Genomics to boost durable leaf rust and stripe rust resistance in hard winter wheat

GENOMICS TO BOOST DURABLE LEAF RUST AND STRIPE RUST RESISTANCE IN HARD WINTER WHEAT

Sponsoring Institution

National Institute of Food and Agriculture

Project Status

ACTIVE

Funding Source

AFRI COMPETITIVE GRANT

Reporting Frequency

Annual

Accession No.

1030056

Grant No.

2023-67014-39298

Cumulative Award Amt.

$264,930.00

Proposal No.

2022-10233

Multistate No.

(N/A)

Project Start Date

May 1, 2023

Project End Date

Apr 30, 2026

Grant Year

2023

Program Code

[A1141]- Plant Health and Production and Plant Products: Plant Breeding for Agricultural Production

Recipient Organization
OKLAHOMA STATE UNIVERSITY
(N/A)
STILLWATER,OK 74078

Performing Department
(N/A)

Non Technical Summary
Leaf rust and stripe rust are the most widespread and devastating wheat diseases globally. The continuous discovery and deployment of rust resistance genes are needed to achieve durable rust resistance. We will explore leaf rust and stripe resistance in US hard red winter wheat. We selected 767 breeding lines and cultivars from recent Regional Germplasm Observation Nursery, Northern Regional Performance Nursery, and Southern Regional Performance Nursery. This germplasm will be evaluated at the seedling stage and adult-plant stage in multiple locations in the US. This germplasm was genotyped using Multiplex Restriction Amplicon Sequencing and 63,888 single nucleotide polymorphism markers were generated. Both phenotypic and genotypic data will be used for genome-wide association studies (GWAS). The GWAS will identify leaf rust and stripe rust resistance genes in current US winter wheat. User-friendly markers associated with the discovered genes will also be developed for use in marker-assisted selection. Linkage mapping is another approach that will be used to identify and characterize leaf rust and stripe rust resistance genes in the hard winter wheat cultivar 'Big Country'. Based on preliminary data, Big Country has novel rust-resistance genes that should be explored. We are developing a double haploid (DH) population from a cross between the rust-resistant cultivar, Big Country, and the rust susceptible cultivar 'Jagalene'. The DH lines will be evaluated for leaf rust and stripe rust at both seedling and adult-plant stages and genotyped using Genotyping-by-Sequencing. Mapping will then be performed to identify rust resistance genes in Big Country.

Animal Health Component

40%

Research Effort Categories

Basic

40%

Applied

40%

Developmental

20%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
216	1540	1080	40%
216	1544	1081	20%
212	1549	1160	40%

Knowledge Area
212 - Pathogens and Nematodes Affecting Plants; 216 - Integrated Pest Management Systems;

Subject Of Investigation
1549 - Wheat, general/other; 1540 - Hard red winter wheat; 1544 - Hard white wheat;

Field Of Science
1160 - Pathology; 1081 - Breeding; 1080 - Genetics;

Keywords

Goals / Objectives
Goals:This project focuses on plant breeding discovery and research and is crucial to wheat cultivar development in the US and globally to counter the most damaging wheat diseases. This project is expected to provide sources of leaf rust and stripe rust in contemporary US hard winter wheat, which will benefit plant breeders and growers. We will also dissect the genetic basis of rust resistance and develop molecular markers for use in marker-assisted selection (MAS) to facilitate the transfer of resistance sources to future cultivars. Since the used genotypes in this project are from both public and private breeding programs, the knowledge gained will be directly translated into wheat genetic improvement.Objectives:Identify sources of leaf rust and stripe rust resistance in contemporary US hard winter wheatMap leaf/stripe rust resistance genes in US hard winter wheat using association mapping and develop markers for use in marker-assisted selectionMap and characterize leaf rust and stripe rust resistance genes in the hard winter wheat cultivar Big Country

