Genetic Diversity and Population Structure Analysis of Drought Tolerance in Walnut (Juglans regia L.) Using SSR Markers

Abstract Walnut biodiversity represents a critical genetic resource for developing climate-resilient cultivars capable of withstanding increasing drought stress under changing environmental conditions. Understanding the genetic basis of drought tolerance mechanisms is essential for sustainable nut production and conservation of valuable genetic resources. This study investigated the genetic and physiological basis of drought tolerance in walnut ( Juglans regia L.) saplings, providing crucial insights for breeding programs aimed at developing climate-resilient cultivars. A comprehensive analysis was conducted using 65 walnut samples collected from diverse geographic locations, employing SSR (simple sequence repeat) markers for genetic characterization combined with detailed morphological and physiological assessments under controlled drought stress conditions. Our findings revealed significant variability in drought stress responses among walnut saplings. Morphological and physiological traits exhibited substantial variation under drought stress, reflecting the genetic diversity present in natural populations. Population structure analysis performed using STRUCTURE software identified three distinct genetic clusters within the studied population, with varying degrees of admixture indicating complex evolutionary relationships and gene flow patterns across different populations. Some individuals showed strong affiliation to a single cluster, while others exhibited significant admixture patterns. Mixed Linear Model analysis identified significant marker-trait associations between SSR markers and drought tolerance traits. Multiple markers showed strong correlations with physiological parameters including chlorophyll content, leaf characteristics, and stress response indicators. Hierarchical clustering and phylogenetic analyses confirmed the genetic relationships among populations and revealed patterns consistent with geographic distribution and environmental adaptation. This research elucidates the genetic architecture underlying drought tolerance in walnuts and demonstrates the effectiveness of SSR markers for identifying drought-responsive genes. The identified marker-trait associations provide valuable tools for marker-assisted selection in breeding programs. Furthermore, the observed genetic diversity and its correlation with drought-related traits offer a foundation for developing more resilient walnut cultivars. These findings contribute to the conservation and utilization of walnut genetic resources while supporting the development of climate-adaptive breeding strategies for sustainable nut production under water-limited conditions.