Screening and Evaluation of Stable Reference Genes for Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) Analysis in Chinese Fir Roots under Water, Phosphorus, and Nitrogen Stresses

Chinese fir (Cunninghamia lanceolata) is an economical important timber species widely planted in southeastern Asia. Decline in yield and productivity during successive rotation is believed to be linked with abiotic stress, such as drought stress and nitrogen (N) and phosphorus (P) starvation. Molecular breeding could be an option to develop tolerant genotypes. For gene expression studies using quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR), stable reference genes are needed for normalization of gene expression under different experimental conditions. However, there is no internal reference genes identified for Chinese fir under abiotic stresses. Thus, nine internal reference genes based on transcriptome data were selected and analyzed in the root of Chinese fir under drought stress and N and P starvation. Data were analyzed using geNorm, NormFinder, and BestKeeper, to screen and identify the best reference genes. The results showed that the UBQ and GAPDH genes were the two most stable genes under drought stress and the Actin1 and GAPDH were the two most stable genes under P starvation. Further, it was discovered that the Actin1 and UBC were the two most stable genes under N starvation among nine candidate reference genes. The gene expression of drought stress induced expression protein 14-3-3-4, the P transporter gene ClPht1;3, and the nitrate transporter gene NRT1.1 were used to verify the stability of the selected reference genes under drought stress and P and N starvation, respectively, and the results revealed that the screened reference genes were sufficient to normalize expression of the target genes. In conclusion, the results demonstrate that the stability of reference genes was closely related to the external conditions and reference genes applied to the roots of Chinese fir under different abiotic stress treatments were different. Our data will facilitate further studies on stress ecology and gene function analysis in Chinese fir.