SSR结合SRAP标记分析油菜菌核病抗性资源遗传多样性

为了更准确、有效地揭示油菜资源遗传多样性,探索SSR和SRAP 2种分子标记在油菜菌核病抗性资源遗传多样性分析中的应用,采用40对SSR核心引物及陕西理工学院生物学院分子与遗传实验室筛选出的40对多态性高、条带清晰的SRAP引物,对陕西省汉中市农科所经过连续3年牙签茎秆接种试验结合多年的田间抗性表现筛选出的43份菌核病抗性较好的油菜材料进行遗传多样性分析,并对2种分子标记揭示的多态性条带数、多态性信息含量(PIC)进行比较。结果表明:2种标记共检测出634个条带,SRAP标记检测的多态性条带数(335)较SSR(287)高,而SSR引物的平均多态性信息含量(PIC)值较SRAP引物高,分别是0.76和0.69。在遗传相似系数0.67处,43份油菜材料被分为Ⅲ类,白菜型油菜(丰油10号白菜型选系)可较好地与甘蓝型油菜区分。主成分分析(Principal component analysis,PCA)、群体结构分析与聚类分析结果相似,说明SSR与SRAP标记结合能准确有效的反映油菜材料的亲缘关系。供试43份油菜材料遗传相似系数分布在0.65~0.81,表明遗传相似性较高,亲缘关系较近。因此,应进一步加强抗源筛选及引进,对现有材料进行遗传改良,拓宽其遗传背景,从而为抗菌核病油菜品种选育奠定基础。     

以半冬性甘蓝型油菜为亲本增强春性甘蓝型油菜杂种优势

以春性恢复系与半冬性品种(系)杂交后选育的16份新春性恢复系及其4个亲本系[2个半冬性甘蓝型油菜品种(系)和2个春性甘蓝型恢复系]、2个春性甘蓝型不育系为材料, 利用SSR、SRAP和AFLP分子标记技术分析材料间的遗传差异, 同时利用以上2个春性不育系分别与12个新春性恢复系和1个春性亲本恢复系Ag-5进行NCII双列杂交, 测定其杂种优势及杂种表现。16份新恢复系中除931和帐23外, 其余的14份新恢复系与2个不育系的遗传距离均大于其春性亲本恢复系与相应不育系的遗传距离, 说明导入半冬性品种遗传成分能扩大春性恢复系与不育系间的遗传差异; 配制的26个杂交组合中, 其双亲中不育系所对应保持系单株产量为高亲值的组合有15个, 其中13个组合单株产量超亲优势都强于所对应不育系与亲本恢复系Ag-5所配杂交组合, 说明导入半冬性品种遗传成分可增强甘蓝型春油菜杂种优势; 12个新恢复系分别与2个不育系所配24个组合中18个组合的单株产量都分别超过所对应不育系与亲本恢复系Ag-5所配杂交组合, 说明导入半冬性品种遗传成分能提高甘蓝型春油菜杂种的产量; 新恢复系与2个不育系杂交后代的抗病性均强于其亲本恢复系与相应不育系杂交后代的抗病性, 说明导入半冬性品种遗传成分能提高春性甘蓝型油菜杂交种抗菌核病的能力。研究结果表明, 半冬性甘蓝型油菜品种可能为春油菜杂交育种提供有价值的遗传资源。     

双低油菜湘油15(B. napus)对菌核病抗性的研究

经苗期菌丝块接种鉴定和花期带菌牙签鉴定表明，双低油菜湘油15对菌核病有高抗性.当病原危害后，湘油15体内酚类物质含量和几丁质酶活性迅速升高，且维持时间长，湘油15与感病品种98C40和中抗品种中油821杂交后代抗性表现为双亲平均值,但偏向抗病亲本.     

