【作者】 李韬；

【导师】 顾铭洪； 辛志勇；

【作者基本信息】 扬州大学 ， 作物遗传育种， 2005， 博士

【摘要】 由小麦专化型白粉菌（Blumeria graminis DC f.sp.tritici）引起的小麦白粉病几乎在所有小麦生产区都有发生，已成为影响小麦生产的主要病害之一。培育抗病品种是防治其流行最有效、最安全的方法，而有利抗性基因资源的挖掘和高效利用则是培育抗病品种的前提，开展遗传分析可为目标基因的高效利用和后续的克隆及其抗病机制研究奠定基础。M53（YAV2/TEZ//Aegilops squarrosa249）是硬粒小麦（Triticum durum，2n=4x=28，AABB）与粗山羊草（Aegilops tauschii，2n=2x=14，DD）的双二倍体人工合成种，其对北京地区白粉菌主流小种15号和扬州地区主流小种表现免疫。本研究通过抗谱分析研究了抗性基因的来源、遗传模式以及与已命名白粉病抗性基因在抗谱上的差异；利用AFLP和SSR标记以及非整倍体分析对目的基因进行了染色体定位和遗传作图；利用抗病基因具有保守功能域的特点，采用同源克隆的方法从M53基因组DNA中分离了抗病基因类似片段（RGA），结果如下：对抗病亲本M53和感病亲本宛7107的正反交F₁以及F₂和F₃分离群体进行了白粉菌接种，结果表明，F₁正反交均表现为抗病，说明抗性受显性核基因控制，而F₂单株则表现出抗感分离，x²测验表明，抗感分离比例符合3∶1，达显著水平，说明抗性受显性单基因控制。F₃代植株的接种鉴定证实了F₂代表型鉴定结果的可靠性。为了追踪抗性基因的来源，我们对M53及其双亲Triticum durum（YAV2／TEZ）和Ae.squarrosa249分别在苗期和成株期进行接种鉴定，结果表明，YAV2／TEZ在两个时期均表现高感，而M53和Ae.squarrosa249则全生育期表现免疫，说明抗性基因来自Ae.squarrosa249，即来自粗山羊草，该基因暂命名为Pm-M53。截至目前，已定名的白粉病抗性基因中，总共有3个位于D组染色体上，分别为Pm2，Pm19和Pm24，对其载体品种Ulka／8^*Cc、XX186和赤鸭草进行了白粉菌15号生理小种接种鉴定，结果表明，Pm-M53在全生育期表现抗病，Pm19和Pm24则在全生育期表现感病；Pm2在苗期表现抗病，而在成株期表现感病，另外14个白粉菌生理小种在苗期接种也显示Pm-M53在抗谱上不同于Pm2、Pm19和Pm24，对其中5个小种表现感病，说明Pm-M53是小种特异性（race-specific）抗病基因，可能是一个新的白粉病抗性基因。分子标记技术已成为培育抗病品种、基因定位、遗传作图及图位克隆基因的主要手段和技术之一，目前已被广泛的应用于白粉病的研究。本研究结合AFLP、SSR和BSA技术，对来自杂交组合M53／宛7107的F₂作图群体进行了分析，在256对AFLP引物中，所有引物对在亲本M53和宛7107间都能扩增出30个以上位点，每对引物能够检测到1～11个多态性位点，在抗感池间筛选到多态性片段16个，其中Apm109和Apm161两个片段在抗亲和抗池中能够稳定重现，而在感亲和感池中无此带，用JoinMapV3.0和Kosambi公式对F₂作图群体的分析结果表明，这两个片段与目的基因紧密连锁，且位于目的基因的两侧，遗传距离分别为1.0和3.0 cM。标记Apm109在F₃的抗病单株中也能够稳定重现。从70个位于D组染色体的SSR标记中，在亲本及抗感池间筛选到重现性好的多态性标记标记5个，分别为Xwmc289，Xgwm583，Xgwm292，Xgwm191和Xwmc161，除Xgwm191位于5DS上外，其余4个标记均位于5DL上。对上述多态性SSR标记在群体中的分布进行了分析，结果表明，在LOD阈值为3.0时，位于5DL上的3个SSR标记Xwmc289、Xgwm583和Xgwm292与目的基因连锁，遗传距离分别为20.0、33.0和24.0 cM，并且分布于目的基因的两侧，初步说明目的基因位于5DL上。其中标记Xwmc289在M53和宛7107各存在两个共迁移片段，这两个共迁移片段在序列上存在很大差异，但均具有1个CT重复域和1个GA重复域，在M53中共迁移的两个目的片段的大小为159bp，在宛7107中对应的两个共迁移片段的大小为161bp，相对应的等位位点之间均有3个碱基的差异，一个为C／G碱基替换，另一个为GA重复域缺失。利用JoinMap V3.0对两个AFLP标记Apm109和Apm161，以及3个SSR标记Xwmc289、Xgwm583和Xgwm292联合进行了遗传作图，结果表明这些标记分布在目的基因Pm-M53的双侧，SSR标记Xgwm583和Xwmc289以及AFLP标记Apm161分布在一侧，Xgwm292和AFLP Apm109分布在目的基因的另一侧。从着丝粒近端（proximal）到远端（distal）的排列次序依次为Xgwm583、Xwmc289、Apm161、Pm-M53、Apm109、Xgwm292。其中3个SSR标记在染色体上的排列顺序与已经建立的小麦整合图谱是一致的，但标记之间的距离存在一定差异。由于两个AFLP标记Apm109和Apm161距离目的基因较近且位于目的基因的双侧，而3个位于5DL上的SSR标记又覆盖目标基因双侧的两个AFLP标记区，进一步说明目的基因Pm-M53位于5DL上，这点也通过距离目的基因最近的AFLP标记Apm109在两个中国春缺-四体材料（CSN5BT5D、CSN5DT5B）及两个双端体材料（CSDT5DS、CSDT5DL）中的扩增情况进行了证实，标记Apm109在CSN5BT5D和CSDT5DL上有目的特异带，而在CSN5DT5B和CSDT5DS上无此条带。在植物-病原菌相互作用中，不论何种植物类型还是与其作用的何种病原菌，植物抗病基因在结构上具有高度的保守性，因此根据R基因保守区设计特异或兼并引物、用PCR的方法从基因组DNA或诱导表达的转录本cDNA中寻找和克隆抗病基因是常用的方法。本研究根据GenBank中粗山羊草的部分抗病序列以及已克隆的大、小麦抗白粉病基因的核苷酸和氨基酸序列设计了非兼并和兼并引物，在M53和宛7107以及F₂单株组成的抗感池基因组DNA中进行了扩增，平均每对引物能够扩增出8个以上的位点。非兼并引物对RGAP-2和RGAP-6分别能够扩增出一条和两条多态性条带，即抗亲M53和抗池中有带，而在感亲和感池中无带，而所有兼并引物对在6％聚丙烯酰胺变性胶上均未扩增出肉眼可辨的多态性条带。我们对RGAP-2和RGAP-6产生的多态性片段分别进行了回收、克隆和测序，RGAP-2多态性片段大小331bp，而RGAP-6多态性片段之一的大小为259bp。对RGAP-2（331bp）和RGAP-6（259bp）的核苷酸序列在NCBI（http://www.ncbi.nih.gov/BLAST/）中进行相似性搜索（BLASTN）时发现，与RGAP-2和RGAP-6目的片段相似性最高的几个序列均来自粗山羊草，且都与抗病有关，具有明显的LZ-NBS-LRR或NBS-LRR抗病结构保守域。利用DNAStar软件的EditSeq模块对RGAP-6和RGAP-2多态性片段进行了核苷酸到氨基酸的序列翻译，RGAP-6（259bp）多态性片段无法翻译成氨基酸，而RGAP-2（331bp）能够翻译成氨基酸，推测RGAP-6（259bp）可能是内含子或者调控序列的一部分，而RGAP-2很可能是表达序列（外显子）的一部分。