【作者】 李伟；

【导师】 翟虎渠； 万建民；

【作者基本信息】 南京农业大学 ， 作物遗传育种， 2008， 博士

【摘要】 稻瘟病菌(无性世代为Pyricularia grisea,有性世代为Magnaporthe grisea)是危害水稻生产的主要病害之一。利用抗性基因培育水稻抗性品种是克服稻瘟病危害最经济有效的措施。由于稻瘟病菌群体结构和致病性的变化,含单抗性基因品种的抗性容易伴随新的致病型菌株的出现而丧失。因此,挖掘和鉴定广谱的抗性主效基因和抗性数量基因座位,通过分子标记辅助选择聚合有利的抗性基因,培育广谱和持久抗性的水稻品种,是水稻抗稻瘟病育种面临的主要工作。本研究分析了两个水稻抗性品种IR24和DV85的抗稻瘟病基因组成,鉴定和定位了3个稻瘟病抗性主效基因和多个抗性QTL。主要研究结果如下：1.IR24抗中国稻瘟病菌株的主效基因主要是Pi20(t)和Pib,其中Pi20(t)基因对北方粳稻区的菌株表现广谱抗性(94.2%),对南方籼稻区的菌株的抗谱中等(52.6%),是水稻抗稻瘟病育种的一个优良抗性基因。本研究从160个中国稻瘟病菌株中鉴定了一个能特异性鉴别Pi20(t)基因的菌株98095,利用该菌株接种Asominori(感)×IR24(抗)的重组自交系和F2群体,借助分子标记进行精细定位,鉴定出5个与Pi20(t)基因紧密连锁的SSR标记,即OSR32、RM1337、RM5364、RM7102和RM28050,其中,3个共分离标记RM1337、RM5364和RM7102对Pi20(t)基因选择效率达100%,2个旁侧标记OSR32和RM28050对Pi20(t)基因选择效率达98%以上。这5个分子标记在Pi20(t)基因的供体亲本IR24和一系列国内推广品种之间具有良好的多态性。Pi20(t)基因可以通过分子标记辅助选择直接应用于粳稻抗稻瘟病改良或与互补抗性基因聚合应用于籼稻抗稻瘟病改良。2.DV85对中国稻瘟病菌株具有高水平的抗性,其中对北方粳稻区的菌株的抗谱高达92.9%,对籼稻区菌株的抗谱为54.0%,在中国也是一个优良的抗源材料。利用Kinmaze(感)×DV85(抗)的重组自交系、BC1F1 (Kinmaze/DV85//Kinmaze)和F2群体对DV85进行抗性基因分析和基因定位,鉴定出一个新的显性主效抗稻瘟病基因Pidv(t),位于1号染色体长臂末端。通过扩大F2作图群体和开发新标记,将Pidv(t)精细定位于Indel标记C4和SSR标记RM12182区间,与两标记的遗传距离分别为0.5cM和0.1cM,两标记之间的物理距离为66.9kb。Pidv(t)的精细定位为该基因的标记辅助选择和图位克隆奠定了基础。3利用91-17-2、97-27-2、59-3、L64-1、CH26和TH16等6个稻瘟病菌株接种两套重组自交系群体—Asominori(?)：感)×IR24(抗)的71个重组自交系(群体Ⅰ)和Kinmaze(?)：感)×DV85(抗)的81个重组自交系(群体Ⅱ),分别从群体Ⅰ和Ⅱ鉴定出58个抗性QTL和59个抗性QTL分布于1-12号染色体上,其中病斑数(LN)、病斑长(LL)、病叶面积(LA)和病级(LD)等四个性状在群体Ⅰ中分别鉴定出16、13、12和17个抗性QTL,在群体Ⅱ中分别鉴定出16、12、14和17个抗性QTL。34个QTL的抗病等位基因源自IR24,24个QTL的抗病等位基因源自Asominori,29个QTL的抗病等位基因源自DV85,30个QTL的抗病等位基因源自Kinmaze。对表型变异贡献率大的QTL位于IR24的Pib座位和DV85的Pidv(t)座位,抗性QTL与主效基因共同构成了IR24和DV85的抗瘟性能力。两个群体鉴定的QTL均存在聚簇和共座位现象,聚簇现象多发生在主效基因座位区域,而同一菌株的不同致病性状鉴定的QTL常共座位。只有少数抗性座位抗2-3个不同的菌株,因而QTL表现了明显的小种特异性。通过比较分析,两个群体分别有17和15个抗性QTL区域对应存在着前人鉴定的抗稻瘟病QTL或主效基因,而两群体鉴定的QTL之间也存在11个共座位的QTL区域,表明水稻基因组存在保守的广谱抗稻瘟病QTL。这些保守的广谱抗性QTL有助于开展QTL的精细定位以进一步标记辅助选择和图位克隆QTL。更多还原

