Bio-based aromatic epoxides are attractive monomers for sustainable polymer synthesis, yet the direct ring-opening homopolymerization of eugenyl glycidyl ether (EPEU) has remained unexplored. Here we report the first direct homopolymerization of EPEU using a cooperative POSS-B8/tetrabutylammonium chloride (TBACl) binary system. Under optimized conditions, EPEU reached >99% conversion in both THF and CH2Cl2, but only low-molar-mass polyethers were obtained (MnGPC = 1.6–3.0 kg·mol-1). Similar discrepancies between theoretical and experimental molar masses were observed in other catalytic/initiation systems, showing that efficient epoxide activation does not ensure sustained chain growth. Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) and mechanistic analysis support a competing propagation–transfer/cleavage–eugenolate reinitiation network. Besides productive alkoxide-mediated propagation, growing chain ends undergo transfer and cleavage processes that generate shorter chains together with eugenol/eugenolate species capable of reinitiating polymerization. These side reactions account for the persistent molar-mass limitation and provide guidance for the controlled polymerization of bio-based aromatic glycidyl ethers.

Black spot disease causes significant yield and quality loss in Brassica vegetables. Therefore, developing effective management strategies is imperative to ensure both productivity and marketable quality in Chinese cabbage cultivation. We surveyed and isolated 396 Alternaria strains from infected Brassica vegetables, and confirmed that A. brassicae is the most pathogenic one. By screening and identifying the resistant and susceptible lines of Chinese cabbage, we performed a the widely targeted metabolomic analysis to explore the metabolic profiling of Chinese cabbage leaf samples in response to A. brassicae infection via a time course experiment. Coumarin-3-carboxylic acid and dicumarol were identified and functionally validated as effective antifungal compounds in vitro and in vivo assays, by activating the phenylpropanoid pathway. Furthermore, cinnamic acid of phenylpropanoid pathway was functionally validated to strongly inhibit the growth of the A. brassicae by in vitro assay. The extensive dataset of candidate metabolic compounds provided valuable insights for exploring A. brassicae -Brassica vegetables interactions, facilitating the for developing green pesticides and disease-resistant cultivars.

Leafy head formation is a crucial developmental process in Brassica crops. Here, an integrative approach combining machine learning and gene regulatory network analysis was employed to identify novel genes involved in leafy head formation in Chinese cabbage (Brassica rapa) and cabbage (Brassica oleracea). Random Forest models, trained with 47 known leafy head-related genes, demonstrated robust performance with mean AUC values of 0.87 and 0.85 for B. rapa and B. oleracea, respectively. By filtering the model predictions, we identified 11 genes predicted with high confidence which were shared between both species. To further reveal the regulatory mechanisms, we constructed gene regulatory networks for genes in both species. By integrating ML predictions with these networks, we identified key regulatory clusters specifically related to leafy head formation. Network centrality analysis revealed many core genes in key clusters, including important transcription factors such as ANT, GRF2, GRF3, and TCX3, suggesting crucial roles in leafy head formation across the two species. The parallel detection of the same genes and similar network structures in the two species supports the validity of our findings. Our integrative approach provides novel insights into the genetic regulation of leafy head formation and sets the stage for future functional studies of Brassica species.

Brassica rapa has been a key oilseed and vegetable crop since the Neolithic era, yet its obscure evolutionary history has hindered understanding of its domestication and the speciation of its allotetraploid descendants, Brassica napus and Brassica juncea. Here we defined pan-blocks comprising all syntenic regions from 21 B. rapa genome assemblies and analyzed 3,330 accessions spanning the three species to reconstruct the evolutionary trajectory of the Brassica A genome. Our findings reveal its origin in Central and West Asia and subsequent spread across Eurasia by means of three routes. An ancient inversion was identified that supports the divergent origins of the A subgenomes in B. napus and B. juncea. Furthermore, the S-locus exhibited exceptional haplotype diversity, with each variant characterized by a unique transposable element barcode pattern essential for the self-incompatibility system. These findings emphasize the significant value of pan-block-integrated 3,330 A genome variants for the Brassica research community.

