【作者】 潘旭浩；

【导师】 李仕贵； 严文贵；

【作者基本信息】 四川农业大学 ， 作物遗传育种， 2013， 博士

【摘要】 水稻是世界上主要的粮食作物之一,在亚洲绝大多数的人口以水稻为主食,因此水稻生产安全是世界粮食安全的重要组成部分。美国的水稻总产量虽然不高但是其绝大部分用作出口,是世界第二大水稻出口商,因此美国水稻生产安全,从一定程度上对世界粮食安全有着重大的意义。美国主要的水稻病害包括：纹枯病、稻瘟病、直穗病、菌核病、叶鞘黑腐病、稻胡麻斑病、粒黑粉病、和立枯病。水稻直穗病是一种生理病害,在世界上多个国家和地区均有发生。感病时主要表现为不育以及颖壳扭曲呈“鹦鹉嘴”形状,在严重感病品种发病时可造成极大的产量损失。直穗病主要发生于美国南部,这里生产的水稻占全美水稻产量的90%以上,而这里的品种多为长粒型热带粳稻,对直穗病抗性普遍偏低,因此,直穗病对美国乃至全世界的水稻生产安全产生极大的威胁。目前最有效的直穗病防治方法为“放水干旱法”,该法需要在水稻田灌水以后一周将水抽干保持干旱直到水稻叶片出现干旱胁迫症状后重新灌水。所以该方法昂贵,对水资源会造成极大的浪费,且会在一定程度上影响水稻产量。抗病育种被认为是预防直穗病最为有效的方法,但是,由于直穗病真正的致病机理不明加上美国本土严重缺乏直穗病抗病种质资源,直到2000年为止抗病育种一直收效甚微。研究发现有许多因素可能造成直穗病,其中砷一直被认为是导致直穗病的重要因素。由于自然发生的直穗病的不稳定性,对直穗病直接进行鉴定十分困难,因此,早从80年代起,甲基砷酸钠除草剂（monosodium methanearsonate,MSMA）就在直穗病育种研究中被当作诱导剂使用。在本研究中,我们构建了两个重组自交系（recombinant inbred line, RIL） F9代群体和一个全球种质资源群体。两个RIL群体用于直穗病相关数量性状基因座（quantitative trait loci, QTL）定位,而全球种质资源群体和两个RIL共用于定位区间内标记与直穗病抗病性的关联性鉴定。结果概括如下：1.两个RIL群体连续两年在大田实验中利用MSMA诱导其直穗病发生并鉴定群体RIL抗病等级,其中一个群体的抗病亲本为Zhe733,感病亲本为R312,它们是从中国引进的籼稻品种,群体中共含有170个RIL,并用136个全基因组覆盖的SSR多态性标记对其进行基因型鉴定,标记多态性频率为26.1%：另一个群体抗病亲本为Jing185是来自中国的籼稻品种,感病亲本为长粒型粳稻品种Cocodrie是美国南部主要栽培品种之一,利用159个SSR多态性标记对群体中的91个RIL进行基因型鉴定,标记多态性频率为37.2%。2.采用连锁作图法对直穗病相关QTL进行初步定位以及进一步的精细定位。结果显示,在两个群体的8号染色体定位的QTL在两个群体中分别解释了46%和67%的直穗病抗病总变异,且两个QTL区间部分重合,证明这是一个直穗病主效QTL,命名为qSH-8。在Zhe733/R312群体中,利用7个区间内SSR多态性标记qSH-8的定位区间从1.0Mbp被缩小到340kb；继而又利用4个新开发的InDel标记将其定位区间最终缩小到290kb,位于RM22573和InDel27之间。3.根据Gramene数据库的信息,在qSH-8精细定位区间内共有36个候选基因,利用Geneinvestigator基因表达数据库查找对应36个候选基因从水稻开花早期到抽穗期蛋白表达水平变化的信息发现,LOC₀8g10240,10340和10380三个蛋白在此期间蛋白表达水平发生明显的变化,且均为表达水平下调,其中LOC₀8g10240和380均为功能未知的表达蛋白,LOC₀8g10340为含OsFBX278-F-box结构域蛋白。4.在目标基因定位区间还发现了两个与直穗病抗病性紧密连锁的标记,其中一个为SSR标记AP3858-1,另一个为InDel11。全球种质资源群体共包含71个材料,且它们对直穗病均表现为抗病或者感病。利用AP3858-1和InDel11对它们进行基因型鉴定后,计算其表型-基因型吻合率并进行χ2独立性检验。结果显示,InDel11的表型-基因型吻合率（78%）略高于AP3858-1（76%）,χ2独立性检验中,InDel11的P值小于0.0014,AP3858-1的P值小于0.0004,说明在两个标记与直穗病呈极显著相关关系。该结果证明AP3858-1和InDel11可以被用于直穗病抗病育种的分子标记辅助育种工作（marker-assisted selection, MAS）。5.在Cocodrie/Jingl85群体中发现了五个抗病自交系包括：RIL369、397、404、407、469和506,它们在AP3858和InDel11位点均表现为抗病亲本基因型,且分析发现它们与美国感病品种Cocodrie遗传相似度在50%以上且部分产量相关农艺性状与Cocodrie的差异不显著甚或更优,因此该五个自交系可作为优良直穗病抗病种质资源运用到美国感病品种改良育种当中。综上,本研究结果对探明水稻直穗病的遗传机理、指导其抗病育种有相当的意义,特别是在如今缺乏有效的对直穗病进行直接鉴定手段情况下,这些标记的发现对于指导该病的分子辅助育种具有极为现实的指导意义。更多还原

