A pipeline for rapid cloning of stem rust resistance genes effective against Ug99 from wild diploid wheat relatives

小麦可以说是世界上最重要的谷类作物，每年产量超过6亿吨，为人类提供了超过19%的膳食热量。然而，小麦有限的遗传多样性使它容易受到新疾病的侵害。小麦茎锈病真菌被称为“农业脊髓灰质炎”，在北美、欧洲、亚洲和澳大利亚的有记录的历史中，它一再造成广泛的作物歉收。在20世纪50年代，绿色革命之父诺曼·博洛格成功培育出了对这种疾病的抗性。这种抗性一直持续到90年代末，当时一种名为Ug99的新型超强毒力小麦茎锈病在非洲出现。Ug99能使世界上90%以上的小麦品种致病。它首先在乌干达被发现，并以惊人的速度在撒哈拉以南非洲和整个阿拉伯半岛蔓延，2008年在伊朗出现。因为通过风传播的锈病孢子可以传播很远的距离，所以这种祸害到达巴基斯坦和印度只是时间问题。巴基斯坦和印度是世界上19%小麦的来源，也是10亿人口的家园。气候变化将使欧洲面临更大的疾病风险。迫切需要新的抗茎锈病来源。虽然传统育种可以将其他相关品种的新基因引入小麦，但这一过程既费力又耗时，而且难以控制，往往会导致在引入所需性状的同时引入有害性状（“连锁拖动”）。此外，当每次部署一个新的抗性基因时，病原体通常会在一两个生长季节内克服抗性基因，使其失效。这个问题可以通过一次引入一个以上的抗性基因来缓解，但事实证明，传统的育种方法即使不是不可能，也是不切实际的。本文提出的工作旨在从小麦的野生亲缘种sharonensis中鉴定新的抗性基因。这种草原产于黎凡特，与小麦的一种祖先密切相关，含有丰富的新的、未开发的抗性基因。我们的长期战略是通过分子克隆从该物种中分离出尽可能多的新的抗性基因，并通过转基因方法将它们组合引入。分子克隆使把几个新基因放在基因组的同一位置成为可能，实际上是直截了当的，允许育种者把它们当作一个“单一”基因来处理。这对抗病育种具有巨大的优势，并且是转基因技术不仅大大优于传统育种，而且确实是可持续粮食安全所必需的一个明确案例。我们的建议有具体的目标：1)克隆这些基因中的第一个，基于已经掌握的初步遗传图谱信息，2)开发一种新的方法来快速识别任何感兴趣的基因在基因组中的位置。这种新方法将结合经典遗传学和“下一代”超高通量测序技术，使几年前昂贵的实验变得可行和负担得起。我们正在开发的平台将作为主要产出小麦的新品系，在单个遗传位点上含有三个或更多新的茎锈病抗性基因。这些品种将提供给公共育种项目，为发展中国家开发新的育种材料，以防止茎锈病的危害。Aegilops还拥有其他有用性状的遗传多样性，包括水和氮的利用效率。因此，我们将在本项目中开发的基因组数据和方法将普遍有利于小麦的改良。

Wheat is arguably the most important cereal crop in the world, with more than 600 million tons produced annually, supplying greater than nineteen percent of human dietary calories. The limited genetic diversity of wheat however renders it vulnerable to new diseases. The wheat stem rust fungus, known as the 'polio of agriculture', has caused repeated widespread crop failures throughout recorded history in North America, Europe, Asia and Australia. In the 1950s Norman Borlaug, father of the Green Revolution, successfully bred for resistance to the disease. This resistance held until the end of the '90's, when a new, super-virulent race of wheat stem rust called Ug99 emerged in Africa. Ug99 is capable of causing disease on greater than ninety percent of the world's wheat varieties. First detected in Uganda, it has spread at an alarming rate through sub-Saharan Africa and across the Arabian Peninsula, appearing in Iran in 2008. Because wind-borne rust spores can travel long distances, it is only a matter of time before this scourge reaches Pakistan and India, the source of nineteen percent of the world's wheat and home to 1 billion people. Changing climate will expose Europe to enhanced risk of the disease. New sources of stem rust resistance are urgently needed. Although traditional breeding can introduce new genes into wheat from other related species, this process is laborious, time-consuming, and difficult to control, often causing the simultaneous introduction of deleterious characteristics along with the desired trait ('linkage drag'). Moreover, when new resistance genes are deployed one-at-a-time, the pathogen typically overcomes the resistance gene within one or two growing seasons, rendering it useless. This problem can be alleviated by introducing more than one resistance gene at a time, but that turns out to be impractical if not impossible by conventional breeding methods. The work proposed here aims to identify new resistance genes from a wild relative of wheat, Aegilops sharonensis. This grass, native to the Levant, is closely related to one of wheat's progenitors, and contains a rich trove of new, unexploited resistance genes. Our long-term strategy is to isolate, by molecular cloning, as many new resistance genes as possible from this species, and introduce them in combinations using GM methods. Molecular cloning makes it possible, indeed straightforward, to put several new genes together in the same location in the genome, allowing breeders to work with them as a 'single' gene. This holds tremendous advantages for disease resistance breeding, and is a clear case where GM technology is not only vastly superior to conventional breeding, but indeed required for sustainable food security. Our proposal has the specific goals of i) cloning the first of these genes, based on preliminary genetic mapping information already in hand, and ii) developing a novel method for quickly identifying the position in the genome of any gene of interest. This novel method will use a combination of classical genetics and 'next-generation' ultra-high-throughput sequencing technology, which now makes experiments that would have been prohibitively expensive only a few years ago both feasible and affordable. The platform we are developing will have as a primary output new lines of wheat that harbour three or more new stem rust resistance genes at a single genetic locus. These lines will be made available to public breeding programs that develop new breeding material for developing countries in harm's way from stem rust. Aegilops also harbors genetic diversity for other useful traits, including water and nitrogen use efficiency. Thus, the genomic data and methodology we will develop in this project will benefit wheat improvement generally.