A natural variation in Ribonuclease H-like gene underlies Rht8 to confer “Green Revolution” trait in wheat

Introduction of gibberellin (GA)-insensitive Reduced height (Rht) genes, Rht-B1b and Rht-D1b, has resulted in the "Green Revolution" in modern wheat cultivars (Triticum aestivum) that has skyrocketed wheat grain yields worldwide since the 1960s (Peng et al., 1999Peng J. Richards D.E. Hartley N.M. Murphy G.P. Devos K.M. Flintham J.E. Beales J. Fish L.J. Worland A.J. Pelica F. et al.'Green revolution' genes encode mutant gibberellin response modulators.Nature. 1999; 400: 256-261Google Scholar; Velde et al., 2021Velde K. Thomas S. Heyse F. Kaspar R. Van Der Straeten D. Rohde A. N-terminal truncated RHT-1 proteins generated by translational reinitiation cause semi-dwarfing of wheat green revolution alleles.Mol. Plant. 2021; 14: 1-9Google Scholar). However, Rht-B1b/D1b also reduce coleoptiles, which is undesired in dryland regions where deep planting is essential for seedling establishment (Rebetzke et al., 1999Rebetzke G.J. Richards R.A. Fischer V.M. Mickelson B.J. Breeding long coleoptile, reduced height wheats.Euphytica. 1999; 106: 159-168Google Scholar, Rebetzke et al., 2001Rebetzke G. Appels R. Morrison A.D. et al.Quantitative trait loci on chromosome 4B for coleoptile length and early vigour in wheat (Triticum aestivum L.).Crop Pasture Sci. 2001; 52: 1221-1234Google Scholar; Ellis et al., 2004Ellis M. Rebetzke G. Chandler P. Bonnett D. Spielmeyer W. Richards R. The effect of different height reducing genes on the early growth of wheat.Funct. Plant Biol. 2004; 31: 583-589Google Scholar). The GA-sensitive Rht8 on the short arm of chromosome 2D reduces plant height without scarifying coleoptile length (Supplemental Figures 1 and 2), thus it has been widely used for wheat semi-dwarfing breeding (Rebetzke et al., 1999Rebetzke G.J. Richards R.A. Fischer V.M. Mickelson B.J. Breeding long coleoptile, reduced height wheats.Euphytica. 1999; 106: 159-168Google Scholar; Worland et al., 2001Worland A.J. Sayers E. Korzun V. Allelic variation at the dwarfing gene Rht8 locus and its significance in international breeding programmes.Euphytica. 2001; 119: 157-161Google Scholar; Ellis et al., 2004Ellis M. Rebetzke G. Chandler P. Bonnett D. Spielmeyer W. Richards R. The effect of different height reducing genes on the early growth of wheat.Funct. Plant Biol. 2004; 31: 583-589Google Scholar; Grover et al., 2018Grover G. Sharma A. Gill H. Srivastava P. Bains N. Rht8 gene as an alternate dwarfing gene in elite Indian spring wheat cultivars.PLoS One. 2018; 13: e0199330Google Scholar). In this study, Rht8 candidate gene was isolated via map-based cloning. Rht8 was first mapped to a 0.58-cM interval between markers STARP2003 and SSR2650 on chromosome 2DS (Supplemental Figure 3) based on the analysis of a segregating population generated from a heterozygous recombinant inbred line (RIL), RIL171, derived from the cross between Yumai8679 (Y8679, semi-dwarf Rht8 allele) and Jing411 (J411, tall rht8 allele) (Chai et al., 2019Chai L. Chen Z. Bian R. Zhai H. Cheng X. Peng H. Yao Y. Hu Z. Xin M. Guo W. et al.Dissection of two quantitative trait loci with pleiotropic effects on plant height and spike length linked in coupling phase on the short arm of chromosome 2D of common wheat (Triticum aestivum L.).Theor. Appl. Genet. 