Inheritance Analysis and Identification of SSR Markers Linked to Late Blight Resistant Gene in Tomato

Late blight caused by Phytophthora infestans is the most serious disease of tomato production in China. Studies on the genetics of resistance and identification of molecular markers are very useful for breeding late blight resistant varieties. The objective of this paper was to study the inheritance of late blight resistance and identify simple sequence repeat （SSR） markers associated with resistance allele in tomato （Lycopersicon esculentum Mill）. The results came from an F2 progeny of 241 plants derived from a cross between 5~ inbred line that is susceptible to late blight and a resistant accession CLN2037E. The late blight responses of F2 plants were tested by artificially inoculation of detached-leaflets in plate and natural infection assayed under greenhouse conditions. Both methods showed that the resistance is dominant and inherited as monogenic trait. Genetic mapping and linkage analysis showed that the late blight resistance gene Ph-ROL was located on chromosome 9 with a genetic distance of 5.7 cM to the SSR marker TOM236.     

DNA markers of the <Emphasis Type="Italic">R1</Emphasis> and <Emphasis Type="Italic">R3</Emphasis> genes as predictors of potato late blight resistance

To investigate whether the presence of R-genes is associated with higher late blight resistance of potato cultivars, we modified an earlier described SCAR marker for the R1 gene and developed a new SCAR marker for the R3 gene. We demonstrated the significant input of R1 transferred from Solanum demissum and probably S. stoloniferum into foliage and tuber resistance; the effect of R3 introgression was less conclusive.     

晚疫病诱导的番茄叶片cDNA文库构建及序列分析

以番茄抗病品系(CLN2037E)为研究材料,应用SMART技术构建晚疫病诱导的番茄叶片cDNA表达文库,并进行生物信息学分析。成功构建的cDNA文库的克隆数为2.3×109,重组率为96%,通过PCR检测,插入片段范围在0.1~2.0 kb之间。从文库中随机挑取110个克隆进行测序,最终得到107条差异表达ESTs序列。GenBank提交的序列号为GT742146-GT742150;GT865998-GT866099。利用BLAST进行同源性分析:75条ESTs与其他物种同源性较高,视为已知基因,功能涉及能量代谢的占42.7%、细胞内生命活动与信号传导的占10.7%、基因转录和翻译的占9.3%以及与抗性相关的占25.3%;另有32条ESTs无同源序列或同源性较低,预测是一些未知功能基因的占30%。本项研究应用SMART技术成功构建了高质量、信息丰富的晚疫病诱导的番茄叶片cDNA文库,为进一步筛选抗病相关候选基因奠定了基础。     

分子标记技术在马铃薯晚疫病研究中的应用

马铃薯是世界上重要的粮食作物之一,晚疫病则是当今危害马铃薯生产最为严重的病害。重点介绍了四种常用的分子标记技术RFLP、RAPD、AFLP和SSR,以及国内外利用这些标记技术在马铃薯晚疫病研究中的应用。分析了马铃薯晚疫病菌的遗传多样性、菌株抗药性、有性杂交后代的遗传分离以及抗病种质资源的遗传多样性,介绍了遗传图谱的构建、抗性基因及与晚疫病抗性相关的QTL定位、体细胞杂种及回交后代的晚疫病抗性检测。这些对今后中国学者开展相关领域的研究具有重要的借鉴意义。     

马铃薯 StBI-1 基因鉴定及其抗疫霉菌的功能探索

Bax inhibitor 1(BI-1）是真核生物中高度保守的细胞死亡调节因子,在植物响应生物与非生物胁迫中扮演重要角色。为探究BI-1是否参与马铃薯响应疫霉菌侵染的免疫应答,首先以拟南芥AtBI-1蛋白序列为模板在马铃薯蛋白数据库中进行BLAST检索。通过比对分析及构建系统进化树,鉴定到与AtBI-1相似度为77.22%的马铃薯蛋白NP_001305552.1,将编码该蛋白的马铃薯基因命名为 StBI-1 ;蛋白结构预测分析表明, StBI-1 包含7个跨膜域和Bax inhibitor-1-like家族结构域。利用实时定量PCR分析 StBI-1 基因在马铃薯响应致病疫霉菌侵染过程中的表达模式,并借助农杆菌介导的烟草瞬时表达体系初步探索 StBI-1 抗疫霉菌的生物学功能。结果显示, StBI-1 响应致病疫霉菌侵染而诱导上调表达,尤其是在侵染后期。同时,过表达 StBI-1 可显著增强本氏烟草对寄生疫霉菌和致病疫霉菌的抗性。综合以上结果,本研究对马铃薯 BI-1基因进行鉴定分析及克隆,并初步揭示 StBI-1 抗疫霉菌的生物学功能,为挖掘马铃薯抗晚疫病新型基因资源及深入解析其抗病分子机理奠定了理论基础。     

