microsatellite markers

The aim of this study was to investigate the genetic structure of Iranian native horse breeds to compare genetic diversity and understanding the relationships between the populations using 11 ISAG microsatellite markers. For this reason, 565 samples of the Iranian Equestrian database from different ages including the Iranian Arab Asil, Caspian, Darehshouri, Kurdish and Turkmen and 113 samples were used for each breed. The number of observed alleles for each locus was 7 to 19 alleles with an average of 10.81 alleles, and it was 19 for ASB17 and 7 for HTG4 locus with the highest and lowest observed alleles ranking respectively. The average observed heterozygosity from the largest to the lowest rank was, Turkmen (0.68±0.11), Caspian (0.67±0.07), Kurdish (0.66±0.06) Darehshouri (0.65±0.07), and Arab Asil (0.62±0.08). Population structure analysis with UPGMA method showed that Caspian and Kurdish populations were grouped as a unit cluster while the other populations grouped as a separate cluster. These results confirmed this hypothesis that the Caspian and Kurdish populations are close to the Nisa horses. In general, the results of this study indicate that the Iranian native horses have got a high genetic diversity, despite of populations have genetic similarity and the other hand genetic clustering of the populations is consistent with their geographic distances. The result of this study shows that the ISAG microsatellite markers are polymorphic and have more efficiency for assignment genetic diversity and genetic structure analysis of Iranian native horse breeds.

Due to having historical value and climatic diverse, Iran has unique horse breeds. Unfortunately, due to the lack of attention and control of the import of foreign breeds into this rich
genetic source, huge damage has been created. Therefore, many horse breeds in the country got crossbred and their racial purity reduced. Knowing the genetic structure of native breeds will play an important role in their safeguarding and ensuring of their survival. Indigenous breeds, due to their unique characteristics, are considered as part of the genetic resources of the country, and their genetic structure will help them to protect and develop eugenic programs.

 This study was conducted on 136 horses including Taleshi (25 samples), Caspian (49 samples) and Arabic (62 samples) breeds from their breeding areas. Taleshi horse samples was captured from local stock in the Guilan province, Caspian samples was captured from horse riding clubs around Tehran and Guilan provinces, and Arabian samples was captured from horse riding clubs around the provinces of Tehran, Khuzestan and Alborz. They were unrelated and selected randomly. The race recognition of horses was based on books published by the Federation of Equestrian and their phenotypic characteristics. After determining of the concentration and uniformity of the concentration of extracted DNA, all individuals under study were conducted for 12 microsatellite markers recommended by the Animal Genetic Association (ISAG) to determine the genotype in order to estimate the parameters such as heterozygosity, inbreeding, Hardy and Weinberg equilibrium, and so on and find an appropriate strategy to maintain these valuable breeds. The number of observed alleles (na), and effective (ne), observed heterozygosity (Ho) and the Expectation case (He), Shannon index (I), inbreeding coefficient (ISF), genetic distance, genetic similarity, Hardy-Weinberg equilibrium, and phylogeny tree between races were calculated using POPGENE 1.31 software.

 All of the used markers were multi-shaped and the ASB17 markers with 12 alleles and HMS6 produced the highest and lowest number of alleles with 6 alleles, respectively.
The highest and lowest expected heterozygosity were calculated in VHL20 (0.78) and ASB23 (0.62) markers, and the average Shannon index for all sites was 1.72. Hardy Weinberg balance
analysis by Chi square test showed that except ASB2 and HMS3 markers in Taleshi breed, ASB17 and HTG4 markers in the Caspian breed and ASB17 markers in the Arabic breed, all markers had a significant deviation from Hardy and Weinberg equilibrium (P <0.05). The highest observed heterozygosity was related to AHT5 marker (0.81) and the lowest observed heterozygote was related to ASB17 and HTG4 markers (0.68). The Shannon index, like the observed heterozygosity, shows the amount of genetic diversity, and because the maximum value is equal to Ln (n), it is useful to express the genetic diversity of multi-formed sites. The highest and lowest values of Shannon index were 1.96 and 1.46, respectively, for VHL20 and HTG4 markers. Given the fact that the VHL20 marker showed the highest and the HTG4 marker showed the least effective allele, so the calculated values for the Shannon index are justified. Phylogeny diagram showed that Caspian and Taleshi horses were placed in one branch and Arab horses in separate branch.

Breeds with fewer populations are more at risk for genetic changes and extinction. Reducing of the genetic diversity of indigenous populations will result in the loss of many useful
genes, especially those that are compatible with different environments and resistant to regional diseases. Undoubtedly, it is very important to pay attention to the genetic diversity of small-sized
population of breeds such as Taleshi and Caspian. The results of this study showed that due to the low number of Caspian and Taleshi horses, genetic diversity is still at a high level, so it would be hoped that by adopting the principled measures, we would prevent their extinction in the long duration.

In some cases, due to overlapping of morphological traits and parents incomplete recording information, it is difficult to determine the breed origin of a horse. The use of molecular methods for assigning individuals to their breed can be a significant help in this regard. The present study was conducted to investigate the genetic structure and the accuracy of the assignment of horses to the origin populations using microsatellite markers. For this purpose, samples from 165 horses including Caspian (35), Arabian (36), Turkoman (30), Thoroughbred (28) and Turkoman - Thoroughbred crossbred population (36) were randomly collected. Polymerase chain reaction was performed using 12 microsatellite markers recommended by the International Society for Animal Genetics (ISAG), and the PCR products were separated by 8% acryl amide gel and stained using silver nitrate staining procedure. Estimation of genetic parameters such as the number of effective alleles and the expected heterozygosity level showed a high genetic variation in the used markers. Among them AHT4 marker showed the highest (11.20 alleles) and the AHT5 showed the lowest (4. 28) effective alleles and AHT4 (0.82) showed the highest and ASB17 (0.67) showed the lowest genetic variation. The highest and lowest genetic distances were observed between Thoroughbred with Arabian and Caspian (0.91) and Thoroughbred with crossbred population (10.0). In conclusion, the markers used in this study, could correctly assign 79% of the individuals to their source populations. Therefore, in some cases microsatellite markers can be a helpful tool for determining the breed origin of the horse.

In this study, the genetic variation in Zandi sheep was investigated using 15 microsatellite markers. Genomic DNA was extracted from 120 Blood samples, using modified salting-out method. All of fifteen loci were amplified successfully and all tested loci were all polymorphic. The Tests of genotype frequencies for deviation from the Hardy-Weinberg equilibrium (HWE) were performed at each locus and revealed significant departure from HWE (P<0.001) due to heterozygote excess.Parameters of variability such as effective number of alleles and gene diversities (Heterozygosity) corroborated the high level of variation frequently displayed by microsatellite markers. Furthermore, other criteria of genetic variation including PIC values and Shanon information index had calculated in this study. The mean of polymorphism information content of the Zandi sheep population was equal to 0.808 and indicate high level of polymorphism. Also the Shannon's information index of the Zandi sheep population was high (1.92 ± 0.09). Based on results of this research, high level of genetic diversity was observed in Zandi breed. This research also was shown that microsatellite markers is a useful tool for evaluation of genetic variation among of domesticated animals.