Selection of Reference Genes of Flower Development in Ludisia discolor

<b>Background:</b> RT-qPCR is a powerful strategy for recognizing the most appropriate reference genes, which can successfully minimize experimental mistakes through accurate normalization. <i>Ludisia discolor</i>, recognized for its ornamental value, features little, distinctive blossoms with twisted lips and gynostemium showing chiral asymmetry, together with striking blood-red fallen leaves periodically marked with golden blood vessels. <b>Methods and Results:</b> To ensure the accuracy of qRT-PCR, selecting appropriate reference genes for quantifying target gene expression levels is essential. This study aims to identify stable reference genes during the development of <i>L. discolor</i>. In this study, the entire floral buds, including the lips and gynostemium from different development stages, were taken as materials. Based upon the transcriptome information of <i>L. discolor</i>, nine housekeeping genes, <i>ACT</i>, <i>HIS</i>, <i>EF1-α1</i>, <i>EF1-α2</i>, <i>PP2A</i>, <i>UBQ1</i>, <i>UBQ2</i>, <i>UBQ3</i>, and <i>TUB</i>, were selected in this research study as prospect interior referral genes. The expression of these nine genes were found by RT-qPCR and afterwards comprehensively examined by four software options: geNorm, NormFinder, BestKeeper, and ΔCt. The outcomes of the analysis showed that <i>ACT</i> was the most steady gene, which could be the most effective inner referral gene for the expression evaluation of flower advancement in <i>L. discolor</i>. <b>Conclusions:</b> The results of this study will contribute to the molecular biology research of flower development in <i>L. discolor</i> and closely related species.