Mapping of isolate-specific QTLs for clubroot resistance in Chinese cabbage (<Emphasis Type="Italic">Brassica rapa</Emphasis> L. ssp. <Emphasis Type="Italic">pekinensis</Emphasis>)

A number of clubroot resistant (CR) Chinese cabbage cultivars have been developed in Japan using resistant genes from CR European fodder turnips (B. rapa ssp. rapifera). Clubroot resistance in European fodder turnips are known to be controlled by the combined action of several dominant resistance genes. We have developed three Chinese cabbage clubroot-resistant doubled haploid (DH) lines-T136-8, K10, and C9-which express resistance in different manners against two isolates of Plasmodiophora brassicae, M85 and K04. Depending on the isolates, we identified two CR loci, CRk and CRc. CRk was identified by quantitative trait loci (QTL) analysis of an F(2) population derived from a cross between K10 and Q5. This locus showed resistance to both isolates and is located close to Crr3 in linkage group R3. The other locus, CRc was identified by QTL analysis of an F(2) population derived from a cross between C9 and susceptible DH line, 6R. This locus was mapped to linkage group R2 and is independent from any published CR loci. We developed sequence-tagged site markers linked to this locus.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation system for large-scale producion of transgenic chinese cabbage (<Emphasis Type="Italic">Brassica rapa</Emphasis> L. ssp.<Emphasis Type="Italic">pekinensis</Emphasis>) plants for insertional mutagenesis

In order to better utilize insertional mutagenesis and functional genomics in Chinese cabbage, we have developed an improved transformation system that more efficiently produces a large number of transgenic plants. Hypocotyl explants were inoculated withAgrobacterium tumefaciens LBA4404. This strain harbors tagging vector pRCV2, which contains a hygromycin-resistance gene, an ampicillin resistance gene, and a bacterial replication origin within the T-DNA. Transformation efficiency was highest when the explants were first co-cultivated for 3 d in a medium supplemented with 5 mg L^-1 acetosyringone, then transferred to a 0.8% agar selection medium containing 10 mg L^-1 hygro-mycin. In addition, maintaining a low pH in the co-cultivation medium was critical to enhancing transformation frequency. A total of 3369 transgenic plants were obtained, with efficiencies ranging from 2.89% to 5.00%. Southern blot analysis and T, progeny tests from 120 transgenic plants confirmed that the transgenes were stably inherited to the next generation. We also conducted plasmid rescue and inverse PCR with some transformants, based on their phenotype, to demonstrate the applicability of T-DNA tagging in Chinese cabbage. The tagged sequences were then analyzed.     

Effects of Endogenous Salicylic Acid During Calcium Deficiency-Induced Tipburn in Chinese Cabbage (<Emphasis Type="Italic">Brassica rapa</Emphasis> L. ssp. <Emphasis Type="Italic">pekinensis</Emphasis>)

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 β-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.     

Integrated analysis of leaf morphological and color traits in different populations of Chinese cabbage (<Emphasis Type="Italic">Brassica rapa</Emphasis> ssp. <Emphasis Type="Italic">pekinensis</Emphasis>)

Key message

QTLs and candidate gene markers associated with leaf morphological and color traits were identified in two immortalized populations of Brassica rapa, which will provide genetic information for marker-assisted breeding.

Abstract

Brassica rapa is an important leafy vegetable consumed worldwide and morphology is a key character for its breeding. To enhance genetic control, quantitative trait loci (QTLs) for leaf color and plant architecture were identified using two immortalized populations with replications of 2 and 4 years. Overall, 158 and 80 QTLs associated with 23 and 14 traits were detected in the DH and RIL populations, respectively. Among them, 23 common robust-QTLs belonging to 12 traits were detected in common loci over the replications. Through comparative analysis, five crucifer genetic blocks corresponding to morphology trait (R, J&U, F and E) and color trait (F, E) were identified in three major linkage groups (A2, A3 and A7). These might be key conserved genomic regions involved with the respective traits. Through synteny analysis with Arabidopsis, 64 candidate genes involved in chlorophyll biosynthesis, cell proliferation and elongation were co-localized within QTL intervals. Among them, SCO3, ABI3, FLU, HCF153, HEMB1, CAB3 were mapped within QTLs for leaf color; and CYCD3;1, CYCB2;4, AN3, ULT1 and ANT were co-localized in QTL regions for leaf size. These robust QTLs and their candidate genes provide useful information for further research into leaf architecture with crop breeding.

