Genetic Architecture of Grain Protein Content in Hulless Barley: Insights from Multi-Environment GWAS

Hulless barley (HB) is a nutrient-rich cereal gaining renewed research interest in the Tibetan regions of China. Grain protein content (GPC) is a quality-determining trait in HB that significantly affects end-use quality and is strongly influenced by environmental factors. Identifying stable association regions and developing breeding-applicable molecular markers are breeding objectives for high-GPC HB variety improvement. This study analyzed 266 HB accessions grown across six environments (2018–2021). Phenotypic GPC ranged from 5.02% to 18.31%, with moderate heritability (H2 = 0.473) and significant G×E interaction. A genome-wide association study (GWAS) using 168,983 GBS-derived SNPs and three models (GLM, MLM, FarmCPU) revealed no Bonferroni-significant SNPs in the BLUP meta-analysis. At the suggestive threshold (p < 1 × 10−5), 40 SNPs (18 loci) showed nominal association, with chromosome 7H harboring the most robust cross-model signal. Two SNPs on chromosome 6H and 7H exceeded Bonferroni correction in Yangma-2018 under GLM and FarmCPU. Candidate gene mining revealed seven functional genes, including 30S ribosomal protein S13 and glutamate-cysteine ligase. The lead SNP S7H_6966377 was located 165 kb from 30S ribosomal protein S13. These findings establish chromosome 7H as a regulatory hub for GPC and provide markers for marker-assisted selection.