Identification of candidate loci for leafing date in Iranian walnut germplasm collection using GBS-GWAS approach and...

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, additional robust markers for leafing date are still needed to enhance breeding efficiency. 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. Leafing date was defined as the stage when 50% of terminal buds reached budburst, quantified as the cumulative number of days since March 1st. Population structure analysis revealed three major genetic clusters corresponding to geographic origin, reflecting the deep evolutionary history of the Iranian walnut. Using 68 K high-quality Single-nucleotide polymorphisms )SNPs(, GWAS consistently identified significant loci on chromosomes 1, 4, 6, 8, 11, and 13. Notably, the most robust associations were consistently detected across the 2017 and 2018 growing seasons, as well as in the multi-year average data, using MLMM, Blink, and FarmCPU models. Based on these stable associations, a SNaPshot marker panel was developed and validated in an independent set of 24 Iranian and Turkish genotypes. Among these, a single marker-JRgwLDch4pos20189439 on chromosome 4-exhibited strong and repeatable discrimination between early- and late-leafing phenotypes across populations. This locus represents a key experimentally validated leafing-date marker for Iranian walnut germplasm and provides a powerful tool for marker-assisted selection (MAS). Our findings reveal the multi-allelic nature of leafing date and demonstrate that markers may vary among geographically distinct germplasm pools. The validated chr4-associated marker offers a promising genomic resource that may support efforts to accelerate breeding of climate-resilient walnut cultivars in Iran and potentially other regions with related genetic backgrounds.