Project Methods
Objective 1. Identify sources of leaf rust and stripe rust resistance in contemporary US hard winter wheat. Hard winter wheat germplasm: Throughout multiple years, the USDA-ARS coordinated evaluations of Regional Germplasm Observation Nursery (RGON), Northern Regional Performance Nursery (NRPN), and Southern Regional Performance Nursery (SRPN) in several locations in the US to multiple diseases and agronomic traits. In this project, we will use breeding lines and cultivars from 2021 and 2022 RGON, SRPN, and NRPN. The breeding lines and cultivars in this germplasm were selected from multiple public and private hard winter wheat breeding programs across the US.Leaf rust and stripe rust data: The RPN (SRPN & NRPN) accessions were phenotyped at the seedling stage using current US Pt races and Pst races. The RPN accessions were also evaluated at an adult-plant stage in field plots in Texas (for leaf rust) and Washington (for stripe rust). Based on available phenotypic data, there were continuous disease distributions appropriate for further analysis. The RPN accessions in 2021 were different from the RPN accessions in 2022, however, there were some common genotypes between years that will be used to calculate adjusted means for each genotype. To reduce environmental effects, rust evaluations of all 2021 and 2022 RPN accessions will be performed this season (the year 2023) in Oklahoma (leaf rust and stripe rust), Kansas (leaf rust and stripe rust), and Washington (stripe rust). The field trials were planted in the fall of 2022 in an augmented design. The individual plot consisted of a single 1-m-long row with ~ 20 cm between adjacent rows. The plots were surrounded with susceptible checks. Artificial inoculations of the spreader rows (susceptible checks) will be performed in these locations using local races (to each location) to increase disease pressure. Details about inoculations and disease ratings will be similar to that described by Aoun et al. (2016) and Liu et al. (2020).The RGON accessions were evaluated for leaf rust only at adult-plant stage in Castroville, TX. Adult-plant evaluations of the 2021 RGON were also performed for stripe rust in Kansas, however, in 2022, stripe rust pressure was low in Kansas due to extreme heat conditions that suppressed the disease establishment. Thus, the 2022 RGON could not be evaluated for stripe rust. Seedling evaluations of the RGON will be done using current US Pt (MNPSD, MPPSD, MBDSD, TNBJS, and MJBJG) and Pst races (PSTv-4, PSTv-14, PSTv-37, PSTv-40, and PSTv-51). Adult-plant stage evaluations of the RGON accessions will be performed in field locations in 2023 as described for the RPN. As many APR genes are temperature sensitive, field evaluations in different geographical locations will provide valuable information on the efficacy of the APR sources under different climate conditions.Objective 2. Map leaf/stripe rust resistance genes in US hard winter wheat using association mapping (GWAS) and develop markers for use in marker-assisted selection.High-throughput genotyping of the GWAS Panel: A total of 767 wheat accessions from 2021 and 2022 RGON, SRPN and NRPN were genotyped using Multiplex Restriction Amplicon Sequencing (MRASeq) (Bernardo et al., 2019) by the USDA Central Small Grain Genotyping Lab in Manhattan, KS. The genotyping generated 63,888 SNPs for this germplasm. Furthermore, this germplasm was genotyped using moderately diagnostic markers for 13 characterized Lr genes and six Yr genes that are known to be present in the US hard winter wheat.GWAS and KASP marker development: The generated phenotypic data in Obj. 1 will be used for GWAS in combination with SNP data generated from MRASeq. Genome Association and Prediction Integrated Tool (Lipka et al., 2012) in R software will be used for the association analysis to identify the genomic regions underlying leaf rust and stripe rust resistance in this US hard winter wheat germplasm. Since diagnostic markers for some known Lr and Yr genes will be used in the GWAS, novel rust resistance genes/quantitative trait loci (QTL) can be distinguished. We will also use the physical locations of significant markers in this project and previously mapped rust resistance genes/QTL to determine the novelty of the discovered genes. SNPs significantly linked with rust response will be converted into competitive allele-specific PCR (KASP) for use in marker-assisted breeding. KASP primers will be designed based on the wheat reference genome sequence. KASP marker development will be similar to that described by Aoun et al. (2017). KASP markers will be developed for novel rust resistance genes and/or for known genes with not yet available diagnostic markers.Objective 3. Map and characterize leaf rust and stripe rust resistance genes in the hard winter wheat cultivar Big Country. Big Country is a hard white winter wheat cultivar from the Oklahoma State University (OSU) released in 2020. Based on multiple-year evaluations in Oklahoma and other states (Big Country was included in the 2018 SRPN), Big Country is resistant to multiple diseases including leaf rust, stripe rust, powdery mildew, Septoria leaf blotch, and soilbornewheatmosaic/wheatspindlestreakmosaic virus. For this project, we will focus only on leaf rust and stripe rust resistance in Big Country. Based on molecular markers and phenotypic data of multiple races, Big Country carries unknown leaf rust and stripe rust resistance gene (s). In contrast to Big Country, 'Jagalene' is susceptible to both leaf rust and stripe rust and was chosen as the susceptible parent to develop a double haploid (DH) population, Big Country/Jagalene.The parents (Big Country and Jagalene) and the 160 DH lines will be tested at the seedling stage in the greenhouse using US Pt and Pst races as described in Obj.1. In addition, the DH lines will be tested in multiple field locations in 2024 in Oklahoma (for leaf rust and stripe rust), Kansas (leaf rust and stripe rust), and Washington (stripe rust). The DNA samples of the DH population will be genotyped at the USDA Central Small Grain Genotyping Lab in Manhattan, KS. Using the Genotyping by Sequencing (GBS) protocol (Poland et al., 2012). SNP genotypes will be called using the TASSEL GBS software version 5 (Glaubitz et al., 2014) and the wheat assembly T. aestivum Chinese Spring RefSeq v2.1 to assign SNP markers to physical positions and chromosomes. After marker filtering for different criteria (e.g., missing data, heterozygosity, redundant markers), linkage groups will be generated using R/qtl package (Broman et al., 2003). QTL analysis will then be performed using different methods such as composite interval mapping in the R/qtl package. To increase mapping resolution, a large F2:3 families Jagalene/Big Country (~ 2,000 individuals) will be developed during this project for future fine mapping/cloning of rust-resistance genes and other disease-resistance genes.Project outcomes: We expect to identify sources of rust resistance in contemporary US hard winter wheat that will be useful to manage the rapidly evolving races of Pt and Pst in the US and globally. This work will provide molecular markers that can be used in marker-assisted selection to breed for durable rust resistance. Novel rust resistance genes could be identified and results from this project will be shared in scientific publications and conferences and made available on databases. This project will also be a training opportunity for students. Rust-resistant SRPN breeding lines, especially those developed by the Wheat Improvement Team at Oklahoma State University, will be included in demonstration plots in Oklahoma in 2024 to pass knowledge to farmers and extension educators. This information will also be shared in extension factsheets, presentations, and SUNUP videos. This should help inform variety selection to reduce yield losses caused by rust diseases.

