Comparison of nine reference genes for real-time quantitative PCR in roots and leaves during five developmental stages in carrot (Daucus carota L.)

Carrot is a biennial plant with a nutritionally healthy composition and has long been recognised as an important model for genetic and molecular studies. The present study aimed to identify suitable reference genes for the normalisation of gene expression in carrot roots and leaves at different developmental stages. Three different software tools were applied to detect the stability of expression of nine candidate reference genes. The results showed that the best reference genes differed across tissues and developmental stages. Actin (ACTIN) was recommended as the most stable gene in carrot roots and leaves at all five developmental stages examined. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) appeared to be the least adequate gene, indicating that it should be avoided as a reference gene in carrot. Thus, we suggest ACTIN or different combinations of ACTIN, elongation factor 1α (EF-1α), eukaryotic translation initiation factor 4α (eIF-4α), tubulin β-7 (TUB), or polyubiquitin 10 (UBQ) for the normalisation of gene expression during carrot developmental processes. These results provide guidelines for future work on relative levels of gene expression in carrot using real-time reverse transcription-quantitative PCR (RT-qPCR).