An AP2-Family Gene Correlates with the Double-Flower Trait in Petunia × hybrida

The double-flower trait is highly valued in ornamental plants due to its unique aesthetic appeal, yet its genetic basis varies significantly across different species. While <i>AGAMOUS</i> (<i>AG</i>) and <i>APETALA2</i> (<i>AP2</i>)-like genes have been demonstrated to play crucial roles in floral organ identity regulation in the model plant <i>Arabidopsis thaliana</i>, the underlying mechanisms governing double-flower formation in many ornamental species remain largely unexplored. In this study, we examined the inheritance pattern of this trait and identified a genetic variant associated with petal number variation. Crosses between the single-flowered cultivar 'Baccarat White' (BW) and the semi-double cultivar 'Duo Lavender' (DL) produced a 1:1 segregation of single and semi-double flowers in the F1 generation, while self-pollination of DL yielded a 1:2:1 segregation of single, semi-double, and double flowers. These results indicate that the double-flower trait follows a single-gene, semi-dominant inheritance model. Whole-genome sequencing of BW and DL followed by sequence analysis of floral organ identity genes revealed no significant differences in B-class (<i>PhGLO1</i>, <i>PhGLO2</i>, <i>PhDEF</i>, and <i>PhTM6</i>) or C-class (<i>pMADS3</i> and <i>FBP6</i>) genes between the two cultivars. Notably, a 10 kb insertion upstream of the <i>miR172</i> target site in the <i>PhBOB</i> gene was detected in DL. PCR genotyping of 192 F1 progenies demonstrated complete co-segregation between this insertion and the double-flower phenotype, suggesting a strong genetic association. Moreover, qRT-PCR analysis showed that <i>PhBOB</i> expression was significantly elevated in DL-exhibiting a 69-fold increase in petals compared to BW-implying that its overexpression disrupts the petal-to-stamen identity transition. Additionally, another <i>AP2</i> family gene, <i>PhROB3</i>, was upregulated in semi-double flowers, with a 10-fold higher expression in the petals and stamens of DL relative to BW, suggesting its potential role in floral organ differentiation. This study elucidates the molecular regulatory mechanism underlying the double-flower trait in petunia, highlighting the role of <i>PhBOB</i> in floral organ identity specification and providing new insights into the potential function of <i>PhROB3</i> in double-flower development.