【Abstract】 Rice blast disease, caused by Pyricularia grisea (Cooke) Sacc., teleomorph Magaporthe grisea (Hebert) Barr, is one of the most destructive diseases worldwide. Utilization of resistance genes in rice breeding programs is considered an effective and economical strategy to control the disease. However, cultivars carrying a single R gene can only last for a short period of time until the advent of new dominant pathogenic isolates due to the high variability and pathogenicity of M. grisea population. Hence, exploiting wide-spectrum R genes and quantitative resistant loci, and pyramiding them into rice cultivars for developing broad-spectrum and durable resistance to rice blast by marker-assisted selection is a priority in rice breeding programs. In this study, we analyzed the genotypes of blast resistance in cv. IR24 and cv. DV85, and mapped three major blast R genes and some quantitative resistant loci. The main results were as follows:1. The resistance of cv. IR24 to Chinese blast isolates are mainly conditioned by R gene Pi20(t) and Pib, among which the Pi20(t) can be used as an superior R gene in rice breeding programs as it confer resistance to a broad spectrum of japonica-isolates (94.2% of 160 isolates) and a moderate spectrum of indica-isolates (52.6% of 160 isolates) in China. The isolates 98095 which could specifically differentiate the Pi20(t) was selected from a total of 160 blast isolates tested, and used to inoculate the recombinant inbred lines(RILs) and F2 population from the cross between Asominori and IR24 to finely map the Pi20(t). As a result, two flanking and three co-segregating SSR markers for Pi20(t), nearby the centromere region of chromosome 12, were identified. These five markers can tag Pi20(t) over 98% accuracy and show high polymorphisms between Pi20(t) donor IR24 and a series of elite cultivars in China. We suggest that these SSR markers can be useful in marker assisted selection for rapidly introducing Pi20(t) into japonica varieties or pyramiding Pi20(t) with other resistance genes into indica varieties.2. The indica cv. DV85 has a high level of resistance to Chinese blast isolates as it confer resistance to 92.9% of 98 japonica-derived isolates and 54.0% of 50 indica-derived isolates, this indicate it can also be used in rice breeding programs for blast resistance. Based on the results of genetic analysis and preliminary gene mapping from RILs, BC1F1(Kinmaze/DV85//Kinmaze) and F2 population derived from the cross of Kinmaze(susceptible) and DV85(resistant), we confirm that DV85 harbors a new dominant R gene conferring resistance to the Chinese japonica-derived isolate 97-27-2. The novel R gene was located on the terminal region of long arm of chromosome 1, tentatively designed as Pidv(t). By using an enlarged mapping population from F2 individuals, and by developing InDel markers combined with the released SSR markers, we further finely map the Pidv(t) gene to an 66.9kb interval defined by two markers, C4 and RM12182, with a genetic distance of 0.5 and 0.1 cM, respectively. The fine-mapping of Pidv(t) is favorable to marker-assisted selection and map-based cloning of the gene.3 Seventy-one RILs of Asominori×IR24 (populationⅠ) and 81 RILs of Kinmaze×DV85 (populationⅡ) were inoculated with six isolates 91-17-2,97-27-2,59-3, L64-1, CH26 and TH16, respectively. Fifty-eight QTLs were detected in the populations I at the LOD 2.5, of which 34 QTLs derived from the alleles of resistant parent IR24,24 QTLs derived from the alleles of susceptible parent Asominori, and 16,13,12 and 17 QTLs were related to lesion number (LN), lesion length (LL), lesion area (LA) and lesion degree (LD), respectively. In the populationⅡ, fifty-nine QTLs were detected at the LOD 2.5, including 29 QTLs derived from the alleles of resistant parent DV85,30 QTLs derived from the alleles of susceptible parent Kinmaze, and 16,12,14 and 17 QTLs related to LN, LL, LA and LD. The QTLs conferring major effects were mainly located in Pib and Pidv(t) region, thus, QTLs together with the major genes are responsible for the broad-spectrum resistance in IR24 and in DV85, respectively. QTLs are often clustered in the region of major genes, meanwhile, most QTLs detected for different traits for the same isolate are co-located in the same loci. Only few QTLs conferred resistance to 2-3 isolates, and no QTLs conferred resistance to more than 3 isolates. By comparative analysis for QTLs from two RILs populations,17 loci from populationⅠand 15 loci from populationⅡwere found to locate within or near to the same region as other QTLs or major genes identified by other researchers using different mapping populations. Eleven QTLs identified from the two RIL populations were located in the same loci in this study, including 5 loci corresponding to the same location of QTLs identified in other mapping populations. This indicates that some consensus QTLs for blast resistance were harbored in rice genome,which are helpful for fine mapping and map-based cloning of quantitative resistance loci.更多还原