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Cytokinins are a class of phytohormones that promote cell division and differentiation and are thought to affect plant immunity to multiple pathogens. However, a comprehensive analysis of cytokinin dehydrogenase/oxidase (CKX) family genes in cabbage has not been reported. In this study, a total of 36 CKX genes were identified using a genome-wide search method. Phylogenetic analysis classified these genes into three groups. They were distributed unevenly across nine chromosomes in B. oleracea, and 15 of them did not contain any introns. The results of colinearity analysis showed that 36 CKX gene in Arabidopsis was present in several copies in the Brassica oleracea genome. An analysis of cis-acting elements indicated that all genes possessed at least one stress or hormone responsive cis-acting element. A heatmap of CKX gene expression showed the patterns of expression of these genes in various tissues and organs. Three genes (Bol028363, Bol031036 and Bol018140) were relatively highly expressed in all of the investigated tissues under normal conditions, showing the expression profile of housekeeping genes. Generally, the expression patterns of CKX genes in Jingfeng 1 and Xiangan 336 were quite different under the same treatment. Notably, three genes (Bol020547, Bol028392 and Bol045724) were significantly down-regulated and up-regulated in the susceptible and resistant material, respectively, after inoculation, which may indicate their crucial roles in resistance to clubroot disease. The results provide insights for better understanding the roles of CKX genes in the B. oleracea–P. brassicae interaction.

Background The aerial organs of most terrestrial plants are covered by cuticular waxes, which impart plants a glaucous appearance and play important roles in protecting against various biotic and abiotic stresses. Despite many glossy green (wax-defective) mutants being well characterized in model plants, little is known about the genetic basis of glossy green mutant in broccoli. Results B156 is a spontaneous broccoli mutant showing a glossy green phenotype. Detection by scanning electron microscopy (SEM) and chromatography-mass spectrometry (GC-MS) revealed that B156 is a cuticular wax-defective mutant, lacking waxes mostly longer than C28. Inheritance analysis revealed that this trait was controlled by a single recessive gene, BoGL5. Whole-genome InDel markers were developed, and a segregating F2 population was constructed to map BoGL5. Ultimately, BoGL5 was mapped to a 94.1 kb interval on C01. The BoCER2 gene, which is homologous to the Arabidopsis CER2 gene, was identified as a candidate of BoGL5 from the target interval. Sequence analyses revealed that Bocer2 in B156 harbored a G-to-T SNP mutation at the 485th nucleotide of the CDS, resulting in a W-to-L transition at the 162nd amino acid, a conserved site adjacent to an HXXXD motif of the deduced protein sequence. Expression analysis revealed that BoCER2 was significantly down-regulated in the leaves, stems, and siliques of B156 mutant than that of B3. Last, ectopic expression of BoCER2 in A. thaliana could, whereas Bocer2 could not, rescue the phenotype of cer2 mutant. Conclusions Overall, this study mapped the locus determining glossy phenotype of B156 and proved BoCER2 is functional gene involved in cuticular wax biosynthesis which would promotes the utilization of BoCER2 to enhance plant resistance to biotic and abiotic stresses, and breeding of B. oleracea cultivars with glossy traits.

To better understand the genetic diversity and population structure of broccoli cultivars planted in China, a total of 161 representative broccoli cultivars in the past 25 years were collected and analysed based on single nucleotide polymorphism (SNP) markers. Ten pairs of primers with good polymorphism and high resolution were screened from 315 pairs of SNP primers by 3 broccoli accessions (inbred lines) with different phenotypes and maturity. The 10 pairs of SNP primers were selected, producing 78 alleles. The diversity analysis indicated that the polymorphism information content (PIC) of SNP primer ranged from 0.64 to 0.90. The observed number of alleles (Na) was 2.00, the effective number of alleles (Ne) was 1.11–2.00, the Nei's gene diversity (H) was 0.10–0.50, and Shannon information index (I) was 0.20–0.70 using PopGene32 software. The clustering results showed that the 161 broccoli cultivars could be divided into 4 major subgroups (A, B, C and D), foreign cultivars were all assigned to subgroup A, and domestic cultivars were assigned to 3 subgroups of B, C, and D. This study indicated that some domestic cultivars and foreign cultivars were similar in genetic background, but most domestic cultivars were still different from the Japanese cultivars. When K = 2, the population structure result presented that 161 broccoli cultivars could be divided into 1 simple group (2 groups) and 1 mixed group. When Q ≥ 0.6, 143(88.82%) broccoli cultivars belonged to the simple groups. In simple groups 68 (42.24%) broccoli cultivars of group 1 were derived from Japan, the United States, Switzerland, the Netherlands, China-Taiwan, and China-Mainland; 75 (46.58%) broccoli cultivars belonged to group 2; when Q

Cabbage (Brassica oleracea var. capitata) is an important vegetable crop widely grown throughout the world, providing plentiful nutrients and health-promoting substances. To facilitate further genetics and genomic studies and crop improvement, we present here a high-quality reference genome for cabbage. We report a de novo genome assembly of the cabbage double-haploid line D134. A combined strategy of single-molecule real-time (SMRT) sequencing, 10× Genomics and chromosome conformation capture (Hi-C) produced a high quality cabbage draft genome. The chromosome-level D134 assembly is 529.92 Mb in size, 135 Mb longer than the current 02-12 reference genome, with scaffold N50 length being raised as high as 38 times. We annotated 44,701 high-quality protein-coding genes, and provided full-length transcripts for 45.59% of the total predicted gene models. Moreover, we identified novel genomic features like underrated TEs, as well as gene families and gene family expansions and contractions during B. oleracea evolution. The D134 draft genome is a cabbage reference genome assembled by SMRT long-read sequencing combined with the 10× Genomics and Hi-C technologies for scaffolding. This high-quality cabbage reference genome provides a valuable tool for improvement of Brassica crops.

Key messageOgura CMS fertility-restored materials, with 18 chromosomes, normal seed setting, stable fertility and closer genetic background to the parent Chinese kale, were successfully developed inB. oleraceavia a triploid strategy for the first time.AbstractOgura cytoplasmic male sterility (CMS) is the most widely used sterile type in seed production for commercial hybrids ofBrassica oleraceavegetables. However, the natural Ogura CMS restorer line has not been found inB. oleraceacrops. In this study, the triploid strategy was used with the aim to create euploidB. oleraceaprogenies with theRfogene. The allotriploid AAC hybrid YL2 was used as a male parent to backcross with Ogura CMS Chinese kale. After successive backcrosses, the BC2Rfo-positive individual 16CMSF2-11 and its BC3progenies, with 18 chromosomes, were developed, which were morphologically identical to the parent Chinese kale. Compared with F1and BC1plants, it showed stable fertility performance, and regular meiosis behavior and could produce seeds normally under natural pollination. The genomic composition analysis ofRfo-positive progenies by using molecular markers showed that more than 87% of the C-genome components of BC3Rfo-progenies recovered to the parent Chinese kale, while most or all of theAn-genome segments were lost in 16CMSF2-11 and its progenies. The results suggested that the genetic background ofRfo-positive individuals was closer to that of the parent Chinese kale along with backcrossing. Hereof, the Ogura CMS fertility-restored materials of Chinese kale were successfully created via triploid strategy for the first time, providing a bridge for utilizing the Ogura CMSB. oleraceagermplasm in the future. Moreover, our study indicates that the triploid strategy is effective for transferring genes fromB. napusintoB. oleracea.

Ornamental kale is popular because of its colorful leaves and few studies have investigated the mechanism of color changes. In this study, an ornamental kale line (S2309) with three leaf colors was developed. Analysis of the anthocyanin, chlorophyll, and carotenoid contents and RNA-seq were performed on the three leaf color types. There was less chlorophyll in the white leaves and purple leaves than in the green leaves, and the anthocyanin content was greatest in the purple leaves. All the downregulated DEGs related to chlorophyll metabolism were detected only in the S2309_G vs. S2309_W comparison, which indicated that the decrease in chlorophyll content was caused mainly by the inhibition of chlorophyll biosynthesis during the leaf color change from green to white. Moreover, the expression of 19 DEGs involved in the anthocyanin biosynthesis pathway was upregulated. These results provide new insight into the mechanisms underlying the three-color formation.

