BrMYB108 confers resistance to Verticillium wilt by activating ROS generation in Brassica rapa.

Increasing plant resistance to Verticillium wilt (VW), which causes massive losses of Brassica rapa crops, is a challenge worldwide. However, few causal genes for VW resistance have been identified by forward genetic approaches, resulting in limited application in breeding. We combine a genome-wide association study in a natural population and quantitative trait locus mapping in an F2 population and identify that the MYB transcription factor BrMYB108 regulates plant resistance to VW. A 179 bp insertion in the BrMYB108 promoter alters its expression pattern during Verticillium longisporum (VL) infection. High BrMYB108 expression leads to high VL resistance, which is confirmed by disease resistance tests using BrMYB108 overexpression and loss-of-function mutants. Furthermore, we verify that BrMYB108 confers VL resistance by regulating reactive oxygen species (ROS) generation through binding to the promoters of respiratory burst oxidase genes (Rboh). A loss-of-function mutant of AtRbohF in Arabidopsis shows significant susceptibility to VL. Thus, BrMYB108 and its target ROS genes could be used as targets for genetic engineering for VL resistance of B. rapa.

Wall-associated kinase BrWAK1 confers resistance to downy mildew in Brassica rapa.

The plant cell wall is the first line of defence against physical damage and pathogen attack. Wall-associated kinase (WAK) has the ability to perceive the changes in the cell wall matrix and transform signals into the cytoplasm, being involved in plant development and the defence response. Downy mildew, caused by Hyaloperonospora brassicae, can result in a massive loss in Chinese cabbage (Brassica rapa L. ssp. pekinensis) production. Herein, we identified a candidate resistant WAK gene, BrWAK1, in a major resistant quantitative trait locus, using a double haploid population derived from resistant inbred line T12-19 and the susceptible line 91-112. The expression of BrWAK1 could be induced by salicylic acid and pathogen inoculation. Expression of BrWAK1 in 91-112 could significantly enhance resistance to the pathogen, while truncating BrWAK1 in T12-19 increased disease susceptibility. Variation in the extracellular galacturonan binding (GUB) domain of BrWAK1 was found to mainly confer resistance to downy mildew in T12-19. Moreover, BrWAK1 was proved to interact with BrBAK1 (brassinosteroid insensitive 1 associated kinase), resulting in the activation of the downstream mitogen-activated protein kinase (MAPK) cascade to trigger the defence response. BrWAK1 is the first identified and thoroughly characterized WAK gene conferring disease resistance in Chinese cabbage, and the plant biomass is not significantly influenced by BrWAK1, which will greatly accelerate Chinese cabbage breeding for downy mildew resistance.

Recent Advancements and Biotechnological Implications of Carotenoid Metabolism of Brassica.

Carotenoids were synthesized in the plant cells involved in photosynthesis and photo-protection. In humans, carotenoids are essential as dietary antioxidants and vitamin A precursors. Brassica crops are the major sources of nutritionally important dietary carotenoids. Recent studies have unraveled the major genetic components in the carotenoid metabolic pathway in Brassica, including the identification of key factors that directly participate or regulate carotenoid biosynthesis. However, recent genetic advances and the complexity of the mechanism and regulation of Brassica carotenoid accumulation have not been reviewed. Herein, we reviewed the recent progress regarding Brassica carotenoids from the perspective of forward genetics, discussed biotechnological implications and provided new perspectives on how to transfer the knowledge of carotenoid research in Brassica to the crop breeding process.

Subgenome dominance and its evolutionary implications in crop domestication and breeding.

