根肿病胁迫下外源SA对不结球白菜抗性诱导和生理生化的影响

以不结球白菜‘新矮青’为试验材料,采用菌土接种法接种根肿病菌,研究不同浓度(0.2~0.8mmol·L~(-1))的外源水杨酸(SA)对根肿病胁迫下不结球白菜植株抗性诱导和感病植株生长的影响,以及植株叶片和根系中防御酶系统的变化规律,探讨外源SA对防治不结球白菜根肿病的可行性。结果显示:(1)根肿病显著影响不结球白菜的生长,加剧植物的膜脂过氧化程度;适宜浓度的外源SA能不同程度地缓解不结球白菜所受根肿病的侵害,并以0.6mmol·L~(-1) SA处理效果最好。(2)外源SA显著降低了根肿病的发病率和病情指数,提高了抗根肿病的诱抗效果,并缓解感病对不结球白菜的生长抑制,显著促进感病植株的生长。(3)外源SA提高了不结球白菜叶片和根系中的超氧化物歧化酶(SOD)、过氧化物酶(POD)和抗坏血酸-过氧化物酶(APX)的抗氧化酶活性,以及多酚氧化酶(PPO)、苯丙氨酸解氨酶(PAL)、谷胱甘肽还原酶(GR)的防御酶活性;显著降低了植株叶片和根系中的MDA、H2O2含量及O-·2产生速率。研究表明,外源SA诱导不结球白菜对根肿病的抗性,缓解根肿病的伤害,且具有剂量效应,以根部灌施0.6mmol·L~(-1)SA的效果最好;在根肿病的胁迫下,SA可提高不结球白菜植株抗氧化酶和防御酶的活性,增强渗透调节能力和细胞活性氧清除能力,降低根肿病对植株的伤害,从而促进生长。     

花生黄曲霉侵染抗性的SCAR标记

利用与花生黄曲霉侵染抗性基因紧密连锁的AFLP标记 “E45/M53-440”, 经PAGE凝胶电泳后回收、克隆、测序, 并根据测序结果设计PCR特异引物, 通过对PCR条件的优化, 成功地将AFLP标记“E45/M53-440”转化为实验结果稳定, 操作更简单的SCAR标记“AFs-412”, 标记与花生黄曲霉侵染抗性间的遗传距离为6.5 cM。利用获得的SCAR标记对抗、感黄曲霉的花生种质资源进行了分子鉴定, 结果表明标记与抗性鉴定结果具有较高的一致性, 证实了该标记应用于研究群体之外的育种潜力。SCAR标记的建立为开展花生黄曲霉侵染抗性的标记辅助选择育种提供了简便实用的鉴定技术。     

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。     

用PCR技术诊断水稻的白叶枯病抗性

植物育种中应用分子标记辅助选择要求分子标记与目的基因紧密连锁,而且分析手段经济简便、重复性好。Ｘａ２１是最近发现的一个具有广谱抗性的水稻白叶枯病抗性基因,利用一个含Ｘａ２１基因的品系ＩＲＢＢ２１分别与２个感病品种杂交获得２个Ｆ_２群体。用４对引物分别对３个亲本进行ＰＣＲ分析,其中一对引物（ＰＢ７８）的ＰＣＲ产物在抗、感病品种间可揭示多态性。对２个Ｆ_２群体进一步的遗传分析表明,ＰＣＲ标记和Ｘａ２１的白叶枯病抗性紧密连锁,其重组率仅为２．４８％。根据该标记选择基因型纯合的抗性植株,其准确率可达１００％。本文还就植物育种中分子标记的检测途径进行了评价。     

不结球白菜晚抽薹BcFLC3基因的克隆及表达分析

根据大白菜BcFLC3基因(GenBank登录号AY036890.1)保守域序列设计引物,扩增不结球白菜晚抽薹BcFLC3基因的核心片段,结合RACE技术获得该基因1 017 bp的全长cDNA序列.序列分析结果表明,该cDNA包含完整开放阅读框,编码197个氨基酸的蛋白质,其分子量为21.62 kD,等电点9.36.荧光定量 PCR分析表明,不结球白菜经4℃低温处理后,BcFLC3基因在叶片中的表达量抽薹前明显高于抽薹后;低温处理后BcFLC3基因在不同部位的表达量存在明显差异,表达量由高到低依次为叶、茎、花蕾、花和根.Southern杂交结果表明,BcFLC3基因在不结球白菜基因组中为多拷贝.     