     

Induction of male sterile cabbage using a tapetum-specific promoter from<Emphasis Type="Italic"> Brassica campestris</Emphasis> L. ssp.<Emphasis Type="Italic"> pekinensis</Emphasis>

The anther (tapetum)-specific gene BcA9 was isolated from Chinese cabbage, Brassica campestris L. ssp. pekinensis cv. Jangwon, using the Arabidopsis tapetum-specific A9 gene as a probe. The DNA and amino acid sequences of the coding region of the BcA9 gene showed high homology with A9 genes from Arabidopsis and B. napus. However, the DNA sequences of the 5 noncoding (promoter) region were different, except for the sequence from –281 to –89. To test the specific activity of this promoter, a plant expression vector, pGR011, was constructed by fusing the BcA9 promoter and the cytotoxic diphtheria toxin A-chain (DTx-A) gene. Several transgenic plants from cabbage, B. oleracea ssp. capitata, were obtained by way of Agrobacterium-mediated transformation. Southern blot analysis indicated that the tapetum-specific BcA9 promoter and DTx-A gene were successfully integrated into the genome of the transgenic cabbage. Under the control of the BcA9 promoter, expression of the cytotoxic DTx-A gene in the tapetal cells of the transgenic plants resulted in male sterile cabbages. Microscopic examination revealed that pollen grains in anthers of the male sterile cabbages had not developed normally, but the vegetative growth and phenotype showed no difference compared to wild-type plants.Abbreviations At Arabidopsis thaliana - Bc Brassica camepstris - Bn Brassica napus - DTx-A Diphtheria toxin A-chain gene - hpt Hygromycin phosphotransferase - PCR Polymerase chain reaction - SDS Sodium dodecyl sulfate - SSC Sodium chloride-sodium citrate bufferThis revised version was published online September 2003 with corrections to Figure 6.Communicated by I.S. Chung     

Impact of <Emphasis Type="Italic">cry1AC</Emphasis>-carrying <Emphasis Type="Italic">Brassica rapa</Emphasis> subsp. <Emphasis Type="Italic">pekinensis</Emphasis> on leaf bacterial community

The effects of Chinese cabbage (Brassica rapa subsp. pekinensis) carrying cry1AC derived from Bacillus thuringiensis (Bt) on leaf bacterial community were examined by analyzing the horizontal transfer of trans-gene fragments from plants to bacteria. The effect of plant pathogenic bacteria on the gene transfer was also examined using Pseudomonas syringae pathovar. maculicola. The frequency of hygromycin-resistant bacteria did not alter in Bt leaves, though slight increase was observed in Pseudomonas-infected Bt leaves with no statistical significance. The analysis of bacterial community profiles using the denaturing gradient gel electrophoresis (DGGE) fingerprinting indicated that there were slight differences between Bt and control Chinese cabbage, and also that infected tissues were dominated by P. syringae pv. maculicola. However, the cultured bacterial pools were not found to contain any transgene fragments. Thus, no direct evidence of immediate gene transfer from plant to bacteria or acquisition of hygromycin resistance could be observed. Still, long-term monitoring on the possibility of gene transfer is necessary to correctly assess the environmental effects of the Bt crop on bacteria.     