Optimal allocation of resources in evaluating current sunflower inbred lines for resistance to Sclerotinia

Sclerotinia sclerotiorum (Lib.) de Bary is a major limiting factor for sunflower (Helianthus annuus) production. This study was conducted to (1) evaluate a large number of current sunflower lines for their reaction to Sclerotinia, and (2) determine the optimum allocation of resources for their selection. A set of 85 inbred lines was screened under artificial leaf infection in three environments. The test revealed ample variation for all resistance traits in the current germplasm. A line selected from NDBLOS, an oilseed sunflower germplasm poo, and its derivatives were most resistant to Sclerotinia. Lines derived from interspecific crosses with the wild species Helianthus tuberosus and Helianthus argophyllus also showed satisfactory Sclerotinia resistance. The evaluation of five plants per plot in two replications and four environments appears to be a reasonable allocation of resources in order to optimize the selection response.     

The residual background genome from a donor within an improved line selected by marker-assisted selection: impact on phenotype and combining ability

A successful marker-assisted selection (MAS)-derived line should express new characteristics derived from the donor, and have a small number of donor chromosomal fragments. Different selection strategies can result in a different proportion of donor residual background within the improved line. The effects of the residual background on phenotype and combining ability mostly remain unknown. In this study, waxy ( wx ) and a restorer gene ( Rf3 ) of S-type cytoplasm sterility were selected as a model for MAS breeding by three strategies, to develop MAS-derived lines with a different level of residual background. Three MAS-derived lines were successfully developed by introgression of the target gene. Furthermore, the genetic background, agriculturally important traits and combining ability of the three MAS-derived lines were assayed. The results suggested that the three MAS-derived lines retained 4–18% of the chromosomal fragment of the donor, respectively, and the residual background impacted on the phenotype of important traits and combining ability of MAS-derived lines. We also showed that whole genome background screening each backcross generation can accelerate recovery of the recurrent parent genome, phenotype and combining ability.     

Breeding of cabbage (Brassica oleracea L. var. capitata) with fusarium wilt resistance based on microspore culture and marker-assisted selection

Cabbage Fusarium wilt (CFW), caused by Fusarium oxysporum f. sp. conglutinans, is one of the most devastating diseases of cabbage worldwide. In recent years, CFW has been spreading across northern Chinad. Thus, development of resistant cultivars is necessary to control CFW. Here we report microspore culture from a cross of two elite cabbage inbred lines, 96–100 (highly resistant to CFW) and 01–20 (highly susceptible to CFW), in order to obtain doubled haploids (DHs) to cultivate cabbage materials for resistance breeding. A total of 196 DHs were obtained in 2011 and 2012 and three elite DH lines were selected from them through agronomic trait evaluation as well as marker-assisted selection. These three elite DH lines were crossed with elite cabbage inbred lines to create hybrids, which were then evaluated for their resistance and agronomic traits. Three excellent hybrids were selected. The use of microspore culture and marker-assisted selection greatly shortened the time required to obtain these excellent hybrids. These resistant varieties may become an effective control of CFW in diseased areas.     

甘蓝型油菜萝卜质雄性不育恢复系的选育

为了获得甘蓝型油菜萝卜细胞质雄性不育(Cytoplasmic male sterility,CMS)高含油量双低品质恢复系,以Ougra-CMS不育胞质杂交种Oug-F1(S(Rfrf))为不育基因和恢复基因供体,高含油量双低亲本D89-44(N(rfrf)为受体,通过杂交、多代同交以及最后自交对恢复基因进行了定向转育...     

