Source: KANSAS STATE UNIV submitted to
PLANT BREEDING PARTNERSHIP: EXPLORING RESISTANCE AND TOLERANCE TO WHEAT STREAK MOSAIC COMPLEX IN WHEAT WILD RELATIVE, AEGILOPS TAUSCHII
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1027901
Grant No.
2022-67013-36204
Cumulative Award Amt.
$556,497.00
Proposal No.
2021-07579
Multistate No.
(N/A)
Project Start Date
Dec 1, 2021
Project End Date
Nov 30, 2025
Grant Year
2022
Program Code
[A1141]- Plant Health and Production and Plant Products: Plant Breeding for Agricultural Production
Project Director
Nouri, S.
Recipient Organization
KANSAS STATE UNIV
(N/A)
MANHATTAN,KS 66506
Performing Department
Plant Pathology - AES
Non Technical Summary
Wheat Streak Mosaic (WSM) is one of the most economically important viral diseases of wheat with an estimated annual loss of 1.5-5.6% of the U.S. wheat crop. While cultural control can mitigate the disease, resistant varieties are sorely needed to reduce the losses and ensure food security and farm profitability into the future. The WSM complex is associated with several documented viruses withWheat streak mosaic virus(WSMV) as the predominant followed by mixed infections withTriticum mosaic virus(TriMV). To date, only three resistant genes have been identified against WSM. Although the use of current resistant varieties is promising, resistance-breaking virus isolates have been recently reported. Hence, it is crucial for breeding programs to continue searching for alternative resistance from different sources including wheat wild relatives.Aegilops tauschii, which is the diploid donor of the D genome of cultivated bread wheat, has known resistance to many pests and diseases. Despite the significant damage of wheat viral diseases, this species has not been explored as a source of resistance for viruses. To address this knowledge gap, this project will explore a large panelAe. tauschiiaccessions for resistance/tolerance to WSMV using association mapping with whole-genome sequencing data to identify resistance loci which can be subsequently used for wheat improvement. To better understand the dynamics and biology of the WSM, we will evaluate resistance/tolerance through phenotypic assessment of disease symptoms combined with the virus accumulation quantification. To disentangle the synergistic impact, identified resistant accessions will be assessed for WSMV in single and mixed infections with TriMV. The identification of resistance sources and specific resistance gene loci along with the detailed understanding of resistance/tolerance to WSM generated from this study will help to improve wheat germplasm for viral resistance.
Animal Health Component
20%
Research Effort Categories
Basic
80%
Applied
20%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2121549110165%
2021549108035%
Knowledge Area
212 - Pathogens and Nematodes Affecting Plants; 202 - Plant Genetic Resources;

Subject Of Investigation
1549 - Wheat, general/other;

