Candidate gene discovery for nitrogen use efficiency in rice based on genome-wide association study

Providing reliable and sufficient food resources for a growing global population is a significant societal challenge, in particular achieving this while keeping nitrogen pollution within safe environmental limits. Therefore, it is crucial to identify nitrogen-use efficient (NUE) accessions and identify candidate genes associated with NUE for sustainable agricultural development. Here, we present analysis on the genetic diversity of 518 accessions of Chinese germplasm and performed a genome-wide association study (GWAS) on 16 traits associated with NUE and yield. We identified a total of 89 significant loci, including 47 associated with NUE and 42 with yield, of which 56 (63 %) were newly discovered. Through association and indica - japonica genetic differentiation analysis, we identified a high-confidence candidate gene - OsNPT4 - that encoding a protein from the POT family. This gene was associated with nitrogen grain production efficiency (NGPE), nitrogen harvest index (NHI), and tillering number (TN). RNA-seq results indicated that OsNPT4 may play a crucial role in effectuating the response of rice plants to nitrogen treatment. Further haplotype analysis revealed significant differences among the various haplotypes of this gene concerning NGPE, NHI, and TN, with accessions carrying Hap1 demonstrating strong NUE and increased yields. RT-qPCR results showed that OsNPT4 expression significantly increased in Hap1-carrying accessions in both leaves and roots upon treatment, while no significant differences were observed in Hap2-carrying accessions. This further confirmed OsNPT4 as a key candidate gene associated with varying NUE. Taken together, our results provide a theoretical foundation for cloning NUE genes and facilitate the design of molecular breeding strategies. • A total of 56 new loci associated with NUE-related traits and yield-related traits were identified, laying a groundwork for further exploration and identification of candidate genes associated with NUE. Furthermore, the methodologies used in this study could easily be applied to relevant research in other crops. • Through association and genetic differentiation analysis, the candidate gene OsNPT4 , encoding a POT family protein, was identified as associated with nitrogen grain production efficiency (NGPE), nitrogen harvest index (NHI), and tillering numbe number (TN). RNA-seq results suggest that OsNPT4 plays a critical role in rice's response to low nitrogen conditions. Further haplotype analysis showed that accessions with Hap1 exhibited high NUE and yield. RT-qPCR revealed significantly increased OsNPT4 expression in Hap1-carrying accessions under low-nitrogen conditions, confirming its role as a key gene for NUE. These findings lay the groundwork for future genetic studies and molecular design aimed at enhancing NUE breeding in rice. • Geographic distribution of OsNPT4 haplotypes shows Hap1 prevalent in Asia and West Africa, suggesting strategic breeding opportunities for nitrogen-efficient rice. • Ten high NUE and yield varieties identified, with six accessions displayed all superior haplotypes of OsNPT4 , OsNRT1.1B , and OsMYB61 , promising for future rice breeding efforts.