Progress 05/01/24 to 04/30/25

Outputs
Target Audience:Researchers: Graduate students, collaborators, and the PI published two articles in the journals of Theoretical and Applied Genetics and The Plant Genome to share knowledge gained from this project with peers. Graduate students and the PI also shared research outcomes from this project with other researchers at national conferences/meetings including ASA, CSSA, SSSA International Annual Meeting, Plant and Animal Genome Conference, North American Cereal Rust Workers Workshop, NCERA 184: Diseases of Small Grains Annual Meeting, Wheat Initiative Webinar: The Control of Wheat Pathogens Expert Working Group. Graduate students: Two graduate students (1 MS and 1 PhD students) worked on this project. Students were trained in leaf rust and stripe rust evaluations in the greenhouse and in the field, GWAS, rust pathology, data analysis, KASP marker development, and manuscript writing. A Masters student graduated in the Summer of 2024. Undergraduate students: An undergraduate student from Universidad de las Fuerzas Armadas -ESPE in Ecuador was hosted by our lab and trained on DNA extraction and development of KASP markers. Wheat breeders: We communicated our project results with hard winter wheat breeders in the US Great Plains to inform them about identified rust resistance sources to be deployed in breeding to enhance rust resistance. Wheat growers and extension professionals: The project results were presented to growers, county educators, and students at extension meetings in Oklahoma, Texas, and South Dakota to educate them about rust diseases and management. These presentations were provided to ~ 272 attendees at the Central Oklahoma Wheat Conference, Oklahoma Wheat Growers Association Annual Meeting, Pre-season Wheat Meeting in Oklahoma, South Dakota State University Extension Crop Hour, Red River Crops Conference in Texas. E-Pest alerts were also provided to growers to inform them about leaf rust and stripe rust situation in Oklahoma to help them take management decisions. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Training activities: The PI trained two graduate students on GWAS, data analysis, KASP marker development, and manuscript writing. A Masters student successfully defended her thesis in the Summer of 2024. An undergraduate student from Universidad de las Fuerzas Armadas -ESPE in Ecuador was hosted by our lab and trained on DNA extraction and development of KASP markers. Professional development: Graduate students and the PI participated in national conferences and workshops including ASA, CSSA, SSSA International Annual Meeting, Plant and Animal Genome Conference, and North American Cereal Rust Workers Workshop. How have the results been disseminated to communities of interest?The results of this project were disseminated in two articles in peer-reviewed journals. We also provided seven poster and oral presentations at conferences/meetings. Outreach activities In 2024, there was a severe stripe rust epidemic in the Great Plains including Oklahoma. The PI provided six wheat disease updates (e-pest alerts) to wheat growers, consultants, county educators, and researchers. The updates provided information on stripe rust and leaf rust situations in Oklahoma to help growers make disease management decisions. The PI also provided five presentations and trainings in Oklahoma, Texas, and South Dakota to educate growers and extension professionals about wheat diseases and management. These presentations discussed some of this project results and updated variety ratings against leaf rust and stripe rust to ~ 272 attendees at the Central Oklahoma Wheat Conference, Oklahoma Wheat Growers Association Annual Meeting, Pre-season Wheat Meeting in Oklahoma, South Dakota State University Extension Crop Hour, Red River Crops Conference in Texas. What do you plan to do during the next reporting period to accomplish the goals?In 2025-2026, we will evaluate the parents and the 224 doubled haploid lines of the biparental cross Big Country/Jagalene at the seedling stage against leaf rust pathogen races. In Fall 2025, this population will be planted in leaf rust nurseries in Oklahoma and Texas and stripe rust nurseries in Oklahoma, Kansas, and Washington for leaf rust/stripe rust evaluations at the adult plant stage. The phenotypic and genotypic data will be used for QTL analysis to map leaf rust and stripe rust resistance genes in Big Country. SNP markers flanking the resistance genes will be converted into KASP markers for use in marker-assisted selection. The development of KASP markers for GWAS-significant SNPs (objective 2) will continue in 2025-2026. For objective 3, the greenhouse seed increase of the doubled haploid lines of the biparental cross Big Country/Jagalene produced a low quantity of seed not enough for field and greenhouse testing that was initially planned in 2024-2025. A second round of seed increase of the doubled haploid population is ongoing in Yuma, AZ and a sufficient quantity of seed should be available by Summer 2025.