Polyploidization or whole-genome duplication (WGD) is a well-known speciation and adaptation mechanism in angiosperms, while subgenome dominance is a crucial phenomenon in allopolyploids, established following polyploidization. The dominant subgenomes contribute more to genome evolution and homoeolog expression bias, both of which confer advantages for short-term phenotypic adaptation and long-term domestication. In this review, we firstly summarize the probable mechanistic basis for subgenome dominance, including the effects of genetic [transposon, genetic incompatibility, and homoeologous exchange (HE)], epigenetic (DNA methylation and histone modification), and developmental and environmental factors on this evolutionary process. We then move to Brassica rapa, a typical allopolyploid with subgenome dominance. Polyploidization provides the B. rapa genome not only with the genomic plasticity for adapting to changeable environments, but also an abundant genetic basis for morphological variation, making it a representative species for subgenome dominance studies. According to the 'two-step theory', B. rapa experienced genome fractionation twice during WGD, in which most of the genes responding to the environmental cues and phytohormones were over-retained, enhancing subgenome dominance and consequent adaption. More than this, the pangenome of 18 B. rapa accessions with different morphotypes recently constructed provides further evidence to reveal the impacts of polyploidization and subgenome dominance on intraspecific diversification in B. rapa. Above and beyond the fundamental understanding of WGD and subgenome dominance in B. rapa and other plants, however, it remains elusive why subgenome dominance has tissue- and spatiotemporal-specific features and could shuffle between homoeologous regions of different subgenomes by environments in allopolyploids. We lastly propose acceleration of the combined application of resynthesized allopolyploids, omics technology, and genome editing tools to deepen mechanistic investigations of subgenome dominance, both genetic and epigenetic, in a variety of species and environments. We believe that the implications of genomic and genetic basis of a variety of ecologically, evolutionarily, and agriculturally interesting traits coupled with subgenome dominance will be uncovered and aid in making new discoveries and crop breeding.

The Adaxial/Abaxial Patterning of Auxin and Auxin Gene in Leaf Veins Functions in Leafy Head Formation of Chinese Cabbage.

Leaf curling is an essential prerequisite for the formation of leafy heads in Chinese cabbage. However, the part or tissue that determines leaf curvature remains largely unclear. In this study, we first introduced the auxin-responsive marker DR5::GUS into the Chinese cabbage genome and visualized its expression during the farming season. We demonstrated that auxin response is adaxially/abaxially distributed in leaf veins. Together with the fact that leaf veins occupy considerable proportions of the Chinese cabbage leaf, we propose that leaf veins play a crucial supporting role as a framework for heading. Then, by combining analyses of QTL mapping and a time-course transcriptome from heading Chinese cabbage and non-heading pak choi during the farming season, we identified the auxin-related gene BrPIN5 as a strong candidate for leafy head formation. PIN5 displays an adaxial/abaxial expression pattern in leaf veins, similar to that of DR5::GUS, revealing an involvement of BrPIN5 in leafy head development. The association of BrPIN5 function with heading was further confirmed by its haplo-specificity to heading individuals in both a natural population and two segregating populations. We thus conclude that the adaxial/abaxial patterning of auxin and auxin genes in leaf veins functions in the formation of the leafy head in Chinese cabbage.

The Carotenoid Esterification Gene BrPYP Controls Pale-Yellow Petal Color in Flowering Chinese Cabbage (Brassica rapa L. subsp. parachinensis).

Carotenoid esterification plays indispensable roles in preventing degradation and maintaining the stability of carotenoids. Although the carotenoid biosynthetic pathway has been well characterized, the molecular mechanisms underlying carotenoid esterification, especially in floral organs, remain poorly understood. In this study, we identified a natural mutant flowering Chinese cabbage (Caixin, Brassica rapa L. subsp. chinensis var. parachinensis) with visually distinguishable pale-yellow petals controlled by a single recessive gene. Transmission electron microscopy (TEM) demonstrated that the chromoplasts in the yellow petals were surrounded by more fully developed plastoglobules compared to the pale-yellow mutant. Carotenoid analyses further revealed that, compared to the pale-yellow petals, the yellow petals contained high levels of esterified carotenoids, including lutein caprate, violaxanthin dilaurate, violaxanthin-myristate-laurate, 5,6epoxy-luttein dilaurate, lutein dilaurate, and lutein laurate. Based on bulked segregation analysis and fine mapping, we subsequently identified the critical role of a phytyl ester synthase 2 protein (PALE YELLOW PETAL, BrPYP) in regulating carotenoid pigmentation in flowering Chinese cabbage petals. Compared to the yellow wild-type, a 1,148 bp deletion was identified in the promoter region of BrPYP in the pale-yellow mutant, resulting in down-regulated expression. Transgenic Arabidopsis plants harboring beta-glucuronidase (GUS) driven by yellow (BrPYP Y ::GUS) and pale-yellow type (BrPYP PY ::GUS) promoters were subsequently constructed, revealing stronger expression of BrPYP Y ::GUS both in the leaves and petals. Furthermore, virus-induced gene silencing of BrPYP significantly altered petal color from yellow to pale yellow. These findings demonstrate the molecular mechanism of carotenoid esterification, suggesting a role of phytyl ester synthase in carotenoid biosynthesis of flowering Chinese cabbage.