Abstract Cis-regulatory elements (CREs) are fundamental to precise gene regulation. Although distal CREs located in intergenic regions have been extensively studied, the functional landscape of those residing in 3′ untranslated regions (UTRs) remains largely unexplored. In this study, we generate comprehensive epigenomic profiles of Brassica rapa leaves at two developmental stages (rosette and reproductive), integrating ATAC-seq, CUT&Tag for five histone modifications (H3K4me3, H3K27ac, H3K27me3, H3K36me3, H3K9me2), and BL-Hi-C data. We identify 6362 CREs within 3’ UTRs, defined as accessible chromatin regions within 0.5 Kb of transcription termination sites. These CREs within 3’ UTRs are functionally classified by histone marks: H3K27ac-marked, H3K27me3-marked, and bivalent CREs. Using BL-Hi-C at resolutions up to 100 bp, we uncover that these CREs within 3’ UTRs contribute to the formation of single-gene domains. Comparative analysis between developmental stages reveals that distal CREs orchestrate stage-specific gene expression by modulating chromatin interaction, a mechanism exemplified by the flowering regulator BrFLC2. The transcriptional activation activity of the distal CRE of BrFLC2 was verified by dual-luciferase reporter assays. Together, our findings establish 3’ UTR-embedded distal CREs as critical regulatory elements that shape gene expression through single-gene domain architecture in B. rapa.

Chinese cabbage forms a leafy head as its main edible organ, a process involving extensive morphological and transcriptional changes. Here, we employed ATAC-seq to profile a time-series chromatin accessibility landscape across key developmental stages of leaf heading. This analysis revealed highly dynamic, stage-specific chromatin accessible regions accompanied by distinct transcription factors activities. Genes associated with adaxial-abaxial polarity and hormone signaling displayed dynamic accessibility patterns, suggesting their critical roles in head formation. BrKAN2 emerged as a key candidate regulator. Mutant analysis in Chinese cabbage and overexpression in Arabidopsis significantly altered leaf morphology and dorsoventral polarity. Population genetic analysis further indicated strong selection on BrKAN2 in heading Brassica rapa. Integration of DAP-seq and ATAC-seq identified BrKAN2.1 target genes enriched in organ development and hormone-related pathways, which were validated by VIGS and EMSA. Auxin responsiveness assays underscored the importance of auxin signaling in heading. Collectively, these findings uncover a dynamic chromatin landscape underlying leaf heading and establish BrKAN2 as a central regulatory factor, offering new insights for genetic improvement of Chinese cabbage.

Leaf vasculature not only acts as a channel for nutrients and signaling information but also influences leaf morphology. It consists of several distinct cell types with specialized functions. Cell type-specific characterizations based on single-cell RNA sequencing technology could aid in understanding the identities of vascular tissues and their roles in leaf morphogenesis in Brassica rapa. Here, we generated a single-cell transcriptome landscape of the Chinese cabbage leaf vasculature. A total of 12 cell clusters covering seven known cell types were identified. Different vascular cell types were characterized by distinct identities. The xylem parenchyma and companion cells exhibited an active expression pattern of amino acid metabolism genes. Tracheary elements and sieve elements were enriched in many genes related to cell wall biosynthesis, and the phloem parenchyma was enriched in many sugar transporter-encoding genes. Pseudo-time analyses revealed the developmental trajectories of the xylem and phloem and the potential roles of auxin and ethylene in xylem development. Furthermore, we identified key candidate regulators along the differentiation trajectory of the sieve elements and companion cells. Most of the homoeologous genes in the syntenic triads from the three subgenomes showed asymmetric gene expression patterns in different vascular cell types. Collectively, our study revealed that Chinese cabbage leaf vasculature cells had highly heterogeneous transcriptomes, providing new insights into the complex processes of leaf vasculature development in B. rapa leafy vegetables and other Brassica crops.

