Identification of reference genes provides insights into the determinants of self-incompatibility in Camellia oleifera

Self-incompatibility (SI) is one of the main reasons leading to the low fruit set of Camellia oleifera and limiting the yield of Camellia oil. It is important to uncover the molecular mechanism of SI for guiding production. Accurate quantification of the expression level of SI determinants is essential, which requires qRT-PCR technology and appropriate reference genes. However, the screening of SI-related reference genes in C. oleifera has not been reported. In this study, multiple transcriptome data were innovatively integrated and candidate reference genes of SI were systematically and rapidly screened in C. oleifera. TIF3H1 was selected as the most stable gene expressed in pistillate tissues of C. oleifera under different pollination treatments based on the comprehensive evaluation using qRT-PCR and statistical algorithms of geNorm, NormFinder, BestKeeper, and RefFinder. The expression of SI-related genes of C. oleifera validated the stability of TIF3H1 as the reference gene. Using TIF3H1 as the reference gene, the expression of the potential SI determinants in compatible and incompatible pistils was accurately quantified, and the high expression of the homologous gene of pectin methylesterase (PME) in incompatible pistils suggested its positive effect on SI of C. oleifera. The identification of TIF3H1 as the reliable reference gene guarantees more accurate qRT-PCR quantification for the study of the SI of C. oleifera, laying a foundation for identifying determinants of SI and exploring its molecular mechanism.