2019; 132: 1815-1831Google Scholar). Continuous screening of the progeny with a recombination in the Rht8 region obtained 65 recombinant near-isogenic lines (NILs) with nine recombination types (NIL1–NIL9). Phenotypic analysis of these NILs mapped Rht8 to a ∼107-kb region between markers INDEL2005 and SSR2650 (Figure 1A and Supplemental Figure 3). Based on the recently updated Chinese Spring (CS) genome assembly, Triticum_aestivum_4.0 (Alonge et al., 2020Alonge M. Shumate A. Puiu D. Zimin A.V. Salzberg S.L. Chromosome-scale assembly of the bread wheat genome reveals thousands of additional gene copies.Genetics. 2020; 216: 599-608Google Scholar), the Rht8 interval contains only two open reading frames (ORFs), ORF1 and ORF2, with almost identical coding sequences that match with the high-confidence gene, TraesCSU02G024900 (the gene is also designated as TraesCSU03G0022100 according to CS RefSeq v2.1; Figure 1A), encoding an unknown protein of 808 amino acids containing a predicted Ribonuclease H-like domain (Figure 1B and Supplemental Figure 4). Therefore, we designated ORF1 as Ribonuclease H-Like 1 (RNHL-D1) and ORF2 as RNHL-D1.2, where D refers to D subgenome. Comparison of RNHL-D1 and RNHL-D1.2 sequences between Rht8- and rht8-harboring accessions identified only one sequence variation (1649CG→T) at position +1649 of RNHL-D1 exon that substitutes a CG in rht8 by a T in Rht8, which caused a frame-shift mutation (RNHL-d1 allele) and resulted in a truncated RNHL-D1 protein with only a small portion of the RNase H-like domain (Figure 1B and Supplemental Figure 5). Notably, the RNHL-D1.2 gene copy also harbors this 1649CG→T mutation (RNHL-d1.2). However, it shows no sequence polymorphism between Rht8 and rht8 genotypes, suggesting that RNHL-D1.2 is not related to Rht8 effect. Therefore, rht8 carries RNHL-D1 and RNHL-d1.2 (designated as D1d1.2), and Rht8 carries RNHL-d1 and RNHL-d1.2 (designated as d1d1.2) haplotypes, by which the mutated RNHL-d1, but not RNHL-d1.2, is most likely the contributor for Rht8. RNHL-D1-GFP protein locates in cell nuclei (Supplemental Figure 6A). The expression of RNHL-D1 was very high in stem and spike tissues before heading, but lower in roots and seedlings and very low in mature tissues after heading (Supplemental Figures 6B and 6C). To validate the in vivo effect of RNHL-D1, we knocked out this gene in Fielder (with RNHL-D1 haplotype) using the CRISPR/Cas9-based genome-editing technology. Two independent rnhl-d1 mutants (#1 and #2) showed significant reduction in plant height and spike length (SL) compared with the non-edited control (Figure 1C and Supplemental Figures 7–9). In addition, the RNHL-D1-overexpressing transgenic lines (OE-1 and OE-2) showed significantly increased plant height and SL (Supplemental Figures 9 and 10). To evaluate possible functions of the RNHL1 homoeologs from A (RNHL-A1, TraesCS2A02G059900) and B (RNHL-B1, TraesCS2B02G073600) subgenomes, we generated wheat plants with one mutation in each homoeolog (rnhl-a1 and rnhl-b1), with both mutations in the two homoeologs (rnhl-a1b1 and rnhl-b1d1) and with triple mutations in all the three homoeologs (rnhl-a1b1d1) (Supplemental Figure 7). As expected, rnhl-a1 and rnhl-b1 plants were all significantly shorter than the non-edited control. Importantly, all the double and triple mutants were much shorter than their single mutated ones, suggesting a significant dosage effect of RNHL1 homoeologs in regulating plant height (Figure 1D and Supplemental Figure 11). RNHL1 gene is conserved in both monocot and dicot plant species (Supplemental Figure 12 and Supplemental Data 1). For example, maize (Zea mays L., B73-329; Zm00001d004164 and Zm00001d025091) and Arabidopsis thaliana (Columbia-0, Col-0; AT1G12380 and AT1G62870) each carry two RNHL1 gene orthologs (Supplemental Figures 13–15). Notably, AT1G12380 and AT1G62870 differ from the three annotated RNase H1 proteins in Arabidopsis, AtRNH1A (AT3G01410), AtRNH1B (AT5G51080), and AtRNH1C (AT1G24090; Supplemental Figure 14) (Kuciński et al., 2020Kuciński J. Chamera S. Kmera A. Rowley M.J. Fujii S. Khurana P. Nowotny M. Wierzbicki A.T. Evolutionary history and activity of RNase H1-like proteins in Arabidopsis thaliana.Plant Cell Physiol. 