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

Abstract

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.

Identification of Reference Genes for qRT‐PCR Gene Expression Studies during Seed Development and under Abiotic Stresses in Cyamopsis tetragonoloba

“…Similarly, ACT 11 was also not found to be a suitable reference gene for qRT-PCR studies in different tissues of poplar (Brunner et al, 2004). However, this gene was not the suitable reference gene under various developmental stages in rice (Pabuayon et al, 2016) and Lilium regale E.H. Wilson (Liu et al, 2016). In a previous report, ACT 7 was the most suitable reference gene for a developmental stage gene expression study in soybean ( Jian et al, 2008).…”

“…In a previous report, ACT 7 was the most suitable reference gene for a developmental stage gene expression study in soybean ( Jian et al, 2008). However, this gene was not the suitable reference gene under various developmental stages in rice (Pabuayon et al, 2016) and Lilium regale E.H. Wilson (Liu et al, 2016).…”

“…In earlier studies, GAPDH and ACT 7 genes were found to be suitable reference genes for qRT-PCR normalization under heat stress conditions in Brachypodium distachyon (Hong et al, 2008). In heat stress studies, GAPDH was also found to be the most suitable reference gene in pigeonpea (Sinha et al, 2015) and Lilium regale (Liu et al, 2016), whereas this gene was not a suitable reference gene in chickpea (Reddy et al, 2016) and maize (Lin et al, 2014). The ACT 7 gene was not the suitable reference gene in Caragana intermedia Kuang & H.C. Fu (Zhu et al, 2013) for qRT-PCR studies in heat stress condition.…”

Identification of reference genes for real-time PCR gene expression studies during seed development and under abiotic stresses in Cyamopsis tetragonoloba (L.) Taub

“…In a previous report, ACT 7 gene was the most suitable reference gene for developmental stage gene expression study in soybean (Jian et al 2008). However, this gene was not the suitable reference gene under various developmental stages in rice (Pabuayon et al 2016) and Lilium regale (Liu et al 2016).…”

“…In the earlier studies, GAPDH and ACT 7 genes were found to be suitable reference genes for qRT-PCR normalization under heat stress condition in Brachypodium distachyon (Hong et al 2008). In heat stress studies, the GAPDH gene was also found to be the most suitable reference gene in pigeonpea (Sinha et al 2015) and Lilium regale (Liu et al 2016) whereas this gene was not a suitable reference gene in cicer (Reddy et al 2016) and maize (Lin et al 2014). The ACT 7 gene was not the suitable reference gene in Caragana intermedia (Zhu et al 2013) for qRT-PCR studies in heat stress condition.…”