A multi-omic dissection of spatial hierarchy-dependent grain development in barley (Hordeum vulgare L.).

key messageGWAS on grain uniformity and transcriptomics across spike shapes/positions reveal checkpoints in barley grainheterogeneity development. Grain uniformity, defined as the consistency of grain size among siblings within a sample, can influence yield performance and malting quality in barley, yet its genetic basis remains poorly understood. Here, we combine genome-wide association analysis of 334 barley accessions with spatiotemporal transcriptomics of two cultivars exhibiting contrasting uniformity. We identified six quantitative trait loci (QTLs) across chromosomes 1H, 2H, 4H, and 5H. The non-uniform cultivar displayed 3,314 differentially expressed genes between apical and basal grains at grain filling stage, versus only 222 in the uniform cultivar. This approximately 15-fold difference was accompanied by positional differences in starch biosynthesis, sugar transport, and trehalose-6-phosphate signaling. We identified four candidate genes proximal to stable GWAS signals on chromosome 1H and developed a dCAPS marker. These findings identify grain filling stage as a key stage associated with grain uniformity and suggest asymmetric carbon metabolism as a candidate mechanism underlying spatial hierarchy-dependent grain development.