Genetic Dissection of Frost Tolerance in Winter Durum Wheat: Three Validated KASP Markers for Marker-Assisted Selection

Winter durum wheat combines the benefits of autumn sowing with high grain quality but remains poorly adapted to temperate regions due to low frost tolerance. To elucidate the genetic basis of winter hardiness and support breeding for improved cold adaptation, a segregating multi-family F₂ panel (n = 270) was developed from crosses among frost-tolerant and frost-susceptible lines. GWAS identified four loci significantly associated with winter survival on chromosomes 1B, 5A, 5B, and 7B, collectively explaining 7.6-21.5% of phenotypic variance. These loci jointly improved model performance (ΔMcFadden R² = 0.230, <i>p</i>-value = 4.76 × 10^-17) without evidence of epistasis, indicating additive inheritance. Predicted survival increased nearly linearly with the number of favorable alleles, highlighting the potential for pyramiding through marker-assisted or genomic selection. Three significant SNPs were converted to KASP assays, providing validated molecular tools for breeding applications. Overall, the study broadens understanding of frost-tolerance genetics in winter durum wheat beyond canonical <i>Fr</i> regions and delivers practical markers for improving winter hardiness in breeding programs targeting continental climates.