Aegilops sharonensis genome-assisted identification of stem rust resistance gene Sr62

Abstract

The wild relatives and progenitors of wheat have been widely used as sources of disease resistance (R) genes. Molecular identification and characterization of these R genes facilitates their manipulation and tracking in breeding programmes. Here, we develop a reference-quality genome assembly of the wild diploid wheat relative Aegilops sharonensis and use positional mapping, mutagenesis, RNA-Seq and transgenesis to identify the stem rust resistance gene Sr62, which has also been transferred to common wheat. This gene encodes a tandem kinase, homologues of which exist across multiple taxa in the plant kingdom. Stable Sr62 transgenic wheat lines show high levels of resistance against diverse isolates of the stem rust pathogen, highlighting the utility of Sr62 for deployment as part of a polygenic stack to maximize the durability of stem rust resistance.

Fig. 1. Positional mapping restricts Sr62 to a 480 kb interval on chromosome 1S^sh.

a Wheat–Ae. sharonensis translocation chromosomes and Ae. sharonensis chromosome 1S^sh. b Genetic map of the region harbouring Sr62 on the short arm of Ae. sharonensis chromosome 1S^sh. c Physical map of the region around Sr62. d Genes in the interval genetically delimiting the presence of Sr62. WTK is presumably an ortholog of Pm24.

Fig. 2. Candidate gene identification by mutagenesis and transcriptome sequencing (MutRNA-Seq).

RNA-Seq reads from the wild-type parent and independently derived EMS mutants are mapped to a reference genome sequence. Annotated genes are inspected (within a mapping interval, if available) for a gene exhibiting a preponderance of single-nucleotide variants (SNVs, red dots) across the mutants.

Fig. 3. Functional validation of Sr62 by EMS mutagenesis and transformation into wheat.

a Structure of Sr62, with predicted nucleotide change caused by EMS-derived loss-of-function mutations. Boxes represent exons and lines represent introns with white boxes representing untranslated regions and black boxes representing the predicted open reading frame. The 11.4-kb portion of the third intron excluded from the binary construct is indicated. b Schematic representation of the Sr62 protein, with the position of the two protein kinase domains and the predicted amino-acid changes caused by the EMS mutations indicated. c The Sr62 sequence used for transformation of wheat cultivar Fielder. CDS, coding DNA sequence. d Reactions of three homozygous independent transgenic lines to four Pgt isolates. The copy number of the hygromycin selectable marker in T₀ plants is indicated.

Fig. 4. Phylogenetic relationship between tandem kinases from cereal crop and wild grasses.

A total of 99 predicted tandem kinases were retrieved from the genomes of bread wheat, durum wheat, maize, barley, sorghum, rice, Ae. tauschii and Ae. sharonensis, along with the five cloned tandem kinase disease resistance genes. Phylogenetic clades and subclades are indicated by different colours and labelled with numbers. a Phylogeny based on the whole tandem kinase coding sequence. b Phylogeny based on the individual protein kinase domain coding sequences.

Fig. 5. Synteny around Sr62.

Genomic regions containing genes orthologous to Sr62 along with surrounding genes reveal micro-synteny. The syntenic block is well conserved within the Triticum spp., Aegilops spp., and barley, but appears to be absent from Brachypodium, rice, sorghum and maize. The synteny alignment was generated through Gramene, except for Ae. sharonensis, which was added manually.