Identification of Suitable Reference Genes for Studying Expression of Cell Wall-Related and Developmental Associated Genes in Mungbean (Vigna radiata (L.) Wilczek)

Spatio-temporal changes in gene expression are associated with certain biological activities. Therefore, identifying reference genes is paramount to elucidate the gene expression using reverse transcription quantitative polymerase chain reaction (RT-qPCR). Therefore, to identify suitable reference genes, we initially screened 15 putative reference genes by studying their gene expression in different tissues of mungbean. Further, ten candidate reference genes were ranked based on stability using algorithms such as GeNorm, NormFinder, Bestkeeper, and RefFinder. The EF-1 ALPHA , CYP1 and GAPDH showed the best stable expression across different tissues as compared to other reference genes. The EF-1ALPHA exhibited the lowest Ct value, the highest expression and most stable (using different algorithms) by the comparative analysis among 10 reference genes after analysing its expression in leaf and stem. Further, the suitability of EF-1 ALPHA as reference genes was validated by checking the expression of PHENYLALANINE AMMONIA LYASE (PAL) , CINNAMOYL ALCOHOL DEHYDROGENASE (CAD), IRREGULAR XYLEM 14 (IRX14) and RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE/OXYGENASE (RuBisCO ) in three different mungbean genotypes and tissue types. We further tested the expression of RuBisCO in seedlings under salt and osmotic stress and found its expression was lower as compared to control plants, which correlated with the phenotype of seedlings. Therefore, we propose EF1-ALPHA is the best reference gene to analyse the expression of genes in different tissues and development stages with and without stress conditions.