Analysis of Genes Involved in Flower Colour Variation in Canna indica (Cannova) cultivars

Abstract

Flower colour development is a complex biological process and a key ornamental trait with high artistic and commercial value. Canna indica, a member of the Cannaceae family, exhibits outstanding diversity in flower pigmentation across its cultivars. In this study, six Canna Cannova cultivars showing distinct flower colours (red, orange, pink, yellow, and lemon) were investigated using integrative molecular approaches combining genomic DNA sequencing, transcriptomic (RNA-seq) analysis, optimization of a transient expression system, and functional expression experiments. Comprehensive genomic and transcriptomic analyses identified key structural genes associated with anthocyanin and carotenoid biosynthetic pathways in the studied cultivars. Several significant single nucleotide polymorphisms (SNPs) were detected, although the corresponding genes remained transcriptionally active. RNA-seq further enabled the differential expression characterization of structural genes involved in pigment biosynthesis. Many differentially expressed genes were identified among the studied Cannova cultivars. A transient gene expression system for Canna indica flowers was successfully optimized for functional validation of the candidate genes. The most effective transient expression system combined Agrobacterium strain EHA105 carrying the Arabidopsis UBI10 promoter and infiltration buffer (MES, MgCl₂, and acetosyringone). Functional assays targeted key anthocyanin biosynthesis genes, CiCHS, CiDFR, and CiANS, which were highly expressed in the red-flowered cultivar, alongside PeMYB2 as a positive control. Although transient expression capability was confirmed by GUS reporter gene activity, anthocyanin pigmentation was not visually detected in the introduced Canna indica petaloid staminodes. This study provides valuable genomic and transcriptomic resources and establishes a transient expression platform for functional gene analysis in Canna indica. The results contribute to advancing molecular studies and the biotechnological improvement of floral colour trait in Canna species.