Evaluation of Brassica germplasm for field resistance against clubroot (Plasmodiophora brassicae Woron)

Of the 124 germplasm accession of oil seed Brassicas screened under field condition against clubroot disease (Plasmodiophora brassicae), 80% were susceptible and 17, 3, 1 and 1 of Brassica juncea, Brassica rapa var. toria, B.rapa var. yellow sarson and B. rapa, respectively, were resistant.     

Transfer of resistance to clubroot (Plasmodiophora brassicae) to swedes {Brassica napus L. var. napobrassica Peterm) from B. rapa

In 1975, tests with UK populations of Plasmodiophora brassicae not only revealed a lack of effective clubroot resistance in swedes (Brassica napus), but also the outstanding resistance of the European Clubroot Differential (ECD)04 (B. rapa). It was, therefore, decided to transfer the resistance genes from ECD04 to swedes, using the most pathogenic UK population of clubroot (C56) available for screening purposes. An autotetraploid form of ECD04 was crossed with tetraploid kale (B. oleracea) using the latter as female parent. One of the euploid, 2n = 38, hybrids secured by embryo rescue in 1976 was crossed to the swede cultivars Marian and Ruta Øtofte. Three further backcrosses of clubroot resistant plants to lines derived from modern swede cultivars were made over the period 1980 to 1982. Selfing commenced in 1983 to produce F₂ populations. From F₃ to F₅ there was family selection for yield and agronomic characters, as well as single plant selection for clubroot resistance. In 1991, the six most promising F₅ families were multiplied for subsequent evaluation in replicated yield trials in Dundee. The most promising family entered official trials at the beginning of 1993 and, 2 years later, was added to the National List as cv. Invitation and granted Plant Breeders' Rights. The first certified seed was sold in 1996, 20 years after the original synthetic B. napus was produced. The breeding programme provided evidence for only one of the three postulated dominant genes in ECD04 being required for resistance to C56 and also good evidence of differential resistance from tests with other clubroot populations. Hence, whilst the differential resistance in cv. Invitation should prove useful in the UK in the immediate future, it may not be durable in the longer term. It is, therefore, argued that the next and more difficult goal to achieve should be to introduce high levels of non-differential resistance from B. oleracea.     

Selection for resistance to clubroot (Plasmodiophora brassicae) in marrowstem kale (Brassica oleracea var. acephala L.)

Ninety-six cultivars of Brassica oleracea were screened for clubroot resistance in a seedling test using two populations of Plasmodiophora brassicae. The most resistant cultivars were kales. Sixteen resistant marrowstem kale cultivars of diverse geographical origin were used to start a selection programme for clubroot resistance. Four generations of selection, involving single plants, half-sib and full-sib families, reduced a disease index averaged over six clubroot populations from 41.2 to 12.5. This was lower than the most resistant cultivar in the original population, cv. Mixti 28.8, and as good as a German landrace of cabbage noted for its resistance, Bohmerwaldkohl 10.5. In comparison, the mean of five kale controls, cvs Bittern, Canson, Condor, Kestrel and Merlin, was 61.1 and the value for the most susceptible control, cabbage cv. Septa, was 89.3. In the final assessment, there were no clubroot population x B. oleracea genotype interactions and in the initial assessment of cultivars there were only small interactions which could be removed by an angular transformation of the data. It was concluded that a high level of non-differential resistance had been achieved and that it may prove durable. It was also concluded from a small field trial that this level of resistance would prevent serious yield losses in practice.     