Interspecific potato hybrids as a resource for late blight resistance genes

Clones of potato hybrids bred from up to seven wild Solanum species were studied with phytopathological methods and DNA markers for late blight resistance genes R1, R3a, and RB. Late blight resistance of these clones was significantly related to the number of DNA markers discerned in particular inter-specific hybrids.     

Potato late blight caused by Phytophthora infestans: From molecular interactions to integrated management strategies

Over 170 years after the infamous Irish Potato Famine, potato late blight (PLB) caused by Phytophthora infestans remains the single most devastating disease of global potato production, causing up to 10 billion USD in yield loss and management costs. Through decades of research, growers and agronomists in the field as well as laboratory scientists have made significant progress in understanding the molecular pathogenesis process of this critical pathosystem and effective management strategies to control PLB. Yet, the need to feed an ever-increasing global population under changing climate demands continued improvement in efficient and sustainable PLB management schemes that can be implemented across a broad economic spectrum. In this review, we briefly summarize the current understanding of the molecular interaction between P. infestans and its host plants, highlight the current integrated pest management strategy to control PLB on local and continental scales, and discuss the potential of further improvement of sustainable PLB control through genetic enhancement of crop resistance and emerging crop protection technologies.     

利用田间抗病基因近等混合系防治马铃薯晚疫病

马铃薯是世界上第四大粮食作物,是我国七大农作物之一。晚疫病是由致病疫霉菌（Phytophthora infestans）引起的毁灭性的病害,19世纪中叶曾造成举世闻名的“爱尔兰大饥荒”,至今仍然是马铃薯最严重的病害。提高抗晚疫病的水平是马铃薯育种的重要目标。马铃薯抗晚疫病育种是通过遗传操作来抵抗植物病害的最早的人类实践之一,但至今收效甚微,从野生种导入的抗病基因在田间很容易失效。马铃薯和致病疫霉菌的互作关系符合经典的“基因对基因”假说,只有马铃薯的抗病基因和致病疫霉菌的非毒力基因同时存在并表达时,抗病反应才能发生。致病疫霉菌是一种进化潜力非常高的病原,可以快速突变本身的非毒力基因,因而造成对应的马铃薯抗病基因的失效。要提高抗病基因的持久性,目前唯一的途径是同时释放多个抗病基因,人为造成田间抗病基因的多态性。马铃薯抗晚疫病基因的克隆取得了快速的发展,为通过转基因的手段创造田间抗晚疫病基因的多态性提供了基础。     

Rapid detection of potato late blight using a loop-mediated isothermal amplification assay

Potato late blight caused by Phytophthora infestans is one of the most destructive plant diseases that threaten global food security. Early and effective diagnosis of P. infestans is required before disease management decisions are made. Here, we developed a quick protocol to detect P. infestans based on a loop-mediated isothermal amplification(LAMP) assay. The P. infestans specific multiple copy DNA sequences(PiSMC), a transposon-like element, provides an ideal target for molecular detection of this pathogen. We designed highly specific and sensitive primers allowing effective LAMP detection of the pathogen at 64℃ in 70 min. In the validation assay, all 15 P. infestans isolates collected from China, Europe and South America could be positively detected, but results of the other 9 Phytophthora species infecting different plants, fungal and bacterial plant pathogens tested were negative. The detection limit of this assay is 1 pg P. infestans DNA. Moreover, the LAMP-PiSMC assay is able to detect P. infestans from infected leaves, tubers and soil. Taken together, this study reports the development of a specific and sensitive LAMP-PiSMC assay for early diagnosis of potato late blight.     

