Selection and Validation of Reference Candidate Genes for qRT-PCR Analysis in the Developing Fruit of Phyllanthus emblica L.

Accurate normalization of target gene expression in qRT-PCR experiments requires the development of stable and efficient housekeeping reference genes, particularly for tissue-specific genes in a given organ. Phyllanthus emblica L., an economically important cash tree, has been applied as medical or functional fruit for years in Asian countries due to its fruit that contains rich and diverse active compounds. Developing housekeeping reference genes is critical to investigate the physiological and molecular regulation of fruit development and ripening for P. emblica genetic improvement in breeding practice. Here, based on the expressional stabilities and efficiencies, expression profiles of 13 candidate genes at various fruit development stages were compared between two accessions using expression levels and multiple statistical methods, including BestKeeper, NormFinder, geNorm, ΔCt, and RefFinder. The validation test was conducted through qRT-PCR analysis of two fruit tissue-specific genes, PeGASA and PeMLP, across the different fruit development stages, combined with the comparison of gene expression consistency between qRT-PCR and transcriptomic data. These analyses identified PeACT and PeUBQ6, two regulators of tissue development and ripening, to be the most suitable housekeeping reference genes. Thus, we recommended PeACT and PeUBQ6 can serve as housekeeping reference genes for conducting qRT-PCR analysis in P. emblica fruit, helpful for investigating gene expression related to fruit development and ripening using the qRT-PCR technique.