Genetic diversity of Bahmaei sheep breed using microsatellite analysis

Introduction In Iran, sheep are one of the important farm animals for a variety of products (milk, meat, and wool), and their important role as a sacrificial animal in many religious ceremonies. Genetic variation in economic traits is the basis of breeding programs in sheep and the lack of diversity in the base population has the limiting effect on selection power to meet unpredictable needs in the future. Generally, microsatellites are considered codominant and highly polymorphic markers and have quickly become one of the best molecular markers to estimate genetic diversity in different livestock species. In recent years, several studies have been reported the use of microsatellites to determine genetic diversity, population structure, genetic differentiation and phylogenetic reconstruction with the aim of identifying endangered populations and also developing genetic conservation strategies in sheep. Therefore, this study aimed to assess the genetic diversity and population genetic structure of the Bahmaei sheep breed in the Kuhgiloyeh and Boyer Ahmad province of Iran using 10 microsatellite markers.
Materials and methods In this study, blood samples were collected from 20 non-related animals of the Bahmaei sheep breed in Kohgiluyeh and Boyer Ahmad province. DNA was extracted from samples using the Takapouzist Kit (BiONEER), following the manufacturer’s instructions. The genetic variation of obtained samples was investigated using 10 different microsatellite markers. Microsatellite loci were selected from different chromosomes of the genome to reduce the possibility of linkage between the loci and to obtain an appropriate genetic variation estimation due to the same dispersion of markers on different chromosomes. PCR amplification was performed at a reaction volume of 25 µL. The final concentration of reaction mixtures contained 1 unit Taq DNA polymerase enzyme, 200 μM of each dNTP, 1.5 mM MgCl2, 18 pm of each primer, 50 ng DNA, and 2.5 mM 10× buffer, for all the microsatellite markers. After performing the PCR, the amplified DNA fragments were separated by 8% polyacrylamide gel electrophoresis. Then, the alleles were characterized by silver nitrate staining. The allele frequencies for all loci, observed (Na) and effective number of alleles (Ne) per locus, expected (He) and observed heterozygosity (Ho), Shannon's information index (I) as a measure of genetic variation, and Deviation from Hardy Weinberg's equilibrium were estimated by Chi-square test and Wright's index (F) using the GenAIEx 6.5 software. The polymorphism information content (PIC) for each locus was estimated using PowerMarker software.
Results and discussions
A total of 108 alleles were found in 10 microsatellite loci. The highest and lowest allele numbers were for BM6444 and ETH10 loci, respectively. In Qashqai Lak sheep, a total of 88 alleles were identified for the same number of 10 loci, and TGLA53 and ETH10 loci showed the maximum and minimum number of alleles with 15 and 2 alleles, respectively (Salehi and Muhaghegh, 2015).
The mean observed heterozygosity was more than expected for all loci. Based on the results, the observed heterozygosity at all loci was 1.0. The expected heterozygosity range in the 10 studied loci was from 0.55 to 0.91, the highest of which is related to BM6444, and the smallest is ETH10. The highest unbiased expected heterozygosity was for BM6444 and the lowest was for ETH10. The mean observed, expected, and unbiased expected heterozygosity were 1, 0.81, and 0.83, respectively, indicating a high level of heterozygosity in the analyzed population. The average value of polymorphism information content (PIC) for all loci was 0.79 for 10 microsatellites. The PIC for all investigated markers was higher than 0.5 in 7 breeds of Montenegrin sheep, ranging from 0.60 (INRA172a) to 0.93 (CP49a), so they can be considered to contain high PIC (Marković et al. 2022). In this study, by comparing the Shannon index among the 10 markers, all of which were polymorphic. The highest value of the Shannon index was related to the BM6444 marker with the highest allele number and the lowest value of the Shannon index was for the ETH10 marker with the lowest allele number. The average Shannon index was also high for all loci, indicating the high polymorphism of these loci and confirming the high genetic variation. In two breeds of Bulgarian sheep, the average Shannon index for both populations was 1.79, and the highest and lowest index values were obtained for McM042 and INRA005 loci, respectively (Odjakova et al. 2023). Negative or near zero values of Wright's index for loci indicate that there is a cross between animals whose kinship relationship is less than the average relative relationship of the corresponding population. Tests of genotype frequencies for deviation from the Hardy-Weinberg equilibrium (HWE) at each locus, revealed a significant departure from HWE for all loci (p<0.001). In 13 Colombian sheep breeds, Hardy-Weinberg equilibrium tests for 11 microsatellite markers showed significant deviations (P<0.05), which indicated heterozygous deficiency in each breed (Ocampo et al. 2016).
Conclusion In this research, the genetic diversity of Bahamei sheep breed was investigated using 10 microsatellite markers. The results of the present research indicate the basic information about the genetic diversity in the Bahamai sheep, one of the native sheep of Kohgiluyeh and Boyer Ahmad provinces. The studied population showed high genetic diversity through the high number of effective alleles, mean number of alleles, heterozygosity and PIC values. Therefore, the results reported in this study can provide useful indicators for developing more in-depth studies with more accurate sampling of sheep and increasing the sample size to determine appropriate conservation and breeding priorities, especially considering its potential economic and cultural importance.
Keywords: Bahmaei sheep, Genetic variation, microsatellite