Reference gene selection for RT-qPCR normalization in the drone testis of honey bee (Apis cerana) during meiosis stages

Drones are the males in the honey bee population that provide high quantity and quality sperm to mate with the queen and ensure colony reproduction. To understand spermatogenesis in the drone testis at the molecular level, reverse transcription quantitative PCR (RT-qPCR) is a standard and useful means to investigate the target genes. However, a lack of appropriate reference genes hinders the accurate normalization of target genes. To identify stable reference genes in the drone testis during the meiosis stages of honey bee, we assessed eight candidate genes using BestKeeper, delta-CT, GeNorm, NormFinder, and RefFinder tools. Moreover, the expression pattern of stat92e and dicer1 was used to validate the stability of the selected reference genes. The results showed that the most stable reference gene in the testis during meiosis stages varied according to different algorithms, and gapdh was the most stable reference gene in the drone testis during meiosis stages when integrating these different algorithms. In addition, gapdh, rps18, and the combination of gapdh & rps18 were optimal reference genes to normalize the expression of the target genes in testis during meiosis stages, as validated by the expression of stat92e and dicer1. Our findings provide a conducive foundation for the accurate quantification of gene expression levels in the testis during the drone meiosis stages of the honey bee.