Scientia Agricultura Sinica ›› 2025, Vol. 58 ›› Issue (23): 5031-5045.doi: 10.3864/j.issn.0578-1752.2025.23.017

• HYBRIDIZATION BREEDING AND GERMPLASM INNOVATION IN PAEONIA • Previous Articles     Next Articles

Genome-Wide Analysis of AP2/ERF Transcription Factors in Peony

XU DuoDuo(), DU QianQian, ZHAO LiXiang, LI Yan, HUANG Gan, LI YongHua, LU JiuXing*()   

  1. College of Landscape Architecture, Henan Agricultural University, Zhengzhou 450002
  • Received:2025-06-09 Accepted:2025-08-15 Online:2025-12-01 Published:2025-12-09
  • Contact: LU JiuXing

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Abstract:

【Objective】 To investigate the roles of the AP2/ERF gene family in peony flower development and provide a theoretical foundation for the precise regulation of flower type breeding in peony. 【Method】 Using the peony genome as a reference, we systematically identified members of the AP2/ERF gene family and performed phylogenetic, gene structure, cis-element, and repeat event analyses. Genome sequences of Arabidopsis thaliana, rice, grape, and sweetgum were retrieved from public databases for interspecific synteny analysis using peony as the reference. RNA-seq datasets from various peony tissue types were obtained from global public repositories, processed by file segmentation and sequence alignment, and subsequently used to construct heatmaps with TBtools for functional prediction of AP2/ERF gene family members. Principal component and hierarchical clustering analyses were conducted to evaluate overall sample correlations. Tissue-specific expression patterns were further validated via quantitative real-time PCR (qRT-PCR) of 12 selected genes. 【Result】 A total of 126 AP2/ERF family members were identified and classified into four subfamilies (AP2, ERF, DREB, and RAV) and one unclassified group (Soloists) based on phylogenetic analysis. Synteny analysis revealed that 122 of these genes were anchored to the five chromosomes of peony, comprising 73 syntenic gene pairs. The number of homologous gene pairs between peony and sweetgum or grape was substantially higher than that between peony and Arabidopsis thaliana or rice, indicating a high degree of subfamily conservation, frequently accompanied by loss of the untranslated region (UTR). Cis-element analysis indicated that AP2/ERF family genes in peony are predominantly involved in plant growth and development, hormone signaling, abiotic stress responses, and light signal regulation. Expression profiling revealed that 48% of the 126 identified AP2/ERF members were associated with flower development. Twelve genes potentially related to flower development were identified, including three from the AP2 subfamily, six from the DREB subfamily, and three from the ERF subfamily. Using roots, stems, leaves, fully bloomed petals, and flower buds at five differentiation stages of the Fengdanbai cultivar as materials, qRT-PCR validation was performed. The results showed that 83% of these genes exhibited expression patterns consistent with RNA-seq predictions. 【Conclusion】 The expansion of the peony AP2/ERF gene family is attributed to both tandem and segmental duplications and occurred subsequent to the divergence between peony and Arabidopsis thaliana. In addition to members of the AP2 subfamily, certain genes from the ERF and DREB subfamilies also contribute to flower development in peony, highlighting a notable functional divergence of AP2/ERF family members in peony compared to other plant species.

Key words: peony, gene identification, AP2/ERF, flower development, genome, principal component analysis, qRT-PCR

Table 1

The sequences of primers used for qRT-PCR"

基因Gene 引物序列Primer sequence (5′-3′)
ubiquitin F：AGCCCATATTCAGGAGGTGT
R：GCAATCTCAGGTACAAGGGG
PoDREB2 F：CCCGACACCCAATTTACCGA
R：ACGTGCCCAACCATATCCTC
PoERF37 F：ACCCACTTCAAAACTCCGCA
R：TTCCGCTACCCATTTTCCCC
PoDREB25 F：GGTTCTCGAGCCATGACCAA
R：ATTGGGCTTGGATCTGGTGG
PoAP2-9 F：ACTTCCTCTCATCAGCCCCT
R：CGCGGGCTATTCGAGTTAGT
PoAP2-5 F：CGCATCATGGCAAGCAGTAC
R：AACACCGGTCGATTGCTGAT
PoERF19 F：CAGAGATCCGAACAAACGCG
R：ACGTCCCGATCTCAAGAGGA
PoERF2 F：CTGGCTTGGTACGTTCGACT
R：GAGGAGGCGACGATGATGTT
PoDREB28 F：ACATCACAACAGCAATGCGG
R：ACCCCATTTCCTTCGTCGAA
PoDREB43 F：TTCGACGCTGCACTCTTTTG
R：GCAGCCTCTTGAATTTCCGG
PoAP2-16 F：AGCATGGAAGATGGCAAGCT
R：TTCTGCAGCTTCCTCTTGGG
PoDREB39 F：AAGGGACCGATGAAAGGCTG
R：AACTTCCCCACTCCCTCTGT
PoDREB21 F：GTGGCCGCATCTAACGAAAG
R：GTTCCTTCCACCCAGCAAGA

Fig. 1

Phylogenetic tree of the Peony AP2/ERF family Different colors represent different subgroups within each subfamily. Blue stars indicate genes in peonies, while red triangles indicate genes in Arabidopsis"

Fig. 2

Co-lineage mapping within the peony genome"

Fig. 3

Co-linearity and homology of PoAP2/ERF genes between peony and other species"

Fig. 4

Distribution of conserved motifs and gene structure of peony AP2/ERF gene A: Phylogenetic tree and motif composition; B: Protein structural domains; C: Exon-intron structure"

Fig. 5

Analysis of cis-regulatory elements of the peony AP2/ERF promoter"

Fig. 6

Heat map of the expression levels of different tissues of peony AP2/ERF gene LF: Leaf; RT: Root; SH: Branch; ST: Stem; BD_1: Stage of sepal differentiation of flower buds; BD_2: Stage of petal differentiation of flower buds; BD_3: Fully differentiated flower buds; PE_1: Unpigmented compact buds; PE_2: Buds slightly pigmented prior to anthesis; PE_ 3: First bloomed flowers; PE_4: Fully opened flowers with anthers exposed. Asterisks refer to the 12 genes for which fluorescence quantification was done"

Fig. 7

Principal component analysis and cluster analysis of samples from RNA-seq datasets of different sites"

Fig. 8

Tissue-specific fluorescence quantitative PCR analysis of 12 AP2/ERF genes RT: Root; ST: Stem; LF: Leaf; S1: Flower buds at the stage of sepal primordial differentiation; S2: Flower buds at the stage of petal primordial differentiation; S3: Flower buds at the stage of stamen primordial differentiation; S4: Flower buds at the stage of pistil primordial differentiation; S5: Flower buds at the stage of completion of differentiation; PE: Flower petals in full bloom. Different letters indicate significant differences at P<0.05"

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