Cabbage Fusarium wilt (CFW) disease, caused by the soil-borne fungus Fusarium oxysporum f. sp. conglutinans (Foc), threatens cabbage production worldwide. We aimed to explore the molecular mechanism of CFW resistance and the avirulence/virulence factors of Foc. The resistant ‘96–100’ and susceptible ‘01–20’ cabbage lines were examined histologically and in RNA-seq analyses. The key differentially expressed genes (DEGs) and pathways of both the host and fungus were determined via bioinformatics databases and tools. After inoculation, Foc began to colonize in the root of 01–20 at approximately 3 dpi and almost covered the root at 9 dpi, while the colonization was inhibited in 96–100. 96,142 and 3152 unigenes were generated for cabbage and Foc, respectively, by de novo assembly. For cabbage, there were 42,056 and 37,346 DEGs in 01–20 and 96–100 at all time points. Plant-pathogen interaction (map04626) was the major enrichment pathway among the DEGs. Many NBS-LRR genes and WRKY transcription factors were identified with different expression levels between 96 and 100 and 01–20. For Foc, 977 upregulated genes and 113 downregulated genes were identified, and the pathway of ribosome (map03010) was greatly enriched. There were 1 potential effectors, 2 elicitors and 6 virulence factors with increased or decreased transcript abundance among Foc DEGs, which deserved further functional validation. The RNA-seq data were further validated by qRT-PCR. Our results provide a distinct dual transcriptomic landscape to reveal the molecular mechanisms of cabbage resistance to Foc, and expand our understanding of the interaction between plant hosts and their fungal pathogens.

From Brassica oleracea genome, 88 anthocyanin biosynthetic genes were identified. They expanded via whole-genome or tandem duplication and showed significant expression differentiation. Functional characterization revealed BoMYB113.1 as positive and BoMYBL2.1 as negative regulators responsible for anthocyanin accumulation. Brassica oleracea produces various health-promoting phytochemicals, including glucosinolates, carotenoids, and vitamins. Despite the anthocyanin biosynthetic pathways in the model plant Arabidopsis thaliana being well characterized, little is known about the genetic basis of anthocyanin biosynthesis in B. oleracea. In this study, we identified 88 B. oleracea anthocyanin biosynthetic genes (BoABGs) representing homologs of 46 Arabidopsis anthocyanin biosynthetic genes (AtABGs). Most anthocyanin biosynthetic genes, having expanded via whole-genome duplication and tandem duplication, retained more than one copy in B. oleracea. Expression analysis revealed diverse expression patterns of BoABGs in different tissues, and BoABG duplications showed significant expression differentiation. Additional expression analysis and functional characterization revealed that the positive regulator BoMYB113.1 and negative regulator BoMYBL2.1 may be key genes responsible for anthocyanin accumulation in red cabbage and ornamental kale by upregulating the expression of structural genes. This study paves the way for a better understanding of anthocyanin biosynthetic genes in B. oleracea and should promote breeding for anthocyanin content.

Background Leaf shape is an important agronomic trait in ornamental kale (Brassica oleracea L. var. acephala). Although some leaf shape-related genes have been reported in ornamental kale, the detailed mechanism underlying leaf shape formation is still unclear. Here, we report a lobed-leaf trait in ornamental kale, aiming to analyze its inheritance and identify the strong candidate gene. Results Genetic analysis of F2 and BC1 populations demonstrate that the lobed-leaf trait in ornamental kale is controlled by a single dominant gene, termed BoLl-1 (Brassica oleracea lobed-leaf). By performing whole-genome resequencing and linkage analyses, the BoLl-1 gene was finely mapped to a 127-kb interval on chromosome C09 flanked by SNP markers SL4 and SL6, with genetic distances of 0.6 cM and 0.6 cM, respectively. Based on annotations of the genes within this interval, Bo9g181710, an orthologous gene of LATE MERISTEM IDENTITY 1 (LMI1) in Arabidopsis, was predicted as the candidate for BoLl-1, and was renamed BoLMI1a. The expression level of BoLMI1a in lobed-leaf parent 18Q2513 was significantly higher compared with unlobed-leaf parent 18Q2515. Sequence analysis of the parental alleles revealed no sequence variations in the coding sequence of BoLMI1a, whereas a 1737-bp deletion, a 92-bp insertion and an SNP were identified within the BoLMI1a promoter region of parent 18Q2513. Verification analyses with BoLMI1a-specific markers corresponding to the promoter variations revealed that the variations were present only in the lobed-leaf ornamental kale inbred lines. Conclusions This study identified a lobed-leaf gene BoLMI1a, which was fine-mapped to a 127-kb fragment. Three variations were identified in the promoter region of BoLMI1a. The transcription level of BoLMI1a between the two parents exhibited great difference, providing new insight into the molecular mechanism underlying leaf shape formation in ornamental kale.

Cabbage (Brassica oleracea L. var. capitata L.) is an important vegetable crop cultivated around the world. Previous studies of cabbage gene transcripts were primarily based on next-generation sequencing (NGS) technology which cannot provide accurate information concerning transcript assembly and structure analysis. To overcome these issues and analyze the whole cabbage transcriptome at the isoform level, PacBio RS II Single-Molecule Real-Time (SMRT) sequencing technology was used for a global survey of the full-length transcriptomes of five cabbage tissue types (root, stem, leaf, flower, and silique). A total of 77,048 isoforms, capturing 18,183 annotated genes, were discovered from the sequencing data generated through SMRT. The patterns of both alternative splicing (AS) and alternative polyadenylation (APA) were comprehensively analyzed. In total, we detected 13,468 genes which had isoforms containing APA sites and 8978 genes which underwent AS events. Moreover, 5272 long non-coding RNAs (lncRNAs) were discovered, and most exhibited tissue-specific expression. In total, 3147 transcription factors (TFs) were detected and 10 significant gene co-expression network modules were identified. In addition, we found that Fusarium wilt, black rot and clubroot infection significantly influenced AS in resistant cabbage. In summary, this study provides abundant cabbage isoform transcriptome data, which promotes reannotation of the cabbage genome, deepens our understanding of their post-transcriptional regulation mechanisms, and can be used for future functional genomic research.

Xanthomonas campestris pv. campestris (Xcc) is the causal agent of black rot for cruciferous vegetables worldwide, especially for the cole crops such as cabbage and cauliflower. Due to the lack of resistant cabbage cultivars, black rot has brought about considerable yield losses in recent years in China. Understanding of the pathogen features is a key step for disease prevention, however, the pathogen diversity, population structure, and virulence are largely unknown. In this study, we studied 50 Xcc strains including 39 Xcc isolates collected from cabbage in 20 regions across China, using multilocus sequence genotyping (MLST), repetitive DNA sequence-based PCR (rep-PCR), and pathogenicity tests. For MLST analysis, a total of 12 allelic profiles (AP) were generated, among which the largest AP was AP1 containing 32 strains. Further cluster analysis of rep-PCR divided all strains into 14 DNA groups, with the largest group DNA I comprising of 34 strains, most of which also belonged to AP1. Inoculation tests showed that the representative Xcc strains collected from diverse regions performed differential virulence against three brassica hosts compared with races 1 and 4. Interestingly, these results indicated that AP1/DNA I was not only the main pathotype in China, but also a novel group that differed from the previously reported type races in both genotype and virulence. To our knowledge, this is the first extensive genetic diversity survey for Xcc strains in China, which provides evidence for cabbage resistance breeding and opens the gate for further cabbage-Xcc interaction studies.

Background: Leaf shape is an important agronomic trait in ornamental kale (Brassica oleracea L. var. acephala). Although some leaf shape-related genes have been reported in ornamental kale, the detailed mechanism underlying leaf shape formation is still unclear. Here, we report a lobed-leaf trait in ornamental kale, aiming to analyze its inheritance and identify the strong candidate gene.Results: Genetic analysis of F2 and BC1 populations demonstrate that the lobed-leaf trait in ornamental kale is controlled by a single dominant gene, termed BoLl-1. By performing whole-genome resequencing and linkage analyses, the BoLl-1 gene was finely mapped to a 127-kb interval on chromosome C09 flanked by SNP markers SL4 and SL6, with genetic distances of 0.6 cM and 0.6 cM, respectively. Based on annotations of the genes within this interval, Bo9g181710, an orthologous gene of LATE MERISTEM IDENTITY 1 (LMI1) in Arabidopsis, was predicted as the candidate for BoLl-1, and was renamed BoLMI1. The expression level of BoLMI1 in lobed-leaf parent 18Q2513 was significantly higher compared with unlobed-leaf parent 18Q2515. Sequence analysis of the parental alleles revealed no sequence variations in the coding sequence of BoLMI1, whereas a 1737-bp deletion, a 92-bp insertion and an SNP were identified within the BoLMI1 promoter region of parent 18Q2513. Verification analyses with BoLMI1-specific markers corresponding to the promoter variations revealed that the variations were present only in the lobed-leaf ornamental kale inbred lines.Conclusions: This study demonstrated that promoter variations strongly enhance the transcription level of BoLMI1 and cause the leaf shape change from unlobed to lobed, providing new insight into the molecular mechanism underlying leaf shape formation in ornamental kale.

