Study of the genetic structure of the Shin Bash sheep population by molecular markers

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.