OCTOPUS regulates BIN2 to control leaf curvature in Chinese cabbage.

Heading is one of the most important agronomic traits for Chinese cabbage crops. During the heading stage, leaf axial growth is an essential process. In the past, most genes predicted to be involved in the heading process have been based on leaf development studies in Arabidopsis. No genes that control leaf axial growth have been mapped and cloned via forward genetics in Chinese cabbage. In this study, we characterize the inward curling mutant ic1 in Brassica rapa ssp. pekinensis and identify a mutation in the OCTOPUS (BrOPS) gene by map-based cloning. OPS is involved in phloem differentiation in Arabidopsis, a functionalization of regulating leaf curvature that is differentiated in Chinese cabbage. In the presence of brassinosteroid (BR) at the early heading stage in ic1, the mutation of BrOPS fails to sequester brassinosteroid insensitive 2 (BrBIN2) from the nucleus, allowing BrBIN2 to phosphorylate and inactivate BrBES1, which in turn relieves the repression of BrAS1 and results in leaf inward curving. Taken together, the results of our findings indicate that BrOPS positively regulates BR signaling by antagonizing BrBIN2 to promote leaf epinastic growth at the early heading stage in Chinese cabbage.

Discovery of a DFR gene that controls anthocyanin accumulation in the spiny Solanum group: roles of a natural promoter variant and alternative splicing.

Anthocyanins are important pigments that impart color in plants. In Solanum, different species display various fruit or flower colors due to varying degrees of anthocyanin accumulation. Here we identified two anthocyanin-free mutants from an ethylmethane sulfonate-induced mutant library and naturally occurring mutants in Solanum melongena, with mutations in the 5' splicing site of the second intron of dihydroflavonol-4-reductase (DFR) - leading to altered splicing. Further study revealed that alternative splicing of the second intron was closely related to anthocyanin accumulation in 17 accessions from three cultivated species: S. melongena, Solanum macrocarpon and Solanum aethiopicum, and their wild related species. Analysis of natural variations of DFR, using an expanded population including 282 accessions belonging to the spiny Solanum group, identified a single-nucleotide polymorphism in the MYB recognition site in the promoter region, which causes differential expression of DFR and affects anthocyanin accumulation in fruits of the detected accessions. Our study suggests that, owing to years of domestication, the natural variation in the DFR promoter region and the alternative splicing of the DFR gene account for altered anthocyanin accumulation during spiny Solanum domestication.

Virus-Induced Gene Silencing (VIGS): A Powerful Tool for Crop Improvement and Its Advancement towards Epigenetics.

Virus-induced gene silencing (VIGS) is an RNA-mediated reverse genetics technology that has evolved into an indispensable approach for analyzing the function of genes. It downregulates endogenous genes by utilizing the posttranscriptional gene silencing (PTGS) machinery of plants to prevent systemic viral infections. Based on recent advances, VIGS can now be used as a high-throughput tool that induces heritable epigenetic modifications in plants through the viral genome by transiently knocking down targeted gene expression. As a result of the progression of DNA methylation induced by VIGS, new stable genotypes with desired traits are being developed in plants. In plants, RNA-directed DNA methylation (RdDM) is a mechanism where epigenetic modifiers are guided to target loci by small RNAs, which play a major role in the silencing of the target gene. In this review, we described the molecular mechanisms of DNA and RNA-based viral vectors and the knowledge obtained through altering the genes in the studied plants that are not usually accessible to transgenic techniques. We showed how VIGS-induced gene silencing can be used to characterize transgenerational gene function(s) and altered epigenetic marks, which can improve future plant breeding programs.

Genome-Wide Identification, Characterization, and Transcriptomic Analysis of the Cyclin Gene Family in Brassica rapa.