栽培小麦Brock中与新抗白粉病基因紧密连锁的AFLP分子标记筛选与分析

本研究用225对引物对农艺性状优良但对白粉菌敏感的栽培小麦京411、抗白粉病栽培小麦Brock以及京411与Brock配制的近等基因系进行AFLP分子标记筛选,结果发现只有2对引物组合Pst GAC/Mse TCT(P1)和Pst AGC/Mse ACC(P2)在上述抗感材料中表现出多态性,分别扩增到2个特异片段,将特异片段克隆并测序发现,P1扩增的特异片段长268bp,P2扩增的特异片段长227bp,命名为AFLP标记P1268和P227.用106个京411×Brock的F2单株进行连锁性分析表明,P1268和P2227与抗白粉病基因的遗传距离分别为3.6和1.9cM,与Brock中的抗白粉病基因呈紧密连锁.该两个AFLP标记对小麦抗白粉病基因的积累和分子标记辅助选择育种有重要意义.     

不结球白菜新种质P70-203雄性不育细胞质类型的鉴定

本研究根据OguraCMS、PolimaCMS的不育性状相关的线粒体基因序列设计特异引物,对不结球白菜雄性不育系新种质P70-203及其保持系P60-27-1进行PCR分析.研究结果表明,Polima引物P3/P4,P5/P6在不育系与可育系中均无扩增条带;Ogura引物P1/P2在不育系中扩增出750 bp的特异片段,但可育系中无扩增条带.将扩增的特异条带回收并测序,将得到的测序结果在NCBI中进行Blastn同源性比较,结果与青花菜Ogura(登录号:EU604643)和萝卜Ogura(登录号:AB055438)细胞质雄性不育同源性均达到99%.从分子角度初步推测:该雄性不育系新种质P70-203具有Ogura细胞质.     

大白菜—结球甘蓝1号二体异附加系分子标记鉴定及其自交后代的性状分析

利用与抗霜霉病、抗芜菁花叶病毒基因连锁的SCAR标记和能够区分大白菜和结球甘蓝FLCs基因的特异引物对大白菜—结球甘蓝1号二体异附加系(AC1d)及其亲本进行分析,结果表明:AC1d及其双亲中均含有与抗霜霉病基因连锁的标记;AC1d和亲本结球甘蓝中含有与抗芜菁花叶病毒基因连锁的标记,亲本大白菜中无该标记;AC1d除具有4个大白菜Br FLCs基因外,同时还添加了结球甘蓝Bo FLC3基因。AC1d自交后代的株高、株展、球高、维生素C含量、可溶性蛋白质及其7种硫苷组分含量超出了亲本大白菜和结球甘蓝;叶形指数、可溶性糖含量和对小菜蛾的抗性等超出了亲本大白菜。为利用AC1d后代选育携有目标性状的易位系提供了依据。     

与棱果沙棘性别相关的RAPD标记

应用RAPD技术筛选与棱果沙棘性别相关的分子标记,对棱果沙棘雌雄株的基因组DNA进行混合分组分析（BSA）,在194条随机引物中有50条引物能够在雌雄DNA反应池间形成多态性条带,应用这50条引物分别对棱果沙棘雌雄个体（雌雄个体各选取5个）进行RAPD分析,其中引物S10扩增得到1个约为1030 bp的与雌性相关的RAPD标记。该标记的获得进一步表明棱果沙棘雌雄株间存在基因水平的差异,为棱果沙棘的性别研究提供分子依据。进一步利用该雌性特异位点设计出更加稳定的SCAR标记,可望用于棱果沙棘的早期性别的准确鉴别。     

无选择标记和载体骨干序列的Xa21转基因水稻的获得

利用双右边界T-DNA载体通过根癌农杆菌介导法将水稻白叶枯病广谱抗性基因Xa21导入杂交稻重要恢复系C418中。T0代共获得27个独立转基因株系,通过田间抗性鉴定与PCR分析,有17个株系的Xa21基因分子鉴定为阳性,且对白叶枯病原菌P6生理小种具有抗性。通过对17个株系的后代植株进行田间抗性鉴定,分子标记辅助选择及Southern杂交分析,结果显示4个株系的T1代植株中能分离出无潮霉素标记基因的Xa21转基因植株。无选择标记Xa21转基因株系的获得率为15%。PCR检测还表明,这些无选择标记的Xa21转基因植株不带有载体骨架序列。通过对转基因后代进一步的抗性鉴定与PCR辅助选择,获得了无选择标记和载体骨架序列的转基因Xa21纯合的抗白叶枯病水稻。     

Development of molecular markers based on CRa gene sequencing of different clubroot disease-resistant cultivars of Chinese cabbage

Background

CRa is a key gene in Chinese cabbage (Brassica rapa ssp. pekinensis) that confers resistance to Plasmodiophora brassicae. In order to efficiently screen the clubroot resistance (CR) gene CRa in breeding, two functional codominant markers of the CRa gene were developed.

