Identification of Suitable Reference Genes for RT-qPCR Assays in Liriodendron chinense (Hemsl.) Sarg

The precision and reliability of reverse transcription quantitative polymerase chain reaction (RT-qPCR) depend mainly on suitable reference genes; however, reference genes have not yet been identified for Liriodendron chinense (Hemsl.) Sarg. In this study, the expression stability of 15 candidate reference genes, ACT7, ACT97, UBQ1, eIF2, eIF3, HIS, BIG, AGD11, EFG, GAPDH, CYP, RPL25, UBC, RPB1, and TUB, was tested across multiple organs of L. chinense using four algorithms, geNorm, NormFinder, BestKeeper, and RefFinder. To understand the difference between the selected reference genes and the unsuitable candidate reference genes, the expression level of a target gene, LcPAT7, was normalized across various plant samples. ACT97 and eIF3 represented the best combination across all samples tested, while AGD11 and UBQ1 were unsuitable for normalization in this case. In the vegetative organ subset, ACT97, ACT7, and GAPDH showed the highest expression stability. For floral organs, UBC and eIF3 were the most stable reference genes. Unsuitable reference genes underestimated the expression levels of a target gene, LcPAT7. This study identified two reference genes (ACT97 and eIF3) for the precise and reliable normalization of L. chinense RT-qPCR data across various organs. Our work provides an effective framework for quantifying gene expression in L. chinense.