与大白菜霜霉病抗性主效QTL连锁的分子标记开发

霜霉病是危害大白菜的三大病害之一,该病的发生会严重影响大白菜的产量及品质,因而研究与霜霉病抗性QTL紧密连锁的分子标记对大白菜抗病新品种培育具有重要意义。该研究在前期工作的基础上,选用高感霜霉病株系91-112、高抗霜霉病株系T12-19以及由二者为双亲构建的DH群体为实验材料,针对大白菜霜霉病抗性主效QTL——BrDW所在的标记区间,利用已有的大白菜基因组信息发展与抗性QTL紧密连锁的分子标记,通过Blast和IMap分析,将与BrDW连锁的RAPD标记K14-1030定位于大白菜KBrB058M10上(位于Contig214上),根据KBrB058M10附近的BAC及BAC-end序列设计引物,结合限制性内切酶酶切及HRM分析方法,筛选得到5个与BrDW连锁的分子标记,包括1个Indel标记Brb062-Indel230,3个CAPS标记Brb094-DraⅠ787、Brb094-AatⅡ666和Brb043-BglⅡ715,1个SNP标记Brh019-SNP137;同时,通过筛选与目标区域具有同源性的Unigene序列得到了1个与BrDW紧密连锁的SSR标记bru1209。标记Brb062-Indel230、Brb094-DraⅠ787、Brb094-AatⅡ666、Brb043-BglⅡ715、Brh019-SNP137和bru1209与RAPD标记K14-1030之间的遗传距离分别为4.3 cM、1.7 cM、5.9 cM、5.9 cM、4.6 cM和0.8 cM,在对DH群体中的抗性株系选择上准确率分别为69.7%、70.9%、72.4%、72.4%、58.3%和74.2%,可应用于分子标记辅助选择,为霜霉病抗性分子育种奠定了良好基础。     

Genetic mapping and localization of a major QTL for seedling resistance to downy mildew in Chinese cabbage (Brassica rapa ssp. pekinensis)

Downy mildew caused by the fungus Peronospora parisitica is a serious threat to members of the Brassicaceae family. Annually, a substantial loss of yield is caused by the widespread presence of this disease in warm and humid climates. The aim of this study was to localize the genetic factors affecting downy mildew resistance in Chinese cabbage (Brassica rapa ssp. pekinensis). To achieve this goal, we improved a preexisting genetic map of a doubled-haploid population derived from a cross between two diverse Chinese cabbage lines, 91-112 and T12-19, via microspore culture. Microsatellite simple sequence repeat (SSR) markers, isozyme markers, sequence-related amplified polymorphism markers, sequence-characterized amplified region markers and sequence-tagged-site markers were integrated into the previously published map to construct a composite Chinese cabbage map. In this way, the identities of linkage groups corresponding to the Brassica A genome reference map were established. The new map contains 519 markers and covers a total length of 1,070 cM, with an average distance between markers of 2.06 cM. All markers were designated as A1–A10 through alignment and orientation using 55 markers anchored to previously published B. rapa or B. napus reference maps. Of the 89 SSR markers mapped, 15 were newly developed from express sequence tags in Genbank. The phenotypic assay indicated that a single major gene controls seedling resistance to downy mildew, and that a major QTL was detected on linkage group A8 by both interval and MQM mapping methods. The RAPD marker K14-1030 and isozyme marker PGM flanked this major QTL in a region spanning 2.9 cM, and the SSR marker Ol12G04 was linked to this QTL by a distance of 4.36 cM. This study identified a potential chromosomal segment and tightly linked markers for use in marker-assisted selection to improve downy mildew resistance in Chinese cabbage.     

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.     

BrpSPL9 (Brassica rapa ssp. pekinensis SPL9) controls the earliness of heading time in Chinese cabbage

The leafy heads of cabbage (Brassica oleracea), Chinese cabbage (Brassica rapa ssp. pekinensis), Brussels sprouts (B. oleracea ssp. gemmifera) and lettuce (Lactuca sativa) comprise extremely incurved leaves that are edible vegetable products. The heading time is important for high quality and yield of these crops. Here, we report that BrpSPL9‐2 (B. rapa ssp. pekinensis SQUAMOSA PROMOTER BINDING‐LIKE 9‐2), a target gene of microRNA brp‐miR156, controls the heading time of Chinese cabbage. Quantitative measurements of leaf shapes, sizes, colour and curvature indicated that heading is a late adult phase of vegetative growth. During the vegetative period, miR156 levels gradually decreased from the seedling stage to the heading one, whereas BrpSPL9‐2 and BrpSPL15‐1 mRNAs increased progressively and reached the highest levels at the heading stage. Overexpression of a mutated miR156‐resistant form of BrpSPL9‐2 caused the significant earliness of heading, concurrent with shortening of the seedling and rosette stages. By contrast, overexpression of miR156 delayed the folding time, concomitant with prolongation of the seedling and rosette stages. Morphological analysis reveals that the significant earliness of heading in the transgenic plants overexpressing BrpSPL9‐2 gene was produced because the juvenile phase was absent and the early adult phase shortened, whereas the significant delay of folding in the transgenic plants overexpressing Brp‐MIR156a was due to prolongation of the juvenile and early adult phases. Thus, miR156 and BrpSPL9 genes are potentially important for genetic improvement of earliness of Chinese cabbage and other crops.     

