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

Abstract

GAGA-25 - GATA transcription factor 25; HDAC9 - histone deacetylase 9; HLM2B - histone-lysine_N-methyltransferase 2B; NtaA - N(alpha)-acetyltransferase 16, NatA auxiliary; pbP - peroxisome biogenesis protein 22-like; RFU1 - RING finger ubiquitin ligase; RT-qPCR reverse transcription qPCR; RUB1 - ubiquitin-NEDD8-like protein RUB1; STAM - Stam binding; TL-OEMC - translocon at the outer membrane of chloroplasts 64; UPF3 - UPF3 regulator of nonsense transcripts homolog UPF3; V - variation. Abstract 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.