Identification and testing of reference genes for qRT-PCR analysis during pear fruit development

Quantitative real-time PCR (qRT-PCR) is currently one of the most reliable and improved tools for analyzing gene expression. Various studies have shown that housekeeping genes vary with cultivars, tissues and treatment. Reliable and stable reference genes were necessarily identified and evaluated according to different experimental requirements. In this study, 10 candidate reference genes were initially screened based on transcriptome sequencing data of four pear fruit development stages for three pear cultivars, including a candidate housekeeping gene PbrTUB. Furthermore, we ranked the expression stability of 10 candidate reference genes using GeNorm, NormFinder, BestKeeper and ReFinder algorithms. The results showed that Pbr028511, Pbr038418 and Pbr041114 were the most stable reference genes in Cuiguan, Housui and Xueqing fruit, respectively. Taken together, these results serve as a useful reference for gene function investigations and molecular mechanism studies in fruit development and ripening for various pear cultivars.