Infection of Chinese cabbage by Plasmodiophora brassicae leads to a stimulation of plant growth: impacts on cell wall metabolism and hormone balance

The importance of plant hormones in clubroot infection has long been recognized. The morphological changes, such as cell division and cell elongation leading to gall formation are triggered in the early stages of infection. We analysed cell expansion by localizing Xyloglucan endoTransglucosylase/Hydrolase (XTH)-action and screened the endogenous concentrations of several classes of phytohormones by mass spectrometry in the early stages of Plasmodiophora brassicae infection in Chinese cabbage (Brassica rapa spp. pekinensis). Infected plants showed a general transient growth promotion early in infection. Furthermore a clear XTH action was visible in the epidermal layer of infected roots. Complex changes in the endogenous phytohormone profile were observed. Initially infection resulted in an increased total auxin pool. The auxin increase, together with an increased XTH action, results in wall loosening and consequently cell expansion. When the first secondary plasmodia are formed, thirteen days after infection (DAI), can be considered a switch point in phytohormone metabolism. Twenty-one DAI the plasmodia might act as a plant hormone sink resulting in a reduction in the active cytokinin pool and a lower indole-3-acetic acid content in the infected plants.     

结球甘蓝根肿菌鉴定和种质抗性评价

采集湖北省长阳县火烧坪乡根肿病重发区的病土和病根,通过病原菌形态和PCR鉴定,确定是芸薹根肿菌,然后利用欧洲ECD鉴别系统确定生理小种为ECD17/31/13,此病原菌致病力极强。采用田间苗期人工接种鉴定,与田间成株期自然诱发鉴定相结合,对88份甘蓝种质进行抗性评价和筛选,结果表明:苗期获得1份高抗,7份抗病,17份耐病材料;成株期获得4份高抗,4份抗病,15份耐病材料。2个时期88份材料群体抗性鉴定级别基本一致,93.18%材料成株期病指比苗期高。CR21在2个时期均为高抗,抗性最强,表现稳定;CR55在苗期发病最严重,病指达到76.19,成株期为74.10;CR54在成株期发病最严重,病指达到81.54,苗期为75.97,2个时期发病率均达到100%。根肿病菌的鉴定和致病力的确定,及甘蓝种质抗性评价为抗病品种选育和抗病机理的研究奠定基础。     

Effects of synthetic plant growth retardants and abscisic acid on root functions of Brassica rapa plants exposed to low root-zone temperature

Possible interactions of two synthetic plant-growth retardants during the short-term response of Brassica rapa L. ssp. oleifera (DC.) Metzger plants to low root-zone temperature were investigated by pretreating with mefluidide or paclobutrazol. Water and solute transfers were studied by measuring xylem sap volume flow (under root pressure exudation) and ion flow from the roots. Relations with nitrate uptake rate were also considered. Root pretreatment with paclobutrazol strongly restricted the cold-inducible processes which normally restore water and solute flow from the root xylem. Paclobutrazol decreased the rates of nitrate uptake and exudation flow from the root xylem (principally by reducing root hydraulic conductivity) with dramatic consequences for ion flow, especially that of nitrate.
The effects of root ABA pretreatment on plant response to root cooling were then studied separately or in association with a pretreatment with paclobutrazol. Despite a slight decrease in nitrate uptake rate, ABA pretreatment of the roots enabled the plant to develop rapid mechanisms for adaptation to cold constraint at the root level. Moreover, this action of exogenous ABA greatly reduced the effect of a simultaneous paclobutrazol pretreatment and partly restored water and solute flows.
Thus, the improvement of plant resistance to cold conditions brought about by treatments with mefluidide and paclobutrazol (previously shown in long-term experiments) cannot simply be explained by their short-term effects.     

The occurrence of free and bound cytokinins in clubroots and Plasmodiophora brassicae infected turnip tissue cultures