利用NCBI中的BLASTX对RGAP-2翻译的氨基酸序列进行同源搜索时发现，所有命中的序列均与抗病有关，且与来自粗山羊草的LZ-NBS-LRR型抗病蛋白序列同源性最高。在与RGAP-2同源性较高氨基酸序列的比较中发现，RGAP-2目的片段及其同源序列在I（V／L）IDD（K／I）WDK、N（N／E）CGSR（I／V）I（T／A）TTRI、PLS、KIL、KCGGVPLAII等序列上高度保守，与已知R基因NB功能域的kinase2、kinase3a和GLPLAII 3个结构域具有一定的相似性。在NCBI中利用rpsblast进行功能域搜索时发现RGAP-2目的片段含有保守域NB-ARC，与来自拟兰芥的R基因RPS-2、RPM1、RPP8和RPP13，番茄的R基因Prf，12C-1和12C-2，亚麻的R基因L6，以及与人和果蝇程序性死亡相关蛋白具有较高同源性，这些结果表明RGAP-2和RGAP-6两个感兴趣片段极可能来自粗山羊草，而RGAP-2很可能与粗山羊草的抗病性有关，但较低的相似性得分与较高的E值暗示这两个序列不同于任何已知的粗山羊草序列。此外，本文还就目的基因Pm-M53与已定名白粉病抗性基因的关系、白粉病的表型鉴定、Pm-M53遗传图谱与已构建遗传图谱的差异、位点特异性标记的转化，以及克隆的RGA片段与禾本科中已克隆R基因的关系、与植物中已克隆的白粉病抗性基因的关系、与小麦和大麦中白粉菌诱导抗性相关基因或候选基因的关系进行了讨论。对麦类白粉菌的生物学特性、白粉病抗性基因的发现及其相关分子标记的开发、白粉病抗性基因的遗传特点和抗白粉病分子育种策略、植物抗病机制及抗病基因的克隆、植物抗病性构筑策略以及AFLP标记技术的原理、发展、完善、优缺点及应用等方面进行了文献综述。更多还原

【Abstract】 Wheat powdery mildew disease caused by Blumeria graminis DC f.sp.tritici is one of themost devastating diseases prevalent in the wheat production area worldwide.Development of resistant cultivars is the most cost efficient and environmental safe wayto prevent its epidemic. Mining effective resistance genes and resources, however, is theprerequisite to developing the resistant cultivars. Genetic dissection of the genes ofinterest provides a foundation for marker-assisted selection, subsequent gene isolationand for understanding of plant disease resistance mechanism.Aegilops tauschii （syn. Aegilops squarrosa, 2n=2x=14, DD） is the diploid donor of Dgenome of allohexaploid bread wheat （Triticum aestivum,2n=6x=42,AABBDD）. Itcarries a wide variety of superior genes which can be transferred to bread wheat withoutmuch effort because the two species share D genome. M53 （YAV2/TEZ//Aegilopssquarrosa249） is the synthetic hexaploid wheat of Aegilops tauschii and tetraploidTriticum durum（2n=4x=28,AABB）. It was introduced from CYMMIT and exhibitedeverlasting immunity to the predominant isolates of Blumeria graminis DC f.sp.tritici（Bgt） in Beijing and Yangzhou wheat fields of China. In this paper, we explored theorigin and the inheritance mode of the gene of interest carried by M53 and the differences of disease response patterns of the target gene from known powdery mildewresistant genes （Pm） through inoculation of an array of isolates of Bgt. Chromosomelocation and integrated genetic mapping of the gene of interest were done employingAFLP, SSR and a set of aneuploids with Chinese Spring （CS） genetic backgrounds.Considering the cloned R genes have functional and structural similarities in proteinsequences irrespective of host type and pest type, we designed both degenerate andspecific PCR primers based on the conserved motifs and isolated the resistant geneanalogs （RGAs） from genomic DNA of M53. The following are the major points:Under infection of isolate No. 15 of Bgt, which is prevalent in wheat fields of Beijing area,both of F₁ reciprocal crosses of M53/Wan7107 demonstrated resistant phenotypes.indicating the gene resides in the nucleus in a dominant form. Further investigation of F₂individuals showed that the gene of interest segregated in a Mendelian factor waysuggested by significant 3:1 ratio of resistant/susceptible at Chi-square level. Randomlysampled F₂ individuals were advanced