Comparative Transcriptome Analysis Reveals a Potential Regulatory Network for Ogura Cytoplasmic Male Sterility in Cabbage (Brassica oleracea L.).

Ogura cytoplasmic male sterility (CMS) lines are widely used breeding materials in cruciferous crops and play important roles in heterosis utilization; however, the sterility mechanism remains unclear. To investigate the microspore development process and gene expression changes after the introduction of orf138 and Rfo, cytological observation and transcriptome analysis were performed using a maintainer line, an Ogura CMS line, and a restorer line. Semithin sections of microspores at different developmental stages showed that the degradation of tapetal cells began at the tetrad stage in the Ogura CMS line, while it occurred at the bicellular microspore stage to the tricellular microspore stage in the maintainer and restorer lines. Therefore, early degradation of tapetal cells may be the cause of pollen abortion. Transcriptome analysis results showed that a total of 1287 DEGs had consistent expression trends in the maintainer line and restorer line, but were significantly up- or down-regulated in the Ogura CMS line, indicating that they may be closely related to pollen abortion. Functional annotation showed that the 1287 core DEGs included a large number of genes related to pollen development, oxidative phosphorylation, carbohydrate, lipid, and protein metabolism. In addition, further verification elucidated that down-regulated expression of genes related to energy metabolism led to decreased ATP content and excessive ROS accumulation in the anthers of Ogura CMS. Based on these results, we propose a transcriptome-mediated induction and regulatory network for cabbage Ogura CMS. Our research provides new insights into the mechanism of pollen abortion and fertility restoration in Ogura CMS.

Global DNA Methylation and mRNA-miRNA Variations Activated by Heat Shock Boost Early Microspore Embryogenesis in Cabbage (Brassica oleracea).

Microspore culture, a type of haploid breeding, is extensively used in the cultivation of cruciferous crops such as cabbage. Heat shock (HS) treatment is essential to improve the embryo rate during the culture process; however, its molecular role in boosting early microspore embryogenesis (ME) remains unknown. Here we combined DNA methylation levels, miRNAs, and transcriptome profiles in isolated microspores of cabbage '01-88' under HS (32 °C for 24 h) and normal temperature (25 °C for 24 h) to investigate the regulatory roles of DNA methylation and miRNA in early ME. Global methylation levels were significantly different in the two pre-treatments, and 508 differentially methylated regions (DMRs) were identified; 59.92% of DMRs were correlated with transcripts, and 39.43% of miRNA locus were associated with methylation levels. Significantly, the association analysis revealed that 31 differentially expressed genes (DEGs) were targeted by methylation and miRNA and were mainly involved in the reactive oxygen species (ROS) response and abscisic acid (ABA) signaling, indicating that HS induced DNA methylation, and miRNA might affect ME by influencing ROS and ABA. This study revealed that DNA methylation and miRNA interfered with ME by modulating key genes and pathways, which could broaden our understanding of the molecular regulation of ME induced by HS pre-treatment.

Mechanism and Utilization of Ogura Cytoplasmic Male Sterility in Cruciferae Crops.

Hybrid production using lines with cytoplasmic male sterility (CMS) has become an important way to utilize heterosis in vegetables. Ogura CMS, with the advantages of complete pollen abortion, ease of transfer and a progeny sterility rate reaching 100%, is widely used in cruciferous crop breeding. The mapping, cloning, mechanism and application of Ogura CMS and fertility restorer genes in Brassica napus, Brassica rapa, Brassica oleracea and other cruciferous crops are reviewed herein, and the existing problems and future research directions in the application of Ogura CMS are discussed.

Transcriptome Analysis Reveals Key Genes and Pathways Associated with the Petal Color Formation in Cabbage (Brassica oleracea L. var. capitata).