Natural variations of BrHISN2 provide a genetic basis for growth-flavour trade-off in different Brassica rapa subspecies.

Selection for yield during B. rapa breeding may have unintended consequences for other traits, such as flavour. LYH-type (light yellow head) Chinese cabbage (Brassica rapa ssp. pekinensis) and wucai (Brassica rapa L. ssp. chinensis var. rosularis) varieties are becoming popular because of their unique flavour and yellow leaves. However, the molecular mechanism underlying the interplay for these traits remains unknown. We conducted a fine mapping and genome-wide exploration analysis of the leaf yellowing of LYH and wucai, including transgenic plants, to identify causal genes. We identified that BrHISN2, a rate-limiting enzyme in histidine biosynthesis, causes leaf yellowing by destroying LYH chloroplasts. Normal growing Brhisn2 mutant plants became etiolated and senesced at the cotyledon-seedling stage. Sequence variations in the promoter confers cold-dependent expression on BrHISN2, probably resulting in leaf yellowing in LYH and wucai. Insertions of two DRE cis elements and the subsequent recruitment of two CBF2 proteins by the DREs to the promoter provided the cold-induced expression plasticity of BrHISN2 in plants. Both LYH and wucai are farmed in the fall, in which the temperature gradually decreases, therefore the CBF2-BrHISN2 module probably maximises the benefits of gene-environment interaction for breeding. We determined the mechanistic connections of chlorophyll synthesis and the growth-flavour trade-off in these B. rapa varieties.

Identification of long noncoding RNAs involved in resistance to downy mildew in Chinese cabbage.

Brassica downy mildew, a severe disease caused by Hyaloperonospora brassicae, can cause enormous economic losses in Chinese cabbage (Brassica rapa L. ssp. pekinensis) production. Although some research has been reported recently concerning the underlying resistance to this disease, no studies have identified or characterized long noncoding RNAs involved in this defense response. In this study, using high-throughput RNA sequencing, we analyzed the disease-responding mRNAs and long noncoding RNAs in two resistant lines (T12-19 and 12-85) and one susceptible line (91-112). Clustering and Gene Ontology analysis of differentially expressed genes (DEGs) showed that more DEGs were involved in the defense response in the two resistant lines than in the susceptible line. Different expression patterns and proposed functions of differentially expressed long noncoding RNAs among T12-19, 12-85, and 91-112 indicated that each has a distinct disease response mechanism. There were significantly more cis- and trans-functional long noncoding RNAs in the resistant lines than in the susceptible line, and the genes regulated by these RNAs mostly participated in the disease defense response. Furthermore, we identified a candidate resistance-related long noncoding RNA, MSTRG.19915, which is a long noncoding natural antisense transcript of a MAPK gene, BrMAPK15. Via an agroinfiltration-mediated transient overexpression system and virus-induced gene silencing technology, BrMAPK15 was indicated to have a greater ability to defend against pathogens. MSTRG.19915-silenced seedlings showed enhanced resistance to downy mildew, probably because of the upregulated expression of BrMAPK15. This research identified and characterized long noncoding RNAs involved in resistance to downy mildew, laying a foundation for future in-depth studies of disease resistance mechanisms in Chinese cabbage.

Genome-wide analysis of changes in miRNA and target gene expression reveals key roles in heterosis for Chinese cabbage biomass.

