Evaluation of reference genes for gene expression studies in senna (Cassia angustifolia Vahl.) using quantitative real-time polymerase chain reaction

Abstract

Quantitative real-time polymerase chain reaction (RT-qPCR) is one of the mostcommon methods used for quantification of gene expression due to its highsensitivity, specificity, accuracy and reproducibility. However, a selection of stableand suitable reference genes is crucial for reliable estimation of target geneexpression under varied experimental conditions. The aim of the present studywas to identify reference gene for gene expression studies in Senna (Cassia angustifolia Vahl.), a medicinal plant with potent laxative properties. Nine genes commonly used as internal controls in plants were selected in this study: Actin(caActin2-7), 18srRNA (ca18s), Glyceraldehyde-3-phosphate dehydrogenase (caGAPDH), Actin11 (caActin11), Alcohol dehydrogenase (caADH), ATP synthase (caATPSyn), β-Tubulin (caβ-TUB), Eukaryotic elongation factor 1-alpha (caEEF1-α) and Eukaryotic elongation factor 1-beta (caEEF1-β) for their suitability as internal controls in RT-qPCR to understand sennoside biosynthesis as well as abiotic stress. The expression stability of reference genes was studied using qRT-PCR in leaf (young and mature), pod (young and mature) and flower tissue of three (Sona,DCA-80 and DCA-149) genotypes of senna differing with sennoside content. All genes studied showed low variability (6.0-15.93% CV of Cq). Gene stability was assessed using the statistical algorithm GeNorm. The reference genes, caActin2-7, caGAPDH, caADH, caATPsyn, caEEF1-α and caEEF1- β are stable (M= <1.5) across all the tissues and genotypes studied thus enabling their wider use as internal controls. The study provides the foundation for reference gene(s) selection and will contribute towards more accurate target gene expression estimates which will help to elucidate the biosynthetic pathways that underlie sennoside production and abiotic stress tolerance in senna.