【作者】 郑燕；

【导师】 吴为人；

【作者基本信息】 浙江大学 ， 作物遗传育种， 2008， 博士

【摘要】 揭示植物与病原菌互作的遗传基础是认识植物抗病和病原物致病机理,培育持久、广谱抗病作物品种的理论基础。植物与病原物的互作是一个复杂的生理生化过程,同时受植物和病原菌许多基因的控制。因此,必须同时对植物和病原菌的基因组进行分析,才能完整地理解其内部机理。本研究以稻瘟病为模式,应用最新发展出的将植物和病原菌作为一个整体的植物与病原菌互作QTL分析方法,并将QTL分析与现有的基因表达数据相结合,同时在水稻和稻瘟菌全基因组中寻找控制水稻和稻瘟菌互作的QTL或基因,初步揭示控制水稻和稻瘟菌互作的跨越水稻和稻瘟菌两个物种基因组的多基因体系。主要研究内容和结果如下:1、以稻瘟菌菌株Guy11和2539为亲本进行杂交,通过随机挑取单个子囊孢子,建立了一个包含229个后代菌株的群体。将该群体接种于水稻品种CO39,证实2539对CO39的无毒性是由无毒基因AVR1-CO39控制的。将该群体接种于另外14个对Guy11感病而对2539抗病的水稻品种上,发现有8个品种的抗病表现与CO39一致,推测2539对这些品种的无毒性也是由AVR1-CO39控制的;而另外6个品种的抗病表现也与CO39相似,推测其各自对应的无毒性与AVR1-CO39紧密连锁,分布在AVR1-CO39两侧约30kb的范围内。2、利用已公布的稻瘟菌全基因组序列,开发了446对SSR引物。426对引物能扩增出条带,其中314对在9个从水稻上分离到的稻瘟病菌株(包括实验菌株2539)中检测到多态,多态率达73.9%。每个SSR座位上检测到的等位基因数在2～9之间,平均为3.3。每对标记的多态信息含量(PIC)变化在0.20～0.89之间,平均为0.53。3、利用Guy11和2539杂交获得的遗传群体,构建了一张含有176个SSR标记的稻瘟菌遗传图谱。该图谱总长1247cM,相邻标记间平均距离为7.1cM,覆盖整个基因组的93%(约35.0Mb)。发现有26个(14.4%的)标记在1%显著水平上表现为偏分离,其中16个标记偏向亲本Guy11,10个标记偏向亲本2539。4、通过对稻瘟菌遗传图谱和物理图谱的比较,发现有5个标记在两种图谱中的位置不相吻合。有11个未知物理位置的标记被定位到遗传图谱上。稻瘟菌基因组中1cM的遗传距离平均相当于28kb。发现了一些重组冷点和重组热点。5、为方便读者利用我们开发的稻瘟菌SSR标记,建立了一个命名为MGM的在线数据库(http://ibi.zju.edu.cn/pgl/MGM/index.html)。该数据库提供了各个SSR标记的详细信息,包括SSR标记的物理位置、引物序列、重复基序、重复数、预期的PCR产物长度、退火温度以及在9个稻瘟病菌中检测到的等位基因数和PIC值。6、用121个不含AVR1-CO39的Guy11×2539的后代菌株对籼稻品种Acc8558和H359杂交产生的131个重组自交(RI)系进行随机接种,以相对病斑面积(RLA)和病级(DG)为指标,同时在水稻和稻瘟菌基因组中对控制水稻与稻瘟菌互作的QTL进行定位分析。在水稻和稻瘟菌中分别检测到3个和2个控制RLA的QTL,其中一个水稻QTL与一个稻瘟菌QTL间存在互作;在水稻和稻瘟菌中各检测到4个控制DG的QTL,其中水稻中有一对QTL存在上位性,稻瘟菌中有3对QTL存在上位性。7、根据已公布的水稻和稻瘟菌全基因组序列,将每个QTL的位置区间标注到物理图谱中,并与已公布的基因芯片和EST数据相比较,为4个水稻QTL找到5个候选基因;为5个稻瘟菌QTL找到9个候选基因。更多还原

