Pre-harvest sprouting (PHS), the germination of seeds within heads before harvest, is a major problem in global wheat (Triticum aestivum L.) production, manifested by the loss of bread-making quality in affected grain. Marker-assisted selection can be a useful tool to select for PHS resistance, especially in years without pre-harvest rain. Genome-wide association studies (GWAS) are required to find markers to be used in selection, especially in a breeding context.

We tested 1000 wheat breeding lines and registered varieties from across Europe in four years, 2017 through 2020. Lines and varieties were genotyped for 10K genome-wide SNP markers using GBS and a commercial SNP-Array. To assess PHS in the field, we challenged lines using irrigation at maturity and subsequently measured Hagberg Falling Number as well as counted the number of sprouted grains. For lab-based challenging, we cut ten heads at maturity before irrigation. These heads were soaked in water, incubated, and visually scored for sprouting progression in the following days. Furthermore, we assessed phenological, agronomic, and quality-related traits to co-locate them with PHS markers. These phenotypes were used to test for association with the genome-wide marker data. From the significant associations, we selected the "strongest" across years based on the number of significant associations. To connect our results to PHS related genes from literature and possibly locate previously unassigned markers to chromosomes, we blasted markers and genes to the IWGSC RefSeq v2.1 as well as ten pan-genomes. To assess the structural variation at PHS hot-spots across the pan-genome, we aligned genomes pair-wise using MUMmer.

Hotspots of strong PHS associations were found on chromosomes 1A, 4A and 5B. Associations on 1A and 5B are possibly novel. While markers on chromosome 5B are linked to phenology and therefore of limited use in a breeding program, associations on chromosome 1A are not linked to other traits and might therefore, be highly useful for selecting PHS resistant lines. Markers on chromosome 4A are located in an interval that is known to harbor the Phs-A1 locus, for which two genes, PM19 and MKK3, were previously reported to be causative. We found three distinct peaks with association to PHS in this region and variable arrangements of markers and genes across genomes, suggesting structural variation as a cause for inconclusive association results in this and previous studies. This result could also be caused by imperfect genome assembly since the wheat genome is notoriously hard to assemble due to its large genome size and high content of repetitive elements. To confirm this, more long-read sequencing data and high quality assemblies are necessary, which are to date not available. However, this approach revealed Phs-A1 as the most stable and effective locus for regulating pre-harvest sprouting independent of phenology or quality related traits in Europe and revealed that Phs-A1 is not only influenced by allelic, but also by structural genome variation.