转双价广谱抗病基因创造甘蓝型油菜抗菌核病新品种的研究

菌核病严重威胁着我国长江中下游地区甘蓝型油菜的生产，几乎每年都会导致甘蓝型油菜的大面积减产。来自于烟草的葡聚糖酶(Glucanase)基因，其编码产物能降解真菌的细胞壁结构成分一葡聚糖。来自于烟草的植物蛋白AP24则属于细胞程序性死亡(PCD)的诱导蛋白，对多种真菌具明显抑制作用。前期已成功地将这两个广谱抗病基因导入甘蓝型油菜，并且通过表型鉴定，在T0和T1代检测到了核盘菌抗性。本实验对转基因T2代和T3代甘蓝型油菜进行分子鉴定和田间抗病性鉴定。经过PCR分子鉴定，从14个转基因T3代甘蓝型油菜株系中筛选到4个外源基因已经纯合的株系。对株系GA02—2的RT—PCR鉴定则进一步证明外源广谱抗病基因在RNA水平上的大量表达。田间抗性鉴定主要利用苗期离体叶接种和成株期茎杆插签接种这两种方法。结果表明，T2代转基因株系中有5个株系表现为抗病基因初步纯合，5个株系表现为隐性纯合，12个株系处于分离当中，其中株系GA02—2的抗病性鉴定结果与RT—PCR鉴定的结果吻合。18个T3代转基因甘蓝型油菜株系中有7个株系表现为抗性纯合，3个株系剖现为隐性纯合，其余8个株系基本上仍在分离中，其中株系GA02—2—1的抗病性鉴定结果与前面的分子鉴定以及上代的抗病性鉴定完全吻合。综合分子鉴定和抗病性鉴定结果，筛选出抗病效果良好的转基因纯合株系应用于抗病育种。     

分子标记辅助选择Xa23基因培育杂交稻抗白叶枯病恢复系

水稻抗白叶枯病新基因Xa23抗谱广、抗性强,被定位在第11染色体上。以携带抗白叶枯病基因Xa23的抗病品系CBB23为抗源,以优良杂交稻亲本9311和1826为受体材料,采用杂交和复交,在分离群体中利用与Xa23紧密连锁的EST标记C189进行分子标记辅助选择,通过苗期分子标记检测和成株期农艺性状选择,获得160份目标基因纯合且农艺性状稳定的株系。使用鉴别菌系P6,采用人工剪叶接种法,在苗期和孕穗期对21份重点株系进行了抗性鉴定,所有株系在苗期和孕穗期都表现抗病。同时对3个不育系与其中5个株系配制的15个杂交组合进行了抗性鉴定,所有组合在苗期表现抗或中抗,在孕穗期表现抗。对其中6个杂交组合进行了品比试验,2个组合产量略低于对照两优培九,其余4个组合的产量均高于两优培九,用于测配的3个株系C6201、C6271和C6351有望作为杂交稻恢复系在生产中应用。研究表明在育种进程中利用与Xa23基因紧密连锁的分子标记C189开展抗白叶枯病分子标记辅助育种是一种有效的途径。     

14个甘蓝型油菜品种对菌核病的抗性初报

【研究目的】为探明不同类型的甘蓝型油菜品种对油菜菌核病的抗性差异,提供筛选抗耐病性的材料的新来源;【方法】选用14个甘蓝型油菜品种为材料,利用人工接种方法和病圃自然发病鉴定,研究了其青角期茎秆接种后病斑的扩展情况和成熟期自然发病情况以及在大田生产中的侵染发病;[结果]研究结果表明,不同甘蓝型油菜品种抗病性存在有明显差异,自然鉴定冬油菜材料Wanyou14的抗耐病要优于其他材料,抗病指数为0.36(对照Zhongyou821抗病指数为0),人工接种春油菜材料Ranger具有较好的病害抗扩展性,抗病指数为0.64,中晚熟的材料抗耐病强于早熟材料;【结论】冬油菜材料的抗耐病要优于春油菜材料,春油菜类型也具有较好的抗耐病性,抗性资源通过转育可提高冬油菜的抗病性;生产中油菜类型的选择以中晚熟的冬油菜品种为主。     

