Genome-anchored transcriptome-based screening and validation of dedicated reference genes for Bougainvillea spp. bracts

Bougainvillea is prized for their colorful bracts, yet accurate gene expression studies in this organ are hampered by the lack of stably expressed reference genes. In this study, we integrated genome-anchored transcriptome screening with multi-algorithm stability analysis to identify and validate reference genes suitable for gene expression studies in bracts, and further used these reference genes to screen candidate CHS genes associated with bract colour development. From three published transcriptome datasets, 10 candidate reference genes were selected based on stability and expression-level criteria. Evaluated by Delta‑Ct, BestKeeper, geNorm, NormFinder and RefFinder, S12‑L , 1F1A and Actin were most stable in non‑bract tissues and total samples, whereas S12‑L , MBF and Actin were most stable in bracts. geNorm analysis indicated that two reference genes sufficient for non‑bract tissues, whereas three are required for bracts. Biological validation confirmed that stable reference genes (e.g. S12‑L ) produced consistent expression patterns for target genes ( LOG3 and LOG5 ), whereas unstable reference genes led to biased expression estimates. Using S12‑L as a normalizer, five of seven candidate CHS genes exhibited a biphasic expression pattern during bract development, accompanied by a colour transition from deep yellow to white. This pattern correlating with anthocyanin reduction, a shift in metabolic flux shift towards colourless flavonols, and chlorophyll degradation. This study provides candidate reference genes for bract research in the tested Bougainvillea cultivar and provides a useful workflow for reference gene screening in specialized organs of ornamental plants.