Impacts
What was accomplished under these goals? 1. Issue/problem that this project addresses:Wheat diseases dramatically reduce yield and grain quality. Stripe rust and leaf rust are the most damaging wheat diseases in the U.S. Central and Southern Great Plains. Growing rust resistant varieties is the most effective management strategy. 2. Who or what will be most immediately helped by your work, and how? Researchers: Knowledge gained from this project was shared in 2 peer-reviewed journal articles. Research outcomes were also presented at national conferences/meetings. Graduate students: This project provided training opportunities for two graduate students (1 MS and 1 PhD students). A Masters student graduated in the Summer of 2024. Undergraduate students: This project provided a training opportunity for an undergraduate student. Wheat breeders: We communicated our project results with wheat breeders in the US Great Plains. Wheat growers and extension professionals: Results from this project were presented to growers and county educators at extension meetings in Oklahoma, Texas, and South Dakota. 3. Major activities completed / experiments conducted; Data collected; and Summary statistics and discussion of results. Objective 1. Identify sources of leaf rust and stripe rust resistance in contemporary US hard winter wheat To identify sources of leaf rust resistance in contemporary US hard winter wheat (HWW), a collection of 732 elite HWW genotypes originating from multiple breeding programs across 13 states were evaluated against USleaf rust pathogen (Puccinia triticina) races at the seedling stage and at the adult plant stage in leaf rust nurseries in Oklahoma, Texas, and Kansas. Depending on the race used, 43%-60% of the genotypes showed susceptible reactions at the seedling stage. We observed higher frequencies of genotypes with leaf rust resistance at the adult plant stage than at the seedling stage, suggesting the presence of adult-plant resistance (APR) in this germplasm. We identified eight elite breeding lines with broad spectrum of all-stage resistance (ASR) to 13 U.S. P. triticina races and in multiple field environments.These resistance sources are useful to enhance leaf rust resistance in HWW breeding programs. To identify sources of stripe rust resistance in US contemporary hard winter wheat, a panel of 459 Great Plains wheat genotypes (a subset of the collection used for leaf rust) was evaluated at the seedling stage against five USstripe rust pathogen (Puccinia striiformisf. sp.tritici[Pst]) races and at the adult plant stage in field environments in Oklahoma, Kansas, and Washington. The results showed that 7-14% of the genotypes were resistant toPstraces at the seedling stage, whereas 32-78% of genotypes were resistant at the adult plant stage across field environments, indicating the presence of APR. Sixteen genotypes displayed a broad spectrum of resistance to all fivePstraces and across all field environments. Broad-spectrum ASR in most of these 16 genotypes (except CO19D304R) was associated with the presence ofthe stripe rust resistance (Yr) genes Yr5,Yr15, andQYr.tamu-2B. Based on molecular markers, the resistance in CO19D304R was not associated with any of the knownYrgenes in HWW. As stripe rust ASR israrein Great Plains hard winter wheat, the gene (s) in CO19D304R will help diversify ASR sources. Since the resistance gene (s) originated from an elite breeding line, its deployment in breeding programs will be easier. Objective 2. Map leaf/stripe rust resistance genes in US hard winter wheat usingassociation mapping and develop markers for use in marker-assisted selection The wheat genotypes (same panel used in objective 1) were genotyped using multiplex restriction amplicon sequencing (MRASeq) and molecular markers linked to the known ASR leaf rust resistance (Lr) genes Lr18,Lr19,Lr21,Lr24,Lr37, andLr42and APR genes Lr34, Lr46, Lr67, Lr68, Lr77, and Lr78. Single nucleotide polymorphism (SNP) markers derived from MRA-Seq, markers linked to the knownLrgenes, and the phenotypic leaf rust data (from objective 1) were used for genome-wide association study (GWAS) to identify markers associated with leaf rust response. Gene postulation based on leaf rust reactions, gene-linked markers, and GWAS confirmed the presence of the characterized ASR genes Lr1, Lr2a, Lr10, Lr14a, Lr16, Lr18, Lr19, Lr21, Lr24, Lr26, Lr37, Lr39, and Lr42 and the APR slow rusting genes Lr34, Lr46, Lr68, Lr77, and Lr78. Furthermore, we identified 59 SNPs associated with leaf rust response, of which 20 were likely associated with novel resistance loci and can be used to diversify and accelerate the deployment of resistance sources. Results from this project were detailed in Lakkakula et al. 2025. Identification of leaf rust resistance loci in hard winter wheat using genome?wide association mapping.The Plant Genome,18(1), p.e20546. Nine SNPs (S1B_621726983, S2B_112250510, S3D_150055006, S4A_90386472, S5A_388167949, S5D_282820006, S6A_611716646, S7A_589056553, and S7A_599211104) identified by the GWAS were prioritized for conversion into kompetitive allele specific PCR (KASP) markers for potential use in marker-assisted breeding. To date, we successfully developed two KASP markers S4A_90386472 and S5D_282820006. For stripe rust, the panel of 459 hard winter wheat was genotyped with MRASeq and molecular markers linked to the known Yr genesYr5,Yr15,Yr17,Yr18,Yr29,Yr36,Yr40,Yr46, andQYr.tamu-2B. GWAS was performed using genotypic and phenotypic data (from objective 1), which identified 110 SNPs and the markers linked toYr15andYr17to be significantly associated with stripe rust response. In addition,Yr5,Yr15,Yr17,Yr18,Yr29, andQYr.tamu-2Bwere detected by their linked markers in this panel. This study identified 40 novel loci associated with stripe rust resistance. Comparative mapping of significant SNPs identified in this study with previously characterized Yr genes/loci indicated that Yr5, Yr10, Yr15, Yr26, Yr53, Yr62, Yr68, Yr84, and Yr85 are likely present in this HWW panel. Results from this project are detailed in Sharma et al. 2025. Genome-wide association mapping for the identification of stripe rust resistance loci in US hard winter wheat.Theoretical and Applied Genetics,138(4), p.67. KASP markers development for significant GWAS SNPs associated with stripe rust response across field environments is ongoing. To date, we have successfully developed two KASP markers for S4A_352174561 and S4B_571886653. Objective 3. Map and characterize leaf rust and stripe rust resistance genes in the hard winter wheat cultivar Big Country In 2024, we completed the development of 224 doubled haploid (DH) lines from the biparental cross Big Country/Jagalene. Genotyping by sequencing of the parents of the DH lines was completed at the USDA-ARS genotyping lab in Manhattan, KS in 2025. Greenhouse and field evaluations of this population will be performed against leaf rust and stripe rust in 2025-2026. The genotypic data and the phenotypic data will then be used to map leaf rust and stripe rust resistance genes/QTL in the wheat cultivar Big Country. SNP markers flanking the discovered rust resistance genes will be converted into KASP markers for use in MAS. 4. Key outcomes or other accomplishments realized This project focuses on plant breeding discovery and research and is crucial to wheat cultivar development in the US and globally to counter the most damaging wheat diseases. This project provided sources of leaf rust and stripe rust in contemporary US hard winter wheat, which will benefit plant breeders and growers. We identified known and novel rust resistance loci in hard winter wheat. KASP markers under development will facilitate marker-assisted selection. This project provided training opportunities for two graduate students and an undergraduate student. ?

