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.

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.

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.

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.

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.

In this article, we address the problem of estimating the state of a class of polynomial systems with sporadically available measurements and external disturbance. To begin with, an observer with sporadically available measurements is designed to estimate the states of the polynomial system. We introduce a timer variable to initiate the arrival of new measurements and examine the whole system in a hybrid systems framework. Then, we establish conditions for exponential stability of the polynomial system by employing the observer. Additionally, the designed observer ensures the exponential input-to-state stability for the hybrid error system in the presence of external disturbances. Finally, we provide an illustrative example to demonstrate the validity of the designed observer in estimating the states of the polynomial system and guaranteeing input-to-state stability under external disturbance.

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.

This paper is concerned with reset controller design and L-2-gain stability of piecewise-affine systems under the framework of hybrid systems. Firstly, stability conditions of piecewise-affine systems under dynamic state-feedback control are established through bilinear matrix inequality conditions. Secondly, a reset controller with reset rules under the hybrid systems framework is proposed in the sense of Lyapunov and sufficient conditions for exponential and L-2-gain stability of the closed-loop systems are provided. Different from the piecewise-affine systems with the dynamic state-feedback controller, the reset controller is designed such that the L-2-gain performance of piecewise-affine systems can be enhanced. Thirdly, an LMI approach is proposed to avoid the difficulty of solving the bilinear matrix inequalities. Furthermore, robustness to inflations of the flow and jump sets is established, and robustness to the norm-bounded uncertainties is proposed. Finally, numerical simulations including a robot arm system, an inverted pendulum system and the Chua's circuit system are provided to illustrate the results.

The continuously refined genome assembly of the Chinese cabbage accession Chiifu is widely recognized as the reference for Brassica rapa. However, the high self-incompatibility of Chiifu limits its broader utilization. In this study, we report the development of self-compatible Chiifu lines through a meticulous marker-assisted selection (MAS) strategy, involving the substitution of the Chiifu allele of MLPK (M-locus protein kinase) with that from the self-compatible Yellow Sarson (YS). A YS-based marker (SC-MLPK) was employed to screen 841 B. rapa accessions, confirming that all eight accessions with the mlpk/mlpk (mm) genotype exhibited self-compatibility. Additionally, we designed 131 High-Resolution Melting (HRM) markers evenly distributed across the B. rapa genome as genomic background selection (GBS) markers to facilitate the introgression of self-compatibility from YS into Chiifu along with SC-MLPK. Genome background screening revealed that the BC3S1 population had a proportion of the recurrent parent genome (PR) ranging from 93.9% to 98.5%. From this population, we identified self-compatible individuals exhibiting a high number of pollen tubes penetrating stigmas (NPT) (>25) and a maximum compatibility index (CI) value of 7.5. Furthermore, we selected two individuals demonstrating significant similarity to Chiifu in both genetic background and morphological appearance, alongside self-compatibility. These selected individuals were self-pollinated to generate two novel lines designated as SC-Chiifu Lines. The development of these self-compatible Chiifu lines, together with the SC-MLPK marker and the set of HRM markers, represents valuable tools for B. rapa genetics and breeding.

Abstract In Brassicaceae, RCO regulates lobed leaves, an important agronomic trait. RCO evolved through duplication, regulatory diversification, and loss. However, how RCO evolved and diversified in different lineages of Brassicaceae is unclear. The RCO locus in B. juncea had been reported to be responsible for lobed leaf formation, but its complexity has largely remained unknown. In this study, we identified 54 RCO homologues in 16 species of Brassicaceae through syntenic analysis. Based on the phylogenetic relationship, we classified these homologues into two types, LMI1-type and RCO-type. Further, we proposed two independent lineage-specific evolution routes for RCO after the divergence from Aethionema arabicum. In Brassiceae species, we revealed that the RCO loci regulating lobed leaf formation were located on the LF subgenomes. In the case of B. juncea (T84-66), we found that the complex RCO locus was formed through the duplication of a large DNA segment comprising Exostosin- LMI1- RCO (E-R-L), resulting in the tandem presence of four RCO homologues on chromosome A10. As further evidence, we mapped the complex RCO locus regulating lobed leaf formation to chromosome A10 using a B. juncea F2 population, which was consistent with the evolutionary analysis results. We further clarified that BjRCO.1 and BjRCO.2 were functional in regulating lobed leaf formation by transcriptome analysis. This study provides valuable information on the control of leaf morphology in the breeding of Brassiceae crops.

