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

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.

This project aims to identify and isolate genes responsible for resistance to the wheat stem rust fungus Puccinia graminis f. sp. tritici, Pgt from the wild wheat relative Aegilops sharonensis. Pgt has become a paramount problem in agriculture because new races of the pathogen (Ug99 and related strains) have appeared that can overcome nearly all genetic resistances currently deployed in wheat. Ae. sharonensis contains several novel resistance (R) genes that function against Ug99. Starting with already-available mapping populations segregating for resistance genes, we propose to genetically map and clone these genes. We will use established map-based cloning methods that take advantage of the synteny among monocot genomes, which we have validated for Ae. sharonensis, barley, and Brachypodium. We also propose to develop a novel method for rapidly fine-mapping R genes based on using oligonucleotide capture and next-generation sequencing. This strategy focuses on the class of nucleotide binding leucine-rich repeat-containing (NB-LRR) genes, which comprise roughly 90% of the known R genes. Specifically, we propose to: i) obtain deep genome sequence coverage of Ae. sharonensis using next-generation (Illumina) sequencing ii) identify and assemble candidate NB-LRR gene sequences from these data iii) design an oligonucleotide capture array that selects for NB-LRR genes iv) create bulked genomic DNA preparations from homozygous resistant and homozygous susceptible individuals derived from a mapping population v) carry out target enrichment using the NB-LRR capture array on the DNA from the bulks vi) perform next-generation sequencing of NB-LRR-enriched DNA from the bulks and identify single nucleotide polymorphisms that co-segregate with the genes of interest vii) test candidate genes identified by stable transformation of wheat

A durable solution to wheat stem rust is a paramount problem in global food security. Because the wheat stem rust pathogen is evolving rapidly and has shown the ability to overcome each new resistance gene that is deployed in agriculture, developing a capability to isolate large numbers of useful genes and deploy them in combinations is critical to successfully overcome this disease. The proposed research aims to do exactly that: develop new methods for rapidly accessing novel resistance genes that can be deployed in wheat breeding programs. Direct beneficiaries of this work include public wheat germplasm improvement programs (such as those at CIMMYT), the country-level breeding efforts that they support in the developing world (including Kenya, Ethiopia, Pakistan, India, and China), growers of wheat, and consumers of wheat in those countries. In the absence of a durable solution to wheat stem rust, wheat agriculture, particularly in Africa and South Asia, will live under the constant threat of epidemics and resulting crop failure caused by this disease. The genetic materials developed in this project, transgenic wheat lines containing multiple resistance genes effective against stem rust, will be available in three to five years. In addition, although stem rust has not been important in recent years in UK or European wheat production, climate change brings a new threat from this disease, which requires relatively warm conditions early in the growing season to have full impact. Although in high-value agriculture the pathogen can be controlled with chemical fungicide application, genetic resistance is vastly preferable from the standpoints of cost and environmental sustainability. Thus, UK and European wheat breeding companies, wheat growers, and consumers are potential direct beneficiaries of this work as well. The timescale for these benefits to be realized is uncertain, because we do not yet know the trajectory of stem rust's progression in Europe and the UK. Aegilops sharonensis, our choice as source for these novel resistance genes, is of interest to the global community of wheat researchers because its genome is closely related to the B genome of hexaploid bread wheat. The progenitors of the other components of the wheat genome are already being sequenced by BGI (China), so our effort in this project to generate a draft sequence of Ae. sharonensis will ideally complement these other resources. The combined tools of deep sequence and vastly improved genetic map of this organism will enable any interested researcher to more easily identify genes associated with useful agronomic traits in Ae sharonenesis. In addition to resistance genes for other diseases, these will include genes for tolerance to abiotic stresses such as heat and drought, and increased efficiency of nutrient utilization. These resources will be available to the research community as soon as we develop them, largely in the first year of the project, so their impact will be felt immediately. The research staff working on this project will develop additional technical expertise in a diverse range of skills, including molecular genetics, bioinformatics, and plant biotechnology, specifically in wheat, an exceedingly important crop in UK agriculture. They will also develop an increased understanding of the application of how basic science discoveries, such as the cloning of new genes envisioned in this project, get translated into practical outcomes for agriculture by working with our downstream deployment partners. This expertise can be applied in future career positions in academia, research institutes, and in companies, both small agricultural biotech firms and global multi-national agriculture companies. These benefits will be realized immediately upon the next career progression steps taken by the individuals involved in the project.