SCAR and CAPS mapping of CRb, a gene conferring resistance to Plasmodiophora brassicae in Chinese cabbage ( Brassica rapa ssp. pekinensis)

Clubroot disease, caused by Plasmodiophora brassicae Wor., is highly damaging for Chinese cabbage. The CR (clubroot resistant) Shinki DH (doubled haploid) line of Chinese cabbage carries a single dominant gene, CRb, which confers resistance to the P. brassicae races 2, 4, and 8. An F₂ population derived from a cross between the CR Shinki DH line and a susceptible line, 94SK, was used to map the CRb gene. Inoculation of F₃ families with SSI (single-spore isolate) resulted in a 1:2:1 segregation ratio. Use of the AFLP technique combined with bulked segregant analysis allowed five co-dominant AFLP markers, and four and seven dominant AFLP markers linked in coupling and repulsion, respectively, to be identified. Six of the 16 AFLP markers showing low frequencies of recombination with the CRb locus among 138 F₂ lines were cloned. A reliable conversion procedure allowed five AFLP markers to be successfully converted into CAPS and SCAR markers. An F₂ population (143 plants) was analyzed with these markers and a previously identified SCAR marker, and a genetic map around CRb covering a total distance of 6.75 cM was constructed. One dominant marker, TCR09, was located 0.78 cM from CRb. The remaining markers (TCR05, TCR01, TCR10, TCR08, and TCR03) were located on the other side of CRb, and the nearest of these was TCR05, at a distance of 1.92 cM.Communicated by R. Hagemann     

Molecular characterization of novel haplotypes of eIF4E family in Chinese cabbage (Brassica rapa L. ssp. pekinensis)

Two genes coding for eukaryotic translation initiation factors, eIF4E.a and eIF4E.c, were isolated from twelve accessions of Chinese cabbage (Brassica rapa L. ssp. pekinensis). Polymorphism analysis revealed that 94 and 142 polymorphic sites were characterized from allele of BraeIF4E.a and BraeIF4E.c which produced complex haplotype structures. Six novel haplotypes were characterized from the two alleles respectively. Among the six novel haplotypes of BraeIF4E.a, three loss-of-function mutations were identified in which a conserved single nucleotide deletion mutation cause the early termination of BraeIF4E.a coding product; while for six new BraeIF4E.c haplotypes, their coding product show amino acid substitution mutations on non-conservative amino acid residues which might affect TuMV infection in Chinese cabbage.     

Fine mapping of BrWax1, a gene controlling cuticular wax biosynthesis in Chinese cabbage (Brassica rapa L. ssp. pekinensis)

The surface of plants is covered with a cuticular wax, which contains a mixture of very-long-chain fatty acid derivatives. This wax layer provides a hydrophobic barrier which reduces non-stomatal water loss and prevents pathogen attack. The biosynthesis pathway of cuticular wax in Arabidopsis is well studied; however, little is known about the synthesis of cuticular wax in Brassica rapa. Genetic analyses indicated that the waxy characteristic is controlled by a single dominant gene. In the present study, preliminary mapping results from an F2 population consisting of 308 recessive individuals showed that the BrWax1 (Brassica Wax) gene is located on linkage group A01. We developed a set of new markers closely linked to the target gene, and used another population of 1,020 recessive F2 individuals to fine-map the BrWax1 gene to a genomic DNA fragment of approximately 86.4 kb. Fifteen genes were identified in this target region. Based on gene annotation, the Bra013809 gene was the candidate for the BrWax1 gene. Quantitative real-time PCR analysis and expression pattern of the two parents showed that the expression level of Bra013809 was much higher in the waxy phenotype than in the glossy phenotype. This result should provide not only important information for functional studies of the BrWax1 gene, but also the starting point for studying the pathway of cuticular wax biosynthesis in Brassica rapa.     

Identification of expressed genes during infection of Chinese cabbage (Brassica rapa subsp. pekinensis) by Plasmodiophora brassicae

Plasmodiophora brassicae is an obligate, biotrophic pathogen causing the club-root disease of crucifers. Despite its importance as a plant pathogen, little is known about P. brassicae at the molecular level as most of its life cycle takes place inside the plant host, and axenic culturing is impossible. Discovery of genes expressed during infection and gene organization are the first steps toward a better understanding of the pathogen-host interaction. Here, suppression subtractive hybridization was used to search for the P. brassicae genes expressed during plant infection. One-hundred and forty ESTs were found of which 49% proved to be P. brassicae genes. Ten novel P. brassicae genes were identified, and the genomic sequences surrounding four of the ESTs were acquired using genome walking. Alignment of the ESTs and the genomic DNA sequences confirmed that P. brassicae genes are intron rich and that the introns are small. These results show that it is possible to discover new P. brassicae genes from a mixed pool of both plant and pathogen cDNA. The results also revealed that some of the P. brassicae genes expressed in Chinese cabbage (Brassica rapa subsp. pekinensis) were identical to the genes expressed in the infection of Arabidopsis plants, indicating that these genes play an important role in P. brassicae infection.     

