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.