Dissecting the genetic basis of DUS characters in Pakistani wheat varieties

Distinctness, Uniformity and Stability (DUS) testing is fundamental to varietal characterization and the protection of Plant Breeders Rights (PBRs), but conventional DUS assessment relies predominantly on morphological characteristics whose expression may be influenced by environmental conditions, developmental stage and observer-related variation. Integrating character-specific molecular markers with conventional DUS assessment may therefore improve the reproducibility and genetic resolution of varietal identification. This study investigated the genetic architecture of DUS characteristics in a panel of 102 historical and modern Pakistani wheat varieties using genome-wide association studies (GWAS). Phenotypic variation was evaluated using distribution, correlation and hierarchical clustering analyses, while the germplasm was genotyped using a wheat genotyping-by-targeted sequencing platform. After quality filtering, 21,670 high-quality single-nucleotide polymorphisms (SNPs) were retained for analyses of genetic diversity, population structure, linkage disequilibrium (LD) and marker-trait associations. Population structure analysis indicated extensive admixture, with cross-entropy supporting K = 9 ancestral components, whereas hierarchical genetic clustering separated the germplasm into five broad groups. The first three principal components explained 8.09%, 7.20% and 5.39% of the total genetic variation, respectively. GWAS using BLINK and FarmCPU identified significant associations for several diagnostically important DUS characteristics. Growth habit was strongly associated with SNP 1B_129176704, with an additional signal on chromosome 5B. Shattering showed its strongest association at 1D_445256324, accompanied by several closely linked SNPs in the same region and a secondary significant locus at 7B_703502819. Flag leaf length was associated with a cluster of SNPs on chromosome 6A, while tiller density showed a major association with 5D_205488820. These findings demonstrate substantial phenotypic and genetic diversity within Pakistani wheat germplasm and identify genomic regions that merit validation as character-specific molecular tools to complement conventional DUS testing and PBR implementation.