USDA-SoyWheOatBar-3K: a robust high-throughput genotyping platform to accelerate molecular breeding in small grains

High-throughput genotyping supports marker-assisted selection of known loci and genomic prediction of complex traits. Yet, routine adoption of these molecular breeding tools remains out of reach for many breeding programs. Here, we present a multi-species single-nucleotide polymorphism (SNP) array designed for standardized genotyping of wheat, barley, and oat. The platform contains 9,468 small-grain SNPs (3,421 wheat, 3,058 barley, and 2,989 oat), strategically selected to provide genome-wide coverage using genetic selection bins of 1 cM for wheat and oat and 0.5 cM for barley. Validation across diverse germplasm collections (>10,000 samples) revealed robust technical performance, with 98.7%, 89.0%, and 91.5% of SNPs in barley, wheat, and oat, respectively, meeting all quality thresholds. A useful feature of the array is the ability to genotype samples in multi-species hybridization mode, thereby lowering the array-processing cost per sample. Multi-species hybridization produced partner-specific signal responses while retaining high genotype-call concordance, ranging from 93.5% to 98.7% across the evaluated dual- and tri-hybridization configurations. Oat signal quality was highest under tri-hybridization, although its performance was only marginally greater than in the oat-wheat dual configuration. Independent applications demonstrated that the array produced genomic prediction accuracies comparable to those obtained using higher-density arrays or sequence-based genotyping platforms and supported the detection of marker-trait associations in genome-wide association studies. By combining species-specific marker sets for wheat, barley, and oat on one platform, the array provides a standardized genomic resource for routine molecular breeding.