Xanthomonas campestris pv. campestris is an important bacterial pathogen that causes black rot and brings about enormous production loss for cruciferous vegetables worldwide. Currently, genome sequences for only a few X. campestris pv. campestris isolates are available, most of which are draft sequences. Based on the next-generation sequencing and single-molecule sequencing in real time technologies, we present here the complete genome sequence of strain WHRI 3811 race 1 of X. campestris pv. campestris, which is a type strain that has been extensively used. The genome data will contribute to our understanding of X. campestris pv. campestris genomic features and pave the way for research on X. campestris pv. campestris–host interactions.

Clubroot, caused by the soil-borne protist Plasmodiophora brassicae, is one of the most destructive disease for Brassica oleracea worldwide. However, the molecular mechanism of clubroot resistance still remains poorly elucidated. Therefore, we aim at identifying key genes responsive to P. brassicae infection and deducing possible molecular mechanism regulating clubroot resistance in cabbage. A clubroot-resistant line (XG) and a clubroot-susceptible line (JF) were employed to conduct histological observation and transcriptome analysis at 7 and 28 DAI (days after inoculation) following inoculation with P. brassicae. Differentially expressed genes (DEGs) obtained by comparing infected roots with mock-infected roots were assigned to Gene Ontology (GO) functions and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways for enrichment analysis. TEM observation showed obvious histological differences of root cells between JF and XG after inoculation with P. brassicae. At 7 DAI, the number of DEGs identified in JF was much higher than that of XG, and most of them were enriched in metabolic pathways, metabolites biosynthesis and starch, sucrose metabolism. More DEGs were identified at 28 DAI compared to 7 DAI in XG, and most of these DEGs involved in biosynthesis of secondary metabolites, plant-pathogen interaction and plant hormone transduction. Genes related to cell wall biosynthesis, pattern recognition receptors (PRRs), disease resistance proteins, SA signal transduction, calcium influx, respiratory burst oxidase homolog (RBOH), MAPK cascades, transcription factors and chitinase were mainly up-regulated in XG at 28 DAI, while most of them were repressed in JF. Our research work suggest drastic and complex defense response to P. brassicae infection at 28 DAI (secondary infection stage) at transcriptional level. Results generated in the present study could provide comprehensive insights into the transcriptomic landscape for better understanding of molecular regulatory mechanism of clubroot resistance in cabbage.

Broccoli is a worldwide vegetable famous for good nutrition that is particularly rich in sulforaphane. The evolution of 95 broccoli cultivars that were widely collected and cultivated in China since 1980 was evaluated using 35 pairs of SSR markers. Thirty-five markers were selected from 960 pairs of SSR markers designed from Brassica oleracea, rapa and napus by two resources with different maturity periods. The results revealed that all 35 pairs of SSR markers were polymorphic, and 167 alleles were amplified with an average of 4.8 per marker. The mean content was 0.79 for polymorphism information, with a range from 0.48 to 0.99. The genetic diversity indexes ranged from 0.6909 to 0.8969, with an average of 0.7809. Genetic diversity analysis indicated the 95 genotypes could be separated into five primary sub-groups at a 0.72 distance. Sub-group A included more than 70% of genotypes with early and mid-early maturating cultivars, and the cultivars covered more than 87% of the genotypes of the 95 materials, including the primary cultivars widely planted that directed the domestication of broccoli in China in the past 37 years. Sub-groups B and C contained one inbred line with high generation and one DH line, respectively, suggesting both were innovative resources in cultivation. Sub-groups D and E were all inbred lines, and sub-group D had six members with similar characteristics: small florets, high-domed head, tall plants with nearly no lateral branches and good heat tolerance. Sub-group E had five members that presented similar wrinkle leaf morphology, and most matured at an intermediate rate, except IVF-B18. In this study, the evolution of broccoli cultivars in China was first summarized overall, together with their characteristics and distributions in China. Our study provides important information on genetic diversity and for cultivar replacement of broccoli in China, in addition to two new germplasms and polymorphism markers for research and molecular breeding.

Background Brassica oleracea exhibits extensive phenotypic diversity. As an important trait, petal color varies among different B. oleracea cultivars, enabling the study of the genetic basis of this trait. In a previous study, the gene responsible for petal color in B. oleracea was mapped to a 503-kb region on chromosome 3, but the candidate gene has not yet been identified. Results In the present study, we report that the candidate gene was further delineated to a 207-kb fragment. BoCCD4, a homolog of the Arabidopsis carotenoid cleavage dioxygenase 4 (CCD4) gene, was selected for evaluation as the candidate gene. Sequence analysis of the YL-1 inbred line revealed three insertions/deletions and 34 single-nucleotide polymorphisms in the coding region of BoCCD4. Functional complementation showed that BoCCD4 from the white-petal inbred line 11–192 can rescue the yellow-petal trait of YL-1. Expression analysis revealed that BoCCD4 is exclusively expressed in petal tissue of white-petal plants, and phylogenetic analysis indicated that CCD4 homologs may share evolutionarily conserved roles in carotenoid metabolism. These findings demonstrate that BoCCD4 is responsible for white/yellow petal color variation in B. oleracea. Conclusions This study demonstrated that function loss of BoCCD4, a homolog of Arabidopsis CCD4, is responsible for yellow petal color in B. oleracea. Electronic supplementary material The online version of this article (10.1186/s12864-019-5596-2) contains supplementary material, which is available to authorized users.

Glucoraphanin is a major secondary metabolite found in Brassicaceae vegetables, especially broccoli, and its degradation product sulforaphane plays an essential role in anticancer. The fine mapping of sulforaphane metabolism quantitative trait loci (QTLs) in broccoli florets is necessary for future marker-assisted selection strategies. In this study, we utilized a doubled haploid population consisting of 176 lines derived from two inbred lines (86,101 and 90,196) with significant differences in sulforaphane content, coupled with extensive genotypic and phenotypic data from two independent environments. A linkage map consisting of 438 simple sequence repeats markers was constructed, covering a length of 1168.26 cM. A total of 18 QTLs for sulforaphane metabolism in broccoli florets were detected, 10 were detected in 2017, and the other 8 were detected in 2018. The LOD values of all QTLs ranged from 3.06 to 14.47, explaining 1.74–7.03% of the biochemical variation between two years. Finally, 6 QTLs (qSF-C3-1, qSF-C3-2, qSF-C3-3, qSF-C3-5, qSF-C3-6 and qSF-C7) were stably detected in more than one environment, each accounting for 4.54–7.03% of the phenotypic variation explained (PVE) and a total of 30.88–34.86% of PVE. Our study provides new insights into sulforaphane metabolism in broccoli florets and marker-assisted selection breeding in Brassica oleracea crops.

Comparative morphological, transcriptomic and phytohormone analyses reveal a defence network leading to PCD involved in cabbage hybrid lethality. Hybrid lethality (HL) plays an essential role in the stability of a population by blocking gene exchange between species, but the molecular mechanism remains largely undetermined. In this study, we performed phenotype, transcriptome and plant hormone analyses of HL in cabbage. Phenotype analysis confirmed that HL is characterised by a typical programmed cell death (PCD) process. A time-resolved RNA-Seq identified 2724 differentially expressed genes (DEGs), and functional annotations analyses revealed that HL was closely associated with the defence response. A defence regulation network was constructed based on the plant–pathogen interaction pathway and MAPK signalling pathway, which comprised DEGs related to Ca2+ and hydrogen peroxide (H2O2) leading to PCD. Moreover, important DEGs involved in hormone signal transduction pathways including salicylic acid (SA) and jasmonic acid (JA) were identified, which were further confirmed by endogenous and exogenous SA and JA measurements. Our results identified key genes and pathways in the regulating network of HL in cabbage, and might open the gate for revealing the molecular mechanism of HL in plants.

Main conclusion Chitinase family genes were involved in the response of Brassica oleracea to Fusarium wilt, powdery mildew, black spot and downy mildew. Abstract Abstract Chitinase, a category of pathogenesis-related proteins, is believed to play an important role in defending against external stress in plants. However, a comprehensive analysis of the chitin-binding gene family has not been reported to date in cabbage (Brassica oleracea L.), especially regarding the roles that chitinases play in response to various diseases. In this study, a total of 20 chitinase genes were identified using a genome-wide search method. Phylogenetic analysis was employed to classify these genes into two groups. The genes were distributed unevenly across six chromosomes in cabbage, and all of them contained few introns (≤ 2). The results of collinear analysis showed that the cabbage genome contained 1–5 copies of each chitinase gene (excluding Bol035470) identified in Arabidopsis. The heatmap of the chitinase gene family showed that these genes were expressed in various tissues and organs. Two genes (Bol023322 and Bol041024) were relatively highly expressed in all of the investigated tissues under normal conditions, exhibiting the expression characteristics of housekeeping genes. In addition, under four different stresses, namely, Fusarium wilt, powdery mildew, black spot and downy mildew, we detected 9, 5, 8 and 8 genes with different expression levels in different treatments, respectively. Our results may help to elucidate the roles played by chitinases in the responses of host plants to various diseases.