云南省马铃薯春作区晚疫病菌对甲霜灵敏感性测定

旨在明确云南省马铃薯春作区晚疫病菌对甲霜灵的敏感性,为马铃薯晚疫病化学防治提供参考。采用菌落直径法对2018-2020年采自云南省春作区的301株马铃薯晚疫病菌进行甲霜灵敏感性测定,结果显示:甲霜灵高抗、中抗、敏感菌株分别占测定菌株的76.41%、21.26%和2.33%。研究结果表明,2018-2020年云南省马铃薯春作区晚疫病菌甲霜灵抗性菌株已高达97.67%,甲霜灵已不能在云南省春作区用于防治晚疫病。     

马铃薯无性系对晚疫病的抗病性配合力分析

本文按16X3双因数交叉式遗传设计配置的48个单交种为材料，连续2年在云南农业大学试验农场进行了家系的抗病性评价试验，试验采用随机区组，4次重复；抗病性采用Jan W.Henfling的方法计算发病叶面积，并进行了配合力分析。结果表明：（1）马铃薯对晚疫病的抗病性主要由本决定。（2）一般配合力对于马铃薯抗晚疫病更为重要。（3）母本393085．5、391002．15、391002．6、391004．4号具有较高的一般配合力，父本一般配合力不高。（4）母本39328058号与父本392639．8具有较高的特殊配合力。本研究结果为抗晚疫病的亲本选配提供了重要依据。     

Population Improvement of Resistance to Late Blight in Tetraploid Potato: A Case Study in Combination with AFLP Marker Assisted Background Selection

Cultivated potato with high level of horizontal resistance against late blight is one of the most important goals of potato breeding.The recurrent selection has been adopted to increase the level of potato horizontal resistance and a B3C1 population without R1-R11 dominant genes has been released by the International Potato Center at the short-day condition of Peru.The present research was carried out to further improve the resistance and the agronomic traits of B3C1 population under long-day condition of Hubei,China,with maximized retention of its genetic diversity.Twenty-seven individual clones of B3C1 were used to generate population B3C2 by in-population crossing with the bulk pollens aiming to elevate the frequency of late blight resistance genotypes and to improve the adaptation to local long-day conditions.The late blight resistance and the main agronomic traits including the maturity,the plant characters and the tuber traits were evaluated for the foreground selection in three years,by which 130 pedigrees were maintained as the basic population of B3C2 for further selection.A total of 312 polymorphic loci detected by 9 AFLP marker combinations were used to monitor the genetic diversity of the populations for the background selection.The B3C2 population of 51 clones was finally selected,of which the frequency of resistant genotypes increased by 23.8％ points and the genetic diversity was maintained by about 96％ as referred to B3C1.Our results strongly suggested that combination of the foreground selection for target traits and the background selection for the genetic diversity is an efficient strategy in the recurrent selection of tetraploid potato to improve quantitative traits.     

番茄第9号染色体上抗晚疫病基因的发掘及表达模式

为提供番茄晚疫病抗病育种候选基因,本研究以高抗晚疫病的CLN2037E自交系和感病5#自交系为实验材料,在前期使用分子标记将CLN2037E中存在的抗晚疫病基因定位在第9号染色体的基础上,利用晚疫病病菌诱导CLN2037E的cDNA文库、番茄基因组CDS数据库、本地Blast程序及实时荧光定量PCR技术,发掘与第9号染色体上EST最佳匹配的CDS以及对晚疫病病菌的响应模式。结果发掘出6条抗晚疫病的候选基因,其中Solyc09g097960、Solyc09g082810和Solyc09g065760在5#自交系和CLN2037E自交系中均响应晚疫病病菌,Solyc09g092030和Solyc09g090430在2个自交系中分别被抑制和不响应晚疫病病菌,Solyc09g008670STBZ的表达在5#自交系中被抑制,而在CLN2037E中被诱导。为验证该基因的功能,成功构建Solyc09g008670基因的过表达和VIGS(Virus Induced Gene Silencing)沉默载体。     

毕节地区主栽马铃薯品种对晚疫病的田间抗性评价

为摸清毕节地区主栽马铃薯品种对晚疫病的抗性情况,2007-2008年,在毕节、威宁、织金、赫章、黔西5县(市)不同生态区域开展了主栽马铃薯品种对晚疫病的田间抗性试验研究,结果表明:16个参试品种抗性存在明显差异,表现较好(中抗以上)的品种有7个(昭绿23号、昭绿4号、BP08、会-2、黔薯1号、威芋4号、鄂薯5号),只占参试品种的43.75%.因此,要不断加强新品种的引进、试验与示范及田间抗病性的监测,以便为品种的合理利用与布局提供科学依据.     