Petal color is an important agronomic trait in cabbage (Brassica oleracea L. var. capitata). Although the key gene BoCCD4 has been functionally characterized, the underlying molecular regulatory mechanism of petal color formation in cabbage is still unclear. In this study, we applied the transcriptome analysis of yellow petals from the cabbage inbred line YL-1 and white petals from the Chinese kale inbred line A192-1 and the BoCCD4-overexpressing transgenic line YF-2 (YL-1 background), which revealed 1928 DEGs common to both the A192-1 vs. YL-1 and the YL-1 vs. YF-2 comparison groups. One key enzyme-encoding gene, BoAAO3, and two key TF-encoding genes, Bo2g151880 (WRKY) and Bo3g024180 (SBP), related to carotenoid biosynthesis were significantly up-regulated in both the A192-1 and YF-2 petals, which was consistent with the expression pattern of BoCCD4. We speculate that these key genes may interact with BoCCD4 to jointly regulate carotenoid biosynthesis in cabbage petals. This study provides new insights into the molecular regulatory mechanism underlying petal color formation in cabbage.

Genetic mapping and candidate gene identification of BoGL5, a gene essential for cuticular wax biosynthesis in broccoli.

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.

Genome-wide characterization and analysis of the anthocyanin biosynthetic genes in Brassica oleracea.

MAIN CONCLUSION: 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.

Genome-wide identification and expression analysis of the Brassica oleracea L. chitin-binding genes and response to pathogens infections.

MAIN CONCLUSION: Chitinase family genes were involved in the response of Brassica oleracea to Fusarium wilt, powdery mildew, black spot and downy mildew. 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.

Identification and validation of an ECERIFERUM2- LIKE gene controlling cuticular wax biosynthesis in cabbage (Brassica oleracea L. var. capitata L.).

KEY MESSAGE: 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.

Global Survey of the Full-Length Cabbage Transcriptome (Brassica oleracea Var. capitata L.) Reveals Key Alternative Splicing Events Involved in Growth and Disease Response.

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.

Organelle Comparative Genome Analysis Reveals Novel Alloplasmic Male Sterility with orf112 in Brassica oleracea L.

B. oleracea Ogura CMS is an alloplasmic male-sterile line introduced from radish by interspecific hybridization and protoplast fusion. The introduction of alien cytoplasm resulted in many undesirable traits, which affected the yield of hybrids. Therefore, it is necessary to identify the composition and reduce the content of alien cytoplasm in B. oleracea Ogura CMS. In the present study, we sequenced, assembled, and compared the organelle genomes of Ogura CMS cabbage and its maintainer line. The chloroplast genome of Ogura-type cabbage was completely derived from normal-type cabbage, whereas the mitochondrial genome was recombined from normal-type cabbage and Ogura-type radish. Nine unique regions derived from radish were identified in the mitochondrial genome of Ogura-type cabbage, and the total length of these nine regions was 35,618 bp, accounting for 13.84% of the mitochondrial genome. Using 32 alloplasmic markers designed according to the sequences of these nine regions, one novel sterile source with less alien cytoplasm was discovered among 305 materials and named Bel CMS. The size of the alien cytoplasm in Bel CMS was 21,587 bp, accounting for 8.93% of its mtDNA, which was much less than that in Ogura CMS. Most importantly, the sterility gene orf138 was replaced by orf112, which had a 78-bp deletion, in Bel CMS. Interestingly, Bel CMS cabbage also maintained 100% sterility, although orf112 had 26 fewer amino acids than orf138. Field phenotypic observation showed that Bel CMS was an excellent sterile source with stable 100% sterility and no withered buds at the early flowering stage, which could replace Ogura CMS in cabbage heterosis utilization.

First Report of Chaetomium globosum Causing Leaf Blight on Brassica oleracea L. in China.

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 ß-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.

Fine-mapping and transcriptome analysis of BoGL-3, a wax-less gene in cabbage (Brassica oleracea L. var. capitata).