Field Of Science
1101 - Virology; 1080 - Genetics;
Goals / Objectives
To date, only three resistant genes have been identified against WSM. Recent reports have documented that at least one WSMV isolate has overcoming resistance associated with one of those genesm, with several additional potential resistance-breaking WSMV and TriMV isolates. Hence, it is crucial for breeding programs to continue searching for alternative resistance from different sources including from wild relatives. The diploid donor of the D genome (Aegilops tauschii) of cultivated bread wheat has known resistance to many pests and diseases. In this project, we evaluate resistance/tolerance of a diversty panel of Aegilops tauschiivia phenotypic assessment of disease symptoms combined with the virus accumulation quantification. In addition, we will identify resistance loci using genome-wide association mapping approaches. The outcomecan be subsequently used for wheat improvement.
Project Methods
We will mechanically inoculate a large panel of Ae. tauschii genotypes with WSMV(242 accessions *3 replicates = 726 plants in total for WSMV infection). We will estimate genotype-specific responses to the virus infection from two variables: (1) symptom development (score-based), and (2) virus accumulations (titers) via absuluate quantification (RT-qPCR).Absolute quantifications of WSMV are determined by running the default setting of CFX Maestro software (Bio-Rad). The standard curve, correlation coefficient of the standard curve, and efficiencies are calculated by the CFX software. The amount of cDNA quantified for each sample is expressed as number of copies of viral RNA/ng of total RNA. The data obtained from symptomdevelopment and viral RNA accumulations are assembled in Microsoft Excel and all correlation analyses will be performed using the Statistical Analysis System.To identify genomic regions association with resistance and tolerance in theAe. tauschiipanel, we will implement GWAS using both SNP variants andk-mer bases analysis.TheAe. tauschiipanel has been sequenced to 10x coverage using Illumina paired end sequencing of 2x150bp reads.For variant calling of single nucleotide polymorphisms (SNPs) and small intertion / deletions (InDel) we will utilize the reference genome assembly of AL8/78.For analysis of the RT-qPCR assessment of virus titer and the phenotypic data scores for disease severity, we will use a mixed linear model to account for the experimental design and to calculate the best linear unbiased estimator (BLUE) for each accession at each time point.For GWAS analyses we will use the R software package 'GAPIT' accounting for multiple testing with a Bonferroni correction with an experimental significance level alpha value of 0.01. Manhattan plots to visualize association analysis results will be done with 'CMplot' package in R software.To validate the results of our greenhouse evaluation as well as genome association mapping, we will select 10-20 most promising resistant or tolerant accessions to WSMV based on symptom severity and virus titer. We will inoculate these lines with WSMV sap with 10 biological replicates under growth chamber condisions. Additionally, for con-infection of WSMV and TriMV,we will select 5-10 genotypes with the highest degree of resistance or tolerance to WSMV from single infection assessmentalong with 2-4 susceptible genotypes.

Progress 12/01/23 to 11/30/24

Outputs
Target Audience:University, USDA, and industry wheat researchers Changes/Problems:None What opportunities for training and professional development has the project provided?The project provided research opportunity for a Postdoc, a PhD student, and an undergraduate. How have the results been disseminated to communities of interest?Results have been shared with researchers and wheat industry partners through presentations/updates at NSF-IUCRC monthly meetings hold by Wheat Genomic Research Center (WGRC). What do you plan to do during the next reporting period to accomplish the goals?Aim 1. Identify novel sources of genetic resistance and tolerance to WSMV in Ae. taushii. Completed. Aim 2. Characterize genetic basis of resistance and tolerance genomic in a diverse panel of Ae. tauschii accessions through genome-wide association studies (GWAS). Complete GWAS using both SNP variants and k-mers from completed set of single and mixed infection screening. Complete analysis of genotyping data for the final report and a manuscript. Aim 3. Detailed evaluations of resistance and tolerance in selected Ae. tauschii to WSMV in single and mixed infections with TriMV. Complete screening of the selected accessions for mixed infections. Complete phenotype data analysis for the final report and a manuscript.

Impacts
What was accomplished under these goals? Specific Aim 1: Identify novel sources of genetic resistance and tolerance to WSMV in Ae. taushii. This aim was completed last year and results were included in the previous project progress report. Specific Aim 2: Characterize genetic basis of resistance and tolerance genomic in a diverse panel of Ae. tauschii accessions through genome-wide association studies (GWAS). Sequenced and assempled the whole genome of 9 Ae. tauschii accessions. These 9 genotypes were used for phenotyping but their whole-genome sequences were not available for GWAS. Organized final collection of complete accessions (n=250) with whole-genome sequencing data, generated k-mer matrix, and variant (SNP) genotyping. Evaluated population structure and geographic origin of accessions in relationship to WSMV single infections tolerance profile. Specific Aim 3: Detailed evaluations of resistance and tolerance in selected Ae. tauschii to WSMV in single and mixed infections with TriMV. Screened 31 accessions of Ae. tauschii already showed tolerance to WSMV single infections for co-infections with TriMV. Screening was performed based on the symptom severity and both WSMV and TriMV titers measured by real-time PCR at 3 time points: early infection, mid infection and late infection. Identified 15 accessions showed tolerance respond to both viruses either as mild symptoms with low titer of viruses or mild symptoms with high titer of viruses or delayed symptoms. We considered thoseAe. tauschiigenotypes that showed mild symptoms despite high titers of WSMV and TriMV as "True Tolerant".