to F₃ generation. The disease response pattern ofF₃ individuals were in concert with the expectation, indicating the reliable scoring of F₂phenotypes. In order to make clear of the origin of the gene of interest, the two parents ofM53, YAV2/TEZ and Aegilops squarrosa249, were inoculated with the same isolate ofBgt. Consequently, YAV2/TEZ exhibits considerable susceptibility and Aegilopssquarrosa249 showed immunity to the tested isolate. These results figured out the factthat the gene is originated from Aegilops squarrosa249, that is, from D genome. Thegene of interest is temporarily named Pm-M53.To date a total of three Pm genes were assigned to D genome of wheat and its wildrelatives. They are Pm2, Pm19 and Pm24. All of these three Pm genes respondeddifferently from Pm-M53 to isolate No. 15 infection either at seedling stage or at adultstage. Pm-M53 was immune at both stages, whereas Pm19 and Pm24 showedsusceptibility at both stages. and Pm2 was resistant at seedling stage and susceptible atadult stage. Further infection with 14 additional isolates of Bgt differentiated Pm-M53from the other three Pm genes. The facts above showed that Pm-M53 is a race-specificand single dominant R gene, and seemed to be different from known Pm genes. Molecular markers are now becoming indispensable tools for marker-assisted selectionbreeding （MAS）, genetic mapping and map-based cloning of agronomically importantgenes. AFLP marker technique is very powerful and popular because of no requirementof sequence information of organisms and being accessible to many loci at one-time PCRamplification. When combined with bulked segregant analysis （BSA）, it is quite effectiveto search for closely-linked markers and to saturate mapping of the target region. SSRmarkers also have distinct advantages of widely and evenly distributions and knownpositions on wheat chromosomes and of displaying co-dominant band patterns which candifferentiate homozygote from heterozygote. Therefore, they have been universally usedfor genetic mapping and gene localization. Application of these powerful molecularmarkers facilitates identification and characterization of wheat Pm genes.In this study, F₂ segregation population from cross of M53/Wan7107 was investigated tomap Pm-M53 by combining employment of AFLP, SSR and BSA. Each of 256 AFLPprimer pairs can produce over thirty bands both in M53 and Wan7107 genomic DNA,and polymorphic loci ranged from one to eleven between M53 and Wan7107, but only asmall fraction of them, a total of 16, were found to repeat well between the bulkedresistant genomic DNA pool and the bulked susceptible genomic DNA pool. Usinglinkage analysis software JoinMap version 3.0 and Kosambi’s function at LODthreshold of 3.0, only two AFLP polymorphic fragments, Apm109 and Apm161, werefound to be closely linked to Pm-M53 with genetic distances of 1.0 and 3.0 cM,respectively. These two fragments flanked the target gene. Apm109 repeat well inresistant F₃ individuals. Amongst of 70 SSR primers sampled from D genome, only fiveprimers, Xwmc289, Xgwm583, Xgwm292, Xgwm191 and Xwme161, were able toproduce required polymorphic loci between bulked resistant genomic