Molecular mapping of a novel non-canonical gibberellin-sensitive dwarfing gene Rht29 in wheat (Triticum aestivum)

Molecular mapping of a novel non-canonical gibberellin-sensitive dwarfing gene Rht29 in wheat (Triticum aestivum) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Molecular mapping of a novel non-canonical gibberellin-sensitive dwarfing gene Rht29 in wheat (Triticum aestivum) Fuqiao Deng, Juanyu Zhang, Yanyan Tang, Furong Huang, Tao Li, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8634203/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 7 You are reading this latest preprint version Abstract Breeding semi-dwarf cultivars has long been a major objective for wheat improvement due to the inseparable association between plant height (PH) and grain yield. Although the utilization of Rht-B1b and Rht-D1b genes successfully achieved semi-dwarfism in the 1960s, these genes were associated with undesirable traits. The current wheat breeding urgently requires continuously exploring PH-controlling genes and their regulatory mechanisms, which will expand the genetic diversity of the PH gene pool to achieve precise PH regulation while maintaining or even increasing the grain yield potential. In this study, we identified a gibberellin (GA) - sensitive dwarf mutant, designated wph3 ( wheat plant height 3 ). It showed GA biosynthesis deficiency and had pleiotropic effects on PH, spike length, grain weight, and grain number per spike. Using Exome Capture Sequencing for Bulked Segregant Analysis and molecular marker mapping, a novel recessive nuclear dwarfing gene was identified and localized into a ~ 3.9 Mb physical interval on chromosome 2B, designated Rht29 ( Reduced height 29 ). Transcriptome analysis and candidate gene mining indicated that Rht29 may not encode a canonical key enzyme for GA biosynthesis, but participate in the GA biosynthesis by regulating the expression level of GA3ox . This study enriches the genetic resources available for wheat dwarfing breeding and establishes a foundation for further molecular characterization of phenotypic regulation by Rht29. Triticum aestivum Plant height Rht genes Gibberellin GA3oxidase Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Common wheat ( Triticum aestivum L.) is one of the most vital staple crops in the world, sustaining over one-third of the global population. Its yield and quality are thus directly linked to global food security. Plant height (PH) is a core but complex agronomic trait governed by both multiple quantitative trait loci (QTLs) and major genes. It critically determines lodging resistance and is intimately associated with harvest index, biomass partitioning, and yield potential (Song et al. 2023 ). Dwarfing alleles such as Rht-B1b and Rht-D1b successfully reduced wheat plant height, significantly improved lodging resistance and harvest index, thereby revolutionized wheat productivity during the Green Revolution in the 1960s (Mohan et al. 2021 , Chai et al. 2022 ). The primary dwarfing genes discovered in wheat were categorized into gibberellin (GA) signal transduction pathway genes (GA-insensitive type) and GA biosynthesis or deactivation pathway genes (GA-sensitive type). Rht-B1b and Rht-D1b belong to GA-insensitive type and encode N-terminally truncated DELLA proteins that conferred resistance to GA-dependent degradation, thereby suppressed stem elongation persistently. Conversely, there are some genes belonging to the GA-sensitive type. For example, Rht12 (Sun et al. 2019 ) and Rht18 (Grant et al. 2018 ) encode GA2-oxidase (GA2ox) A13 and GA2oxA9, respectively. The up-regulated expression of Rht12 and Rht18 significantly reduced endogenous GA levels, thereby reducing plant height (Ford et al. 2018 , Buss et al. 2020 ). Apart from genes directly participating in the GA biosynthesis and signaling pathways, dwarfing genes indirectly influencing these pathways have also been identified. The GA-sensitive gene Rht8 encoding a ribonuclease (RNase) H-like protein modulated bioactive GA ratios through regulating the expressions of GA biosynthetic enzyme genes. The Rht8 mutations