Cyclins are involved in cell division and proliferation by activating enzymes required for the cell cycle progression. Our genome-wide analysis identified 76 cyclin genes in Brassica rapa, which were divided into nine different types (A-, B-, C-, D-, H-, L-, P-, T-, and SDS-type). Cyclin genes were unevenly scattered on all chromosomes, with a maximum of 10 on A08 and a minimum of 2 on A04. The gene structure and conserved motif analysis showed that the cyclins which belonged to the same type or subgroup have a comparable intron/exon pattern or motif. A total of 14 collinear gene pairs suggested that the B. rapa cyclin genes experienced a mass of segmental duplication. The Ka/Ks analysis revealed that the Brcyclin gene family has undergone an extensive purifying pressure. By analyzing the cis-elements in the promoters, we identified 11 cis-elements and five of them are related to the hormone response. We observed 48 potential miRNAs targeting 44 Brcyclin genes, which highlighted the involvement of miRNAs in the regulation of cyclin genes. An association analysis between the leaf size and SNPs in mutants and a transcriptome analysis of two Chinese cabbage-cabbage translocation lines also showed that the Brcyclin gene family was involved in the development of the leaves. The functional characterization of the B. rapa cyclin gene family will provide the foundation for future physiological and genetic studies in the regulation of leaf growth.

Transcriptional Regulation and Gene Mapping of Internode Elongation and Late Budding in the Chinese Cabbage Mutant lcc.

Two important traits of Chinese cabbage, internode length and budding time, destroy the maintenance of rosette leaves in the vegetative growth stage and affect flowering in the reproductive growth stage. Internodes have received much attention and research in rice due to their effect on lodging resistance, but they are rarely studied in Chinese cabbage. In Chinese cabbage, internode elongation affects not only the maintenance of rosette leaves but also bolting and yield. Budding is also an important characteristic of Chinese cabbage entering reproductive growth. Although many studies have reported on flowering and bolting, studies on bud emergence and the timing of budding are scarce. In this study, the mutant lcc induced by EMS (Ethyl Methane Sulfonate) was used to study internode elongation in the seedling stage and late budding in the budding stage. By comparing the gene expression patterns of mutant lcc and wild-type A03, 2280 differentially expressed genes were identified in the seedling stage, 714 differentially expressed genes were identified in the early budding stage, and 1052 differentially expressed genes were identified in the budding stage. Here, the transcript expression patterns of genes in the plant hormone signaling and clock rhythm pathways were investigated in relation to the regulation of internode elongation and budding in Chinese cabbage. In addition, an F2 population was constructed with the mutants lcc and R500. A high-density genetic map with 1602 marker loci was created, and QTLs for internode length and budding time were identified. Specifically, five QTLs for internode length and five QTLs for budding time were obtained. According to transcriptome data analysis, the internode length candidate gene BraA02g005840.3C (PIN8) and budding time candidate genes BraA02g003870.3C (HY5-1) and BraA02g005190.3C (CHS-1) were identified. These findings provide insight into the regulation of internode length and budding time in Chinese cabbage.

Multispectral detection of dietary fiber content in Chinese cabbage leaves across different growth periods.

Multispectral imaging, combined with stoichiometric values, was used to construct a prediction model to measure changes in dietary fiber (DF) content in Chinese cabbage leaves across different growth periods. Based on all the spectral bands (365-970 nm) and characteristic spectral bands (430, 880, 590, 490, 690 nm), eight quantitative prediction models were established using four machine learning algorithms, namely random forest (RF), backpropagation neural network, radial basis function, and multiple linear regression. Finally, a quantitative prediction model of RF learning algorithm is constructed based on all spectral bands, which has good prediction accuracy and model robustness, prediction performance with R2 of 0.9023, root mean square error (RMSE) of 2.7182 g/100 g, residual predictive deviation (RPD) of 3.1220 > 3.0. In summary, this model efficiently detects changes in DF content across different growth periods of Chinese cabbage, which offers technical support for vegetable sorting and grading in the field.

Generation and identification of Brassica alboglabra-Brassica campestris monosomic alien addition lines.

Four monosomic alien addition lines (MAALs) for Brassica alboglabra-Brassica campestris were developed through digenomic triploid (ACC) backcrossing with the recurrent parent B. alboglabra (CC). The objectives of this study were to compare morphological traits, microsatellite markers (simple sequence repeats), chromosomal karyotypes, and meiotic behaviors. Based on the new chromosome nomenclature system established for Brassica, we preliminarily identified these MAALs as CC+A1, CC+A3, CC+A6, and CC+A7. Their alien chromosomes were transmittable through both female and male gametes at rates of 11.46%-26.53% and 4.88%-12.90%, respectively.

Transcriptome analysis of two pepper genotypes infected with pepper mild mottle virus.

