Gene

Agriculture

Ecology

Biotechnology

Biology

Computer science

Botany

Staple food

Computational biology

Genetics

Coleoptile

Crop

Rootstock

Seedling

Green Revolution

Cloning (programming)

Dwarfing

Agronomy

Grain yield

Horticulture

Programming language

Molecular Plant

Wheat (Triticum aestivum) is a major staple crop in the world and provides ∼20% of the food calories for human consumption (Appels et al., 2018Appels R. Eversole K. Feuillet C. Keller B. Rogers J. Stein N. Pozniak C.J. Stein N. Choulet F. Distelfeld A. et al.Shifting the limits in wheat research and breeding using a fully annotated reference genome.Science. 2018; 361: eaar7191Google Scholar). During the 1960s and 1970s, the introduction of two semi-dwarfing genes Rht-B1b and Rht-D1b in wheat varieties strikingly improved lodging resistance and harvest index, and therefore substantially increased grain yield, resulting in the well-known "Green Revolution" (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). However, plants carrying Rht-B1b and Rht-D1b suffer from several drawbacks, such as reduced coleoptile length and weak seedling vigor, which limit their cultivation in warm and dry environments (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). Thus, continuous efforts have been made to search for "Green Revolution" replacement genes in wheat. Rht8 is a semi-dwarfing gene that complements well the shortcomings of Rht-B1b and Rht-D1b, and thus has been widely introgressed into wheat varieties across the globe since its introduction from the Japanese variety Akakomugi into European wheat in the 1930s (Worland et al., 1998Worland J.A. Korzun V. Röder S.M. Ganal W.M. Law N.C. Genetic analysis of the dwarfing gene Rht8 in wheat. Part II. The distribution and adaptive significance of allelic variants at the Rht8 locus of wheat as revealed by microsatellite screening.Theor. Appl. Genet. 1998; 96: 1110-1120Google Scholar, Worland et al., 2001Worland A.J. Sayers E.J. Korzun V. Allelic variation at the dwarfing gene Rht8 locus and its significance in international breeding programmes.Euphytica. 2001; 119: 155-159Google Scholar; Zhang et al., 2006Zhang X.K. Yang S.J. Zhou Y. He Z.H. Xia X.C. Distribution of the Rht-B1b, Rht-D1b and Rht8 reduced height genes in autumn-sown Chinese wheats detected by molecular markers.Euphytica. 2006; 152: 109-116Google Scholar). The underlying causal gene of Rht8 was mapped to a genetic interval of 1.29 cM on chromosome 2DS in 2012 (Gasperini et al., 2012Gasperini D. Greenland A. Hedden P. Dreos R. Harwood W. Griffiths S. Genetic and physiological analysis of Rht8 in bread wheat: an alternative source of semi-dwarfism with a reduced sensitivity to brassinosteroids.J. Exp. Bot. 2012; 63: 4419-4436Google Scholar); however, cloning of Rht8 has remained an arduous task due to the complex nature of the wheat genome. In this study, we identified two new semi-dwarfing mutants named je0199 and je0105 in the wild-type (WT) background of Jing411 from our in-house wheat mutant library generated via ethyl methanesulfonate (EMS) treatment. Our phenotyping and genetic mapping analyses collectively suggested that these two newly identified EMS-induced mutants are allelic to Rht8, thus we renamed them Rht8-2 and Rht8-3, respectively. Rht8-2 and Rht8-3 effectively reduced plant height ∼20% and had no obvious negative impact on grain yield (Supplemental Figure 1). To identify the underlying gene, we developed two large independent mapping populations of Rht8-2 and Rht8-3. Using bulked segregant analysis and newly developed molecular markers, we fine-mapped Rht8-2 and Rht8-3 to a genomic region of ∼700 kb and 170 kb on chromosome 2DS, respectively (Figure 1A , Supplemental Figures 2 and 3). Unfortunately, the presence of a gap region of unknown size in the reference genome of Chinese Spring v1.0 precludes us from pinpointing the gene. Thus, we assembled the genome of Jing411 using PacBio Sequel I long read sequencing. The result showed that the mapped region contained two copies of TraesCSU03G0022100 (CS RefSeq v1.1 gene ID: TraesCSU02G024900). Intriguingly, one copy of them is likely a pseudogene with a reading frame shift, while the other copy retains an intact open reading frame, and thus likely encodes a function protein (Figure 1A). To verify the copy number of TraesCSU03G0022100 in this region, we examined the recently published reference genomes of Chinese Spring 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) and Fielder (Sato et al., 2021Sato K. Abe F. Mascher M. Haberer G. Gundlach H. Spannagl M. Shirasawa K. Isobe S. Chromosome-scale genome assembly of the transformation-amenable common wheat cultivar 'Fielder'.DNA Res. 2021; 28: dsab008Google Scholar). Intriguingly, Chinese Spring contained two copies, whereas Fielder contained three copies of TraesCSU03G0022100 (arranged in a tandem repeat fashion) in the mapped region. The difference in copy number of TraesCSU03G0022100 in Fielder versus Chinese Spring could be assembling errors as multiple gaps are contained in this region (Supplemental Figure 4). Gene BLAST of Rht8 against different wheat reference genomes also indicates errors easily occurred for assembly of Rht8 copies (Supplemental Data 1). Therefore, the actual copy number of TraesCSU03G0022100 awaits further experimental verification. TraesCSU03G0022100 encodes an