Natural variation of the MIR168a promoter influences its activity and miR168a abundance in rice

miR168a, a plant-specific microRNA (miRNA) derived from the MIR168a gene, plays a pivotal role in modulating rice blast disease resistance and critical agronomic traits such as flowering time and yield. However, the regulatory mechanisms governing the MIR168a promoter remain poorly understood. This study identified a 1661 bp upstream fragment of the mature miR168a as highly active in promoter function. Sequence alignments revealed substantial variation in MIR168 promoters across plant species. Analysis of over 4000 rice accessions showed that while the MIR168a promoter exhibited abundant SNPs and InDels, miR168a itself had no such polymorphisms. Based on promoter polymorphisms, the MIR168a promoter was classified into three haplotypes, with Hap2 and Hap3 showing higher activity than Hap1. Through DNA fragment swapping and site-directed mutagenesis, the T site in Hap2 and the A site in Hap3 were identified as critical determinants of promoter activity. Rice accessions containing these sites exhibited significantly higher miR168a abundance compared to Hap1 accessions. Population genetic and evolutionary analyses revealed that highly active MIR168a promoters in Hap2 and Hap3 predominantly occur in indica accessions and trace their origins to wild rice. Furthermore, the nucleotide diversity of the MIR168a promoter in cultivated rice was markedly lower than in wild rice, likely reflecting artificial selection during domestication and artificial selection. Breeders may have favored rice lines harboring MIR168a promoter variants with reduced activity, such as Hap1 accessions, underscoring its potential for breeding programs. Additionally, miR168a expression was induced in all three haplotypes following infection with Magnaporthe oryzae. These findings illuminate the natural variation in MIR168a promoter sequences and their influence on miR168a expression activity, offering new insights for rice improvement strategies.