effectively reduced plant height (Chai et al. 2022 , Xiong et al. 2022 ). Rht-13 is a nucleotide-binding site/leucine-rich repeat ( NB-LRR ) gene and is GA-sensitive. A point mutation in the semi-dwarfing Rht-B13b alle resulted in autoactivation of NB-LRR genes and up-regulation of pathogenesis-related genes, thereby enhancing cell wall cross-linking and ultimately inhibiting cell elongation (Borrill et al. 2022 ). Rht22 reduced plant height by disrupting cell proliferation and decreasing the number of internode cells, thereby shortening internodes(Wang et al. 2022 ). A recent study has revealed that GA-sensitive dwarfing gene Rht25 encodes a PLATZ transcription factor regulating wheat plant height through directly interacting with DELLA. Consequently, loss of function mutations in PLATZ-A1 reduced plant height (Zhang et al. 2023 ). Despite the remarkable success of these dwarfing genes, their widespread deployment has also revealed limitations and potential drawbacks, including reduced coleoptile length, diminished early growth vigor, decreased grain size, and potentially increased susceptibility to certain diseases (Guo et al. 2022 ). Furthermore, modern breeding's heavy reliance on this limited set of major loci has narrowed genetic diversity. However, less attention has been paid to plant height of wheat in recent years due to the widespread perception that the lodging seemed solved by the Green Revolution. In the face of intensifying global climate change, increased frequency of extreme weather events, and escalating threats from pests and diseases, as well as the unknown weakness of the existing repertoire of dwarfing genes, exploring new genes regulating plant height is still necessary. In this study, a plant height-reduced mutant was identified from an EMS-mutagenized population of an elite wheat variety Kechengmai 1 (K1), designated as wheat plant height 3 ( wph3 ). Phenotypical and genetic characterization of wph3 were performed. The Exome Capture Sequencing based Bulked Segregant Analysis (BSE) and subsequent molecular mapping were conducted to localize causal gene for wph3 in an approximately 3.9 Mb interval at the end of the long arm of chromosome 2B, representing a genetically novel dwarfing gene, designed as Rht29. We further analyzed its response to exogenous GA 3 application and endogenous GA content. Candidate gene and its potential mechanism in controlling plant height were also inferred and discussed by combining variant and transcriptome data. This research provided a novel dwarfing gene with breeding potential and laid the foundation for future cloning and functional characterization of Rht29 . Materials and methods Plant Materials Two elite wheat varieties, K1 and Kechengmai 6 (K6), were both developed and conserved by our lab. The dwarf mutant wph3 was generated by EMS mutagenesis of K1(Zhang et al. 2022 ). For genetic analysis, reciprocal crosses were performed between wph3 and K1, as well as between wph3 and K6. The hybrid seeds were collected and sown for summer propagation in Maerkang, Sichuan, China (31°54’25.99''N, 102°10'34.72''E, alt. 3025 m). Seeds of all F 1 of wph3 × K6 and K6 × wph3 were harvested and planted in Shifang, Sichuan, China (31°06'37.07''N, 104°09'09.70''E, alt. 521 m) to generate the F 2 genetic population RK6-F 2 for inheritance investigation and molecular mapping. Measurements of agronomic traits At the jointing stage, ten randomly selected plants of K1, K6, and wph3 , and all individuals of the population were phenotyped in the field for plant height (PH), spike length (SL), spikelet number per spike (SNS), and tiller number (TN). After seed harvest, thousand grain weight (TGW), grain length (GL), grain width (GW), and grain number per spike (GNS) were measured by using SC-G software (WSeen, Hangzhou, Zhejiang, China). All data were analyzed by Student’s t -test ( P < 0.05) or ANOVA. Exogenous GA treatment and endogenous GA content measurement GA 3 (MeilunBio ® ) was initially dissolved in a small amount of ethanol to formulate the 100 mM stock solution and then stored at 4℃. Thirty plants each of wph3 and K1 