A high-resolution karyotype of <Emphasis Type="Italic">Brassica rapa</Emphasis> ssp. <Emphasis Type="Italic">pekinensis</Emphasis> revealed by pachytene analysis and multicolor fluorescence in situ hybridization

A molecular cytogenetic map of Chinese cabbage (Brassica rapa ssp. pekinensis, 2n=20) was constructed based on the 4-6-diamino-2-phenylindole dihydrochloride-stained mitotic metaphase and pachytene chromosomes and multicolor fluorescence in situ hybridization (McFISH), using three repetitive DNA sequences, 5S rDNA, 45S rDNA, and C11-350H. The lengths of mitotic metaphase chromosomes ranged from 1.46 m to 3.30 m. Five 45S and three 5S rDNA loci identified were assigned to different chromosomes. The C11-350H loci were located on all the mitotic metaphase chromosomes, except chromosomes 2 and 4. The pachytene karyotype consisted of two metacentric (chromosomes 1 and 6), five submetacentric (chromosomes 3, 4, 5, 9 and 10), two subtelocentric (chromosomes 7 and 8), and one acrocentric (chromosome 2) chromosome(s). The mean lengths of ten pachytene chromosomes ranged from 23.7 m to 51.3 m, with a total of 385.3 m, which is 17.5-fold longer than that of the mitotic metaphase chromosomes. In the proposed pachytene karyotype, all the chromosomes of B. rapa ssp. pekinensis can be identified on the basis of chromosome length, centromere position, heterochromatin pattern, and the location of the three repetitive sequences. Moreover, the precise locations of the earlier reported loci of 5S rDNA, 45S rDNA, and Chinese cabbage tandem DNA repeat C11-350H were established using McFISH analysis. We also identified a 5S rDNA locus on the long arm of pachytene bivalent 7, which could not be detected in the mitotic metaphase chromosomes in the present and earlier studies. The deduced karyotype will be useful for structural and functional genomic studies in B. rapa.     

Expression of a glutathione reductase from <Emphasis Type="Italic">Brassica rapa</Emphasis> subsp. <Emphasis Type="Italic">pekinensis</Emphasis> enhanced cellular redox homeostasis by modulating antioxidant proteins in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Glutathione reductase (GR) is an enzyme that recycles a key cellular antioxidant molecule glutathione (GSH) from its oxidized form (GSSG) thus maintaining cellular redox homeostasis. A recombinant plasmid to overexpress a GR of Brassica rapa subsp. pekinensis (BrGR) in E. coli BL21 (DE3) was constructed using an expression vector pKM260. Expression of the introduced gene was confirmed by semiquantitative RT-PCR, immunoblotting and enzyme assays. Purification of the BrGR protein was performed by IMAC method and indicated that the BrGR was a dimmer. The BrGR required NADPH as a cofactor and specific activity was approximately 458 U. The BrGR-expressing E. coli cells showed increased GR activity and tolerance to H₂O₂, menadione, and heavy metal (CdCl₂, ZnCl₂ and AlCl₂)-mediated growth inhibition. The ectopic expression of BrGR provoked the co-regulation of a variety of antioxidant enzymes including catalase, superoxide dismutase, glutathione peroxidase, and glucose-6-phosphate dehydrogenase. Consequently, the transformed cells showed decreased hydroperoxide levels when exposed to stressful conditions. A proteomic analysis demonstrated the higher level of induction of proteins involved in glycolysis, detoxification/oxidative stress response, protein folding, transport/binding proteins, cell envelope/porins, and protein translation and modification when exposed to H₂O₂ stress. Taken together, these results indicate that the plant GR protein is functional in a cooperative way in the E. coli system to protect cells against oxidative stress.     

Genome-wide characterization and stress-responsive expression profiling of <Emphasis Type="Italic">MCM</Emphasis> genes in <Emphasis Type="Italic">Brassica oleracea</Emphasis> and <Emphasis Type="Italic">Brassica rapa</Emphasis>

