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The SPL gene is a plant-specific transcription factor involved in the regulation of plant growth and development, which have been identified in woody plants. The process of floral bud differentiation affects the timing of flowering and fruit set and regulates plant growth, however, the mechanism of regulation of flower development by SPL genes is less studied. In this study, 56 VcSPL genes were identified in the tetraploid blueberry. The VcSPL gene family was classified into six subfamilies, and analysis of cis-elements showed that VcSPL genes were regulated by light, phytohormones (abscisic acid, MeJA), and low temperature. In the evolutionary analysis, segmental replication may play an important role in VcSPL gene amplification. Interestingly, we also studied diploid blueberry (Bilberry), in which 24 SPL genes were identified, and 36 homologous pairs were found, suggesting a high degree of convergence in the syntenic relationship between blueberry (Vaccinium corymbosum L) and bilberry (Vaccinium darrowii). Based on the expression profile, VcSPL genes were expressed at high levels in flowers, shoots, and roots, indicating a diversity of gene functions. Then we selected 20 differentially-expressed SPL genes to further investigate the role of VcSPL in floral induction and initiation. It showed that the genes VcSPL40, VcSPL35, VcSPL45, and VcSPL53 may play a crucial role in the blueberry floral transition phase (from vegetative growth to flower initiation). These results provided important information for understanding and exploring the role of VcSPLs in flower morphogenesis and plant growth. [ABSTRACT FROM AUTHOR]