Identification and Expression Analysis of Anthocyanin-associated R2R3-MYB Genes in Canna indica

doi:10.13560/j.cnki.biotech.bull.1985.2025-0507

Abstract

Abstract:

Objective To identify the R2R3-MYB gene family in Canna indica, analyze its expression patterns in different flower colors, and provide a theoretical reference for understanding the molecular regulatory mechanism of flower color and breeding new varieties of C. indica. Method Using R2R3-MYB protein sequences from Arabidopsis thaliana as reference, the members of the R2R3-MYB gene family were identified from the C. indica genome through HMM modeling and BLAST searches, and their physicochemical properties, subcellular localization, phylogenetic relationships, chromosomal distribution, conserved motifs, and promoter cis-acting elements were analyzed. Based on pre-existing transcriptome data from eight C. indica varieties within the research group, TBtools was used to visualize their expression patterns in fully open petals. Petals from four varieties with distinct flower colors (orange 'Yinli', pink 'Grand Due', red 'Anwang', yellow 'Sajin') were selected, and the expression patterns of 11 candidate CiMYB genes were detected via quantitative real-time PCR (RT-qPCR), and the relationship between gene expression and petal anthocyanin/flavonoid content was analyzed. Protein-protein interactions (PPI) in C. indica were redicted using Arabidopsis protein. Result A total of 185 R2R3-MYB genes were identified in C. indica. The encoded proteins are hydrophilic and nuclear-localized, distributed across 9 chromosomes. Approximately 94% of members contained the core conserved motifs (Motif1, Motif2, Motif3), and promoter regions are enriched with light-responsive elements. Phylogenetic analysis grouped them with Arabidopsis homologs into 30 subgroups, with 11 genes homologous to MYB proteins regulating anthocyanin synthesis in Arabidopsis. RNA-seq results showed differential expression of these 11 candidate genes across canna varieties with different flower colors. PPI network prediction indicated that CiMYB65 significantly co-expressed with DFR-A (a key anthocyanin synthesis gene) and directly regulated the anthocyanin synthesis RT-qPCR analysis revealed that CiMYB21, CiMYB39, CiMYB65, and CiMYB20 had the highest expressions in 'Grand Due', 'Sajin', 'Anwang', and 'Yinli', respectively. Gene expressions were significantly positively correlated with both anthocyanin and flavonol content. Conclusion The C. indica'sgenome contains 185 R2R3-MYB genes. Among them, genes such as CiMYB20, CiMYB39, and CiMYB65 participate in regulating C. indica flower color formation by modulating anthocyanin synthesis.

Key words: Canna indica, R2R3-MYB transcription factors, flower color, anthocyanin synthesis, phylogenetic analysis, RT-qPCR, gene function

YANG Chen-xin, LI Meng-xiu, JIANG Tang, ZHANG Wen-e, PAN Xue-jun. Identification and Expression Analysis of Anthocyanin-associated R2R3-MYB Genes in Canna indica[J]. Biotechnology Bulletin, 2025, 41(12): 201-213.

Figures/Tables 12

Fig. 1 Phenotypes of tested Canna lily samplesA: RNA sequencing sample. B: Genomic DNA sample. C: RT-qPCR sample

Table 1 Primers for CiMYBs genes

基因名称 Gene name	上游引物 Forward primer （5′-3′）	下游引物 Reverse primer （5′-3′）
18S	ATTCTATGGGTGGTGGTGC	CCATCCAATCGGTAGGAGC
CiMYB20	AGCAGGGCTTTAACAAGGGG	GCTCCTCGGGCATCGATTTA
CiMYB39	GAATGCAGGACTGCTGAGGT	GGTTGACGATGGTGTCGTCT
CiMYB65	TGTTCCACCACCGCCATATC	CCAATCGGAGAACTCGCTGT
CiMYB116	CGTCGTCAGTAGTACCGCTC	CTCCTCGTTGGGATCGACAG
CiMYB16	GGCAGCCAACAAAAGGCTAC	GTCTCTGGATCTGCGTTGCT
CiMYB179	GCTCATAGGCAACAGGTGGT	GAGCTGAGCTTTGACCGAGT
CiMYB8	CCAGCAGCTCAAATCCTCCA	ACCAAGTCCTGCATGCTCTC
CiMYB80	ACGGAGACTTGTTCGGTGTC	TGGCCATGTTGGGTTGTGAT
CiMYB21	TTTGCTGACTTCAATGCGGC	CCTCCTCGATGAATCTCGCC
CiMYB171	CGGAGGAAACTTACGGCACT	CTCCTCGGAGGCAATGAGTC
CiMYB68	CCAGCCCTTGCTGTTACTGA	CCAGCAGCTCAAATCCTCCA

Fig. 2 Phylogenetic tree of R2R3-MYB proteins from Canna indica and Arabidopsis thaliana

Fig. 3 Localization of R2R3-MYB subfamily on Canna lilychromosomes

Fig. 4 Collinearity analysis of protein sequences of R2R3-MYB gene family members in Canna lily

Fig. 5 Collinearity analysis of protein sequences of the R2R3-MYB gene family in C. indica, A. thaliana,and O. sativa

Fig. 6 Gene structures of R2R3-MYB genes in Canna lilyA: Motif analysis. B: Gene domain analysis. C: Gene structure analysis

Fig. 7 Analysis of cis-acting elements of R2R3-MYB promoter in Canna lily

Fig. 8 Expression patterns of R2R3-MYB genes in Canna lilya: Anwang. b: Yinli. c: Progeny 1 of aquatic Canna. d: Progeny 2 of aquatic Canna. e: Progeny 3 of aquatic Canna. f: Progeny 4 of aquatic Canna. g: Progeny 1 of Canna generalis. h: Progeny 2 of Canna generalis

Fig. 9 Interaction analysis between anthocyanin-related Canna lilyThe connecting lines indicate co-expression relationships between genes, with color coding reflecting the nature of interactions. Red lines indicate significantly positive correlations (synergistic regulation), blue lines denote significantly negative correlations (repressive or antagonistic effects), and the thickness of the lines is positively correlated with the strength of co-expression or statistical confidence

Fig. 10 Contents of total anthocyanin and total flavonol in Canna lilyDifferent lowercase letters indicate significant differences among different treatments (P<0.05). The same below

Fig. 11 Relative expression patterns and correlation analysis of candidate R2R3-MYB genes in four cultivars of Canna lily

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