Reference gene selection and expression analyses of anthocyanin biosynthetic genes in flower and vegetative tissues of Clivia miniata L.

Pigmentation is one of the most variable traits in plants, with flowers typically being the main tissue that displays such diversity. Unlike other horticulturally valuable plants, the genetic basis of flower pigmentation in Clivia species remains poorly understood. Therefore, this study aimed to analyze the expression of numerous anthocyanin biosynthetic genes in flower and vegetative tissues of the most popular Clivia species, Clivia miniata. Such information can facilitate the breeding and biotechnological use of Clivia in the global horticulture value-chain. Initially, we mined a previously assembled C. miniata flower transcriptome for WDR transcripts that are homologous to key pigmentation genes from maize (PAC1) and Arabidopsis thaliana (TTG1). Subsequently, we identified and tested eight candidate reference genes (18S, ACT, EF1α, G6PDH, PP2A, RNPII, TUBα and UBQ) for expression stability in Clivia tissues using geNorm, NormFinder, BestKeeper, and the comparative Delta-Ct method. Homology analysis revealed a transcript encoding a partial protein with specific motifs that are common in pigmentation-related WDR proteins. Further, three reference genes (CmiPP2A, CmiEF1α and Cmi18S) were most stable in tested Clivia tissues. Quantitative real-time PCR (qPCR) analyses of ten anthocyanin biosynthetic genes (CmiDFR, CmiF3H, CmiF3’H, CmiUF3GT, CmibHLH001, CmibHLH002, CmiMYB001, CmiMYB002, CmiMYB003 and CmiWDR001) normalized against the three reference genes revealed that most had relatively higher expression levels in flower than vegetative tissues. Also, expression was generally higher during anthesis than in buds, but some genes remarkably showed marked expression in roots too. Overall, this is the first study to systematically select and validate reference genes for expression analysis of anthocyanin biosynthetic genes in Clivia.