Heterosis is a complex phenomenon in which hybrids show better phenotypic characteristics than their parents do. Chinese cabbage (Brassica rapa L. spp. pekinensis) is a popular leafy crop species, hybrids of which are widely used in commercial production; however, the molecular basis of heterosis for biomass of Chinese cabbage is poorly understood. We characterized heterosis in a Chinese cabbage F1 hybrid cultivar and its parental lines from the seedling stage to the heading stage; marked heterosis of leaf weight and biomass yield were observed. Small RNA sequencing revealed 63 and 50 differentially expressed microRNAs (DEMs) at the seedling and early-heading stages, respectively. The expression levels of the majority of miRNA clusters in the F1 hybrid were lower than the mid-parent values (MPVs). Using degradome sequencing, we identified 1,819 miRNA target genes. Gene ontology (GO) analyses demonstrated that the target genes of the MPV-DEMs and low parental expression level dominance (ELD) miRNAs were significantly enriched in leaf morphogenesis, leaf development, and leaf shaping. Transcriptome analysis revealed that the expression levels of photosynthesis and chlorophyll synthesis-related MPV-DEGs (differentially expressed genes) were significantly different in the F1 hybrid compared to the parental lines, resulting in increased photosynthesis capacity and chlorophyll content in the former. Furthermore, expression of genes known to regulate leaf development was also observed at the seedling stage. Arabidopsis plants overexpressing BrGRF4.2 and bra-miR396 presented increased and decreased leaf sizes, respectively. These results provide new insight into the regulation of target genes and miRNA expression patterns in leaf size and heterosis for biomass of B. rapa.

Assembly of the non-heading pak choi genome and comparison with the genomes of heading Chinese cabbage and the oilseed yellow sarson.

Brassica rapa displays a wide range of morphological diversity which is exploited for a variety of food crops. Here we present a high-quality genome assembly for pak choi (Brassica rapa L. subsp. chinensis), an important non-heading leafy vegetable, and comparison with the genomes of heading type Chinese cabbage and the oilseed form, yellow sarson. Gene presence-absence variation (PAV) and genomic structural variations (SV) were identified, together with single nucleotide polymorphisms (SNPs). The structure and expression of genes for leaf morphology and flowering were compared between the three morphotypes revealing candidate genes for these traits in B. rapa. The pak choi genome assembly and its comparison with other B. rapa genome assemblies provides a valuable resource for the genetic improvement of this important vegetable crop and as a model to understand the diversity of morphological variation across Brassica species.

A histone H4 gene prevents drought-induced bolting in Chinese cabbage by attenuating the expression of flowering genes.

Flowering is an important trait in Chinese cabbage, because premature flowering reduces yield and quality of the harvested products. Water deficit, caused by drought or other environmental conditions, induces early flowering. Drought resistance involves global reprogramming of transcription, hormone signaling, and chromatin modification. We show that a histone H4 protein, BrHIS4.A04, physically interacts with a homeodomain protein BrVIN3.1, which was selected during the domestication of late-bolting Chinese cabbage. Over-expression of BrHIS4.A04 resulted in premature flowering under normal growth conditions, but prevented further premature bolting in response to drought. We show that the expression of key abscisic acid (ABA) signaling genes, and also photoperiodic flowering genes was attenuated in BrHIS4.A04-overexpressing (BrHIS4.A04OE) plants under drought conditions. Furthermore, the relative change in H4-acetylation at these gene loci was reduced in BrHIS4.A04OE plants. We suggest that BrHIS4.A04 prevents premature bolting by attenuating the expression of photoperiodic flowering genes under drought conditions, through the ABA signaling pathway. Since BrHIS4.A04OE plants displayed no phenotype related to vegetative or reproductive development under laboratory-induced drought conditions, our findings contribute to the potential fine-tuning of flowering time in crops through genetic engineering without any growth penalty, although more data are necessary under field drought conditions.

Genome-wide analysis of mRNA and lncRNA expression and mitochondrial genome sequencing provide insights into the mechanisms underlying a novel cytoplasmic male sterility system, BVRC-CMS96, in Brassicarapa.

KEY MESSAGE: Characterization of a novel and valuable CMS system in Brassicarapa. Cytoplasmic male sterility (CMS) is extensively used to produce F1 hybrid seeds in a variety of crops. However, it has not been successfully used in Chinese cabbage (Brassicarapa L. ssp. pekinensis) because of degeneration or temperature sensitivity. Here, we characterize a novel CMS system, BVRC-CMS96, which originated in B.napus cybrid obtained from INRAE, France and transferred by us to B.rapa. Floral morphology and agronomic characteristics indicate that BVRC-CMS96 plants are 100% male sterile and show no degeneration in the BC7 generation, confirming its suitability for commercial use. We also sequenced the BVRC-CMS96 and maintainer line 18BCM mitochondrial genomes. Genomic analyses showed the presence of syntenic blocks and distinct structures between BVRC-CMS96 and 18BCM and the other known CMS systems. We found that BVRC-CMS96 has one orf222 from 'Nap'-type CMS and two copies of orf138 from 'Ogu'-type CMS. We analyzed expression of orf222, orf138, orf261b, and the mitochondrial energy genes (atp6, atp9, and cox1) in flower bud developmental stages S1-S5 and in four floral organs. orf138 and orf222 were both highly expressed in S4, S5-stage buds, calyx, and the stamen. RNA-seq identified differentially expressed mRNAs and lncRNAs (long non-coding RNAs) that were significantly enriched in pollen wall assembly, pollen development, and pollen coat. Our findings suggest that an energy supply disorder caused by orf222/orf138/orf261b may inhibit a series of nuclear pollen development-related genes. Our study shows that BVRC-CMS96 is a valuable CMS system, and our detailed molecular analysis will facilitate its application in Chinese cabbage breeding.