This paper is concerned with reset control and L2-gain stability of piecewise-affine systems with input saturation and external disturbance under the framework of hybrid systems. Firstly, we present a reset controller and establish exponential stability conditions of piecewise-affine systems with input saturation under the reset controller. Secondly, sufficient conditions for L2-gain stability of the closed-loop systems with input saturation and external disturbance are provided and a reset controller is designed to enhance the L2-gain performance of piecewise-affine systems with input saturation. Furthermore, robustness to inflations of the flow and jump sets is established. Finally, a numerical simulation is provided to illustrate the results.

Lineage-specific evolution of RCO was described in Brassicaceae. BjRCO.1 and BjRCO.2 within the complex locus regulated highly lobed-leaf formation in Brassica juncea. RCO regulates the formation of lobed leaves in Brassicaceae species. RCO originated from the duplication of LMI1-type sequences and evolved through gene duplication and loss within the Brassicaceae. However, the evolutionary process and diversification of RCO in different lineages of Brassicaceae remain unclear. Although the RCO locus in B. juncea has been associated with lobed-leaf formation, its complexity has remained largely unknown. This study involved the identification of 55 LMI1-like genes in 16 species of Brassicaceae through syntenic analysis. We classified these LMI1-like genes into two types, namely LMI1-type and RCO-type, based on their phylogenetic relationship. Additionally, we proposed two independent lineage-specific evolution routes for RCO following the divergence of Aethionema. Our findings revealed that the LMI1-like loci responsible for lobed-leaf formation in Brassica species are located on the LF subgenomes. For B. juncea (T84-66V2), we discovered that the complex locus underwent duplication through segments of nucleic acid sequence containing Exostosin-LMI1-RCO (E-R-L), resulting in the tandem presence of two RCO-type and two LMI1-type genes on chromosome A10. As additional evidence, we successfully mapped the complex locus responsible for highly lobed-leaf formation to chromosome A10 using a B. juncea F2 population, which corroborated the results of our evolutionary analysis. Furthermore, through transcriptome analysis, we clarified that BjRCO.1 and BjRCO.2 within the complex locus are functional genes involved in the regulation of highly lobed-leaf formation. The findings of this study offer valuable insights into the regulation of leaf morphology for the breeding of Brassica crops.

The species Brassica rapa includes enormous leafy vegetables with extreme leaf morphological diversity. Leaf traits such as size, shape, weight, and ratio of the leaf blade to the petiole contribute to yield, appearance, and desirability to consumers. These leaf-related traits are controlled by quantitative trait loci (QTLs). The construction of high-density bin maps using low-coverage sequencing is a powerful method for QTL fine-mapping and gene identification. In this study, we performed whole-genome re-sequencing of Wutacai ‘Zhongbaye’ and Chinese cabbage ‘HN53’ and 150 F2 individuals to construct a high-density bin map for QTL mapping of 11 leaf-related traits. The parental lines and F2 population were re-sequenced at 10x and 1x coverage, respectively. A map containing 565 bin markers was constructed based on parental single-nucleotide polymorphisms and a modified sliding window approach. The total map length was 944.6 cM and the average distance of the bins was 1.65 cM. In total, 60 significant QTLs controlling 11 leaf-related traits were detected. We further identified candidate genes responsible for these complex leaf-related traits. These findings suggest that this cost-effective bin-mapping approach is capable of rapid identification of QTLs and candidate genes, and will thus facilitate the dissection of the underlying molecular basis of leaf morphological variations and accelerate the improvement of B. rapa vegetable breeding.

Brassica crops encompass a diverse array, including vegetables, oil crops, ornamentals, and condiments [...]