Cabbage (Brassica oleracea var. capitata), a member of the Brassicaceae family, is an important vegetable crop grown worldwide. Self-incompatibility (SI) in cabbage is a key trait that prevents self-fertilization and inbreeding, thereby maintaining genetic diversity within populations. Although several genes related to SI have been reported, its genetic control remains unclear. In this study, we developed an F2 population from the highly self-compatible (SC) cabbage line 87-534 and the highly self-incompatible (SI) line 01-20, both of which exhibit the S5 haplotype. The segregation analysis of the F2 population revealed the possible control of SI by a major gene with additional modifying genetic factors. Bulk segregant analysis sequencing (BSA-Seq) and RNA sequencing (RNA-Seq) were performed on SI and SC samples selected from the F2 population. BSA-Seq revealed a candidate region on chromosome 7 (C07: 7.45 Mb to 8.93 Mb), including 32 differentially expressed genes (DEGs). RNA-Seq identified a total of 2400 DEGs between the two pools, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses suggested that plant hormone biosynthesis and signaling, plant immune response were significantly enriched and may be involved in SI. The combined analysis of BSA-Seq and RNA-Seq identified six candidate genes associated with SI, and their expression was confirmed using quantitative real-time PCR (qRT-PCR). Among them, Bol023956 encodes fructokinase, Bol023986 is involved in plant defense response, Bol024018 is related to pollen development, Bol024012 encodes a transport protein for phytohormones, Bol023943 encodes chorismate mutase 3, and Bol012515 is an important regulatory gene for chloroplast synthesis. These six genes, potentially linked to SI, should be targets for further validation. These findings provide insights into the molecular mechanisms of SI in cabbage and the selection of superior cabbage varieties.

Black rot (BR), caused by Xanthomonas campestris pv. campestris (Xcc), severely hampers Brassica production worldwide. Type III effectors (T3Es), which include transcription activator-like effectors (TALEs) and Xanthomonas outer proteins (Xops), are virulence factors for Xcc in host crops, such as rice, pepper, and cassava. However, their effects in Brassica remain unclear. Here, we analyzed 70 Xcc strains collected from locations worldwide and evaluated their pathogenicity in 5 Brassica accessions, identifying 6 highly aggressive strains. Southern blotting revealed TALEs in only ∼5% of Chinese Xcc strains, and Δtal mutants retained full virulence, indicating that these TALEs are dispensable for BR occurrence in Brassica crops. Whole-genome sequencing of 5 strains identified 33 Xops. Moreover, targeted mutagenesis of xopK, xopQ, xopX-1, xopAM, and xopN indicated host-dependent functions in cabbage. Deletion of xopQ, xopX-1, xopAM, or xopN increased disease indices by more than 10% in cabbage line G1180 (ΔxopN >20%), while bacterial counts also increased. In cabbage line G87-534, deletion of ΔxopK or ΔxopN reduced both disease indices and bacterial counts. Transient expression of XopQ, XopX-1, and XopN triggered a hypersensitive response in cabbage, and reverse transcription quantitative PCR (RT-qPCR) analyses revealed that 5 effectors suppressed the expression of pathogenesis-related protein 1 (PR1) or WRKY transcription factor (WRKY) genes associated with pattern-triggered immunity. Our study revealed that Xops, rather than TALEs, dominate virulence in Xcc‒Brassica interactions, in striking contrast to the pattern in other Xanthomonas pathosystems. This comprehensive T3E profile of Chinese Xcc provides a framework for developing Brassica crops with targeted resistance to BR.