Publications

Type: Peer Reviewed Journal Articles Status: Published Year Published: 2025 Citation: Lakkakula, I.P., Kolmer, J.A., Sharma, R., St. Amand, P., Bernardo, A., Bai, G., Ibrahim, A., Bowden, R.L., Carver, B.F., Boehm Jr, J.D. and Aoun, M., 2025. Identification of leaf rust resistance loci in hard winter wheat using genome?wide association mapping. The Plant Genome, 18(1), p.e20546.
Type: Peer Reviewed Journal Articles Status: Published Year Published: 2025 Citation: Sharma, R., Wang, M., Chen, X., Lakkakula, I.P., Amand, P.S., Bernardo, A., Bai, G., Bowden, R.L., Carver, B.F., Boehm Jr, J.D. and Aoun, M., 2025. Genome-wide association mapping for the identification of stripe rust resistance loci in US hard winter wheat. Theoretical and Applied Genetics, 138(4), p.67.
Type: Theses/Dissertations Status: Published Year Published: 2024 Citation: Lakkakula, I.P., 2024. Unraveling the Genetic Basis of Leaf Rust Resistance in Hard Winter Wheat (Master's thesis, Oklahoma State University).
Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: 13. Sharma, R. and Aoun, M. 2024. Development and validation of KASP markers based on GWAS for enhancing stripe rust resistance in hard winter wheat. ASA, CSSA, SSSA International Annual Meeting. San Antonio, Texas. November 1013, 2024 (Oral and Poster presentations).
Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Aoun, M. 2025. Stripe rust resistance in hard winter wheat. North American Cereal Rust Workshop 2025, Fargo, ND. March 12-13, 2025 (Oral presentation).
Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Aoun, M. 2025. Stripe rust resistance in hard winter wheat. Wheat Initiative Webinar: The Control of Wheat Pathogens Expert Working Group (Oral presentation: https://www.wheatinitiative.org/ewg-pathogens). March 6, 2025
Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Aoun, M., Sharma, R., Wang, M., Chen, X., Lakkakula, I.P., Amand, St., P., Bernardo, A., Bai, G. Bowden, R., Carver, B.F., Boehm Jr., J. D. 2025. Genome-wide association mapping and KASP marker development for stripe rust resistance loci in Great Plains hard winter wheat. Plant and Animal Genome Conference. Plant Disease Resistance Workshop. San Diego, CA. January 1015, 2025 (Oral and Poster presentation).
Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Aoun, M. 2025. Overview of Oklahoma's Disease Landscape in 2024. NCERA-184: Diseases of Small Grains Annual Meeting. Pensacola, FL. Feb 24, 2025

