How to perform RT-qPCR accurately in plant species? A case study on flower colour gene expression in an azalea (Rhododendron simsii hybrids) mapping population

摘要: Background: Flower colour variation is one of the most crucial selection criteria in breeding a flowering pot plant, as also case for azalea (Rhododendron simsii hybrids). Flavonoid biosynthesis was studied intensively several species. In azalea, flower can be described by means 3-gene model. However, this model does not clarify pink-coloration. The last decade gene expression studies have been implemented widely studying colour. methods used were often only semi-quantitative or quantification done according to MIQE-guidelines. We aimed develop an accurate protocol RT-qPCR and validate study mapping population. Results: An had established. RNA quality evaluated combined approach different techniques e.g. SPUD-assay Experion-analysis. demonstrated importance testing noRT-samples all genes under detect contaminating DNA. spite limited sequence information available, we prepared set 11 reference which validated petals; combination three optimal. Finally plasmids construction standard curves. This allowed us calculate gene-specific PCR efficiencies every assure quantification. validity six flavonoid pathway. No correlations found between individual profiles. early pathway (CHS, F3H, F3'H FLS) clearly related co-pigmentation with flavonols. late DFR ANS are minor extent involved differentiating coloured white flowers. Concerning pink coloration, could demonstrate that lower intensity type flowers correlated F3'H. Conclusions: Currently plant research, qualitative protocols still rare. on species expression. able correlate regulation structural genes, both branch differentiate colours broader genetic background so far studies. These data will now eQTL comprehend even more