Brassica species evolved through recurrent polyploidization and chromosomal rearrangements, forming diploid progenitors that hybridize into allopolyploids. These plants exhibit remarkable morphological diversity, with specialized edible organs including leaf-, stem-, root-, and oil-type cultivars, yet cross-species multi-organ transcriptomic studies elucidating their gene expression similarities and divergences remain lacking. To address this gap, we analyzed publicly available transcriptomes (downloaded from NCBI SRA) from eight organs (embryo, seed coat, silique, root, stem, leaf, flower and seedling) across six U’s Triangle species (Brassica rapa, B. nigra, B. oleracea, B. juncea, B. napus, B. carinata), revealing that (1) reproductive organs show higher gene expression conservation (GEC), particularly embryos (p < 0.05); (2) lineage-specific subgenome dominance patterns (BnaC/BjuB/BcaC) persist across organs; and (3) ancestral subgenomes functionally specialize, with MF2-subgenome transcription factors (YABBY/GRF) regulating embryogenesis and LF/MF1-subgenome MYBs controlling seed coat development. Comparative analyses demonstrate floral GEC exceeds that of the Arabidopsis thaliana homologs, while also exhibiting seed-specific divergence patterns. This study establishes a comprehensive Brassica multispecies expression atlas, elucidating organ-specific evolutionary conservation principles and providing molecular insights into subgenome functional partitioning, which offers valuable perspectives for understanding Brassica evolutionary mechanisms and crop improvement strategies.

Polyploidy, or whole-genome duplication, is an important evolutionary process that has shaped the genomes and traits of many plants, including numerous important crops. The Brassica genus, which includes diverse vegetables and oilseeds, is a key model system for studying how polyploidy affects plant diversification and domestication. This review summarizes the current understanding of how multiple rounds of ancient and more recent polyploidization events laid the foundation for the wide diversity seen in Brassica. We discuss the key outcomes through which polyploidy facilitates the accumulation of genetic variation, including genomic buffering that enables mutation retention. Furthermore, we explore the significant roles of interspecies and interploidy introgression in introducing external genetic novelty. We highlight homoeologous exchange (HE) as a critical mechanism unique to allopolyploids, driving substantial genomic rearrangements including presence-absence variations and gene dosage alterations that directly contribute to significant phenotypic innovation and adaptation in Brassica. Together, these polyploidy-associated processes have led to the extensive range of genomic variations that shaped great morphological diversification in the domestication of Brassica. By integrating insights from genomics, genetics, and evolutionary biology, this review shows how polyploidy has been central to Brassica's success and agricultural value. We also suggest future research areas to better understand polyploid evolution and improve crop breeding.

Cabbage (Brassica oleracea var. capitata), a world-widely cultivated leafy vegetable, is characterized by its dense-leaved head formation. Premature bolting, where flowering occurs before head formation significantly reduces cabbage quality and yield. In this study, we employed bulked segregant analysis (BSA) and linkage analysis to identify the quantitative trait loci (QTL) associated with flowering time variation in cabbage. Our analysis revealed a major QTL-designed as qFT2.1, located on chromosome C02. The QTL spans a 12.8 kb region that encompasses five genes. Further analysis identified BoFLC2 (an ortholog of AtFLC) as the candidate gene for qFT2.1. Comparative sequence analysis revealed a 215 bp-insertion in the first intron of BoFLC2 in the early-flowering parental line, along with a 3 bp-insertion and three missense mutations in the second exon. RNA-sequencing revealed that BoFLC2 expression in non-flowering individuals (extremely late flowering) was significantly higher than that in the early-flowering individuals. Analysis of the BoFLC2 sequence across 37 published B. oleracea genomes revealed six haplotypes that are likely associated with flowering time domestication in B. oleracea. Our results suggest that BoFLC2 as a major regulator in flowering time control, has been under selection during the domestication of B. oleracea.

Brassica juncea var. tumida, commonly known as Zha Cai, is a pickled stem mustard widely cultivated in southern China. Its most distinctive trait is the swollen stem, which serves as the primary economic organ for harvest. However, the origin and domestication history of tumida remain unclear, hindering genetic improvement and molecular breeding efforts. Here, we assembled a chromosome-level genome of the landrace 'YAXY' from Chongqing—the center of tumida diversity—totaling 909.1 Mb with a contig N50 of 4.17 Mb. We also collected and resequenced 203 tumida accessions across southern China. By integrating the 'YAXY' reference genome with population data, we generated the first comprehensive tumida variation dataset, comprising 1.38 million single-nucleotide polymorphisms (SNPs) and 0.27 million insertions and deletions (InDels). Joint analysis of the newly sequenced tumida population and 504 public B. juncea datasets revealed that tumida and leafy types from southern China share a common origin from local oilseed mustard. Tumida domestication was accompanied by a strong genetic bottleneck. Additionally, we conducted genome-wide association studies (GWAS) for 21 agronomic traits and identified candidate genes linked to key domestication traits in tumida. For the swollen stem trait, selective sweep and GWAS analyses jointly identified candidate genes likely involved in lignification. Transcriptome data showed consistent differential expression of BjuA05g15010, the Arabidopsis SAGL1 ortholog, across all swelling stages, suggesting a key role in stem morphogenesis. Collectively, our findings shed light on tumida evolution and provide valuable genomic resources and candidate genes to support genetic research and breeding in B. juncea.