【Abstract】 Rice（oryza sativa L.）, is one of the most important food crops in the world. In Asia where has the largest population around the world, most pepole live here are feeded by rice, in this case, rice production security is important component of world food security. Rice growing in USA originated in South of Carolina state in1690. First rice produce record was in1895, at that time there was hundreds of thousands of hectares rice planting area. Then rice growing area kept increasing. Until now, it maintains in about1.2million hectares in USA. Rice produce area is distributed in California in west coast, and several states in Southern USA. Most of the varieties grown in USA are japonica, and according to USA market standard they were classified into three groups, including:long-grain rice, medium-grain rice and short-grain rice. The total rice production of USA is not very high, but most of rice produced here is for exportation, which made the USA as the world’s second largest exporter, therefore, rice production security in USA is one of the important part of world food security. The major rice diseases happened in USA inculding:sheath blight, blast, sclerotinia, the leaf sheaths black rot, rice brown spot disease, kernel smut, blight and straighthead.Straighthead, a physiological disorder characterized by sterile florets and distorted spikelets, causes significant yield losses in rice, and occurs in many countries. Straighthead happens frequently in southen USA where about90%of USA rice is produced, however, most of rice varieties grown here are susceptible to straighthead. In this case, straighthead is a huge threat for rice production in USA and worldwide. The current control method of draining paddies early in the season stresses plants, is costly, and wastes water. Development of resistant cultivar is regarded as the most efficient way for its control. However, because of uncertain of straighthead causal factor and lacking of resistant germplasm, a little progress was made in resistant breeding until2000. Although a lot of factors were reported to involved in straighthead disorder by previous studies, arsenic is considered as the most important one. Straighthead is very difficult to be directly evaluated, because instability of heritance of nature straighthead. Since the1980s, the breeding community has used soil incorporation of As in the form of MSMA as a common practice for evaluating rice susceptibility to straighthead. In this study, two recombinant inbred line （RIL） F9populations were developed for linkage mapping and fine mapping of straighthead resistance gene. Result showed that:1) Two RIL F9populations were phenotyped over two years using monosodium methanearsonate （MSMA） to induce the symptoms. One population of170RILs was genotyped with136SSRs, polymorphsm frequency is26.1%, the other population of91RILs was genotyped with159SSRs, polymorphsm frequency is37.2%.2) We fine mapped a gene for straighthead resistance by linkage mapping and fine mapping. A major QTL qSH-8was identified in an overlapping region in both populations, and explained46%of total variation in one and67%in another population for straighthead resistance. qSH-8was fine mapped from1.0Mbp to340kb using7SSR markers and further mapped to290kb in a population between RM22573and InDel27using4InDel markers. A total of36putative genes were detected in this region.3) According to the information from Gramene data base, a total of36putative genes were found in the fine mapped region. According to the information from Geneinvestigator data base, protein expression level of the36putative genes at different rice growning stage was investigated. The result showed that:3putative genes, LOC₀8g10240.10340and10380, their protein expression level were detected to decrease significantly.4) Two molecular markers, SSR AP3858-1and InDel11, were within the fine mapped region, and co-segregated with straighthead resistance in both RIL populations. Then we constructed a world germplasm collection which contian72accessions selected from USDA core collection for marker verification. These accessions were selected based on their representation of global diversity and diverse straighthead responses. The χ2test result showed that P value of two markers, InDel11and AP3858-1, were0.0014and0.0004, respectively; in InDel11,78%of the genotypes matching the phenotypes among those global accessions, and in AP3858-1was76%. These results demonstrated that AP3858-1and InDel11significantly associated with straighthead resistance and can be used for marker-assisted selection （MAS） for straighthead resistant cultivars, which is especially important because there is no effective way to directly evaluate straighthead resistance.5) Five straighthead resistance RILs from Cocodire/Jing185F9population, RIL369,397,404,407、469and506, which showed genotype of resistant parent at AP3858-1and InDel11loci. Furthermore, they were found to have more than50%of similarity with Cocodrie, moreover, some of their agronomic traits showed no significant differnce from Cocodrie or even better. In this case, these RILs can be used to improve straighthead resistance for susceptible varieties in southern USA.更多还原