GBS-based SNP dataset of Iranian walnut germplasm
Late-spring frost is increasingly damaging walnut orchards worldwide, underscoring the urgent need for late-leafing cultivars adapted to climate instability. Despite extensive genomic resources in Juglans regia, robust and transferable markers for leafing date remain scarce. Here, we combine high-density genotyping-by-sequencing (GBS) with multi-model genome-wide association analysis (GWAS) to dissect the genetic architecture of leafing date in a uniquely diverse panel of 85 Iranian walnut genotypes and commercial cultivars evaluated over three consecutive years. Population structure analysis revealed three major genetic clusters corresponding to geographic origin, reflecting the deep evolutionary history of Iranian walnut. Using 68K high-quality SNPs, GWAS consistently identified significant loci on chromosomes 1, 4, 6, 8, 11, and 13. A SNaPshot marker panel was developed based on the most stable associations, and validation was performed in an independent set of 24 Iranian and Turkish genotypes. A single marker—JRgwLDch4pos20189439 on chromosome 4—exhibited strong and repeatable discrimination between early- and late-leafing phenotypes across populations. This locus represents the first experimentally validated leafing-date marker for Iranian walnut and provides a powerful tool for marker-assisted selection (MAS). Our findings reveal the multi-allelic nature of leafing time and demonstrate that markers may vary among geographically distinct germplasm pools. The validated chr4-associated marker offers an immediately deployable genomic resource for accelerating breeding of climate-resilient walnut cultivars in Iran and other regions with comparable genetic backgrounds.