Black spot, a fungal disease caused by Alternaria brassicae infection, inflicts severe damage on Chinese cabbage. Through comparative transcriptomic analysis, this study investigated the molecular mechanisms underlying Chinese cabbage's defense responses to A. brassicae infection. Notably, we found that the expression of BrERF109 was induced by A. brassicae infection. Silencing of BrERF109 by an optimized virus-induced gene silencing (VIGS) assay in Chinese cabbage diminished disease resistance, while BrERF109-overexpression in Arabidopsis enhanced it. Additionally, BrERF109 silencing in Chinese cabbage suppressed indolic glucosinolates gene expression, substantially reducing indolic glucosinolates levels, whereas BrERF109-overexpression in Arabidopsis promoted their accumulation. BrERF109 directly interacts with the BrIGMT4 promoter, thereby facilitating indolic glucosinolates accumulation and enhancing defense against A. brassicae. This study elucidates the BrERF109-BrIGMT4 regulatory module in Chinese cabbage's defense against A. brassicae infection, while providing valuable data for further investigation of plant-A. brassicae interactions.

Many temperate plants require vernalization, a prolonged low-temperature period, to accelerate flowering. Vernalization is a quantitative process whereby extended cold exposure establishes a stable transcriptional repression, with the degree of silencing correlating with the length of cold treatment. While much is known about the genes regulating this process, the expression dynamics at the single-cell level remain elusive. Using single-cell RNA sequencing, we analyze the vernalization response in Brassica rapa. Our data show that mesophyll cells exhibit the most significant changes in gene expression at low temperatures, whereas vasculature exhibits higher expression levels of flowering-related genes. Mesophyll trajectory analyses suggest that B. rapa plants undergo a biphasic response to chill stress during vernalization. Tissue-wide BrFLC expression changes result from variations in the proportion of expressing cells, supporting the quantitative nature of vernalization through digital cell responses. This study provides valuable resources and insights into the spatiotemporal regulation of flowering during vernalization.

Genetic degeneration is a striking feature of Y chromosomes, often involving losses of many genes carried on the X chromosome. However, the time course of gene losses remains unclear. Sex chromosomes of plants evolved more recently than animals' highly degenerated ones, making them ideal for studying degeneration timing. To investigate Spinacia sex chromosome evolution and the time course of degeneration, we compared genome sequences of cultivated Spinacia oleracea, with a small Y-linked region on Chr4, with its two wild relatives. In spinach and its closest relative Spinacia turkestanica, the Y duplication region (YDR) introduced a male-determining factor into Chr4's low-recombining pericentromeric region. In other words, a turnover event occurred in these species' recent common ancestor. The homologous Chr4 of the more distantly related S. tetrandra has a c. 133 Mb completely sex-linked and partially degenerated region, possibly reflecting the ancestral state. Sequence divergence analysis suggests that two 'evolutionary strata' evolved shortly before the two Spinacia lineages split. Consistent with the turnover hypothesis, the YDR of the other two Spinacia species is not within the S. tetrandra older stratum. We discuss the unexpected findings in S. tetrandra that genetic degeneration, genomic rearrangements, and repetitive sequence density are all greatest in the younger stratum.

Cultivated spinach (Spinacia oleracea) is a dioecious species. We report high-quality genome sequences for its two closest wild relatives, Spinacia turkestanica and Spinacia tetrandra, which are also dioecious, and are used to study the genetics of spinach domestication. Using a combination of genomic approaches, we assembled genomes of both these species and analyzed them in comparison with the previously assembled S. oleracea genome. These species diverged c. 6.3 million years ago (Ma), while cultivated spinach split from S. turkestanica 0.8 Ma. In all three species, all six chromosomes include very large gene-poor, repeat-rich regions, which, in S. oleracea, are pericentromeric regions with very low recombination rates in both male and female genetic maps. We describe population genomic evidence that the similar regions in the wild species also recombine rarely. We characterized 282 structural variants (SVs) that have been selected during domestication. These regions include genes associated with leaf margin type and flowering time. We also describe evidence that the downy mildew resistance loci of cultivated spinach are derived from introgression from both wild spinach species. Collectively, this study reveals the genome architecture of spinach assemblies and highlights the importance of SVs during the domestication of cultivated spinach.

