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Identification of perfect housekeeping genes for gene expression studies in pigeonpea by quantitative real-time PCR
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Identification of perfect housekeeping genes for gene expression studies in pigeonpea by quantitative real-time PCR

  1. Inclusive Market-Oriented Development (IMOD) – our approach to bringing prosperity in the drylands. ICRISAT is a member of the CGIAR Consortium. Identification of perfect housekeeping genes for gene expression studies in pigeonpea by quantitative real-time PCR Pallavi Sinha, Rachit K. Saxena, Vikas K. Singh, V. Suryanarayana, L. Krishnamurthy, Rajeev K. Varshney* International Crop Research Institute for the Semi-Arid Tropics (ICRISAT), Hyderabad, India *Address of corresponding author: r.k.varshney@cgiar.org Gene expression analysis using quantitative 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 as reference genes 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 results obtained confirm the ranking of the reference genes identified in the study. Abstract Gene name Gene description EF1α Elongation factor Tu GTP binding domain UBQ10 Ubiquitin family GAPDH Glyceraldehyde 3-phosphate dehydrogenase 18SrRNA 18S ribosomal RNA 25SrRNA 25S ribosomal RNA TUB6 Tubulin/FtsZ family, GTPase domain ACT1 Actin 1 IF4α Initiation factor 4a UBC Ubiquitin-conjugating enzyme HSP90 Heat shock protein 90 Approach Gene expression stability graph Financial support from USAID is gratefully acknowledged. This work has been undertaken as part of the CGIAR Research Program on Grain Legumes. Summary The present study revealed that for drought stress condition IF4α followed by TUB6 and HSP90, for heat stress condition EF1α and HSP90 and for salinity stress GAPDH followed by UBC and ACT1 were the most stable reference genes. Therefore, our study will facilitate selection of the suitable housekeeping genes for transcript profiling using qRT-PCR for specific abiotic stress condition for functional genomics studies in pigeonpea . Drought Vegetative / reproductive Heat Vegetative / reproductive Salinity Vegetative / reproductive cDNA synthesis qRT-PCR Gene stability Analysis RNA isolation Root, shoot and leaves were collected Stresses /Stages Asha Gene Validation Stresses /Stages Acknowledgement Validation of identified genes Trait Drought Heat Salinity Genes/ Tools geNorm NormFinder geNorm NormFinder geNorm NormFinder M- value Rank S- value Rank M- value Rank S- value Rank M- value Rank S- value Rank EF1α 0.924 7 0.043 6 0.000 1 0.015 1 0.555 5 0.723 7 UBQ10 0.842 6 0.055 8 1.486 9 0.142 8 0.650 6 0.643 6 GAPDH 0.757 4 0.034 5 0.575 3 0.028 4 0.384 1 0.192 1 18srRNA 1.193 9 0.127 10 1.090 7 0.151 9 0.943 8 1.345 9 25srRNA 1.090 8 0.122 9 1.196 8 0.153 10 0.819 7 1.179 8 TUB6 0.504 1 0.032 4 0.934 6 0.061 7 1.046 9 1.353 10 ACT1 0.682 3 0.029 3 0.702 4 0.029 5 0.505 4 0.491 3 IF4α 0.504 1 0.027 1 0.840 5 0.048 6 0.474 3 0.613 5 UBC 0.566 2 0.049 7 0.419 2 0.015 3 0.384 1 0.210 2 HSP90 0.786 5 0.028 2 0.000 1 0.015 2 0.422 2 0.544 4 Results: Ranking of genes based on stability Plant tissues Foldexpressionlevel
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