Selection and Validation of Housekeeping Genes as Reference for Gene Expression Studies in Pigeonpea (Cajanus cajan) under Heat and Salt Stress Conditions

Abstract

To identify stable housekeeping genes as a reference for expression analysis under heat and salt stress conditions in pigeonpea, the relative expression variation for 10 commonly used housekeeping genes (EF1α, UBQ10, GAPDH, 18Sr RNA, 25Sr RNA, TUB6, ACT1, IF4α, UBC, and HSP90) was studied in root, stem, and leaves tissues of Asha (ICPL 87119), a leading pigeonpea variety. Three statistical algorithms geNorm, NormFinder, and BestKeeper were used to define the stability of candidate genes. Under heat stress, UBC, HSP90, and GAPDH were found to be the most stable reference genes. In the case of salinity stress, GAPDH followed by UBC and HSP90 were identified to be the most stable reference genes. Subsequently, the above identified genes were validated using qRT-PCR based gene expression analysis of two universal stress-resposive genes namely uspA and uspB. The relative quantification of these two genes varied according to the internal controls (most stable, least stable, and combination of most stable and least stable housekeeping genes) and thus confirmed the choice as well as validation of internal controls in such experiments. The identified and validated housekeeping genes will facilitate gene expression studies under heat and salt stress conditions in pigeonpea.

Keywords: heat stress; housekeeping genes; quantitative real-time PCR; salt stress.

FIGURE 1

Ct variation of tested housekeeping genes among different tissue samples under heat and salt stresses. (A) Boxplot depicting absolute Ct values of heat imposed/control samples and (B) Boxplot depicting absolute Ct values of salt imposed/control samples. Lower and upper boxes indicate the 25th and 75th percentile, respectively. The median is depicted by the horizontal line in the box.

FIGURE 2

Ranking of housekeeping genes for heat stress conditions. Gene expression studies for identification of most stable housekeeping genes under heat stress condition using two software programs. The direction of arrow indicates the most and least stable housekeeping genes in graphs (A) Gene expression stability graph of housekeeping gene using geNorm program based on average expression stability value (M), which is based on stepwise exclusion process. M-value is inversely related to gene stability (B) Gene expression stability graph using NormFinder program based on stability value and lower the stability value indicates higher stability of the housekeeping genes.

FIGURE 3

Ranking of housekeeping genes for salinity stress conditions. Gene expression studies for identification of most stable housekeeping genes under salt stress condition using two software programs. The direction of arrow indicates the most and least stable housekeeping genes in graphs (A) Gene expression stability graph of housekeeping gene using geNorm program based on an average expression stability value (M), which is based on stepwise exclusion process. M-value is inversely related to gene stability (B) Gene expression stability graph using NormFinder program based on stability value and lower the stability value indicates higher stability of the housekeeping genes.

FIGURE 4

Validation of reference genes under heat stress conditions. Expression profiling of candidate gene (A) uspA and (B) uspB in heat imposed tissues (root, stem, and leaves) and normalized with (i) UBC (ii) HSP90 (iii) GAPDH (iv) UBC + HSP90 (v) UBC + GAPDH (vi) UBC + HSP90 + GAPDH (vii) UBQ10 and (viii) ACT1. The analysis was completed in two different stages with six different tissues. The relative quantification values of selected drought responsive candidate gene were obtained after scaling to control samples. EHR, vegetative root stressed; LHR, reproductive root stressed; EHS, vegetative stem stressed; LHS, reproductive stem stressed; EHL, vegetative leaves stressed; LHL, reproductive leaves stressed.

FIGURE 5

Validation of reference genes under salt stress conditions. Expression profiling of candidate gene (A) uspA and (B) uspB in salt imposed tissues (root, stem, and leaves) and normalized with (1) GAPDH (ii) UBC (iii) HSP90 (iv) GAPDH + UBC (v) GAPDH + HSP90 (vi) GAPDH + HSP90 + UBC (vii) ACT1 and (viii) TUB6. The analysis was completed in two different stages with six different tissues. The relative quantification values of selected drought responsive candidate gene were obtained after scaling to control samples. ESR, vegetative root stressed; LSR, reproductive root stressed; ESS, vegetative stem stressed; LSS, reproductive stem stressed; ESL, vegetative leaves stressed; LSL, reproductive leaves stressed.