A single nucleotide mutation of BoCER2 is the primary cause of the wax deficiency in cabbage. An effective allele-specific KASP marker was developed for marker-assisted selection of glossiness. TL28-1 is a novel spontaneous wax-deficient mutant with a glossy phenotype identified from cabbage. In this study, the genetic analysis suggested that the wax-deficient trait of TL28-1 was controlled by a single recessive gene. All wax monomers longer than 28 carbons were significantly decreased in TL28-1. Fine-mapping results showed that the wax-deficient locus wdtl28 was located at an 80-kb interval between BOL01-20 and BOL01-24 markers on chromosome 1. According to the genome annotation of B. oleracea, the ECERIFERUM2- LIKE (CER2-LIKE) gene, BoCER2, was identified as the candidate gene. Phylogenetic analysis showed that BoCER2 and other CER2-LIKEs from vascular plants formed a clade within the BAHD superfamily of acyltransferases. The BoCER2 transcript was detected in various tissues, including stem, leaf, flower, and silique, but not in the cabbage roots. Subcellular localization indicated that BoCER2 protein functions in the endoplasmic reticulum. Further sequence analysis showed that a single nucleotide mutation (G to A) is present in the BoCER2 coding sequence in TL28-1, leading to a stop codon (TGA), hence premature translation termination. Linkage analysis showed that the homozygotic mutational BoCER2 co-segregated with wax deficiency. Moreover, the complementation test suggested that BoCER2 from wild type can rescue the wax deficiency of TL28-1. These results indicate that BoCER2 mutation hinders the elongation of very-long-chain fatty acid precursors in TL28-1, leading to wax deficiency. The allele-specific KASP marker designed in this study could be effective for marker-assisted selection of glossiness.

Cabbage is a globally cultivated vegetable with obvious heterosis. The main method used for cabbage breeding is the creation of hybrid combinations with inbred lines, and how to combine the parents plays a crucial role in cabbage breeding. Therefore, it is urgent to classify heterotic groups by molecular markers. In this study, single nucleotide polymorphism (SNP) markers were developed on the basis of whole-genome resequencing data of 50 inbred lines. A total of 2.54 × 106 SNP loci were obtained by aligning resequencing data to the reference genome of cabbage. Finally, five hundred SNP markers among 50 re-sequenced genotypes were further selected on the basis of a high polymorphism information content, a high minor allele frequency, a wide average distribution and low heterozygosity. Then, 442 SNP markers were successfully converted into kompetitive allele-specific PCR (KASP) markers, accounting for 88.4% of all 500 SNP markers. The cabbage inbred lines included three ecotypes: spring cabbage, autumn cabbage and winter cabbage. Based on the KASP genotyping data, the genetic similarity among inbred lines was analyzed, and the heterotic groups within each ecotype were classified. Seven heterotic groups were identified for spring cabbage (77); six, for autumn cabbage (70); and five, for winter cabbage (97). In addition, we found parents of 48 developed elite combinations were all from different heterotic groups or subgroups, which verified that the group classification was accurate and useful. Therefore, we suggest that the parents of combinations should be selected from different heterotic groups or subgroups, which will guide parental selection in cabbage breeding and improve breeding efficiency.

Background: Male sterility contributes strongly to hybrid seed purity and production in Brassica crops. To detect plant phenotypes and the gene expression patterns involved in bioprocess of an Ogura cytoplasmic male sterility line (Ogura-CMS), a dominant genic male sterility line (DGMS) and their maintainer line, we analyzed the transcriptomes of broccoli florets among the isogenic Ogura-CMS (T54C), DGMS (T54M) and inbred lines (T54S).Results: There were respectively 505, 585 and 469 upregulated genes, and 1109, 1073 and 543 downregulated genes in the comparison groups of T54C and T54M, T54C and T54S, and T54M and T54S. The head weight and head width showed stronger performance in the Ogura-CMS than the DGMS line or maintainer line. The Ogura-CMS line showed poorer performance in seed yield and seed germination than the DGMS line or maintainer line. The DGMS line had longer maturation and flowering periods than the Ogura-CMS and maintainer lines. The plant hormone genes, auxin (TIR1, GH3 and SAUR) and salicylic acid (SA) (NPR1), and adenosine triphosphate-binding cassette (ABC) transporters-related genes (ABCB1 and ATM) were highly up-regulated in T54C compared with T54M or T54S. The brassinosteroid upregulated gene CYCD3 related to the function of late-flowering and delaying senescence in plants, was detected in T54M over two seasons.Conclusions: Among the isogenic Ogura-CMS, DGMS and inbred lines of broccoli, the Ogura-CMS line showed strong performance in head yield than the DGMS line or the inbred line. The DGMS line had longer days to flowering than the Ogura-CMS and inbred lines. However, the inbred line presented a higher seed yield and seed germination rate than the DGMS or Ogura-CMS line. This study found that some potential plant hormone genes, auxin (TIR1, GH3 and SAUR) and SA (NPR1), and ABC transporters related genes (ABCB1 and ATM) might play a key role in regulation of the developmental trait in the Ogura-CMS line. The BR- upregulated gene CYCD3 may function in late-flowering and delaying senescence of broccoli consistent with the investigations conducted over two seasons. Our findings provided a possible explanation for physiological and developmental differences of broccoli among the Ogura-CMS, the DGMS and the inbred lines.

Background: Virus-induced gene silencing (VIGS), a posttranscriptional gene silencing method, represents an effective technology for the analysis gene functions in plants. However, no VIGS vectors are available for Brassica oleracea till now. The gene silencing vectors TRV, pTYs and CaLCuV were chose to improve the VIGS system in cabbage, using phytoene desaturase (PDS) gene as an efficient visual indicator for VIGS.Results: We successfully silenced the expression of PDS and observed the photobleaching phenomenon in cabbage by pTYs and CaLCuV, with the latter displaying higher efficiency and lower cost. Then, the factors potentially affecting the silencing efficiency of VIGS by CaLCuV in cabbage, including targeting fragment strategy, inoculation method and incubation temperature, were compared. The optimized CaLCuV-based VIGS system is as follows: approximately 500 bp insert sequence, Agrobacterium OD600=1.0, vacuum osmosis method at the bud stage, and the incubation temperature of 22 °C, through which we achieved a stable silencing efficiency of 65%. To further test the effectiveness of the system, we selected two other genes in cabbage including Mg-chelataseH subunit (ChlH) and lobed-leaf 1 (LL1) to knock down their expression, and we observed the expected yellow or lobed leaves. In addition, we successfully applied the CaLCuV-based VIGS system in two other representative Brassica crops including B. rapa and B. nigra and thus expanded its application scope.Conclusion: These results demonstrate that CaLCuV-based VIGS effectively work in cabbage and can be used for analyzing gene function. Our VIGS system described here will contribute to efficient functional genomics research in Brassica crops.

Background The planting density of broccoli can directly affect the yield and overall health of plants. So there is necessary to reveal the regulatory genes of planting density in broccoli by QTL mapping. In this study, the important planting density-dependent factors of broccoli, plant height (PH), maximum outer petiole length (PL) and leaf width (LW), were investigated during 2017 and 2018. The mapping of QTLs for PH, PL and LW were performed, and the interaction between QTLs and the environment was also analyzed by a DH population constructed with 176 genotypes derived from F1 obtained by crossing the broccoli inbred lines 86101 (P1) and 90196 (P2).Results A linkage group including a total of 438 SSR markers were constructed covering a length of 1168.26 cM using QTL IciMapping 4.0 software. Finally, there were mainly four QTLs (phc1, phc2, phc4-1, phc4-2), one QTL (plc6), and two QTLs (lwc1, lwc3) corresponding for PH, PL and LW recurred during the two years. In three environments, inclusive composite interval mapping (ICIM) analysis showed that there was a major QTL for PH at 7.20 cM on chromosome 1 between molecular markers 8C024 and sf4482 with a high explanatory contribution rate of 20.05%. The QTL at the 11.10 cM position of chromosome 6 was located for the PL with a high explanatory contribution rate of 20.02% between the molecular markers sc2170 and sf43960. The QTL at the 147.00 cM position of chromosome 3 was located on LW with a high explanatory contribution rate of 19.97% between molecular markers of Sc52751 and RA2-E12.Conclusions According to the QTL results of planting density in broccoli by a DH population, the possible positions of candidate genes were screened to provide a basis for further locating and cloning genes for plant height, maximum outer petiole and leaf width.