马铃薯晚疫病抗性相关基因定位及QTL位点分析

【目的】寻找与马铃薯晚疫病水平抗性紧密相关的候选基因,以期作为分子标记辅助选择的重要标记。【方法】以BCT和PCC1两个马铃薯群体为材料,对38个晚疫病菌诱导表达的ESTs和基因进行定位,再将定位结果与已定位的QTL位点进行比对。【结果】11个候选基因的引物在两个群体中扩增出13个多态性位点,其中12个多态性位点定位到遗传连锁图谱上。定位结果与QTL进行比较显示,07-F08-P1-564位于晚疫病QTL区域。【结论】07-F08-P1-564与马铃薯晚疫病水平抗性紧密相关。定位的候选基因丰富了马铃薯的连锁群,可作为遗传连锁图谱构建的桥梁,同时也为筛选重要抗性候选基因奠定了基础。     

马铃薯晚疫病抗性相关基因定位及QTL位点分析

【目的】寻找与马铃薯晚疫病水平抗性紧密相关的候选基因,以期作为分子标记辅助选择的重要标记。【方法】以BCT和PCC1两个马铃薯群体为材料,对38个晚疫病菌诱导表达的ESTs和基因进行定位,再将定位结果与已定位的QTL位点进行比对。【结果】11个候选基因的引物在两个群体中扩增出13个多态性位点,其中12 个多态性位点定位到遗传连锁图谱上。定位结果与QTL进行比较显示,07-F08-P1-564位于晚疫病QTL区域。【结论】07-F08-P1-564与马铃薯晚疫病水平抗性紧密相关。定位的候选基因丰富了马铃薯的连锁群,可作为遗传连锁图谱构建的桥梁,同时也为筛选重要抗性候选基因奠定了基础。     

转无毒基因马铃薯抗晚疫病的研究

晚疫病菌(Phytophthora infestans)引起的马铃薯晚疫病是危害全球马铃薯生产的严重病害.通过基因工程方法把外源基因导入植物体内以增强抗病性被证明是一条行之有效的途径.应用植物基因工程技术将具有能激活植物自身防御系统的无毒基因与适合于植物背景、非专一性的病原物诱导启动子组合成嵌合基因构建到植物表达载体中.通过农杆菌或基因枪的介导转化植物,可筛选出高效广谱的抗真菌和细菌病害的转基因植株.本研究从病原细菌Pseudomonas syringae PV.tomato中获得的无毒基因avrD(0*93kb)和从病原真菌Phytophthora parasitica中获得的无毒基因Elicitin(0.294kb)分别与非专一性病原物诱导启动子Pill和BG组成含2个嵌合基因(Pill-avrD,BG-Elicitin)的植物表达载体pYH144和pYHEt.通过农杆菌LBA4404介导转化马铃薯,其中用pYH144载体转化2个品种(克新1号,2号),用pYHEt载体转化3个品种(Desiree,克新2号,4号),通过组织培养分别获得潮霉素(HygromycinB)标记的转基因马铃薯试管苗.将转基因试管苗扩繁,应用马铃薯脱毒微型种薯生产技术获得转无毒基因微型薯,在温室(15～25℃和湿度高)条件下,观察转无毒基因马铃薯植株中对晚疫病菌自然感染的抗性.1998年和1999年(每年的3-5月)的温室试验初步表明用avrD和elicitin基因分别转化的转无毒基因马铃薯植株都具有较明显的对晚疫病菌侵染的抗性,大部分转基因植株不表现或表现轻微的感病症状,对照植株(未转化)则表现明显的感病症状.转基因植株生长正常,且在感染后期(恢复期)生长良好.对照植株在恢复期生长弱和缓慢.在获得较多数量的转无毒基因马铃薯微型种薯的时候,将进行人工接种晚疫病菌和田间种植试验,从中筛选出抗真菌病和细菌病的转基因马铃薯株系.     