The great majority of terrestrial plants produce epicuticular wax that is used to protect plants from a variety of biotic and abiotic stresses. Cabbage epicuticular wax is a white crystalline compound of various lipids. Wax-less cabbage has the characteristics of lustrous green leaves and beautiful exterior, which facilitates the brilliant green cabbage breeding. CGL-3 is a spontaneous wax-less mutant identified from cabbage. Genetic analysis indicated that the waxy deficiency of the mutant was controlled by a single dominant gene. To clarify the mechanism of the waxy deficiency, fine-mapping and transcriptome analysis of the wax-less gene, BoGL-3, were carried out in this study. The result of fine mapping showed that the wax-less gene, BoGL-3, was delimited in a 33.5-kb interval which is between the flanking marker C08-98 and the end of chromosome 8. Two cDNA libraries, constructed with wax-less cabbage CGL-3 and the wild-type cabbage WT, were sequenced for screening of the target gene BoGL-3. A total of 8340 genes were identified with significant differential expression between CGL-3 and WT. Among these genes, 3187 were up-regulated and 5153 were down-regulated in CGL-3. With homologous analysis, four differential expressed genes related to wax metabolism were obtained. Among these four genes, only Bol018504 is located within the region of fine-mapping. Bol08504 is homologous to CER1, which encodes fatty acid hydroxylase and plays an important role in wax synthesis in Arabidopsis. However, there was no difference of Bol08504 sequence between CGL-3 and WT. We suggested that Bol018504 was regulated by BoGL-3. The suppression of Bol018504 leads to the reduction of wax. These findings will be helpful to reveal the mechanism of the wax metabolism in cabbage and develop lustrous green cabbage germplasm material.

Recessive male sterility in cabbage (Brassica oleracea var. capitata) caused by loss of function of BoCYP704B1 due to the insertion of a LTR-retrotransposon.

KEY MESSAGE: The LTR-retrotransposon insertion in BoCYP704B1 is proved to be the primary cause of the male sterility in cabbage. Effective allele-specific markers were developed for marker-assisted selection of male sterile gene. 83121A is a spontaneous male sterile mutant identified from cabbage. Genetic analysis indicated that male sterility is controlled by a single recessive gene. Pollen wall formation in the 83121A mutant was severely defective, with a lack of sporopollenin or exine. To understand the mechanisms of male sterility in 83121A, transcription analysis using RNA-Seq was carried out in the buds of the male sterile line 83121A and the male fertile line 83121B, which are near-isogenic lines differing only in the fertility trait. Via expression analysis of differentially expressed genes involved in pollen exine development before the bicellular pollen stage, BoCYP704B1 was identified as a candidate gene, which was approximately downregulated 30-fold in 83121A. BoCYP704B1 is a member of the evolutionarily conserved CYP704B family, which is essential for sporopollenin formation. The BoCYP704B1 transcript is specifically detected in the developing anthers of wild-type cabbage. Further sequence analysis revealed that a 5424-bp long terminal repeat-retrotransposon (LTR-RT) was inserted into the first exon of BoCYP704B1 in 83121A, which is not found in wild-type plants. The insertion of LTR-RT not only reduced the expression of BoCYP704B1 but also altered structure of protein encoded by BoCYP704B1. Moreover, linkage analysis showed that the homozygotic mutational BoCYP704B1 always cosegregated with male sterility. These data suggest that the LTR-RT insertion in BoCYP704B1 hinders sporopollenin formation in 83121A leading to male sterility. The allele-specific markers developed in this study were effective for marker-assisted selection of the male sterile gene.

Plant non-coding RNAs: The new frontier for the regulation of plant development and adaptation to stress.

Most plant transcriptomes constitute functional non-coding RNAs (ncRNAs) that lack the ability to encode proteins. In recent years, more research has demonstrated that ncRNAs play important regulatory roles in almost all plant biological processes by modulating gene expression. Thus, it is important to study the biogenesis and function of ncRNAs, particularly in plant growth and development and stress tolerance. In this review, we systematically explore the process of formation and regulatory mechanisms of ncRNAs, particularly those of microRNAs (miRNAs), small interfering RNAs (siRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs). Additionally, we provide a comprehensive overview of the recent advancements in ncRNAs research, including their regulation of plant growth and development (seed germination, root growth, leaf morphogenesis, floral development, and fruit and seed development) and responses to abiotic and biotic stress (drought, heat, cold, salinity, pathogens and insects). We also discuss research challenges and provide recommendations to advance the understanding of the roles of ncRNAs in agronomic applications.