抗根肿病甘蓝型油菜新品种华油杂62R的选育

我国油菜根肿病发病面积在66.7万公顷左右,约占总生产面积的10%,已严重威胁到油菜的安全生产。基于此,本研究以含有CRb抗根肿病位点的大白菜材料CR Shinki为供体亲本,以甘蓝型油菜国审杂交种华油杂62的父本Pol.CMS恢复系Bing409为受体亲本,通过杂交、回交及自交等育种程序,结合前景和遗传背景筛选,将CRb抗病位点导入到Bing409中。在BC3F2代获得了遗传背景高度接近Bing409且含CRb抗病位点的抗根肿病新恢复系Bing409R,进而成功选育了我国首个抗根肿病杂交油菜新品种华油杂62R。CRb抗病位点在甘蓝型油菜背景中表现为单基因显性遗传,根肿病抗性遗传改良的同时并未对Bing409R及由其配制的杂交种华油杂62R的产量、品质造成不良影响,Bing409R及华油杂62R对我国四川、湖北、安徽等地区根肿菌生理小种具有免疫抗性。本研究的开展为我国油菜抗根肿病育种提供了宝贵资源,为我国抵抗油菜根肿病的威胁提供了重要保障。     

以器官和发育特异性抗病双基因转化油菜

菌核病是我国油菜主产区的主要病害，系由核盘菌(Sclerotinia sclerotiorum)的子囊孢子在晚春直接侵染开花植株衰老的叶片，或先侵染飘落在衰老叶片上的花瓣后再产生菌丝侵入叶片和植株本体。几丁质是核盘菌菌丝体细胞壁的主要组成成分，几丁质酶破坏真菌菌丝尖端新合成的几丁质而抑制病菌的生长。草酸是核盘菌侵染寄主植物后分泌的毒素，草酸氧化酶能够分解草酸，缓解核盘菌在油菜体内的毒性。我们将几丁质酶基因和草酸氧化酶基因与拟南芥衰老叶片启动子PSAG12嵌合在一起构建成表达载体，使两外源抗病基因仅在转基因油菜的衰老叶片中表达，在发挥抗病作用的同时最大限度地节约了植物的能量消耗。本实验采用in planta的转化方法，转化效率在0．1％左右，每个转基因植株的插入位点为1——3个拷贝。对T1代转基因植株的RT-PCR分析表明，两个外源抗病基因在衰老叶片中的表达强于幼嫩叶片。转基因植株叶片尤其是衰老叶片对核盘菌的抗性及对草酸毒素的耐性均得到了增强。从转基因植株上飘落的花瓣也表现出对核盘菌抗性的提高。抗病双基因在叶片和花瓣发育的特定时期表达不仅提高了油菜对核盘菌侵染的针对性，还可能减轻转基因植株的能源消耗和生理扰乱。对转基因植株后代的农艺学和生理学研究正在进行之中。     

农杆菌介导脂滴包被蛋白基因8-3转化烟草的研究

为开发新的抗菌核病基因,针对抗病相关的脂滴包被蛋白基因8-3,设计了分别带有酶切位点Xba I和BamH I的一对引物,从克隆载体pMD19-T-8-3中扩增到基因8-3,双酶切8-3并连接到植物表达载体pBI 121上;将重组质粒导入根癌农杆菌(Agrobacterium tumefaciens) LBA4404中,通过其介导将8-3基因转化到烟草中;获得了35株抗性转化株,PCR及测序分析鉴定阳性植株为21株,RT-PCR检测到基因8-3在其中能够表达;通过接种核盘菌和草酸处理试验,表明8-3基因可使烟草提高了草酸耐受水平,从而对菌核病具备一定的抗性。本实验研究了潜在功能基因8-3的抗病性,为植物抗菌核病的研究奠定了基础。     

江苏省油菜菌核病菌对多菌灵和菌核净的抗药性监测

2006-2010年从江苏省12个地级市的油菜产区采集油菜菌核,经分离纯化共获得6017株油菜菌核病菌。采用区分剂量法测定其对多菌灵和菌核净的抗药性。结果表明,2006-2010年间江苏省各地均有多菌灵抗性菌株的分布且抗药性水平较高,抗药性频率分别为63.9%、24.0%、17.1%、16.7%和9.8%,呈逐年下降的趋势。同时经室内研究发现,多菌灵抗性菌株与敏感菌株在菌丝生长、菌核产生、以及致病力等方面无显著差异。而2006-2010年间江苏省油菜菌核病菌对菌核净表现敏感,平均抗性频率仅为0.10%,5年内只在盐城、常州、连云港三地监测到共6株低抗菌株,且菌核净低抗菌株在菌丝生长、菌核产生等方面明显弱于敏感菌株。     

