Development of liquid chip for functional genes in foxtail millet (Setaria italica) and association analysis of related traits

Foxtail millet ( Setaria italica ) is a nutrient-rich, drought-tolerant cereal crop with significant potential for promoting sustainable agriculture. However, the lack of specialized, cost-effective genotyping tools has limited the development of breeding technology. We developed a liquid-phase functional single nucleotide polymorphism (SNP) chip utilizing Genotyping by Target Sequencing (GBTS) technology. This customized chip integrates 59 functionally verified SNPs from 30 key genes governing critical agronomic traits, including selenium accumulation, plant architecture, yield, stress tolerance, and grain quality. We validated the approach on a panel of 192 foxtail millet accessions. The chip demonstrated a high average call rate of 98.53% and revealed robust genotype-phenotype associations for traits such as fertility, leaf color, leaf sheath color, and seed color. Furthermore, population structure analysis clearly delineated modern cultivars from landraces and admixed groups, highlighting its utility in genetic diversity studies. This robust, scalable, and cost-effective genotyping tool is poised to significantly accelerate marker-assisted selection and precision breeding programs in foxtail millet, with broader implications for genetic research in other Poaceae species.