Pepper mild mottle virus (PMMoV) poses a significant threat to pepper production because it is highly contagious and extremely persistent in soil. Despite this threat, little is known about the molecular processes that underlie plant responses to pepper mild mottle virus. Here, we performed RNA sequencing of tolerant ("17-p63") and susceptible ("16-217") pepper genotypes after pepper mild mottle virus or mock inoculation. Viral accumulation in systemic leaves was lower in the pepper mild mottle virus-resistant 17-p63 genotype than in the pepper mild mottle virus-sensitive 16-217 genotype, and infection symptoms were more apparent in systemic leaves of 16-217 than in those of 17-p63 at the same timepoints during the infection process. We identified 2,959 and 2,159 differentially expressed genes (DEGs) in systemic leaves of infected 16-217 and 17-p63, respectively. Through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of differentially expressed genes from both genotypes revealed significant enrichment of the MAPK signaling pathway, plant-pathogen interaction, and flavonoid biosynthesis. A number of differentially expressed genes showed opposite trends in relation to stress resistance and disease defense in the two genotypes. We also performed weighted gene co-expression network analysis (WGCNA) of all samples and identified modules associated with resistance to pepper mild mottle virus, as well as seven hub genes. These results identify candidate virus resistance genes and provide insight into pepper defense mechanisms against pepper mild mottle virus.

Hormones and carbohydrates synergistically regulate the formation of swollen roots in a Chinese cabbage translocation line.

The genus Brassica contains a rich diversity of species and morphological types, including leaf, root, and oil crops, all of which show substantial phenotypic variation. Both Chinese cabbage and cabbage are typical leaf-type crops with normal roots. We created translocation lines based on interspecific crosses between Chinese cabbage and cabbage and identified qdh225, which exhibited a swollen-root phenotype. The swollen root of qdh225 contained a large number of granular substances, and the formation of its irregular morphological tissue was caused by a thickening of the phloem. Transcriptomic and metabolomic data suggested that differential expression of genes encoding nine types of enzymes involved in starch and sucrose metabolism caused changes in starch synthesis and degradation in the swollen root. These genes jointly regulated sucrose and starch levels, leading to significant enrichment of starch and soluble proteins in the swollen root and a reduction in the content of soluble sugars such as d-glucose and trehalose 6-phosphate. A significant increase in auxin (IAA) and abscisic acid (ABA) contents and a decrease in gibberellin (GA) content in the swollen root likely promoted the differential expression of genes associated with hormone signal transduction, thereby regulating the development of the swollen root. Taken together, our data suggest that accumulation of IAA and ABA and reduction in GA promote swollen root formation by regulating hormone-mediated signaling, leading to a thickening of phloem, root enlargement, and substantial accumulation of starch and soluble proteins. The latter provide materials, energy, and nutrient sources for the development of swollen roots.

Molecular and genetic regulations of fleshy fruit shape and lessons from Arabidopsis and rice.

Fleshy fruit shape is an important external quality trait influencing the usage of fruits and consumer preference. Thus, modification of fruit shape has become one of the major objectives for crop improvement. However, the underlying mechanisms of fruit shape regulation are poorly understood. In this review we summarize recent progress in the genetic basis of fleshy fruit shape regulation using tomato, cucumber, and peach as examples. Comparative analyses suggest that the OFP-TRM (OVATE Family Protein - TONNEAU1 Recruiting Motif) and IQD (IQ67 domain) pathways are probably conserved in regulating fruit shape by primarily modulating cell division patterns across fleshy fruit species. Interestingly, cucumber homologs of FRUITFULL (FUL1), CRABS CLAW (CRC) and 1-aminocyclopropane-1-carboxylate synthase 2 (ACS2) were found to regulate fruit elongation. We also outline the recent progress in fruit shape regulation mediated by OFP-TRM and IQD pathways in Arabidopsis and rice, and propose that the OFP-TRM pathway and IQD pathway coordinate regulate fruit shape through integration of phytohormones, including brassinosteroids, gibberellic acids, and auxin, and microtubule organization. In addition, functional redundancy and divergence of the members of each of the OFP, TRM, and IQD families are also shown. This review provides a general overview of current knowledge in fruit shape regulation and discusses the possible mechanisms that need to be addressed in future studies.

Identification of candidate genes associated with less-photosensitive anthocyanin phenotype using an EMS mutant (pind) in eggplant (Solanum melongena L.).