were grown in the greenhouse under a 16-h light/8-h dark photoperiod, with the temperature maintained at 24 ℃. At the initiation of the jointing stage, GA 3 stock solution was diluted 1000-fold then evenly sprayed onto the leaves and stems of both wph3 and K1. The control group was sprayed with the solution containing 0.1% ethanol. Both treatments were performed in three replicates, with each replicate consisting of five plants. For endogenous GA content measurement, thirty plants each of wph3 and K1 were planted in the greenhouse. The peduncle tissues of wph3 and K1 were collected during the jointing stage when the peduncles were about 2-3cm. Three biological replicates were performed, with each replicate consisting of samples from at least five uniformly growing plants. Fresh plant samples were immediately frozen in liquid nitrogen, ground into powder, and stored at -80 ℃ until needed. 50 mg sample from each replicate was weighed and transferred into a 2 mL microcentrifuge tube. GAs was extracted with 1 mL methanol/H 2 O/formic acid (15:4:1, v/v/v) and quantified by MetWare (Wuhan, Hubei, China) based on the AB Sciex QTRAP®6500 + LC-MS/MS platform. Exome capture sequencing based bulked segregant analysis (BSE) The BSE was employed to identify key loci responsible for reduced plant height in wph3 . At the flowering stage, leaves of the 30 individuals in RK6-F 2 population with extreme plant height were collected in equal amounts to construct two extreme phenotype bulks: a wild-type (WT) bulk and a dwarf bulk. Simultaneously, two parental pools were constructed using equal quantities leaf tissue from 30 individuals each of wph3 and K6. Library preparation and exome capture sequencing were performed by Tcuni Technology Co., Ltd. (Chengdu, Sichuan, China). The SpeedSeq pipeline was utilized for reads mapping and variant calling (Chiang et al. 2015 ). Bulk segregant analysis was conducted using the Gene Mapping module of the Tcuni platform ( https://www.wheatgmap.org ). Molecular marker development and gene mapping Single nucleotide polymorphisms (SNPs) within the target region were selected to design molecular markers, including kompetitive allele-specific PCR (KASP) markers and Sanger sequencing markers for genotyping. Marker design and application followed the procedures described previously (Pu et al. 2021 ). KASP markers were subsequently used to genotype a subset of the F 2 population to construct a genetic linkage map. Once a primary genetic interval was determined, the fine mapping was performed by designing more markers within this interval to genotype more individuals in the mapping population. The key recombinants were validated by performing genotyping and phenotyping on their progeny. RNA-seq analysis The peduncle tissues of K1 and wph3 were sampled when they elongated to 2–3 cm. The whole peduncles dissected from the plant was immediately flash-frozen in liquid nitrogen and subsequently stored at -80°C until further processing. To minimize experimental error, all samples were collected at the same time point. Three biological replicates were collected with each replicate consisting of samples from the main tiller of at least 10 individuals with similar growth status(Zhang et al. 2022 ). RNA extraction, library preparation, RNA-seq and subsequent analysis were performed by Biomarker Technologies Co., Ltd. (Beijing, China). Paired-end sequencing was conducted on the Illumina NovaSeq platform. HISAT2 (Kim et al. 2015 ) was used to align the clean reads to the Chinese Spring reference genome (IWGSC RefSeq v2.1) (Zhu et al. 2021 ). Gene expression levels were quantified as Fragments Per Kilobase of transcript per Million mapped reads (FPKM). Differentially expressed genes (DEGs) between wph3 and K1 were identified using DESeq2 (Love et al. 2014 ) with thresholds of false discovery rate (FDR) < 0.05 and absolute fold change ≥ 1.5. Finally, DEGs were functionally annotated via homology search against the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (Kanehisa et al. 2004 ) using DIAMOND (Buchfink et al. 2015 ). Results Phenotypic Characterization of