Selection of reference genes for quantitative real-time PCR normalization in Narcissus pseudonarcissus in different cultivars and different organs

Quantitative real-time PCR (qRT-PCR) has been a widely used accurate technique for gene expression analysis in various species. However, its results require data normalization by reliable reference genes. Despite the horticultural importance of Narcissus pseudonarcissus, and genome sequence has become available for the species, no gene expression study based on the stability of reference genes in qRT-PCR has been conducted. To boost the use of qRT-PCR in N. pseudonarcissus, we uncovered eight commonly used candidate reference genes for their stability. The expression levels of the eight genes were detected for the normalization in five different organs (bulbs, scapes, leaves, perianths and coronas) of three N. pseudonarcissus cultivars ('Marieke', 'Pinza' and 'Slim Whitman') by qRT-PCR. Subsequently, three commonly used computational programs were applied for evaluating the stability of the candidate reference gene's expressions. It turned out that for all the samples and most subgroups, ACT and GAPDH were the most suitable reference genes for normalization. However, the best reference genes were found not always the same one across diverse samples by different computational programs. Our study was the first reference gene evaluation in N. pseudonarcissus and will promote future studies on gene expression levels of N. pseudonarcissus.