Identification and fine mapping of qSB.A09, a major QTL that controls shoot branching in Brassica rapa ssp. chinensis Makino.

KEY MESSAGE: QTL mapping plus bulked segregant analysis revealed a major QTL for shoot branching in non-heading Chinese cabbage. The candidate gene was then identified using sequence alignment and expression analysis. Shoot branching is a complex quantitative trait that contributes to plant architecture and ultimately yield. Although many studies have examined branching in grain crops, the genetic control of shoot branching in vegetable crops such as Brassica rapa L. ssp. chinensis remains poorly understood. In this study, we used bulked segregant analysis (BSA) of an F2 population to detect a major quantitative trait locus (QTL) for shoot branching, designated shoot branching 9 (qSB.A09) on the long arm of chromosome A09 in Brassica rapa L. ssp. chinensis. In addition, traditional QTL mapping of the F2 population revealed six QTLs in different regions. Of these, the mapping region on chromosome A09 was consistent with the results of BSA-seq analysis, as well as being stable over the 2-year study period, explaining 19.37% and 22.18% of the phenotypic variation across multiple genetic backgrounds. Using extreme recombinants, qSB.A09 was further delimited to a 127-kb genomic region harboring 28 annotated genes. We subsequently identified the GRAS transcription factor gene Bra007056 as a potential candidate gene; Bra007056 is an ortholog of MONOCULM 1 (MOC1), the key gene that controls tillering in rice. Quantitative RT-PCR further revealed that expression of Bra007056 was positively correlated with the shoot branching phenotype. Furthermore, an insertion/deletion marker specific to Bra007056 co-segregated with the shoot branching trait in the F2 populations. Overall, these results provide the basis for elucidating the molecular mechanism of shoot branching in Brassica rapa ssp. chinensis Makino.

Natural variation in a calreticulin gene causes reduced resistance to Ca2+ deficiency-induced tipburn in Chinese cabbage (Brassica rapa ssp. pekinensis).

Tipburn is an irreversible physiological disorder of Chinese cabbage that decreases crop value. Because of a strong environmental component, tipburn-resistant cultivars are the only solution, although tipburn resistance genes are unknown in Chinese cabbage. We studied three populations of Chinese cabbage over four growing seasons under field conditions: (a) 194 diverse inbred lines, (b) a doubled haploid (DH100) population, and (c) an F2 population. The 194 lines were genotyped using single nucleotide polymorphism markers, and genome-wide-association mapping showed that 24 gQTLs were significantly associated with tipburn disease index. Analysis of the DH100 and F2 populations identified a shared tipburn-associated locus, gqbTRA06, that was found to cover the region defined by one of the 24 gQTLs. Of 35 genes predicted in the 0.14-Mb quantitative trait locus region, Bra018575 (calreticulin family protein, BrCRT2) showed higher expression levels during disease development. We cloned the two BrCRT2 alleles from tipburn-resistant (BrCRT2R ) and tipburn-susceptible (BrCRT2S ) lines and identified a 51-bp deletion in BrCRT2S . Overexpression of BrCRT2R increased Ca2+ storage in the Arabidopsis crt2 mutant and also reduced cell death in leaf tips and margins under Ca2+ -depleted conditions. Our results suggest that BrCRT2 is a possible candidate gene for controlling tipburn in Chinese cabbage.

BrLAS, a GRAS Transcription Factor From Brassica rapa, Is Involved in Drought Stress Tolerance in Transgenic Arabidopsis.

