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

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.

Fig 1. Expression levels of candidate reference genes across all samples.

Lines across the boxes depict the medians. Boxes indicate the interquartile range. Whiskers represent 95% confidence intervals, black dot indicate the presence of outliers. Coefficient of variance (CV) of each gene among all samples is given in percentage.

Fig 2. geNorm analysis.

(A-D) Average expression stability and ranking of all 25 candidate reference genes: A lower value of average expression stability (M) indicates more stable expression. (E-H) Determination of the optimal number of reference genes for normalization by pairwise variation: The pairwise variation (Vn/Vn+1) was analyzed between normalization factors NFn and NFn+1 by geNorm algorithm to determine (V<0.15) the optimal number of reference genes. Error bars show standard deviation of relative expression of target genes in three biological replicates.

Fig 3. Relative quantification of aquaporin genes PIP1;4 and TIP3;1 to validate selected reference genes under drought stress conditions.

Expression of PIP1;4 and TIP3;1 genes in high VPD treated chickpea leaf sample of three genotypes were relatively quantified by comparing with their control counterparts, with expression levels normalized with two most stable (ABCT and UCP) and least stable (CYP and SKIP16) reference genes.