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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。
Phytophthora infestans and Colorado potato beetle (CPB) are the most serious pests in potato production. Breeding potato varieties resistant to late blight and beetle is an important part of Canadian potato breeding. At present, our laboratory has identified new genes that are resistant to potato late blight and beetle in diploid 1EBN Mexican wild species and successfully transferred them to cultivars using protoplast fusion techniques. However, developing new potato varieties resistant to late blight and anti-beetle remains a difficult and complicated task. In order to speed up the isolation of resistance genes, it is of crucial importance to establish DNA molecular markers that are closely related to resistance genes. In this study, the susceptible diploid potato S.cardiophyllum was used as the male parent to cross with S.pinnatisectum, a Mexican wild species with resistance gene. The resistance of F1 and BC1 populations was tested by in vitro identification of leaves to screen for plants resistant to late blight and anti-beetle. The US-8 / A2 mating pathogen test showed that all F1 plants showed late blight resistance compared to a 1: 1 ratio of resistant to susceptible plants in the BC1 population. This result proves that there is a single dominance gene Rpi1 resistant to late blight in S.pinnatisectum, a Mexican wild species. In the test of potato beetle resistance, the ratio of insect-resistant isolates in the BC1 population was 1: 3. This indicates that their resistance to beetles is genetically controlled by polygenes. Fine mapping of the late blight resistance gene Rpi1 in S. pinnatisectum using molecular markers in combination with group segregation analysis (BSA) in F1 and BC1 populations. According to the sequence information of EST and STS markers between RFLP markers TG20A and CP56 on chromosome 7 of the potato, 27 pairs of specific PCR primers were synthesized. Some new DNA markers associated with the late blight gene Rpi1 were obtained. Analysis of a large number of individual plants in the BC1 population showed that the two markers S1c9 and GP127-300 flanking the resistance to late blight gene Rpi1 on the chromosome 7 of the potato had genetic distances of 1.17 cM and Rpi1, respectively 3.89cM. These markers were used to screen two Bacterial Artificial Chromosome (BAC) libraries and to isolate BAC clones of 90-125 kb associated with late blight resistance, which clones will be used in a subsequent work by map cloning Late blight resistance gene was isolated. Work is also underway to separate molecular markers closely related to beetle resistance.