wph3 A dwarf mutant, designated wph3 , was derived from EMS-induced mutagenesis of K1. From the appearance, the plant height and spike length were significantly decreased in wph3 compared to K1 (Fig. 1 a, b). The length of all stem internodes of wph3 was reduced and resulted in the 38.41% decrease in plant height (Fig. 1 c, d). The uppermost internode (peduncle) contributed the most. Measurement of agronomic traits indicated that compared to the WT, the SL of the wph3 decreased by 19.30% (Fig. 1 e), while there was no significant change in the SNS, GL and GW ( P > 0.05) (Fig. 1 f- 1 h). Additionally, the TGW, TN and GNS significantly decreased in wph3 (Fig. 1 i- 1 k). These results indicated that the mutation in wph3 showed pleiotropic effects on agronomic traits. Exogenous GA treatment and endogenous GA levels analysis Given that most dwarfing genes participate in GA biosynthesis or signaling pathways, we exogenously applied GA 3 to wph3 and the WT plants during the jointing stage. Our results indicated that both wph3 and the WT were responsive to GA 3 , and the dwarf phenotype of wph3 could be completely rescued by GA 3 treatment (Fig. 2 a, b), showing that wph3 is a GA-sensitive dwarf mutant. This result suggested potential endogenous bioactive GA deficiency in wph3 . Therefore, we further quantified the levels of endogenous bioactive GAs, along with precursors, intermediates and catabolites in the elongating peduncle. Compared to the WT, levels of bioactive GA 1 and GA 3 , synthesized through the early-13-hydroxylation (13-OH) pathway, were dramatically reduced by 68.71% and 28.22% in the wph3 , respectively. (Fig. 2 c). Meanwhile, the intermediates of GA 1 and GA 3, including GA 20 , GA 19 , GA 44 , and GA 53 in the 13-OH pathway were all decreased in wph3 , while GA 5 showed a slight increase. Consistently, the level of GA 8 , the catabolite of GA 1 , was lower in wph3 than in K1. Furthermore, the levels of bioactive GA 4 and its intermediates GA 15 and GA 24 and catabolite GA 34 in the non-13-hydroxylation (13-H) pathway were not detectable or decreased in wph3 , whereas the GA 9 slightly increased in wph3 . GA 12 , the precursor for bioactive GAs was not detectable in both whp3 and K1. These results suggested significant suppression of GA biosynthesis in wph3 . *, **, and *** indicated p < = 0.05, 0.01, and 0.001, respectively. n.s. indicated no significance. ND means not detectable. Genetic Analysis and molecular mapping of reduced plant height phenotype in wph3 Reciprocal crosses were performed between wph3 and each of two elite wheat varieties K1 and K6. All F 1 progeny from all crosses exhibited plant height comparable to that of the WT parents (K1 or K6) (Fig. 3 a, b). Meanwhile, the PH in the RK6-F 2 population was continuously distributed within the range of 40 to 110 cm, with most values concentrated between 60 and 90 cm (Fig. 3 c). Therefore, we hypothesize that multiple genetic loci regulating plant height may be present in the RK6-F 2 population. To identify the mutant gene in wph3 , we used the RK6-F 2 as the mapping population and performed BSE analysis with QTL-seq software (Takagi et al. 2013 ). A sharp peak of G ' value was identified on chromosome 2B, indicating that a candidate chromosome region of approximately 750.0-817.0 Mb was responsible for the dwarf phenotype (Fig. 3 d). We developed five KASP markers ( M1 - M5 , Table S1 ) to genotype a total of 200 F 2 individuals (Table S2) from the population, and constructed a genetic linkage map (Fig. 3 e). This map localized the target to an interval between markers M3 and M5 , corresponding to 799.5-807.3 Mb of chromosome 2B. Additional 562 F 2 individuals were further genotyped using markers M3 and M5 . As a result, 172 recombination events between the two markers were identified (Table S3). Five markers ( M6 - M10 , Table S4) were further developed to genotype the recombinants and their F 2:3 progeny, thereby further narrowing the candidate region. Finally, the candidate gene was mapped to a 3.9 Mb segment (799.5-803.4 Mb) on chromosome 2B, flanked by markers M8 and M10 (Fig. 3 f). The