Multi-Location Evaluation of GWAS-Derived PACE Markers for Yield-Related Traits in Spring Barley

Barley productivity is controlled by complex quantitative traits that are strongly influenced by genotype-by-environment interactions, requiring validation of candidate markers across representative target locations before their use in marker-assisted selection. A panel of 83 two-rowed spring barley accessions was evaluated for 11 agronomic traits at three contrasting locations in Kazakhstan during the 2025 growing season. The panel was genotyped using 103 PACE assays targeting previously reported QTLs. Location-specific and across-location phenotypes were analyzed using BLUPs, ANOVA, PCA, population-structure-corrected linear regression, marker-assisted breeding value analysis, and positional candidate-gene annotation. Of 70 polymorphic markers with minor allele frequency above 0.05, 27 were associated with eight traits, producing 51 marker–trait associations at FDR < 0.05. Twenty-six associations involving 17 markers were retained for preliminary breeding-utility assessment. Two associations involving ipbb_hv50K_046 were supported at two or more locations, 19 were detected at one location and in the BLUP analysis, and five were BLUP-specific. Fourteen markers reproduced previously reported GWAS associations at the trait or trait-group level. Marker ipbb_hv50K_046 showed the strongest spatially reproducible effects and was associated with an 81.1% increase in the number of kernels per spike, a 31.4% increase in kernel weight per spike, and a 58.3% reduction in spike rachis internode length. Positional analysis of the Morex v3 reference genome identified candidate genes for all 17 retained markers, with 12 SNPs located within high-confidence gene models. These markers represent promising candidates for marker-assisted selection in barley breeding programs across diverse agro-climatic conditions in Kazakhstan and comparable environments.