Selection of the optimal reference genes for transcript expression analysis of lipid biosynthesis-related genes in Okra (Abelmoschus esculentus)

Okra (Abelmoschus esculentus) is a well-known nutritional and health-benefit vegetable crop worldwide. Quantitative real-time PCR (qPCR) is an effective approach to characterize gene functions, and for such analysis, it is crucial to select the optimal reference genes. However, no systematic evaluation of reference genes was performed in okra. Here, to characterize expression profiles of genes involved in lipid biosynthesis and oil accumulation in okra, nine okra genes including ACT4, EF1a, MZA, PP2A, FBX, RAN, UBQ7, TUBα, and UBC selected from transcriptome sequencing data were examined with respect to their suitability as reliable reference genes. The expression stability of the nine candidates was assessed by four statistical algorithms (geNorm, NormFinder, BestKeeper and RankAggreg). Notably, MZA and PP2A were identified as the stable reference genes for different developmental seeds while EF1a was the optimal reference gene for different okra tissues. RAN was the highest-ranked reference gene for seed samples of different okra genotypes. Conversely, TUBα was not suitable reference gene in all the experimental conditions. The expression profiles of six lipid biosynthesis-related genes (AeFAD2, AeSAD, AeDGAT1, AeDGAT2, AePDAT1 and AePDAT2) were investigated by qPCR to evaluate the effectiveness and reliability of the reference genes identified. When MZA, PP2A and EF1a, the most suitable reference genes, were employed to normalize the expression data of these six genes in okra, the expression patterns of the six genes were consistent with the results of transcriptome. The present findings provide valuable data for further research on molecular mechanism of lipid biosynthesis and also the basis to select the reliable reference genes for gene expression and functional genomics studies in okra.