【Objective】 Inter-sectional hybrids (Paeonia Itoh Group) produced by crossing between tree peonies and herbaceous peonies are important emerging ornamental plants. No previous studies have found ploidy other than triploid (2n=3x=15, ABC), or translocation between A and B genomes in intersectional hybrids. By employing the wider range of materials and the improved karyotyping technology, this research aimed to test whether there is other ploidy or inter-genome translocation in intersectional hybrids, and to explore how the intersectional hybrid traits are related to chromosome compositions, which could lay a theoretical foundation for the generation mechanism of inter-sectional hybrids in genus Paeonia and the genetic rules of their traits. 【Method】 In this study, we did molecular karyotype analysis on 11 intersectional hybrids from P. lactiflora Fen Yun Fei He (2n=2x=10, CC) × P. × lemoinei ‘L’ Esperance’ (2n=2x=10, AB) by bicolor genomic in situ hybridization (GISH) and rDNA fluorescence in situ hybridization (FISH). The relationship between morphology and ploidy of leaves from the inter-sectional hybrid Jing Hua Huan Cai was examined by flow cytometry. 【Result】 The results showed that 9 of them were triploids (2n=3x=15, ABC), in 2 of which chromosomal translocations between A and B genomes were found, including reciprocal translocations between 2A and 2B, or 3A and 3B chromosomes, and non-reciprocal translocations between 3A and 3B chromosomes. In the rest two intersectional hybrids, Jing Rui Zi was diploid (2n=2x=10, AC), but Jing Hua Huan Cai were diploid (2n=2x=10, AC), or near triploid (2n=3x-1=14, AB-1C) or in more other ploidy, which were found to exist independently or in chimeric state in different divided seedlings. Therein, the unlobed leaves of Jing Hua Huan Cai were all diploids, while the lobed leaves were near triploid, near diploid, or chimera involving near triploid. The intersectional hybrids with different ploidy had highly consistent traits, including herbaceous life form, leathery flower disc and high sterility of both pistils and stamens. However, leaf morphology, flower color and flowering time were significantly different among different ploidy. 【Conclusion】 The results above confirm that triploidization is the main pathway for the formation of intersectional hybrids, and the newly discovered few diploids might be formed by the loss of B genome chromosomes during mitosis of somatic cells in triploids or near triploids. The traits of intersectional hybrids are strongly influenced by their ploidy level. The three subgenomes from the parents were all closely related to the trait formation of intersectional hybrids. These findings reveal the diversity of chromosome karyotypes and the formation mechanism of intersectional hybrids in Paeonia.
【Objective】 Paeonia ludlowii, an endemic wild tree peony species in Tibet, possesses superior characteristics including tall plant stature, pure yellow flowers, and scarlet autumn foliage. However, the distant hybridization barrier with cultivated peonies has prevented the effective utilization of its superior genes. This study aims to elucidate the molecular mechanisms underlying the stigma response to distant hybridization after pollination, clarify the regulatory network of pollen-stigma recognition barriers, and provide a theoretical basis for overcoming pre-fertilization barriers in distant hybridization of peonies. 【Method】 Hybridization experiments were conducted using the cultivated variety Paeonia ostii Fengdan as the maternal parent and Paeonia ludlowii as the paternal parent. Three experimental groups were established: self-pollination of Fengdan (CK), ‘Fengdan’ × P. ludlowii (DH), and Fengdan × P. ludlowii with stigmas pretreated with 2 mg·L-1 KCl (KH). Fluorescence microscopy was used to dynamically observe pollen germination and pollen tube growth from 0 to 4 h after pollination. Stigma transcriptome analysis (RNA-seq) was performed using the Illumina high-throughput sequencing platform, combined with UPLC-QTOF-MS-based untargeted metabolomics to systematically identify differentially expressed genes (DEGs) and differentially accumulated metabolites (DAMs). Key metabolic pathways were further analyzed through KEGG and GO enrichment. 【Result】 Phenotypic observations revealed that in the CK group, a large number of pollen germination occurred at 2 h after pollination, polar growth of pollen tubes was observed at 4 h. Only a few pollen grains germinated 4 h after pollination in the DH group, while the KH group exhibited significantly higher pollen germination rates than the DH group. During pollen-pistil interactions, high concentrations of O2- and H2O2 were found to inhibit pollen germination. KEGG enrichment indicated that DEGs and DAMs were primarily involved in sugar metabolism, cell wall organization or biogenesis, and flavonoid biosynthesis. In interspecific hybridization of tree peony, the widespread downregulation of carbohydrates results in insufficient energy supply, while the reduction in flavonoid compounds disrupts reactive oxygen species (ROS) homeostasis these constitute the key inhibitory factors for pollen germination. 【Conclusion】 Reactive oxygen species (ROS) dynamic imbalance mediated by Rboh, insufficient energy supply induced by glucose metabolism disorder, abnormal flavonoid metabolism, and disrupted hormone signal transduction collectively impede pollen germination in the distant hybridization of Paeonia ludlowii, ultimately resulting in the formation of prezygotic barriers.
【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.
【Objective】 Tree peony, renowned for its ornamental flowers, also serves as a spring foliage plant due to the striking purple-red coloration of its young leaves. Elucidating the physiological mechanisms and key genes underlying red color leaf traits provides a theoretical basis for breeding ornamental foliage cultivars. 【Method】 Leaf samples were collected at six developmental stages from Paeonia qiui and P. Luoyang Hong. Leaf color parameters were measured using a colorimeter. Anthocyanin and chlorophyll contents were quantified through enzymatic methods. The composition of anthocyanins was analyzed by high performance liquid chromatography (HPLC). Furthermore, quantitative real-time PCR (qPCR) was utilized to examine the expression levels of structural genes involved in anthocyanin biosynthesis. 【Result】 Phenotypic observations and colorimetric analyses revealed that P. qiui exhibits a higher redness and a prolonged red-leaf period that persists until flowering. Conversely, P. Luoyang Hong displays red coloration in its young leaves during the early stages, but this red hue rapidly transitions to green following leaf expansion. HPLC analysis revealed that both tree peonies had peonidin-3,5-diglucoside (Pn3G5G) as the main anthocyanin component in leaves. The total anthocyanin content in P. qiui was significantly higher than that in P. Luoyang Hong, with the most pronounced difference observed during the S4 stage. qPCR results showed that the expression levels of CHS, DFR, and ANS in P. qiui were consistent with the trend in anthocyanin content. Correlation analysis further revealed a highly significant positive correlation between DFR expression levels and anthocyanin content, as well as a significant positive correlation for ANS. These findings underscore the critical role of DFR and ANS as key structural genes in anthocyanin biosynthesis. Similarly, in P. Luoyang Hong, the expression levels of CHS, F3H, DFR, and ANS generally aligned with the trends in anthocyanin content. Correlation analysis confirmed that both DFR and ANS expression levels were significantly positively correlated with anthocyanin content. 【Conclusion】 The primary pigment responsible for the red coloration in the leaves of P. qiui and P. Luoyang Hong is peonidin-3,5-diglucoside. DFR and ANS are key structural genes in the anthocyanin biosynthesis pathway of tree peony. Their expression levels are closely associated with anthocyanin accumulation, which in turn influences the formation of red leaf coloration in peonies.
