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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 familyGAPDH Glyceraldehyde 3-phosphate

dehydrogenase18SrRNA 18S ribosomal RNA25SrRNA 25S ribosomal RNATUB6 Tubulin/FtsZ family, GTPase domainACT1 Actin 1IF4α Initiation factor 4aUBC Ubiquitin-conjugating enzymeHSP90 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.

SummaryThe 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 SalinityGenes/ 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

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