核盘菌诱导下甘蓝型油菜防御相关基因表达差异分析

菌核病是油菜主要病害, 至今尚未在油菜及相关植物中找到抗性基因。本研究利用qRT-PCR法比较了抗病品种宁RS-1和感病品种APL01在接种核盘菌后0~48 h内11个防御相关基因的表达差异, 以揭示抗病品种宁RS-1的抗病机制。结果表明, 4个基因(PGIP、Cu/ZnSOD、OXO和GLP)在核盘菌诱导前后抗、感品种内表达量均较高, 且抗病品种的表达量显著高于感病品种, 尤其是PGIP基因, 抗病品种宁RS-1在24 h的表达量为诱导前的170.4倍, 而感病品种仅为诱导前的3.5倍, 该时期抗病品种PGIP的表达量为感病品种的1299.4倍;2个基因(LOX2和PDF1.2)在诱导前后抗、感品种内的表达量均较低, 但抗、感品种间表达量差异显著;5个基因(FeSOD、PAL、EDS1、PR1和EIN3)诱导前后抗、感品种内的表达量均较低, 且抗、感品种间的表达量差异不显著。推测抗病品种宁RS-1对菌核病的抗性可能是由于PGIP的上调表达, 抑制了核盘菌PG蛋白对侵染部位油菜组织细胞壁的降解, 从而抑制了油菜菌核病的发生与蔓延。     

抗根肿病甘蓝型油菜新品种华油杂62R的选育

The rapeseed clubroot disease incidence in China is about 0.67 million hectare, accounting for 10% of the canola production area, which become a serious threat for the safety of Brassica napus industry. Based on this, we used CR Shinki, a Chinese cabbage material containing CRb clubroot disease resistance locus, as the donor parent, and Pol.CMS restorer line Bing409, the parent of Brassica napus national approved varieties Huayouza 62, as the recipient parent, and the CRb resistance locus was introduced into Bing409 by breeding programs such as crossing, backcrossing, self-cross with the foreground and genetic background selection. In the BC₃F₂ generation, a new restorer line Bing409R with a genetic background close to Bing409 containing CRb resistance locus was obtained, and Huayouza 62R, the first rapeseed hybrid resistant to clubroot disease in China was successfully developed. The results were as follows: CRb disease resistance locus appeared as a dominant single-gene inheritance in B. napus background, and the genetic improvement of resistance to clubroot disease did not at the expense of yield and quality losses for new restorer line Bing409R and its hybrid Huayouza 62R. Bing409R and Huayouza 62R were showed immune-resistance to physiological races of Plasmodiophora brassicae in Sichuan, Hubei, and Anhui provinces in China. This study will provide valuable resources for the breeding of rapeseed in China, and supplemented important support to overcome the threat of rapeseed clubroot disease.     

Breeding cowpea for resistance to Striga gesnerioides in the Nigerian dry savannas using marker‐assisted selection

Historically, conventional breeding has been the primary strategy used to develop a number of Striga‐resistant varieties currently grown in the Sahel of Western Africa. In this study, we have successfully developed and applied a marker‐assisted selection strategy that employs a single backcross programme to introgress Striga resistance into farmer preferred varieties of cowpea for the Nigeria savannas. In this strategy, we have introduced the Striga resistance gene from the donor parent IT97K‐499‐35 into an elite farmer preferred cowpea cultivar ‘Borno Brown’. The selected 47 BC₁F₂ populations confirmed the recombinants with desirable progeny having Striga resistance gene(s). The 28 lines selected in the BC₁F_2:4 generation with large seed size, brown seed coat colour and carrying marker alleles were evaluated in the field for resistance to Striga resistance. This led to the selection of a number of desirable improved lines that were immune to Striga having local genetic background with higher yield than those of their parents and standard varieties.     

