Wild barley (Hordeum vulgare subsp. spontaneum), the progenitor of cultivated barley, is an invaluable genetic resource for enhancing crop resilience, particularly in drought-prone regions. Its natural adaptation to water-limited environments makes it an ideal candidate for studying mechanisms of drought tolerance. This study aims to investigate the genetic basis of drought tolerance by examining the correlation between molecular markers and root traits across a diverse collection of wild barley genotypes. This study evaluated the relationship between molecular markers and root traits in 114 wild barley genotypes collected from the natural distributional range in western Iran. The genotypes were sub- jected to normal (90%–95% field capacity) and water-stress (50%–55% field capacity) conditions. Root, physiological and seedling traits were carefully measured, and the genotypes were analyzed using 35 molecular markers, including simple sequence repeats (SSRs) and expressed sequence tag-SSRs (EST-SSRs). Statistical association analyses were performed to assess the correlation between markers and root traits. The study revealed significant genetic diversity among the 114 wild barley genotypes, reflecting distinct environmental pressures in their regions of origin. Several molecular markers, especially BMAG0603 and GBM1126, consistently exhibited strong associations with desirable root traits, such as increased root length, root density, and seedling vigor under both normal and water-stressed conditions. These markers are valuable for marker-assisted selection (MAS) in breeding programs aimed at improving drought tolerance. Specific chromosomal regions critical for root trait development were identified, offering insights into the genetic control of drought tolerance in barley. The results highlight the importance of using molecular markers to enhance drought tolerance in barley. The identification of key markers associated with beneficial root traits offers a valuable resource for breeding programs focused on drought resilience. Further research should explore marker-trait associations under various stress conditions to optimize the genetic potential of wild barley for crop improvement strategies.
