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

Reddy, D S and Bhatnagar-Mathur, P and Reddy, P S and Cindhuri, K S and Ganesh, A S and Sharma, K K (2016) Identification and Validation of Reference Genes and Their Impact on Normalized Gene Expression Studies across Cultivated and Wild Cicer Species. PLOS ONE, 11 (02). 01-19. ISSN 1932-6203

PDF (It is an Open Access article) - Published Version
Download (838kB) | Preview


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.

Item Type: Article
Divisions: RP-Grain Legumes
CRP: CGIAR Research Program on Grain Legumes
Uncontrolled Keywords: Gene expression, Gene expression studies, Plant materials, Stress treatments, Chickpea, Functional Genomics, Genome Sequence
Subjects: Mandate crops > Chickpea
Others > Genetics and Genomics
Depositing User: Mr Ramesh K
Date Deposited: 17 Feb 2016 08:11
Last Modified: 22 Dec 2016 04:32
Official URL:
Funders: This work has received funding from the CGIAR research program on grain legumes
Acknowledgement: The authors acknowledge the technical assistance from Kanaka Reddy and Rahul. This work was undertaken as a part of the CGIAR Research Program on Grain Legumes. Conceived and designed the experiments: PBM DSR KKS. Performed the experiments: DSR KSC ASG. Analyzed the data: DSR PBM PSR. Contributed reagents/materials/analysis tools: PBM KKS. Wrote the paper: DSR PBM KKS.
View Statistics

Actions (login required)

View Item View Item