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Normalization to reference genes is the most common method to avoid bias in real-time quantitative PCR (qPCR), which has been widely used for quantification of gene expression. Despite several studies on gene expression,  Lilium , and particularly  L. regale , has not been fully investigated regarding the evaluation of reference genes suitable for normalization. In this study, nine putative reference genes, namely  18S rRNA ,  ACT ,  BHLH ,  CLA ,  CYP ,  EF1 ,  GAPDH ,  SAND  and  TIP41 , were analyzed for accurate quantitative PCR normalization at different developmental stages and under different stress conditions, including biotic ( Botrytis elliptica ), drought, salinity, cold and heat stress. All these genes showed a wide variation in their Cq (quantification Cycle) values, and their stabilities were calculated by geNorm, NormFinder and BestKeeper. In a combination of the results from the three algorithms,  BHLH  was superior to the other candidates when all the experimental treatments were analyzed together;  CLA  and  EF1  were also recommended by two of the three algorithms. As for specific conditions,  EF1  under various developmental stages,  SAND  under biotic stress,  CYP/GAPDH  under drought stress, and  TIP41  under salinity stress were generally considered suitable. All the algorithms agreed on the stability of  SAND  and  GAPDH  under cold stress, while only  CYP  was selected under heat stress by all of them. Additionally, the selection of optimal reference genes under biotic stress was further verified by analyzing the expression level of  LrLOX  in leaves inoculated with  B. elliptica . Our study would be beneficial for future studies on gene expression and molecular breeding of  Lilium .

Evaluation of putative reference genes for quantitative real-time PCR normalization inLilium regaleduring development and under stress