Progress 06/01/20 to 05/31/23
Outputs
Target Audience: This project sought to mine a wild wheat relative for rust resistance genes to advance the breeding of more resilient wheat cultivars, with a target audiences of: the scientific community interested in plant genetics, plant pathology, plant breeding, plant genetic resources, and host- pathogen interactions researchers interested in accessing effective resistance genes from a wheat relative - wild emmer wheat, Triticum turgidum ssp. dicoccoides bioinformaticists interested in better software tools to identify genes from larger datasets where there is no reference genome. Our project sought to identify new genes for resistance to stripe, stem and leaf rust in wild emmer by sequencing the genomes of 200 diverse specimens, assessing their resistance to the three diseases, and using computational techniques to discover useful genes. The whole-genome sequence data we generate will be available to researchers seeking genes that are useful to improve disease resistance, as well as other traits. Changes/Problems:The SARS-CoV2 pandemic resulted in delays in some aspects of the project. Assembly and increase of the wild emmer accessions took additional time, and seed shipments were delayed. We did not progress to cloning specific resistance genes as a result of the delays. What opportunities for training and professional development has the project provided?Our NIFA-funded research has provided additional training opportunities for several grant and non-grant funded scientists in cereal rust pathology and the cultivation of wild cereal relatives at the U of M. This includes assistant research professor Pablo Olivera; post-doctoral research associates Ahmad Sallam and Oadi Matny; Researcher 2 scientists Tamas Szinyei and Emmery Hartwig; and graduate students Rae Page, Yoonjung Lee, and Mitchell Ritzinger. There were also several undergraduate students who assisted on this project in various capacities, the recent ones included Emma Leff, Zviad Chakhnaskvili, and Annie Russell-Pribnow. All of these individuals were trained in the methodology for working with rust pathogens, including production and storage of inoculum; inoculation techniques; and disease severity scoring. How have the results been disseminated to communities of interest?A poster was presented by Pablo Olivera at the Durum Wheat Conference in Italy. We are currently working on initial publications that include genotyping of the WGS panel, population structure, diversity, and the phenotypic responses of the panel against different races of leaf rust, stem rust, and stripe rust. Upon publication, the WGS data of the WEW panel will be deposited in the public repository database, the Sequence Read Archive (SRA) of the National Center for Biotechnology Information (NCBI), to ensure accessibility of the sequencing data to all researchers. Prior to that time access will be granted to anyone who contacts us for prepublication access. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Goal 1: We assembled a Wild Emmer Diversity Collection (WEDC) comprised of 290 Triticum turgidum ssp. dicoccoides derived from four genebanks, 90 more than proposed. The panel was constructed to cover the broadest geographic distribution of the species. All accessions were increased from single plants grown in the greenhouse and were selfed at least once, and in most cases multiple generations. Thus, WEDC represents a refined germplasm collection with a high level of homozygosity useful for many types of experiments. Goal 2: We phenotyped the 