RNase H-like protein carrying a zinc finger BED-type motif (Supplemental Figure 5). We found that Rht8-2 and Rht8-3 possessed distinct mutations that ultimately lead to premature stop codons in TraesCSU03G0022100 (Figure 1B, Supplemental Figure 3B). Intriguingly, we found that the original Rht8 variety Akakomugi harbored an identical mutation to Rht8-2. The presence of background-specific nucleotide sequences in Jing411, Rht8-2, and Akakomugi precluded the possibility of cross contamination of Rht8-2 by the original Rht8 locus (Supplemental Figure 6). CRISPR/Cas9 knockout mutants of TraesCSU03G0022100 exhibited reduced plant height, verifying that TraesCSU03G0022100 is the causal gene of Rht8 (Figure 1C, Supplemental Figure 7). Quantitative reverse transcriptase (RT)-PCR analysis showed that Rht8 was mainly expressed in the nodes of stem (Figure 1D) and its protein was localized to the nucleus (Figure 1E). Rht8 plants are gibberellic acid (GA) sensitive (Ellis et al., 2005Ellis M.H. Rebetzke G.J. Azanza F. Richards R.A. Spielmeyer W. Molecular mapping of gibberellin-responsive dwarfing genes in bread wheat.Theor. Appl. Genet. 2005; 111: 423-430Google Scholar). Consistently, we showed that treatment with exogenous GA3 could effectively restore the semi-dwarf phenotype of Rht8-2 and Rht8-3 (Figure 1F, Supplemental Figure 8), hinting at a role of Rht8 in regulating GA biosynthesis. We further demonstrated that GA biosynthetic genes, such as GA20ox-2 and GA13ox, were significantly downregulated in both mutants (Figure 1G, Supplemental Figure 9). We also analyzed the contents of various GAs among the two pathways for biosynthesis of GA1/GA3 and GA4/GA7 in the internode of Rht8-2 and Rht8-3 from the booting stage. The results showed that the level of bioactive GA3 was significantly decreased while the level of GA4 was significantly increased in the two mutants, compared with the WT plants (Figure 1H). GA13ox encodes an enzyme that catalyzes the convention of GA12 to GA53 for production of bioactive GA1 and GA3. The lower expression of this gene in the two mutants is consistent with the significantly decreased amount of GA3, which leads to the high amount of GA4. Therefore, we identified a novel gene that participates in GA biosynthesis regulation, thereby modulating the ratio of bioactive GA3 and GA4, and hence plant height. To analyze the effects of Rht8 on plant height and yield components, we backcrossed Rht8-2 to WT three times and obtained six near-isogenic lines by marker-assisted selection. The average plant height of WT lines ranged from 88 to 95 cm, while that of mutant genotypes ranged from 77.6 to 80 cm. The plants carrying Rht8-2 had significantly reduced plant height compared with the WT plants (Figure 1I). Despite being shorter, Rht8-2-carrying plants showed no significant reduction in thousand grain weight or kernels per spike (Figures 1J and 1K). To assess how Rht8 has been used in wheat breeding, we sequenced the Rht8 locus in 365 Chinese modern varieties, and found high frequency of Rht8 in the major wheat cultivation regions, such as up to 77.5% in the Yellow and Huai River Valleys, the most important wheat cultivation area producing ∼45% of total wheat grains in China (Supplemental Table 1). Notably, we found that the dwarfing variety Aikang 58, a high-yielding wheat cultivar widely cultivated in China with superior lodging resistance (Jia et al., 2021Jia J. Xie Y. Cheng J. Kong C. Wang M. Gao L. Zhao F. Guo J. Wang K. Li G. et al.Homology-mediated inter-chromosomal interactions in hexaploid wheat lead to specific subgenome territories following polyploidization and introgression.Genome Biol. 2021; 22: 26Google Scholar), harbors three Rht loci: Rht-D1b, Rht24, and Rht8. These results highlight the value of stacking Rht8 with other Rht genes in modern wheat breeding. Rht8 is located on the D subgenome in wheat, which is likely derived from a progenitor called Aegilops tauschii (diploid D). To track the evolutionary origin of Rht8, we analyzed the sequences of TraesCSU03G0022100 in 18 A. tauschii accessions and the results showed that none of them contained Rht8 allele, suggesting that the Rht8 allele in Akakomugi occurred after hexaploidization of common wheat. To sum up, the present work identifies two new semi-dwarf wheat mutants that are allelic to Rht8, and reveals the complexity and evolutionarily history of Rht8 in common wheat. Rht8 encodes a protein containing a zinc finger BED-type motif and an RNase H-like domain that regulates plant height via influencing bioactive GA biosynthesis. Modern wheat breeding has extensively utilized Rht8 itself, or stacked it together with other Rht loci to maximize lodging resistance and yield. Therefore, our results provide critical information for efficient application of Rht8 by marker-assisted and geographical-based breeding of environmentally resilient wheat cultivars. This work was financially supported by the National Key Research and Development Program of China (grant no. 2020YFE0202300 ), the National Natural Science Foundation of China (grant no. 31801346 and 32172040 ), the China Agriculture Research System of MOF and MARA (grant no. CARS-03 ), and the Agricultural Science and Technology Innovation Program (grant no. CAAS-ZDRW202109 ).

Cloning and functional characterization of Rht8, a “Green Revolution” replacement gene in wheat