苦瓜SSR标记开发、分子遗传图谱构建及白粉病抗性QTL定位

The powdery mildew resistance of bitter gourd is an important character for bitter gourd cultivation both in field and greenhouse and disease-resistant breeding. Its genetic mechanism and mapping QTL is still an important problem which is still not solved. In this research, the crosses and segregation population will be made with the bitter gourd strain hai09-2 which has been identified to own high resistance to powdery mildew and strain hai09-1 which is strongly powdery mildew-susceptible, the statistical analysis about the individual plant infection rate and the chlorophyll content will be done after powdery mildew inoculated to make genetic analysis for their powdery mildew resistance; more saturated molecular genetic map of bitter gourd will be constructed by developing the SSR markers of bitter gourd independently by using EST sequence, magnetic beads enrichment and selective amplification of microsatellite(SAM) technology, combining with the other SSR markers relevant with the other melon crops' powdery mildew which can be used in bitter gourd; the QTL map of powdery mildew resistance and chlorophyll content will be done by using the SSR genetic mapping of bitter gourd which has been constructed. The results will lay the foundation for breeding new powdery mildew resistance varieties of bitter gourd by using molecular markers, cloning important characteristic gene, studying the molecular mechanism about powdery mildew resistance and chlorophyll, analysis of QTL and consolidation of genetic map; they are also significant to the molecular breeding of bitter gourd and the study of comparative genomics of cucurbit plant.

苦瓜白粉病抗性是苦瓜露地和设施栽培及抗病育种的重要性状，其遗传机制和QTL定位是尚未解决的重要科学问题。本项目拟以鉴定得到的高抗白粉病的苦瓜品系hai09-2为试材，利用高感白粉病的苦瓜品系hai09-1与其配制杂交组合和分离群体，接种白粉病菌后统计单株的发病情况和叶绿素含量，对其白粉病抗性进行遗传分析；并利用EST序列、磁珠富集法、选择性扩增微卫星等技术自主开发苦瓜的SSR标记，结合已筛选的可在苦瓜上利用的其他瓜类的与白粉病相关的SSR标记，构建较为饱和的苦瓜分子遗传图谱；利用该遗传图谱，将白粉病抗性和叶绿素含量进行QTL定位。该结果将为使用分子标记培育抗白粉病苦瓜新品种、为苦瓜重要性状基因克隆、白粉病抗性和叶绿素相关的分子机制研究及 QTL分析和遗传图谱整合奠定基础；对苦瓜的分子育种及葫芦科作物比较基因组学研究有重要意义。

苦瓜白粉病抗性是苦瓜露地和设施栽培及抗病育种的重要性状，其遗传机制和QTL定位是尚未解决的重要科学问题。项目组收集了大量的国内外苦瓜种质资源，经过连续2年的田间和温室两种环境接种白粉病菌后发现不同来源的苦瓜品系之间白粉病发病程度表现出较大的差异和多样性，28.57% 的苦瓜种质资源表现为抗病，9.53% 表现为高感；对苦瓜抗感品系的组成型防御机制和诱导型防御机制研究发现，叶绿素、可溶性糖质量分数和POD、PAL、PPO和APX活性、叶片蜡质含量、叶背面气孔及茸毛密度均可作为苦瓜对白粉病抗性早期鉴定的辅助指标；纯化了海南苦瓜主产区的苦瓜白粉菌，经形态和分子系统学鉴定为单囊壳属单囊壳种白粉菌(Sphaerotheca fuliginea )，利用白粉菌生理小种的国际通用寄主鉴定最终确定海南苦瓜白粉菌的生理小种为2（2France）；发现苦瓜对白粉病抗性遗传符合2对加性-显性-上位性主基因+加性-显性多基因模型（E-1），抗病对感病为不完全隐性；对苦瓜基因组DNA提取方法及ISSR、SSR、MSAP等标记进行了系统优化，并利用简化基因组测序技术和已发表的苦瓜全基因组序列信息开发了48566对SSR标记，苦瓜的 SSR 标记在其他葫芦科作物中具有一定的通用性，并且在不同瓜类间扩增多态性存在明显差异；构建了一张含120个多态性标记（ISSR、SRAP、SSR）的遗传连锁图谱，总长为732.1 cM，包含116个位点，平均图距为6.31 cM和一张覆盖基因组长度545.9cM，平均间距7.58cM的SSR标记遗传图谱，2年中有6个QTL在F2（2011年秋季）和F2:3家系（2012年春季）分别定位在了相同的基因座。利用SSR遗传连锁图谱，在F2:3代家系获得了到1个QTL位点，能解释11.5%的遗传变异，在第2连锁群检测到1个叶绿素含量的QTL位点，支持区域为0-2.7cM。本研究结果为苦瓜重要性状基因克隆、白粉病抗性和叶绿素相关的分子机制研究及遗传图谱整合奠定基础，对苦瓜的分子标记辅助育种具有重要意义。项目组在重要学术刊物上发表相关研究论文15篇，出版专著1部，培养硕士研究生3人，其中1名研究生的论文获得“海南省优秀硕士论文”称号。