墨西哥野生马铃薯Solanum pinnatisectum抗晚疫病及抗马铃薯甲虫新基因的遗传分析与分子标记(英文)

马铃薯晚疫病(Phytophthora infestans)和科罗拉多马铃薯甲虫(CPB)是马铃薯生产中最为严重的病虫害。培育高抗晚疫病和甲虫的马铃薯品种是加拿大马铃薯育种工作的重要组成部分。目前,我们实验室在二倍体1EBN墨西哥野生种中已鉴定出抗马铃薯晚疫病和甲虫的新基因,并利用原生质体融合技术成功的将其转移到栽培品种中。但是,培育出抗晚疫病和抗甲虫的马铃薯新品种仍然是一项艰难而繁杂的工作。为了加快分离抗性基因,建立与抗性基因紧密关联的DNA分子标记至关重要。本研究以感病的二倍体马铃薯品种S.cardiophyllum作为父本,与带有抗性基因的墨西哥野生种S.pinnatisectum杂交。用叶片离体鉴定的方法测试F1和BC1代群体的抗病性,从而筛选抗晚疫病和抗甲虫的植株。US-8/A2交配型病菌测试显示所有的F1代植株都表现出抗晚疫病,而在BC1群体中抗病与感病植株的比例为1:1。这个结果证明,在墨西哥野生种S.pinnatisectum中存在一个抗晚疫病的单显性基因Rpi1。马铃薯甲虫抗性检测中,BC1群体的抗虫性分离比例为1:3.这表明其对甲虫的抗性是由多基因遗传控制的。在F1和BC1群体中利用分子标记结合集团分离分析法(BSA)对S.pinnatisectum中的晚疫病抗性基因Rpi1进行精细作图。根据马铃薯第7条染色体上RFLP标记TG20A和CP56之间的EST和STS标记的序列信息,合成了27对特异性PCR引物。获得一些与抗晚疫病基因Rpi1相关联的新的DNA标记。对BC1群体中大量的个体植株进行的分析表明,在马铃薯第7条染色体上位于抗晚疫病基因Rpi1两侧的两个标记S1c9和GP127-300,它们与Rpi1基因的遗传距离分别为1.17cM和3.89cM。这些标记被用来筛选两个细菌人工染色体(BAC)文库,并分离出与晚疫病抗性相关的90-125kb的BAC克隆,这些克隆将在后续的工作中通过图位克隆的方法而用于分离晚疫病抗性基因。同时分离与甲虫抗性紧密相关的分子标记的工作正在进行中。

Inheritance and Molecular Mapping of New Genes Conferring Late Blight and CPB Resistance in Mexican Wild Potato Species Solanum pinnatisectum

Late blight (Phytophthora infestans) and Colorado potato beetle (CPB) are the most disastrous disease and insect problem of potatoes. Development of high levels of late blight and CPB resistant potatoes has become a high priority for the Canadian potato breeding program. New genes for late blight and CPB resistance have been identified in a wild 1EBN diploid Mexican species Solanum pinnatisectum which has been successfully transferred via protoplast fusion to the cultivated potato background. However, development of new potato cultivars with late blight and CPB resistance has been a difficult and cumbersome task. To accelerate the isolation of the resistant genes, molecular DNA markers tightly linked to the resistance are needed. In this study, a susceptible diploid potato S. Cardiophyllum was selected as the male parent to cross with S. Pin-natisectum. The F1 and BC1 populations were assessed for resistance to late blight and CPB by the detached leaf method. Dis-ease test using US-8/A2 mating type isolate revealed that all of the F1 individuals were resistant to the late blight. The ratio of late blight resistant plants to susceptible plants was 1 : 1 in BC1 populations. The results confirmed that a single dominant gene Rpil for late blight resistance was present in S. Pinnatisectum. For CPB resistance, a 1:3 resistant to susceptible ratio in BC1 populations confirmed polygenic inheritance. Molecular marker analysis combined with bulked segregating analysis (BSA) was carried out in the F1 and BC1 populations for fine mapping late blight resistant gene Rpi1 in S. Pinnatisectum. Twenty seven specific PCR primers were designed from sequence information of EST and STS markers located between RFLP markers TG20A and CP56 on potato chromosome Ⅶ. Several new DNA markers showed the linkage relation with the late blight resistant gene Rpi1. The analysis of a large number of individual plants from BC1 populations indicated that two mark-ers S1c9 and GP127～300 on chromosome Ⅶ are flanking the late blight resistant gene Rpi1 with a genetic distance of 1.17 cM and 3.89 cM, respectively. These markers were used to screen two bacterial artificial chromosome (BAC) libraries. Several BAG clones 90～125 kb in size linked with late blight resistance were isolated which will be used to isolate late blight resis-tance genes through the map-based cloning strategy. Development of molecular matkers closely linked with CPB resistance is on going.