2020; 61: 1107-1119Google Scholar), suggesting that the wheat RNHL1 and its orthologs in other plants are new, atypical Rnase H-like proteins. In maize, Zm00001d004164-knockout lines (Zm164-KO#1 and Zm164-KO#3) and Zm00001d025091-knockout lines (Zm091-KO#1, Zm091KO#2, and Zm091KO#3) all exhibited significant reduction in both plant and ear height compared with their non-edited controls (Figure 1E, Supplemental Figures 16 and 17). Similarly, Arabidopsis at1g12380/at1g62870 double mutants (#1 and #2) also showed reduced stature and shorter silique (Figure 1F, Supplemental Figures 16 and 17). These results support the highly conserved functions of RNHL1 orthologs in regulating plant growth across plant species. RNA sequencing revealed 747 differentially expressed genes (DEGs) between NIL-Rht8 and NIL-rht8 (Supplemental Figure 18 and Supplemental Data 2). Gene ontology (GO) analysis revealed that the genes for gibberellin (GA) biosynthetic process (GO: 0009686) and response to light stimulus (GO: 0009416) were downregulated in NIL-Rht8 (Figure 1G and Supplemental Data 3). Indeed, a gene encoding the key bioactive GA biosynthesis enzyme GA 3-β-dioxygenase 2 (TaGA3ox2, TraesCS3D02G124500) was dramatically downregulated by Rht8 (Figure 1H). However, genes for response to ethylene (ET) process (GO: 0009723), response to abscisic acid (ABA) (GO:0009737), and cell wall biogenesis (GO: 0042546) were most enriched among the upregulated DEGs (Figure 1G and Supplemental Data 3). Indeed, most of the ET- and ABA-responsive APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) genes were largely upregulated in NIL-Rht8 relative to NIL-rht8 (Figures 1H and 1I, Supplemental Figure 19). These results indicate the genetic repression effect of Rht8 on GA3ox2 transcription, but the promotion effect of Rht8 on AP2/ERF expression. We developed a cleaved amplified polymorphic sequence (CAPS) marker, CAPS-Rht8, as a diagnostic gene marker for selecting Rht8-allele (Supplemental Figure 20). Screening the world collection of 951 wheat accessions identified 310 accessions (32.60%) with Rht8 allele and 641 accessions with the rht8 allele (67.40%) (Figure 1J, Supplemental Figure 21, Supplemental Data 4). Geographic distribution analysis of the Chinese accessions found significantly higher frequency of Rht8 in modern cultivars than those in landraces (Figure 1K, Supplemental Data 5), suggesting a preferential selection of Rht8 during wheat breeding. In summary, we have isolated the candidate gene for Rht8 via map-based gene cloning, and confirmed that loss of RNHL-D1 is responsible for semi-dwarf trait in Rht8-carrying wheat plants (Figure 1L). The functional conservativeness of RNHL1 among wheat subgenomes suggests the possibility of designing plants with desirable height by engineering RNHL-D1 and its homoeologs. Furthermore, wheat RNHL-D1 and its orthologs could be used as the targets for improving other crops because of their sequence and possible function conservation across monocot and dicot species. This work was supported by the grants from the National Natural Science Foundation of China (grants 91935302 and 31991210 ) and Hainan Yazhou Bay Seed Laboratory ( B21HJ0111 ).