Publications


    Progress 12/01/22 to 11/30/23

    Outputs
    Target Audience:Some of the generated data were presented at the APS annual meeting with Plant Pathologists as the major target audiences. We have also presented/shared our generated data with researchers (Plant breeders and Genetisists) and industry partners at WGRC-IUCRC montly meetings. Changes/Problems:No major changes What opportunities for training and professional development has the project provided?The project has provided training opportunities for a postodc, a grdaute student and undergrads. A graduate student was recently recruited to conduct the experiments related to objective 3. Addiotionally,Nouri lab at KSU was the host of an undergraduate student for the Research and Extension Experience for Undergraduates (REEU) program for 8 weeks in summer 2023. The undergraduate was trained and performed some of the experiments related to objective 1 under the supervision of the PI Nouri and the assigned Postdoc. How have the results been disseminated to communities of interest?Presenting at meetings such as APS as well ascollaborative meetings between the Wheat Genomic Research Center scientists and wheat industry partners (WGRC-IUCRC). What do you plan to do during the next reporting period to accomplish the goals?Aim 1. Identify novel sources of genetic resistance and tolerance to WSMV inAe. taushii. We have completed this aim. Aim 2. Characterize genetic basis of resistance and tolerance genomic in a diverse panel ofAe. tauschiiaccessions through genome-wide association studies (GWAS). Curate final collection of complete accessions (n=250) with whole-genome sequencing data, generated k-mer matrix, and variant (SNP) genotyping. GWAS using both SNP variants and k-mers from preliminary completed set of WSMV screening (n~150 accessions) Evaluate population structure and geographic origin of accessions in relationship to WSMV resistance/tolerance profile. GWAS using both SNP variants and k-mers from preliminary completed set of mixed infection screening. Aim 3. Detailed evaluations of resistance and tolerance in selectedAe. tauschiito WSMV in single and mixed infections with TriMV. Our plan is to screen selectedAe. tauschiiaccessions from Aim 1 showed potential tolerance to WSMV single infections for the mixed-infections of WSMV+TriMV. Our criteria for the evaluation will be the same as Aim 1, symptom severity and the titers of both viruses in infected plants.

    Impacts
    What was accomplished under these goals? Specific Aim 1:Identify novel sources of genetic resistance and tolerance to WSMV inAe. taushii. We screened extra 90 accessions ofAe. tauschiifor WSMV single infections under the controlled conditions to complete screening of the total of 250 accessions which was the goal of this aim. The seeds ofAe. tauschiigenotypes received from the Wheat Genomic Research Center (WGRC) at KSU. Our methods to perform experiments were explained in details in our last year report. Based on both symptom severity and the virus titers measured by the RT-quantitative PCR (RT-qPCR), we identified 52 accessions ofAe. tauschiias potential tolerant to WSMV single infections. We had previously identified 13 tolerant genotypes. Therefore, in total 65 accessions were determined as potential tolerant. We did not identify any complete resistant genotypes among our screenedAe. tauschiigenotypes. Specific Aim 2: Characterize genetic basis of resistance and tolerance genomic in a diverse panel ofAe. tauschiiaccessions through genome-wide association studies (GWAS). The phenotypic data including symptom severity scores and WSMV titers from all screenedAe. tauschiihave been shared with the Co-PI Poland`s research group at KAUST for conducting GWAS. Specific Aim 3: Detailed evaluations of resistance and tolerance in selectedAe. tauschiito WSMV in single and mixed infections with TriMV. "Tomahawk" wheat plants have been inoculated with both WSMV and TriMV to provide the source of mixed-infections. The standard curve has been created for TriMV which will be used to calculate the copy number of the genome.

