with 10 sequences of different breeds and isolates of horses around world. The reference sequences used were: NC_001640.1, EU939445.3, EF597512.1, EF597513.1, EF597514.1, F038160.1, X79547.1, AP013078.1, X97337.1 and JX312729.1. The results showed that all 24 tested horses belonging to Arabian and Thoroughbred horses are grouped with 8 out of 10 references which are Equus caballus and separated from the other two references; JX312729.1 (Equus burchellii) and X97337.1 (Equus asinus) (Fig. 3). This result declared that all tested Arabian and Thoroughbred horses are belonging to Equus caballus subspecies.

Figure 3: Phylogeny tree of 24 tested samples for ATP6 gene with ten reference sequences.   Click here to View figure

ARA: Arabian in red,

TH: Thoroughbred in blue and

KF038160, EF597514, EU939445, AP013078, EF597513, X79547, NC_001640, EF597512, X97337 and JX312729: accession number of references in green

MT-ND2 gene

ND2 gene is a mitochondrial gene encoding NADH dehydrogenase subunit 2 protein.²¹ MT-ND2 is located in mitochondrial DNA and produces a 39 kDa protein composed of 347 amino acids.²² MT-ND2 is a subunit of the respiratory chain complex which belongs to the minimal assembly for core protein required to catalyze NADH dehydrogenase.²³ The identification of genetic variations in this gene through different horse breeds has a great interest due to its role in the physiological characteristics associated with race performance in horses.

The primer used in this study amplified 362-bp fragments from ND2 gene in Arabian and Thoroughbred horses. These amplified fragments were sequenced and the nucleotide sequences of 20 samples belonging to these two horse breeds were aligned to identify the sequence variation and polymorphic sites in the aligned sequences.

The results showed the presence of 6 polymorphic sites leading to the construction of 5 different haplotypes (Fig. 4). The sequences of detected haplotypes of horse ND2 gene were submitted to GenBank database with the accession numbers KX396591-KX396595. The six Arabian samples showed 2 haplotypes with one polymorphic site whereas the 14 Thoroughbred horses showed 4 haplotypes with 5 polymorphic sites. Fig. 5 declared the presence of a major cluster containing most of the tested samples; 4 from Arabian and 11 from Thoroughbred horses. The rest of tested samples are belonged to 4 small clusters; one is specific for Arabian horses (2 samples) and the other three clusters are specific for Thoroughbred horses containing one sample for each.

Figure 4: The sequences of five detected haplotypes of ND2 genes with 6 polymorphic sites in red   Click here to View figure

Figure 5: Phylogeny tree of 20 tested samples for ND2 gene showed their segregation in 5 clusters.   Click here to View figure

ARA: Arabian in red,

TH: Thoroughbred in blue

The haplotype diversities (HD) in Arabian and Thoroughbred horses were 0.5333 and 0.3956 and the average nos. of nucleotide diversities (K) were 0.533 and 0.714, respectively. The HD and K in all 20 tested samples were 0.4421 and 0.689, respectively. The nucleotide diversity was 0.00147 for Arabian, 0.00197 for Thoroughbred and 0.0019 for all tested horses (Table 3). The statistical analysis of haplotypes diversity of Arabian and Thoroughbred horses showed that the average number of nucleotide differences between two populations was 0.96, the average number of nucleotide substitutions per site between populations (Dxy) was 0.00191 and the number of net nucleotide substitutions per site between populations (Da) was 0.00018.

Figure 6: Phylogeny tree of 20 tested samples for ND2 gene with ten references.   Click here to View figure

ARA: Arabian in red,

TH: Thoroughbred in blue and

KF038160, EF597514, EU939445, AP013078, EF597513, X79547, NC_001640, EF597512, X97337 and JX312729: accession number of references in green

Table 3: The genetic diversity data of ND2 gene

Neighbor-joining (Phylogeny) tree was constructed using the Mega 5.0 software. The sequences of the 20 tested samples of ND2 gene were aligned with 10 sequences of different breeds and isolates of horses around world which were mentioned above. The results – confirmed the findings of ATP6 gene – where all tested horses belonging to Arabian and Thoroughbred horses are grouped with 8 out of 10 references which are Equus caballus and separated from the other two references; JX312729.1 (Equus burchellii) and X97337.1 (Equus asinus) (Fig. 6).

In conclusion, the identification of genetic variations and SNPs in horse mitochondrial genes like ATP6 and ND2 genes are of great interest because they have highly significant effect and play important roles in different characteristics associated with speed and force which constitute race performance efficiency. ATP6 gene is considered more informative marker that ND2 for genetic biodiversity and affinities of different horse breeds.

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