CHARACTERIZATION AND UTILIZATION OF FUSARIUM HEAD BLIGHT RESISTANCE IN WHEAT

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1020608
• Project Start Date: Nov 20, 2019
• Project End Date: Sep 30, 2024
• Program Code: [(N/A)]- (N/A)

Recipient Organization
NORTH DAKOTA STATE UNIV
1310 BOLLEY DR
FARGO,ND 58105-5750

Performing Department
Plant Pathology

Non Technical Summary
Our overall goal is to characterize novel sources of resistance to the Fusarium head blight (FHB) diseasein spring wheat and durum wheatat both genetic and molecular levels. Therefore, the specific objectives of this proposal are to 1)To identify and map novel quantitative trait loci (QTL) for resistance to Fusarium head blight (FHB) and deoxynivalenol (DON) accumulation.2)To develop adapted wheat germplasm with improved FHB resistance.3)To isolate and functionally characterize the major quantitative trait locus (QTL) for FHB resistance in the wheat line PI 277012.we will identify and map the genes (QTL) for resistance to FHB and DON accumulation in new sources of FHB resistance, and develop DNA markers associated with the resistance QTL for use in spring wheat and durum wheat breeding programs. Through marker-assisted selection and speed breeding method, we will introgress the FHB resistance QTL from the new sources of FHB resistance into adapted spring wheat and durum wheat varieties. Furthermore, we willclone and characterize the major FHB resistance QTL in the wheat line PI 277012 using genomics and functional genomics approaches. Identification of novel QTL for FHB resistance and DNA markers associated with the disease resistance geneswill facilitate the transfer of the FHB resistance from the original sources (with undesirable traits) into adapted wheat germplasm (with high yield and quality). Isolation and functional characterization of genes for FHB resistance will help us to elucidate the molecular mechanisms involved in the resistance. The research project proposed will not only facilitate the development of new varieties with better resistance to FHB, but also provide basic information about the function and mechanism of FHB resistance genes or QTL so that we can develop novel approaches for management of the disease.

Animal Health Component

30%

Research Effort Categories

Basic

60%

Applied

30%

Developmental

10%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
212	1549	1080	100%

Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
1549 - Wheat, general/other;

Field Of Science
1080 - Genetics;

Keywords

fusarium head blight

spring wheat

durum wheat

disease resistance gene

quantitative trait loci

dna markers

Goals / Objectives
Goals:The geneal goals of this project areto characterize new sources of Fsarium head blight (FHB) resistance in spring wheat and durum wheat at genetic and molecular level and to introgress the gene loci conferring FHB resistance into adapted wheat germplasm.Objectives:1. To identify and map novel quantitative trait loci (QTL) for resistance to Fusarium head blight (FHB) and deoxynivalenol (DON) accumulation.2. To develop adapted wheat germplasm with improved FHB resistance.3. To isolate and functionally characterize the major quantitative trait locus (QTL) for FHB resistance in the wheat line PI 277012.