DNA pool andbulked susceptible genomic DNA pool. Interestingly, all of these five markers werelocated to the chromosome 5D with Xgwm191 on the short ann and the remaining fouron the long ann. These five polymorphic primers were subsequently used to analyze F₂mapping population, as a result, three markers, Xwmc289, Xgwm583 and Xgwm292,were linked to and flanked Pm-M53 with genetic distances of 20.0, 33.0 and 24.0 cM,respectively, indicating that Pm-M53 was probably located on the long arm of chromosome 5D. Unexpectedly, when the band of interest of marker Xwmc289 fromM53 and its ’allele band’ from Wan7107 was respectively excised, cloned and sequenced,each band was found to be involved with two distinct types of nucleotide sequences ofsame size. That is, one band, two loci. The size of co-migration band from M53 was 159bp and its ’allele band’ from Wan7107 was 161 bp. Two-nucleotide motifs of CT and GAreside in each co-migration band, whereas each type of sequence from M53 has acountpart in Wan7107 with polymorphisms of a C/G substitution and a GA-unitdeletion/insertion.Integrated genetic map encompassing Pm-M53 was constructed with two screened AFLPmarkers and three SSR markers. Markers Apm161, Xgwm583 and Xwmc289 wereplaced at one side, and Apm109 and Xgwm292 at the other side of Pm-M53. Thearrangement of the screened markers and Pm-M53 on 5DL, from proximal to distal,follows the sequence of Xgwm583, Xwmc289, Apm161, Pm-M53, Apm109 andXgwm292. The sequence of the three SSR markers on our genetic map was consistentwith the reference map. The fact that flanking SSR and AFLP markers covered the targetregion further indicated that the target gene was located on 5DL. This assignation ofPm-M53 to 5DL was also enhanced by chromosome location using a set of CSaneuploids. Closely linked AFLP marker Apm109 was present both innullisomic-tetrasomic line CSN5BT5D and ditelosomic line CSDT5DL, but absent innullisomic-tetrasomic line CSN5DT5B and ditelosomic line CSDT5DS.Cloned R genes share a strong functional and structural similarity in protein sequence.One or more conserved domains and motifs are present in cloned R genes irrespective ofhost type and pest type. These domains are typically categorized into four types,NBS-LRR, extracellular LRR, receptor kinase and cytosomic protein kinase. Each ofdomains has one or more highly conserved motifs. Therefore, PCR amplification wereoften utilized to search for candidate R genes or defense response related genes, usingdegenerate or specific primers which were designed based on amino acid or nucleotidesequence of conserved motifs. In this study, both degenerate and specific primers weredesigned based on the conserved amino acid or nucleotide sequences of NBS-LRR likeprotein from Aegilops tauschii and cloned Pm genes from wheat and barley. For M53. Wan7107, bulked resistant genomic DNA pool and bulked susceptible genomic DNApool, both types of primers can produce over eight loci. Only two specific primers,RGAP-2 and RGAP-6, amplified required polymorphic bands, present in M53 andbulked resistant genomic DNA pool, but absent in W7107 and bulked susceptiblegenomic DNA pool. None of degenerate