Screening for quantitative real-time PCR reference genes with high stable expression using the mRNA-sequencing data for pear

Abstract

Housekeeping genes were considered to have a constant expression without spatio-temporal difference. They are commonly used as the internal reference for PCR-based gene expression analysis. However, increasing evidence revealed that the expression of housekeeping genes also varied with tissues and treatments. Previous studies for reference gene identification were usually limited to a small number of housekeeping genes based on their expression stability comparison. In this study, reference genes for pear fruit were initially screened on the transcriptome level with 10 messenger RNA (mRNA)-seq datasets from the fruit of two pear species across important developmental stages. Ten potential reference genes were chosen for validation using the mRNA-seq data from fruit skin libraries. Five potential reference genes are proposed that can be used as suitable controls for PCR-based expression studies in pear fruit. Four of the selected genes had homologous annotation of important roles in the basic biological processes. Sulfhydryl oxidase 2 (SOX2) and protein phosphatase (PP) 2A also showed high stable expression in both pear fruit and other tissues and should be more widely applicable as reference genes in different tissues or species. This study provided a comprehensive view for transcriptional analysis in pear fruit, which will help researchers to select reference genes for normalization against target genes by qRT-PCR across different developmental stages of the fruit as well as for different tissues and species.