Evaluation and validation of housekeeping genes as reference for gene expression studies in pigeonpea (Cajanus cajan) under drought stress conditions

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.

Fig 1. Ct variation and expression stability analysis of each candidate reference gene among different tissue samples using geNorm.

(a) Boxplot depicting absolute Ct values, which was calculated using GenEx program. Lower and upper boxes indicate the 25th and 75th percentile, respectively. The median is depicted by the line and all outliers are indicated by dots (b) Gene expression stability graph based on average expression stability values (M-value), using stepwise exclusion process. The lower the M-value indicates, higher the stability of gene. The direction of the arrow indicates the most and least stable housekeeping genes.

Fig 2. Ct variation and expression stability analysis of each candidate reference gene among different tissue samples using NormFinder.

(a) Boxplot depicting absolute Ct values, which was calculated using GenEx program. Lower and upper boxes indicate the 25th and 75th percentile, respectively. The median is depicted by the line and all outliers are indicated by dots. (b) Gene expression stability graph using NormFinder algorithm based on stability values. Lower the stability value indicates higher stability of the housekeeping gene. The direction of the arrow indicates the most and least stable housekeeping genes.

Fig 3. Validation of selected housekeeping genes under drought stress conditions.

Expression profiling of candidate gene responsible for universal stress protein A-like (uspA) protein in drought imposed tissues (root, stem and leaves). The expression value of candidate gene was normalized with stable, combination of stable and least stable genes namely (i) IF4α (ii) TUB6 (iii) HSP90 (iv) IF4α + TUB6 (v) IF4α + HSP90 (vi) IF4α + TUB6+HSP90 (vii) ACT1 and (viii) 18SrRNA. The relative quantitative values of selected drought responsive candidate gene were obtained after scaling to control samples. EDR: vegetative root stressed; LDR: reproductive root stressed; EDS: vegetative stem stressed; LDS: reproductive stem stressed; EDL: vegetative leaves stressed; LDL: reproductive leaves stressed.