Haplotype-defining NGR5 sequence variation identified through multiple bioinformatic approaches provides a potential mechanistic basis for differential NGR5 expression among the haplotypes

NITROGEN-MEDIATED TILLER GROWTH RESPONSE 5 ( NGR5 ) encodes an AP2/ERF-family transcription factor, which recruits Polycomb Repressive Complex 2 (PRC2) to branching-inhibitory loci, promoting histone 3 lysine 27 trimethylation (H3K27me3), leading to transcriptional repression of branching-inhibitory genes. Higher NGR5 protein levels can lead to increased tiller growth and enhanced grain yield even under low soil nitrogen conditions. In this study, we employed multiple bioinformatic approaches to mine the NGR5 sequence variation derived primarily from the 3K Rice Genomes (3KRG) and publicly available de novo assembled rice genomes.. These approaches combinedly identified 53 SNPs (promoter + gene body) and nine structural variants in the promoter region. Haplotyping of this collective NGR5 sequence variation yielded 10 major haplotypes. Using about three representative accessions from each haplotype, grown under three diverse nitrogen regimes (N0, N50, and N100), we carried out NGR5 expression analysis through quantitative PCR (qPCR) approach. We show that across nitrogen regimes, mean NGR5 expression levels vary among haplotypes, among genotypes within a haplotype, and between haplotypes. Based on overall performance across the nitrogen regimes, we identified HAP2 as the most consistent and superior haplotype for NGR5 expression, closely followed by HAP4b and HAP3a. Notably, the NGR5 sequence variation identified in this study distinguishes these relatively superior haplotypes from the others, providing a potential mechanistic basis for differential NGR5 expression among haplotypes. We also identified a predicted differential targeting of NGR5 mRNA by a miRNA (osr-miR5071) among the haplotypes as another potential cause for varying NGR5 mRNA levels. Our proposed NGR5 sequence variation-mediated differential NGR5 expression among the haplotypes includes both genetic (cis-regulatory elements-based) and epigenetic (cytosine methylation-mediated) regulatory mechanisms. Furthermore, we show that the genotype x growth medium nitrogen status interaction also affects NGR5 expression levels. The study also provides sources of potentially superior NGR5 alleles and a focused approach for mining them from unknown rice germplasm, which can be exploited to enhance rice grain yield under low-nitrogen conditions. Lastly, the NGR5 polymorphic regions identified in this study are promising genome-editing targets for enhancing NGR5 expression levels.