Genome-Wide Association Study and RNA Sequencing Identify Candidate Genes Regulating Nitrogen Use Efficiency and Associated Traits in Sorghum (Sorghum bicolor (L.) Moench)

Sorghum (Sorghum bicolor L. Moench) is a resilient cereal crop with remarkable adaptability to diverse environments. Nitrogen use efficiency (NUE) is critical for improving sorghum yields, resource utilization, livelihood security, and environmental sustainability of the target ecologies. To dissect the physio-genetic variation of sorghum for NUE/Nitrogen (N) stress tolerance, a set of 186 diverse sorghum accessions was evaluated for 15 agro-physiological and NUE-related traits under three N regimes (0%, 50%, and 100% of the recommended [90 kg ha⁻¹] dose) across two seasons. Genotyping-by-sequencing (GbS) SNP data enabled genome-wide association studies (GWAS). A total of 1369 marker-trait associations (MTAs) were detected across sorghum chromosomes, along with 69 candidate genes linked to N metabolism, including glutamine synthetase (GS), nitrate transporters, and sucrose-phosphate synthase. Transcriptome analysis of contrasting sorghum accessions detected 2229 (shoot) and 8661 (root) differentially expressed genes (DEGs). Integration of GWAS and transcriptomic data identified 10 key candidate genes such as the master N-regulators: the NIN-like protein (NLP), AP2/ERF transcription factor, ABC transporter, glutamine synthetase (GS), amino acid selective channel protein, F-box protein (FBP), SWEET transporter, glucose-1-phosphate adenylyltransferase (AGPase), and phosphofructokinase (PFK). Analysis of identified homologous gene groups across major cereals revealed evolutionary relationships and genetic conservation. Furthermore, this study identified contrasting sorghum accessions for N stress tolerance. The identified candidate genes and contrasting genetic stocks provide a foundation for the molecular dissection of NUE-related traits, offering clear targets for crop improvement via genomics-assisted breeding and gene editing technologies.