This paper deals with the quantitative determination of free and bound cytokinins in clubroot tissue and in Plasmodiophora brassicae Woron, infected Brassica campestris L. callus tissue. The fractions were separated in a butanol soluble fraction containing the free cytokinins such as zeatin and zeatin riboside and a water soluble fraction containing the bound cytokinins. The butanol fraction was extensively purified and analysed by high pressure liquid chromatography (HPLC). The butanol fraction contained cytokinins which cochromatographed with zeatin and zeatin riboside and not with dihydrozeatin. Zeatin and zeatin riboside were quantitatively determined by HPLC. Recovery of the cytokinins varied between 30–50%. Clubs contained 50–160 ng zeatin and 210–300 ng zeatin riboside per g dry weight. Callus tissue contained 133 ng zeatin and 169 ng zeatin riboside per g dry weight. Clubs, callus as well as healthy tissue contain large amounts of bound cytokinins. Upon treatment of the water soluble fraction first with alkaline phosphatase and then with β-glucosidase biologically active fractions were found which coeluted with zeatin and zeatin riboside on Sephadex LH₂₀ in 20% ethanol. Evidence is presented for a novel cytokinin in the water soluble fraction which yields free zeatin and glucose-6-phosphate after treatment with β-glucosidase.     

Peroxidase and chitinase isoenzyme activities during root infection of Chinese cabbage with Plasmodiophora brassicae

Roots of two Chinese cabbage (Brassica campestris L. ssp. pekinensis) varieties, one tolerant and one susceptible, were inoculated with Plasmodiophora brassicae in liquid medium and in soil. Chitinase and peroxidase activities were determined in roots and shoots 1–21 days after inoculation with resting spores of Plasmodiophora and the enzyme activities compared with healthy tissue of the same age. In infected roots of the susceptible variety ‘Granat’ chitinase activity was higher than in the control 10 days after inoculation with spores. In the tolerant variety ‘Parkin’ we detected an increase in chitinase activity at the same time, which was about twice that of ‘Granat’. Chitinase activity in ‘Granat’ was also enhanced on day 13, 14 and 17 after inoculation, whereas chitinase activity in ‘Parkin’ was lower in the infected roots than in the controls during that period. In the shoots no correlation between chitinase activity and infection in the two varieties was observed. Chitinase from Chinese cabbage was further characterized and showed a pH optimum at pH 4.5–5.5 and a temperature optimum at 35–45°C. After isoelectric focusing 7 isoenzymes were discovered, but there were almost no differences between infected and healthy root extracts. Two isoenzymes with pI 8.7 and 8.8 showed cross-reactivity with an antiserum against bean chitinases. The molecular mass of these isoenzymes was determined as 33 kDa. Total peroxidase activity was generally higher in root tissue of both varieties than in the shoots. Peroxidase activity was increased most prominently in infected ‘Granat’ roots on day 13 after inoculation and of both varieties on day 17 compared to the controls. In clubbed tissue of ‘Granat’ a specific peroxidase isoenzyme appeared the first time 21 days after inoculation and was most prominent 28–30 days after inoculation. This isoenzyme had a molecular mass of ca 24 kDa and a pI of ca 8.8. With respect to our results the strategy of the Plasmodiophorales for plant attack is discussed.     

Biomass, reproductive output, and physiological responses of rapid-cycling Brassica (Brassica rapa) to ozone and modified root temperature

Brassica rapa L. (rapid-cycling Brassica), was grown in environmentally controlled chambers to determine the interactive effects of ozone (O₃) and increased root temperature (RT) on biomass, reproductive output, and photosynthesis. Plants were grown with or without an average treatment of 63 ppb O₃. RT treatments were 13°C (LRT) and 18°C (HRT). Air temperatures were 25°C/15°C day/night for all RT treatments.
Ozone affected plant biomass more than did root temperature. Plants in O₃ had significantly smaller total plant d. wt, shoot weight, leaf weight, leaf area and leaf number than plants grown without O₃. LRT plants tended to have slightly smaller total plant d. wt, shoot weight, root weight, leaf weight, leaf area, and leaf number than HRT plants. For all variables, LRT plants grown in O₃ had the smallest biomass, and plants grown in HRT without O₃ had the largest biomass.
Ozone reduced both fruit weight and fruit number; LRT also reduced fruit weight but had no effect on fruit number. Ozone reduced photosynthesis but RT had no effect. Conductance and internal CO₂ were unaffected by O₃ or RT.
These studies indicate that plant growth with LRT might be more reduced in the presence of O₃ than growth in plants with HRT, which might be able to compensate for O₃-caused reductions in photosynthesis to avoid decreased biomass and reproductive output.     

