Location: Cereal Crops Research
Project Number: 3060-21000-046-005-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Jul 1, 2020
End Date: Jun 30, 2025
Objective:
Spring wheat is a major small grain crop in the Northern Great Plains and is vulnerable to the fungal diseases fusarium head blight (FHB), caused by Fusarium graminearum and stem rust, caused by Puccinia graminis f. sp. tritici,. The best method to control these pathogens is to utilize varieties with genetic resistance. Breeding for resistance has been limited due to the lack of strong quantitative trait loci (QTL), useful molecular markers, and overall genomic information.
The objective of this project is to enhance our knowledge of fungal disease resistance in wheat. With a better understanding of known resistance genes and the identification of new genes, we can accelerate the breeding of cultivars that inherently combat the disease. We will investigate two diseases with different modes of resistance. Sub-Objective 1 will focus on quantitative resistance of fusarium head blight by improving the genomics foundation with an applied wheat pangenome. Sub-Objective 2 will focus on qualitative resistance of stem rust with association mapping of several tetraploid wheat bi-parental populations. Sub-Objective 3 will be the development of a targeted long-read sequencing approach to assist the other aims by filling in sequencing gaps and refining genomic loci that confer resistance.
Approach:
Sub-Objective 1 will focus on developing a wheat pangenome from de novo assembled genomes of foundational FHB-resistant lines. The approach consists of high-molecular weight DNA extraction, PacBio HiFi sequencing of 20-25 kb fragments to obtain approximately 25X read coverage, assembly of the reads into contigs with Canu2, and scaffolding of the contigs into pseudomolecules with Hi-C sequencing. The pangenome will be annotated by PacBio IsoSeq sequencing of mRNA extracted from various tissues types. This evidence data will be combined with the plant-specific gene-prediction software (Maker-P) for structural annotation, and alignment with related grass transcriptomes will be used for functional annotation.
Sub-Objective 2 focuses on the identification of new QTL with association mapping in four durum x emmer populations. The four emmer parent lines have been shown to be resistant to various prevalent races of stem rust, including the Ug99 race-types that pose great threats to the global wheat supply. These have been crossed to the stem-rust sensitive cultivar “Rusty” and 192 F2 progeny of each cross have been advanced to recombinant inbred lines (RIL) with single seed descent. F5 seedlings will be screened for stem rust resistance, genome-wide genotyping markers will be identified, and linkage maps will be generated using software such as Joinmap and Mapdistro. QTL mapping will be performed with the linkage maps and linearized disease ratings with Qgene and r/QTL.
Sub-Objective 3 is the development of a targeted long-read sequencing approach. To do this, we will extract HMW DNA from the lines of interest and enrich for the large fragments that include these QTL. To enrich, we will utilize a CRISPR-Cas9 technology that uses a specific guide RNA to add sequencer-specific adapters to only the desired region. Once enriched, we will pool barcoded samples and submit to in-house Nanopore sequencing with GPU-enabled direct base-calling. The combination of library-preparation and computational enrichment is expected to enhance the multiplexing capacity of the platform and allow for the simultaneous screening of entire populations at a low per-sample costs.
Amendment:
Sub-Objective 2C. Hybrid lines crossed in Sub-Obj2B will be screened for presence of the QTL, backcrossed and selfed to generate BC2F2 families for screening, with lines possessing and average genome compositions that are 1/8 originating from PI 192711. There lines will be screened for tan-spot resistance.
SubObj 2b. Preliminary analysis with RIL lines of the 4 tetraploid populations in Sub-Objective 2 identified several distinct QTLs conferring resistance to different rust isolates. To fine-map the resistance gene regions arising from PI 192711 and evaluate dominance of the genes, individual populations segregating for a single QTL will be developed by crossing F5 progeny carrying a single QTL to Rusty. Hybrid lines will be inbred and rust resistance at F2 stage will be conducted to determine dominance. Through single-seed descent, lines will be expanded to F2:5 and re-evaluated for rust resistance. Genotyping and QTL mapping will be performed to identify refine QTL regions.
