Reference gene selection for qRT-PCR analysis of flower development in Lagerstroemia indica and L. speciosa.

Tangchun Zheng,Ming Cai,Huitang Pan,Zhilin Chen,Yiqian Ju,Han Zhang,Qixiang Zhang

doi:10.1371/journal.pone.0195004

Abstract

Quantitative real-time polymerase chain reaction (qRT-PCR) is a prevalent method for gene expression analysis, depending on the stability of the reference genes for data normalization. Lagerstroemia indica and L. speciosa are popular ornamental plants which are famous for the long flowering period. However, no systematic studies on reference genes in Lagerstroemia have yet been conducted. In the present study, we selected nine candidate reference genes (GAPDH, TUA, TUB, 18S, RPII, EF-1α, ATC, EIF5A and CYP) and evaluated their expression stability in different tissues during floral development of L. indica and L. speciosa using four algorithms (geNorm, NormFinder, BestKeeper and, RefFinder). Results showed that RPII and EF-1α were the most stably expressed and suitable reference genes for both of Lagerstroemia species. Moreover, ACT exhibited high expression stability in L. indica and GAPDH was a suitable reference gene for L. speciosa in different flower development stages. TUB was an unsuitable reference gene for gene expression normalization due to significant variations in expression across all samples. Finally, we verified the reliability of the selected candidate reference genes by amplifying an AGAMOUS homolog (LsAG1) of Arabidopsis thaliana. This study provides a list of suitable reference genes, thereby broadening the genetic basis of the gene expression patterns in Lagerstroemia species.

Highlights

QRT-PCR is a popular method to study gene expression with high sensitivity, specificity and adaptability to high throughput analyses [1,2,3]
Expression profiling of candidate reference genes Quantitative real-time polymerase chain reaction (qRT-PCR) for transcript profiling was performed with primers of the nine candidate reference genes in the 16 sample sets of L. indica and L. speciosa
The qRT-PCR method has become a popular and powerful tool to analyze the expression patterns of target genes, it requires proper reference genes for normalization [26]. qRT-PCR is convenient and fast to analyze the genetic regulation of plant growth and development, especially the floral development which is the key to diversification in plant kingdom

Summary

Introduction

QRT-PCR is a popular method to study gene expression with high sensitivity, specificity and adaptability to high throughput analyses [1,2,3]. It bases on the use of reference gene as the normalization of gene transcription levels requires the stability of the selected reference genes [4,5].

Methods

Results

Conclusion