甘蓝型油菜抗菌核病研究进展

油菜是主要油料作物之一,由核盘菌(Sclerotiniasclerotiorum(Lib.)deBary)引起的菌核病(sclerotiniastemrot)是油菜的主要病害。针对当前与菌核病相关研究的新进展,本研究从4个方面对其进行了概括：（1）核盘菌的侵染方式以及在侵染过程中核盘菌分泌的草酸与寄主中钙离子的动态关系;（2）油菜通过合成植保素、酚类化合物、木质素、几丁质酶和β-1,3-葡聚糖酶等来抵抗核盘菌入侵的抗病机理;（3）现有综合防治技术中抗性种质筛选、无花瓣育种和生物防治;（4）利用与草酸代谢相关基因、抗病相关基因、防御相关转录因子基因和抗菌肽基因开展的油菜基因工程研究成果。并且进一步提出了未来油菜抗菌核病研究的可能方向,这些总结与建议为今后油菜抗菌核病研究提供了有益参考。     

Resistance to Sclerotinia sclerotiorum of 'high oleic' sunflower inbred lines

Cultivation of sunflower (Helianthus annuus L.) is strongly affected by Sclerotinia sclerotiorum. To identify new sources of genetic diversity for sunflower breeding 25 sunflower inbred lines were analysed using eight Amplified Fragment Length Polymorphism (AFLP) primer combinations and their genetic similarities (GS) were estimated. Data were used to develop a Unweighted Pair Group Method using Arithmetic Averages (UPGMA) dendrogram. GS values of 0.58‐0.98 were observed but with no separate groupings dependant on oil quality. The inbred lines were screened for their reaction to inoculation with Sclerotinia sclerotiorum (Lib.) de Bary. Sunflower heads were artificially inoculated with S. sclerotiorum in three environments. Head infection was monitored after 1 week (lesion length) and 2 weeks (head rot). The F5 generation of a cross between a resistant (SWS‐B‐04) and a susceptible inbred line (SWS‐B‐01) was also tested for sclerotinia reaction across three environments. Significant differences in sclerotinia resistance, moderate heritabilities and a high correlation between the two assessments were observed. Inbred lines with a high level of resistance could be identified. These lines can be used for further breeding to improve sunflower sclerotinia resistance and to develop superior new hybrids.     

Introgression of bacterial blight resistance genes Xa7, Xa21, Xa22 and Xa23 into hybrid rice restorer lines by molecular marker-assisted selection

Four bacterial blight (BB) resistance genes, Xa7, Xa21, Xa22 and Xa23, were introgressed into an elite hybrid rice restorer line Huahui 1035, by marker-assisted selection (MAS). Ten promising BB resistant lines identified by MAS approach in Huahui 1035 restorer background and their respective F₁ hybrids with a cytoplasmic male sterile line i.e. Jinke 1A, three BB resistant gene donors and their recurrent parent were evaluated for BB resistance by inoculating them with eleven representative races of Xanthomonas oryzae pv. oryzae (Xoo) in China. Further, the ten marker assisted selection of BB (MAS-BB) resistant restorer lines and their F₁ hybrids were also characterized for agro-morphological traits and grain yield. Results revealed that restorer lines with Xa23 introgression i.e. HBQ809 and HBQ810 were found to be resistant to all eleven Chinese representative Xoo races, showing broad spectrum resistance to BB. However, the lines possessing Xa7 or Xa7+Xa21 were resistant to ten out of eleven Xoo races. While restorer lines with Xa21 were resistant to nine out of eleven Xoo races. Interestingly, the F₁ hybrids with Xa23, Xa7 or Xa7+Xa21 were resistant to two severe epidemic Xoo races of China. Restorer lines i.e. HBQ807 and HBQ808 possessing Xa22 were only resistant to six or five out of inoculated eleven Xoo races. Lesion length comparisons between restorer lines and their F₁ hybrids for the above four BB resistance genes showed them to be dominant in heterozygote genotype. Three promising high yielding F₁ hybrids with BB resistance were identified for immediate exploitation for hybrid rice production in China.