Selection of reference genes in Hedysarum coronarium under various stresses and stages of development

The cultivation of Hedysarum coronarium has generated interest recently for its high yield as a fodder crop, its high protein content, and the presence of condensed tannins in its leaf and stem tissues. Gene expression studies can lead to a better understanding of the biological processes of live organisms. Specifically, reverse transcription followed by quantitative polymerase chain reaction (PCR) represents the most powerful technology for comparing the expression profiles of target genes. The use of reference genes as internal controls to normalize messenger RNA (mRNA) levels is a requirement of quantitative PCR (qPCR). Few studies on reference genes have been performed in plants, and no studies have been performed in H. coronarium. Therefore, the aim of this study was to identify and evaluate reference genes to use in qPCR in H. coronarium. Sulla tissues under two conditions of abiotic stress and at various stages of development were studied to determine adequate reference genes. To optimize the identity and number of reference genes, geNorm and BestKeeper software programs were employed. Based on the results of both analyses, TUA1, TUA2, and UBQ were found to be the most suitable reference genes, and the combination of these three genes was suggested for the accurate normalization of gene expression in sulla tissues.