【作者】 杨晓慧；

【导师】 杜永臣；

【作者基本信息】 中国农业科学院 ， 蔬菜学， 2012， 博士

【摘要】 由番茄黄化曲叶病毒（Tomato yellow leaf curl virus, TYLCV）引起的番茄黄化曲叶病是番茄生产中世界性的重要病害之一。利用野生资源中的抗性基因选育抗病品种是最根本的解决办法。目前从野生番茄中定位到5个抗性基因：Ty-1、Ty-2、Ty-3、Ty-4和Ty-5，并通过分子标记辅助选择培育出了一些抗病品种；但是由于目前得到的分子标记与目的基因的遗传距离仍然较远，在育种中存在不良性状连锁、假阳性和抗源材料丢失等问题。因此，对抗性基因进一步精细定位并克隆，对于明确番茄抗病机制和抗病育种都具有重要的意义。含有单一抗性基因的品种在病害大规模发生时抗性容易下降，研究不同基因的聚合效应对于抗病品种选育具有重要意义。Ty-2基因是来源于野生多毛番茄‘B6013’的单个显性基因，最初被定位到番茄第11号染色体长臂末端TG36和TG393之间约19cM的距离内，后来被进一步定位到标记C2_At2g28250和T0302之间6.7cM范围内。本研究在前人研究的基础上，对Ty-2基因进行了精细定位，以期为克隆该基因和提高标记辅助育种效率提供分子依据。同时对Ty-2、Ty-3和Ty-4不同基因的抗性水平进行了研究，为获得高抗TYLCV品种选育提供技术支撑和理论依据。主要研究结果如下：1.基于番茄基因组相关序列，利用BatchPrimer设计了242对引物，同时合成了Tomato-EXPEN遗传图谱上已知标记引物18对，对抗病纯合体E951-7、抗病杂合体TyQueen和感病纯合体Horizon进行基因型验证，筛选得到56个多态性标记，其中包括16个SCAR标记和40个CAPS标记。选取在染色体上均匀分布的21个标记，构建了目的区域的高密度遗传图谱，标记之间的平均遗传距离为0.05cM。2．以来源于H24杂交种H9205的11000个F4代单株为材料，筛选出在侧翼分子标记C2_At1g07960和T0302之间发生重组的单株157个，对这些重组单株的后代进行基因型和烟粉虱接种鉴定，其结果将Ty-2基因精细定位到分子标记UP8和M1之间，遗传距离0.4cM。在精细定位的过程中，获得了一个与Ty-2基因紧密连锁的共显性SCAR标记P1-16，在抗、感病对照材料中分别可扩增出300bp和600bp的片段，并利用48个不同的番茄转育品系对该标记进行了验证，发现P1-16的带型与TYLCV抗性一致。该标记可以用于Ty-2基因的辅助选择。3.以测序番茄品种S.lycopersicum H1706的基因组为参考序列，在Ty-2基因侧翼标记UP8和M1之间300kb的区域预测到了35个基因。其中Solyc11g069620.1、Solyc11g069660.1、Solyc11g069670.1、Solyc11g069920.1和Solyc11g069930.1具有植物抗病基因典型的NB-ARC结构，位于重组发生的位点，将这5个基因作为Ty-2的候选基因。4.利用栽培番茄8083、含有Ty-2基因纯合片段的近等基因系E846和同时含有Ty-3和Ty-4纯合片段的材料Fla.726构建群体，从中筛选出了14个含有不同抗性基因组合的材料，并用TYLCV-IL株系进行了抗性鉴定。结果表明，只含有Ty-2、Ty-3、Ty-4基因的植株分别表现为抗、中抗和感病；同时含有Ty-2和Ty-3或者Ty-2和Ty-4基因的植株均表现抗病；同时含有Ty-3和Ty-4基因的植株表现中抗；不同基因之间的抗性表现出累加效应，含有这3个基因的材料抗病效果最好。更多还原