芜菁SSR引物的开发及多态性分析

目的:用于芜菁的简单序列重复(SSR)标记数量有限,初步探索多态性较好SSR的结构特性,开发更多的SSR引物,有助于芜菁的研究。方法:开发680对新的SSR引物,以2个芜菁品种为模板进行多态性扩增,筛选出多态性较好的引物。结果:565(83.1%)对引物在2种芜菁之间能够有效扩增,有多态性的引物有141对(20.7%);SSR的长度与其多态性水平之间没有直接的线性关系,但SSR基序碱基数与重复次数和多态性水平之间存在一定的联系,即3碱基为基序、7次重复的SSR多态性较好。结论:SSR的类型与引物的多态性之间有一定的联系。     

‘津田’芜菁Transparent Testa Glabra 1（BrTTG1）基因的克隆及表达分析

TTG1（Transparent Testa Glabra 1）蛋白是一种WD40类蛋白,参与植物的生长和发育。采用RT-PCR方法从芜菁品种‘津田＇中克隆了BrTTG1 cDNA序列（GenBank登录号HM208590）。该基因cDNA开放阅读框长度为1 014 bp,编码一个由337个氨基酸残基组成的蛋白,该蛋白分子量为37.28 kDa,理论等电点为4.66。与其他植物中的TTG1蛋白进行同源性比对结果显示,BrTTG1与甘蓝型油菜的TTG1同源性最高。BrTTG1蛋白在31～337位氨基酸处含有WD40超家族的保守结构域。荧光定量PCR检测BrTTG1在‘津田＇芜菁不同组织中的表达结果表明,该基因在有花青素合成的红色‘津田＇芜菁根皮中表达量最高。     

Fitness of hybrids between rapeseed (Brassica napus) and wild Brassica rapa in natural habitats

Fitness of hybrids between genetically modified (GM) crops and wild relatives influences the likelihood of ecological harm. We measured fitness components in spontaneous (non-GM) rapeseed x Brassica rapa hybrids in natural populations. The F1 hybrids yielded 46.9% seed output of B. rapa, were 16.9% as effective as males on B. rapa and exhibited increased self-pollination. Assuming 100% GM rapeseed cultivation, we conservatively predict < 7000 second-generation transgenic hybrids annually in the United Kingdom (i.e. approximately 20% of F1 hybrids). Conversely, whilst reduced hybrid fitness improves feasibility of bio-containment, stage projection matrices suggests broad scope for some transgenes to offset this effect by enhancing fitness.     

白菜和白菜型油菜角果相关性状遗传分析

以种荚差异显著的白菜自交不亲和系（P1）和白菜型油菜自交系（P2）为亲本及杂交获得的4个基本世代（P1、P2、F1、F2）为材料,应用植物数量性状主基因+多基因混合遗传模型对其种荚相关性状进行遗传分析。结果表明,芸薹种作物的种荚长度(SPL)性状及喙长（SBL）性状均受加性-显性-上位性多基因控制（C-0模型）,多基因遗传率分别为83.16%和68.67%;种荚宽度(SPW)性状受2对加性-显性-上位性主基因+加性-显性-上位性多基因控制（E-0模型）,其主基因遗传率为20.40%,多基因遗传率为78.34%。种荚相关各性状均以多基因遗传为主,种荚宽度性状受环境因素影响较小,为1.26%;种荚长度、喙长受环境因素影响较大,分别达16.89%和25.36%。针对芸薹种作物种荚性状的改良要以多基因为主,并注意环境条件影响。以种荚差异显著的白菜自交不亲和系（P1）和白菜型油菜自交系（P2）为亲本及杂交获得的4个基本世代（P1、P2、F1、F2）为材料,应用植物数量性状主基因+多基因混合遗传模型对其种荚相关性状进行遗传分析。结果表明,芸薹种作物的种荚长度(SPL)性状及喙长（SBL）性状均受加性-显性-上位性多基因控制（C-0模型）,多基因遗传率分别为83.16%和68.67%;种荚宽度(SPW)性状受2对加性-显性-上位性主基因+加性-显性-上位性多基因控制（E-0模型）,其主基因遗传率为20.40%,多基因遗传率为78.34%。种荚相关各性状均以多基因遗传为主,种荚宽度性状受环境因素影响较小,为1.26%;种荚长度、喙长受环境因素影响较大,分别达16.89%和25.36%。针对芸薹种作物种荚性状的改良要以多基因为主,并注意环境条件影响。     

