Genome-wide identification and testing of superior reference genes for transcript normalization during analyses of flesh development in Asian pear cultivars

Abstract Quantitative real-time polymerase chain reaction (qRT-PCR) is currently the best available option for analyzing gene expression and transcriptomes. In this context, it is important to use ideal reference genes (RGs) to normalize the expression data for the samples to be compared. Thus, the present study was established to identify and validate superior RGs for qRT-PCR analyses of fruit development in Asian pear cultivars. A total of seven “commonly used” RGs, seven “traditional” housekeeping genes (HKGs), and four novel genes were selected as candidate RGs based on 35 publicly available transcriptome libraries that include data for fruit development in five Asian pear cultivars. Transcriptomic and qRT-PCR analyses consistently revealed that the novel RGs were expressed more stably than “commonly used” RGs and “traditional” HKGs. Among the novel RGs, BPS1 and ICDH1 were the most highly and stably expressed genes. Moreover, these two genes formed the optimal RG combination for normalizing gene expression data during analyses of pear flesh development. These findings may help researchers optimize their selection of RGs for future investigations of gene expression during flesh development in Asian pear cultivars.