Identification and Validation of Reference Genes and Their Impact on Normalized Gene Expression Studies across Cultivated and Wild Cicer Species.

Dumbala Srinivas Reddy,Pooja Bhatnagar-Mathur,Kiran Kumar Sharma,Katamreddy Sri Cindhuri,Palakolanu Sudhakar Reddy,Adusumalli Sivaji Ganesh,Zhulong Chan

doi:10.1371/journal.pone.0148451

Dumbala Srinivas Reddy, Pooja Bhatnagar-Mathur + Show 5 more

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https://doi.org/10.1371/journal.pone.0148451

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Abstract

Quantitative Real-Time PCR (qPCR) is a preferred and reliable method for accurate quantification of gene expression to understand precise gene functions. A total of 25 candidate reference genes including traditional and new generation reference genes were selected and evaluated in a diverse set of chickpea samples. The samples used in this study included nine chickpea genotypes (Cicer spp.) comprising of cultivated and wild species, six abiotic stress treatments (drought, salinity, high vapor pressure deficit, abscisic acid, cold and heat shock), and five diverse tissues (leaf, root, flower, seedlings and seed). The geNorm, NormFinder and RefFinder algorithms used to identify stably expressed genes in four sample sets revealed stable expression of UCP and G6PD genes across genotypes, while TIP41 and CAC were highly stable under abiotic stress conditions. While PP2A and ABCT genes were ranked as best for different tissues, ABCT, UCP and CAC were most stable across all samples. This study demonstrated the usefulness of new generation reference genes for more accurate qPCR based gene expression quantification in cultivated as well as wild chickpea species. Validation of the best reference genes was carried out by studying their impact on normalization of aquaporin genes PIP1;4 and TIP3;1, in three contrasting chickpea genotypes under high vapor pressure deficit (VPD) treatment. The chickpea TIP3;1 gene got significantly up regulated under high VPD conditions with higher relative expression in the drought susceptible genotype, confirming the suitability of the selected reference genes for expression analysis. This is the first comprehensive study on the stability of the new generation reference genes for qPCR studies in chickpea across species, different tissues and abiotic stresses.

Highlights

Gene expression studies have become extremely important to obtain insights into gene function and to understand the molecular mechanisms
We have evaluated a set of reference genes including traditional and new generation reference genes in a diverse set of biological samples of chickpea, including nine genotypes representing cultivated and wild species across primary, secondary and tertiary gene pools, plant tissues from various developmental stages, and six abiotic stress treatments
The candidate reference genes selected in this study represent different functional classes and gene families, including traditional and new generation reference genes based on reports in Arabidopsis, soybean, peanut and chickpea

Summary

Introduction

Gene expression studies have become extremely important to obtain insights into gene function and to understand the molecular mechanisms. Since several studies have shown that no single universal gene has consistent expression under all experimental conditions, the evaluation of reference gene(s) under specific experimental conditions is essential for reliability of qPCR analysis [7, 8]. Several algorithms such as geNorm [9], NormFinder [10], BestKeeper [11] and comparative ΔCt method [12] have been developed to evaluate the most stable reference gene(s) from set of candidate genes

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