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

马铃薯对多种病虫害都很敏感，尤其是由马铃薯晚疫病菌引起的晚疫病。这种疾病每年在全球造成至少60亿美元的损失，在英国每年造成5000万英镑的损失，并在19世纪40年代导致爱尔兰土豆饥荒。土豆块茎也容易出现问题。在阴凉的条件下储存会提高还原糖的水平，在高温烹饪后，如油炸或烘焙，这些糖可以转化为有毒的丙烯酰胺。马铃薯块茎如果掉落或被压碎，会因瘀伤而受损，导致酶反应变色，导致严重的食物浪费。最后，马铃薯Y病毒可导致马铃薯种薯的严重减产或排斥，给马铃薯产业带来额外的负担和费用。我们克隆了3个非常有效的抗性基因RPI-vnt1、RPI-amr3和RPI-amr1，并表明当它们一起部署在转基因马铃薯上时，它们在田间对目前正在流通的所有LB小种都具有完全抗性。这应该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，这些基因可以降低酶的水平，这些酶可以提高还原糖的水平，并在瘀伤时调节变色，从而缓解问题。在目前正在进行的一项拨款中，我们利用农杆菌将3个RPI基因和块茎质量性状引入英国青睐的品种Maris Piper(MP)，创造了PiperPlus 1.0。然而，我们产生的所有线路都容易受到PVY的影响，这极大地削弱了我们为更广泛的田间试验增加容量的能力。PVY是通过蚜虫传播的，很难控制，特别是在用来控制传播PVY的蚜虫的杀虫剂日益受到限制的情况下，全球变暖意味着蚜虫和PVY是苏格兰马铃薯种薯生产的一个日益严重的问题。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方种植马铃薯种子。我们与同事一起克隆了一种极其有效的PVY抗性基因Ry-sto。在这个项目中，我们的目标是将PVY抗性与PiperPlus 1.0的LB抗性和块茎质量性状相结合，创造PiperPlus 2.0。我们已经构建了一个DNA结构，通过农杆菌将所有性状都导入到一个T-DNA上，并正在使用转基因方法生产转基因Maris Piper品系。我们预计将于2022年夏季在诺里奇进行约20条PiperPlus 2.0候选线路的现场试验，并于2023年夏季再试验约20条。我们可以很容易地鉴定出Ry和所有3个RPI基因表达的品系，我们还将选择那些块茎品质性状得到改善的植株品系。2022年Norwich试验第1批的最佳品系将于2023年夏季在剑桥NIAB进行田间试验，2023年夏季Norwich第2批的最佳品系将在2024年夏季NIAB试验中倍增，以选出表现最好的PiperPlus 2.0品系，这些品系与普通Maris Piper几乎没有区别，但它们将是抗LB、抗PVY并具有改良块茎品质性状的品系。还将对这些品系进行评估，以验证与正常Maris Piper品系的产量可比，并确保转基因方法的使用不会导致任何有害性状的获得，如类固醇糖类生物碱产量增加。我们还将确定我们添加的DNA在马铃薯基因组中插入的确切位置，以及最终选择的PiperPlus 2.0品系具有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的品系，我们预计到2024年，监管框架的变化将使这一改良品系能够被批准用于商业生产和公众消费。

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.