Eggplant (Solanum melongena L.) is a highly nutritious and economically important vegetable crop. However, the fruit peel of eggplant often shows poor coloration owing to low-light intensity during cultivation, especially in the winter. The less-photosensitive varieties produce anthocyanin in low light or even dark conditions, making them valuable breeding materials. Nevertheless, genes responsible for anthocyanin biosynthesis in less-photosensitive eggplant varieties are not characterized. In this study, an EMS mutant, named purple in the dark (pind), was used to identify the key genes responsible for less-photosensitive coloration. Under natural conditions, the peel color and anthocyanin content in pind fruits were similar to that of wildtype '14-345'. The bagged pind fruits were light purple, whereas those of '14-345' were white; and the anthocyanin content in the pind fruit peel was significantly higher than that in '14-345'. Genetic analysis revealed that the less-photosensitive trait was controlled by a single dominant gene. The candidate gene was mapped on chromosome 10 in the region 7.72 Mb to 11.71 Mb. Thirty-five differentially expressed genes, including 12 structural genes, such as CHS, CHI, F3H, DFR, ANS, and UFGT, and three transcription factors MYB113, GL3, and TTG2, were identified in pind using RNA-seq. Four candidate genes EGP21875 (myb domain protein 113), EGP21950 (unknown protein), EGP21953 (CAAX amino-terminal protease family protein), and EGP21961 (CAAX amino-terminal protease family protein) were identified as putative genes associated with less-photosensitive anthocyanin biosynthesis in pind. These findings may clarify the molecular mechanisms underlying less-photosensitive anthocyanin biosynthesis in eggplant.

Metabolomic and transcriptomic analyses identify quinic acid protecting eggplant from damage caused by western flower thrips.

BACKGROUND: Western flower thrips are considered the major insect pest of horticultural crops worldwide, causing economic and yield loss to Solanaceae crops. The eggplant (Solanum melongena L.) resistance against thrips remains largely unexplored. This work aims to identify thrips-resistant eggplants and dissect the molecular mechanisms underlying this resistance using the integrated metabolomic and transcriptomic analyses of thrips-resistant and -susceptible cultivars. RESULTS: We developed a micro-cage thrips bioassay to identify thrips-resistant eggplant cultivars, and highly resistant cultivars were identified from wild eggplant relatives. Metabolomic profiles of thrips-resistant and -susceptible eggplant were compared using the gas chromatography-mass spectrometry (GC-MS)-based approach, resulting in the identification of a higher amount of quinic acid in thrips-resistant eggplant compared to the thrips-susceptible plant. RNA-sequencing analysis identified differentially expressed genes (DEGs) by comparing genome-wide gene expression changes between thrips-resistant and -susceptible eggplants. Consistent with metabolomic analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of DEGs revealed that the starch and sucrose metabolic pathway in which quinic acid is a metabolic by-product was highly enriched. External application of quinic acid enhances the resistance of susceptible eggplant to thrips. CONCLUSION: Our results showed that quinic acid plays a key role in the resistance to thrips. These findings highlight a potential application of quinic acid as a biocontrol agent to manage thrips and expand our knowledge to breed thrips-resistant eggplant. © 2022 Society of Chemical Industry.

Construction of a high-density mutant population of Chinese cabbage facilitates the genetic dissection of agronomic traits.

Chinese cabbage (Brassica rapa ssp. pekinensis) is an economically important vegetable crop throughout the world, especially in Asia. High-quality genome sequences are available for Chinese cabbage, but gene functional studies remain challenging. To promote functional genomic studies of Chinese cabbage, we generated an ethyl methane sulfonate (EMS) mutant population of ∼8000 M2 plants using the double haploid inbred line A03 as the parent. The genome of A03 was sequenced and used as a reference for high-throughput functional characterization of gene mutations at the whole-genome level. A total of 300 M2 to M5 EMS mutants were phenotypically screened and then sequenced, revealing 750 629 SNPs and 46 272 InDel mutations that cover 98.27% of all predicted genes in the A03 genome. A forward-genetics approach was successfully used to identify two genes with chloroplast-related functions that are responsible for the yellow leaf mutant trait. A reverse-genetics approach was also used to identify associations between mutations in five genes of the glucosinolate biosynthetic pathway and variations in glucosinolate content of the mutant plants. In addition, we built the Chinese cabbage EMS mutation database (CCEMD, www.bioinformaticslab.cn/EMSmutation/home) to increase the usability of this mutant population resource. In summary, we performed large-scale screening of a heading Chinese cabbage EMS mutant collection at the phenotypic and genotypic levels, which will facilitate gene mining of Chinese cabbage and might also be useful for the study of other Brassica crops.