表达 HarpinEa基因的转基因马铃薯的晚疫病抗性分析

 通过根癌农杆菌介导,用Harpin_Ea基因转化马铃薯四倍体栽培种大西洋（Atlantic）,获得107个马铃薯转化株系。经过卡那霉素的抗性鉴定和PCR分子检测,有59株PCR检测成阳性,占所有转化植株的55.14%.实验对部分转基因植株进行室内抗病性评价,从24个PCR阳性株系中筛选到9个株系的抗病性与对照相比有显著差异(P<0.05),其中7株的抗病性与对照相比差异极显著(P<0.001)。结果初步表明Harpin_Ea基因已整合到马铃薯的基因组中并得到表达,提高了植株的晚疫病抗性。     

部分马铃薯主栽品种田间抗晚疫病性初步鉴定

通过对9个主栽马铃薯品种的抗晚疫病性田间自然诱发初步鉴定试验结果表明,中薯3号的产量高、抗性好,是适合在当地大面积推广的最佳品种.费乌瑞它虽然较感病,但产量最高,若结合防治,也是生产上推广的理想品种.     

分子标记辅助甜瓜抗蔓枯病基因的聚合及品种改良

【目的】蔓枯病（gummy stem blight）是一种真菌性土传病害,是危害甜瓜生产的严重病害之一,其存在生理小种的分化,品种即使仅携带单个抗病基因,仍会导致甜瓜抗性逐步降低甚至丢失。建立甜瓜抗蔓枯病聚合育种的分子标记辅助选择体系,选育抗性高且不再分离的聚合抗源自交系和品质优良且高抗甜瓜蔓枯病的改良白皮脆品种（品系）,为甜瓜优质、抗病和高产育种提供一种简单、快捷的选择方法和重要的中间材料。【方法】利用5份甜瓜单一抗源材料PI140471、PI157082、PI511890、PI482398和PI420145两两杂交获得8份聚合抗源（082-471、082-890、082-398、145-471、145-082、145-890、145-398和890-398）。利用3个不同浓度的蔓枯病病菌孢子液（5×10⁵个/mL、5×10⁷个/mL、5×10⁹个/mL）对8份聚合抗源的F₁植株幼苗进行接种鉴定。结合春、秋两季的接种鉴定结果,筛选得到2份聚合后抗性显著提高的基因组合145-471和145-398。一方面,以145-471和145-398为材料,利用苗期梯度接种鉴定、分子标记辅助选择以及农艺性状观察,对其自交后代进行逐代筛选至F₇。另一方面,选用组合PI420145和PI140471以及PI420145和PI482398作为蔓枯病抗性基因的供体亲本,综合性状优良的感病甜瓜品种‘白皮脆’为抗性基因受体亲本。先分别单向回交5代,在单个基因回交转移时,利用分子标记检测结合农艺性状观察对回交后代进行筛选。当回交后代的遗传背景得到一定程度恢复后,再将2个方向的回交后代进行杂交,后自交3代稳定。【结果】聚合抗源对不同浓度的蔓枯病菌均表现为抗,而单一抗源对不同浓度蔓枯病菌表现出选择性抗性且抗性水平低于聚合抗源。SSR标记CMCT505和CMTA170a在PI140471和PI482398上可分别扩增出189 bp和121 bp的特异性片段,SCAR标记SGSB₁₈₀₀可以在PI420145上扩增出1 800 bp的特异性片段,而145-471（145-398）的聚合单株可以同时扩增出189 bp和1 800 bp（121 bp和1 800 bp）两条特异片段。聚合抗源145-471（或145-398）的自交F₇世代以及改良白皮脆BC₅F₄世代群体的分子标记鉴定结果显示,各单株已经成功聚合了Gsb-1和Gsb-6（或Gsb-4和Gsb-6）两个抗病基因,抗性不再有分离且农艺性状稳定一致。初步建立了甜瓜抗蔓枯病聚合育种的分子标记辅助选择体系,获得了两份表现高抗且抗性不再分离的聚合抗源自交系F₇和2个以优质品种‘白皮脆’为受体亲本的改良白皮脆BC₅F₄品种（系）。改良白皮脆BC₅F₄世代表现为高抗甜瓜蔓枯病,且在单果质量、果形指数、果实脆度、果肉质地、果肉厚度和可溶性固形物含量等农艺性状方面与白皮脆并无显著差异。【结论】创建的3个分子标记CMCT505、CMTA170a和SGSB₁₈₀₀对抗病基因Gsb-1、Gsb-4和Gsb-6的选择具有较高的准确性。初步建立了甜瓜抗蔓枯病聚合育种的分子标记辅助选择体系,为甜瓜优质、抗病和高产育种提供了一种简单、快捷的选择方法,将大大提高育种的效率。改良‘白皮脆’作为抗病育种的新材料,为甜瓜抗病品种的选育和抗病基因进一步聚合提供了材料。