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Validation of reference genes for RT-qPCR studies of gene expression in banana fruit under different experimental conditions

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Abstract

Reverse transcription quantitative real-time PCR (RT-qPCR) is a sensitive technique for quantifying gene expression, but its success depends on the stability of the reference gene(s) used for data normalization. Only a few studies on validation of reference genes have been conducted in fruit trees and none in banana yet. In the present work, 20 candidate reference genes were selected, and their expression stability in 144 banana samples were evaluated and analyzed using two algorithms, geNorm and NormFinder. The samples consisted of eight sample sets collected under different experimental conditions, including various tissues, developmental stages, postharvest ripening, stresses (chilling, high temperature, and pathogen), and hormone treatments. Our results showed that different suitable reference gene(s) or combination of reference genes for normalization should be selected depending on the experimental conditions. The RPS2 and UBQ2 genes were validated as the most suitable reference genes across all tested samples. More importantly, our data further showed that the widely used reference genes, ACT and GAPDH, were not the most suitable reference genes in many banana sample sets. In addition, the expression of MaEBF1, a gene of interest that plays an important role in regulating fruit ripening, under different experimental conditions was used to further confirm the validated reference genes. Taken together, our results provide guidelines for reference gene(s) selection under different experimental conditions and a foundation for more accurate and widespread use of RT-qPCR in banana.

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Abbreviations

ACT :

Actin

APT :

Adenine phosphoribosyltransferase

CAC :

Clathrin adaptor complexes medium

CYP :

Cyclophilin

DNAJ :

DnaJ-like protein

EBF :

EIN3-binding F-box protein

EF1α :

Elongation factor 1-alpha

EIF5A :

Eukaryotic initiation factor 5A

GAPDH :

Glyceraldehyde-3-phosphate dehydrogenase

RAN :

GTP-binding nuclear protein

RPL :

Ribosomal protein L

RPS :

Ribosomal protein S

RT-qPCR:

Reverse transcription quantitative real-time PCR

SAMDC :

s-Adenosyl methionine decarboxylase

TIP4I :

TIP4I-like family protein

TUA :

Alpha-tubulin

UBCE :

Ubiquitin conjugating enzyme

UBQ :

Ubiquitin

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Acknowledgments

We thank Dr. Yuhai Cui (Agriculture and Agri-Food Canada, Southern Crop Protection and Food Research Centre, London, Canada) for his helpful discussion and revising. We also thank the three reviewers for their helpful suggestions. This study was financially supported by the National Natural Science Foundation of China (Grant Nos. 30800772; 30972068) and Modern Agro-industry Technology Research System (Grant No. nycytx-33).

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Authors and Affiliations

  1. Guangdong Key Laboratory for Postharvest Science, College of Horticultural Science, South China Agricultural University, Guangzhou, 510642, People’s Republic of China

    Lei Chen, Hai-ying Zhong, Jian-fei Kuang, Wang-jin Lu & Jian-ye Chen

  2. China Litchi Research Center, South China Agricultural University, Guangzhou, 510642, People’s Republic of China

    Jian-guo Li

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  1. Lei Chen
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  2. Hai-ying Zhong
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Correspondence to Jian-ye Chen.

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Chen, L., Zhong, Hy., Kuang, Jf. et al. Validation of reference genes for RT-qPCR studies of gene expression in banana fruit under different experimental conditions. Planta 234, 377–390 (2011). https://doi.org/10.1007/s00425-011-1410-3

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