Abstract: Bombus pyrosoma, a bumblebee species endemic to China, plays a vital role as one of the most abundant pollinators for many wild flowers and crops in North China. We report for the first time  the isolation and characterization of microsatellite markers within this population in order to support investigations into its genetic structure and evolution. Magnetic bead-based enrichment, PCR and bioinformatics were used to isolate microsatellite markers from B. pyrosoma. The species specificity of the isolated primers was confirmed through tests with seven other bumblebee species of the subgenus Melanobombus also known from China. Our results show that four dinucleotide probes have different hybridization rates to the microsatellite DNA sequence. The highest hybridization rate (82%) was obtained with the probe TC, the moderate hybridization rate (28%) with TG, and the lowest hybridization rates (0%) with AT and GC. For 31 polymorphic microsatellite loci isolated from B. pyrosoma, four types of microsatellite can be found by sequence analysis, i.e., 18 perfect types (58.1%), 10 imperfect types (32.3%), 1 compound perfect type (3.2%) and 2 compound imperfect types (6.4%). The repeat unit of the microsatellite core sequence is different in each probe. The highest repeat unit for TC and TG probes is 28 and 15, respectively, and the lowest repeat unit for TC and TG probes is 7 and 11, respectively. The primer specificity test on 31 primer pairs showed that products can be amplified from all seven bumblebee species with 26 primer pairs, and the other 5 primer pairs were found to be specific for some of the seven species, of which one primer (BPM5) was found to be specific for B. pyrosoma. These novel microsatellite markers will be useful not only for future studies on the genetic structure, molecular evolution and resource conservation of this endemic species, but also for population genetic studies of other Melanobombus species.

Key words:  Bumblebee, Bombus pyrosoma, molecular marker, microsatellite markers, magnetic bead enrichment, primer design, species specificity