The Minichromosome maintenance protein [MCM (2-7)] complex is associated with helicase activity for replication fork formation during DNA replication. We identified and characterized each 12 putative MCM genes from Brassica oleracea and Brassica rapa. MCM genes were classified into nine groups according to their evolutionary relationships. A high number of syntenic regions were present on chromosomes C03 and A03 in B. oleracea and B. rapa, respectively, compared to the other chromosomes. Expression analysis showed that most of the MCM(2-7) helicase-subunit genes and their coregulating MCM genes were upregulated during hydroxyurea (HU) induced stress in B. oleracea. In B. rapa, MCM(2-7) helicase genes BrMCM2_2, BrMCM7_1, BrMCM7_2 and their co-regulating genes were upregulated during replication stress. During cold stress, BoMCM6 in B. oleracea and BrMCM5 in B. rapa were remarkably upregulated. During salt stress, BoMCM6_2, BoMCM7_1, BoMCM8, BoMCM9, and BoMCM10 were markedly upregulated in B. oleracea. Hence, our study identified the candidate MCM family genes those possess abiotic stress-responsive behavior and DNA replication stress tolerance. As the first genome-wide analysis of MCM genes in B. oleracea and B. rapa, this work provides a foundation to develop stress responsive plants. Further functional and molecular studies on MCM genes will be helpful to enhance stress tolerance in plants.     

Characterization of directly transformed weedy <Emphasis Type="Italic">Brassica rapa</Emphasis> and introgressed <Emphasis Type="Italic">B. rapa</Emphasis> with Bt <Emphasis Type="Italic">cry1Ac</Emphasis> and <Emphasis Type="Italic">gfp</Emphasis> genes

Crop to weed transgene flow, which could result in more competitive weed populations, is an agricultural biosafety concern. Crop Brassica napus to weedy Brassica rapa hybridization has been extensively characterized to better understand the transgene flow and its consequences. In this study, weedy accessions of B. rapa were transformed with Bacillus thuringiensis (Bt) cry1Ac- and green fluorescence protein (gfp)-coding transgenes using Agrobacterium to assess ecological performance of the wild biotype relative to introgressed hybrids in which the transgenic parent was the crop. Regenerated transgenic B. rapa events were characterized by progeny analysis, Bt protein enzyme-linked immunosorbent assay (ELISA), Southern blot analysis, and GFP expression assay. GFP expression level and Bt protein concentration were significantly different between independent transgenic B. rapa events. Similar reproductive productivity was observed in comparison between transgenic B. rapa events and B. rapa × B. napus introgressed hybrids in greenhouse and field experiments. In the greenhouse, Bt transgenic plants experienced significantly less herbivory damage from the diamondback moth (Plutella xylostella). No differences were found in the field experiment under ambient, low, herbivore pressure. Directly transformed transgenic B. rapa plants should be a helpful experimental control to better understand crop genetic load in introgressed transgenic weeds.     

Accumulation and detoxification of cadmium in <Emphasis Type="Italic">Brassica pekinensis</Emphasis> and <Emphasis Type="Italic">B. chinensis</Emphasis>

The effect of excessive Cd on the growth and metal uptake by leafy vegetables Brassica chinensis L. (cv. Wuyueman) and Brassica pekinensis (Lour.) Rupr. (cv. Qingyan 87-114) were studied in hydroponic solution culture. The Cd concentration higher than 10 μM significantly decreased the root elongation and leaf chlorophyll contents of both plant species. The shoots of B. pekinensis had significantly higher concentrations of total and water-soluble Cd than B. chinensis. The roots of both species accumulated more Cd than the shoots in all the Cd treatments. Most of the Cd in the roots was found in the cell walls. The shoot/root ratio of Cd concentrations in B. pekinensis was always greater than that in B. chinensis. But, the concentration and proportion of Cd in the cell walls in B. chinensis were higher than that in B. pekinensis. Cadmium treatments also increased the concentrations of total non-protein thiols (NPT) in the shoots of the both species. A significant correlation was found between the concentrations of soluble Cd and NPT in plant shoots.     