plants carrying genotypes of the two markers derived from wph3 exhibited a dwarf phenotype, indicating that the identified locus was derived from wph3 . No GA-sensitive dwarf genes or their homologues had been reported in this region previously. Therefore, we proposed that it is a novel locus regulating plant height, designated Reduced height 29 ( Rht29 ). Candidate gene mining Based on the Chinese Spring reference genome annotation (IWGSC RefSeq Annotation v2.1) and BSE results, a total of 26 high-confidence genes carrying high or moderate variants were identified within the target region (Table S5). Given that wph3 is an EMS-induced mutant of K1, the causal variant in wph3 should be unique, and not present in Chinese Spring, K6 or K1. Therefore, whole-genome resequencing data of K1 was integrated with the reference genome (Li et al. 2021 ) to filter out the non-causal SNPs in wph3 . Meanwhile, we focused on the typical EMS-induced guanine (G) → adenine (A) or cytosine (C) → thymine (T) transition. This integrated analysis resulted in only two candidate SNPs in two genes TraesCS2B03G1539200 and TraesCS2B03G1527800 (Table 1 ). TraesCS2B03G1539200 encodes a peroxidase and TraesCS2B03G1527800 encodes the SKP1-interacting partner 15 (SKIP15) (Table 1 ). The candidate SNPs on these two genes resulted in amino acid changes or splice region variants. Thus, they were predicted as the potential causal genes for Rht29 . Table 1 Candidate SNPs in the target interval. Position SNP Type Gene Variant DNA Variant Protein 800072182 C/T missense_variant TraesCS2B03G1527800 G1214A Gly405Asp 803273626 C/T missense_variant & splice_region_variant TraesCS2B03G1539200 G215A Gly72Asp Transcriptome analysis To further mine the genes and transcription network responsible for the dwarf phenotype of the wph3 , we collected peduncle samples of wph3 and K1 during the jointing stage for RNA sequencing (RNA-seq). Principal Component Analysis (PCA) revealed that the K1 and wph3 groups showed a clear clustering pattern in the 3D PCA space. Sample points within each group were relatively tightly clustered, indicating good consistency in transcriptome expression patterns among biological replicates of the same group (Fig. 4 a). Quantification of gene expression level using FPKM values was performed. From this dataset, we found that the candidate gene TraesCS2B03G1539200 was not expressed in either K1 or wph3 , while TraesCS2B03G1527800 exhibited no significant expression difference between wph3 and K1 (Fig. 4 c). This result indicated that TraesCS2B03G1539200 could be excluded from candidate genes. Based on the threshold of FDR < 0.05 and fold change ≥ 1.5, a total of 3479 DEGs, including 2228 up-regulated and 1251 down-regulated, were identified (Fig. 4 b). Since exogenous GA application could restore the PH phenotype of wph3 and wph3 exhibited apparent deficiency in bioactive GAs, as well as their intermediates and catabolites, we focused on DEGs related to the GA biosynthesis pathway. Several key enzymes, such as GA 13-oxidase (GA13ox), GA 20-oxidase (GA20ox) and GA3-oxidase (GA3ox), participate in converting the common precursor GA 12 to several intermediates and the bioactive GAs through 13-OH pathway (GA 1 and GA 3 ) and the 13-H pathway (GA 4 and GA 7 ) (Shani et al. 2024 ). The bioactive GAs could also be further deactivated by GA2-oxidase (GA2ox). According to our transcriptome data, seven genes encoding key enzymes of GA biosynthesis were detected to express in the peduncle and exhibit differential expression levels (Fig. 4 d). One DEG encoding GA20ox2 was up-regulated in wph3 , and four GA2oxs were identified showing down-regulated expression in wph3 compared to K1. Interestingly, two genes, annotated as GA3ox2-2 and GA3ox2-3 , encoding GA3ox, which catalyzes the key step for bioactive GA biosynthesis, exhibited significantly down-regulated expression in wph3 (Fig. 4 d). Effects of the Rht29 on agronomic traits To evaluate effects of Rht29 on agronomic traits in different genetic background, we genotyped RK6-F 2:3 population using a molecular marker M4 tightly linked with Rht29 