290 accession panel against 5 races of each of the rust pathogens: Puccinia graminis f. sp. tritici (stem), Puccinia triticina (leaf) and Puccinia striiformis f. sp. tritici (stripe). The five stem rust races represent the four main genetic clades of this rust pathogen (TTKSK, JRCQC, TKTTF, and TTRTF) and the most virulent race in the US (TTTTF): Table 1. Number of wild emmer accession exhibiting resistant, moderately resistant, and susceptible reactions to five races of Puccinia graminis f. sp. tritici. TTKSK JRCQC TKTTF TTRTF TTTTF Resistant 106 60 65 71 90 Moderately Resistant 24 22 17 15 25 Susceptible 160 208 208 204 175 Total 290 290 290 290 290 Of the five races of the leaf rust pathogen (Puccinia triticina) tested in phenotyping trials, races TBBGS and TFBGQ are US races, virulent on hexaploid wheat (Triticum aestivum) and BBBQD is virulent on durum wheat (T. turgidum ssp. durum): Table 2. Number of wild emmer accession exhibiting resistant, moderately resistant, and susceptible reactions to five races of Puccinia triticina (some accessions had missing data). BBBQD TBBGS TFBGQ TNBGJ MFPSB Resistant 151 84 91 120 91 Moderately Resistant 1 3 4 2 2 Susceptible 131 196 188 167 196 Total 283 283 283 289 289 Table 3. Number of wild emmer accession exhibiting resistant, moderately resistant, and susceptible reactions to five races of stripe rust, Puccinia striiformis f. sp. tritici. Pstv-221 Pstv-14 Pstv-143 Pstv-37 5006 Resistant 69 58 83 123 100 Moderately Resistant 117 125 94 103 75 Susceptible 97 101 109 64 111 Total 283 284 286 290 286 All accessions were sequenced to 7.5x depth coverage with Illumina 150 bp paired end reads. The sequences were subjected to quality control and backed up in the Kansas State University Beocat High Performance Compute Cluster. We mapped the sequences from each accession against the genome assembly of the reference accession Zavitan (WEW_v1.0; Avni et al., 2017), called single nucleotide polymorphisms (SNPs), and generated a species-wide phylogeny. Overall, ~11- 16% of accessions were resistant to all five races of each rust pathogen, indicating that some genes or gene combinations may be broadly effective against individual rust types. Additionally, ~0.5-4% of accesssions showed resistance to two rust types, but no accesssions showed resistance to all three rusts. Goal 3. In collaboration with the group of Prof. Xin Gao at KAUST, Saudi Arabia, we developed a new, computationally efficient k-mer counting and sorting pipeline in C++ and used it to generate a 878 Gb non-redundant k-mer matrix from all the 290 accessions on the KAUST Shaheen Super Computer. We conducted k-mer-based genome-wide association studies (kGWAS) with a 7.4% subset of the k-mers. We used the accessions TA1045 (resistant to stem rust), TA1054 (resistant to leaf rust), and Zavitan to map k-mers positively associated with stem, leaf and stripe rust resistance, respectively. For stem rust resistance to TTKSK (Ug99), we identified discreet association blocks on chromosomes 1B, 3B, 5B, 6B and 7A ranging in size from 0.1 to 2.0 Mb. For leaf rust resistance to TBBGS, we identified discreet blocks on chromosomes 1B, 2A, 3B 4A and 5A ranging from 0.18 to 2.39 Mb, and with highly significant - log10 (p) values of up to 20. For stripe rust resistance to Pstv-221, we identified discreet blocks on chromosomes 1A, 2A, 4A, 4B, and 6A ranging from 0.24 to 1.0 Mb with -log10 (p) values of ~20. We are currently annotating the gene content and expression in the association blocks and shortlisting candidate genes for functional validation.
