Genome-wide identification and validation of optimal reference genes for gene expression normalization in pear peel

Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) is the preferred method for gene expression research, but normalization based on suitable reference genes (RGs) is the key to obtaining reliable gene expression results. In this study, we selected six “commonly used” RGs, two “traditional” housekeeping genes (HKGs), and four novel genes as candidate RGs based on 54 publicly available peel transcriptome libraries that include data from development, bagging, and post-harvest cryopreservation studies of four pear cultivars. The results of this multifaceted assessment from transcriptome and qRT-PCR analyses consistently revealed that ACT6/7/8/9 had the best expression stability among all candidate RGs, and expression of the novel RGs showed greater stability compared with the other “commonly used” RGs and “traditional” HKGs. Among the candidate RGs, ACT6/7/8/9 and Nucleosome Assembly Protein 1 (NAP1) showed superior expression stability and abundance, and they were recommended as the optimal RG combination for gene expression normalization in pear peel. These genome-wide findings provide more reasonable RG usage specifications, and additional useful and reliable RGs as resources for accurate study of gene expression in pear peel studies of different cultivars.