Evaluation and Validation of Housekeeping Genes as Reference for Gene Expression Studies in Pigeonpea (Cajanus cajan) Under Drought Stress Conditions

Sinha, P and Singh, V K and Suryanarayana, V and Krishnamurthy, L and Saxena, R K and Varshney, R K (2015) Evaluation and Validation of Housekeeping Genes as Reference for Gene Expression Studies in Pigeonpea (Cajanus cajan) Under Drought Stress Conditions. PLOS One, 10 (4). pp. 1-15. ISSN 1932-6203

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Abstract

Gene expression analysis using quantitative real-time PCR (qRT-PCR) is a very sensitive technique and its sensitivity depends on the stable performance of reference gene(s) used in the study. A number of housekeeping genes have been used in various expression studies in many crops however, their expression were found to be inconsistent under different stress conditions. As a result, species specific housekeeping genes have been recommended for different expression studies in several crop species. However, such specific housekeeping genes have not been reported in the case of pigeonpea (Cajanus cajan) despite the fact that genome sequence has become available for the crop. To identify the stable housekeeping genes in pigeonpea for expression analysis under drought stress conditions, the relative expression variations of 10 commonly used housekeeping genes (EF1α, UBQ10, GAPDH, 18SrRNA, 25SrRNA, TUB6, ACT1, IF4α, UBC and HSP90) were studied on root, stem and leaves tissues of Asha (ICPL 87119). Three statistical algorithms geNorm, NormFinder and BestKeeper were used to define the stability of candidate genes. geNorm analysis identified IF4α and TUB6 as the most stable housekeeping genes however, NormFinder analysis determined IF4α and HSP90 as the most stable housekeeping genes under drought stress conditions. Subsequently validation of the identified candidate genes was undertaken in qRT-PCR based gene expression analysis of uspA gene which plays an important role for drought stress conditions in pigeonpea. The relative quantification of the uspA gene varied according to the internal controls (stable and least stable genes), thus highlighting the importance of the choice of as well as validation of internal controls in such experiments. The identified stable and validated housekeeping genes will facilitate gene expression studies in pigeonpea especially under drought stress conditions.

Item Type: Article
Divisions: RP-Grain Legumes
CRPS: CGIAR Research Program on Grain Legumes
Uncontrolled Keywords: Gene expression; Polymerase chain reaction; Algorithms; Crop genetics; Gene targeting; Complementary DNA; Gene amplification; Housekeeping genes ; Pigeonpea, Drought stress
Subjects: Mandate crops > Pigeonpea
Others > Genetics and Genomics
Depositing User: Mr Ramesh K
Date Deposited: 10 Apr 2015 05:02
Last Modified: 16 Dec 2015 08:46
URI: http://oar.icrisat.org/id/eprint/8658
Official URL: http://journals.plos.org/plosone/article?id=10.137...
Projects: CGIAR Research Program on Grain Legumes
Funders: CGIAR Generation Challenge Programme (GCP) and United States Agency for International Development (USAID)
Acknowledgement: The authors thank the CGIAR Generation Challenge Programme (GCP) and United States Agency for International Development (USAID) for financial support for the research work. This work has been undertaken as part of the CGIAR Research Program on Grain Legumes. ICRISAT is a member of CGIAR Consortium. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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