Location: Sunflower Improvement Research
Project Number: 3060-21000-047-022-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Aug 1, 2025
End Date: Dec 30, 2026
Objective:
The overall goal of this project is to develop new genetic and genomic tools to facilitate research on the sunflower stem canker pathogens Diaporthe helianthi and D. gulyae. Primary objectives for the project will be, first, to assemble reference quality, chromosome-level genomes for four plant-pathogenic Diaporthe species, including D. helianthi and D. gulyae, along with the soybean pathogens D. caulivora and D. aspalathi. A second aim is to complete genome assemblies for additional isolates of these Diaporthe species using existing short-read sequence data. The final objective for the project is to identify D. helianthi isolates of opposite mating types and exhibiting differential virulence on previously tested sunflower lines and to evaluate media and culture conditions to facilitate in vitro mating and crossing of D. helianthi isolates for future mapping and identification of virulence-associated genes. Specific objectives to be completed in the first year of the project are to: 1) Assemble existing D. helianthi and D. gulyae genome sequence data; 2) Prepare tissue from selected D. helianthi, D. gulyae, D. caulivora, and D. aspalathi isolates for additional sequencing; 3) Evaluate mating type for 20 D. helianthi isolates previously tested for aggressiveness on 15 sunflower lines.
Approach:
Existing short-read sequence data at approximately 100x coverage for multiple isolates of Diaporthe helianthi, D. gulyae, D. caulivora, and D. aspalathi will initially be assembled using the Spades assembly tool. To facilitate development of chromosome-level genome assemblies, single isolates of the four Diaporthe species will be sequenced by an external provider using two complementary long-read sequencing technologies. Long-read sequence data will be assembled using the Canu assembly tool and assemblies will be evaluated for quality and completeness using BUSCO (benchmarking universal single-copy orthologs) and the QUAST genome quality assessment tool. Mating types will be determined for 20 Diaporthe helianthi isolates using previously designed mat-a and mat-alpha primers. In vitro mating and production of perithecia will be attempted by adapting methods previously described for other Diaporthe species. Media including water agar and oatmeal agar supplemented with autoclaved wild fennel stems or sunflower hypocotyls placed on the media surface will be tested for induction of perithecia to facilitate future development of cross populations for mapping genes encoding virulence factors such as proteinaceous effectors and toxic secondary metabolites.