【Abstract】 Revealing the genetic basis of interaction between plant and pathogen would provide the theoretical foundation for understanding the mechanisms of plant resistance and pathogen pathogenesis and breeding crop varieties with durable and broad-spectrum resistance.Plant-pathogen interaction is a complicated biochemical and physiological process,in which many genes in plant and pathogen genomes are involved.Therefore,simultaneous analysis of plant and pathogen genomes is needed for full understanding of the internal mechanisms of plant-pathogen interaction.With rice blast as a model,this study aimed to reveal the across-species polygenic system controlling the interaction between rice and Magnaporthe grisea by scanning for related QTLs/genes in the rice and M.grisea genomes simulataneously,using the recently proposed QTL mapping method for plant-pathogen interaction,which takes the plant and pathogen as an integrated system,and combining QTL analysis with gene expression data.The main contents and results are as follows:1.A cross between two M.grisea strains,Guy11 and 2539,was performed and 229 progeny strains were obtained by randomly isolating ascospores.By inoculating this population on rice cultivar CO39,it was comfirmed that the avirulence of 2539 to CO39 was controlled by the avirulence gene AVR1-CO39.After inoculating this population on other 14 rice cultivars,it was found that 8 rice cultivars showed the same responses to the inoculations as CO39,suggesting that the avirulence of 2539 to these 8 cultivars should be also controlled by AVR1-CO39;the responses of the other 6 rice cultivars were also similar to CO39,suggesting that the avirulence of 2539 to these 6 cultivars might be controlled by different avirulence genes other than but closely linked to AVR1-CO39,and linkage analysis indicated that these gene are located around AVR1-CO39 within a range of 30kb.2.Based on the released genome sequence data of M.grisea,we designed primers for 446 simple sequence repeat(SSR)loci.426 pairs of primers could obtain amplification products,of which 314 pairs showed polymorphisms among 9 M. grisea strains isolated from rice(including a laboratory strain 2539).The polymorphism percentage is 73.9%.The number of alleles of each SSR marker ranged 2～9 with an average of 3.3.The polymorphic information content(PIC)of each marker ranged 0.20～0.89 with an average of 0.530.3.Using the progeny population of Guy11×2539,a genetic map of M.grisea consisting of 176 SSR markers was constructed.The map covers a total length of 1247 cM with an average distance of 7.1 cM between adjacent markers,equivalent to a physical length of about 35.0 Mb or 93%of the genome.Among these SSR markers, 26(14.4%)markers showed the genetic segregation distortion at the 1%significance level.Among them,16 markers deviated toward Guy11 and 10 markers deviated toward 2539.4.Comparison between the genetic map and the physical map indicated that the positions of five markers were not consistent in the two maps.Eleven markers with unknown physical positions were mapped in the genetic map.In the M.grisea genome,1cM is approximately equivalent to 28kb on average.Some recombination hot spots and cold spots were identified.5.For the convenience of readers to utilize the SSR markers developed in this study,we have established a web-based database named MGM,which is accessible at website http://ibi.zju.edu.cn/pgl/MGM/index.html.Detailed information of each SSR is provided,including position,primers,repeat motif,expected PCR product length, anneal temperature,number of alleles detected in the nine M.grisea isolates and PIC value.6.One hundred and twenty-one progeny strains of Guy11×2539 not carrying AVR1-CO39 were used to randomly inoculate 131 recombinant inbred lines(RILs) derived from a cross between two indica rice cultivars,Acc8558 and H359.With relative lesion area(RLA)and disease grade(DG)as index,QTL mapping was performed for rice-M,grisea interaction in both the rice and the M.grisea genomes simultenously.Three and two QTLs underlying RLA were detected in the rice genome and the M.grisea genome,respectively,of which a QTL of rice was found to interact with a QTL of M.grisea.Four QTLs each conferring DG were found in the rice genome and the M.grisea genome,respectively.Epistatic effects were detected between one pair of QTLs in rice and three pairs of QTLs in M.grisea.7.The positional interval of each QTL in the physical map of rice and M.grisea was identified according to their released genome sequences,and was compared with the microarray and EST data.Five candidate genes were found for 4 rice QTLs and 9 candidate genes were found for 5 M.grisea QTLs.更多还原