Progress 05/01/23 to 04/30/24

Outputs
Target Audience:Researchers: Graduate students and PI presented research outcomes from this project to other researchers at different national conferences/meetings including Norman E. Borlaug International Dialogue at Des Moines, Iowa; Plant Health 2023 at Denver, Colorado; NCERA 184: Diseases of Small Grains Annual Meeting at Pensacola, FL, Plant and Animal Genome Conference at San Diego, CA; North American Cereal Rust Workers Workshop at Saint Paul, MN. Graduate students: Two graduate students (1 MS and 1 PhD students) are working on this project. Students were trained onleaf rust and stripe rust evaluations in the greenhouse and in the field, GWAS, rust pathology, data analysis, and manuscript writing. The PI provided lectures related to breeding for rust resistance in wheatat different institutions including Oklahoma State University, Cornell University, and Assiut University (Egypt). Undergraduate students: Two undergraduate students from Oklahoma State University and Universidad de las Fuerzas Armadas -ESPE in Ecuador were involved in this project. They were trained on rust evaluations and DNA extraction. Visiting scholar: The PI lab hosted a visiting scholar supported by Egypt Cultural and Educational Bureau US-AID. The visiting scholar was trained on different aspects ofstripe rust pathology. Wheat breeders: We communicated our project results (identified rust resistance sources) with wheat breeders in the US Great Plains. Wheat growers and extension professionals: Results from this project were presented to growers and county educators at extension meetings in Oklahoma including Oklahoma State University Winter Crops School, Oklahoma Wheat Commission, and Oklahoma Genetics Inc. The PI also talked about research at a stripe rust nursery during wheat field days at Chickasha. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Graduate students: Two graduate students (1 MS and 1 PhD students) are working on this project. Students were trained in leaf rust and stripe rust evaluations in the greenhouse and in the field, GWAS, rust pathology, data analysis, and manuscript writing. The PI provided lectures related to breeding for rust resistance at different institutions including Oklahoma State University, Cornell University, and Assiut University in Egypt. ? Undergraduate students: This project provided training opportunities for two undergraduate students from Oklahoma State University and Universidad de las Fuerzas Armadas -ESPE -Ecuador. Visiting scholar: The PI lab hosted a visiting scholar supported by Egypt Cultural and Educational Bureau US-AID. The visiting scholar worked on stripe rust seedling evaluations in the greenhouse and stripe rust field evaluations in Oklahoma (Chickasha) and Washington State (Pullman) Growers and countyeducators: extension presentation and CEU How have the results been disseminated to communities of interest?In 2023, we provided six wheat disease updates(e-pest alerts) to wheat growers, consultants, county educators and researchers, with secondary distribution by the USDA-ARS Cereal Disease Lab (Minnesota) and the OSU Plant and Soil Sciences Department. These disease updates were also presented in eight wheat field days to ~367 wheat growers and county educators and disseminated through mass media, newspaper articles, and social media. We published a current report (CR-2143) that summarized wheat disease overview in Oklahoma in 2023. We contributed to handbooks summarizing data from Oklahoma small grains variety performance tests. We provided wheat disease loss estimates in Oklahoma and rust samples to our national collaborators who used our data in fact sheets/newsletters. Further, we gave eight presentations/trainings in extension meetings to educate Oklahoma growers and extension professionals about wheat disease (including rusts) diagnosis and integrated disease management. We also conducted consultations for Oklahoma wheat producers about stripe rust management in 2024. What do you plan to do during the next reporting period to accomplish the goals?1- Publish two manuscripts related to GWAS for leaf rust and stripe rust resistance in hard winter wheat in 2024 (Objective 1 &2). 2- Develop KASP markers associated with leaf rust and stripe rust resistance 2- Perform genotyping, leaf rust/stripe rust phenotyping, and QTL analysis for the biparental population Big County/Jagalene to map leaf rust and stripe rust resistance genes in Big Country.