    Publications


      Progress 12/01/21 to 11/30/22

      Outputs
      Target Audience:The goal of this research project is to screen the genotypes of a wheat wild relative for identifying new sources of virus resistance/tolerance. The research communities in Plant Breeding, Plant Virology, and Plant Genetics were reached asthe target audience of this research. The wheat industry also benefited from the results of this research. Changes/Problems:None What opportunities for training and professional development has the project provided?The project has provided research opportunity for a Postdoc in PI Nouri`s lab.A second postdoc at KAUST is now working on the project for genomics and association mapping.The postdoc will gain further train in these areas. How have the results been disseminated to communities of interest?Some results were disseminated through presentations at meetings such as a collaborative meeting between the Wheat Genomic Research Center scientists and wheat industry partners. What do you plan to do during the next reporting period to accomplish the goals?Aim 1. Identify novel sources of genetic resistance and tolerance to WSMV inAe. taushii. We will continue screening moreAe. tauschiiaccessions for WSMV single infections based on both symptoms and virus titer measured by the real-time PCR. We will update our database constantly. We will also share our results with the Co-PI Poland at KAUST. The PI Nouri lab has advertised the current research to recruit an REEU student for summer 2023. Nouri`s lab will also recruit a graduate student for this project in fall 2023. Aim 2. Characterize genetic basis of resistance and tolerance genomic in a diverse panel ofAe. tauschiiaccessions through genome-wide association studies (GWAS). We have organized the SNP variants and k-mers for association mapping and will complete preliminary analysis confiming the overall analysis pipeline.With the completed datasets for symptom severity and virus copy counts, we will run the association mapping for the complete panel. Aim 3. Detailed evaluations of resistance and tolerance in selectedAe. tauschiito WSMV in single and mixed infections with TriMV. Our plan is to complete the Aim 1 first. Then, promising accessions will be selected to get screened for double infections of WSMV and TriMV.

      Impacts
      What was accomplished under these goals? Specific Aim 1:Identify novel sources of genetic resistance and tolerance to WSMV inAe. taushii. We have screened 70 accessions ofAe. tauschiiso far for WSMV infection under controlled conditions. The seeds have been received from the Wheat Genomic Research Center (WGRC) at KSU. For infecting wild wheat varieties, 100mg of WSMV infected Tomahawk (susceptible variety), which was stored at -80°C, was crushed in 1.5 ml of 0.2 M phosphate buffer pH 7 in ice cold and sterile mortar-pestle. Carborundum was added in to this sap followed by mechanical inoculation with cotton bud on two leaves (three leaf stage). For control, mixture of phosphate buffer-carborundum was used. Symptom of infection was monitored till 31 days post inoculation (dpi) and scored 1-4, where 1 was given for only yellowing, 2 for mild mosaic/streak; 3, sever mosaic/streak; and 4, stunting with sever mosaic/streak symptoms. To know the exact copy number of viral RNA in mechanically infected plants, absolute quantification of WSMV coat protein RNA was conducted from leaf samples collected at 14, 21 and 31 dpi. For that, Total RNA was isolated and converted in to cDNA followed by absolute quantification with coat protein primers using the real-time PCR machine. In our earlier preliminary screens, we found that line TA2431 is a susceptible accession based on symptom and high titer.Therefore, we used this line as positive control and compared symptoms and titer of WSMV tested genotypes with symptoms and titer of WSMV of TA2431. Ae. tauschiiaccessions which showed mild symptoms (scores 1 and 2) either with high or low titer of the virus have been identified as "Tolerant"; while the accessions that showed mild symptoms (scores 1 and 2) with high virus titer have been selected as "True Tolerant" accessions. We also consider "delayed symptoms" as the tolerance response. Based on this criteria, we have identified15Ae. tauschiigenotypesso far as "Tolerant" and "True Tolerant". Specific Aim 2: Characterize genetic basis of resistance and tolerance genomic in a diverse panel ofAe. tauschiiaccessions through genome-wide association studies (GWAS). The phenotypic data including symptom severity scores and WSMV titers from 70 screenedAe. tauschiihave been shared with the Co-PI, Dr. Jesse Poland.We have organized phenotypes for association mapping. The genotyes for association mapping have been organized for theAe. tauschiipanel.This includes SNP variants and k-mer matrix.We are initializing association mapping for both symptom severity and virus copy number. Specific Aim 3: Detailed evaluations of resistance and tolerance in selectedAe. tauschiito WSMV in single and mixed infections with TriMV. Experiments related to this Aim will be conducted after completing the first Aim.

      Publications