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Evaluation And Validation of Reliable Reference Genes For Quantitative Real-Time PCR Analysis of The Gene Expression In Macadamia Integrifolia


 Background: Macadamia integrifolia, a new economically important crop, the kernel oil is rich in bioactive compound and monounsaturated fatty acid. Gene expression analysis of qRT-PCR is beneficial to understand the complex regulatory networks of macadamia.Results: In this study, the expression stability of 11 traditional housekeeping genes including α-tubulin (TUBa), β–tubulin (TUBb), malate dehydrogenase (MDH), 18S ribosome 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) were accessed by qRT-PCR in macadamia seedlings under different experimental conditions and tissues. The expression stability of the 11 reference genes was evaluated by the online tool RefFinder, which include ΔCt, geNorm, NormFinder, BestKeeper four commonly software, and then determinated a comprehensive expression stability ranking by integrating above four ranking results based on the geometric mean. Our results show that ACT was the best stable genes for all samples, cold stress, NaCl sress, PEG stress, ABA treatment, MeJA treatment, stem and leaf tissue samples; EF1b is the most stable gene in GA treatment and heat stress samples; UBC and CYP were respectively ranked top in ethylene treatment and root tissue samples. Finally, the reliability of these results was further validated with a target gene SAD by qRT-PCR. Conclusions: In summary, this study evaluated and validated the suitable reference genes for qRT-PCR under different experiment treatment and tissues, and will be useful for further gene expression studies on the molecular mechanisms in Macadamia.