Evaluation and Validation of Reliable Reference Genes for Quantitative Real-Time PCR Analysis of the Gene Expression in Macadamia integrifolia

Macadamia is an economically significant crop, with its kernel oil being abundant in monounsaturated fatty acids (MUFA). Analyzing the expression of genes related to MUFA biosynthesis is essential for understanding the complex regulatory networks in Macadamia. However, there are few reports on the identification of suitable reference genes for use as internal controls in this species. Consequently, selecting a reliable reference gene for gene expression studies under various conditions is critical. In this study, we evaluated the expression stability of 11 traditional housekeeping genes: α-tubulin (TUBa), β-tubulin (TUBb), malate dehydrogenase (MDH), 18S ribosomal RNA (18S), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), α-elongation factor 1 (EF1a), β-elongation factor 1 (EF1b), ubiquitin (UBQ), ubiquitin-conjugating enzyme (UBC), cyclophilin (CYP), and actin (ACT) under abiotic stresses, hormonal treatments and in variety of plant tissues using the online tool RefFinder, which integrates four commonly used software programs: ΔCt, geNorm (version 3.4), NormFinder (version 0953), and BestKeeper (version 1.0). A comprehensive expression stability ranking was established by integrating results from these four methods based on the geometric mean. The findings indicated that ACT was the most stable gene across all samples, including those subjected to cold stress, NaCl stress, PEG stress, ABA treatment, MeJA treatment, and both stem and leaf tissues. EF1b was identified as the most stable gene in GA treatment and heat stress samples, while UBC and CYP were ranked highest in ethrel treatment and root tissue samples, respectively. Finally, the reliability of these findings was further validated using the target gene SAD through qRT-PCR. In summary, this study evaluated and validated appropriate reference genes for qRT-PCR, which will facilitate future investigations into the molecular mechanisms in Macadamia.