Impacts
What was accomplished under these goals? Issue/problem that this project addresses Global food production needs to be dramatically improved to feed a growing human population that is expected to reach 9.7 billion people in 2050. Wheat is a major food and feed crop throughout the world and ranks third among U.S. field crops in planted acreage, production, and gross farm receipts, behind corn and soybeans. However, wheat diseases reduce yield and grain quality. Over the last five years (2018-2022), wheat diseases caused annual yield losses in the US estimated at 675 million U.S. dollars on average, of which 153 million dollars are due to leaf rust and stripe rust damages. Therefore, continuous efforts to manage rust diseases are critical. Who or what will be most immediately helped by your work, and how? Researchers: Graduate students and PI presented research outcomes from this project to other researchers at different national conferences/meetings. Graduate students: This project provided training opportunities for two graduate students (1 MS and 1 PhD students). Undergraduate students: This project provided training opportunities for two undergraduate students from Oklahoma State University and Universidad de las Fuerzas Armadas -ESPE -Ecuador. Visiting scholar: The PI lab hosted a visiting scholar supported by Egypt Cultural and Educational Bureau US-AID. The visiting scholar worked on stripe rust seedling evaluations in the greenhouse and stripe rust field evaluations in Oklahoma (Chickasha) and Washington State (Pullman). Wheat breeders: We communicated our project results (identified rust resistance sources) with wheat breeders in the US Great Plains. Wheat growers and extension professionals: Results from this project were presented to growers and county educators at extension meetings in Oklahoma. Major activities completed /experiments conducted; Data collected; and Summary statistics and discussion of results. Objective 1. Identify sources of leaf rust and stripe rust resistance in contemporary US hard winter wheat In 2022-2023, we initiated a multi-state project in collaboration with the USDA-ARS to identify rust resistance genes in current U.S. Great Plains hard winter wheat (HWW) varieties originated from multiple breeding programs across 13 states. We selected 459 breeding lines and varieties from the 2021 and 2022 Northern Regional Performance Nursery (NRPN), Southern Regional Performance Nursery (SRPN), and the Regional Germplasm Observation Nursery (RGON). In 2023, this germplasm was evaluated for reactions to leaf rust and stripe rust at the adult plant stage in the field in Oklahoma (Stillwater and Chickasha), Kansas (Rossville), and Washington (Pullman and Mt. Vernon). We also conducted seedling tests in the greenhouse using five U.S. leaf rust pathogen races (i.e., MNPSD, MBDSD, TNBJS, MPPSD, and MJBJG) and five U.S. stripe rust pathogen races (PSTv-4, PSTv-14, PSTv-37, PSTv-40, and PSTv-52). Sources of seedling resistance (also known as all-stage resistance (ASR) or race-specific resistance) and APR (also known as non-race specific resistance) were identified in this germplasm to both leaf rust and stripe rust. At the seedling stage, 39- 59% of the lines were moderately to highly resistant to leaf rust (depending on the race), whereas 66% of the lines were highly resistant to leaf rust (coefficient of infection ≤ 20%) at the adult plant stage in Stillwater. However, leaf rust pressure was not high in 2023 in Oklahoma due to drought. Therefore, additional leaf rust testing was performed in a leaf rust nursery at Stillwater, OK in Spring 2024. At the seedling stage, low frequencies of moderately to highly resistant lines to stripe rust were identified (4-7% depending on the race). Stripe rust resistance in Great Plains HWW was primarily due to adult plant resistance (32-78% of lines were moderately to highly resistant, depending on the location). We identified a total of 11 breeding lines from the 2022 RGON that were highly resistant to stripe rust across all tested races at the seedling stage and across different locations (Oklahoma, Kansas, and Washington) at the adult plant stage.We also identified 140 lines that are sources of adult plant resistance to stripe rust. Objective 2. Map leaf/stripe rust resistance genes in US hard winter wheat using association mapping and develop markers for use in marker-assisted selection This germplasm was genotyped by USDA-ARS using moderately diagnostic molecular markers for known leaf rust (Lr) and stripe rust (Yr) resistance genes (i.e., Lr18, Lr19, Lr21, Lr24, Lr34/Yr18, Lr37/Yr17, Lr42, Lr46/Yr29, Lr67/Yr46, Lr68, Lr77, Lr78, Yr5, Yr15, Yr36, and QYr.tamu-2B). The studied germplasm was found to carry Lr21, Lr24, Lr34/Yr18, Lr37/Yr17, Lr46/Yr29, Lr68, Lr77, Lr78, Yr5, Yr15, and QYr.tamu-2B. This germplasm was also genotyped at USDA-ARS using a high-density single nucleotide polymorphism (SNP) marker platform (multiplex restriction amplicon sequencing), and 9,858 SNP markers distributed across the wheat genome were generated. Genome-wide association studies (GWAS) were performed using phenotypic rust data and genotypic data. A total of 26 SNP markers were significantly associated with leaf rust responses at the seedling stage. The SNP marker S3D_150055006 on chromosome 3D at 150 Mb (million base pair) was associated with leaf rust responses to all five races. The SNP marker S6A_611716646 on chromosome 6A at 612 Mb was associated with leaf rust response to three races (i.e., MBDSD, MJBJG, and TNBJS). A total of 12 SNP markers were associated with leaf rust response at the adult plant stage in Stillwater. The SNP marker S3A_568983726 on chromosome 3A at 569 Mb was the most significant marker associated with leaf rust response at the adult plant stage. For stripe rust, GWAS identified 46 significant SNP markers associated with stripe rust response at the seedling stage. Four of these markers (S1B_141655194, S2B_458606839, S2B_598744752, and S2B_706506028) were associated with seedling response to at least three of the five tested stripe rust pathogen races. A total of 16 significant markers were associated with infection types and disease severity at the adult plant stage across field environments. Six of these markers, S2A_480477702, S2A_638603325, S2B_48062121, S3A_261175449, S4B_571886653, and S7D_518590833 had relatively larger effects across environments and will be prioritized for the development of KASP markers. A few of the associated SNP markers with stripe rust response were in proximity to known Yr genes, including Yr5, Yr10, Yr26, Yr53, Yr62, Yr68, Yr84, and Yr85. Most of the genomic regions that are associated with leaf rust and stripe rust responses in this study were not previously known. Objective 3. Map and characterize leaf rust and stripe rust resistance genes in the hard winter wheat cultivar Big Country The OSU variety Big Country is resistant to leaf rust and stripe. Based on prior phenotypic and molecular markers linked to known leaf rust/stripe rust resistance genes, Big Country carries unknown rust resistance genes. We developed 160 doubled haploid lines from the cross Big Country/Jagalene. Seed increase of the 160 DHs is ongoing for leaf rust and stripe rust evaluations in Oklahoma, Kansas, and Washington in 2024-2025. Big Country is resistant at the seedling stage to multiple P. triticina races; thus, leaf rust seedling evaluations will also be performed for the DH population Big Country/Jagalene in 2024-25. The parents and the double haploid population will be genotyped using GBS at the USDA-ARS genotyping lab in Manhattan, KS in Summer/fall 2024. QTL analysis will then be performed using the phenotypic rust data and SNP data. Key outcomes? We identified seedling and adult plant resistance lines to leaf rust and stripe rust in different environments and to different races. Using GWAS, we identified SNP markers associated with leaf rust and stripe rust resistance.