Clubroot disease, a major plant root disease caused byPlasmodiophora brassicae, has become one of the most destructive diseases among cultivated cruciferous vegetables. However, clubroot-resistantBrassica oleraceamaterials are rare. A few clubroot-resistant cabbage varieties are available on the market, but all are Ogura cytoplasmic male sterile (CMS) types. Therefore, in this study, to reutilize the clubroot-resistant Ogura CMS germplasm of cabbage, a new fertility-restored Ogura CMS material, 16Q2-11, was used as a bridge to transfer the clubroot resistance (CR) gene from the Ogura CMS cytoplasm to the normal cytoplasm by a two-step method (a fertility restoration and cytoplasm replacement method). In the first cross for fertility restoration of Ogura CMS clubroot-resistant cabbage (FRCRC), 16Q2-11 was used as a restorer to cross with Ogura CMS materials containing the CR geneCRb2. ElevenRfo-positive progenies were generated, of which four containedCRb2: F8-514, F8-620, F8-732 and F8-839. After inoculation with race 4 ofP. brassicae, these fourCRb2-positive individuals showed resistance. Furthermore, F8-514 and F8-839 were then used as male parents in the second cross of FRCRC to cross with cabbage inbred lines, resulting in the successful introgression of theCRb2gene into the inbred lines. All offspring produced from this step of cross, which had a normal cytoplasm, showed a high resistance to race 4 ofP. brassicaeand could be utilized for the breeding of clubroot-resistant cabbage varieties in the future. This is the first time that the Ogura CMS restorer has been used to restore the fertility of Ogura CMS clubroot-resistant cabbages, which could improve germplasm diversity in cabbage and provide a reference method for using CMS germplasm inBrassicacrops.

Microspore embryogenesis (ME), a widely used haploid breeding method that can considerably shorten the breeding cycle, provides an efficient mean of cultivating many important Brassica crops, such as cabbage, Chinese cabbage, and oilseed rape. For cabbage, in many cases, short-term heat shock treatment can strongly increase the embryogenesis rate, however, the underlying mechanism of this effect has not been elucidated. In this study, we compared the proteomics of isolated microspores with samples pretreated at 32 °C for 24 h and 25 °C for 24 h using two cabbage accessions (Zhonggan 628 and 87–534) showing highly different embryogenic rates. The embryo yield was 19.7 embryos/bud in Zhonggan 628 after 32 °C treatment, while no embryoid was observed in Zhonggan 628 after 25 °C treatment as well as in 87–534 at both temperatures. We identified a total of 363 and 282 differentially expressed proteins (DEPs) for Zhonggan 628 and 87–534 via a label-free proteomics technology. There were 97 DEPs specifically identified only in Zhonggan 628 but not in 87–534 after 32 °C heat-shock treatment that may be related to heat shock-induced embryogenesis in vitro culture. These DEPs were primarily enriched in carbon metabolic process, protein synthesis and degradation process, and signal transduction. Based on protein-protein interaction and pathway enrichment analyses, we proposed that SGT1 homolog A and B(SGT1), heat shock 70 kDa protein 5 (HSP70), cell division control protein 48 homolog A (CDC48) and fatty acyl-CoA reductase (FAR) might play important roles in microspore embryogenesis. This proteomic study may contribute to our molecular understanding of cabbage microspore embryogenesis and help to build a high-efficiency haploid breeding system.

Brassica species include many economically important crops that provide nutrition and health-promoting substances to humans worldwide. However, as with all crops, their production is constantly threatened by emerging viral, bacterial, and fungal diseases, whose incidence has increased in recent years. Traditional methods of control are often costly, present limited effectiveness, and cause environmental damage; instead, the ideal approach is to mine and utilize the resistance genes of the Brassica crop hosts themselves. Fortunately, the development of genomics, molecular genetics, and biological techniques enables us to rapidly discover and apply resistance (R) genes. Herein, the R genes identified in Brassica crops are summarized, including their mapping and cloning, possible molecular mechanisms, and application in resistance breeding. Future perspectives concerning how to accurately discover additional R gene resources and efficiently utilize these genes in the genomic era are also discussed.

TIFY, a plant-specific gene family with the conserved motif TIF[F/Y]XG, plays important roles in various plant biological processes. Here, a total of 36 TIFY genes were identified in the Brassica oleracea genome and classified into JAZ (22 genes), TIFY (7 genes), ZML (5 genes), and PPD (2 genes) subfamilies based on their conserved motifs, which were distributed unevenly across nine chromosomes with different lengths (339&ndash, 1077 bp) and exon numbers (1&ndash, 8). Following phylogenetic analysis with A. thaliana and B. rapa TIFY proteins, ten clades were obtained. The expression of these TIFY genes was organ-specific, with thirteen JAZ genes and two PPD genes showing the highest expression in roots and leaves, respectively. More importantly, the JAZs showed divergent responses to various pathogen infections and different phytohormone treatments. Compared with the susceptible line, most JAZs were activated after Plasmodiophora brassicae infection, while there were both induced and inhibited JAZs after Fusarium oxysporum or Xanthomonas campestris infection in the resistance line, indicating their probably distinct roles in disease resistance or susceptibility. Further, the JAZs were all upregulated after MeJA treatment, but were mostly downregulated after SA/ET treatment. In summary, these results contribute to our understanding of the TIFY gene family, revealing that JAZs may play crucial and divergent roles in phytohormone crosstalk and plant defense.

The genus Brassica includes many economically important crops providing nutrition as well as health-promoting substances. Most cultivars of the Brassica vegetables are F1 hybrids, and breeding system was successfully established by effectively applying the phenomenon of heterosis or self-incompatibility. However, their production is constantly threatened by abiotic and biotic stresses such as the increasing numbers of races and isolates of pathogens, inappropriate cropping systems, and changing climate. Traditional methods of control are often costly and environmentally damaging, while the ideal way is to mine and use the abiotic or biotic resistance from the crop hosts. Fortunately, genomics and molecular genetics enables the rapid discover and application of plant breeding to improve adaptation to environmental conditions and abiotic or biotic resistance. Herein, we have summarized the important characteristics for breeding of the Brassica vegetables, including the trials for understanding the molecular mechanisms with genetic and epigenetic approaches. Some future perspectives are also given concerning how to efficiently use these genes and overcome global climate change.

The ms3 gene responsible for a male-sterile phenotype in cabbage was mapped to a 187.4-kb genomic fragment. The gene BoTPD1, a homolog of Arabidopsis TPD1, was identified as a strong candidate gene. Cabbage 51S is a spontaneous male-sterile mutant. Phenotypic investigation revealed defects in anther cell differentiation, with failure to form the tapetum layer and complete abortion of microsporocytes before the tetrad stage. Genetic analysis indicated that this male sterility was controlled by a single recessive gene, ms3. Using an F2 population, we mapped ms3 to a 187.4-kb interval. BoTPD1 was identified as a candidate from this interval. Sequence analysis revealed an intronic 182-bp insertion in 51S that interrupted the conserved motif at the 5′ splicing site of the third intron, possibly resulting in a truncated transcript. Analyses of BoTPD1 homologous proteins revealed evolutionarily conserved roles in anther cell fate determination during reproductive development. RT-PCR showed that BoTPD1 was expressed in various tissues, excluding the root, and high expression levels were detected in anthers and buds. A BoTPD1-specific marker based on the 182-bp insertion cosegregated with male sterility and can be used for marker-assisted selection.

Background: cytokinins have important functions in regulating plant growth and response to abiotic stress. cytokinin family genes have been described in several plant species, but a comprehensive analysis of the cytokinin family genes in Brassica oleracea has not been reported to date, especially their roles in dealing with the invasion of P. brassicae. Results: Cytokinins are a class of phytohormones that promote cell division and differentiation and are thought to affect plant immunity to multiple pathogens. To reveal the mechanisms of the Brassica oleracea cytokinin family genes in response to clubroot disease, a total of 36 cytokinin genes were identified using a genome-wide search method. Phylogenetic analysis classified these genes into three groups. They were distributed unevenly across nine chromosomes in B. oleracea, and 15 of them did not contain introns. The results of colinear analysis showed that each cytokinin gene in the B. oleracea genome had at least one homologous gene in the Arabidopsis genome. A cis-element analysis indicated that these genes possessed several stress response cis-elements. The heatmap of the cytokinin gene family showed that these genes were expressed in various tissues and organs. Five and eight genes were up- and downregulated, respectively, in the susceptible material after inoculation. In addition, two and one genes were up- and downregulated, respectively, in resistant material. This may indicate that these cytokinin genes play important roles in the host plant response to clubroot disease. In addition, the results provide insights for better understanding the role of cytokinin in the B. oleracea–P. brassicae interaction. Conclusions: Our results are helpful to elucidate the role of cytokinin family genes in cabbage response to infection by P. brassicae, and lay a foundation for further study on the function of these genes. Keywords: Brassica oleracea, genome-wide, cytokinin family genes, clubroot

