a. javanrouh

Horse has been of great interest to humans due to its speed, strength, and endurance. Based on its role in human history and civilization, it is considered the most important domesticated animal. Iranian horse breeds are bred in the north, south, and west regions of the country. Arabian horse is one of the international breeds in the world, which is bred in more than 60 countries. Preliminary evidence shows that the Arabian horse breed was established about 3000 years ago and was bred in small populations in many countries of the Middle East, including Egypt, Iran, Saudi Arabia, and Syria. These separate populations have led to the development of different maternal strains of the Arabian horse. Genetic diversity is necessary for genetic conservation and survival of the breeds. Molecular markers are used to assess the structure and genetic diversity of different populations. Therefore, one of the suitable markers is a microsatellite. Regarding the importance of genetic diversity in the survival of the breed, and designing the genetic conservation and breeding programs, the purpose of this study was to investigate the population structure and genetic diversity of Arabian horse breeds using microsatellite markers.
In this study, hair root samples were obtained from 8673 (5602 mares and 3071 stallions) Arabian horses from Khuzestan, Yazd, Kerman, Isfahan, Lorestan, and Alborz provinces. DNA samples were extracted from hair roots using the Direct PCR Kit (Thermo Fisher Scientific, Vilnius, Lithuania). The quantity and quality of the extracted DNA were controlled by spectrophotometry and agarose gel methods. The number of 10 microsatellite markers including AHT04, AHT05, ASB02, ASB17, ASB23, HMS03, HMS06, HMS07, HTG10, and VHL20 were used based on ISAG recommendation. Then, the amplification of genomic fragments and the genotype of samples was determined by COrDIS Horse Reagent Kit (Moscow, Russia). Forward primers were labeled with fluorescent dye at 5'-end. These microsatellite loci were amplified by the multiplex PCR method. Then, the PCR products were genotyped by the Genetic Analyzer system and the capillary electrophoresis method. Obtained data were used to estimate demographic parameters. The number of effective alleles, the number of observed alleles, the observed heterozygosity, the expected heterozygosity, and the Shannon index were calculated by GenAlex 6.5 software. Genpop 4.7.5 software was used to calculate the FIS statistic or Fixation index in the population.
The results showed that the lowest and highest number of observed alleles were related to ASB17 (17 alleles), HMS06 (eight alleles), and HMS07 (eight alleles) markers, respectively. The total number of alleles observed at all loci was 113 alleles. The average number of observed alleles was estimated to be 11.3, which indicates high allelic diversity in the Persian Arabian horse. The mean number of effective alleles, Shannon index, observed and expected heterozygosity and Fixation index were 3.97, 1.58, 0.721, 0.736, and 0.021, respectively. The mean genetic diversity in the Arabian horse’s population was calculated to be 0.736. The highest and lowest genetic diversity was observed in VHL20 (0.803) and ASB02 (0.620) markers, respectively. The average number of effective alleles per locus in studies conducted in different countries on Arabian horses, including Egypt, Syria, and Western countries, with Persian Arabian horses in this research, showed that number of effective alleles is higher in Persian Arabian horses. The average Shannon's index has been reported to be slightly lower in previous studies of Persian Arabian horses, which is probably due to the small number of samples in these studies compared to the present study.  The highest and lowest observed heterozygosities in this study were related to AHT4 (0.788) and ASB02 (0.613) markers, which were consistent with the results of the research conducted on Arabian horses of Iran, Syria, and Egypt. The positive FIS value in Arabian horses made a decrease in heterozygosity in the population, an increase in homozygosity and consequently inbreeding in Arabian horse populations. In this research, all studied loci showed a significant deviation from the Hardy-Weinberg equilibrium. Except for the AHT04 marker, which deviated from the Hardy-Weinberg equilibrium at P<0.01, the others have deviated at P<0.001. Deviation from the Hardy-Weinberg equilibrium can indicate the presence of some factors disturbing Hardy-Weinberg equilibrium, such as migration and selection, which in Persian Arabian horses, the entry of stallions from outside the herd and the gene flow between different maternal strains and also the existence of a selection program among breeders are the main factors of deviation from Hari-Weinberg equilibrium.