Nuclear Factor Y (NF-Y) transcription factors play pivotal roles in plant adaptation to abiotic stress, yet their genomic landscape and functional mechanisms in cabbage (Brassica oleracea var. capitata L.) remain underexplored. Here, we performed a genome-wide identification of the NF-Ys in cabbage, identifying 53 BoNF-Ys classified into three subfamilies: 20 BoNF-YAs, 22 BoNF-YBs, and 11 BoNF-YCs. Phylogenetic clustering revealed evolutionary conservation with their Arabidopsis orthologs. Domain analysis revealed that all BoNF-YA members contain the CBF_NF-YA domain, while all BoNF-YB and BoNF-YC members possess the CBFD_NFYB_HMF conserved domain. The BoNF-Y genes were named according to their chromosomal locations. Bioinformatic analysis showed that BoNF-Y proteins range in size from 131 to 642 amino acids, with molecular weights of 14.82-73.18 kDa, theoretical pI values of 4.57-9.96, instability indices between 33.02 and 73.48, aliphatic indices of 45.3-86.26, and grand average of hydropathicity (GRAVY) values ranging from -1.139 to -0.367. Promoter cis-element profiling uncovered stress- and hormone-responsive motifs, including abscisic acid-responsive elements (ABREs), TC-rich repeats, and ethylene-responsive elements (EREs). RNA sequencing (RNA-seq) and quantitative reverse transcription polymerase chain reaction (qRT-PCR) conducted under salt stress (256 mM) identified three salt-responsive candidate genes (BoNF-YA14, BoNF-YB9, and BoNF-YC8). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses highlighted significantly expressed genes' roles in MAPK signaling, proline metabolism, and phytohormone transduction pathways. This study conducted a comprehensive survey of the BoNF-Y gene family in cabbage. It could serve as a theoretical foundation for further functional identification and utilization of BoNF-Y family members and their role in the interaction between cabbage and salt stress.

Cabbage (Brassica oleracea var. capitata) is a leafy Brassica vegetable crop whose leaf color and morphology critically influence yield, photosynthetic performance, and market quality. In this study, we identified a natural cabbage mutant, namely, 1180mu, that exhibits virescent-malformed leaves, reduced thylakoid content, decreased fertility, and a relatively low seed-setting rate. Physiological analyses further revealed reduced chlorophyll accumulation, impaired photosynthetic capacity, and stunted growth in 1180mu compared with the wild type (WT). Genetic analysis and map-based cloning demonstrated that BoVML1, a dominant gene homologous to Arabidopsis RER3, is causal; a 44-bp deletion in BoVML1 disrupts its function. CRISPR/Cas9 knockout of BoVML1 in the WT produced a phenotype similar to that of 1180mu, whereas BoVML1 complementation restored normal leaf color, chloroplast ultrastructure, and plant morphology in 1180mu plants. Subcellular localization assays revealed that BoVML1 is targeted to chloroplasts. Transcriptomic profiling uncovered extensive gene downregulation in 1180mu and enrichment of differentially expressed genes (DEGs) in secondary metabolic, phenylpropanoid, and hormone signaling pathways, indicating broad transcriptional reprogramming associated with the virescent phenotype. Moreover, yeast two-hybrid (Y2H) and coimmunoprecipitation assays (Co-IP) demonstrated that BoVML1 interacts with BoPMD1 and BoNAC62, both of which are associated with stress responses. Together, these findings identify BoVML1 as a key positive regulator of chloroplast development, leaf color and leaf morphology in cabbage and provide a mechanistic framework and a practical genetic target for improving leaf color and plant morphogenesis in Brassica breeding programs.

Cabbage is a widely cultivated leafy vegetable valued for both biomass and nutritional quality. Brassinosteroids (BRs) are essential plant hormones that regulate growth and metabolism, yet the molecular mechanisms linking BR signaling with cabbage head development and nutrient accumulation remain unclear. Here, we generated transgenic cabbage lines overexpressing BoBZR1d, a constitutively active form of the transcription factor BoBZR1 lacking the 14-3-3 binding site, to investigate its role in coordinating growth and nutritional metabolism. BoBZR1d-overexpressing lines exhibited significantly increased plant height, leaf expansion, head fresh weight, and enhanced photosynthetic performance. Metabolomic profiling revealed elevated levels of vitamin C, riboflavin (vitamin B2), glucose, fructose, sucrose, and raffinose, whereas flavonols kaempferol and quercetin were reduced. Transcriptomic analysis revealed differential expression of genes involved in chlorophyll biosynthesis, auxin metabolism, vitamin biosynthesis, and starch and sucrose metabolism, suggesting potential crosstalk between BR signaling and auxin-related processes; however, direct targeting of auxin-related genes by BoBZR1 remains to be determined. Yeast one-hybrid assays indicated potential interactions between BoBZR1 and the promoters of several candidate genes involved in vitamin C and sugar metabolism, including BoDHAR, BoGGP, BoGLA, BoINV, and BoHK. Collectively, these findings indicate that constitutive activation of BoBZR1 is associated with enhanced photosynthetic capacity and nutrient accumulation in cabbage. Our findings offer valuable insights for breeding high-yield, nutrient-dense leafy vegetable cultivars and highlight BoBZR1 as a promising genetic target for horticultural improvement.