Project Methods
MethodsObjective 1. To identify and map novel quantitative trait loci (QTL) for resistance to Fusarium head blight (FHB) and deoxynivalenol (DON) accumulation1.1 Plant materials: Three mapping populations consisting of recombinant inbred lines have been developed from the cross between PI 185843 and Wheaton (susceptible to FHB), the cross between PI 644467 and Wheaton, and the cross between Divide and PI 254188, respectively. They will be used in the phenotyping and genotyping experiments.1.2 Greenhouse evaluation for FHB:The mapping populations (with 200 RILs for each) and their parents along with resistant and susceptible checks will be evaluated in the greenhouse for Type II resistance using the point inoculation methoddescribed by Stack (1989).Three replicates will be used for each line/season in a randomized complete block design. At 14- and 21-days post-inoculation, the blighted spikelets in each of the inoculated spikes will be counted and the total number of spikelets in each of the inoculated spikes will be used to calculate the disease severity based on the percentage of infected spikeles.1.3 Field evaluation for FHB:The mapping populations and their parents along with resistant and susceptible checks will be planted and evaluated for Type I (resistance to primary infection) and Type II (resistance to fungal spread in a spike) resistance in field nurseries at Fargo, North Dakota and other disease nurseries (Prosper, ND).Three replications (hill plots) will be planted for each line in a randomized complete block design. FHB-infected corn kernels will be spread in the field twice at the rate of 60 g per plot for inoculation. The field will be misted for 3 min (or 5 min when it is extremely dry) every 30 min for 12 h per day mostly during the night.FHB incidence (percentage of diseased spikes) and FHB severity (ratio of infected spikelets on diseased spikes) will be recorded at 21 days after anthesisusinga visual scale described by Stack and McMullen (1998). The grains will be harvested from each line and sent to a service lab for measurement of DON content.1.4 Genetic analysis and identification of QTL for FHB resistance: The analysis of variance, mean comparison and other statistic tests for FHB incidence, severity and DON content will be performed using the SAS program (SAS correlations and the Institute, Inc., 1990). For QTL mapping, a genetic linkage map will be developed for each mapping population using single nucleotide polymorphism (SNP) markers. The recombinant inbred lines (RILs) and their parents will be genotyped using the wheat 90K-SNP arrays and polymorphic SNP markers will be identified and used for genetic map construction. Linkage analysis and map construction will be performed using MapDisto (Lorieux 2012) with a minimum LOD threshold of 3.0 and the Kosambi mapping function.For QTL analysis, composite interval mapping (CIM) will be performed using the software Windows QTL Cartographer Version 2.5 (Wang et al.2012) to determine the genomic regions (i.e., marker intervals) associated with FHB resistance.A chromosome region identified to be associated with FHB resistance will also be saturated with additional markers developed from the target genome sequence and semi-thermal asymmetric reverse PCR (STARP) markers will be developed for use in MAS in the wheat breeding programs.Objective 2. To develop adapted wheat germplasm with improved FHB resistance2.1 Plant materials: For spring wheat, PI 277012, PI 185843 and PI 644141 will be used as the FHB resistance donor parents to cross with wheat cultivars that are commercially grown in North Dakota. The progenies with improved agronomic traits and a high level of FHB resistance will be selected and backcrossed to the wheat cultivars. For durum wheat, we will use PI 254188 and the FHB resistant RILs derived from the cross between Joppa and 10Ae564 (Zhao et al. 2018b) as donor parents to cross and backcross with newly released durum cultivars (ND Riveland and Grano). Marker-assisted selection will be performed in each backcross generation for both spring wheat and durum wheat.2.2. Marker-assisted selection and speed breeding.The F2plants and progenies from the backcrosses will be genotyped with DNA markers associated with the FHB resistance QTL. Those plants with the favorable alleles will be selected and used for the next run of backcrosses to the recurrent wheat parents. To speed up the process, the Speed Breeding (SB) method (Ghosh et al. 2018) will be used to shorten the time for each generation. Using SB, 5-6 generations can be achieved and thus stable improved germplasm can be developed within one and half to two years.2.3 Greenhouse and field evaluation:The materials selected by marker genotypes and SB will be evaluated in greenhouse for Type II resistance using the point inoculation method asdescribed by Stack (1989). Also, they will be evaluated in FHB nurseries at Fargo, ND, using the methods as described above (see Objective 1). The improved wheat lines with a combination of FHB resistance and good agronomic traits will be provided to wheat breeding programs for cultivar development.Objective 3. To isolate and functionally characterize major quantitative trait locus (QTL) for Fusarium head blight (FHB) resistance in the wheat line PI 277102?3.1 Plant materials: The hexaploid wheat line PI 277012 will be used as the source of DNA for bacterial artificial chromosome (BAC) library construction. PI 277012 carries two QTL for FHB resistance and DNA markers closely linked to the QTL have been identified (Chu et al. 2011; Zhao et al. 2015; Zhao et al. unpublished data).3.2 Construction of BAC library:High molecular-weight (HMW) DNA will be isolated from young leaves harvested from PI 277012 and used to make the BAC librray according tothe method described by Peterson et al. (2000). 3.3 BAC library screening: To identify the BAC clones covering the QTL-containing region on chromosome 5AL, we will use the DNA markers flanking and within the target region to screen the BAC library pools by adopting the scheme outlined by Xia et al. (2014). Positive clones of interest will be identified by colony PCR. DNAs of the selected BACs will be isolated using Large-construct kit from Qiagen according to the manufacturer's instructions. The BACs will be fingerprinted with 4-bp and 6-bp cutter restriction enzymes and separated on 1.2% agarose gels following protocols described by Mathewson et al. (2007).3.4 BAC sequencing and sequence assembly:The individual unique BACs identified will be barcoded and sequenced by the PacBio sequencing or NanoPore Sequencing methods in a Sequencing Facility. De Novo DNA sequence assembly will be done with the software provided by PacBio (Menlo Park, CA) or NanoPore (New York, NY).3.5 Contig construction and gene annotation:Since the reference genome sequence of the wheat variety Chinese Spring (CS) is available, we will use the chromosome 5AL sequence of CS as a template to build up the contig for the 5AL QTL region in PI 277012.F-GENESH will be used for gene annotation with default setting parameters. BLASTn and BLASTp searches in GenBank and Gene Ontology searches will be performed to predict the tentative function of the annotated genes.3.6 Generation of ethyl-methane sulfonate (EMS) mutants and validation of candidate gene for FHB resistance:EMS mutants will be produced from the seeds of PI 277012 according to the method described by Rawat et al (2016). The M2 plants will be screened for FHB reactions in the greenhouse and susceptible individuals will be selected for PCR to search for SNPs in the candidate genes in comparison with the wild type. Missense, nonsense and intronic mutants obtained will be further characterized for FHB phenotype. The candidate gene will also be verified by gene transformation if necessary.