Regulation of Brassica rapa chloroplast proliferation in vivo and in cultured leaf disks

Summary. To understand the regulatory mechanisms of chloroplast proliferation, chloroplast replication was studied in cultured leaf disks cut from plants of 25 species. In leaf disks from Brassica rapa var. perviridis, the number of chloroplasts per cell increased remarkably in culture. We examined chloroplast replication in this plant in vivo and in culture media with and without benzyladenine, a cytokinin. In whole plants, leaf cells undergo two phases from leaf emergence to full expansion: an early proliferative stage, in which mitosis occurs, and a differentiational stage after mitosis has diminished. During the proliferative stage, chloroplast replication keeps pace with cell division. In the differentiational phase, cell division ceases but chloroplast replication continues for two or three more cycles, with the number of chloroplasts per cell reaching about 60. In the leaf disks, the number of chloroplasts per cell increased from about 18 to 300 without benzyladenine, and to over 600 with benzyladenine, indicating that this cytokinin enhances chloroplast replication in cultured tissue. We also studied changes in ploidy and cell volume between in vivo cells and cells grown in culture with and without benzyladenine. Ploidy and cell volume increased in a manner very similar to that of the number of chloroplasts, suggesting a relationship between these phenomena.Correspondence and reprints: Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo, Tokyo 113-0033, Japan.     

基于非变性聚丙烯酰胺凝胶电泳的菜心SCoT分析

以菜心(Brassica rapa var. parachinensis)抗小菜蛾品种Caixin65和感小菜蛾品种Caixin69及6份F2株系为材料,利用非变性聚丙烯酰胺凝胶电泳检测菜心抗虫株系的SCoT多态性,同时进行SCoT多态性的非变性聚丙烯酰胺凝胶电泳检测效率、遗传多样性分析和差异条带克隆分析。结果表明,非变性聚丙烯酰胺凝胶能检测出亲本与子代间的SCoT多态性和遗传多样性变化,条带数量较多且清晰,提高了SCoT标记检测效率。选取亲本的10条非变性聚丙烯酰胺凝胶差异片段克隆测序,获得感虫序列4条、抗虫序列6条,GENSCAN预测其中8条具有启动子、终止子、阅读框等基因结构序列。同源性检索分析表明,感虫序列分别与泛素羧基末端水解酶mRNA序列、大白菜克隆序列、白菜线粒体丙酮酸载体蛋白序列、甘蓝基因组编码未知蛋白的HDEM序列同源,抗虫序列分别与白菜RNA假尿苷合酶4线粒体mRNA序列、京水菜线粒体DNA序列、白菜未知蛋白mRNA序列、白菜4-香豆酸：辅酶A连接酶mRNA序列、大白菜克隆序列、哈茨木霉mRNA序列同源。本研究提高了SCoT标记清晰度、遗传多样性检测水平和差异片段克隆的精准性,使得SCoT成为批量克隆差异片段的高效工具,有助于挖掘SCoT功能性标记信息,开展初步的功能基因组学研究,提高优异株系筛选鉴定效率,加快育种进程,为菜心抗虫性机制的进一步研究提供理论基础。     