Cabbage is one of the most important cruciferous vegetables worldwide including China. Clubroot, caused by Plasmodiophora brassicae, occurs on most cruciferous crops and causes great yield losses. Utilization of resistant cultivar is the principal way to combat clubroot. To provide resistant sources for breeding, 102 cabbage genotypes were evaluated for seedling resistance to P. brassicae race 4, which is the predominant race in China. Results of repetitive identifications showed that Xiangan336 was highly resistant to clubroot, and four resistant genotypes including Verheul, Bindsachsener, Zhouyebai and 2358 were identified as resistant. Marker analysis suggested that Xiangan336 carried the clubroot resistance gene CRb. To clarify the infection characteristic of P. brassicae and deduce the resistance mechanism of cabbage, the infection process in Xiangan336 and Jingfeng No.1 (highly susceptible) were observed and compared. The significantly lower level of primary infection in Xiangan336 than that in Jingfeng No.1 at 4 dai (days after inoculation) indicated the early defense against P. brassicae in resistant genotype. However, the accumulated secondary plasmodia in Xiangan336 were more abundant than in Jingfeng No.1 from 7 to 21 dai and then declined significantly after 28 dai, suggesting that the clubroot-specific resistance mechanism may restrict the proliferation of P. brassicae after 28 dai. Histological observation verified that the development of P. brassicae from secondary plasmodia to resting spores was restricted within cortex cells in Xiangan336. The identified resistant genotypes will be of great value for resistance breeding and the analysis of infection process will further improve our understanding of resistance mechanism.

Winter cabbage is an important crop cultivated through winter in the region near the Yangtze River, enabling the supply of fresh cabbage there at that time of year. However, a problem has emerged regarding the newly developed parents of winter cabbage, which is completely different from spring and autumn cabbage, namely, how to combine these parents to breed an elite hybrid. To classify the heterotic groups and improve the efficiency of parent selection in winter cabbage breeding, 20 polymorphic SSR markers were selected to screen 63 winter cabbage inbred lines. Seventeen pairs among the 20 SSR markers amplified polymorphic bands. These primers amplified two to six bands, with an average of 2.8 bands per primer, and a total of 47 polymorphic bands were generated in the 63 inbred lines. These lines included flat-headed morphotype and round-headed morphotype, thus they were separately classified into heterotic groups based on the SSR markers. The flat-headed morphotype contained 21 inbred lines and was classified into three heterotic groups, named Hanchun 4, Jiali, and Dongsheng, in accordance with the representative germplasm contained in each group. The round-headed morphotype contained 42 inbred lines and was classified into five heterotic groups, named Parte, Bejo1039, YK-143, SCA002, and Golden B90. Meanwhile, parent analysis of 20 developed elite combinations showed that their parents were all distributed in different heterotic groups, indicating that the group classification was reasonable, which can provide a basis for further parent selection in winter cabbage breeding. Furthermore, polymorphic SSR primers were successfully used to identify the hybrid purity of three elite varieties. Keywords: winter cabbage, head morphotype, SSR marker, heterotic group, hybrid purity identification

Plant male reproductive development is a very complex biological process that involves multiple metabolic pathways. To reveal novel insights into male reproductive development, we conducted an integrated profiling of gene activity in the developing buds of a cabbage recessive genetic male sterile mutant. Using RNA-Seq and label-free quantitative proteomics, 2881 transcripts and 1245 protein species were identified with significant differential abundance between the male sterile line 83121A and its isogenic maintainer line 83121B. Analyses of function annotations and correlations between transcriptome and proteome and protein interaction networks were also conducted, which suggested that the male sterility involves a complex regulatory pattern. Moreover, several key biological processes, such as fatty acid metabolism, tapetosome biosynthesis, amino acid metabolism and protein synthesis and degradation were identified as being of relevance to male reproductive development. A large number of protein species involved in sporopollenin synthesis, amino acid synthesis, ribosome assembly, protein processing in endoplasmic reticulum and lipid transfer were observed to be significantly down-accumulated in 83121A buds, indicating their potential roles in the regulation of cabbage microspore abortion. In summary, the conjoint analysis of the transcriptome and proteome provided a global picture regarding the molecular dynamics in male sterile buds of 83121A. Biological significance Male sterile mutants are excellent materials for the study of plant male reproductive development. This study revealed the molecular dynamics of recessive male sterility in cabbage at the transcriptome and proteome levels, which deepens our understanding of the metabolic pathways involved in male development. Moreover, the male sterility-related genes identified in this study could provide a reference for the artificial regulation of cabbage fertility by using genetic engineering technology, which may result in potential applications in agriculture such as production of hybrid seeds using male sterility.

The content and component of glucosinolates in edible stems and leaves of eight Chinese kale varieties from Japan and eight varieties from China were determined by HPLC-MS. Simultaneously, the expression levels of glucosinolate biosynthesis pathway genes from four varieties with high and low total glucosinolate contents were analyzed by the qRT-PCR method. Four types of aliphatic glucosinolates (A-GLSs: GRA, SIN, GNA and GER) and indole glucosinolates (I-GLSs: 4-HGBS, GBS, 4-MGBS and NGBS) were detected in the stems and leaves of 16 varieties, and no aromatic glucosinolates (R-GLSs) were detected. A-GLSs account for more than 80.69% of the total content of total glucosinolates (T-GLSs), in which GNA and GRA are the main components of stems and leaves. Among Japanese varieties, QB1 has higher content of A- and T-GLSs, while that of XLB was lower, however, the corresponding varieties were ZH and DSHH in Chinese varieties. Among the above four varieties, the expression levels of SOT16, CYP83B1, SOT17, CYP83A1 and MAM1 genes were significantly higher in the varieties with higher GLSs, the expression levels of SOT16 and CYP83B1 were consistent with the content of I-GLSs, and SOT17, CYP83A1 and MAM1 expression levels were consistent with A-GLSs content. At the same time, the expression levels of SOT16 and CYP83B1 in the leaves were higher than those in the stems. CYP83A1 and MAM1 genes were less expressed in the leaves than in the stems of lower content varieties. It is speculated that these genes may be the key genes regulating GLS biosynthesis in Chinese kale.

s Xanthomonas campestris pv. campestris (Xcc) is the causal agent of cabbage (Brassica oleracea var. capitata) black rot (BR) that leads to severe yield losses worldwide. Cultivation and application of resistant cultivars is the most desirable way to control BR. However, due to the lack of BR-resistant germplasm resources and an unclear understanding of the resistance inheritance as well as the controlling gene, the breeding process has been seriously hindered. To identify new BR resistance sources to Xcc race 1, 162 cabbage accessions collected worldwide were evaluated using the spray inoculation method. In total, 64.2% of them were susceptible, and only four germplasms were highly resistant, including two inbred lines (‘05-574-323’ and ‘MD219’) and two hybrids (‘Qinglian’ and ‘Dadilv 2’). Further, to define the genetic characteristics of resistance, a mixed major gene plus polygene inheritance model was used to analyze BR resistance in six populations (P1, P2, F1, B1, B2, and F2) derived from the resistant parent ‘05-574-323’ and the susceptible parent ‘02-359’. The results showed that BR resistance was controlled by a pair of additive major genes and additive-dominant multiple genes (D-2 model), and the main gene heritabilities of B1, B2, and F2 were 49.29%, 43.53%, and 50.35%, respectively. The resistant accessions obtained in this study provide new resources for cabbage BR-resistance breeding, and the inheritance analysis opens the gate for further mapping and application of the resistance gene.

Broccoli (Brassica oleracea L. var. italica) is one of the most important vegetable crops cultivated worldwide. The market demand for broccoli is still increasing due to its richness in vitamins, anthocyanins, mineral substances, fiber, secondary metabolites and other nutrients. The famous secondary metabolites, glucosinolates, sulforaphane and selenium have protective effects against cancer. Significant progress has been made in fine-mapping and cloning genes that are responsible for important traits; this progress provides a foundation for marker-assisted selection (MAS) in broccoli breeding. Genetic engineering by the well-developed Agrobacterium tumefaciens-mediated transformation in broccoli has contributed to the improvement of quality; postharvest life; glucosinolate and sulforaphane content; and resistance to insects, pathogens and abiotic stresses. Here, we review recent progress in the genetics and molecular breeding of broccoli. Future perspectives for improving broccoli are also briefly discussed.

