Extended Data Fig. 1: Allelic variation at the GRF4 locus affects GRF4 mRNA abundance and root \({}^{{\bf{15}}}{{\bf{NH}}}_{{\bf{4}}}^{{\boldsymbol{+}}}\) uptake.

a, Positional cloning indicates the equivalence of GRF4 with qNGR2 (nitrogen-mediated growth response 2). Successive maps show progressive narrowing of focus of qNGR2 (red dot, using recombination break points and linked DNA markers) to an approximately 2.7-kb region on chromosome 2 flanked by molecular markers L17 and L18 and overlapping candidate gene LOC_02g47280 (also known as GRF4). The start ATG (nucleotide 1) and close TGA (nucleotide 3385) of GRF4 are shown, together with the protein-coding DNA sequence (thick black bars). The target site for miR396 is indicated by an asterisk. The structure of a CRISPR–Cas9-generated grf4 mutant 91-bp deletion allele spanning parts of exon 1 and intron 1 is shown. b, \({}^{15}{{\rm{NH}}}_{4}^{+}\) uptake rates of roots of BC₂F₂ progeny (derived from a NJ6 × NM73 cross) homozygous or heterozygous for GRF4^NGR2 or GRF4^ngr2 grown in high nitrogen supply (1.25 mM NH₄NO₃). Data are mean ± s.e.m. (n = 9). Different letters denote significant differences (P < 0.05) from a Duncan’s multiple range test. c, GRF4 mRNA abundance in plants (genotypes as shown) relative to the abundance in NJ6 (set to one). Data are mean ± s.e.m. (n = 3). Different letters denote significant differences (P < 0.05) from a Duncan’s multiple range test. d, Natural varietal GRF4 allelic variation. Nucleotide position relative to the GRF4 start ATG is shown in a. SNPs shared between varieties NM73, RD23 and TZZL1 are highlighted. Sequences representative of GRF4 promoter haplotypes A, B and C (see main text) are shown. e, GRF4 mRNA abundance in various rice varieties under the high nitrogen conditions (1.25 mM NH₄NO₃), GRF4 promoter haplotypes are indicated. Abundance data are all relative to the abundance of rice Actin2 mRNA. Data are mean ± s.e.m. (n = 3). Different letters denote significant differences (P < 0.05) from a Duncan’s multiple range test. f, Comparisons of GRF4 mRNA abundance in selected rice varieties grown in between high (HN, 1.25 mM NH₄NO₃) and low (LN, 0.375 mM NH₄NO₃) nitrogen conditions. Data are mean ± s.e.m. (n = 3). Abundance data are all relative to the high nitrogen condition (set to one). **P < 0.05 compared to high nitrogen in a two-sided Student’s t-test. g, Relative abundances of rice miR396 family members in NJ6 plants grown at different levels of nitrogen supply (0.15N, 0.1875 mM NH₄NO₃; 0.3N, 0.375 mM NH₄NO₃; 0.6N, 0.75 mM NH₄NO₃; 1N, 1.25 mM NH₄NO₃), shown relative to the abundance in plants grown in 1N conditions (set to one). Data are mean ± s.e.m. (n = 3). Different letters denote significant differences (P < 0.05) from a Duncan’s multiple range test.