GRAS proteins belong to a plant-specific transcription factor family and play roles in diverse physiological processes and environmental signals. In this study, we identified and characterized a GRAS transcription factor gene in Brassica rapa, BrLAS, an ortholog of Arabidopsis AtLAS. BrLAS was primarily expressed in the roots and axillary meristems, and localized exclusively in the nucleus of B. rapa protoplast cells. qRT-PCR analysis indicated that BrLAS was upregulated by exogenous abscisic acid (ABA) and abiotic stress treatment [polyethylene glycol (PEG), NaCl, and H2O2]. BrLAS-overexpressing Arabidopsis plants exhibited pleiotropic characteristics, including morphological changes, delayed bolting and flowering time, reduced fertility and delayed senescence. Transgenic plants also displayed significantly enhanced drought resistance with decreased accumulation of ROS and increased antioxidant enzyme activity under drought treatment compared with the wild-type. Increased sensitivity to exogenous ABA was also observed in the transgenic plants. qRT-PCR analysis further showed that expression of several genes involved in stress responses and associated with leaf senescence were also modified. These findings suggest that BrLAS encodes a stress-responsive GRASs transcription factor that positively regulates drought stress tolerance, suggesting a role in breeding programs aimed at improving drought tolerance in plants.

BrRLP48, Encoding a Receptor-Like Protein, Involved in Downy Mildew Resistance in Brassica rapa.

Downy mildew, caused by Hyaloperonospora parasitica, is a major disease of Brassica rapa that causes large economic losses in many B. rapa-growing regions of the world. The genotype used in this study was based on a double haploid population derived from a cross between the Chinese cabbage line BY and a European turnip line MM, susceptible and resistant to downy mildew, respectively. We initially located a locus Br-DM04 for downy mildew resistance in a region about 2.7 Mb on chromosome A04, which accounts for 22.3% of the phenotypic variation. Using a large F2 mapping population (1156 individuals) we further mapped Br-DM04 within a 160 kb region, containing 17 genes encoding proteins. Based on sequence annotations for these genes, four candidate genes related to disease resistance, BrLRR1, BrLRR2, BrRLP47, and BrRLP48 were identified. Overexpression of both BrRLP47 and BrRLP48 using a transient expression system significantly enhanced the downy mildew resistance of the susceptible line BY. But only the leaves infiltrated with RNAi construct of BrRLP48 could significantly reduce the disease resistance in resistant line MM. Furthermore, promoter sequence analysis showed that one salicylic acid (SA) and two jasmonic acid-responsive transcript elements were found in BrRLP48 from the resistant line, but not in the susceptible one. Real-time PCR analysis showed that the expression level of BrRLP48 was significantly induced by inoculation with downy mildew or SA treatment in the resistant line MM. Based on these findings, we concluded that BrRLP48 was involved in disease resistant response and the disease-inducible expression of BrRLP48 contributed to the downy mildew resistance. These findings led to a new understanding of the mechanisms of resistance and lay the foundation for marker-assisted selection to improve downy mildew resistance in Brassica rapa.

A Genomic Variation Map Provides Insights into the Genetic Basis of Spring Chinese Cabbage (Brassica rapa ssp. pekinensis) Selection.

Chinese cabbage is the most consumed leafy crop in East Asian countries. However, premature bolting induced by continuous low temperatures severely decreases the yield and quality of the Chinese cabbage, and therefore restricts its planting season and geographic distribution. In the past 40 years, spring Chinese cabbage with strong winterness has been selected to meet the market demand. Here, we report a genome variation map of Chinese cabbage generated from the resequencing data of 194 geographically diverse accessions of three ecotypes. In-depth analyses of the selection sweeps and genome-wide patterns revealed that spring Chinese cabbage was selected from a specific population of autumn Chinese cabbage around the area of Shandong peninsula in northern China. We identified 23 genomic loci that underwent intensive selection, and further demonstrated by gene expression and haplotype analyses that the incorporation of elite alleles of VERNALISATION INSENTIVE 3.1 (BrVIN3.1) and FLOWER LOCUS C 1 (BrFLC1) is a determinant genetic source of variation during selection. Moreover, we showed that the quantitative response of BrVIN3.1 to cold due to the sequence variations in the cis elements of the BrVIN3.1 promoter significantly contributes to bolting-time variation in Chinese cabbage. Collectively, our study provides valuable insights into the genetic basis of spring Chinese cabbage selection and will facilitate the breeding of bolting-resistant varieties by molecular-marker-assisted selection, transgenic or gene editing approaches.

