This study analyzed the genetic diversity and structure of two Bulgarian indigenous sheep (Ovis aries) breeds -Kotel and Teteven, each presented by 4 flocks, using 15 microsatellite markers. A total of 195 alleles were identified. Among the markers used, the most informative were INRA5 and OarFCB20 with PIC values of 0.87. Both breeds showed a high level of genetic diversity with average values of He and Ho, respectively 0.74 and 0.76. The observed high level of genetic diversity is combined with the absence of heterozygous deficiency reported as Fis values of 0.04 and 0.07, respectively for Kotel and Teteven breeds. Low level of genetic differentiation between the two breeds was observed, as evidenced by the low value of Fst of 0.09, and analysis of variance (ANOVA) data showing that only 9% of the total variation is due to variation between breeds, whereas 91% is due to variation within breeds. Furthermore, we revealed a breeding practice that comprises an exchange of animals between the breeds which reflects in the clustering obtained by STRUCTURE analysis. According to it, Kotel breed is a more homogenous population and forms cluster 1, whereas Teteven breed is more heterogenous and consists of two subpopulations that form two distinct clusters. The results obtained in the present study confirm the high potential of microsatellite markers in studies related to both breed affiliation and purity and can serve as a basis for the implementation of future management and conservation programs of these indigenous breeds.

This study attempts to provide a deeper insight into the current genetic status of 12 Bulgarian autochthonous sheep breeds using microsatellite (SSR) markers. A total of 600 individuals from 50 flocks were analyzed using a panel of 13 SSR markers. In total, 228 alleles were found in the studied microsatellite loci. The mean number of alleles, the effective number of alleles, and the polymorphic information content (PIC) values per locus were 17.54, 5.250, and 0.799, respectively. The expected heterozygosity (He) for all breeds ranged from 0.70 to 0.82. The within-population heterozygote deficit (Fis) varied from −0.03 to 0.1, reflecting significant levels for 10 of the 12 breeds. The average genetic differentiation (Fst) was 0.046, revealing a low discrimination between the breeds. The genetic distance, principal coordinate analysis, and the structure analysis showed that two of the studied breeds—Local Stara Zagora/SZ/ and Local Karnobat/MK/—were the most distinct sheep populations. The Bayesian clustering approach suggested poor breed differentiation for the remaining 10 sheep breeds. The results suggest that proper management strategies and specific breeding policies need to be implemented in Bulgaria to avoid the intermixing of breeds and to reduce the erosion of breed purity observed in some breeds.