Abstract
Gene expression analysis usingquantitative real-time PCR(qRT-PCR) is a very sensitive technique which completely depends on stable performance of reference genes used in the study. A number of housekeeping genes have been used in various expression studies however, they found to be varying under different stress conditions. To identify the suitable housekeeping genes in pigeonpea for expression analysis under different abiotic stress conditions (drought, heat and salinity) we have compared relative expression variations of 10 commonly used housekeeping genes (EF1α, UBQ10, GAPDH, 18srRNA, 25srRNA, TUB6, ACT1, IF4α, UBC and HSP90) on various tissues (root, stem and leaves) of Asha (ICPL 87119). Based on analysis of drought stress conditions IF4α followed by TUB6 and HSP90 were identified as most stable reference genes. For heat stress conditions EF1α, UBC and HSP90 were found most stable reference genes. Similarly, for salinity stress conditions, GAPDH followed by UBC and ACT1 were identified as the most stable reference genes. Stable and most unstable genes identified in the study were validated to estimate the expression level of candidate gene in pigeonpea (Cc_cds_33874) for drought tolerance and result obtained confirm the ranking of the reference genes identified in the study.