primers could produce any polymorphic bandsbetween them. The polymorphie bands were then excised, cloned and sequenced. Thesizes of RGAP-2 and RGAP-6 fragments are 331bp and 259bp, respectively.Interestingly, when searching for similarities in GenBank non-redundant nucleotidedatabase using NCBI BLASTN function, the top three or four hits were all originatedfrom Aegilops tauschii and related to disease resistance. RGAP-2 fragment shared ahigher homology with top hits. Moreover, RGAP-2 fragment could be translated intoprotein sequence without interruption, whereas RGAP-6 fragment could not, resulting inthe prediction that RGAP-2 fragment may be a partial sequence of a functionallyunknown gene, while RGAP-6 fragment may be an intron or a regulatory sequence.When searching for similarities in non-redundant protein database of GenBank usingNCBI BLASTX function, all of the hits were related to plant disease resistance withobvious NB or kinase domains and motifs. The top four hits share a strong homologywith LZ-NBS-LRR type of protein sequences originated from Aegilops tauschii.RGAP-2 fragment shared with top 10 hits several conserved amino acid motifs likeI（V/L）IDD（K/I）WDK, PLS, KIL, KCGGVPLAII and N（N/E）CGSR（I/V）I（T/A）TTRI,amongst of which, I（V/L）IDD（K/I）WDK, N（N/E）CGSR（I/V）I（T/A）TTRI andKCGGVPLAII were correspondingly similar to kinase2, kinase3a and conservedhydrophobic GLPLAII motifs of cloned R genes. Further ’domain search’ with NCBIRPSBLAST function in GenBank showed that RGAP-2 fragment possesses a NB-ARCdomain which shared by cloned R genes of Arobidopsis thaliana-derived RPS-2, RPM1,RPP8 and RPP13, and tomato-derived Prf, I2C-1 and I2C-2, and flax-derived L6, andprogrammed cell death related proteins from human and fruit fly. These results showedthat RGAP-2 fragment was probably originated from Aegilops tauschii, and may be apartial sequence of a functional R gene. But relative low score of identity and high Evalue suggested that RGAP-2 and RGAP-6 were distinct sequences from known Aegilops tauschii-derived sequences in GenBank.In this paper, the following issues were discussed: The relationship of Pm-M53 withknown Pm genes; scoring system of powdery mildew inducible phenotype; conversion ofAFLP markers into locus-specific markers; the possible relationship of RGAP-2fragment with the cloned R genes in cereals, and with the cloned Pm genes in plant, andwith powdery mildew inducible defense response genes or candidate genes in wheat andbarley, respectively. Additionally, the advances of relevant issues were mini-reviewed inthe beginning pages of this paper: The biological characteristics of Blumeria graminisDC f.sp.tritici; identification and characterization of known Pm genes and QTLs;exploitation of closely linked or genie markers of known Pm genes; the inheritancemodes of powdery mildew resistance genes or QTLs; molecular breeding strategies ofdevelopment of powdery mildew resistance cultivars; mechanism of plant-pathogeninteraction and R gene cloning; strategies of engineering enhanced plant resistance.Finally, the disadvantages and disadvantages, progress and optimization of AFLPsystems were thoroughly reviewed.更多还原