Selection and Validation of Reference Genes for qRT-PCR Analysis in Neocinnamomum caudatum

Neocinnamomum caudatum (Nees) Merr. is an underutilized woody oil plant with seeds rich in long-chain fatty acids and polyunsaturated fatty acids. Reliable quantitative real-time PCR (RT-qPCR) analysis is essential for investigating the molecular mechanisms underlying seed oil biosynthesis, but suitable reference genes have not yet been validated in this species. Here, seven candidate reference genes, namely EF-1α, ACT2, ACT11, UBQ11, TUA, F-BOX, and GAPDH, were selected from transcriptomic data and evaluated in leaves, flowers, and developing seeds of N. caudatum. Their expression stability was assessed using geNorm, NormFinder, and BestKeeper, followed by comprehensive ranking with RankAggreg. Among all tested samples (leaves, flowers and developing seeds combined), GAPDH was identified as the most stable reference gene, whereas EF-1α was the least stable. For developing seeds alone, TUA showed the highest stability, while EF-1α exhibited poor stability. In leaf and flower samples, ACT11 was the most stable gene, whereas TUA was unsuitable for normalization. The expression patterns of NcFAD2 and NcFatB, two genes involved in fatty acid biosynthesis, were used to validate the selected reference genes. Stable reference genes and the optimized multi-gene combination generated consistent expression profiles, while unstable reference genes caused evident distortion. This study provides the first systematic evaluation of reference genes for qRT-PCR analysis in N. caudatum and offers a practical foundation for future functional studies of lipid metabolism in this woody oil plant.