Genetic Diversity in Paternal Line of Iranian Horses Using Partial Sequencing of Chromosome Y

 Among domesticated animals, the horse can be considered as one of the most influential animals in the process of human life modernization. Based on archaeological evidence, the presence of horses in Iran goes back to 3000 BC. Today, over 300 horses are recognized worldwide and a great number that is indigenous to Iran. The statistics provided by FAOSTAT indicate that there are about 140,000 horses. Due to climate diversity in Iran, there are various horse breeds in different parts of the country, such as Turkmen, Kordi, Drehshuri, Arab, Sistani, Northwest native and Caspian horses. Study of variations in Y chromosome and mtDNA make it possible to the characterization of genetic diversity in the maternal and paternal lines, respectively. Despite the high diversity in the genome of mitochondria in horses, but variation in Y chromosome is in low level. Low variation in Y chromosome of horses can be due to small male effective population size and loss of variations due to bottleneck during domestication. Several studies on Chinese indigenous and European modern horses revealed that native horses have more Y chromosomal variation in compared with modern breeds. So, the aim of present study was an assessment of genetic diversity in paternal line of Iranian horses using Y chromosome. Identified the genetic structure is important for appropriate breeding programs.

 Blood samples were collected from Jugular vein using 4 ml tubes containing EDTA (1 mg/ml) of 81 horse belonging to six different populations including Ardebil’s stables horses (24 samples), Northwest horses (17 samples), Kordi horses (15 samples), Arab horses (10 samples), Darehshuri horses (10 samples) and Qarebagh horses (5 samples). Total genomic DNA was extracted from whole blood. A total of the 264-bp fragment (locus: Y-50869) of Y chromosome was amplified using a pair of 20-nucleotide primer (Genbank accession number: JX646950). Polymerase Chain Reaction (PCR) was carried out and then, products of PCR sequenced using Sanger sequencing method. Alignment of sequences was performed by MEGA 6.0 software. Also, MEGA 6.0 used for segregating sites identification and also phylogeny tree construction based on identified haplotypes. DnaSP5 was used to the estimation of haplotype diversity (Hd), nucleotide diversity (π), genetic distance (Dxy) and an average number of differences (k). Finally, the median-joining network was generated using the Network 5 program to present the possible relationships among haplotypes.

 Alignment of sequences led to the identification of six segregating sites and consequently nine haplotypes based on Y chromosome sequences. Three haplotypes (H_1, H_2, and H_6) are widely distributed among under study samples, so that, 65 of 81 (more than 80 %) individuals have placed in three haplotypes. Among haplotypes with more than one individual, there is no special haplotype for any of breeds.  Haplotype diversity values ranged from 0.6 for Arab breed to 0.86 for Kordi breed. The nucleotide diversity values ranged from 0.00288 for Arab breed to 0.01442 for the Qarebaghg breed. Average values for nucleotide diversity and haplotype diversity were 0.0067 and 0.81 respectively. Comparison of present results with the previous study on mtDNA diversity of Iranian horses revealed that maternal line of Iranian horses is more divers from paternal line.  Among Iranian breeds, Dareshuri breed has the lowest nucleotide diversity 0.00481 and haplotype diversity 0.00481 and 0.8 respectively. Assessment of genetic distance among breeds revealed that Ardebil and Qarebagh horses have the highest distance (0.00822), while Arab and Dareshuri horses showed the lowest genetic distance (0.00327). Obtained results indicated that, unlike Arab breed, Qarebagh horses had low influence in Iranian horses. Phylogeny tree based on haplotypes of Iranian horses showed that divided into two branches. Generally, 73 individual (90.12% of all horses) and 8 individuals (9.88% of all samples belongs to each of the two branches.

 The number of haplotypes which was found for Iranian native horses was placed among three haplogroups, which indicate moderate genetic variety and numerous maternal lines in native horses of Iran. It seems that the presence of the accurate pedigree information along genetic studies can help us to better categorize Iranian horses. In fact, to designing effective breeding strategies, we need to identify the precise genetic structure of Iranian horses. What we learned from this study was that Iranian horses in compared with European pure breeds are more diverse. Obtained results from this study showed that Stalions of Arab breed had high impact in Iranian horses.