In this study, we explored a more efficient method for genotyping honeybee colonies using the contents of the queen bee's spermatheca instead of individual offspring (worker bees). This method saves time and provides insight into the composition of paternal lines immediately after mating. To obtain genetic information from spermatozoa in the spermatheca, we investigated different methods for short-term storage of spermatheca and DNA isolation. We tested six different storage methods coupled with two DNA isolation methods. Their success was verified by analyzing DNA, RNA, and protein concentrations, as well as DNA fragmentation and suitability of DNA quality for microsatellite (MS) analysis. We also investigated the use of MS analysis and the csd gene sequence to determine paternal lines in the bee colony. Our results showed that the range of medians of DNA concentrations from the spermathecae isolated before storage was generally lower than the range of medians of DNA concentrations from those isolated after storage. However, the storage method or DNA isolation method did not significantly affect the success of MS amplification. For transport at room temperature, we recommend ethanol as a storage medium due to its availability, low cost, ease of use in the field and laboratory, and the ability to preserve samples well for DNA analysis. The results for determining paternal lines with analysis of the contents of the spermatheca were found to be equivalent to te results with analysis ot the brood, indicating that the use of spermatheca is more suitable than brood. Preliminary testing of the method for determining paternal lines using the csd gene sequence in the spermatheca showed potential usefulness.
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