【Abstract】 Tomato yellow leaf curl disease became one of the most prevalent and damaging tomato diseasesin tomato production in the world. The best way to reduce this damage is by breeding resistant ortolerant tomato cultivars. So far, five resistance genes have been mapped, i.e. Ty-1, Ty-2, Ty-3, Ty-4andTy-5, derived from several wild tomato species. The breeding programs have been based on the transferof these resistance genes into the cultivated tomatoes by marker-assisted selection. But resistance toTYLCV was still associated with some undesirable traits due to the longer genetic distance of theknown linked molecular markers. So it is necessary to develop tightly linked molecular markers andfurther fine mapping TYLCV resistance genes, which is very significant to develop tomato cultivarswith high resistance to TYLCV and provide academic guidance to analysis disease resistancemechanisms. So far, most of developed cultivars only have a single resistance gene, which makeresistance reduced under the high disease pressure. So it is important and promising to pyramid allresistant genes in a single genotype to reach the maximum level of resistance.The Ty-2gene, which is a single dominant gene derived from S. habrochiates f. glabratumaccession ‘B6013’, was previously mapped to a19cM region on the long arm of chromosome11delimited by molecular markers TG36and TG393. Later, Ty-2gene was located to a6.7cM intervalbetween C2_At2g28250and T0302. The objective of this present research is to fine mapping of Ty-2gene based on the previous research. Resistance levels of different genes were also discussed throughpyramiding resistances from different wild tomato species in a tomato line in this study. The mainconclusions were as follows:1. Two hundred forty-two pairs of primers were designed according to the tomato genome sequencebetween markers C2_At2g28250and T0302on chromosome11by a tool of BatchPrimer. Theseprimers, in addition to the primers of18markers already in Tomato-EXPEN map, allowed amplificationamong homozygous resistance control E951-7, heterozygous resistance control TyQueen, andhomozygous susceptible control Horizon. Fifty-six polymorphic molecular markers were developed,including16SCAR markers and40CAPS markers. With21polymorphic markers, a high-resolutiongenetic map of Ty-2gene was constructed, in which, average genetic distance was0.05cM.2. From over11000plants derived from F1hybrid H9205from H24, nearly157plants were selected forcarrying a recombined introgression between flanking markers C2_At1g07960and T0302. The progenyof each recombinant was genotyped with additional molecular markers developed from the tomatopublic genomic sequence within the target region and evaluated by TYLCV. The results indicated thatTy-2was located between markers UP8and M1, a distance of0.4cM and an interval of300,000baseson a single scaffold of the tomato assembly. In this screening, a PCR-based co-dominant SCAR markertightly linked to the Ty-2locus, P1-16, can be used in the breeding program for efficient selection ofhigh-levels of begomovirus resistance. The PCR fragment sizes for resistant and susceptible controlwere300bp and600bp, respectively. 3. Thirty-five genes were predicted within around300kb of Ty-2gene target region based on thegenome sequence of S.lycopersicum H1706. Five genes, i.e. Solyc11g069620.1, Solyc11g069660.1,Solyc11g069920.1, Solyc11g069670.1and Solyc11g069930.1, which contained the conserved domainsof NB-ARC class and located in the recombinant area, were selected as Ty-2candidate genes.4. Fourteen materials harboring different resistance genes were selected from the population developedwith susceptible cultivar8083, near-isogenic line E846(Ty-2+), and Fla.726(Ty-3+Ty-4+), and evaluatedwith TYLCV disease. Results indicated that the material with single Ty-2gene showed high resistanceto TYLCV, and the material with single Ty-3was tolerance to TYLCV; while the material with singleTy-4gene exhibited sever susceptible symptoms. The plants containing both Ty-2and Ty-3or Ty-2andTy-4were all resistant to TYLCV, while the plants containing both Ty-3and Ty-4were all tolerance. Thedifferent genes showed the additive effect and the plant containing Ty-2, Ty-3and Ty-4genes exhibitedthe highest resistance to TYLCV.更多还原