Plant cytochrome P450 monooxygenases (CYPs) make up a large superfamily of heme-thiolate enzymes involving in many primary and secondary metabolism reactions. In this study, a total of 279 Brassica oleracea CYP (BoCYP) genes were identified in the cabbage genome. These BoCYPs were classified into eight distinct clans with 44 families. Expression analyses of the BoCYP genes of different cabbage tissues suggested that some BoCYPs may play significant roles in reproduction. Recently, BoCYP704B1 has been identified as a candidate gene associated with male sterility in the cabbage line 83121A. Here, the functional mechanism of BoCYP704B1 was explored. Subcellular localization showed that BoCYP704B1 is located in the endoplasmic reticulum. RNA in situ hybridization indicated that BoCYP704B1 is predominantly expressed in the tapetum of cabbage anther, and the expression was maximal at the tetrad and early monocyte stage and weakened to the termination. Furthermore, functional complementation test results showed that BoCYP704B1 from wild type could rescue the male sterility of 83121A mutant. The results of this study increase our understanding of the functional mechanism of cabbage CYP genes and will facilitate the application of the BoCYP704B1 gene in cabbage molecular breeding.

Indole-3-carbinol is a potential anti-cancer agent, and it has been found rich in Brassica vegetables. However, there was few good extraction method of indole-3-carbinol in cabbage and broccoli. In this work, the wavelength and the ration of mobile phase in determination method, as well as different temperature and pH values in extraction method were all investigated some cultivars of cabbage and broccoli were collected and validated the determination method. A sensitive and rapid high-performance liquid chromatography method has been established and used for the determination of indole-3-carbinol in cabbage (Brassica oleracea L. var. capitata) and broccoli (Brassica oleracea L. var. italica). The method was proved to be sensitive, selective, rapid and reproducible, with a good recovery of 99.25%. There was a fast retention time (4.88 min) and good linearity (R2=0.9991) in the system. Meanwhile, significant differences of indole-3-carbinol contents were detected among all the materials (P

Brassica oleracea comprises several important subspecies, including cabbage, broccoli, cauliflower, Chinese kale, and kohlrabi. The petal color of Chinese kale is mostly white and sometimes yellow. To explore the genetic basis of petal color variation in Chinese kale, F2 and BC1 (backcross) populations were constructed from the cross of two inbred lines, 2114 (yellow petal) and 2116 (white petal). Genetic analysis of the F2 and BC1 populations demonstrated that yellow petal color was controlled by a single recessive nuclear gene, termed cpc-2. Insertion-deletion (InDel) markers, designed based on the parental resequencing data, were used to map cpc-2. The fine mapping results indicated that the cpc-2 gene was located in a 569-kb interval on chromosome C03 flanked by InDel markers ZB636 and ZB692, with genetic distances of 0.3 cM and 0.6 cM, respectively. By analyzing the nucleotide variations and annotations of the genes in this interval, a CCD4 family gene was predicted to be a candidate for cpc-2 and renamed BoCCD4.2. In addition, insertion of the CACTA-like transposable element (TE3) interrupted the function of the BoCCD4 gene, which may have resulted in the loss of function of BoCCD4 and the petal color transition from white to yellow. The TE3 insertion in the BoCCD4 gene was also present in 63 cabbage inbred lines among 159 accessions, which revealed that the TE3-type null allele of BoCCD4 formed before the divergence of the two subspecies cabbage and Chinese kale and that Chinese kale evolved much earlier than cabbage. This study lays the foundation for cloning BoCCD4.2 and revealing the molecular mechanism underlying petal color formation in Chinese kale.

Plant dwarf mutants generally exhibit delayed growth, delayed development, short internodes, and abnormal leaves and flowers and are ideal materials to explore the molecular mechanism of plant growth and development. In the current study, we first discovered a spontaneous cabbage (Brassica oleracea) dwarf mutant 99–198dw, which exhibits a dwarf stature, wrinkled leaves, non-heading, and substantially reduced self-fertility compared with the wild-type 99–198; however, the underlying molecular mechanism of its dwarfism is unknown. Here, we performed comparative phenotype, transcriptome and phytohormone analyses between 99–198 and 99–198dw. Cytological analysis showed that an increase in cell size, a reduction in cell layers, chloroplast degradation and a reduction in mitochondria were observed in 99–198dw. RNA-Seq showed that a total of 3801 differentially expressed genes (DEGs) were identified, including 2203 upregulated and 1598 downregulated genes in the dwarf mutant. Key genes in stress-resistant pathways were mostly upregulated, including salicylic acid (SA), jasmonic acid (JA), abscisic acid (ABA), ethylene (ET), etc., while the DEGs reported to be related to plant height, such as those involved in the gibberellin (GA), brassinolide (BR), indole-3-acetic acid (IAA), and strigolactone (SL) pathways were mostly downregulated. In addition, the DEGs in the cell division pathway were all downregulated, which is consistent with the cytokinesis defects detected by cytological analysis. The changes in the GA4, JA, ET, SA and ABA contents measured by liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) absolute quantification were consistent with the transcriptome analysis. Further hormone treatment tests showed that the exogenous application of GA, BR, 6BA, paclobutrazol (PC), etc. did not rescue the phenotype, implying that the change in phytohormones is due to but not the cause of the dwarf trait. It was speculated that mutation of certain DEG related to cell division or participating in signalling pathway of phytohormones like GA, BR, IAA, and SL were the cause of dwarf. These results are informative for the elucidation of the underlying regulatory network in 99–198dw and enrich our understanding of plant dwarf traits at the molecular level.

Seed number per silique is a major factor that affects cabbage seed yield. To identify quantitative trait loci (QTL) related to seed number per silique and develop linked markers for cabbage molecular marker-assisted breeding, a double haploid (DH) population with 100 individuals, derived from two founder parents (01-20 and 96-100) with notable differences in seed number per silique, was subjected to whole-genome resequencing. Genotyping by sequencing (GBS) technology was applied to construct a high-density SNP-based genetic linkage group with 1465 bin markers, covering 1285.77 cM of genome length, with an average genetic distance of 0.88 cM between adjacent bin markers. Finally, one significant QTL, qSNS6.1, on chromosome 6 was repeatedly detected in two experimental years, with a mean contribute rate of 15.2% and LOD value of 3.1. The major QTL qSNS6.1 will facilitate further understanding of the genetic basis of seed number per silique, and the development of genetic markers linked with seed number per silique would save time and reduce labor intensity by allowing selection of this trait at the seeding stage of cabbage.

Broccoli is an important Brassica vegetable that shows obvious heterosis. The main method for hybrid broccoli production is to use cytoplasmic male sterility (CMS) and pollination by bees. Therefore, the evaluation and selection of high quality sources of CMS is important for broccoli breeding. We aimed to evaluate the effects of different CMS sources on the morphology and size of floral organs, the attractiveness to pollinators and yield of hybrid seeds. Using broccoli CMS lines generated from different CMS sources with the same maintainer lines, we studied morphophysiological characteristics of flowers, such as flower shape and size, nectar production and sugar content, as well as the number of bee visits and seed yield. The degree of bud abortion, floral organs shape, degree of flower opening, nectar production, bee visits, and hybrid yield differed in CMS lines with same recurrent parent. When the CMS source was same and the recurrent parents were different, the degrees of bud abortion were different among the CMS lines, while the flowering characteristics were similar. Generally, larger floral organs and higher volumes of nectar were associated with higher frequency of bee visits and higher seed yield. Based on the morphological characteristics and size of floral organs, nectar secretion, bee-attracting ability and hybrid yield, we selected four CMS sources suitable for future use in broccoli breeding. Our results will provide an important reference for breeding high-quality CMS lines in broccoli and provide valuable CMS sources for breeding other cruciferous crops.

Cabbage 79-399-3 is a spontaneous male-sterile mutant controlled by the dominant genic gene (DGMS) Ms-cd1. Dozens of DGMS lines in cabbage, broccoli, kohlrabi, and Chinese kale have been created using 79-399-3 as a donor. To realize fast and accurate marker-assisted selection (MAS) and build a high-efficiency breeding system based on DMGS, a high-throughput, user-friendly marker is needed. In this study, we resequenced the DGMS line DGMS01-20 (backcross lines with over 20 generations) and its maintainer line 01-20 and developed four markers linked to Ms-cd1 using a segregating population of DGMS01-20. Kompetitive allele specific PCR (KASP) markers K13 and K23 were the flanking markers with genetic distances of 0.76 cM and 1.09 cM, respectively. K6 cosegregated with Ms-cd1 in all 919 individuals of the population. K13 and K6 were tested for their accuracy and applicability using 18 different DGMS lines. K13 was not applicable in five broccoli, kohlrabi, and Chinese kale DGMS lines, resulting in 72.2% accuracy, whereas K6 showed 100% accuracy in these DGMS lines and in an additional assessment using 35 inbred lines. Moreover, K6 can be used to genotype the self-pollination progenies of heterozygous plants (heterozygous means one Ms-cd1 allele and one wild-type allele; such plants produce pollen grains in very rare cases). The results showed that K6 is a reliable marker for accurately and rapidly identifying Ms-cd1 loci in Brassica oleracea; thus, it is beneficial for the DGMS-based breeding program in B. oleracea.