Methods and results

In this study, through comparing the CRa allele sequences in resistant and susceptible cultivars of Chinese cabbage, we found two insertion and deletion of sequence variations in the fourth exon between resistant and susceptible cultivars. Two functional codominant markers for CRa gene were obtained based on the variations, namely, CRaEX04-1 and CRaEX04-3. The lengths of the extended fragment of CRaEX04-1 marker were 321 bp and 186 bp in resistant and susceptible cultivars, respectively. In contrast, those of CRaEX04-3 were 704 bp and 413 bp, respectively. We verified the genetic stability between the developed markers and CRa gene using 57 Chinese cabbage cultivars with known resistance and two genetic populations. The results showed that the marker identification was completely consistent with the known phenotypes in 57 cultivars. The marker identification results followed the 3:1 of Mendel’s first law in the F₂ population, and the 1:1 of Mendel’s first law in the BC₁.

Conclusions

CRaEX04-1 and CRaEX04-3 can be used as a practical molecular marker for breeding and germplasm resource creation of clubroot disease-resistant Chinese cabbage.

     

大白菜骨干自交系的苗期抗病性评价

为明确大白菜骨干自交系的抗病性,本研究于2012-2014年,对课题组保存和创制的203个大白菜自交系进行了霜霉病、病毒病、黑腐病、黄萎病和根肿病的苗期抗性评价。结果显示,高抗上述病害的自交系分别有7、9、0、31和12个;只抗其中一种病害的自交系82个;兼抗两种病害的有61个,兼抗三种病害的自交系有28个,兼抗四种病害的自交系有4个。自交系11-234、04-622、12-85、13-108和09-894综合抗病性最优。此外,春大白菜、夏大白菜和秋大白菜三种生态类群间,以及四种叶球抱合类群间的抗病性表现出明显差异。     

The New Clubroot Resistance Locus Is Located on Chromosome A05 in Chinese Cabbage (<Emphasis Type="Italic">Brassica rapa</Emphasis> L.)

Clubroot disease, which is caused by Plasmodiophora brassicae Wor., a soil-borne microorganism, is one of the most severe diseases of Brassica crops. Combining of two and more dominant resistance loci is an efficient method in breeding for clubroot resistance. Several clubroot resistance loci were earlier identified on linkage groups 1, 2, 3, 6, and 8 of Brassica rapa by different research groups. In our previous studies, we found a dominant monogenic resistance locus in an inbred line 20-2ccl of Chinese cabbage. In this study, a SCAR marker tau_cBrCR404 tightly linked to clubroot resistance locus (2.9 cM) was identified by a bulked segregant analysis (BSA) of a backcross population (BC₁). The position of this clubroot resistance locus, named CrrA5, was determined on the linkage group 5 of B. rapa genome using genetic mapping. The efficiency of the tau_cBrCR404 marker in marker-assisted selection was validated using a collection of different Chinese cabbage accessions.     

Molecular characterization of the CRa gene conferring clubroot resistance in Brassica rapa

Clubroot disease is one of the major diseases affecting Brassicaceae crops, and a number of these crops grown commercially, such as Chinese cabbage (Brassica rapa L. ssp. pekinensis), are known to be highly susceptible to clubroot disease. To provide protection from this disease, plant breeders have introduced genes for resistance to clubroot from the European turnip into susceptible lines. The CRa gene confers specific resistance to the clubroot pathogen Plasmodiophora brassicae isolate M85. Fine mapping of the CRa locus using synteny to the Arabidopsis thaliana genome and partial genome sequences of B. rapa revealed a candidate gene encoding a TIR-NBS-LRR protein. Several structural differences in this candidate gene were found between susceptible and resistant lines, and CRa expression was observed only in the resistant line. Four mutant lines lacking clubroot resistance were obtained by the UV irradiation of pollen from a resistant line, and all of these mutant lines carried independent mutations in the candidate TIR-NBS-LRR gene. This genetic and molecular evidence strongly suggests that the identified gene is CRa. This is the first report on the molecular characterization of a clubroot Resistance gene in Brassicaceae and of the disease resistance gene in B. rapa.     

