# Resequencing and Functional Analysis Revealed That BsDFR4 Could Cause the Formation of Different Flower Colors in Bletilla striata (Orchidaceae)

## TL;DR

This study identifies a gene, BsDFR4, responsible for different flower colors in Bletilla striata by affecting anthocyanin production.

## Contribution

The study reveals BsDFR4 as a key gene influencing flower color variation through functional and resequencing analyses in Bletilla striata.

## Key findings

- Purple flowers had significantly higher flavonoid and anthocyanin levels than pink flowers.
- A premature stop codon in BsDFR4 disrupts anthocyanin production and causes color variation.
- BsMYB36 and BsMYB51 transcription factors regulate BsDFR4, influencing flower color formation.

## Abstract

The formation of flower color is closely related to anthocyanin synthesis. In this study, flowers of Bletilla striata (Orchidaceae) exhibiting distinct color morphs were collected and analyzed. The HPLC results showed significantly higher total flavonoid and anthocyanin contents in purple flowers compared to pink counterparts, with increases of 2.20-fold (p < 0.01) and 15.22-fold (p < 0.01), respectively. Cyanidin was the predominant anthocyanin in B. striata. Resequencing analyses highlighted SNP as the primary variation associated with color divergence. A comprehensive screen identified 61 genes encoding enzymes critical to the flavonoid and anthocyanin biosynthesis pathways in B. striata. Among these, 16 flower-specific genes exhibited high expression levels and harbored SNP variations. Notably, a premature stop codon was identified in a gene encoding dihydroflavonol 4-reductase (DFR), leading to truncated protein synthesis and potential disruption of anthocyanin production. Further, the heterologous overexpression of BsDFR4 in Phalaenopsis aphrodite changed petal color from white to yellow-green, demonstrating that it indeed played a regulatory role in the formation of flower color. Furthermore, yeast one-hybrid assays confirmed that transcription factors BsMYB36 and BsMYB51 could directly bind to the BsDFR4 promoter, suggesting their synergistic regulation of anthocyanin biosynthesis. These results provided a conceptual basis for insights into the formation of different flower colors in Orchidaceae.