关键词: 牡丹, 远缘杂交, 受精前障碍, 苯胺蓝染色, 花粉管伸长

Abstract:

【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.

Key words: tree peony, distant hybridization, prezygotic barriers, aniline blue staining, extension of pollen tube