and randomly selected 84 homozygous K6-type individuals and 73 mutant individuals for phenotyping. Compared to K6-type, wph3 -type showed average 34.13% decrease in PH, 15.35% decrease in SL, but similar SNS and TN (Fig. 5 a-d). For the grain-related traits, GL, GW, and TGW decreased by 5.99%, 6.58%, and 11.51% in wph3 , respectively (Fig. 5 e-g). Similar to that in the genetic background of K1, the 30.41% decrease of GNS was found in the wph3 -type plants (Fig. 5 h). PH segregation in the RK6-F 2 population did not show monogenic inheritance, suggesting potential additional dwarf gene(s) besides Rht29 between K6 and wph3 . Based on our exome sequencing data, we found a premature termination variant in K6 and a missense variant in wph3 of Rht-B1 , corresponding to alleles Rht-B1b and Rht-B1i , respectively (Table S6). We therefore analyzed interaction between Rht-B1 and Rht29 . We used molecular marker KASP_Rht-B1 linked with Rht-B1 (Li et al. 2022 ) and those linked with Rht29 to identify F 2 individuals homozygous at both loci. Under the background of Rht-B1b or Rht-B1i , mutant allele of Rht-29 ( Rht-B29b ) caused approximately 23% PH decrease. A greater reduction (32.67%) in PH could be observed when comparing the combination of Rht-B1b and Rht-B29b to the combination of Rht-B1i and WT Rht-29 ( Rht-B29a ) (Fig. 5 i). Discussion Rht29 is a novel wheat dwarfing gene In this study, a dwarfing gene was identified within the physical interval of 799.5-803.4 Mb on the long arm of chromosome 2B of wheat. Several Rht genes have been cloned or localized on the specific chromosomes. For example, Rht1 and Rht2 were located on chromosomes 4B and 4D, respectively (Peng et al. 1999 ); Rht5 was on chromosome 3B (Cui et al. 2022 ); Rht14 , Rht18 , Rht24 , and Rht25 were all located on chromosome 6A (Haque et al. 2011 , Ford et al. 2018 , Tian et al. 2022 , Zhang et al. 2023 ). Several Rht genes have been reported to be derived from the group 2 homoeologous chromosomes. Rht8 was derived from chromosome 2D and its homologous locus Rht8-2B was located on the short arm of chromosome 2B (Chai et al. 2022 ). Rht28 (Liu et al. 2025 ) was located at 558.90-562.08Mb on chromosome 2A, which was not collinear with the interval we defined for Rht29 . The homologues of the predicted candidate genes of Rht28 on chromosome 2B, TraesCS2B03G0888600 (495,354,127–495,360,583) and TraesCS2B03G0889100 (496,131,191–496,141,611), were far apart from Rht29 . Rht4 was the dwarfing gene closest to Rht29 on chromosome 2B (770–773 Mb) (Qiao 2020 ). However, it still did not overlap with the Rht29 interval. Its candidate gene, TraesCS2B03G1429200 , exhibited no variant and expression in K6 and wph3 . Additionally, other known genes associated with plant height on chromosome 2B, such as TaAP1-3-2B , TaAP1-2-2B , and TaARF12-2B (Li et al. 2019 , Li et al. 2022 ), also fall outside the Rht29 region. Therefore, Rht29 is a novel dwarfing gene. Rht29 is potentially a non-canonical GA-sensitive dwarfing gene In this study, Rht29 was characterized as a GA-sensitive dwarfing gene due to the two aspects: First, the PH of wph3 is sensitive to exogenous GA 3 application, which can rescue the dwarf phenotype of wph3. Second, wph3 showed apparent GA biosynthesis deficiency (Fig. 2 ). Quantitative analysis of GAs revealed that both the 13-OH and 13-H pathways were suppressed in the wph3 . The intermediates of the 13-OH pathway (e.g. GA 53 , GA 44 , GA 19 , GA 20 ) exhibited a more pronounced reduction compared to those of the 13-H pathway. This observation was consistent with previous study showing that GA biosynthesis in wheat mainly relies on the 13-OH pathway, with GA 1 as the major bioactive GA. In line with this, 13-hydroxylated GAs, such as GA 1 , GA 20 , and GA 19 , predominantly accumulate in vegetative tissues of wheat, whereas GA 4 from the 13-H pathway is present at very low levels in these tissues (Appleford et al. 2006 ). Typical GA-sensitive dwarfing genes usually encode key enzymes for bioactive GA synthesis or deactivation. For example, rice ‘green revolution’ gene sd1 encodes a GA20ox (Sasaki et al. 