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首页> 外文期刊>BMC Biotechnology >Development of late blight resistant potatoes by cisgene stacking
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Development of late blight resistant potatoes by cisgene stacking

机译：通过顺基因堆叠技术开发抗晚疫病马铃薯

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Background Phytophthora infestans, causing late blight in potato, remains one of the most devastating pathogens in potato production and late blight resistance is a top priority in potato breeding. The introduction of multiple resistance ( R ) genes with different spectra from crossable species into potato varieties is required. Cisgenesis is a promising approach that introduces native genes from the crops own gene pool using GM technology, thereby retaining favourable characteristics of established varieties. Results We pursued a cisgenesis approach to introduce two broad spectrum potato late blight R genes, Rpi-sto1 and Rpi-vnt1.1 from the crossable species Solanum stoloniferum and Solanum venturii, respectively, into three different potato varieties. First, single R gene-containing transgenic plants were produced for all varieties to be used as references for the resistance levels and spectra to be expected in the respective genetic backgrounds. Next, a construct containing both cisgenic late blight R genes ( Rpi-vnt1.1 and Rpi-sto1 ), but lacking the bacterial kanamycin resistance selection marker ( NPTII ) was transformed to the three selected potato varieties using Agrobacterium -mediated transformation. Gene transfer events were selected by PCR among regenerated shoots. Through further analyses involving morphological evaluations in the greenhouse, responsiveness to Avr genes and late blight resistance in detached leaf assays, the selection was narrowed down to eight independent events. These cisgenic events were selected because they showed broad spectrum late blight resistance due to the activity of both introduced R genes. The marker-free transformation was compared to kanamycin resistance assisted transformation in terms of T-DNA and vector backbone integration frequency. Also, differences in regeneration time and genotype dependency were evaluated. Conclusions We developed a marker-free transformation pipeline to select potato plants functionally expressing a stack of late blight R genes. Marker-free transformation is less genotype dependent and less prone to vector backbone integration as compared to marker-assisted transformation. Thereby, this study provides an important tool for the successful deployment of R genes in agriculture and contributes to the production of potentially durable late blight resistant potatoes.
机译：背景技术疫霉疫霉在马铃薯中引起晚疫病，仍然是马铃薯生产中最具破坏性的病原体之一，晚疫病抗性是马铃薯育种中的重中之重。需要将具有不同光谱的多重抗性（R）基因从可杂交物种引入马铃薯品种。同基因发生是一种有前途的方法，该方法使用转基因技术从作物自身的基因库中引入天然基因，从而保留了已有品种的有利特征。结果我们采取了一种同基因形成方法，分别将来自可交叉品种Solanum stoloniferum和Solanum venturii的两个广谱马铃薯晚疫病R基因Rpi-sto1和Rpi-vnt1.1引入三个不同的马铃薯品种。首先，为所有品种生产了单一的含R基因的转基因植物，以用作在各个遗传背景中预期的抗性水平和光谱的参考。接下来，使用农杆菌介导的转化将同时包含顺生晚疫病R基因（Rpi-vnt1.1和Rpi-sto1）但缺乏细菌卡那霉素抗性选择标记（NPTII）的构建体转化为三个选择的马铃薯品种。通过PCR在再生枝中选择基因转移事件。通过进一步分析，包括温室形态评估，离体叶片分析中对Avr基因的响应性和晚疫病抗性，选择范围缩小到八个独立事件。选择这些顺生事件是因为由于两个引入的R基因的活性，它们显示了广谱的晚疫病抗性。在T-DNA和载体骨架整合频率方面，将无标记的转化与卡那霉素抗性辅助转化进行了比较。同样，评估了再生时间和基因型依赖性的差异。结论我们开发了无标记转化管道，以选择功能性表达一堆晚疫病R基因的马铃薯植株。与标记辅助的转化相比，无标记的转化对基因型的依赖性更小，并且载体骨架整合的可能性更小。因此，这项研究为成功地将R基因应用于农业提供了重要的工具，并有助于生产潜在的耐用晚疫病抗性马铃薯。

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