Selection and validation reference genes for qRT-PCR normalization in different cultivars during fruit ripening and softening of peach (Prunus persica)

Shuanghong You,Weichao Fang,Lirong Wang,Ke Cao,Xinwei Wang,Gengrui Zhu,Changwen Chen,Jinlong Wu,Yong Li

doi:10.1038/s41598-021-86755-5

Abstract

Quantitative real-time PCR (qRT-PCR) has been emerged as an effective method to explore the gene function and regulatory mechanisms. However, selecting appropriate reference gene (s) is a prerequisite for obtaining accurate qRT-PCR results. Peach is one of important fruit in Rosaceae and is widely cultivated worldwide. In this study, to explore reliable reference gene (s) in peach with different types during fruit ripening and softening (S1–S4), nine candidate reference genes (EF-1α, GAPDH, TBP, UBC, eIF-4α, TUB-A, TUB-B, ACTIN, and HIS) were selected from the whole-genome data. Then, the expression levels of the nine selected genes were detected using qRT-PCR in three peach types, including ‘Hakuho’ (melting type), ‘Xiacui’ (stony hard type), ‘Fantasia’ and ‘NJC108’ (non-melting type) cultivars were detected using qRT-PCR. Four software (geNorm, NormFinder, BestKeeper and RefFinder) were applied to evaluate the expression stability of these candidate reference genes. Gene expression was characterized in different peach types during fruit ripening and softening stages. The overall performance of each candidate in all samples was evaluated. The Actin gene (ACTIN) was a suitable reference gene and displayed excellent stability in ‘Total’ set, ‘Hakuho’ samples, S3 and S4 fruit developmental stages. Ubiquitin C gene (UBC) showed the best stability in most independent samples, including ‘Fantasia’, ‘NJC108’, S2 sets. Elongation factor-1α gene (EF-1α) was the most unstable gene across the set of all samples, ‘NJC108’ and S2 sets, while showed the highest stability in ‘Xiacui’ samples. The stability of candidate reference genes was further verified by analyzing the relative expression level of ethylene synthase gene of Prunus persica (PpACS1) in fruit ripening and softening periods of ‘Hakuho’. Taken together, the results from this study provide a basis for future research on the mining of important functional genes, expression patterns and regulatory mechanisms in peach.

Highlights

Quantitative real-time PCR is widely used for expression analysis of genes due to its fast, sensitive, specific and accurate c haracteristics[1,2]
Using the least stable gene TBP as internal control, the expression level of PpACS1 showed significant difference with the data when normalized using other optimal reference genes individually or in combination (Fig. 6b) and the results were in accordance with expression level of PpACS1 when the least stable reference genes: Elongation factor-1α gene (EF-1α), HIS and eIF-4α were used for normalization in S2, S3 and S4 stage, respectively (Fig. 6c–e). These results indicate that using inappropriate internal reference genes for normalization may lead to in accuracy Quantitative real-time PCR (qRT-PCR) results
Accurate results of qRT-PCR are closely related to the normalization of certain suitable reference gene, and using of inappropriate reference gene will lead to deviated analysis and even wrong conclusions[32,33,34]

Summary

Introduction

Quantitative real-time PCR (qRT-PCR) is widely used for expression analysis of genes due to its fast, sensitive, specific and accurate c haracteristics[1,2]. Due to the importance of reference genes in gene expression analysis, many studies on the screening of reference genes with stable expression in higher plants have been conducted and the results showed different reference genes have been applied in different experimental materials and conditions. UBC is the best reference gene for all samples and different cultivars in Osmanthus fragrans, while ACTIN is the best reference gene for different flower development stages and different temperature treatments[16] All these studies showed that the reference genes should be screened, evaluated and verified accurately according to the plant cultivars, different tissues and conditions. It is necessary to accurately select the appropriate reference genes in different flesh texture cultivars during peach fruit ripening and softening. The suitable reference genes obtained in this study provided a theoretical basis for further exploring the gene expression and regulation mechanisms in peach

Methods

Results

Conclusion