丝瓜抗黄瓜花叶病毒（CMV）基因的精细定位

Sponge gourd is one of the main vegetables in slack season of autumn. Cucumber mosaic virus (CMV) has become an important virus disease in production. The genetic basis and molecular mechanism of resistant to CMV are not clear now. High efficient MAS technique depends on high density genetic map and accurate positioning CMV resistance gene. Therefore, high density genetic map construction and fine mapping of CMV resistance gene in sponge gourd are very important. In this study, SNP and Indel markers will be developed based on SLAF-seq technology. The high genetic map will be constructed based on a F2 population crossed from SG-R1 (CMV resistant line) and SG-S1 (CMV susceptible line) by using these molecular markers. The resistance levels of segregate populations will be identified by inoculate CMV virus. Subsequently, based on the phenotypic data of each F2 segregating population, the CMV resistant genes will be preliminarily mapped on the genetic linkage map of sponge gourd. Furthermore, using greater F2 population as materials, the flanking markers linked to CMV resistant gene are used to screen recombinant plants. Eventually, fine mapping of CMV resistant gene is achieved and will be application in variety improvement. The results of this study will not only lay a solid foundation for marker-assisted selection and gene cloning, but also provide a reference for the gene mapping of other agronomic traits, which has important theoretical and practical significance.

丝瓜是我国主要的渡秋淡蔬菜之一。黄瓜花叶病毒导致的病毒病是危害丝瓜的重要病害。目前缺乏对丝瓜抗CMV的遗传基础和分子机制的了解，而高效的分子标记辅助育种技术则依赖于饱和的遗传图谱和抗CMV基因的准确定位。因此，丝瓜饱和遗传图谱的构建和抗CMV基因的精细定位十分重要。本研究以高抗丝瓜CMV自交系SG-R1与高感自交系SG-S1构建的F2群体为材料，利用SLAF-seq技术，开发SNP及InDel标记，构建丝瓜高密度遗传图谱。人工接种CMV，利用F2分离群体各单株抗性的表型数据，在丝瓜高密度遗传图谱上初步定位丝瓜CMV抗性基因。进一步以更大的次级分离群体为材料，利用初步定位获得的与抗CMV基因两侧连锁的标记进行重组株筛选，最终实现该基因的精细定位并应用于品种改良。本研究的结果不仅为丝瓜分子标记辅助选择育种、基因克隆等研究奠定基础，而且为其他农艺性状基因定位提供参考，具有重要的理论和实践意义。

丝瓜是一种非常有经济价值的鲜食和加工型蔬菜作物。由于缺乏足够的分子标记，迄今还未有高密度的分子遗传图谱发表。随着新一代测序技术的发展，促进了SNP标记的开发和大群体上高通量基因分型的发展。本研究通过使用SLAF测序技术，采用丝瓜抗、感CMV亲本杂交构建的130株F2群体的基础上，构建了丝瓜的高密度遗传图谱，共获得271.01 M reads数据，双亲的测序深度平均为86.19X，F2子代测序平均深度为14.57X。过滤掉低质量的SLAF标签，本项目共开发100,077个SLAF标签，其中多态性标签为7,405个。最终，有3,701个多态性性标记上图，构建为13个连锁群。总图距为1518.56 cM，相邻标记的平均距离为0.41cM. 遗传分析表明，丝瓜抗CMV的遗传为数量遗传，基于构建的高密度遗传图谱，在染色体1和4上定位到了丝瓜抗CMV基因的两个连锁区间，分别为100.072 ~ 100.457 cM 和42.475 ~ 44.398 cM。为了研究抗病机制，采用RNA-seq技术，对抗、感CMV双亲，进行转录组测序。利用鉴定到的精细定位标记作为锚定引物，对定位区间的基因功能进行预测，初步推测丝瓜抗CMV的基因功能为转座子逆转录蛋白相关的Pol蛋白。