Effects of Endogenous Salicylic Acid During Calcium Deficiency-Induced Tipburn in Chinese Cabbage (Brassica rapa L. ssp. pekinensis).

By cultivating tipburn-susceptible plants in modified Hoagland's medium containing of gradient exogenous calcium (Ca2+), we have shown that Ca2+ deficiency is one of the main causes of tipburn in Chinese cabbage (Brassica rapa L. ssp. pekinensis). The effect of endogenous plant Ca2+ concentrations on tipburn was also studied in a doubled haploid (DH) population consisting of 100 individuals, but no correlation was found. We then examined the expression of 12 Ca2+ transporter genes that function in cytosolic Ca2+ homeostasis in both tipburn-susceptible and tipburn-resistant plants under normal and tipburn-inducing conditions. Expression patterns for most of these genes differed between the two types of plants. Salicylic acid (SA) accumulated in response to conditions of calcium deficiency in our study, and both total SA and SA ß-glucoside (SAG) in tipburn-susceptible plants was ∼3-fold higher than it was in resistant plants following Ca2+ deficiency treatment. Also, the changes observed in SA levels correlated well with cell death patterns revealed by trypan blue staining. Therefore, we speculate that the cytoplasmic Ca2+ fluctuation-induced downstream signaling events, as well as SA signaling or other biological events, are involved in the plant defense response to tipburn in Chinese cabbage.

[Development of molecular markers linked to the resistant QTL for downy mildew in Brassica rapa L. ssp. pekinensis].

Downy mildew, caused by the oomycete Hyaloperonospora parasitica Constant. (Pers. ex Fr.), is one of the most severe diseases in Chinese cabbage, leading to reduction of yield and quality of the harvested products. Therefore, identifying molecular markers linked to the major QTL for downy mildew resistance will be helpful in breeding resistant varieties of Chinese cabbage. Here, one highly susceptible line 91-112, one highly resistant line T12-19, and the derived DH population were employed to develop linked molecular markers for the major QTL, BrDW, for downy mildew. With BLAST and IMap analysis, the RAPD marker K14-1030 linked to BrDW was anchored on KBrB058M10 (on Contig214). On the basis of the BAC and BAC-end sequences around KBrB058M10, a set of PCR primers were designed, and the methods of restriction analysis and HRM analysis were used to develop molecular makers. Finally, five polymorphism markers were developed, containing one Indel marker named Brb062-Indel230, three CAPS markers named Brb094-DraⅠ787, Brb094-AatⅡ666 and Brb043-BglⅡ715, and one SNP marker named Brh019-SNP137. In addition, one SSR marker from Unigene sequence homologous with KBrB058M10 (known as bru1209) was developed. The map distances between the six markers and RAPD marker K14-1030 were 4.3 cM, 1.7 cM, 5.9 cM, 5.9 cM, 4.6 cM, and 0.8 cM, respectively. The percentage of accuracy in selecting for downy mildew-resistant lines from the DH population were 69.7%, 70.9%, 72.4%, 72.4%, 58.3%, and 74.2%. These markers could be used in marker assisted selection to improve downy mildew resistance in Chinese cabbage.

[cDNA-AFLP analysis on transcripts associated with bolting in Brassica rapa L. ssp. pekinensis].

Premature bolting, caused by low temperature in spring and summer cultivation in low land and high land respectively, leads to reduction of the yield and quality of the harvested products in Chinese cabbage. Therefore, exploring genes involved in vernalization response is important to the improvement of Chinese cabbage varieties. Here, one extremely early bolting line (DH-54) and one extremely late bolting line (DH-43) were employed, and the cDNA-AFLP approach was used to identify key components involved in the low-temperature required vernalization response. Of 256 primer recombinations screened, a total of 191 differential expressed transcript-derived fragments (TDFs) were identified, and 82 TDFs were sequenced. BLAST and alignments showed that 52 candidate TDFs shared high levels of similarity with genes of known function, 22 TDFs of unknown function and 8 novel ESTs. The TDFs of known function were involved in genes encoding enzymes working in metabolism, proteins related to stress and defense, signal transduction, and transcription regulation, etc.