Selection and Validation of Reliable Reference Genes for Gene Expression Studies in Different Genotypes and TRV-Infected Fruits of Peach (Prunus persica L. Batsch) during Ripening.

Ze Xu,Dong Zhang,Weijing Su,Kamran Shah,Juanjuan Ma,Jieyu Dai,Haixia Wu,Caiping Zhao,Libo Xing

doi:10.3390/genes13010160

Abstract

Real-time quantitative PCR (RT-qPCR) is a powerful tool to detect and quantify transcription abundance, and the stability of the reference gene determines its success. However, the most suitable reference gene for different genotypes and tobacco rattle virus (TRV) infected fruits was unclear in peach (Prunus persica L. Batsch). In this study, 10 reference genes were selected and gene expression was characterized by RT-qPCR across all samples, including different genotypes and TRV-infected fruits during ripening. Four statistical algorithms (geNorm, NormFinder, BestKeeper, and RefFinder) were used to calculate the stability of 10 reference genes. The geNorm analysis indicated that two suitable reference genes should be used for gene expression normalization. In general, the best combination of reference genes was CYP2 and Tua5 for TRV-infected fruits and CYP2 and Tub1 for different genotypes. In 18S, GADPH, and TEF2, there is an unacceptable variability of gene expression in all experimental conditions. Furthermore, to confirm the validity of the reference genes, the expression levels of PpACO1, PpEIN2, and PpPL were normalized at different fruit storage periods. In summary, our results provide guidelines for selecting reliable reference genes in different genotypes and TRV-infected fruits and lay the foundation for accurate evaluation of gene expression for RT-qPCR analysis in peach.

Highlights

Gene expression analysis is becoming increasingly important for understanding the signaling and metabolic pathways during cell development and death [1,2]
There is a literature for screening reference genes in plants, such as peach [11], tomato [39,40], Arabidopsis [41], potato [42], soybean [43,44], banana [45], and longan [16], finding that the expression level of the internal reference is unstable under every experimental condition
We aim to select reference genes that could be stably expressed as internal controls for RT-qPCR studies of gene expression in peach fruit with different genotypes and tobacco rattle virus (TRV)-infected fruits

Summary

Introduction

Gene expression analysis is becoming increasingly important for understanding the signaling and metabolic pathways during cell development and death [1,2]. In RT-qPCR, using a reference gene to normalize gene expression is a simple, popular method with wide application [6]. The reference gene, known as the housekeeping gene, is stably expressed in various tissues and experimental conditions and maintains the minimal life function of cells [7,8]. Normalization can lead to incorrect results if there are large fluctuations in the expression of reference genes [6]. Selecting an optimal reference gene with a steady expression pattern is an essential pre-requisite before normalization

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Discussion

Conclusion