2002 ). In wheat, Rht12 (Sun et al. 2019 ) and Rht18 (Grant et al. 2018 ) encode GA2oxA13 and GA2oxA9, respectively. However, some non-canonical GA-sensitive dwarfing genes have also been identified. Rht8 encodes an RNase H-like protein which can modulate bioactive GA ratios by regulating the expressions of GA13ox and GA20ox (Xiong et al. 2022 ). GA-sensitive Rht13 encodes an NB-LRR protein (Borrill et al. 2022 ) and Rht25 encodes a PLATZ transcription factor, which regulated wheat PH via interacting with DELLA (Zhang et al. 2023 ). Two high-confidence genes, TraesCS2B03G1527800 and TraesCS2B03G1539200 , carrying wph3 -specific SNPs were identified within the Rht29 interval. Our transcriptome data showed that TraesCS2B03G1539200 was not expressed either in wph3 or in K1(Table S7). We also queried the spatiotemporal gene expression dataset of Chinese Spring (Consortium 2014 ) and found that it did not express in the stem. Thus, TraesCS2B03G1527800 is considered to be the most possible candidate gene for Rht29. However, it did not encode the known key enzymes for GA synthesis or metabolism, suggesting that Rht29 might be a non-canonical GA-sensitive dwarfing gene. Potential functions of Rht29 in GA-dependent PH modulation Previous studies have shown that expression of GA20ox and GA3ox genes is under negative feedback control of GA, whereas that of GA2ox genes is under positive feedforward control (Hedden and Phillips 2000 , Olszewski et al. 2002 ). Consistent with these reports, a GA20ox gene and four GA2ox genes of wph3 showed up-regulated and down-regulated expression in responsive to GA deficiency, respectively (Fig. 4 d). However, the expression of two detectable GA3ox genes in wph3 was significantly suppressed, which contradicted the negative feedback regulation pattern of GA. Therefore, we speculated that Rht29 may function in bioactive GA biosynthesis through regulating GA3ox expression. The candidate gene TraesCS2B03G1527800 is homologous to BAF1 of Arabidopsis , which encodes an F-box family E3 ubiquitin ligase. BAF1 interacts with BRI1-EMS-SUPPRESSOR1 (BES1), the core transcription factor of the brassinosteroid signaling pathway, and mediates its ubiquitination and degradation (Wang et al. 2021 ). Notably, BES1 plays a key role in the crosstalk between Brassinosteroids (BRs) and GA biosynthesis pathways. It has been reported that BES1 was able to interact with the promoter of GA3ox1D and activate its expression to promote fiber cell elongation in cotton (Hou et al. 2025 ). Therefore, it is possible that the mutation in BAF1 of wph3 may enhance the degradation of BES1, thereby preventing the activation of GA3ox expression. This model proposed the functional relevance between TraesCS2B03G1527800 and the dwarf phenotype of wph3 . Rht29 has pleiotropic effects on agronomic traits By comparing with WT and analyzing its effects in RK6 population, Rht29 was found to cause more than 30% PH and more than 15% SL decrease (Fig. 1 and Fig. 5 ). This combined effect was consistent with the GA-sensitive Rht8 (Chai et al. 2022 ) and Rht25 (Zhang et al. 2023 ), as well as GA-insensitive Rht-B1 (Song et al. 2023 ), indicating that this is a common feature of GA-related dwarfing genes. Rht29 showed relatively large degree of PH reduction under semi-dwarf genetic background and exhibited additive effects with different Rht-B1 alleles. Such effect may originate from the significant suppression of GA3ox and the consequent extensive inhibition of synthesis of bioactive GA 1 , GA 3 , and GA 4 (Fig. 2 ). This also led to a significant decrease in biomass, which may contribute to the decrease of the grain weight. In contrast, Rht8 functions in modulating the ratio of GA 3 and GA 4 by regulating the expression of GA20ox and GA13ox. Such a relatively mild change in GA synthesis generated ~ 20% PH decrease and had no obvious negative impact on grain yield (Xiong et al. 2022 ). Additionally, GA is closely associated with TN and inflorescence development. For example, the wheat ga3ox2 mutant showed approximately 3-fold lower concentration of the bioactive GA 1 compared to the