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.

Identification of Reference Genes for qRT‐PCR Gene Expression Studies during Seed Development and under Abiotic Stresses in Cyamopsis tetragonoloba

“…Hence, for this study, a most stable reference gene was selected on the basis of comprehensive ranking. A similar approach has also been reported in Salicornia europaea L. by Xiao et al (2015) and in chickpea (Cicer arietinum L.) by Reddy et al (2016).…”

“…In the earlier studies, the CYP gene was also found to be a suitable reference gene for qRT-PCR normalization in different tissues of soybean ( Jian et al, 2008) and peanut (Reddy et al, 2013). However, this gene was not a suitable reference gene in poplar (Brunner et al, 2004), cucumber (Cucumus sativus L.; Wan et al, 2010), and chickpea (Reddy et al, 2016). Similarly, ACT 11 was also not found to be a suitable reference gene for qRT-PCR studies in different tissues of poplar (Brunner et al, 2004).…”

“…In earlier studies, GAPDH and ACT 7 genes were found to be suitable reference genes for qRT-PCR normalization under heat stress conditions in Brachypodium distachyon (Hong et al, 2008). In heat stress studies, GAPDH was also found to be the most suitable reference gene in pigeonpea (Sinha et al, 2015) and Lilium regale (Liu et al, 2016), whereas this gene was not a suitable reference gene in chickpea (Reddy et al, 2016) and maize (Lin et al, 2014). The ACT 7 gene was not the suitable reference gene in Caragana intermedia Kuang & H.C. Fu (Zhu et al, 2013) for qRT-PCR studies in heat stress condition.…”

Selection and Validation of Reference Genes for Gene Expression Analysis in Vigna angularis Using Quantitative Real-Time RT-PCR

“…Although ACT , GAPDH , EF , or 18s rRNA have been reported as reference genes for gene expression analysis [30–32], adzuki bean is not involved in these studies. Therefore, nine genes were selected as candidate reference genes in current study and three excel-based approaches BestKeeper, geNorm and NormFinder were used to evaluate the expression stability.…”

Genome-wide identification and transcriptional analyses of MATE transporter genes in root tips of wildCicerspp. under aluminium stress

“…RT-qPCR reaction was performed using the ViiA7 Real-Time PCR System (Thermo Fisher, USA) in 384-well plates. The previously tested chickpea CaCAC gene was used as a reference gene 48 . Three replicates were included for each sample.…”