Assessment of Suitable Reference Genes for Quantitative Gene Expression Studies in Melon Fruits.

Qiusheng Kong,Lingyun Gao,Hameed Saba,Yue Liu,Lei Cao,Zhilong Bie,Yuan Huang

doi:10.3389/fpls.2016.01178

Abstract

Melon (Cucumis melo L.) is an attractive model plant for investigating fruit development because of its morphological, physiological, and biochemical diversity. Quantification of gene expression by quantitative reverse transcription polymerase chain reaction (qRT-PCR) with stably expressed reference genes for normalization can effectively elucidate the biological functions of genes that regulate fruit development. However, the reference genes for data normalization in melon fruits have not yet been systematically validated. This study aims to assess the suitability of 20 genes for their potential use as reference genes in melon fruits. Expression variations of these genes were measured in 24 samples that represented different developmental stages of fertilized and parthenocarpic melon fruits by qRT-PCR analysis. GeNorm identified ribosomal protein L (CmRPL) and cytosolic ribosomal protein S15 (CmRPS15) as the best pair of reference genes, and as many as five genes including CmRPL, CmRPS15, TIP41-like family protein (CmTIP41), cyclophilin ROC7 (CmCYP7), and ADP ribosylation factor 1 (CmADP) were required for more reliable normalization. NormFinder ranked CmRPS15 as the best single reference gene, and RAN GTPase gene family (CmRAN) and TATA-box binding protein (CmTBP2) as the best combination of reference genes in melon fruits. Their effectiveness was further validated by parallel analyses on the activities of soluble acid invertase and sucrose phosphate synthase, and expression profiles of their respective encoding genes CmAIN2 and CmSPS1, as well as sucrose contents during melon fruit ripening. The validated reference genes will help to improve the accuracy of gene expression studies in melon fruits.

Highlights

Melon (Cucumis melo L.) is a cucurbitaceous crop cultivated worldwide, and it is one of the most important fleshy fruits used for fresh consumption
The results showed that only the expected products were amplified with cDNA templates and no products were amplified with genomic DNA (gDNA) templates for CmACT, CmCAC, CmPP2A, CmRP2, CmRPS15, CmSAND, CmTBP2, and CmTIP41
Among the six previously identified reference genes in melon, CmACT7, CmCYP2, CmL2, and CmRPL amplified the same products on both gDNA and cDNA templates, whereas, CmADP had no products and CmUBI generated a larger product on gDNA templates, respectively

Summary

Introduction

Melon (Cucumis melo L.) is a cucurbitaceous crop cultivated worldwide, and it is one of the most important fleshy fruits used for fresh consumption. Melon fruits have significant variations in their ripening physiology; the fruits can be categorized as either climacteric or non-climacteric types based on their ripening-related respiration rate and ethylene evolution profiles (Clepet et al, 2011; Leida et al, 2015; Saladié et al, 2015). These diverse traits can be exploited to reveal the underlying biological processes and Reference Genes in Melon Fruits mechanisms regulating fruit development. Extensive molecular and genetic studies have been conducted on melon in recent years to understand the regulatory mechanisms of fruit development and ripening, with the aim of improving its fruit quality (Moreno et al, 2008; Dai et al, 2011; Portnoy et al, 2011; Vegas et al, 2013; Díaz et al, 2014; Saladié et al, 2015)

Objectives

Methods

Results

Discussion

Conclusion