Mutation in a chlorophyll-binding motif of Brassica ferrochelatase enhances both heme and chlorophyll biosynthesis.

The heme branch of tetrapyrrole biosynthesis contributes to the regulation of chlorophyll levels. However, the mechanism underlying the balance between chlorophyll and heme synthesis remains elusive. Here, we identify a dark green leaf mutant, dg, from an ethyl methanesulfonate (EMS)-induced mutant library of Chinese cabbage. The dg phenotype is caused by an amino acid substitution in the conserved chlorophyll a/b-binding motif (CAB) of ferrochelatase 2 (BrFC2). This mutation increases the formation of BrFC2 homodimer to promote heme production. Moreover, wild-type BrFC2 and dBrFC2 interact with protochlorophyllide (Pchlide) oxidoreductase B1 and B2 (BrPORB1 and BrPORB2), and dBrFC2 exhibits higher binding ability to substrate Pchlide, thereby promoting BrPORBs-catalyzed production of chlorophyllide (Chlide), which can be directly converted into chlorophyll. Our results show that dBrFC2 is a gain-of-function mutation contributing to balancing heme and chlorophyll synthesis via a regulatory mechanism in which dBrFC2 promotes BrPORB enzymatic reaction to enhance chlorophyll synthesis.

Genetic analysis of the "head top shape" quality trait of Chinese cabbage and its association with rosette leaf variation.

The agricultural and consumer quality of Chinese cabbage is determined by its shape. The shape is defined by the folding of the heading leaves, which defines the head top shape (HTS). The overlapping HTS, in which the heading leaves curve inward and overlap at the top, is the shape preferred by consumers. To understand the genetic regulation of HTS, we generated a large segregating F2 population from a cross between pak choi and Chinese cabbage, with phenotypes ranging from nonheading to heading with either outward curving or inward curving overlapping heading leaves. HTS was correlated with plant height, outer/rosette leaf length, and petiole length. A high-density genetic map was constructed. Quantitative trait locus (QTL) analysis resulted in the identification of 22 QTLs for leafy head-related traits, which included five HTS QTLs. Bulked segregant analysis (BSA) was used to confirm HTS QTLs and identify candidate genes based on informative single-nucleotide polymorphisms. Interestingly, the HTS QTLs colocalized with QTLs for plant height, outer/rosette leaf, and petiole length, consistent with the observed phenotypic correlations. Combined QTL analysis and BSA laid a foundation for molecular marker-assisted breeding of Chinese cabbage HTS and directions for further research on the genetic regulation of this trait.

Development and Application of SSR Markers Related to Genes Involved in Leaf Adaxial-Abaxial Polarity Establishment in Chinese Cabbage (Brassica rapa L. ssp. pekinensis).

In Chinese cabbage (Brassica rapa L. ssp. pekinensis), leaf adaxial-abaxial (ad-ab) polarity is tightly related to leaf incurvature, an essential factor for the formation of leafy heads. Therefore, identification of the genes responsible for leaf ad-ab polarity and studying their genetic variation may clarify the mechanism of leafy head formation. By comparing the sequences of the genes regulating leaf ad-ab polarity development in Arabidopsis thaliana (A. thaliana), 41 candidate genes distributed on 10 chromosomes were found to be responsible for the establishment of ad-ab polarity in Chinese cabbage. Orthologous genes, including 10 single copies, 14 double copies, and one triple copies, were detected in the Chinese cabbage. The gene structure and conserved domain analyses showed that the number of exons of the 41 candidate genes range from one to 25, and that most genes share the conserved motifs 1, 6, and 10. Based on the 41 candidate genes, 341 simple sequence repeats (SSRs) were detected, including five replicated types: single, double, triple, quintuple, and sextuple nucleotide replications. Among these sequence repeat (SSR) loci, 323 loci were used to design 969 specific primers, and 362 primer pairs were selected randomly and evaluated using 12 Chinese cabbage accessions with different heading types. 23 primer pairs resulting with clear, polymorphic bands, combined with other 127 markers, was used to construct a linkage map by using an F2 population containing 214 lines derived from the hybrid of the overlapping heading Chinese cabbage "14Q-141" and the outward curling heading Chinese cabbage "14Q-279." The result showed that the sequences of markers in the genetic linkage map and the physical map was consistent in general. Our study could help to accelerate the breeding process of leafy head quality in Chinese cabbage.