Publications

Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Lakkakula, I.P., Kolmer, J.A., St. Amand, P., Bernardo, A., Bai, G., Sharma, R., Bergbower, F., Aoun, M. 2023. Genome-wide association mapping of seedling and adult-plant leaf rust resistance in hard winter wheat. Plant Health 2023. Denver, Colorado, August 12-16, 2023 (Poster)
Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Sharma, R., Wang, M., Chen, X., St. Amand, P., Bernardo, A., Bai, G., Aoun, M. 2023. Association mapping for the identification of stripe rust resistance loci in hard winter wheat. Plant Health 2023. Denver, Colorado, August 12-16, 2023 (Poster).
Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Aoun, M., Sharma, R., Lakkakula, I.P., Wang, M., Chen, X., Bowden, R., Kolmer, J.A., St. Amand, P., Bernardo, A., Bai, G. 2024. Genomics to boost durable leaf rust and stripe rust resistance in the U.S. Great Plains wheat. Plant and Animal Genome Conference. San Diego, CA. January 12-17, 2024. International Wheat Genetics Sequencing Consortium - From Structural to Functional Wheat Genomics" Workshop (Oral + Poster presentation).
Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Sharma, R., Wang, M., Chen, X., Bowden, R., St. Amand, P., Bernardo, A., Bai, G., Lakkakula, I.P., Aoun, M. 2024. Identification of stripe rust resistance loci in Great Plains hard winter wheat. Plant and Animal Genome Conference. San Diego, CA. January 12-17, 2024 (Poster).
Type: Conference Papers and Presentations Status: Submitted Year Published: 2023 Citation: Aoun, M., 2024. Reflecting on Years of Collaboration with Dr. James A. Kolmer: Leaf Rust Resistance in Wheat. 2024 North American Cereal Rust Workers Workshop. University of Minnesota, Saint Paul, MN. March 27-28, 2024 (Oral).
Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Lakkakula, I., Kolmer, J.A., St. Amand, Paul., Bernardo, A., Bai, G., Sharma, R., Aoun, M. 2023. Genome-wide association mapping for the identification of leaf rust resistance loci in hard winter wheat. The Norman E. Borlaug International Dialogue. Des Moines, Iowa, October 24-26, 2023 (Oral and Poster presentations).
Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Sharma, R., Wang, M., Chen, X., Bowden, R., St. Amand, P., Bernardo, A., Bai, G., Lakkakula, I.P. Aoun, M. 2023. Genome-wide association mapping of seedling and adult plant stripe rust resistance in hard winter wheat. The Norman E. Borlaug International Dialogue. Des Moines, Iowa, October 24-26, 2023 (Oral + Poster presentation).
Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Aoun, M., Sharma, R., Lakkakula, I.P., Wang, M., Chen, X., Bowden, R., Kolmer, J.A., St. Amand, P., Bernardo, A., Bai, G. 2024. Unravelling genomic regions conferring stripe rust resistance in hard winter wheat. NCERA-184. Management of Small Grain Diseases. March 4, 2024 (Oral).