New UK potato varieties with late blight and potato cyst nematode resistance, reduced bruising and improved processing quality

土豆是英国和世界上极其重要的农作物，在世界粮食安全中发挥着关键作用。然而，由于现有品种的几个缺点，产量以及加工和消费的适宜性大大降低。对许多疾病和害虫的易感性，特别是由马铃薯晚疫病菌引起的晚疫病(导致爱尔兰马铃薯饥荒)和由马铃薯孢囊线虫引起的根损伤是主要问题。将土豆储存在阴凉的环境中会提高还原糖的水平(冷诱导甜味)，导致在油炸或烘烤后产生潜在损害健康的丙烯酰胺(氨基酸天冬酰胺含量较高)。土豆也容易因瘀伤而变黑，造成相当大的浪费，增加了消费者的成本。植物抗病基因的功能是通过检测病原体的分子，然后激活防御机制。尽管土豆携带100个抗性基因，但栽培品种缺乏能够检测和击败晚疫病的基因。幸运的是，一些野生种的马铃薯表现出了对Lb的抗性，我们已经从其中一些亲缘关系中追踪并分离了Lb抗性基因。然后，可以使用转基因方法将这些基因导入马铃薯，从而产生具有有效抗性的品种。为了确保这种抗性是持久的，在这个项目中，我们的目标是结合三种不同的LB抗性基因，使LB病原菌更难绕过这种保护。当土豆储存在阴凉的条件下时，它们会积累高水平的还原糖，如葡萄糖，也会积累高水平的天冬酰胺。在油炸过程中，这些物质被转化为一种名为丙烯酰胺的有毒分子。通过沉默块茎中的两种植物基因(转化酶和天冬酰胺合成酶)，美国Simplot公司降低了块茎中葡萄糖和天冬酰胺的水平，从而降低了丙烯酰胺，减少了油炸时的发黑。导致瘀伤的褐变是由一种酶(PPO)与细胞酚类化合物相互作用引起的。再一次，使用沉默，Simplot实现了更低的PPO水平，特别是在块茎中，并降低了块茎对瘀伤损害的敏感性。在美国，Simplot正处于具有这些低丙烯酰胺和瘀伤控制基因(LABC)特征的马铃薯品种商业化的后期阶段。美国利兹的研究人员已经用土豆的根表达了抑制PCN的蛋白质。一种是干扰PCN神经系统的小肽。另一种是一种蛋白酶的抑制剂，线虫用来定植植物的根。在这个项目中，我们的目标是将这些特性结合到广受欢迎的马铃薯品种Maris Piper和Agria中。我们将在一个T-DNA上将三个抗性基因工程到一个植物基因可选标记上进行转化，并在田间进行药效试验。我们还将把抗性基因和LABC基因结合起来进行田间试验，最后把抗性基因和LABC基因与PCN抗性基因结合起来进行田间试验。与Simplot合作，我们将为每个构建培育至少100株植物，并对它们进行筛选，以寻找携带简单DNA插入的品系和所有交付的基因的功能。目标是选择适合随后商业化的植物品系。在这些实验中，我们还将产生非常有用的信息，即多个基因(在我们的情况下，多达9个基因)可以在一个事件中传递，而所有基因仍然发挥功能的效率。这些信息将对其他打算通过转基因方法同时在其他作物中传递多个有用基因的人有用。

Potatoes are an extremely important UK and world crop with a key role in world food security. However yields, and suitability for processing and consumption, are much reduced by several shortcomings in currently available varieties. Susceptibility to many diseases and pests, particularly late blight caused by Phytophthora infestans (cause of the Irish potato famine) and root damage caused by potato cyst nematodes, are major problems. Storing potatoes in cool conditions elevates levels of reducing sugars (cold-induced sweetening), resulting (in lines with high levels of the amino acid asparagine) in potentially health-damaging acrylamide production after frying or roasting. Potatoes are also susceptible to blackening upon bruising, resulting in considerable waste, raising costs to consumers. Plant disease resistance genes function by detecting molecules from a pathogen and then activating defence mechanisms. Although potatoes carry 100s of resistance genes, cultivated varieties lack those enabling it to detect and defeat the late blight (LB) organism. Fortunately, some wild species of potato show resistance to LB, and we have tracked down and isolated LB resistance genes from some of these relatives. Such genes can then be introduced into potato using GM methods, resulting in varieties with effective resistance. To ensure this resistance is long lasting, in this project we aim to combine three different LB resistance genes, to make it more difficult for the LB pathogen to by-pass this protection. When potatoes are stored in cool conditions, they can accumulate high levels of reducing sugars such as glucose, and also high levels of asparagine. During frying, these are converted to a toxic molecule called acrylamide. By silencing two plant genes (invertase and asparagine synthetase) specifically in tubers, the American company Simplot has reduced levels of glucose and asparagine in tubers, resulting in lower acrylamide and less blackening upon frying. The browning that results upon bruising is caused by the interaction of an enzyme (PPO) with cellular phenolic compounds. Again, using silencing, Simplot have achieved lower PPO levels specifically in tubers and reduced susceptibility of tubers to bruising damage. Simplot are in advanced stages of commercialising potato varieties with these low acrylamide and bruise control genes (LABC) traits in the US.Researchers in U. Leeds have engineered potatoes with roots that express proteins that inhibit PCN. One is a small peptide that interferes with the PCN nervous system. The other is an inhibitor of a protease used by the nematode to colonize the plant root. In combination, these proteins can greatly reduce losses to nematodes even on heavily infested land.In this project we aim to combine these traits in the popular potato varieties Maris Piper and Agria. We will engineer the three genes for LB resistance on one T-DNA, linked to a plant gene selectable marker for transformation, and test efficacy in the field. We will also combine LB resistance genes with LABC genes and test in the field, and finally we will combine LB resistance and LABC with PCN resistance genes and test in the field. In partnership with Simplot, we will produce at least 100 plants for each construct and screen them for lines that carry simple DNA inserts and for functionality of all the genes that were delivered. The goal is to select plant lines that are suitable for subsequent commercialization. During these experiments, we will also generate very useful information about the efficiency with which multiple genes (in our case, up to 9 genes) can be delivered in one event, and still all function. This information will be useful to others setting out to simultaneously deliver multiple useful genes by GM methods in other crops.