Determination of n+1 gamete transmission rate of trisomics and location of gene controlling 2n gamete formation in Chinese cabbage (Brassica rapa).

A set of trisomics of Chinese cabbage was used for determining the n+1 gamete transmission rate and locating the gene controlling 2n gamete formation on the corresponding chromosome. The results showed that the transmission rates of extra chromosomes in different trisomics varied from 0% to 15.38% by male gametes and from 0% to 17.39% by female gametes. Of the nine F(2) populations derived from the hybridizations between each trisomic and Bp058 (2n gamete material), only Tri-4xBp058 showed that the segregation ratio of plants without 2n gamete formation to plants with 2n gamete formation was 10.38:1, which fitted the expected segregation ratio of the trisomics (AAa) based on the 7.37% of n+1 gamete transmission through female and 5.88% through male. In other populations the segregation ratios varied from 2.48:1 to 3.72:1, which fitted the expected 3:1 segregation ratio of the bisomics (Aa). These results suggested that the gene controlling 2n gamete formation in Chinese cabbage Bp058 was located on chromosome 4. Further trisomic analysis based on the chromosome segregation and the incomplete stochastic chromatid segregation indicated that the gene locus was tightly linked to the centromere.

Characterization of Non-heading Mutation in Heading Chinese Cabbage (Brassica rapa L. ssp. pekinensis).

Heading is a key agronomic trait of Chinese cabbage. A non-heading mutant with flat growth of heading leaves (fg-1) was isolated from an EMS-induced mutant population of the heading Chinese cabbage inbred line A03. In fg-1 mutant plants, the heading leaves are flat similar to rosette leaves. The epidermal cells on the adaxial surface of these leaves are significantly smaller, while those on the abaxial surface are much larger than in A03 plants. The segregation of the heading phenotype in the F2 and BC1 population suggests that the mutant trait is controlled by a pair of recessive alleles. Phytohormone analysis at the early heading stage showed significant decreases in IAA, ABA, JA and SA, with increases in methyl IAA and trans-Zeatin levels, suggesting they may coordinate leaf adaxial-abaxial polarity, development and morphology in fg-1. RNA-sequencing analysis at the early heading stage showed a decrease in expression levels of several auxin transport (BrAUX1, BrLAXs, and BrPINs) and responsive genes. Transcript levels of important ABA responsive genes, including BrABF3, were up-regulated in mid-leaf sections suggesting that both auxin and ABA signaling pathways play important roles in regulating leaf heading. In addition, a significant reduction in BrIAMT1 transcripts in fg-1 might contribute to leaf epinastic growth. The expression profiles of 19 genes with known roles in leaf polarity were significantly different in fg-1 leaves compared to wild type, suggesting that these genes might also regulate leaf heading in Chinese cabbage. In conclusion, leaf heading in Chinese cabbage is controlled through a complex network of hormone signaling and abaxial-adaxial patterning pathways. These findings increase our understanding of the molecular basis of head formation in Chinese cabbage.

Microspore Induced Doubled Haploids Production from Ethyl Methanesulfonate (EMS) Soaked Flower Buds Is an Efficient Strategy for Mutagenesis in Chinese Cabbage.

Chinese cabbage buds were soaked with Ethyl methanesulfonate (EMS) to induce mutagenesis. The influence of different EMS concentrations and treatment durations on microspore development, embryo production rate and seedling rate were evaluated in five Chinese cabbage genotypes. Mutations in four color-related genes were identified using high resolution melting (HRM) curves of their PCR products. The greatest embryo production and seedling rates were observed when buds were treated with 0.03 to 0.1% EMS for 5 to 10 min, while EMS concentrations greater than 0.1% were lethal to the microspores. In total, 142 mutants with distinct variations in leaf shape, leaf color, corolla size, flower color, bolting time and downy mildew resistance were identified from 475 microspore culture derived Doubled Haploids. Our results demonstrate that microspore derived Doubled Haploids from EMS soaked buds represents an efficient approach to rapidly generate homozygous Chinese cabbage mutants.