DNA methylation is an essential epigenetic modification. However, the dynamics of DNA methylation during cabbage bolting remain poorly understood. To investigate changes in DNA methylation during cabbage bolting, we performed whole-genome bisulfite sequencing (WGBS) on both vegetative leaves of non-bolting plants and reproductive leaves of bolting plants in two cabbage accessions. Across the whole genome, the DNA methylation levels on each chromosome showed a negative correlation with gene density, and the methylation levels in reproductive leaves after bolting were higher than those in vegetative leaves before bolting. We identified a total of 10,159 differentially methylated regions (DMRs). GO enrichment analysis revealed that differentially methylated genes were significantly enriched in pathways related to reproductive process and floral organ development. At the subgenome level, MF2 exhibited a higher DNA methylation advantage, and the dominance of all three subgenomes was greater in reproductive leaves than in vegetative leaves. Integrating the transcriptomic data, we found that genes with DNA methylation modifications showed markedly lower expression levels than those without methylation. Two meristem-development-related genes, STM and WUS, displayed reduced DNA methylation and significantly elevated expression during bolting, suggesting that their expression was regulated by methylation. In summary, our study revealed a comprehensive DNA methylation profile of cabbage bolting and highlighted the critical role of DNA methylation in this process. These results provide a theoretical foundation for further molecular studies on bolting in Brassicaceae plants.

The globular buds and stems are the main edible organs of broccoli. Bolting is an important agronomic trait, and the timing of its occurrence is particularly critical when breeding and domesticating broccoli. The molecular mechanism that regulates broccoli bolting time is not well-understood. In this study, the apical flower bud and leaf tissues of two broccoli varieties with different bolting intensities were selected for metabolome and transcriptome analyses. In the apical flower buds of early-bolting B2554 and late-bolting B2557, 1094 differentially expressed genes and 206 differentially accumulated metabolites were identified. In the leaves, 487 differentially expressed genes and 40 differentially accumulated metabolites were identified. In the floral pathway, the expression of FLC (FLOWERING LOCUS C) was significantly upregulated, and that of FT (FLOWERING LOCUS T) was significantly downregulated in the late-bolting plants, indicating their possible role in suppressing bolting. In addition, significant differences were identified in the sucrose synthesis and transport, hormone synthesis, and signal transduction processes in early-bolting B2554 and late-bolting B2557. Sucrose accumulation in the leaves and apical flower buds of the early-bolting plants was about 1.3 times higher than in the late-bolting plants. Indole-3-acetic acid (IAA) and abscisic acid (ABA) accumulation in the apical flower buds of the late-bolting plants was more than twice that in the early-bolting plants. Jasmonic acid (JA) accumulation in the apical flower buds of the late-bolting plants was more than ten times higher than in the early-bolting plants. Phenolic acids may affect the bolting time of broccoli. This study offers new insights into the regulation mechanism of broccoli bolting and provides some potential molecular targets to include in breeding methods that regulate bolting time.

Cabbage (Brassica oleracea var. capitata), a member of the genus Brassica, is a significant economic crop worldwide. Black rot (BR) caused by Xanthomonas campestris pv. campestris (Xcc) severely threatens cabbage yield. Therefore, there is an urgent need to breed resistant varieties. To screen resistant germplasms, 171 cabbage inbred lines were inoculated, of which only three were highly resistant materials, namely, 'M202', 'MY', and 'YC280'. The biomass of Xcc in highly resistant 'MY' and highly susceptible 'LY' cabbage lines at different periods after inoculation revealed that 24-72 h after inoculation was the critical period for bacterial proliferation. Inheritance analysis of 'MY', 'LY' and their constructed populations (P1, P2, F1, B1, B2, and F2) fit the MX2-ADI-ADI model, suggesting that the genetic control of BR resistance in MY was controlled by two pairs of additivedominant-superior major genes plus additive-dominant-superior polygenes. The major gene heritabilities of B1, B2, and F2 were 33.52 %, 46.66 %, and 52.78 %, respectively. These results increased the number of resources about the BR resistance of cabbage germplasms, elucidated the critical proliferation period and resistance inheritance of this pathogen, and provided a theoretical basis for the breeding of BR-resistant plants, which could expedite the cabbage breeding process.

