Muhammad Tanveer Altaf ¹ Pablo Federico Cavagnaro ² Kagan Kokten ¹ Amjad Ali ¹ Andrés Morales ³ Muhammed Tatar ¹ Mehmet Bedir ¹ Muhammad Azhar Nadeem ¹ MUHAMMAD AASIM ¹ Nafiz Celiktas ⁴ Sheikh Mansoor Shafi ⁵ Faheem Shehzad Baloch ^6*

• ¹ Sivas bilim ve teknoloji fakültesi, Sivas, Türkiye
• ² National Institute of Agricultural Technology (INTA), Agricultural Experimental Station, Mendoza, Mendoza, Argentina
• ³ Instituto Nacional de Tecnología Agropecuaria (INTA), Estación Experimental Agropecuaria La Consulta, La Consulta, Argentina
• ⁴ Mustafa Kemal University, Antakya, Hatay, Türkiye
• ⁵ Jeju National University, Jeju City, Jeju, Republic of Korea
• ⁶ Mersin University, Mersin, Mersin, Türkiye

Orchardgrass (Dactylis glomerata L.), a widely cultivated cool-season perennial, is an important forage crop due to its adaptability, high nutritional value, and substantial biomass. Understanding its genetic diversity and population structure is crucial for developing resilient cultivars that can withstand climate change, diseases, and resource limitations. Despite its global significance in fodder production, the genetic potential of many regional accessions remains unexplored, limiting breeding efforts. This study investigates the genetic diversity (GD) and population structure of 91 accessions of D. glomerata from Turkey and Iran using genotyping-by-sequencing based single nucleotide polymorphism (SNP) markers. A total of 2913 high-quality SNP markers revealed substantial genetic variability across provinces. Notably, accessions from Erzurum exhibited the highest GD (mean GD: 0.26; He: 0.5328), while provinces such as Bursa and Muğla demonstrated lower GD (mean GD: 0.15; He < 0.22), suggesting potential genetic bottlenecks. Population structure analysis using Bayesian clustering, PCoA and UPGMA dendrograms divided the accessions into three distinct clusters, with cluster membership largely reflecting geographical origins, and dry biomass content. Cluster II revealed higher GD, associated with enhanced biomass production (128 g/plant), the most important agronomic trait in forage species, supporting the notion of heterosis in breeding programs. The majority of the genetic variation (85.8%) was observed within clusters, with minimal differentiation among clusters (FST = 0.007). Genome-wide association studies (GWAS) identified significant marker-trait associations for dry biomass weight, a critical agronomic trait, with markers DArT-100715788, DArT-101043591, and DArT-101171265 and DArT-101090822 located on Chromosomes 1, 6, and 7 respectively. These findings highlight the importance of regional diversity for maintaining adaptive potential in future breeding programs.