Publications
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Progress 06/01/21 to 05/31/22
Outputs
Target Audience:The target audiences for our efforts in this reporting period are: 1. the scientific community interested in plant genetics, plant pathology, plant breeding, plant genetic resources, and host-pathogen interactions, in particular: - researchers interested in accessing genes from a key wheat relative - wild emmer wheat, Triticum turgidum ssp. dicoccoides. - bioinformaticists interested in better software tools to identify genes from larger datasets where there is no reference genome. Our project aims to identify new genes for resistance to stripe, stem and leaf rust in wild emmer by sequencing the genomes of 200 diverse specimens, assessing their resistance to the three diseases, and using computational techniques to discover useful genes. The whole-genome sequence data we generate will be available to researchers seeking genes that are useful to improve other wheat traits, and the sequences of useful genes can be introduced into cultivated wheat through breeding or biotechnology. . This investment into understanding a wild wheat relative will lead to more resilient wheat cultivars, reduced disease losses, and greater profit for farmers, while contributing to global food security. Changes/Problems:The SARS-CoV2 pandemic resulted in delays in some aspects of the project. Assembly and increase of the wild emmer accessions took additional time, and seed shipments were delayed. GWAS and development of transgenic lines is behind schedule as a result. What opportunities for training and professional development has the project provided?Our NIFA-funded research has provided additional training opportunities for several grant and non-grant funded scientists in cereal rust pathology and the cultivation of wild cereal relativesat the U of M. This includes assistant research professor Pablo Olivera; post-doctoral research associates Ahmad Sallam and Oadi Matny; Researcher 2 scientists Tamas Szinyei and Emmery Hartwig; and graduate students Rae Page, Yoonjung Lee, and Mitchell Ritzinger. There were also several undergraduate students who assisted on this project in various capacities, the recent ones included Emma Leff, Zviad Chakhnaskvili, and Annie Russell-Pribnow. All of these individuals were trained in the methodology for working with rust pathogens, including production and storage of inoculum; inoculation techniques; and disease severity scoring. How have the results been disseminated to communities of interest?The work is still in progress and results will be disseminated once collected. A poster will be presented by Pablo Olivera this Fall at the Durum wheat conference in Italy. What do you plan to do during the next reporting period to accomplish the goals?The collection will be evaluated for resistance to two more leaf rust races and five stripe rust races: PSTv-14, PSTv 37, PSTv-143, PSTv-218, PSTv-221. Phenotyping will be completed in October 2022. We plan to generate 20-fold coverage with HiFi reads. Extrapolating from recent sequencing projects in our groups we expect to obtain assemblies with N50 values of >25 Mb. These assemblies will be used for SNP and k-mer-based genome-wide association studies (GWAS) as previously described (Gaurav et al., 2022) to identify candidate wheat rust resistance genes. We are currently exploring further opportunities for streamlining the k-mer GWAS pipeline to handle this large NGS dataset (24 Tb). These include optimizing the computational algorithms, in particular generation of the k-mer matrix, as well as installing the k-mer GWAS pipeline on the KAUST Shaheen super computer, which is among the top 20 largest and most powerful computers in the world.
Impacts
What was accomplished under these goals?
Goal 1: We assembled a Wild Emmer Diversity Collection (WEDC) comprised of 295 Triticum turgidum ssp. dicoccoides derived from four genebanks, as described in our last annual report (2021, Table 1). The original proposal called for sequencing 200 of these accessions. We contributed additional funds from King Abdullah University of Science and Technology (KAUST, co-PIs B. Wulff and J. Poland) to sequence an additional 95 accessions. A larger sequence-configured panel increases the power for association mapping, identification of rare alleles and achieving greater resolution of associated resistance genes. The panel was constructed with selected accessions to cover the broadest geographic distribution of the species (presented in Table 2 of the 2021 progress report). With respect to accessions from the NSGC, previous evaluations for stem rust resistance were also considered in the final selection process. All accessions were increased from single plants grown in the greenhouse and were selfed at least once but in most cases multiple generations. Thus, WEDC represents a refined germplasm collection with a high level of homozygosity useful for many types of experiments. Goal 2: We have so far phenotyped 292 accessions of the panel against 5 races of the stem