Fusarium wilt (FW) and black rot (BR) are the most devastating diseases affecting cabbage (Brassica oleracea L. var. capitata), severely impacting the global cabbage yield, and the breeding of dual-resistant cabbage is an urgent necessity. However, Ogura cytoplasmic male sterility (CMS) poses significant challenges for reusing most commercial varieties with multiple disease resistance in breeding programs. XG5 is an Ogura CMS commercial hybrid that is resistant to both FW and BR. We designed two steps to achieve the reapplication of XG5 in breeding. During the first stage, XG5 fertility was restored using two methods: crossing XG5 with the restorer line CB40 and transferring the restorer gene (Rfo) into XG5 via transgenic technology. Based on marker-assisted selection, field performance, and resistance identification, three individuals, F1-5, F1-9, and F1-21, from the restorer line cross and two individuals, T0-4 and T0-5, from transformation were selected and further crossed with the elite inbred line S01, which served as the recurrent backcross parent to generate the BC2 generation, successfully integrating resistance into the inbred line. During the second stage, to rapidly obtain the homozygous line, two techniques were used to generate double haploid (DH) lines: isolated microspore culture and haploid induction using the inducer line dmp9. Through in vitro/in vivo induction and colchicine treatment, a total of twenty DH lines were generated. Among these DH lines, D9, D10, and D16 presented normal fertility, dual resistance, and excellent agronomic characteristics. Using a two-by-two method, we reused FW- and BR-resistant germplasms, ultimately generating cabbage lines with dual major disease resistance and normal fertility. These results provide a new approach to the reapplication of Ogura CMS commercial crop hybrids via modern breeding techniques.

Callose synthase (CALS) genes are known to play critical roles in microspore development and in plant responses to diverse biotic and abiotic stresses. While the role of CALS genes has been extensively characterized in several plant species, their homologs in Brassica oleracea (BoCALS) remain understudied. In this study, 15 BoCALS genes were identified in B. oleracea genome, distributed across eight chromosomes. All BoCALS proteins contain Glucan-synthase and Fks1 domains. Phylogenetic analysis grouped BoCALS and their homologs from Arabidopsis thaliana and Brassica rapa into three distinct Clusters (Ⅰ–Ⅲ), revealing conserved evolutionary relationships within the Brassicaceae family. Collinearity analysis showed that AtCALS genes of Arabidopsis have multiple orthologs in B. oleracea. Analysis of RNA-Seq data from public databases suggested that most of the BoCALS genes exhibit tissue-specific expression patterns, indicating their potential roles in organ differentiation and development. QRT-PCR analysis elucidated a different expression level of BoCALS genes in response to Hyaloperonospora parasitica infection. Notably, BoCALS6 expression was significantly higher in resistant varieties compared to susceptible varieties and further up-regulated following H. parasitica infection, indicating its potential role in downy mildew resistance. This study presents the first comprehensive characterization of BoCALS gene family in B. oleracea and provides a foundation for further functional investigations into their roles in downy mildew resistance.

The HI-Edit system combines haploid induction and CRISPR/Cas-based genome editing to provide a promising way to design crops with desired traits in a rapid, precise and transgene-free manner. HI-Edit was applied to produce cabbages with desired anthocyanin contents.

4-coumarate-CoA ligase (4CL) plays a crucial role in the phenylpropanoid metabolic pathway and is a key enzyme involved in plant growth and stress responses. Black rot, caused by Xanthomonas campestris pv. campestris (Xcc) is a major bacterial disease affecting the production of global cruciferous crop-like cabbage (Brassica oleracea var. capitata). However, the role of 4CL genes in cabbage resistance to black rot remains unclear. In this study, transcriptome sequencing was conducted using resistant cabbage MY and susceptible cabbage LY at 0, 6, 24, and 48 h post-inoculation. KEGG analysis identified the enrichment of the phenylpropanoid biosynthesis pathway, and significant expression changes of 4CL genes were determined through the expression heat map. Further genome-wide analysis revealed 43 Bol4CL gene family members on the cabbage genome distributed across nine chromosomes. Gene structure and protein motif analysis revealed similarities in motifs within the same evolutionary branch, but variations in gene structure. A combination of Bol4CL gene expression profiles and differentially expressed genes (DEGs) from the transcriptome identified Bol4CL41 as a key gene for further study. Inoculation of overexpressed Bol4CL41 T2 generation stably expressed cabbage seedlings demonstrated significantly larger lesion areas compared to wild type cabbage, indicating that Bol4CL41 negatively regulates resistance to black rot in cabbage. The analysis of multi-time point transcriptomes in cabbage and the functional study of the Bol4CL gene family enhance our understanding of the mechanisms underlying plant disease resistance. This provides compelling evidence and experimental support for elucidating the mechanisms of black rot resistance in cabbage.