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.     

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 cloning and characterization of ascorbate oxidase gene in non-heading Chinese cabbage

The cDNA clone of ascorbate oxidase gene was isolated from non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino, cv. Suzhouqing) and characterized. Sequence analysis showed that there was a high similarity between this sequence (named BcAO) and its homologues in other plant species. Southern blotting indicated that more than one nuclear gene encoded this enzyme in non-heading Chinese cabbage. The mRNA level of the BcAO gene in leaves was monitored by real-time PCR at different developmental stages and under different stress conditions. Results showed that the expression of BcAO was upregulated by light, and the BcAO gene responded to copper stress as well. After inoculation with Alternaria brassicae, the expression of BcAO in the leaves was increased in general and peaked at 12 and 72 h post inoculation, with much higher expression at the later date. Cloning the BcAO gene will enable us to further understand its function and would provide useful information for resistance breeding program for non-heading Chinese cabbage.     

Molecular cloning and characterization of a PR-5 like protein gene from <Emphasis Type="Italic">Brassica campestris</Emphasis> ssp. <Emphasis Type="Italic">chinensis</Emphasis>

Downy mildew caused by Hyaloperonospora parasitica is a serious fungal disease in non-heading Chinese cabbage (Brassica campestris L. ssp. chinensis Makino). Pathogenesis-related 5 (PR-5) genes play an important role in plant resistance to disease invasion. In this study, a gene encoding pathogenesis-related 5-like (PR-5L) protein, named BcPR-5L, was successfully cloned from non-heading Chinese cabbage. The cDNA sequence of BcPR-5L was 747 bp in length. It encoded a protein of molecular mass of 25.78 kDa, an isoelectric point of 4.42, and containing 248 amino acids. Multiple sequence alignment indicated that BcPR-5L protein was highly homologous to other PR-5L proteins identified in 13 different species, with the highest homology to Brassica rapa. We analyzed the subcellular localization of BcPR-5L protein by using onion epidermal cells and found that it was localized in the membrane. Real time quantitative PCR analyses revealed that the expression of BcPR-5L gene was significantly upregulated after H. parasitica infection, and the expression in the resistant cultivar was higher than that in the susceptible cultivar. In summary, our data suggest that BcPR-5L gene may play an important role in the resistance of non-heading Chinese cabbage to H. parasitica infection.     

甘蓝种质资源根肿病抗性鉴定及CRa、Crr1a同源基因分析

利用来源于湖北长阳、陕西太白、河南新野3个地方的根肿病菌对22份不同甘蓝材料进行抗病性鉴定。采用同源比对的方法,对甘蓝基因组中的大白菜抗根肿病同源基因CRa和Crr1a进行分析;同时对不同抗、感根肿病甘蓝材料中的CRa和Crr1a同源基因序列进行了扩增、测序和比对分析。结果表明:供试22份甘蓝材料对3份根肿病菌存在较大的抗感差异,推测来源于3个地区的菌种可能不是同一个生理小种;筛选出的抗性品种BDH3、Chou hybride Tekila、SW-110、CGL-8、先正达品种、SW-109将来可用作根肿病抗源和抗性基因挖掘;在甘蓝7号染色体上存在3个预测基因为CRa的同源基因,分别是Bo7g107710、Bo7g107730和Bo7g107740,其中,Bo7g107730基因在抗病材料SW-110存在较大的序列变异,推测可能与根肿病抗性相关;在甘蓝3号染色体上存在1个预测基因Bo3g164040为Crr1a的同源基因,所分析的抗、感病材料中Bo3g164040基因序列一致性极高,没有发现与抗根肿病有关的位点,说明甘蓝中Bo3g164040基因可能没有根肿病抗性功能。     

抗根肿病大白菜的花药培养

大白菜是中国北方的重要蔬菜之一,根肿病是危害大白菜生产世界性病害,利用花药培养可以大大加速抗根肿病大白菜杂交育种工作的进程。对33份抗根肿病大白菜品种(品系)进行花药培养,有24个品种诱导出胚,品种诱导率为72.7%,以东方皇冠×C11的诱导率最高,为1.86胚/蕾。对大部分基因型来说,在培养基中添加0.4 g/L谷氨酰胺的诱导效果最好。研究还发现生长在温室中的供体植株更容易诱导出胚状体。变绿的子叶型胚转到B5+0.2 mg/L BA+0.1 mg/L NAA+0.1%活性炭的胚分化培养基上可诱导生芽。