rust (Puccinia graminis f. sp. tritici) and 3 races of leaf rust (Puccinia triticina) pathogens of wheat. The five stem rust races (Tables 1) represent the four main genetic clades of this rust pathogen (TTKSK, JRCQC, TKTTF, and TTRTF) and the most virulent race in the US (TTTTF). Table 1. Number of wild emmer accession exhibiting resistant, moderately resistant, and susceptible reactions to five races of Puccinia graminis f. sp. tritici at the seedling stage. TTKSK JRCQC TKTTF TTRTF TTTTF Resistant 108 60 65 73 89 Moderately resistant 23 22 16 15 25 Susceptible 152 200 203 196 169 Missing 9 10 8 8 9 TOTAL 292 292 292 292 292 Of the three races of the leaf rust pathogen (Puccinia triticina) tested in phenotyping trials (Table 2), races TBBGS and TFBGQ are US races, virulent on hexaploid wheat (Triticum aestivum) and BBBQD is virulent on durum wheat (T. turgidum ssp. durum). Additional races MFPSBand TNBGJ have been recently increased, and phenotyping against them will be conducted shortly. Table 2. Number of wild emmer accession exhibiting resistant, moderately resistant, and susceptible reactions to three races of Puccinia triticina at the seedling stage. TBBGS TFBGQ BBBQD Resistant 86 92 153 Moderately resistant 3 4 1 Susceptible 196 186 131 Missing 7 10 7 TOTAL 292 292 292 As per our proposal, all accessions were sequenced to 7.5x depth coverage with Illumina 150 bp paired end reads. The sequences were subjected to quality control and backed up in the Kansas State University Beocat High Performance Compute Cluster. We mapped the sequences from each accession against the genome assembly of the reference accession Zavitan (WEW_v1.0; Avni et al., 2017), called single nucleotide polymorphisms (SNPs), and generated a species-wide phylogeny. The architecture of the phylogeny conforms with previously generated phylogenies using other marker systems thus confirming the overall quality of our dataset. From this phylogeny we selected four accessions with good resistance to wheat stem, stripe and/or leaf rust whilst striving, along with reference accession Zavitan, to represent the maximum genetic diversity and rust resistance diversity of the species. Seed of these four accessions are being shipped to KAUST for high molecular weight DNA extraction and PacBio high fidelity (HiFi) long read sequencing.
Publications
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Progress 06/01/20 to 05/31/21
Outputs
Target Audience:The target audiences for our efforts in this grant are: 1. the scientific community interested in plant genetics, plant pathology, plant breeding, plant genetic resources, and host-pathogen interactions, in particular: - researchers interested in accessing genes from a key wheat relative - wild emmer wheat, Triticum turgidum ssp. dicoccoides. - bioinformaticists interested in better software tools to identify genes from larger datasets where there is no reference genome. 2. Small grain cereal production commodity groups 3. Wheat growers 4. Food security/ food system specialists and policy makers Our project aims to identify new genes for resistance to stripe, stem and leaf rust in wild emmer by sequencing the genomes of 200 diverse specimens, assessing their resistance to the three diseases, and using computational techniques to discover useful genes. Once the sequences of these genes are known, they can be introduced into cultivated wheat through breeding or biotechnology. The whole-genome sequence data we generate will also be available to other researchers seeking genes that are useful to improve other wheat traits. This investment into understanding a wild wheat relative will lead to more resilient wheat cultivars, reduced disease losses, and greater profit for farmers, while contributing to global food security. Changes/Problems:The SARS-CoV2 pandemic resulted in delays in some aspects of the project. Assembly and increase of the wild emmer accessions took additional time, delaying seed shipments to Kansas State. What opportunities for training and professional development has the project provided?At the UM and KSU, four undergraduate students helped to plant, process, and catalog the wild emmer accessions. At the UM a new assistant professor (Pablo Olivera) has joined the project to help lead the rust phenotyping effort. How have the results been disseminated to communities of interest?The work is in progress and results will be disseminated once collected. What do you plan to do during the next reporting period to accomplish the goals?In the next period, we will: - sequence the remaining 112 accessions of the WEDC panel in late September - curate and map all the sequence data - perform rigorous phenotyping of the full 295 accessions following infection with five diverse races of each of the three rust pathogens under study in the proposal. Stem rust phenotypes will be prioritized for completion by the end of December. A portion of the stripe rust and leaf rust testing will be pursued as well. Phenotyping is expected to be completed in April.
Impacts
What was accomplished under these goals?
