Map-based cloning and functional analysis of a major quantitative trait locus, BolC.Pb9.1, controlling clubroot resistance in a wild Brassica relative (Brassica macrocarpa).

KEY MESSAGE: A causal gene BoUGT76C2, conferring clubroot resistance in wild Brassica oleracea, was identified and functionally characterized. Clubroot is a devastating soil-borne disease caused by the obligate biotrophic pathogen Plasmodiophora brassica (P. brassicae), which poses a great threat to Brassica oleracea (B. oleracea) production. Although several QTLs associated with clubroot resistance (CR) have been mapped in cultivated B. oleracea, none have been cloned in B. oleracea. Previously, we found that the wild B. oleracea B2013 showed high resistance to clubroot. In this study, we constructed populations using B2013 and broccoli line 90196. CR in B2013 is quantitatively inherited, and a major QTL, BolC.Pb9.1, was identified on C09 using QTL-seq and linkage analysis. The BolC.Pb9.1 was finely mapped to a 56 kb genomic region using F2:3 populations. From the target region, the candidate BoUGT76C2 showed nucleotide variations between the parents, and was inducible in response to P. brassicae infection. We generated BoUGT76C2 overexpression lines in the 90196 background, which showed significantly enhanced resistance to P. brassicae compared to the WT line, suggesting that BoUGT76C2 corresponds to the resistance gene BolC.Pb.9.1. This is the first report on the CR gene map-based cloning and functional analysis from wild relatives, which provides a theoretical basis to the understanding of the molecular mechanism of CR, and lays a foundation to improve the CR of cultivated B. oleracea.

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.

Map-based cloning and promoter variation analysis of the lobed leaf gene BoLMI1a in ornamental kale (Brassica oleracea L. var. acephala).

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.

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.

Pigment variation and transcriptional response of the pigment synthesis pathway in the S2309 triple-color ornamental kale (Brassica oleracea L. var. acephala) line.

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.

Creation of fertility-restored materials for Ogura CMS in Brassica oleracea by introducing Rfo gene from Brassica napus via an allotriploid strategy.

KEY MESSAGE: Ogura CMS fertility-restored materials, with 18 chromosomes, normal seed setting, stable fertility and closer genetic background to the parent Chinese kale, were successfully developed in B. oleracea via a triploid strategy for the first time. Ogura cytoplasmic male sterility (CMS) is the most widely used sterile type in seed production for commercial hybrids of Brassica oleracea vegetables. However, the natural Ogura CMS restorer line has not been found in B. oleracea crops. In this study, the triploid strategy was used with the aim to create euploid B. oleracea progenies with the Rfo gene. The allotriploid AAC hybrid YL2 was used as a male parent to backcross with Ogura CMS Chinese kale. After successive backcrosses, the BC2 Rfo-positive individual 16CMSF2-11 and its BC3 progenies, with 18 chromosomes, were developed, which were morphologically identical to the parent Chinese kale. Compared with F1 and BC1 plants, it showed stable fertility performance, and regular meiosis behavior and could produce seeds normally under natural pollination. The genomic composition analysis of Rfo-positive progenies by using molecular markers showed that more than 87% of the C-genome components of BC3 Rfo-progenies recovered to the parent Chinese kale, while most or all of the An-genome segments were lost in 16CMSF2-11 and its progenies. The results suggested that the genetic background of Rfo-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 CMS B. oleracea germplasm in the future. Moreover, our study indicates that the triploid strategy is effective for transferring genes from B. napus into B. oleracea.

Map-based cloning and characterization of BoCCD4, a gene responsible for white/yellow petal color in B. oleracea.

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.

Fine mapping and candidate gene identification of the genic male-sterile gene ms3 in cabbage 51S.

KEY MESSAGE: 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.

iTRAQ-Based Proteomic Analysis of Ogura-CMS Cabbage and Its Maintainer Line.

Ogura cytoplasmic male sterility (CMS) contributes considerably to hybrid seed production in Brassica crops. To detect the key protein species and pathways involved in Ogura-CMS, we analysed the proteome of the cabbage Ogura-CMS line CMS01-20 and its corresponding maintainer line F01-20 using the isobaric tags for the relative and absolute quantitation (iTRAQ) approach. In total, 162 differential abundance protein species (DAPs) were identified between the two lines, of which 92 were down-accumulated and 70 were up-accumulated in CMS01-20. For energy metabolism in the mitochondrion, eight DAPs involved in oxidative phosphorylation were down-accumulated in CMS01-20, whereas in the tricarboxylic acid (TCA) cycle, five DAPs were up-accumulated, which may compensate for the decreased respiration capacity and may be associated with the elevated O2 consumption rate in Ogura-CMS plants. Other key protein species and pathways involved in pollen wall assembly and programmed cell death (PCD) were also identified as being male-sterility related. Transcriptome profiling revealed 3247 differentially expressed genes between the CMS line and the fertile line. In a conjoint analysis of the proteome and transcriptome data, 30 and 9 protein species/genes showed the same and opposite accumulation patterns, respectively. Nine noteworthy genes involved in sporopollenin synthesis, callose wall degeneration, and oxidative phosphorylation were presumably associated with the processes leading to male sterility, and their expression levels were validated by qRT-PCR analysis. This study will improve our understanding of the protein species involved in pollen development and the molecular mechanisms underlying Ogura-CMS.