Evaluation of the genetic structure of Arabian horses of Iran and comparison with other Arabian horses in the world, including those in the Middle East region, showed that Arabian horses of Iran have a higher genetic diversity, which is probably due to the presence of different maternal strains in Iran, high gene flow among the different strains, a large import of Arabian horses and crossbreeding with Arabian horses of Iran, as well as a high number of samples and so identification of new alleles in this research. On the other hand, the rate of inbreeding was positive according to the FIS in Iran Arabian horses, indicating a risk of genetic diversity loss and increasing inbreeding in these horses. Therefore, management of genetic diversity and prevention of mating among related animals should be considered.

West Azerbaijan province is the second most populous sheep province in Iran. In this province, different breeds of sheep such as Makui, Herki, Ghezel, Afshar, and Shin Bash are bred. Shin Bash sheep are bred in the south of West Azerbaijan province, especially in the cities of Mahabad and Piranshahr, and its population is about 200,000. Nowadays, the management of genetic resources and the study of the risk of genetic diversity of populations has become very important. The need to preserve the genetic resources of native livestock and use these genetic resources in the future determines the genetic structure of populations, and the study of the genetic diversity within each population can help manage genetic resources and provide good information for breeding programs. With the development of molecular techniques and the use of molecular markers as a tool to assess genetic diversity, useful information has been provided at various levels such as population structure, gene flow rate, phylogenetic relationships, and genealogy tests. Identification of livestock using various molecular techniques is highly accurate and the results of studies can be used in breeding and management programs. The purpose of this study was to study the genetic structure of Shin Bash sheep using microsatellite markers on the nuclear genome and SNP markers on mitochondrial DNA (mtDNA) and to introduce a lesser-known population.
To study the genetic structure of the Shin Bash sheep population, 75 blood samples were collected from their geographic regions. Genomic DNA was extracted by using a modified Salting-Out method. Ten microsatellite markers and a control region (CR) of D-Loop belonging to mitochondrial DNA (mtDNA) were studied. Microsatellite loci were amplified in a multiplex PCR. Selected primers were labeled and genotyping was conducted using the Genetic Analyzer system. To analyze the data obtained from microsatellite markers, population parameters include: the Hardy-Weinberg equilibrium test, number of alleles per site, number of effective alleles, observed and expected heterozygosity, Shannon index, and F-statistic were calculated using POPGENE software version 3.1 and GENALEX version 6.5. In this research, Chromas ver. 2.33 (http://www.technelysium.com.au/chromas.html) was used to sort the sequencing data. Thus, the nucleotide sequence of each individual in this software was called and saved after sorting in the FASTA format. Also, to ensure the correct reading of the nucleotides, all sequences were examined using Blast online software at the NCBI site, indicating that this sequence is related to sheep mtDNA. To analyze the data obtained from sequencing in the control region of sheep mtDNA, MEGA version 7.0 and DnaSP version 6.12 were used.
   A Total of 84 alleles were identified; thus, the mean number of alleles per locus was 8.4. A total of 10 microsatellite loci were studied, seven were at Hardy-Weinberg equilibrium and three had significant deviations from Hardy-Weinberg equilibrium. Hardy-Weinberg disequilibrium can be caused by an increase in homozygotes vs. heterozygotes or, conversely, a high mutation rate, the formation of new alleles, and the presence of null alleles. The mean expected heterozygosity and observed heterozygosity in this population were 0.724 ± 0.042 and 0.80 ± 0.058, respectively. The FIS value for this population was -0.108 which showed low inbreeding and considerable diversity in the studied population. The results of the control region (CR) of mtDNA showed that haplotype diversity and percentage of the polymorphic site were 0.938 ± 0.039 and 4.59, respectively. A total of 24 sequenced individuals of the control region (CR) of mtDNA and 17 haplotypes were identified in the studied population. The amount of nucleotide diversity in the Shin Bash population was 0.0131 ±0.0013 per site. The results of this study showed that 50% of the Shin Bash population has haplogroup A, 29.2% haplogroup B, and 20.8% haplogroup C.
The results of this study, using microsatellite markers, showed that the population of Shin Bash sheep has significant genetic diversity. The negative FIS index indicates the observed heterozygosity superiority over the expected heterozygosity and thus indicates non-inbreeding and the existence of acceptable diversity within the Shin Bash sheep population. The results of mtDNA control region sequencing also showed the presence of haplotypic diversity and higher nucleotide diversity in the Shin Bash sheep population. On the other hand, the results of determining haplotype groups showed that this population has all three types of haplotype groups A, B, and C.