Systematic selection and validation of appropriate reference genes for gene expression studies by quantitative real-time PCR in pear

RT-qPCR is a widely used method in gene expression and transcriptome studies. Normalization based on reference genes is necessary to accurately analyze RT-qPCR data. Thus, an accurate and systematic evaluation of these reference genes before experiments are conducted is necessary. In this study, 18 candidate reference genes were evaluated under various experimental conditions covering a range of tissue types and cultivars, NaCl, CaCl 2 and temperature treatments, hormones (6-BA, ABA and NAA) and a set of osmotic stress (mannitol and PEG6000) treatments. Gene expression across 48 pear samples was evaluated using geNorm, NormFinder and BestKeeper statistical algorithms. Actin2/7 ( ACT2/7 ), ubiquitin extension protein ( UBI ) and Yellow-leaf-specific gene 8 ( YLS8 ) exhibited the most stable expression across all the pear samples tested. While in the other experimental groups, different sets of samples had their own best reference genes. In addition, the gene expression of PbCBL7 , a member of the calcineurin B-like protein, was measured across all the 48 samples using the best three reference genes, it displayed variation in gene expression across different tissues and cultivars, and exhibited diverse up- or down-regulated expression patterns under various treatments, which indicate that PbCBL7 may play a role in response to specific abiotic stress in pear. These results are valuable for future research on gene expression and abiotic stress tolerance in pear.