Selection and validation of reference genes for RT-qPCR analysis in potato under abiotic stress

Xun Tang,Alejandro Calderón-Urrea,Huaijun Si,Ning Zhang

doi:10.1186/s13007-017-0238-7

Xun Tang, Alejandro Calderón-Urrea + Show 2 more

Open Access

https://doi.org/10.1186/s13007-017-0238-7

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Abstract

BackgroundReal-time quantitative PCR (RT-qPCR) is the most commonly used method for accurately detecting gene expression patterns. As part of RT-qPCR analysis, normalization of the data requires internal control gene(s) that display uniform expression under different biological conditions. However, no invariable internal control gene exists, and therefore more than one reference gene is needed to normalize RT-qPCR results. Identification of stable reference genes in potato will improve assay accuracy for selecting stress-tolerance genes and identifying molecular mechanisms conferring stress tolerance in this species.ResultsIn the experiment, we assessed the expression of eight candidate internal control genes, namely elongation factor-1alpha (EF1α), actin, tubulin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), adenine phosphoribosyl transferase (APRT), 60S ribosomal protein L8 (L8), Cullin 3A (CUL3A), and exocyst complex component sec3 (sec3), in a diverse set of potato samples representing drought stress and osmotic stress challenges, and using geNorm, NormFinder, BestKeeper and RefFinder softwares.ConclusionsThe results indicated that EF1α and sec3 were the most stably expressed genes in the potato under drought and osmotic stress conditions. This work will facilitate future work on gene expression studies in potato and also benefit other species of the Solanaceae, such as tomato.

Highlights

Real-time quantitative PCR (RT-qPCR) is the most commonly used method for accurately detecting gene expression patterns
Modern potato (Solanum tuberosum L.) varieties are considered to be sensitive to drought, but they have different morphological and physiological responses to water deficit compared to other crops [2]
Assessment of primer specificity and PCR amplification efficiency The sequences of Cullin 3A (CUL3A), EF1α, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), sec3, tubulin, L8, adenine phosphoribosyl transferase (APRT) and actin in potato were targeted by using specific primers on a template cDNA

Summary

Introduction

Real-time quantitative PCR (RT-qPCR) is the most commonly used method for accurately detecting gene expression patterns. As part of RT-qPCR analysis, normalization of the data requires internal control gene(s) that display uniform expression under different biological conditions. No invariable internal control gene exists, and more than one reference gene is needed to normalize RT-qPCR results. Identification of stable reference genes in potato will improve assay accuracy for selecting stress-tolerance genes and identifying molecular mecha‐ nisms conferring stress tolerance in this species. Drought and osmotic stress can seriously limit plant growth and productivity. Plants have developed multiple strategies to cope with drought and osmotic stress. These normally involve a mixture of stress avoidance and tolerance adaptations, which produce a range of changes at the morphological, physiological, cellular, and molecular levels [1].

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