Identification of Suitable Reference Genes for qRT-PCR Normalization in Tilia miqueliana Maxim

Quantitative real-time polymerase chain reaction (qRT-PCR) is a rapid and effective approach toward detecting the expression patterns of target genes. The selection of a stable reference gene under specific test condition is essential for expressing levels of target genes accurately. Tilia miqueliana, considered endangered, is a prominent native ornamental and honey tree in East China. No study has evaluated the optimal endogenous reference gene for qRT-PCR analysis in T. miqueliana systematically. In this study, fifteen commonly used reference genes were selected as candidate genes, and the stabilities of their expressions were assessed using four algorithms (GeNorm, NormFiner, BestKeeper, and DeltaCt) in nine experimental datasets. The final integrated evaluation was performed using a comprehensive analysis algorithm (RefFinder). Finally, a target MYB transcription factor gene (TmMYB) was used to verify the accuracy of the candidate reference genes. The results showed that PP2 alpha was the most stable in tissue set, while RPS13 and SAMCD were optimal for heat and cold stress, respectively. Under waterlogging stress, PP2 alpha and TUB were the most stable genes in the leaves and roots, respectively. EF1 alpha and PP2 alpha were optimal for drying stress in leaf and root tissues. TUB and ACT7 were the most stable genes in the leaf and root tissues under salt stress. This is the first systematic evaluation of candidate reference genes in T. miqueliana, and it will benefit future studies on expression and functional analysis of target genes in T. miqueliana.