Identification of causal mutations in barley and wheat is hampered by their large genomes and suppressed recombination. To overcome these obstacles, we have developed MutChromSeq, a complexity reduction approach based on flow sorting and sequencing of mutant chromosomes, to identify induced mutations by comparison to parental chromosomes. We apply MutChromSeq to six mutants each of the barley Eceriferum-q gene and the wheat Pm2 genes. This approach unambiguously identified single candidate genes that were verified by Sanger sequencing of additional mutants. MutChromSeq enables reference-free forward genetics in barley and wheat, thus opening up their pan-genomes to functional genomics.

Summary: There is a large diversity of genetically defined resistance genes in bread wheat against the powdery mildew pathogen Blumeria graminis (B. g.) f. sp. tritici. Many confer race‐specific resistance to this pathogen, but until now only the mildew avirulence gene AvrPm3^a2/f2 that is recognized by Pm3a/f was known molecularly. We performed map‐based cloning and genome‐wide association studies to isolate a candidate for the mildew avirulence gene AvrPm2. We then used transient expression assays in Nicotiana benthamiana to demonstrate specific and strong recognition of AvrPm2 by Pm2. The virulent AvrPm2 allele arose from a conserved 12 kb deletion, while there is no protein sequence diversity in the gene pool of avirulent B. g. tritici isolates. We found one polymorphic AvrPm2 allele in B. g. triticale and one orthologue in B. g. secalis and both are recognized by Pm2. AvrPm2 belongs to a small gene family encoding structurally conserved RNase‐like effectors, including Avr_a13 from B. g. hordei, the cognate Avr of the barley resistance gene Mla13. These results demonstrate the conservation of functional avirulence genes in two cereal powdery mildews specialized on different hosts, thus providing a possible explanation for successful introgression of resistance genes from rye or other grass relatives to wheat.

Plasma membrane-associated and intracellular proteins and protein complexes play a pivotal role in pathogen recognition and disease resistance signaling in plants and animals. The two predominant protein families perceiving plant pathogens are receptor-like kinases and nucleotide binding-leucine-rich repeat receptors (NLR), which often confer race-specific resistance. Leaf rust is one of the most prevalent and most devastating wheat diseases. Here, we clone the race-specific leaf rust resistance gene Lr14a from hexaploid wheat. The cloning of Lr14a is aided by the recently published genome assembly of ArinaLrFor, an Lr14a-containing wheat line. Lr14a encodes a membrane-localized protein containing twelve ankyrin (ANK) repeats and structural similarities to Ca²⁺-permeable non-selective cation channels. Transcriptome analyses reveal an induction of genes associated with calcium ion binding in the presence of Lr14a. Haplotype analyses indicate that Lr14a-containing chromosome segments were introgressed multiple times into the bread wheat gene pool, but we find no variation in the Lr14a coding sequence itself. Our work demonstrates the involvement of an ANK-transmembrane (TM)-like type of gene family in race-specific disease resistance in wheat. This forms the basis to explore ANK-TM-like genes in disease resistance breeding.

Recent progress in large-scale sequencing, genomics, and rapid gene isolation techniques has accelerated the identification of race-specific resistance (R) genes and their corresponding avirulence (Avr) genes in wheat, barley, rye, and their wild relatives. Here, we describe the growing repertoire of identified R and Avr genes with special emphasis on novel R gene architectures, revealing that there is a large diversity of proteins encoded by race-specific resistance genes that extends beyond the canonical nucleotide-binding domain leucine-rich repeat proteins. Immune receptors with unique domain architectures controlling race-specific resistance possibly reveal novel aspects on the biology of host-pathogen interactions. We conclude that the polyploid cereal genomes have a large evolutionary potential to generate diverse types of resistance genes.