Reference gene selection for qPCR analysis in cineraria developing flowers

Abstract Cineraria ( Senecio cruentus Masson ex L’Herit) is a groundsel perennial herbaceous plant that has a variety of colors and is an ideal model used to research the mechanism of flower color variation. Anthocyanin is the major component that determines the flower color, and studying the regulation of endogenous anthocyanin biosynthesis-related genes is important for understanding the molecular mechanism of flower color variation. Selecting suitable reference genes is a prerequisite for identifying the accurate expression pattern of these genes. We analyzed the stability of eight reference genes, glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ), protein phosphatase 2A ( PP2A ), β-tubulin , Actin , TIP41-like family protein ( TIP41 ), elongation factor 1 α ( EF-1α ), SAND family protein ( SAND ) and S-adenosylmethionine synthetase ( SAMS ) in different flower developmental stages of four different color lines in cineraria. The results showed that SAND and Actin were the most stable genes in the blue and pink lines, while the TIP41 and Actin genes performed best in the white line; however, the PP2A and Actin genes were the least variable in the carmine line. SAND and Actin genes were the most stable gene in all samples. SAMS was the least stable gene in all samples, so it is not suitable as a reference gene. In addition, the relative expression level of Senecio cruentus Flavanone 3-hydroxylase gene ( ScF3H ), the upstream gene in the anthocyanin biosynthesis pathway, in the different developmental stages of the flower in the carmine line was analyzed to verify the reference genes selected in this study. We recommended the combination of SAND , Actin and PP2A were suitable for normalization the target genes in different color lines of cineraria during their flower developmental stages. This study presented the first systematic study of reference genes stability and provided a guide to research of the molecular mechanism of the anthocyanin biosynthesis pathway and flower development in cineraria.