Genetic variation and genome-wide association analysis of nitrogen use efficiency-related traits under combined heat and nitrogen-deficient stress in an Aegilops tauschii-derived wheat population

Heat stress and nitrogen (N) deficiency increasingly limit global wheat ( Triticum aestivum L.) yields, highlighting the need to improve nitrogen use efficiency (NUE) under combined stresses for sustainable production. We assessed 145 multiple-synthetic-derivative (MSD) lines, carrying alleles from diverse Ae. tauschii , crossed and backcrossed into ‘Norin 61’, together with three checks across six field environments combining heat stress and either optimal (86 kg N ha - ¹; HS-HN) or zero (HS-LN) N supply in central Sudan. Eighteen agronomic and physiological traits were recorded, and best linear unbiased estimates were used for genome-wide association analysis (GWAS) with 31,362 high-quality DArTseq and GRAS-Di markers. HS-LN reduced mean grain yield (GY) and grain N uptake (GNUp) by 14% and 28%, respectively, but increased thousand-kernel weight and harvest index, indicating resource re-allocation to grain filling. The MSD lines showed wide variation, and some lines maintained high GY under either HS-HN (e.g., MSD053 and MSD450) or HS-LN (e.g., MSD192 and MSD383). The MSD lines MSD026, MSD181, and MSD485 ranked among the top five for GY under HS-LN, HS-HN conditions, and across the six environments. GWAS identified 34 marker-trait associations (MTAs) on 12 chromosomes; 62% resided in the D subgenome. A pleiotropic locus on 5A (rs987242) affected grain growth rate and GY, whereas a novel locus on 3D (rs1071033) explained 88% of the variation in GNUp relative performance. Candidate genes included mitogen-activated protein kinases, DELLA (Rht-1), MADS-box, and DnaJ homologues linked to stress signaling or N metabolism. Our results uncover genetic variants and germplasm that enhance NUE and yield stability under concurrent heat and N stress, providing immediately deployable resources for climate-resilient wheat breeding.