Differentially Expressed Genes Associated with the Cabbage Yellow-Green-Leaf Mutant in the ygl-1 Mapping Interval with Recombination Suppression.

Although the genetics and preliminary mapping of the cabbage yellow-green-leaf mutant YL-1 has been extensively studied, transcriptome profiling associated with the yellow-green-leaf mutant of YL-1 has not been discovered. Positional mapping with two populations showed that the yellow-green-leaf gene ygl-1 is located in a recombination-suppressed genomic region. Then, a bulk segregant RNA-seq (BSR) was applied to identify differentially expressed genes (DEGs) using an F3 population (YL-1 × 11-192) and a BC2 population (YL-1 × 01-20). Among the 37,286 unique genes, 5730 and 4118 DEGs were detected between the yellow-leaf and normal-leaf pools from the F3 and BC2 populations. BSR analysis with four pools greatly reduced the number of common DEGs from 4924 to 1112. In the ygl-1 gene mapping region with suppressed recombination, 43 common DEGs were identified. Five of the DEGs were related to chloroplasts, including the down-regulated Bo1g087310, Bo1g094360, and Bo1g098630 and the up-regulated Bo1g059170 and Bo1g098440. The Bo1g098440 and Bo1g098630 genes were excluded by qRT-PCR. Hence, we inferred that these three DEGs (Bo1g094360, Bo1g087310, and Bo1g059170) in the mapping interval may be tightly associated with the development of the yellow-green-leaf mutant phenotype.

Genetics and fine mapping of a purple leaf gene, BoPr, in ornamental kale (Brassica oleracea L. var. acephala).

BACKGROUND: Due to its variegated and colorful leaves, ornamental kale (Brassica oleracea L. var. acephala) has become a popular ornamental plant. In this study, we report the fine mapping and analysis of a candidate purple leaf gene using a backcross population and an F2 population derived from two parental lines: W1827 (with white leaves) and P1835 (with purple leaves). RESULTS: Genetic analysis indicated that the purple leaf trait is controlled by a single dominant gene, which we named BoPr. Using markers developed based on the reference genome '02-12', the BoPr gene was preliminarily mapped to a 280-kb interval of chromosome C09, with flanking markers M17 and BoID4714 at genetic distances of 4.3 cM and 1.5 cM, respectively. The recombination rate within this interval is almost 12 times higher than the usual level, which could be caused by assembly error for reference genome '02-12' at this interval. Primers were designed based on 'TO1000', another B. oleracea reference genome. Among the newly designed InDel markers, BRID485 and BRID490 were found to be the closest to BoPr, flanking the gene at genetic distances of 0.1 cM and 0.2 cM, respectively; the interval between the two markers is 44.8 kb (reference genome 'TO1000'). Seven annotated genes are located within the 44.8 kb genomic region, of which only Bo9g058630 shows high homology to AT5G42800 (dihydroflavonol reductase), which was identified as a candidate gene for BoPr. Blast analysis revealed that this 44.8 kb interval is located on an unanchored scaffold (Scaffold000035_P2) of '02-12', confirming the existence of assembly error at the interval between M17 and BoID4714 for reference genome '02-12'. CONCLUSIONS: This study identified a candidate gene for BoPr and lays a foundation for the cloning and functional analysis of this gene.

Development of a novel allele-specific Rfo marker and creation of Ogura CMS fertility-restored interspecific hybrids in Brassica oleracea.