Progress 11/20/19 to 09/30/20

Outputs
Target Audience:1. Wheat breeders and geneticists; 2. Wheat growers; 3. Plant pathologists; 4. Extension agents; 5. Agricultural administrators/members. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Participants include Shaobin Zhong (PI), Yueqiang Leng (Research associate), Dandan Li (Postdoctoral Research Associate), Bikash Powdel (Ph.D. graduate student), and Anil (Ph.D. graduate student). The PI and other participants have attended three seminars and two conferences. How have the results been disseminated to communities of interest?The results were presented in conferences. FHB resistant wheat lines and DNA markers developed for FHB resistance have been provided to wheat breeders and research geneticists for development of FHB resistant varieties or germplasm. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The major highlights of the progress we have made for the report period include: 1. One novel major QTL for FHB resistance was detected on chromosome 2A in the recombinant inbred line (RIL) population derived from the cross between the emmer wheat line PI 254188 and durum wheat cultivar Divide. This major QTL will provide a useful source for developing durum wheat varieties with enhanced FHB resistance. 2. The two major FHB resistance QTL (Qfhb.rwg-5A.2 and Fhb1) were introduced into various spring wheat cultivars and breeding lines by backcrossing, marker-assisted selection and speed breeding approach and progenies carrying the QTL at F1BC2 or F1BC3 generations have been generated. 3. The 5A chromosomes of both spring wheat line PI 277012 and cultivar Grandin were isolated by flow cytometer technology and sequenced. The sequence assembly will facilitate the isolation of the FHB resistance gene on this chromosome. Several FHB susceptible Ethyl methanesulfonate (EMS) mutants were identified from PI 277012, which will be useful for validation of the candidate gene(s) to be cloned. To identify and map novel QTL in the emmer wheat line PI 254188 (Objective 1), we evaluated ~200 RILs (F2:7) derived from the cross between Divide and PI 254188 for FHB resistance in greenhouse and field in the summer season of 2020. FHB phenotype data were collected from two greenhouse experiments and two field experiments at the Fargo location. The RIL mapping population segregated for FHB resistance in both greenhouse and field evaluations; Some RILs had a high level of FHB resistance similar to the resistant parent PI 254188 while some other RILs were very susceptible. We also finished genotyping of the RIL population using the genotyping by sequencing (GBS) approach, which generated over 8,000 SNP markers. After quality filtration, a total of 4,192 SNP markers were used to construct a genetic linkage map, which consisted of 16 linkage groups associated with 14 chromosomes of the durum wheat genome. The total map size was 2653.47 cM. Genetic analysis detected a major QTL for FHB resistance from PI 254188 on chromosome 2A based on data from two greenhouse experiments and one field experiment. Another QTL located on chromosome 5A was detected only in one field experiment. To develop adapted wheat germplasm with improved FHB resistance (Objective 2), we have introgressed the two major QTL, Qfhb.rwg-5A.2 and Fhb1, into 11 elite spring wheat cultivars or breeding lines (Glenn, Alsen, ND VitPro, ND828, Linkert, Lang-MN, MN10201, Surpass, SD4539, WA8283, and Dayn) developed by spring wheat breeders at NDSU, University of Minnesota, SDSU, and WSU, respectively. GP112, a wheat line derived from the cross between PI 277012 and Grandin, was used as the donor of Qfhb.rwg-5A.2. Alsen was used as the donor of Fhb1. We first crossed the 11 elite wheat varieties or lines with GP112 and Alsen, separately. The F1 plants were backcrossed to the recipient parents. By backcrossing, marker-assisted selection and speed breeding approach, we obtained F1BC3 progenies each carrying one of the FHB resistance QTL (Qfhb.rwg-5A.2 or Fhb1). We will continue the