Candidate gene prediction for a petal degeneration mutant, <Emphasis Type="Italic">pdm</Emphasis>, of the Chinese cabbage (<Emphasis Type="Italic">Brassica campestris</Emphasis> ssp. <Emphasis Type="Italic">pekinensis</Emphasis>) by using fine mapping and transcriptome analysis

A stably inherited petal degeneration mutant pdm of the Chinese cabbage was obtained from its wild-type ‘FT’ by radiation treatment (⁶⁰Co γ-rays) and isolated microspore culture. Petals of the pdm mutant were observed to be shriveled, degenerated, not fully expanded, and darker at the flowering stage than those of ‘FT.’ The pdm mutant phenotype was found to be controlled by a single recessive nuclear gene. For linkage analysis and gene mapping, 1419 recessive homozygous individuals with the pdm phenotype of the F₂ generation were investigated as the mapping population. Results showed that the pdm was located between markers Indelhsn26 and SSRhsn123 at a genetic distance of 0.04 and 0.04 cM, respectively, on linkage group A01. Physical distance between Indelhsn26 and SSRhsn123, the two most closely linked markers, was estimated to be approximately 285.2 kb. Twenty-eight genes were predicted in the target region. Using RNA-seq, Bra040093 was predicted to be the most likely candidate gene for pdm. Based on gene annotation, Bra040093 encodes a peroxisomal acyl-coenzyme A oxidase 1 (ACX1). Comparison of the sequences in pdm and ‘FT’ revealed two single-nucleotide polymorphisms in pdm. Expression patterns of Bra040093 between pdm and ‘FT’ were analyzed using quantitative real-time PCR, and the expression level was dramatically higher in ‘FT’ than in pdm. These findings provide a solid foundation and valuable resources for map-based cloning, identification, and functional analysis of pdm and facilitate the understanding of floral development processes in the Chinese cabbage.     

Denaturing gradient gel electrophoresis analysis of bacterial community profiles in the rhizosphere of <Emphasis Type="Italic">cry1AC-carrying Brassica rapa</Emphasis> subsp. <Emphasis Type="Italic">pekinensis</Emphasis>

The effect of genetically modified (GM) Brassica rapa subsp. pekinensis (Chinese cabbage) expressing Bt toxin gene (cry1AC) to the rhizosphere bacterial community was examined using the denaturing gradient gel electrophoresis (DGGE) fingerprinting method. From the visual comparison of the DGGE profiles, there were no significant differences between the profiles of Bt and control rhizosphere in both Suwon and Yesan samples. From the sequence analysis of the individual bands, Sphingomonas sp. of Alphaproteobacteria and several actinobacterial members were identified as the main bacterial taxa in both Suwon and Yesan samples. In the multiple correspondence analysis, no clear separation between Bt and control rhizosphere was seen in both Suwon and Yesan datasets. The profiles of bulk soils were separated from those of rhizosphere. The DGGE fingerprinting analyses indicated that Bt crops did not significantly alter the genetic composition of rhizosphere bacterial communities.     

Metabolism-mediated induction of zinc tolerance in <Emphasis Type="Italic">Brassica rapa</Emphasis> by <Emphasis Type="Italic">Burkholderia cepacia</Emphasis> CS2-1

Brassica rapa (Chinese cabbage) is an essential component of traditional Korean food. However, the crop is often subject to zinc (Zn⁺) toxicity from contaminated irrigation water, which, as a result, compromises plant growth and production, as well as the health of human consumers. The present study investigated the bioaccumulation of Zn⁺ by Burkholderia cepacia CS2-1 and its effect on the heavy metal tolerance of Chinese cabbage. Strain CS2-1 was identified and characterized on the basis of 16S rRNA sequences and phylogenetic analysis. The strain actively produced indole-3-acetic acid (3.08 ± 0.21 μg/ml) and was also able to produce siderophore, solubilize minerals, and tolerate various concentrations of Zn⁺. The heavy metal tolerance of B. rapa plants was enhanced by CS2-1 inoculation, as indicated by growth attributes, Zn⁺ uptake, amino acid synthesis, antioxidant levels, and endogenous hormone (ABA and SA) synthesis. Without inoculation, the application of Zn⁺ negatively affected the growth and physiology of B. rapa plants. However, CS2-1 inoculation improved plant growth, lowered Zn⁺ uptake, altered both amino acid regulation and levels of flavonoids and phenolics, and significantly decreased levels of superoxide dismutase, endogenous abscisic acid, and salicylic acid. These findings indicate that B. cepacia CS2-1 is suitable for bioremediation against Zn⁺-induced oxidative stress.