Aegilops tauschii, the diploid wild progenitor of the D subgenome of bread wheat, is a reservoir of genetic diversity for improving bread wheat performance and environmental resilience. Here we sequenced 242 Ae. tauschii accessions and compared them to the wheat D subgenome to characterize genomic diversity. We found that a rare lineage of Ae. tauschii geographically restricted to present-day Georgia contributed to the wheat D subgenome in the independent hybridizations that gave rise to modern bread wheat. Through k-mer-based association mapping, we identified discrete genomic regions with candidate genes for disease and pest resistance and demonstrated their functional transfer into wheat by transgenesis and wide crossing, including the generation of a library of hexaploids incorporating diverse Ae. tauschii genomes. Exploiting the genomic diversity of the Ae. tauschii ancestral diploid genome permits rapid trait discovery and functional genetic validation in a hexaploid background amenable to breeding.

Summary Leaf rust, caused by Puccinia hordei, is a devastating fungal disease affecting barley (Hordeum vulgare subsp. vulgare) production globally. Despite the effectiveness of genetic resistance, the deployment of single genes often compromises durability due to the emergence of virulent P. hordei races, prompting the search for new sources of resistance. Here we report on the cloning of Rph15, a resistance gene derived from barley’s wild progenitor H. vulgare subsp. spontaneum. We demonstrate using introgression mapping, mutation and complementation that the Rph15 gene from the near‐isogenic line (NIL) Bowman + Rph15 (referred to as BW719) encodes a coiled‐coil nucleotide‐binding leucine‐rich repeat (NLR) protein with an integrated Zinc finger BED (ZF‐BED) domain. A predicted KASP marker was developed and validated across a collection of Australian cultivars and a series of introgression lines in the Bowman background known to carry the Rph15 resistance. Rph16 from HS‐680, another wild barley derived leaf rust resistance gene, was previously mapped to the same genomic region on chromosome 2H and was assumed to be allelic with Rph15 based on genetic studies. Both sequence analysis, race specificity and the identification of a knockout mutant in the HS‐680 background suggest that Rph15‐ and Rph16‐mediated resistances are in fact the same and not allelic as previously thought. The cloning of Rph15 now permits efficient gene deployment and the production of resistance gene cassettes for sustained leaf rust control.

Genetic resistance is the most economic and environmentally sustainable approach for crop disease protection. Disease resistance (R) genes from wild relatives are a valuable resource for breeding resistant crops. However, introgression of R genes into crops is a lengthy process often associated with co-integration of deleterious linked genes1, 2 and pathogens can rapidly evolve to overcome R genes when deployed singly3. Introducing multiple cloned R genes into crops as a stack would avoid linkage drag and delay emergence of resistance-breaking pathogen races4. However, current R gene cloning methods require segregating or mutant progenies5–10, which are difficult to generate for many wild relatives due to poor agronomic traits. We exploited natural pan-genome variation in a wild diploid wheat by combining association genetics with R gene enrichment sequencing (AgRenSeq) to clone four stem rust resistance genes in <6 months. RenSeq combined with diversity panels is therefore a major advance in isolating R genes for engineering broad-spectrum resistance in crops.

Aegilops tauschii, the diploid wild progenitor of the D-subgenome of bread wheat, constitutes a reservoir of genetic diversity for improving bread wheat performance and environmental resilience. To better define and understand this diversity, we sequenced 242 Ae. tauschii accessions and compared them to the wheat D-subgenome. We characterized a rare, geographically-restricted lineage of Ae. tauschii and discovered that it contributed to the wheat D-subgenome, thereby elucidating the origin of bread wheat from at least two independent hybridizations. We then used k-mer-based association mapping to identify discrete genomic regions with candidate genes for disease and pest resistance and demonstrated their functional transfer into wheat by transgenesis and wide crossing, including the generation of a library of ‘synthetic’ hexaploids incorporating diverse Ae. tauschii genomes. This pipeline permits rapid trait discovery in the diploid ancestor through to functional genetic validation in a hexaploid background amenable to breeding.

Generation of genetic diversity is crucial for the evolution of new traits, with mutation and sexual 34 recombination as the main drivers of diversity in most eukaryotes. However, many species in the 35 fungal kingdom can propagate asexually for extended periods and therefore understanding alternative 36 mechanisms contributing to genetic diversity in asexual populations has been of great interest 1,2 . Some 37 fungi can use a parasexual mechanism to exchange genetic material independently of meiosis 2 . This 38 process involves anastomosis of haploid hyphae and fusion of two nuclei to generate a single diploid 39 nucleus, which subsequently undergoes progressive chromosome loss to generate recombinant haploid 40 offspring. Parasexuality has been described in members of the ascomycete phylum (64% of described 41 fungal species) in which the dominant asexually propagating form is haploid 3 . However, in 42 basidiomycete fungi (34% of described species), the predominant life stage is generally dikaryotic, 43 with two different haploid nuclei maintained within each individual 3 . The role of non-sexual genetic 44 exchange between such dikaryons in generating genetic diversity is not known. 45 Basidiomycetes include many fungi with critical ecosystem functions, such as wood decay and 46 plant symbiosis, as well as agents of important human and plant diseases 1 . Rust fungi (subphylum 47 Pucciniomycotina) comprise over 8,000 species including many pathogens of major agricultural and 48 ecological significance 4 . These organisms are obligate parasites with complex life cycles that can 49 include indefinite asexual reproduction through infectious dikaryotic urediniospores. Early researchers 50 speculated that rust fungi can exchange genetic material during the asexual phase 5-8 , but these 51 hypotheses could not be confirmed molecularly. Some naturally occurring rust pathotypes have been 52 suggested to have arisen by somatic hybridisation and genetic exchange based on limited molecular 53 evidence of shared isozyme or random amplified polymorphic DNA (RAPD) markers 9,10 . Mechanisms 54 underlying genetic exchange are unknown, but may involve hyphal anastomosis followed by nuclear 55 exchange and/or nuclear fusion and recombination 11 . Recent advances in assembling complete karyon 56 sequences in rust fungi 12,13 provide the opportunity to definitively detect and discriminate between 57 nuclear exchange and recombination. 58 4The Ug99 strain (race TTKSK) of the wheat stem rust pathogen Puccinia graminis f. sp. tritici 59 (Pgt) presents a significant threat to global wheat production 14 . It was first detected in Uganda in 1998 60 and described in 1999 15 , and has since given rise to an asexual lineage that has spread through Africa 61 and the Middle East causing devastating epidemics 14 . The origin of the Ug99 lineage is unknown, 62 although it is genetically distinct from other Pgt races 16,17 . To resolve the genetic makeup of Ug99, we 63 generated a haplotype-phased genome reference for the origi...