biologia plantarum

Biologia plantarum 66:103-111, 2022 | DOI: 10.32615/bp.2021.066

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

F. LI^{1, 2}, J.H. SUN^{1, 2}, J.L. MEN^{1, 2}, H.L. LI^{1, 2}, G. WANG^{1, 2}, S.J. WANG^{1, 2}, J.B. WANG^{1, 2, *}

¹ Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou, Hainan 571101, P.R. China.

² Danzhou Scientific Observing and Experimental Station of Agro-Environment, Ministry of Agriculture and Rural Affairs, Danzhou, Hainan 571737, P.R. China.

Real-time reverse transcription quantitative PCR (RT-qPCR) is an important tool for gene expression analysis. Suitable reference genes are the basis of accurate and reliable RT-qPCR results. Litchi (Litchi chinensis Sonn.) is a commercially important tropical and subtropical fruit, but rapid pericarp browning is a substantial negative impact on its commercial use. Reference gene validation could help in the screening for genes involved in the browning mechanism. We assessed 15 new candidate reference genes from litchi transcriptome to determine stable reference genes for RT-qPCR analysis of pericarps from different cultivars, with differing postharvest storage, and under pathogenic stress. Ct values, geNorm, Normfinder, and RefFinder algorithms, were used to identify genes with the most stable transcription. GAGA-25 was the gene with the most stable transcription for comparing different varieties of the fresh pericarp. HDAC9 was the gene with the most stable transcription for postharvest pericarp. STAM was the gene with the most stable transcription for inoculated pericarp. Of the candidate reference genes, GAGA-25 was the most stable reference gene across the complete sample set. This study evaluated reference gene stability for RT-qPCR in litchi pericarp. This work provides a foundation for using qPCR to study gene function and molecular mechanism studies of litchi pericarp browning.

Keywords: Litchi chinensis Sonn., pericarp, reference genes, RT-qPCR, gene validation.

Received: February 19, 2021; Revised: November 4, 2021; Accepted: November 10, 2021; Published online: April 7, 2022  Show citation