Identification and validation of stable reference genes via transcriptome analysis for studying sex differentiation in tung tree (Vernicia fordii)

Tung oil, obtained from the seeds of the tung tree (Vernicia fordii), holds significant economic value. Nevertheless, the low proportion of female flowers in tung trees limits tung oil yield. Studying the expression of genes involved in sex differentiation provides a promising strategy for increasing the female flower ratio through molecular breeding. Accurate normalization is critical for stable gene expression quantification in qRT-PCR. This study identified stable reference genes for sex differentiation research in the tung tree. Using transcriptome data from diverse floral samples of tung tree, we identified 10 candidate genes and subsequently assessed their expression stability across various tissues using multiple algorithms. Among them, ERF3A and OLA1 exhibited the highest stability in floral tissues. Validation experiments demonstrated that using these stable genes for normalization yielded consistent expression patterns for target genes AP3 and SEP2-2, whereas unstable references led to significant misinterpretation. Thus, ERF3A and OLA1 are proposed for use as reference genes in qRT-PCR when investigating sex differentiation in the tung tree. This study lays a solid foundation for elucidating the molecular mechanisms of sex differentiation in the tung tree.