Development of Reference Genes for Horticultural Plants

Abstract Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is extensively applied technique to investigate the transcript abundance of target genes in various organisms. Selection of appropriate reference genes (RGs) for qRT-PCR normalization is a crucial prerequisite for accurately quantifying gene expression level. RGs should exhibit minimal variation in gene expression. However, the actual expression stability of RGs fluctuates greatly in different species or under different experimental conditions. Due to rapid advancements in next-generation sequencing (NGS) technology, it is no longer difficult to get massive transcriptome data, which has greatly promoted the development of RGs. In this review, we elaborate on the strategies for developing RGs using Northern blotting, expressed sequence tags (ESTs), qRT-PCR, and high-throughput technologies such as microarray and RNA-sequencing (RNA-Seq). The process for developing RGs based on RNA-Seq is further addressed, including processing and normalization of RNA-Seq data, evaluation of gene expression stability, and screening and validation of RGs. The most frequently used RGs in horticultural plants are summarized, and the strategies for developing these RGs are introduced in detail. The information provided here will help to design effective strategies for the development of RGs in horticultural plants, with a focus on using big data generated by RNA-Seq.