Genetic structure of Apis mellifera carnica in Slovakia based on microsatellite DNA polymorphism

Carniolan honeybee (Apis mellifera carnica) is considered an endangered subspecies of Apis mellifera in Slovakia due to its illegal crossbreeding with allochthonous subspecies. Despite more than a century-long tradition of Slovak beekeeping, genetic research of Slovak honeybees has not yet been done. The aim of our study was to develop a molecular approach to characterize Carniolan honeybee population using polymorphic microsatellite markers. A total of 79 samples of Slovak Carniolan honeybee from 19 regions of Slovakia were analyzed together with 85 reference samples of Apis mellifera mellifera, Apis mellifera macedonica, Apis mellifera ligustica and Apis mellifera hybrid (Buckfast). Ten highly informative (PIC > 0.5) microsatellite markers running in two multiplex PCR reactions were used for molecular analysis of bees. The mean number of alleles per locus ranged from 4.6 (A. m. mellifera) to 11.5 (A. m. carnica). Bayesian and frequency-based methods implemented in software ‘GeneClass’ (v 2.0.h) have been used to assign individuals to populations. We were able to successfully assign 95% of individuals in actual sample size. After simulating 100,000 individuals using Bayesian Markov chain Monte-Carlo resampling, we correctly assigned 88% individuals to five tested populations. Population genetic structure was inferred through the Bayesian clustering method incorporated in software ‘Structure’ (v 2.3.4). Overall, 92% of the individuals were assigned to the same subspecies as originally declared. According to Structure analysis, Slovak population of A. m. carnica appears to be separated into two clusters, showing the relatively high level of gene flow from A. mellifera hybrid (Buckfast) and A. m. macedonica subspecies. It might be caused by rebuilding the Slovak population after colony losses using imported subspecies of the honeybee.