Freesia hybrida cultivar ‘Shangnong Golden Queen’ was used as the plant material in the study. Six different tissues or organs including tepal, pistil and stamen (mixed samples), flower scape, leaf, corm and root, corms at five developmental stages and corms under treatments with three exogenous hormones, were collected, respectively. qRT-PCR was used to analyze the gene expression level of selected eight commonly used housekeeping genes, including Actin, Glyceraldehyde-3-phosphate (GAPDH), β-tubulin (TUB), 18S rRNA, elongation factor 1 beta (EF-1β), ubiquinol-cytochrome C reductase (QCR), cyclophilin (CYP) and S-adenosylmethionine decarboxylase (SAMDC). Three widely-used software, including geNorm, NormFinder and BestKeeper, were used to make a comprehensive analysis on the stability of gene expression.

The eight candidate housekeeping genes could be expressed in diffe-rent tissues and organs of Freesia hybrida ‘Shangnong Golden Queen’ with individual expression richness. Among those, two genes, 18S rRNA and Actin were the most stable, while EF???1β was the most unstable gene based on the analysis with the three software. In the corms at five developmental stages, three candidate genes, QCR, Actin and TUB presented a stable expression level, which can be selected as reference genes in future studies on the gene expression involving corm development. While CYP and 18S rRNAwere not stable, these two genes were not introduced to analyze gene expression in corms at different developmental stages. In corms under treatments with exogenous hormones, the expression of CYP and 18S rRNA was quite stable, while EF?1β was the most unstable gene in this experimental condition.

This is the first time reference genes were screened for a given set of experimental conditions in Freesia hybrida. Taken together, 18S rRNAand Actin were the preferred internal reference genes for gene expression analysis in different tissues of Freesia. QCR，Actin and TUB were the optimized genes in corm development, while for the gene expression analysis under hormone induction, the preferred reference genes were 18S rRNA and CYP. Our findings provide a guideline for any future work on gene expression in Freesia by using qRT-PCR.