Selection and validation of reference genes for RT-qPCR analysis in Litchi chinensis Sonn. pericarp

Abstract

Background: Quantitative real time PCR (qRT-PCR) is an important tool for gene expression analysis and function identification. Suitable reference genes are the basis of accurate and reliable qPCR results. Litchi ( Litchi chinensis Sonn.) is a commercially important tropical and subtropical fruit crop, rapid pericarp browning is the major negative impact on the industry. Reference gene validation would help screen for genes involved in the browning mechanism.Results: In this study, fifteen new candidate reference genes, identified with transcriptome data, were assessed to determine stable reference genes for qRT-PCR analysis of litchi pericarps from different varieties, with differing postharvest storage, and under pathogenic inoculation. Ct values, Genorm, Normfind, and Reffinder algorithms, were used to identify the most stable genes. The results showed that GAGA-25 was the most stable gene for comparing different varieties of fresh pericarp, HDAC9 was the most stable gene for postharvest pericarp, STAM was the most stable for inoculated pericarp. Among the candidate reference genes, GAGA-25 was the most stable reference gene across the complete sample set.Conclusion: This study evaluated reference gene stability for qRT-PCR with litchi pericarp. This work supplies a foundation for qPCR use in future gene function and molecular mechanism studies of litchi pericarp browning.