We assembled a Wild Emmer Diversity Collection (WEDC) comprised of 295 Triticum turgidum ssp. dicoccoides accessions derived from four genebanks (Table 1). The selection of accessions was made to cover the broadest geographic distribution of the species (Table 2). With respect to accessions from the USDA-ARS NSGC, previous evaluations for stem rust resistance were also considered in the final selection process. All accessions were increased from single plants grown in the greenhouse and were self-fertilized at least once, and in most cases multiple generations. Thus, WEDC represents a refined germplasm collection with a high level of homozygosity - genetic consistency at a given point in the genome - useful for many types of experiments. Table 1: Sources of Triticum turgidum ssp. dicoccoides accessions comprising the WEDC. Germplasm bank Origin # Accessions USDA-ARS National Small Grains Collection (NSGC) Aberdeen, ID USA 78 Wheat Genetics Resource Center at Kansas State University (WGRC) Manhattan, KS USA 139 National Bioresource Project Komugi, Kyoto, Japan 20 Council for Agricultural Research and Agricultural Economics Analysis (CREA) Fiorenzuola d'Arda, Italy 58 Table 2: Country of origin of wild emmer accessions used in this study. Country of origin Number of accessions Iran 4 Iraq 18 Israel 136 Lebanon 27 Palestinian territories 14 Syria 32 Turkey 57 Unknown* 7 *Reported origin from countries away from the geographic distribution of the species. Our proposal focuses on sequencing 200 accessions of wild emmer wheat. We are expanding the collection to 300, and to date we have added a further 95 accessions that are of interest for phenotyping. Sequencing of the additional accessions is beyond the scope of the current budget, but we are seeking funding to continue the characterization of the additional accessions. Whole-genome sequencing of this collection is in progress, along with the increase and curation of seed stocks that can be distributed to the larger community. Seed increases were carried out for all accessions at University of Minnesota (UM) and Kansas State (KSU). At KSU, the lines were grown out and additional phenotyping completed for some physical traits, including plant height and glume color. Work is in progress to measure seed morphology including seed length and width, and seed shape. To date, tissue was harvested and DNA extracted for sequencing for 88 accessions. DNA of these accessions has been delivered to Novogene for sequencing and completed Illumina sequencing paired-end 150bp sequencing (May 2021). The data were delivered to KSU and JIC with storage on the computing cluster for subsequent analysis. At JIC, Dr. Wulff worked with Drs Matthew Hartley and Burkhard Steuernagel in the John Innes Centre Scientific Computing group to develop a software infrastructure for optimized mining of genomes lacking a reference sequence. A fast programming language and a data structure based on minimizers enables CPU- and memory-efficient interrogation of data sets. These improvements allow more efficiently handling of a large dataset as in the current project. All accessions will be phenotyped against at least five races each of the stem rust (Puccinia graminis f. sp. tritici), stripe rust (Puccinia striiformis f. sp. tritici), and leaf rust (Puccinia triticina) pathogens (Table 3). Table 3. List of isolates of the stem rust (Puccinia graminis f. sp. tritici), stripe rust (Puccinia striiformis f. sp. tritici), and leaf rust (Puccinia triticina) pathogens of wheat to be used evaluate the WEDC. Pathogen Isolate Race Puccinia graminis f. sp. tritici 04KEN156/04 TTKSK Puccinia graminis f. sp. tritici 14GEO189-1 TTRTF Puccinia graminis f. sp. tritici 13ETH18-1 TKTTF Puccinia graminis f. sp. tritici 01MN84A-1-2 TTTTF Puccinia graminis f. sp. tritici 09ETH08-3 JRCQC Puccinia striiformis f. sp. tritici -- PSHv-33 Puccinia striiformis f. sp. tritici -- PSHv-54 Puccinia striiformis f. sp. tritici -- PSHv-71 Puccinia striiformis f. sp. tritici -- PSHv-72 Puccinia striiformis f. sp. tritici -- PSHv-81 Puccinia triticina -- THBJG Puccinia triticina -- BBBD Puccinia triticina -- MPPSD Puccinia triticina -- MBDSD Puccinia triticina -- PBLRG
Publications
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