Nitrogen deficiency has been identified as the most severe abiotic stress affecting the growth, development, and yield of bread wheat (Triticum aestivum L.). To investigate the genetic basis underlying the nitrogen use efficiency (NUE) in bread wheat, a genome-wide association (GWAS) study was conducted for N-use responsive morpho-physiological traits like biomass, photosynthetic pigments, and protein content; five biochemical traits (important N and C metabolizing enzyme activities) and two indexed traits (Stress Susceptible/Tolerance Index: SSI/STI) at the seedling stage under contrasting N conditions.

A panel of 317 diverse bread wheat genotypes spanning diverse ecogeographic regions was genotyped using a breeders’ 35K Axiom Array, and a set of 22,650 filtered single nucleotide polymorphism (SNP) markers were used to study marker-trait associations (MTAs). Population structure and diversity analysis indicated two sub-populations with significant diversity. A large whole genome LD block size of 5.12 MB was obtained. 145 MTAs were identified using the different association mapping algorithms viz., BLINK, FarmCPU, and MLM.

Among them, 14 pleiotropic markers were also identified. We found phenotypic variance up to 24.8% in the case of GOGAT-STI followed by ICDH-SSI (16.8%), and NR enzyme activity (10.2%) in N-stress condition. A total of 12567 genes were retrieved from the 145 SNPs ± 5.12Mb region. 318 differentially expressed genes were identified by integrating GWAS and transcriptome analyses of N-efficient and N-inefficient genotypes.

Finally, nine genes (TraesCS4B02G394500, TraesCS7D02G008700, TraesCS1A02G295600, TraesCS2B02G521600, TraesCS2B02G600200, TraesCS5B02G426300, TraesCS6A02G061300, TraesCS2A02G066700, TraesCS5B02G426500) which expressed differentially in the N-efficient genotype were defined as more valuable candidate genes for low-nitrogen tolerance.

This is the first attempt to use GWAS for the identification of N-use-related biochemical and morpho-physiological traits QTLs in common wheat. This study will provide strong evidence for N regulation mechanisms, and reliable genetic resources for growth and NUE genetic improvement in wheat.