Growth, productivity, and competitiveness of introgressed weedy Brassica rapa hybrids selected for the presence of Bt cry1Ac and gfp transgenes

Concerns exist that transgenic crop x weed hybrid populations will be more vigorous and competitive with crops compared with the parental weed species. Hydroponic, glasshouse, and field experiments were performed to evaluate the effects of introgression of Bacillus thuringiensis (Bt) cry1Ac and green fluorescent protein (GFP) transgenes on hybrid productivity and competitiveness in four experimental Brassica rapa x transgenic Brassica napus hybrid generations (F1, BC1F1, BC2F1 and BC2F2). The average vegetative growth and nitrogen (N) use efficiency of transgenic hybrid generations grown under high N hydroponic conditions were lower than that of the weed parent (Brassica rapa, AA, 2n = 20), but similar to the transgenic crop parent, oilseed rape (Brassica napus, AACC, 2n = 38). No generational differences were detected under low N conditions. In two noncompetitive glasshouse experiments, both transgenic and nontransgenic BC2F2 hybrids had on average less vegetative growth and seed production than B. rapa. In two high intraspecific competition field experiments with varied herbivore pressure, BC2F2 hybrids produced less vegetative dry weight than B. rapa. The competitive ability of transgenic and nontransgenic BC2F2 hybrids against a neighbouring crop species were quantified in competition experiments that assayed wheat (Triticum aestivum) yield reductions under agronomic field conditions. The hybrids were the least competitive with wheat compared with parental Brassica competitors, although differences between transgenic and nontransgenic hybrids varied with location. Hybridization, with or without transgene introgression, resulted in less productive and competitive populations.     

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。     

Expression of GFP and Bt transgenes in Brassica napus and hybridization with Brassica rapa

It is possible to monitor the movement of transgenes by tagging them with green fluorescent protein (GFP). In order to develop a model to study transgene flow, canola (Brassica napus cv Westar) was transformed with two GFP constructs, mGFP5er (GFP only) and pSAM 12 [GFP linked to a synthetic Bacillus thuringiensis (Bt) cry1Ac endotoxin gene]. Transformed callus sectors that fluoresced green were preferentially selected in the tissue culture process. Four independent GFP canola events and 12 events of GFP/Bt canola were regenerated through tissue culture. GFP fluorescence was macroscopically detectable throughout the entire life cycle of canola. The GFP/Bt events were insecticidal to neonate corn earworm (Helicoverpa zea) larvae and prevented herbivory damage. Fluorescence intensity at 508 nm varied between the independent transformation events, and ranged from 7.6×10⁵ to 13.8×10⁵ (counts per second) in contrast with the wild-type at 5.3×10⁵ cps. Nine GFP/Bt and three GFP events were hybridized with three wild accessions of B. rapa. The resultant hybrids fluoresced green and were insecticidal to neonate corn earworm larvae to the same degree as the transgenic canola parents. However, fluorescence intensities of the hemizygous F₁ hybrid lines were lower than the respective original homozygous canola parents. Each F₁ hybrid line was backcrossed by hand onto the B. rapa parent, and transgenic backcrosses were produced at rates ranging from 15% to 34%. These data suggest that GFP can be used as a tool to monitor transgene flow from crop species to wild relatives. Received: 11 September 2000 / Accepted: 1 February 2001     

Detection and analysis of QTLs based on RAPD markers for polygenic resistance to Plasmodiophora brassicae Woron in Brassica oleracea L.

Resistance to Plasmodiophora brassicae Woron, the causal fungus of clubroot, was examined in an F₂ population of a cross between a clubroot-resistant kale (Brassica oleracea L. var. acephala) and a susceptible cauliflower (Brassica oleracea L. var. botrytis). QTL detection was performed with RAPD markers. Two resistance notations, carried out at different times after inoculation, were used. Three markers were associated with these two notations and three were specifically linked to only one notation. QTL analysis suggests the existence of at least two genetic mechanisms implicated in the resistance phenomenon.