【作者】 侯静；

【导师】 徐明良；

【作者基本信息】 中国农业大学 ， 作物遗传育种， 2013， 博士

【摘要】 弯孢叶斑病是20世纪90年代在我国华北地区发生的一种新的玉米病害,主要侵染叶片,如遇适温高湿气候,病斑会迅速蔓延至整个植株,造成干枯,影响产量。研究表明,玉米对弯孢病的抗性属数量性状遗传,同时受基因加性、显性和上位性效应控制。开展玉米弯孢菌叶斑病的抗性遗传研究,获得与抗性相关的候选基因,发掘与抗病基因位点连锁的分子标记,有助于抗病机理研究和分子标记辅助抗性改良,从遗传上解决玉米弯孢叶斑病的危害。本文围绕玉米弯孢叶斑病抗性QTL的定位及遗传分析开展研究,结果如下：1.以玉米抗弯孢叶斑病自交系沈137和感弯孢叶斑病自交系黄早4组配成F2：3分离群体,选取117个家系进行基因型测定和表型分析。从514对SSR引物中选取稳定性好的150个多态标记构建连锁图。利用Mapmaker3.0作图软件,构建了一张玉米分子标记连锁图,与已发表的玉米分子遗传图谱基本一致。图谱总长度为1824.5cM,相邻标记间的平均距离为12.2cM。采用复合区间作图法,结合2008年和2009的表型鉴定结果(均设二次重复),定位玉米抗弯孢叶斑病的QTL。2008年的数据分析显示,两重复间共检测到3个QTL,其中bin6.05上的QTL在两重复间都能检测到,且具有相同的置信区间,可解释的表型变异为7.6～12.5%。2009年的数据分析显示,两重复间共检测到6个QTL,其中bin10.04上的QTL在两重复间均被检测到,位于标记umc1061和标记umc1239之间,可解释表型变异的12.1-28.9%。2.利用抗病自交系沈137和感病自交系黄早4组配的822株F2分离群体,选取62个高抗和59个高感单株组成极端个体的亚群体,检测抗病QTL。选取亲本间稳定性好的145个多态标记,利用Mapmaker3.0作图软件构建分子标记连锁图谱,总长度为1545.8cM,相邻标记的平均间距为10.7cM。结合表型数据,采用复合区间作图法共检测到5个QTL,其中位于bin10.04上的QTL可解释表型变异的19.5%,位于标记umc1061和标记umc1239之间,这一QTL位置与2009年检测到的结果一致。3.利用感病亲本黄早4作为轮回亲本构建一个F3BC4回交群体,采用全基因组背景分析和AFLP标记相结合的方法,发掘与弯孢叶斑病抗病相关的区段。选取群体中的40个单株进行全基因组扫描,检测到的杂合染色体区段主要集中于bin2.08、bin3.04～3.05、bin4.0～4.02、bin5.01、 bin7.05, bin8.03～8.05和bin9.07。利用群体中3个高抗和3个高感植株及相应的亲本自交系筛选多态的AFLP标记,从64对AFLP引物中共检测到8个在抗感单株间表现出多态的AFLP条带。经测序和分析后发现,其中的5个多态AFLP片段,E51M14-331、E52M14-470、E52M20-273、 E53M30-678和E59M41-263,均来自反转录转座子,E59M14-331与S受体激酶高度同源,E52M11-254和E59M30-221为两个未知功能的蛋白序列。E52M11-254位于bin4.01,与F3BC4家系背景检测中杂合染色体区段bin4.0-4.02相一致,推测可能与弯孢叶斑病抗性相关。更多还原

【Abstract】 Curvularia leaf spot, caused by the casual fungal pathogen Curvularia lunata, is a disease of corn firstly identified in Northern region of China in1980s. The leaf spot fungus mainly infects leaves. The infection could rapidly spread to the whole plant under favorable hot-wet weather, and causes severe yield loss. Resistance to the disease was reported to be quantitative in nature, and jointly controlled by additive, dominant, and epitastic genetic effects. The solution to genetically contain curvularia leaf spot could be normally accomplished by studying disease resistance mechanism, screening candidate resistance genes, identifying markers linked to disease-resistance genes, and applying molecular markers to assist breeding to improve disease resistance.The thesis mainly focuses on identification and chracterization of quantitative trait loci for resistance to curvularia leaf spot in maize, and the results were listed as follows:1. A segregating F2：3population was developed from a cross between the resistant inbred line Shen137and the susceptible inbred line Huangzao4, in which a selective F2:3population consisting of117F2:3families were subjected to both genotyping and phenotyping. Of the514SSR markers,150of them which were reliable and polymorphic between two parents were selected to construct a genetic linkage map. The map was1824.5cM in length with an average of12.2cM every two adjacent markers and in agreement with the published reference genetic linkage map.QTL analysis was conducted in years2008and2009(each year with two replicates) with Composite interval mapping (CIM) method. In2008, three QTL have been detected, and one of them, located on bin6.05, was consistently detected in two replicates. The QTL shared the common confidence interval and explained variance ranging from7.6～12.5%. In2008, six QTL were detected, and one QTL located on bin10.04was common in two replicates, flanked by two markers umc1061and umc1239. This resistance QTL accounted for12.1-28.9%of the total phenotypic variation.2. Four F1plants derived from the cross of Shen137×Huangzao4was self-pollinated to produce822F2plants. A selective genotyping population was formed by selecting the62extremely resistant and59completely susceptible F2plants. Totally,145SSR markers were used to genotype each selective F2plant. The genetic linkage map, constructed by Mapmaker3.0, was1545.8cM in length with an average distance of10.7cM every two adjacent markers. QTL analysis was performed using CIM to reveal five QTLs. One of these five QTL, located on chromosome bin10.04, accounted for19.5%of the total phenotypic variation, flanked by two markers umc1061and umc1239. Interestingly, this QTL was also detected in the previous F2:3population in2009.3. The F3BC4population was obtained by continuously backcrossing to the susceptible inbred line, Huangzao4. A combined whole-genome background screening method and AFLP technology was applied to dig those genome segments involved in maize resistance to curvularia leaf spot. A total of40individuals were used to investigate whole-genome background, and found that several chromosome segments were introduced from the donor parent. The donor fragments were scattered in bin2.08, bin3.04-3.05, bin4.0-4.02, bin5.01, bin7.05, bin8.03-8.05and bin9.07. The three extremely resistant and three completely susceptible individuals, together with two parents, were subjected to AFLP analysis. Totally,64AFLP primer combinations were used to search for polymorphic AFLP bands, resulting in eight AFLP bands polymorphic between resistant and susceptible individuals. Sequence analysis revealed that five polymorphic AFLP fragments, E51M14-331, E52M14-470, E52M20-273, E53M30-678and E59M41-263, were amplified from retrotransposons. Another three polymorphic AFLP bands were related to segments of coding sequences of three proteins. Among them, the AFLP fragment E59M14-331on bin2.02belongs to an S-locus receptor-like kinase. The other two AFLP fragments, E52M11-254(bin4.01) and E59M30-221(bin4.04), were amplified from two unknown proteins. Intriguely, E52M11-254overlapped with polymorphic region reveled by the whole genome screening. It was speculated that the AFLP band, E52M11-254, may be associated with maize resistance to curvularia leaf spot.更多还原