Reference gene selection for qRT-PCR normalization of gene expression analysis in Melaleuca bracteata F. Muell. under abiotic stresses and hormonal stimuli

Melaleuca bracteata F. Muell. is among the most significant aromatic plants belonging to the Myrtaceae family, and its essential oil has various biological activities. However, the key enzymes involved in essential oil biosynthesis and their expression patterns are not clearly understood. Appropriate reference genes are required for quantitative real-time polymerase chain reaction (qRT-PCR), sensitive for gene expression studies, to obtain reliable results. In this study, 14 candidate genes, namely ACT, 18S, α-TUB, β-TUB, GAPDH, EF1-α, UBQ, UBC, CYP, EIF-4A, DNAJ, PP2A, TIP41, and UBX, were selected from the M. bracteata transcriptome datasets for analysis under abiotic stresses and hormonal stimuli. The stability of these genes was analyzed using three widely used software programs (geNorm, NormFinder, and BestKeeper). Their results were integrated into a comprehensive ranking by geometric mean. According to the study results, TIP41 and CYP were the most stable genes for abiotic stresses, while TIP41 and UBQ were the most stable genes for hormonal stimulation. Further, TIP41 and CYP were the most stable reference genes across all tested samples, whereas ACT was the least stable. Based on the expression of two target genes (COMT1 and COMT2, which are involved in methyleugenol biosynthesis), the suitability of the selected reference genes for qRT-PCR normalization was further verified. The study results could offer a foundation for gene expression analysis and functional studies in M. bracteata and other Melaleuca species.