KEY MESSAGE: A novel allele-specific Rfo marker was developed and proved to be effective for MAS of Rfo gene in B. oleracea background and six Ogu-CMS fertility-restored interspecific hybrids were created for the first time. Ogura cytoplasmic male sterility (Ogu-CMS) has been extensively used for Brassica oleracea hybrid production. However, because of maternal inheritance, all the hybrids produced by CMS lines are male sterile and cannot be self-pollinated, which prohibits germplasm maintenance and innovation. This problem can be overcome by using the Ogu-CMS restorer line, but restorer material is absent in B. oleracea crops. Here, Rfo, a fertility-restored gene of Ogu-CMS, was transferred from rapeseed restorer lines into a Chinese kale Ogu-CMS line using interspecific hybridization combined with embryo rescue. Nine interspecific, triploid plant progenies were identified at morphological and ploidy level, with phenotypes intermediate between those of rapeseed and Chinese kale. Because the Rfo marker (Hu et al., Mol Breeding 22:663-674, 2008) cannot distinguish the Rfo and its homologies under a B. oleracea background, a novel allele-specific Rfo marker was developed based on the BLAST analysis of highly homologous Rfo sequences in B. oleracea. Screening using the novel Rfo marker found that six interspecific hybrids carrying Rfo were also fertile, although fertility varied during different flowering periods. Furthermore, BC1 offsprings with the Rfo gene were selected with the allele-specific Rfo marker and showed restored fertility. These results indicated that the novel allele-specific marker could be used for the MAS of Rfo gene in B. oleracea, and this study lays the foundation for the development of Ogu-CMS restorer material in cabbage and its related other subspecies.

One-step creation of CMS lines using a BoCENH3-based haploid induction system in Brassica crop.

Heterosis utilization in a large proportion of crops depends on the use of cytoplasmic male sterility (CMS) tools, requiring the development of homozygous fertile lines and CMS lines1. Although doubled haploid (DH) technology has been developed for several crops to rapidly generate fertile lines2,3, CMS lines are generally created by multiple rounds of backcrossing, which is time consuming and expensive4. Here we describe a method for generating both homozygous fertile and CMS lines through in vivo paternal haploid induction (HI). We generated in-frame deletion and restored frameshift mutants of BoCENH3 in Brassica oleracea using the CRISPR/Cas9 system. The mutants induced paternal haploids by outcrossing. We subsequently generated HI lines with CMS cytoplasm, which enabled the generation of homozygous CMS lines in one step. The BoCENH3-based HI system provides a new DH technology to accelerate breeding in Brassica and other crops.

Advances in and Perspectives on Transgenic Technology and CRISPR-Cas9 Gene Editing in Broccoli.

Broccoli, a popular international Brassica oleracea crop, is an important export vegetable in China. Broccoli is not only rich in protein, vitamins, and minerals but also has anticancer and antiviral activities. Recently, an Agrobacterium-mediated transformation system has been established and optimized in broccoli, and transgenic transformation and CRISPR-Cas9 gene editing techniques have been applied to improve broccoli quality, postharvest shelf life, glucoraphanin accumulation, and disease and stress resistance, among other factors. The construction and application of genetic transformation technology systems have led to rapid development in broccoli worldwide, which is also good for functional gene identification of some potential traits in broccoli. This review comprehensively summarizes the progress in transgenic technology and CRISPR-Cas9 gene editing for broccoli over the past four decades. Moreover, it explores the potential for future integration of digital and smart technologies into genetic transformation processes, thus demonstrating the promise of even more sophisticated and targeted crop improvements. As the field continues to evolve, these innovations are expected to play a pivotal role in the sustainable production of broccoli and the enhancement of its nutritional and health benefits.

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.

A highly efficient genetic transformation system for broccoli and subcellular localization.

Introduction: Agrobacterium-mediated genetic transformation has been widely used for the identification of functional genes and regulatory and developmental mechanisms in plants. However, there are still some problems of low genetic transformation efficiency and high genotype dependence in cruciferous crops. Methods: In this study, broccoli, a worldwide Brassica crop, was used to investigate the effects of genotype, explant type, concentration of hygromycin B used during seedling selection, overexpression vector type, RNAi and CRISPR/cas9 on the genetic transformation efficiency. At the same time, two vectors, PHG-031350 and PHG-CRa, were used for subcellular localization of the glucoraphanin synthesis-related gene FMOGS-OX5 and clubroot resistance gene by a PEG-Ca2+-mediated transient transformation system for broccoli protoplasts. Finally, the Agrobacterium-mediated genetic transformation system of broccoli was optimized and improved. Results and Discussion: This study showed that hypocotyl explants are more suitable for Agrobacterium-mediated transgene and CRISPR/Cas9 gene editing of broccoli. In contrast to previous studies, we found that 5 mg/L hygromycin B was more advantageous for the selection of resistant broccoli sprouts, and genotype 19B42 reached the highest transformation rate of 26.96%, which is higher than that in Brassica oleracea crops. In addition, the inbred line 19B42 successfully achieved high genetic transformation of overexpression, RNAi and CRISPR/Cas9 vectors; thus, it is powerful recipient material for the genetic transformation of broccoli. Subcellular localization proved that the glucoraphanin metabolism-related gene Bol031350 and clubroot resistance gene CRa were both expressed in the cytoplasm and nucleus, which provided a scientific basis for studying the regulation of glucosinolate metabolism and clubroot resistance in cruciferous crops. Therefore, these findings will provide new insight into the improvement of the genetic transformation and molecular breeding of Brassica oleracea crops.

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.

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.