Selection and Validation of Appropriate Reference Genes for Quantitative Real-Time PCR Normalization in Staminate and Perfect Flowers of Andromonoecious Taihangia rupestris.

Weiguo Li,Yandi Zhang,Dangyu Song,Guodong Wang,Lihui Zhang,Yanwen Zhang

doi:10.3389/fpls.2017.00729

Weiguo Li, Yandi Zhang + Show 4 more

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https://doi.org/10.3389/fpls.2017.00729

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Abstract

Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is the most commonly used and powerful method for gene expression analysis due to its high sensitivity, specificity, and high throughput, and the accuracy of this approach depends on the stability of reference genes used for normalization. Taihangia rupestris Yu and Li (Rosaceae), an andromonoecious plant, produces both bisexual flowers and unisexual male flowers within the same individual. Using qRT-PCR technique, investigation of the gene expression profiling in staminate and perfect flowers would improve our understanding of the molecular mechanism in regulation of flower formation and sex differentiation in andromonoecious T. rupestris. To accurate normalize the gene expression level in Taihangia flower, 16 candidate reference genes, including 10 traditional housekeeping genes, and 6 newly stable genes, were selected based on transcriptome sequence data and previous studies. The expressions of these genes were assessed by qRT-PCR analysis in 51 samples, including 30 staminate and perfect flower samples across developmental stages and 21 different floral tissue samples from mature flowers. By using geNorm, NormFinder, BestKeeper, and comprehensive RefFinder algorithms, ADF3 combined with UFD1 were identified as the optimal reference genes for staminate flowers, while the combination of HIS3/ADF3 was the most accurate reference genes for perfect floral samples. For floral tissues, HIS3, UFD1, and TMP50 were the most suitable reference genes. Furthermore, two target genes, TruPI, and TruFBP24, involved in floral organ identity were selected to validate the most and least stable reference genes in staminate flowers, perfect flowers, and different floral tissues, indicating that the use of inappropriate reference genes for normalization will lead to the adverse results. The reference genes identified in this study will improve the accuracy of qRT-PCR quantification of target gene expression in andromonoecious T. rupestris flowers, and will facilitate the functional genomics studies on flower development and sex differentiation in the future.

Highlights

Quantitative real-time reverse transcription-polymerase chain reaction has become the most common and powerful method for detecting and measuring transcripts abundance due to its high sensitivity, specificity, accuracy, and high throughput (Gachon et al, 2004)
Based on Nr annotation, we identified nine traditional housekeeping genes, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), EF-1α, UBQ, and ACT, common used as reference genes for flower development (E-value ≤ 10−50)
The results showed that 16 candidate reference genes involved in many aspects of primary metabolism or other basic cellular processes such as cytoskeleton (ACT, ACT2, actin-depolymerizing factor 3 (ADF3), and TUA), transport of ions (THS), transport in vacuoles (VAP32) or membranes (TMP50), glycolysis (GAPDH), energy metabolism (ISP), RNA processing and modification (SPF), protein translation (EF-1α), protein posttranslational modification (UBC, ubiquitin fusion degradation protein 1 (UFD1), and UBQ), cell signaling (PP2A), and chromatin structure, and dynamics (HIS3)

Summary

Introduction

Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) has become the most common and powerful method for detecting and measuring transcripts abundance due to its high sensitivity, specificity, accuracy, and high throughput (Gachon et al, 2004). Relative quantification is a widely accepted procedure to evaluate gene expression by normalization with one or more internal reference genes, which are required for stable expression regardless of experimental conditions (Bustin et al, 2005; Thellin et al, 2009). Numerous studies have reported that the expression levels of traditional housekeeping genes vary considerably alterations in different biological sample and variable experimental condition (Hu et al, 2009; Zhang et al, 2017). The use of improper traditional housekeeping genes for normalization in relative quantification of gene expression could result in bias of expression data and incorrect conclusion (Gutierrez et al, 2008)

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