backcross and marker-assisted selection process until F1BC7 (seventh generation) in order to develop near isogenic lines with 99.6 % identical to their respective parents genetically but each carrying the FHB resistance QTL. To clone the major FHB resistance QTL Qfhb.rwg-5A.2 in the wheat line PI 277012 (Objective 3), 5A chromosomes were sorted and collected from both PI 277012 and Grandin using the chromosome sorting technology in collaboration with Dr. Jaroslav Dolezel's group at Institute of Experimental Botany, Czech Republic. DNA samples were extracted from the sorted 5A chromosomes and used for Illumina sequencing. A total of 122.3 and 206.4 million paired-end reads (250 bp and 150 bp) were generated from the Grandin 5A chromosomes, which were assembled into an 872.38 Mb scaffold using the Chinese Spring 5A as reference. A total of 313.6 million paired-end reads (150 bp) were generated from the PI 277012 5A chromosomes, which were assembled into a 933.66 Mb scaffold using the Chinese Spring 5A as reference. Analysis of 5A sequences in the QTL region flanked by the marker 2375 and 2620 identified 237 and 289 genes from PI 277012 and Grandin, respectively. Comparative genomic analysis showed 71 genes have polymorphism in the target region between the two genotypes. These genes will be further characterized for marker development and candidate gene identification for the QTL. Screening approximately 400 M2 progenies from M1 plants derived from EMS treated seeds of PI 277012 in greenhouse identified five mutants that were much more susceptible to FHB compared to the parent PI 277012. These mutants will be used to validate the candidate FHB resistance genes. Also, fifteen additional PCR-based SNP markers were developed in the 5AL QTL region of PI 277012 and have been used in selection of the QTL in the process of introgression of the FHB resistance into adapted wheat cultivars.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2020 Citation: Shrestha, S., Poudel, R. S., and *Zhong, S. 2020. Identification of fungal species associated with crown and root rots of wheat and evaluation of plant reactions to the pathogens in North Dakota. Please Disease (accepted).
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Leng, Y., Poudel, B., Bernardo, A., Bian, R., Karmacharya, A., Mullins, J., Bai, G., Xu, S.S., Zhong, S. 2020. Identification and molecular mapping of a major QTL on chromosome 2A conferring resistance to Fusarium head blight in emmer wheat (p19, Poster #7). In: Canty, S., A. Hoffstetter, and R. Dill-Macky (Eds.), Proceedings of the 20120 National Fusarium Head Blight Forum. East Lansing, MI: U.S. Wheat & Barley Scab Initiative.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Ren, S., Zhu, X., Leng, Y., Zhang, W., Talukder, Z., Zhong, S., Fiedler, J., Qi, L., Cai, X. 2020. Toward a better understanding of the hexaploid wheat-derived Fusarium head blight resistance in durum wheat (p20, Poster #8). In: Canty, S., A. Hoffstetter, and R. Dill-Macky (Eds.), Proceedings of the 20120 National Fusarium Head Blight Forum. East Lansing, MI: U.S. Wheat & Barley Scab Initiative.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Wang, R., Hegstad, J., Xu, S., Elias, E., Zhong, S., Li, X. 2020. Developing durum wheat FHB resistant germplasm using interspecific crosses and phenotypic selection at early generations (p22, Poster #9). In: Canty, S., A. Hoffstetter, and R. Dill-Macky (Eds.), Proceedings of the 20120 National Fusarium Head Blight Forum. East Lansing, MI: U.S. Wheat & Barley Scab Initiative.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Jiang, H., Wan, J., Zhong, S., Schwarz, P., Chen, B., Rao, J. 2020. Clove oil-in-water nanoemulsion mitigates growth of Fusarium graminearum and trichothecene mycotoxin production during the malting of Fusarium infected barley (p57, Poster #25). In: Canty, S., A. Hoffstetter, and R. Dill-Macky (Eds.), Proceedings of the 20120 National Fusarium Head Blight Forum. East Lansing, MI: U.S. Wheat & Barley Scab Initiative.