ing and comparative sequence analysis of powdery mildew and leaf rust resistance gene complements in wild barley
Authors: Steffenson B. J. , Mayrose I , Muehlbauer Gary J. , Sharon A. ,   University of Minnesota,   United States-Israel Binational Agricultural Research and Development Fund
Published: October 2023

Our overall, long-term goal is to exploit the genetic diversity present in cereal wild relatives for the development of cultivars with durable disease resistance. Our specific objectives for this proposal were to: 1) Utilize Association Genetics Resistance Gene Enrichment Sequencing (AgRenSeq) to identify and clone powdery mildew and leaf rust resistance gene complements in wild barley and 2) Conduct comparative sequence analyses of the cloned resistance genes to elucidate the basis of their specificity and evolution. The deployment of resistant cultivars is the most effective, economically efficient, and environmentally sound means of controlling plant diseases, especially in small grain cereals. The systems selected for study in this proposal are barley (Hordeum vulgare ssp. vulgare, Hvv), its wild progenitor (Hordeum vulgare ssp. spontaneum, Hvs) and the powdery mildew (Blumeria graminis f. sp. hordei, Bgs) and leaf rust (Puccinia hordei, Ph) pathogens. We compiled a diverse panel of Hvs accessions (the Wild Barley Diversity Collection or WBDC; N = 314) from across its native range and evaluated it to 40 isolates of Bgs and 12 isolates of Ph. We obtained genomic DNA sequences enriched for Nucleotide Binding Site-Leucine Rich Repeat (NLR) type resistance genes for 203 WBDC accessions, plus cultivar Morex for which the first reference genome sequence of barley was based. We assembled the 250 bp Illumina sequencing reads into contigs using CLC assembly cell. From this effort, we successfully assembled the sequences of 201 WBDC accessions plus Morex and used NLR Parser to identify contigs containing NLR genes. AgRenSeq was then used to identify k-mers (short oligonucleotide sequences of length k) that were associated with resistance to each isolate of the two pathogens. This analysis was performed individually for all WBDC accessions and each individual pathogen race (9,898 host accession x pathogen race combinations). We visualized the results from these analyses in Manhattan plots and identified 311 and 144 peaks for powdery mildew and leaf rust resistance, respectively. The next step in the analysis was to identify the contigs associated with the peaks in the Manhattan plots. BLAST (Basic Local Alignment Search Tool) searches were employed to identify closely related contigs in other WBDC accessions or in Morex. We identified two candidate R genes that were only present in resistant WBDC accessions. One of these was present in seven WBDC lines and was associated with resistance to four leaf rust isolates. BLAST analysis of this gene revealed that it was Rph15, one of the most widely effective leaf rust resistance genes reported in Hordeum. This gene was cloned and functionally validated in association with our Australian colleagues (Cheng et al., 2021). We are currently in the process of cloning six of other resistance genes: four for powdery mildew and two for leaf rust. As the contigs do not contain much of the promoter sequences, we have employed a genome walking approach to identify 2,500 bp of promoter sequence. To speed up and simplify the cloning of resistance genes from the WBDC, the PI established the International Wild Barley Sequencing Consortium (IWBSC; https://iwbsc.umn.edu/) comprised of over 60 researchers from 14 different countries and raised over $150,000 through crowdfunding to pay for 10X depth sequence coverage. Genome-wide association study (GWAS) of whole genome sequencing (WGS) data identified extremely strong and clear signals of association for several resistance genes which will facilitate gene cloning in concert with a wild barley pan-genome currently under construction. The cloning of multiple resistance gene can facilitate the development of durably resistant cultivars by inserting, through transgenesis, cassettes of multiple resistance genes.