Chloroplast C-to-U editing, regulated by a PPR protein BoYgl-2, is important for chlorophyll biosynthesis in cabbage.

Leaf color is an important agronomic trait in cabbage (Brassica oleracea L. var. capitata), but the detailed mechanism underlying leaf color formation remains unclear. In this study, we characterized a Brassica oleracea yellow-green leaf 2 (BoYgl-2) mutant 4036Y, which has significantly reduced chlorophyll content and abnormal chloroplasts during early leaf development. Genetic analysis revealed that the yellow-green leaf trait is controlled by a single recessive gene. Map-based cloning revealed that BoYgl-2 encodes a novel nuclear-targeted P-type PPR protein, which is absent in the 4036Y mutant. Functional complementation showed that BoYgl-2 from the normal-green leaf 4036G can rescue the yellow-green leaf phenotype of 4036Y. The C-to-U editing efficiency and expression levels of atpF, rps14, petL and ndhD were significantly reduced in 4036Y than that in 4036G, and significantly increased in BoYgl-2 overexpression lines than that in 4036Y. The expression levels of many plastid- and nuclear-encoded genes associated with chloroplast development in BoYgl-2 mutant were also significantly altered. These results suggest that BoYgl-2 participates in chloroplast C-to-U editing and development, which provides rare insight into the molecular mechanism underlying leaf color formation in cabbage.

Large-scale gene expression alterations introduced by structural variation drive morphotype diversification in Brassica oleracea.

Brassica oleracea, globally cultivated for its vegetable crops, consists of very diverse morphotypes, characterized by specialized enlarged organs as harvested products. This makes B. oleracea an ideal model for studying rapid evolution and domestication. We constructed a B. oleracea pan-genome from 27 high-quality genomes representing all morphotypes and their wild relatives. We identified structural variations (SVs) among these genomes and characterized these in 704 B. oleracea accessions using graph-based genome tools. We show that SVs exert bidirectional effects on the expression of numerous genes, either suppressing through DNA methylation or promoting probably by harboring transcription factor-binding elements. The following examples illustrate the role of SVs modulating gene expression: SVs promoting BoPNY and suppressing BoCKX3 in cauliflower/broccoli, suppressing BoKAN1 and BoACS4 in cabbage and promoting BoMYBtf in ornamental kale. These results provide solid evidence for the role of SVs as dosage regulators of gene expression, driving B. oleracea domestication and diversification.

Transcriptome Analysis Reveals Key Genes and Pathways Associated with the Regulation of Flowering Time in Cabbage (Brassica oleracea L. var. capitata).

Flowering time is an important agronomic trait in cabbage (Brassica oleracea L. var. capitata), but the molecular regulatory mechanism underlying flowering time regulation in cabbage remains unclear. In this study, transcriptome analysis was performed using two sets of cabbage materials: (1) the early-flowering inbred line C491 (P1) and late-flowering inbred line B602 (P2), (2) the early-flowering individuals F2-B and late-flowering individuals F2-NB from the F2 population. The analysis revealed 9508 differentially expressed genes (DEGs) common to both C491_VS_ B602 and F2-B_VS_F2-NB. The Kyoto Encyclopedia of Genes and Genomes (KEGGs) analysis showed that plant hormone signal transduction and the MAPK signaling pathway were mainly enriched in up-regulated genes, and ribosome and DNA replication were mainly enriched in down-regulated genes. We identified 321 homologues of Arabidopsis flowering time genes (Ft) in cabbage. Among them, 25 DEGs (11 up-regulated and 14 down-regulated genes) were detected in the two comparison groups, and 12 gene expression patterns closely corresponded with the different flowering times in the two sets of materials. Two genes encoding MADS-box proteins, Bo1g157450 (BoSEP2-1) and Bo5g152700 (BoSEP2-2), showed significantly reduced expression in the late-flowering parent B602 compared with the early-flowering parent C491 via qRT-PCR analysis, which was consistent with the RNA-seq data. Next, the expression levels of Bo1g157450 (BoSEP2-1) and Bo5g152700 (BoSEP2-2) were analyzed in two other groups of early-flowering and late-flowering inbred lines, which showed that their expression patterns were consistent with those in the parents. Sequence analysis revealed that three and one SNPs between B602 and C491 were identified in Bo1g157450 (BoSEP2-1) and Bo5g152700 (BoSEP2-2), respectively. Therefore, BoSEP2-1 and BoSEP2-2 were designated as candidates for flowering time regulation through a potential new regulatory pathway. These results provide new insights into the molecular mechanisms underlying flowering time regulation in cabbage.

