Tree peony (Paeonia suffruticosa Andr.) is a woody plant with high ornamental, medicinal, and oil values. However, its low rooting rate and poor rooting quality are bottleneck issues in the micropropagation of P. ostii. The WUSCHEL-related homeobox (WOX) family plays a crucial role in root development. In this study, based on the screening of the genome and root transcriptome database, we identified ten WOX members in P. ostii. Phylogenetic analysis revealed that the ten PoWOX proteins clustered into three major clades, the WUS, intermediate, and ancient clade, respectively. The conserved motifs and tertiary structures of PoWOX proteins located in the same clade exhibited higher similarity. The analysis of cis-regulatory elements in the promoter indicated that PoWOX genes are involved in plant growth and development, phytohormones, and stress responses. The expression analysis revealed that PoWOX genes are expressed in distinct tissues. PoWOX4, PoWOX5, PoWOX11, and PoWOX13b are preferentially expressed in roots at the early stage of root primordium formation, suggesting their role in the initiation and development of roots. These results will provide a comprehensive reference for the evolution and potential function of the WOX family and offer guidance for further study on the root development of tree peony.

Calcium-dependent protein kinases (CDPKs) function as essential sensors and transducers of intracellular calcium signals, playing crucial regulatory roles in various physiological processes. Nevertheless, studies on CDPK genes in Paeonia ostii remain limited. In this study, 16 PoCDPK genes containing a complete Pkinase domain and four EF-hand domains, distributed across five chromosomes, were identified in P. ostii. Phylogenetic analysis classified these genes into four distinct groups (I–IV). Promoter cis-element analysis suggested a potential involvement of PoCDPK genes in plant growth and development, phytohormone signaling, light, circadian, and stress responses. Transcription levels of PoCDPKs were highest at the early stage of root primordium formation (Gmfq) among the four stages examined. Expression analysis of the 14 PoCDPK genes detected in the transcriptome revealed that nine PoCDPKs were highly expressed in flowers, two in stems, PoCDPK10 in leaves, PoCDPK13 in roots, and PoCDPK1 in roots, stems, and leaves. Moreover, PoCDPK3, PoCDPK12, and PoCDPK13 from Group I, as well as PoCDPK2 and PoCDPK8 from Group III, were highly expressed at 5 d of rooting induction, suggesting their potential involvement in adventitious rooting. These findings provide a comprehensive reference for the potential functions of the CDPK family in the adventitious rooting of tree peony.