Selection of Suitable Reference Genes for RT-qPCR Normalization under Abiotic Stresses and Hormone Stimulation in Persimmon (Diospyros kaki Thunb).

Peihong Wang,Chao Sun,Zhihong Gao,Xinyi Yu,Aisheng Xiong,Yingjun Hou,Man Li,Shenchun Qu,Prasanta K Subudhi

doi:10.1371/journal.pone.0160885

Abstract

The success of quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) to quantify gene expression depends on the stability of the reference genes used for data normalization. To date, systematic screening for reference genes in persimmon (Diospyros kaki Thunb) has never been reported. In this study, 13 candidate reference genes were cloned from 'Nantongxiaofangshi' using information available in the transcriptome database. Their expression stability was assessed by geNorm and NormFinder algorithms under abiotic stress and hormone stimulation. Our results showed that the most suitable reference genes across all samples were UBC and GAPDH, and not the commonly used persimmon reference gene ACT. In addition, UBC combined with RPII or TUA were found to be appropriate for the "abiotic stress" group and α-TUB combined with PP2A were found to be appropriate for the "hormone stimuli" group. For further validation, the transcript level of the DkDREB2C homologue under heat stress was studied with the selected genes (CYP, GAPDH, TUA, UBC, α-TUB, and EF1-α). The results suggested that it is necessary to choose appropriate reference genes according to the test materials or experimental conditions. Our study will be useful for future studies on gene expression in persimmon.

Highlights

Persimmon (Diospyros kaki Thunb.), which is prevalent worldwide, originated in Eastern Asia and was mainly cultivated in China, Korea, and Japan [1]
Selection of Suitable Reference Genes in Persimmon (SSAP) was applied to reveal the genes associated with deastringency in persimmon [3]; genome-wide transcriptome analysis was performed to identify the primary genes involved in proanthocyanidin (PA) biosynthesis in persimmon [2]
The transcript level of DkDREB2C peaked at 1 h and 6 h. These results further suggested that CYP, GAPDH, TUA, and UBC are suitable for RT-qPCR under heat treatment in persimmon, while α-TUB and EF1-α were not suitable

Summary

Introduction

Persimmon (Diospyros kaki Thunb.), which is prevalent worldwide, originated in Eastern Asia and was mainly cultivated in China, Korea, and Japan [1]. Selection of Suitable Reference Genes in Persimmon (SSAP) was applied to reveal the genes associated with deastringency in persimmon [3]; genome-wide transcriptome analysis was performed to identify the primary genes involved in proanthocyanidin (PA) biosynthesis in persimmon [2]. For these molecular biological studies, gene expression analysis is an effective and widely used approach commonly performed by relying on methods such as Northern blotting, microarray analysis, and quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR)

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