Combining late blight resistance and better tuber quality with resistance to potato virus Y (PVY) to improve Maris Piper potato

马铃薯作物易受许多病虫害的影响，特别是由疫霉引起的晚疫病。这种疾病每年在全球范围内造成至少60亿美元的损失，在英国每年造成5000万英镑的损失，并导致了19世纪40年代爱尔兰的马铃薯饥荒。马铃薯块茎也容易出问题。在凉爽的条件下储存会提高还原糖的含量，在高温烹饪（如油炸或烘烤）后，这些糖会转化为有毒的丙烯酰胺。如果马铃薯块茎掉落或压碎，块茎会因擦伤而受损，导致酶反应变色，导致大量食物浪费。块茎在储存过程中也可能因块茎枯萎病而丢失。最后，马铃薯Y病毒可导致严重的产量损失或种薯块茎拒收，给马铃薯产业带来额外的负担和费用。我们克隆了三个非常有效的LB抗性基因，分别为Rpi-vnt1、Rpi-amr3和Rpi-amr1，并表明当它们一起部署在转基因马铃薯中时，它们在田间对所有当前流行的LB品种具有完全的抗性。这应该能为英国的晚疫病问题提供一个持久的解决方案。我们还从美国马铃薯公司Simplot Inc .获得了商业部署和验证的基因，这些基因可以降低提高还原糖的酶水平，从而缓解这个问题。在目前正在进行的一项资助中，我们利用农杆菌将3个Rpi基因和块茎品质性状引入英国受欢迎的品种Maris Piper (MP)，创建了PiperPlus 1.0。然而，我们生成的所有品系都容易受到PVY的影响，这极大地削弱了我们进行更广泛的田间试验的能力。PVY是由蚜虫传播的，很难控制，特别是用于控制传播PVY的蚜虫的杀虫剂越来越少，全球变暖意味着蚜虫和PVY是苏格兰种薯生产中日益严重的问题。此外，苏格兰仍然反对种植转基因作物，这意味着需要在苏格兰以外的地区生产种子马铃薯。我们和同事克隆了一种非常有效的抗PVY基因，Ry-sto。在这个项目中，我们的目标是将PiperPlus 1.0的抗PVY性与抗LB性和块茎品质特性结合起来，创建PiperPlus 2.0。我们已经通过农杆菌构建了一个DNA结构，将所有性状一起引入到一个T-DNA上，并正在使用转基因方法生产转基因Maris Piper品系。我们预计2022年夏季能够在Norwich进行约20条PiperPlus 2.0候选管线的现场试验，2023年夏季将进行另外20条管线的现场试验。我们可以很容易地识别出表达Ry和3个Rpi基因的品系，我们也将选择那些块茎品质性状改善的品系。2022年诺里奇第1批试验的最佳品系将在2023年夏季在剑桥NIAB进行大田试验，2023年诺里奇第2批试验的最佳品系将在2024年夏季NIAB试验中进行繁殖，以选择表现最佳的PiperPlus 2.0品系，这些品系将与普通的Maris Piper没有区别，除了它们具有抗LB、抗PVY和改进的鳞茎质量性状。还将对这些品系进行评估，以验证与普通Maris Piper的产量相当，并确保使用转基因方法不会导致获得任何有害性状，例如甾体糖生物碱的产量增加。我们还将准确定义我们添加的DNA在马铃薯基因组中的插入位置，以及最终选择的PiperPlus 2.0系携带一个简单的单拷贝插入事件。因此，该项目将使我们能够在2024年之前制造出具有这些附加特性的商业可行性生产线，我们预计到2024年，监管框架的变化将使这条改进的生产线能够被批准用于商业生产和公共消费。

The potato crop is susceptible to many diseases and pests, notably late blight (LB) caused by Phytophthora infestans. This disease causes at least $6B in losses worldwide every year, costs £50M per year in the UK and led to the Irish potato famine in the 1840s.Potato tubers are also prone to problems. Storage in cool conditions elevates levels of reducing sugars, and after high temperature cooking such as frying or roasting, these sugars can be converted into toxic acrylamide. Potato tubers are damaged by bruising if dropped or crushed, resulting in discolouration by an enzyme reaction, leading to significant food wastage. Tubers can also be lost to tuber blight in storage.Finally, potato virus Y can result in severe yield losses or rejection of seed potato tubers, imposing an additional burden and expense on the potato industry.We cloned three very effective LB Resistance genes called Rpi-vnt1, Rpi-amr3 and Rpi-amr1 and have shown that when deployed together in GM potato, they confer complete resistance in the field to all currently circulating LB races. This should provide a durable solution to the late blight problem in the UK.We also obtained commercially deployed and validated genes from the American potato company Simplot Inc that reduce levels of the enzymes that elevate reducing sugars and that mediate the discolouration upon bruising, thus alleviating the problem. In a currently running grant, we introduced the 3 Rpi genes and the tuber quality traits, into UK-favoured variety Maris Piper (MP), using Agrobacterium, creating PiperPlus 1.0. However, all lines that we generated are susceptible to PVY, greatly impairing our capacity to bulk them up for more extensive field trials. PVY is aphid transmitted, and difficult to control, especially as insecticides used to control PVY-transmitting aphids are being increasingly restricted, and global warming means that aphids and PVY are an increasing problem for seed potato production in Scotland. Also, Scotland is still opposed to planting GM crops, meaning that seed potato production outside of Scotland will be needed.With colleagues, we cloned an extremely effective PVY resistance gene, Ry-sto. We aim in this project to combine PVY resistance with the LB resistance and tuber quality traits of PiperPlus 1.0, to create PiperPlus 2.0. We have made a DNA construct to introduce all traits together on one T-DNA via Agrobacterium and are producing transgenic Maris Piper lines using the GM method. We expect to be able to field-trial ~20 PiperPlus 2.0 candidate lines in Norwich in summer 2022, and another ~20 in summer 2023. We can easily identify lines in which Ry and all 3 Rpi genes are expressed and we will also select those plant lines with the improved tuber quality traits. The best lines from batch 1 in the Norwich trial in 2022 will be multiplied up for field trials in summer 2023 at NIAB in Cambridge, and the best lines from batch 2 in Norwich 2023 will be multiplied up for NIAB trials summer 2024, to select the best performing PiperPlus 2.0 lines that will be indistinguishable from normal Maris Piper, except that they will be LB resistant, PVY resistant and have the improved tuber quality traits. These lines will also be evaluated to verify comparable yield to that of normal Maris Piper, and to ensure that use of the GM method has not resulted in acquisition of any deleterious traits, such as elevated production of steroidal glycoalkaloids. We will also define exactly where in the potato genome the DNA that we added has inserted, and that the final selected PiperPlus 2.0 line carries a simple, single copy insertion event.The project will thus enable us to make by 2024 a commercially viable line with these added traits, and we anticipate that by 2024 changes in the regulatory framework will enable this improved line to be approved for commercial production and public consumption.