Glucosinolate Biosynthetic Genes of Cabbage: Genome-Wide Identification, Evolution, and Expression Analysis.

Cabbage (Brassica oleracea var. capitata) is a vegetable rich in glucosinolates (GSLs) that have proven health benefits. To gain insights into the synthesis of GSLs in cabbage, we systematically analyzed GSLs biosynthetic genes (GBGs) in the entire cabbage genome. In total, 193 cabbage GBGs were identified, which were homologous to 106 GBGs in Arabidopsis thaliana. Most GBGs in cabbage have undergone negative selection. Many homologous GBGs in cabbage and Chinese cabbage differed in expression patterns indicating the unique functions of these homologous GBGs. Spraying five exogenous hormones significantly altered expression levels of GBGs in cabbage. For example, MeJA significantly upregulated side chain extension genes BoIPMILSU1-1 and BoBCAT-3-1, and the expression of core structure construction genes BoCYP83A1 and BoST5C-1, while ETH significantly repressed the expression of side chain extension genes such as BoIPMILSU1-1, BoCYP79B2-1, and BoMAMI-1, and some transcription factors, namely BoMYB28-1, BoMYB34-1, BoMYB76-1, BoCYP79B2-1, and BoMAMI-1. Phylogenetically, the CYP83 family and CYP79B and CYP79F subfamilies may only be involved in GSL synthesis in cruciferous plants. Our unprecedented identification and analysis of GBGs in cabbage at the genome-wide level lays a foundation for the regulation of GSLs synthesis through gene editing and overexpression.

Map-based cloning and CRISPR/Cas9-based editing uncover BoNA1 as the causal gene for the no-anthocyanin-accumulation phenotype in curly kale (Brassica oleracea var. sabellica).

Brassica oleracea comprises several important vegetable and ornamental crops, including curly kale, ornamental kale, cabbage, broccoli, and others. The accumulation of anthocyanins, important secondary metabolites valuable to human health, in these plants varies widely and is responsible for their pink to dark purple colors. Some curly kale varieties lack anthocyanins, making these plants completely green. The genetic basis of this trait is still unknown. We crossed the curly kale inbred line BK2019 (without anthocyanins) with the cabbage inbred line YL1 (with anthocyanins) and the Chinese kale inbred line TO1000 (with anthocyanins) to generate segregating populations. The no-anthocyanin trait was genetically controlled by a recessive gene, bona1. We generated a linkage map and mapped bona1 to a 256-kb interval on C09. We identified one candidate gene, Bo9g058630, in the target genomic region; this gene is homologous to AT5G42800, which encodes a dihydroflavonol-4-reductase-like (DFR-like) protein in Arabidopsis. In BK2019, a 1-bp insertion was observed in the second exon of Bo9g058630 and directly produced a stop codon. To verify the candidate gene function, CRISPR/Cas9 gene editing technology was applied to knock out Bo9g058630. We generated three bona1 mutants, two of which were completely green with no anthocyanins, confirming that Bo9g058630 corresponds to BoNA1. Different insertion/deletion mutations in BoNA1 exons were found in all six of the other no-anthocyanin kale varieties examined, supporting that independent disruption of BoNA1 resulted in no-anthocyanin varieties of B. oleracea. This study improves the understanding of the regulation mechanism of anthocyanin accumulation in B. oleracea subspecies.