High-throughput Chromatin Conformation Capture (Hi-C) technologies can be used to investigate the three-dimensional genomic structure of plants. However, the practical utility of these technologies is impeded by significant background noise, hindering their capability in detecting fine 3D genomic structures. In this study, we optimized the Bridge Linker Hi-C technology (BL-Hi-C) to comprehensively investigate the 3D chromatin landscape of Brassica rapa and Brassica oleracea. The Bouquet configuration of both B. rapa and B. oleracea was elucidated through the construction of a 3D genome simulation. The optimized BL-Hi-C exhibited lower background noise compared to conventional Hi-C methods. Taking this advantage, we used BL-Hi-C to identify FLC gene loops in Arabidopsis, B. rapa, and B. oleracea. We observed that gene loops of FLC2 exhibited conservation across Arabidopsis, B. rapa, and B. oleracea. While gene loops of syntenic FLCs exhibited conservation across B. rapa and B. oleracea, variations in gene loops were evident among multiple paralogs FLCs within the same species. Collectively, our findings highlight the high sensitivity of optimized BL-Hi-C as a powerful tool for investigating the fine 3D genomic organization.

Polyploidization plays a crucial role in plant evolution and is becoming increasingly important in breeding. Structural variations and epigenomic repatterning have been observed in synthetic polyploidizations. However, the mechanisms underlying the occurrence and their effects on gene expression and phenotype remain unknown. Here, we investigated genome-wide large deletion/duplication regions (DelDups) and genomic methylation dynamics in leaf organs of progeny from the first eight generations of synthetic tetraploids derived from Chinese cabbage (Brassica rapa L. ssp. pekinensis) and cabbage (Brassica oleracea L. var. capitata). One- or two-copy DelDups, with a mean size of 5.70 Mb (400 kb to 65.85 Mb), occurred from the first generation of selfing and thereafter. The duplication of a fragment in one subgenome consistently coincided with the deletion of its syntenic fragment in the other subgenome, and vice versa, indicating that these DelDups were generated by homoeologous exchanges (HEs). Interestingly, the larger the genomic syntenic region, the higher the frequency of DelDups, further suggesting that the pairing of large homoeologous fragments is crucial for HEs. Moreover, we found that the active transcription of continuously distributed genes in local regions is positively associated with the occurrence of HE breakpoints. In addition, the expression of genes within DelDups exhibited a dosage effect, and plants with extra parental genomic fragments generally displayed phenotypes biased toward the corresponding parent. Genome-wide methylation fluctuated remarkably, which did not clearly affect gene expression on a large scale. Our findings provide insights into the early evolution of polyploid genomes, offering valuable knowledge for polyploidization-based breeding. A comprehensive multi-omics landscape across synthetic Brassica napus tetraploids reveals active transcription of homoeologous exchange-related genes.

Summary Structural variations (SVs) are major genetic variants that can be involved in the origin, adaptation and domestication of species. However, the identification and characterization of SVs in Spinacia species are rare due to the lack of a pan‐genome. Here, we report eight chromosome‐scale assemblies of cultivated spinach and its two wild species. After integration with five existing assemblies, we constructed a comprehensive Spinacia pan‐genome and identified 193 661 pan‐SVs, which were genotyped in 452 Spinacia accessions. Our pan‐SVs enabled genome‐wide association study identified signals associated with sex and clarified the evolutionary direction of spinach. Most sex‐linked SVs (86%) were biased to occur on the Y chromosome during the evolution of the sex‐linked region, resulting in reduced Y‐linked gene expression. The frequency of pan‐SVs among Spinacia accessions further illustrated the contribution of these SVs to domestication, such as bolting time and seed dormancy. Furthermore, compared with SNPs, pan‐SVs act as efficient variants in genomic selection (GS) because of their ability to capture missing heritability information and higher prediction accuracy. Overall, this study provides a valuable resource for spinach genomics and highlights the potential utility of pan‐SV in crop improvement and breeding programmes.