Location: Cereal Crops Research
Project Number: 3060-21000-038-34-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Jul 1, 2020
End Date: Jun 30, 2025
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,. In bad years, these diseases can have a devastating impact on the producers by reducing yield and lowering grain end-use quality. The best method to control these pathogens is to utilize varieties with genetic resistance. Breeding for FHB resistance has been limited due to the lack of strong quantitative trait loci (QTL), useful molecular markers, and overall genomic information. Stem rust resistance is difficult to maintain because populations can rapidly change to evade race-specific QTL and spores travel far in the wind. 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. 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.
Sub-Objective 1 will focus on developing new genomic resources by assembling a wheat pangenome from de novo assembled genomes of foundational FHB-resistant lines. The approach consists of high-molecular 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 2-6 botanical and developmental 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. Several known QTL for FHB resistance are present in the lines going into the pangenome, and the platform developed in sub-objective 3 will assist in ensuring complete data in these regions. Structural analysis of the pangenome will uncover new variation that can be used to improve previous mapping efforts to identify new loci that influence resistance. 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 at the USDA-ARS Cereal Diseases Lab and the NDSU Plant Pathology department. Sequence-based genome-wide genotyping markers will be developed 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. If QTL are discovered, the long-read approach being developed in sub-objective 3 will assist in ensuring strong sequence information throughout the QTL, so high-throughput molecular markers can be developed to aid in future gene cloning and introgression efforts. 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 known 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. An additional feature of this sequencing platform is the “Read Until” function that will reject DNA molecules if the first portion of the read doesn’t match the expected sequence. 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.