The ECERIFERUM (CER) gene family is essential for the biosynthesis of plant cuticular wax. In this study, 32 BoCER genes were identified in cabbage through genome-wide analysis. We found that the BoCER genes are highly conserved with their homologous counterparts in Arabidopsis thaliana. However, there was a significant divergence in the expression pattern among the BoCER paralogs, which suggests the occurrence of functional specialization during evolution. The expression analysis also showed that most of the BoCER genes are expressed in the aboveground part. Cis-regulatory element analysis suggested that BoCER genes could potentially be regulated through coordinated light and hormonal signaling. Furthermore, the abscisic acid and drought treatments markedly upregulated multiple BoCER genes, highlighting their involvement in abiotic stress responses. The functional analysis using CRISPR/Cas9-mediated knockout showed that BoCER4.1 governs the biosynthesis of alcohol. In situ hybridization localized the expression of BoCER4.1 to the tapetum, microspores, stem epidermis, and vascular bundles, while subcellular localization assays indicated its location in the endoplasmic reticulum, which aligns it with the biosynthetic machinery for wax. A phenotypic analysis revealed that the cuticles of the BoCER4.1 mutants were more permeable, and this was characterized by accelerated water loss and chlorophyll leaching. Correspondingly, the drought resistance of cababge with BoCER4.1 knockout was significantly reduced, accompanied by increased malondialdehyde content and decreased proline content under drought condition. This study provides new insights into the role of BoCERs in wax biosynthesis of cabbage, and also provides scientific basis for genetic improvement of drought resistance in cabbage.

Embryo defective 1923 (EMB1923) is important for plant embryo development, but its other functions and associated molecular mechanisms remain unclear. Here, we characterized a yellow-green leaf (ygl) mutant in Brassica oleracea, which has significantly reduced chlorophyll levels and abnormal chloroplasts compared to the wild type. Map-based cloning revealed that a 15-bp deletion in the coding region of BoEMB1923 (Brassica oleracea embryo defective 1923) causes the ygl phenotype. Functional analysis demonstrated that BoEMB1923 positively regulates chlorophyll biosynthesis and localizes in chloroplasts. A yeast two-hybrid assay showed that BoEMB1923 and yBoEMB1923 (mutant-type) interact with BoPORB1 and BoPORB2, and these interactions were further confirmed by BiFC and Co-IP assays. The BoEMB1923 mutation reduced BoPORB enzyme activity by 40% and chlorophyllide a (Chlide a) production by 33% in ygl, ultimately resulting in impaired chlorophyll biosynthesis. Taken together, our results reveal the function of BoEMB1923 in regulating chlorophyll biosynthesis by interacting with BoPORBs, which provides rare insight into the molecular mechanisms underlying leaf color formation in cabbage.

Using an optimized CRISPR/Cas9 system to knock out the BTB-POZ and MATH domain gene BoBPM6 and the DOWNY MILDEW RESISTANCE 6 gene in Brassica oleracea resulted in new lines with broad-spectrum disease resistance.

Hybrid breeding based on male sterility requires the removal of male parents, which is time- and labor-intensive; however, the use of female sterile male parent can solve this problem. In the offspring of distant hybridization between Brassica oleracea and Brassica napus, we obtained a mutant, 5GH12-279, which not only fails to generate gynoecium (thereby causing female sterility) but also has serrated leaves that could be used as a phenotypic marker in seedling screening. Genetic analysis revealed that this trait was controlled by a single dominant gene. Further analysis revealed that Bo2g005230, an orthologous gene of LATE MERISTEM IDENTITY1 (LMI1) in Arabidopsis, was predicted as the candidate gene and was renamed BoLMI1c. Sequence analysis revealed that homoeologous exchange (HE) occurred within the BoLMI1c gene body of 5GH12-279, which resulted in the generation of a novel fusion transcript. Two pairs of primers, N5230-1F/1R and N5230-2F/2R, were designed and successfully used for the identification of different genotypes of BoLMI1c. Transcriptome analysis revealed that BoLMI1c orchestrates the expression of several related biological processes and transcription factors. Furthermore, we found that self-pollination with mason bees produced no seeds in 5GH12-279, whereas the near-isogenic line 5GH12-170 produced seeds that were normal. Therefore, a new labor-saving hybrid seed production system with no need to remove the male parents, which is especially important for mechanized harvest in the future, has been proposed. Our study provides a valuable source of dominant female sterility and suggests the potential utilization of the female sterile line in hybrid breeding for mechanized harvest.