Cabbage Fusarium wilt (CFW) caused by Fusarium oxysporum f. sp. conglutinans (FOC) is known to significantly affect yield and quality of cabbages worldwide. CFW was first detected in New York, NY, United States, and has now spread to almost all cabbage-planting areas, including a recent outbreak of the disease in China. However, it was unknown whether the FOC strains emerged in China differed from the strains in other areas of the world. From 2009 to 2018, we collected Chinese FOC isolates and compared them to the races 1 and 2 strains in other areas to define their characteristics. Race tests indicated that most of the Chinese FOC strains belonged to race 1 and were more virulent than type strain 52557. To evaluate the genome level diversity, we performed next-generation sequencing and genome assembly for the race 2 strain 58385. Based on the assembled genome, we discovered abundant single-nucleotide polymorphisms and 645 insertion-deletions (InDels) compared with the race 1 strain FGL03-6 by comparative genomic analysis and showed that all FOC race 1 strains have a low genetic variability, with a genomic background distinct from 58385. Furthermore, the internal transcribed spacer, elongation factor-1α, and whole-genome InDel variation studies suggested that the last might be a powerful tool in phylogenetic as well as evolution analysis for F. oxysporum Schlechtend.: Fr. The race, virulence, and genome-based variation profiles could contribute to our knowledge of FOC diversity and support the studies of pathogen characterization in genomic era and also provide clues for CFW-resistance breeding. To our knowledge, this is the first extensive survey conducted for FOC strains.

Background Heat shock proteins have important functions in regulating plant growth and response to abiotic stress. HSP70 family genes have been described in several plant species, but a comprehensive analysis of the HSP70 family genes in cabbage has not been reported to date, especially their roles in floral development. Results In this study, we identified 52 BoHSP70 genes in cabbage. The gene structures, motifs, and chromosome locations of the BoHSP70 genes were analyzed. The genes were divided into seven classes using a phylogenetic analysis. An expression analysis showed that the BoHSP70 genes were highly expressed in actively growing tissues, including buds and calluses. In addition, six BoHSP70 genes were highly expressed in the binuclear-pollen-stage buds of a male fertile line compared with its near isogenic sterile line. These results were further verified using qRT-PCR. Subcellular localization analysis of the bud-specific gene BoHSP70–5 showed that it was localized in the cytoplasm. Conclusions Our results help to elucidate the involvement of the BoHSP70 family genes in cabbage floral development and establish the groundwork for future research on the functions of these genes. Electronic supplementary material The online version of this article (10.1186/s12864-019-5757-3) contains supplementary material, which is available to authorized users.

Cabbage hybrids, which clearly present heterosis vigor, are widely used in agricultural production. We compared two S5 haplotype (Class II) cabbage inbred-lines 87–534 and 94–182: the former is highly SC while the latter is highly SI; sequence analysis of SI-related genes including SCR, SRK, ARC1, THL1, and MLPK indicates the some SNPs in ARC1 and SRK of 87–534; semi-quantitative analysis indicated that the SI-related genes were transcribed normally from DNA to mRNA. To unravel the genetic basis of SC, we performed whole-genome mapping of the quantitative trait loci (QTLs) governing self-compatibility using an F2 population derived from 87–534 × 96–100. Eight QTLs were detected, and high contribution rates (CRs) were observed for three QTLs: qSC7.2 (54.8%), qSC9.1 (14.1%) and qSC5.1 (11.2%). 06–88 (CB201 × 96–100) yielded an excellent hybrid. However, F1 seeds cannot be produced at the anthesis stage because the parents share the same S-haplotype (S57, class I). To overcome crossing incompatibility, we performed rapid introgression of the self-compatibility trait from 87–534 to 96–100 using two self-compatibility-QTL-specific markers, BoID0709 and BoID0992, as well as 36 genome-wide markers that were evenly distributed along nine chromosomes for background analysis in recurrent back-crossing (BC). The transfer process showed that the proportion of recurrent parent genome (PRPG) in BC4F1 was greater than 94%, and the ratio of individual SC plants in BC4F1 reached 100%. The newly created line, which was designated SC96–100 and exhibited both agronomic traits that were similar to those of 96–100 and a compatibility index (CI) greater than 5.0, was successfully used in the production of the commercial hybrid 06–88. The study herein provides new insight into the genetic basis of self-compatibility in cabbage and facilitates cabbage breeding using SC lines in the male-sterile (MS) system.

Cabbage (Brassica oleracea) is an important vegetable crop that is cultivated worldwide. Previously, we reported the identification of two dominant complementary hybrid lethality (HL) genes in cabbage that could result in the death of hybrids. To avoid such losses in the breeding process, we attempted to develop molecular markers to identify HL lines. Among 54 previous mapping markers closely linked to BoHL1 or BoHL2, only six markers for BoHL2 were available in eight cabbage lines (two BoHL1 lines, three BoHL2 lines, three lines without BoHL), however, they were neither universal nor user-friendly in more inbred lines. To develop more accurate markers, these cabbage lines were resequenced at an ~20× depth to obtain more nucleotide variations in the mapping regions. Then, an InDel in BoHL1 and a single-nucleotide polymorphism (SNP) in BoHL2 were identified, and the corresponding InDel marker MBoHL1 and the competitive allele-specific PCR (KASP) marker KBoHL2 were developed and showed 100% accuracy in eight inbred lines. Moreover, we identified 138 cabbage lines using the two markers, among which one inbred line carried BoHL1 and 11 inbred lines carried BoHL2. All of the lethal line genotypes obtained with the two markers matched the phenotype. Two markers were highly reliable for the rapid identification of HL genes in cabbage.

Cabbage (Brassica oleracea var. capitata L.) is widely cultivated in China and be of important economic value. In October 2019, all the plants of cabbage inbred line '2358' cultivated in greenhouses of the Chinese Academy of Agricultural Sciences (Beijing) were showing symptoms of leaf wilt. It usually took two weeks for the leaves to get completed wilted and the plants gradually died. It was approximately 550 plants affected, and 30 plants were collected and processed as samples. Symptomatic leaves were cut into small pieces (5×5 mm), surface sterilized with 75% ethanol for 30 s, and then sterilized with 8% NaClO for 3 mins, rinsed three times in sterile distilled water, plated on complete medium (CM: 3g Casein Enzymatic Hydrolysate + 3g Casein Acid Hydrolysate + 6g Yeast Extract+10g Sucrose + 15g Agar + 1L dH2O) and incubated at 27℃ for 6 days. Subsequently, the purified culture was obtained by tissue isolation and single-spored on CM. The colony on CM was up to about 50mm and 70mm in diameter after 4 and 7 days, respectively (Supplementary Fig. 2). At the same time, the edge of the colony gradually turned brown. Microscopic observation found that the diameter of the mycelium was about 5 µm (Supplementary Fig. 3). The conidia were transparent white and ovoid-shaped, about 0.3-0.5 µm in diameter (Supplementary Fig. 4). Fungus in liquid CM were spherical with surface hairs (Supplementary Fig. 1), which was consistent with the morphological characteristics of Chaetomium globosum. The rDNA internal transcribed spacer (ITS) of isolate ZM2019 was amplified using primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'- TCCTCCGCTTATTGATATGC-3') (Hong et al. 2013). The amplified product was sequenced and deposited in GenBank. The 573-bp amplicon (GenBank accession no. MN833403) showed a 100% homology to C. globosum isolate CES5 (MN173145). According to the morphological and molecular characterization, the fungus causing leaf blight on '2358' was identified as C. globosum. In order to further distinguish the pathogen species, the β-tubulin (tub2) gene region were amplified using primers T-F (5'-CCCCTGAACTACCCCACC-3') and T-R (5'-TATTTGACCCGACTGACC-3') and sequenced. Finally, we further identified this pathogen as C. globosum CBS 148.51 (Wang et al. 2016), according to the blast result of the sequence (GenBank accession no. MW252170) in National Center for Biotechnology Information (NCBI). To confirm the pathogenicity of this fungus, the leaves of 12 healthy seedlings of '2358' were inoculated by spraying 106 conidia/ml suspension and 12 seedlings inoculated with sterile water served as controls. All plants were incubated in a growth chamber maintained at 27℃. Ten days after inoculation, the leaves of all plants became wilted, which were consistent with the symptom in the natural state, while the control plants remained disease-free (Supplementary Fig. 6b and Fig. 6c). Subsequently, C. globosum was isolated from the inoculated seedlings again. The entire process from isolation to inoculation was repeated again and the same results were obtained. Re-isolation of C. globosum and inoculation of the host fulfiled Koch's postulates. C. globosum has been reported previously to occur on many horticulture plants such as Punica granatum (Guo et al. 2015) and Cannabis sativa (Chaffin et al. 2020); but no brassica species has been reported so far as susceptible to C. globosum. In this sense, this is the first report of leaf blight caused by C. globosum on cabbage in China, in greenhouse condition.