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 (BrCRT2^R) and tipburn‐susceptible (BrCRT2^S) lines and identified a 51‐bp deletion in BrCRT2^S. Overexpression of BrCRT2^R 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.     

Analysis of genomic DNA methylation and gene expression in Chinese cabbage ( Brassica rapa L. ssp. pekinensis ) after continuous seedling breeding

Vernalization plays a key role in the bolting and flowering of Chinese cabbage (Brassica rapa L. ssp. pekinensis). Plants can switch from vegetative to reproductive growth and then bolt and flower under low temperature induction. The economic benefits of Chinese cabbage will decline significantly when the bolting happens before the vegetative body fully grows due to a lack of the edible value. It was found that continuous seedling breeding reduced the heading of Chinese cabbage and led to bolt and flower more easily. In the present study, two inbred lines, termed A161 and A105, were used as experiment materials. These two lines were subjected to vernalization and formed four types: seeds-seedling breeding once, seedling breeding twice, seedling breeding thrice and normal type. Differences in plant phenotype were compared. DNA methylation analysis was performed based on MSAP method. The differential fragments were cloned and analyzed by qPCR. Results showed that plants after seedling breeding thrice had a loosen heading leaves, elongated center axis and were easier to bolt and flower. It is suggested that continuous seedling breeding had a weaker winterness. It was observed that genome methylation level decreased with increasing generation. Four differential genes were identified, short for BraAPC1, BraEMP3, BraUBC26 and BraAL5. Fluorescent qPCR analysis showed that expression of four genes varied at different reproduction modes and different vernalization time. It is indicated that these genes might be involve in the development and regulation of bolting and flowering of plants. Herein, the molecular mechanism that continuous seedling breeding caused weaker winterness was analyzed preliminarily. It plays an important guiding significance for Chinese cabbage breeding.     

Fine mapping and identification of candidate Br-or gene controlling orange head of Chinese cabbage (Brassica rapa L. ssp. pekinensis)

Orange head Chinese cabbage accumulates significant amounts of carotenoids with enhanced nutritional quality. To develop molecular markers for breeding of Chinese cabbage lines with high carotenoid content and to isolate the candidate gene underlying carotenoid synthesis, we performed fine mapping of the orange locus in a F₂S₄ mapping population. Genetic analysis revealed that the phenotype of the orange head trait was controlled by a single recessive gene, Br-or. The F₂S₄ mapping population consisting of 1,724 individuals was developed from the cross between parental lines 11J16 and 11S39-2 by continuous selfing of a single heterozygous individual. Twenty-one tightly linked simple sequence repeat (SSR) and insertion/deletion polymorphism (InDel) markers were obtained. High-resolution genetic mapping of these markers in the F₂S₄ mapping population placed Br-InDel2 and Br-InDel1 at genetic distances of 0.1 and 0.2 cM, respectively, on either side of the Br-or locus. Based on comparison of these two marker sequences with the fully sequenced Brassica rapa genome, the Br-or locus was delimited to a 16.7 kb genomic region. Three open reading frames (ORFs) were predicted in the target region. ORF1 encoded carotenoid isomerase, which is involved in the isomerization of carotenoids. ORF1 was found to be co-segregated with the Br-or locus and was thus the most likely candidate gene for Br-or. The information obtained here will facilitate the breeding of nutrient-enriched Chinese cabbage through marker-assisted selection and provide a platform for gaining a better understanding of the regulation of carotenoid biosynthesis in these plants.     

Control of Lepidopteran insect pests in transgenic Chinese cabbage (Brassica rapa ssp. pekinensis) transformed with a synthetic Bacillus thuringiensis cry1C gene

 A synthetic Bacillus thuringiensis cry1C gene was transferred to three Korean cultivars of Chinese cabbage via Agrobacterium tumefaciens-mediated transformation of hypocotyl explants. Hygromycin resistance served as an efficient selective marker. The transformation efficiency ranged from 5% to 9%. Transformation was confirmed by Southern blot analysis, PCR, Northern analysis, and progeny tests. Many transgenic plants of the closed-head types (lines Olympic and Samjin) flowered in vitro. Over 50 hygromycin-resistant plants were successfully transferred to soil. The transgenic plants and their progeny were resistant to diamondback moths (DBM, Plutella xylostella), the major insect pest of crucifers world-wide, as well as to cabbage loopers (Trichoplusia ni) and imported cabbage worms (Pieris rapae). Both susceptible Geneva DBM and a DBM population resistant to Cry1A protein were controlled by the Cry1C-transgenic plants. The efficient and reproducible transformation system described may be useful for the transfer of other agriculturally important genes into Chinese cabbage. Received: 12 June 2000 / Revision received: 21 August 2000 / Accepted: 22 August 2000