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Iris germanica  L. is a perennial herbaceous plant that has been widely cultivated worldwide and is popular for its elegant and vibrantly colorful flowers. Selection of appropriate reference genes is the prerequisite for accurate normalization of target gene expression by quantitative real-time PCR. However, to date, the most suitable reference genes for flowering stages have not been elucidated in  I. germanica . In this study, eight candidate reference genes were examined for the normalization of RT-qPCR in three  I. germanica  cultivars, and their stability were evaluated by four different algorithms (GeNorm, NormFinder, BestKeeper, and Ref-finder). The results revealed that  IgUBC  and  IgGAPDH  were the most stable reference genes in ‘00246’ and ‘Elizabeth’, and  IgTUB  and  IgUBC  showed stable expression in ‘2010200’.  IgUBC  and  IgGAPDH  were the most stable in all samples, while  IgUBQ  showed the least stability. Finally, to validate the reliability of the selected reference genes, the expression patterns of  IgFT  ( Flowering Locus T  gene) was analyzed and emphasized the importance of appropriate reference gene selection. This work presented the first systematic study of reference genes selection during flower bud development and provided guidance to research of the molecular mechanisms of flowering stages in  I. germanica .

Selection and validation of appropriate reference genes for RT-qPCR analysis of flowering stages and different genotypes of Iris germanica L