Identification of suitable reference genes in Taxodium 'Zhongshanshan' under abiotic stresses

This work provides a guideline for application of Taxodium 'Zhongshanshan' reference gene usage in the qRT-PCR system, and also for reference gene selection for other Taxodiaceae species. Quantitative real-time PCR (qRT-PCR) is a practical method which is frequently used for analysis of gene expression. However, many factors can affect the qRT-PCR analysis by influencing the quantitative measurement of the expression of genes. To avoid erroneous experimental analyses, it is necessary to select suitable reference genes for normalization of the variation. Taxodium 'Zhongshanshan' (T. 'Zhongshanshan') is an economically and ecologically important tree species in China for its extreme tolerance to several stresses. Although some responses of T. 'Zhongshanshan' to abiotic stresses have been studied, reference genes suitable for the normalization of qRT-PCR data under stresses have not previously been identified. Ten candidate reference genes were used to validate expression stability in this study, including actin (ACT), adenine phosphoribosyl transferase (APRT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), protein phosphatase 2A (PP2A), tubulin (TUB), 18S ribosomal RNA (18S rRNA), 25S ribosomal RNA (25S rRNA), ubiquitin extension protein (UBI), ubiquitin (UBQ), and SKP1/ASK-interacting protein (SKIP). To select appropriate reference genes, we selected a diverse set of samples representing different stress conditions. GeNorm and NormFinder were made to evaluate the result systematically. The results indicated that no single reference gene had an optimal performance across all the experimental systems, while, PP2A and APRT can be proposed as good starting points for gene-expression studies. The qRT-PCR analysis of pyruvate decarboxylase (PDC) expression patterns in response to half-waterlogging stress confirmed the reliability of selected stable reference genes. Our results have provided a guideline for application of T. 'Zhongshanshan' reference gene usage in the qRT-PCR system.