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，年产量超过6亿吨，提供了人类饮食卡路里的19%以上。然而，小麦的遗传多样性有限，使其容易受到新疾病的影响。小麦茎锈菌被称为“农业小儿麻痹症”，在北美、欧洲、亚洲和澳大利亚的有记录的历史中，它一再导致大范围的农作物歉收。20世纪50年代，绿色革命之父诺曼·博洛格成功培育出对这种疾病的抵抗力。这种抗药性一直持续到90年代末的S，当时非洲出现了一种新的、超强毒力的小麦茎锈菌小种，名为UG99。UG99能够在世界上90%以上的小麦品种上致病。它首先在乌干达发现，然后以惊人的速度在撒哈拉以南非洲和阿拉伯半岛蔓延，并于2008年出现在伊朗。由于风传播的锈菌孢子可以远距离传播，这种祸害到达巴基斯坦和印度只是个时间问题，这两个国家是世界上19%的小麦的产地，有10亿人口。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源。虽然传统育种可以从其他近缘物种将新基因导入小麦，但这一过程费时费力，难以控制，往往会导致有害性状与所需性状同时导入(连锁拖累)。此外，当新的抗性基因一次一个地部署时，病原菌通常会在一个或两个生长季节内克服抗性基因，使其毫无用处。这个问题可以通过一次导入一个以上的抗性基因来缓解，但事实证明，如果用传统的育种方法不是不可能的话，这是不切实际的。这项工作的目的是从小麦的野生近亲沙龙山羊草中鉴定新的抗性基因。这种草原产于黎凡特，与小麦的一个祖先有密切的亲缘关系，并含有丰富的新的、未开发的抗性基因。我们的长期战略是，通过分子克隆，从这个物种中分离出尽可能多的新的抗性基因，并使用转基因方法将它们组合引入。分子克隆使得将几个新基因放在基因组中的同一位置成为可能，这确实很简单，允许饲养者将它们作为一个单独的基因进行工作。这对抗病育种具有巨大的优势，这是一个明显的例子，表明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。我们的建议的具体目标是：i)根据已有的初步遗传图谱信息克隆第一个这些基因，以及ii)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，使几年前还贵得令人望而却步的实验变得既可行又负担得起。我们正在开发的平台将拥有作为初级产量的新小麦品系，这些品系在一个遗传座位上含有三个或更多新的抗茎锈病基因。这些品系将提供给公共育种项目，为遭受茎锈病危害的发展中国家开发新的育种材料。山羊草还具有其他有用特征的遗传多样性，包括水和氮的利用效率。因此，我们将在该项目中开发的基因组数据和方法学总体上将有利于小麦的改良。

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.