Reference genes selection for qRT-PCR analysis in various flowering transition events of Magnolia ×soulangeana ‘Changchun’

•This is the first comprehensive and systematic study analyzing the reference genes appropriate for qPCR analysis of various flowering transition events in Magnolia ×soulangeana ‘Changchun’.•The selection of reference genes suitable for flowering events in ‘Changchun’ is developmental stage-dependent.•In general, UBC+GBP is recommended as the excellent reference gene combination for qPCR analysis of the integrative flowering events in ‘Changchun’. The appropriate reference genes are the prerequisite for gene expression analysis by qPCR, and their stability can affect the delicate and reliable results directly. Magnolia ×soulangeana ‘Changchun’ is a representative ornamental Magnolia that possesses a complex annual flowering cycle, including floral transition, dormancy, and twice blooming, and it is of great value and significance in investigating the flowering regulation of magnolias. In order to screen the appropriate reference genes related to various flowering transition events in ‘Changchun’, nine candidate reference genes were selected for qPCR analysis based on transcriptome data. Here, the expression stability of these nine genes in 63 different ‘Changchun’ samples was evaluated by four statistical algorithms (geNorm, NormFinder, BestKeeper and RefFinder). The results showed that GBP, UBQ, UBC and GAPDH were identified as the most stable gene in the floral transition, summer flowering initiation, spring flowering initiation and dormancy, respectively. Notably, UBQ showed the highest expression stability in the integrative flowering events set while UBC was suitable for the all samples set. Particularly, GBP was the relatively stable one in all sets. Further gene expression verification indicated that qPCR results using UBC+GBP as the reference genes were the most highly correlated with high throughput sequencing data. In general, the present study suggests that reference genes selection is developmental stage-dependent, and UBC+GBP is recommended as the best reference gene combination applied in qPCR assays of the integrative flowering events in ‘Changchun’. These findings will provide technical support for further molecular regulation studies of flowering events or other biological processes in magnolias, and even can be extended to other species.