title: 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.
authors: Genetic Transformation Laboratory, International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru-502324, Telangana, India.
authors: Dumbala Srinivas
authors: DS
authors: Reddy
authors: Genetic Transformation Laboratory, International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru-502324, Telangana, India.
authors: Pooja
authors: P
authors: Bhatnagar-Mathur
authors: Genetic Transformation Laboratory, International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru-502324, Telangana, India.
authors: Palakolanu Sudhakar
authors: PS
authors: Reddy
authors: Genetic Transformation Laboratory, International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru-502324, Telangana, India.
authors: Katamreddy
authors: K
authors: Sri Cindhuri
authors: Genetic Transformation Laboratory, International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru-502324, Telangana, India.
authors: Adusumalli
authors: A
authors: Sivaji Ganesh
authors: Genetic Transformation Laboratory, International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru-502324, Telangana, India.
authors: Kiran Kumar
authors: KK
authors: Sharma
mesh_terms: D000040:Abscisic Acid / Q000494:pharmacology; D000222:Adaptation, Physiological / Q000235:genetics*; D029863:Cicer / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development; D003080:Cold Temperature; D055864:Droughts; D035264:Flowers / Q000187:drug effects / Q000235:genetics / Q000254:growth & development; D018507:Gene Expression Regulation, Developmental; D018506:Gene Expression Regulation, Plant; D020043:Genes, Essential; D017343:Genes, Plant; D005838:Genotype; D006358:Hot Temperature; D000069600:Plant Breeding; D018515:Plant Leaves / Q000187:drug effects / Q000235:genetics / Q000254:growth & development; D018517:Plant Roots / Q000187:drug effects / Q000235:genetics / Q000254:growth & development; D060888:Real-Time Polymerase Chain Reaction / Q000592:standards*; D012015:Reference Standards; D054712:Salinity; D036226:Seedlings / Q000187:drug effects / Q000235:genetics / Q000254:growth & development; D012639:Seeds / Q000187:drug effects / Q000235:genetics / Q000254:growth & development; D012965:Sodium Chloride / Q000494:pharmacology; D013312:Stress, Physiological
chemical_list: D012965:Sodium Chloride; D000040:Abscisic Acid