Validation of Reference Genes for Quantitative Real-Time PCR during Bicolor Tepal Development in Asiatic Hybrid Lilies (Lilium spp.).

Leifeng Xu,Yayan Feng,Hua Xu,Panpan Yang,Yuchao Tang,Jun Ming,Yuwei Cao,Suxia Yuan

doi:10.3389/fpls.2017.00669

Abstract

Quantitative real-time PCR (qRT-PCR) is a reliable and high-throughput technique for gene expression studies, but its accuracy depends on the expression stability of reference genes. To date, several reliable reference gene identifications have been reported in Lilium spp., but none has been obtained for lily tepals at different developmental stages. In this study, ten candidate reference genes were selected and evaluated for their expression stability in Lilium ‘Tiny Padhye’ during the process of bicolor tepal development. The expression stability of these candidates was evaluated by three software programs (geNorm, NormFinder, and BestKeeper) and the comparative ΔCt method, and comprehensive stability rankings were generated by RefFinder. As a result, TIP41-like family gene (TIP41) and actin (ACT) were the best combination of reference genes for tepals at different developmental stages; TIP41 and F-box family gene (F-box) for tepals under shading treatment; ACT, actin11 (ACT11), and elongation factor 1-α (EF1-α) for different tissues; and ACT, TIP41, and ACT11 for all samples. The selected optimal reference genes were further verified by analyzing the expression levels of flavonoid 3′-hydroxylase (LhF3′H) and anthocyanidin 3-O-glucosyltransfersae (LhUFGT) in tepals at different developmental stages. This study provides useful information for gene expression characterization in lilies under different experimental conditions, and can serve as a basis for similar research in other closely related species.

Highlights

(Lilium spp.) is one of the most important ornamental plants because of their various flower colors and color patterns
A total of 10 candidate reference genes were selected from the transcriptome of the Asiatic lily cultivar ‘Tiny Padhye’ for Quantitative real-time PCR (qRT-PCR) analysis
The amplification efficiency (E) of all PCR reactions ranged from 89.59% for ACT11 to 102.25% for 18S (Table 1), suggesting that these genes are suitable for further gene expression analysis

Summary

Introduction

(Lilium spp.) is one of the most important ornamental plants because of their various flower colors (yellows, oranges, pinks, reds, and whites) and color patterns (spots and bicolors). The molecular mechanisms underlying a bicolor appearance in lilies remain largely unknown. Gene expression analysis plays an important role in elucidating the molecular mechanisms underlying various biological processes (Bustin et al, 2005). QRT-PCR has been widely used as a powerful technique for monitoring gene expression profiles in different samples, due to its high sensitivity, accuracy, specificity, throughput capability, and cost-effectiveness The accuracy of relative quantification in qRT-PCR is always affected by many variable factors (RNA quality, reverse transcription efficiency, and amplification efficiency), which may cause inaccuracies in the gene expression data (Nolan et al, 2006). It is necessary to use one or more stable reference genes to normalize the expression data of target genes

Methods

Results

Conclusion