WT, and exhibited significantly more TN, fewer spikelets, and complete sterility (Phillips et al. 2025 ). In this study, wph3 showed significantly down-regulated expression of GA3ox genes and a similar reduction of GA 1 , but exhibited different performance on tiller and inflorescence development compared to the ga3ox2 mutant. We observed ~ 30% TN reduction in wph3 compared with that in K1 (Fig. 1 k). However, this negative impact was undetectable in RK6 population, indicating that the influence of Rht29 on TN may be genetic background-dependent. Additionally, wph3 showed no difference in SNS under different backgrounds (Fig. 1 g and Fig. 5 c) and significantly decreased GNS (Fig. 1 k and Fig. 5 h), suggesting that Rht29 does not affect differentiation of spikelet. Although it remains unclear that whether Rht29 affects GNS by regulating the floret number or floret fertility, our results indicated that the pleiotropy of Rht29 on agronomic traits is not equivalent to that of the GA3ox mutant. Thus, further causal gene cloning, functional validation, and molecular mechanism dissection are worth conducting, which will facilitate gaining deeper insights into the mechanisms underlying the transcriptional regulation of GA3ox2 genes and the maintenance of bioactive GA homeostasis, as well as their pleiotropic effects on agronomic traits. This will lay the foundation for breaking the linkage between Rht29 and unfavorable traits and expanding wheat yield potential through fine tuning plant height in breeding. Author contribution statement GD and HL conceived and designed the study. YT developed mapping population, FD, YT, FH and GD undertook the field and greenhouse trials, sampling, phenotyping and genotyping, JZ, TL, JW, JL and HZ assisted in genetic mapping, FD, JZ, and HL analyzed data, drafted and revised the manuscript, and all authors reviewed the manuscript. Declarations Conflict of interest All authors declare that they have no conflict of interest. Funding This work is supported National Natural Science Foundation of China (32272125, 32301790), the National Key R&D Program of China (2024YFD1201200), Sichuan Science and Technology Program, China (2022ZDZX0014), and Sichuan Provincial Agricultural Department Innovative Research Team (SCCXTD-2024-11). Author Contribution GD and HL conceived and designed the study. YT developed mapping population, FD, YT, FH and GD undertook the field and greenhouse trials, sampling, phenotyping and genotyping, JZ, TL, JW, JL and HZ assisted in genetic mapping, FD, JZ, and HL analyzed data, drafted and revised the manuscript, and all authors reviewed the manuscript. Data Availability The BSA data and transcriptome data generated and used in this study have been deposited in The Genome Sequence Archive (GSA) (Accession No. XXXXXXXXX, under checked), Beijing Institute of Genomics, Chinese Academy of Sciences, and are publicly available. References Appleford, N. E. J., D. J. Evans, J. R. Lenton, P. Gaskin, S. J. Croker, K. M. Devos, A. L. Phillips and P. Hedden (2006). "Function and transcript analysis of gibberellin-biosynthetic enzymes in wheat." Planta 223(3): 568–582. Borrill, P., R. Mago, T. Xu, B. Ford, S. J. Williams, A. Derkx, W. D. Bovill, J. Hyles, D. Bhatt, X. Xia, C. MacMillan, R. White, W. Buss, I. Molnar, S. Walkowiak, O.-A. Olsen, J. Dolezel, C. J. 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Supplementary Files Table1CandidateSNPsinthetargetinterval.xlsx Supportingmaterial.xlsx Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 06 May, 2026 Reviews received at journal 21 Feb, 2026 Reviewers agreed at journal 13 Feb, 2026 Reviewers invited by journal 12 Feb, 2026 Editor assigned by journal 22 Jan, 2026 Submission checks completed at journal 19 Jan, 2026 First submitted to journal 18 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8634203","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":590859527,"identity":"e5d0a293-25fa-400a-9404-310fd3b11ec2","order_by":0,"name":"Fuqiao Deng","email":"","orcid":"","institution":"Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Fuqiao","middleName":"","lastName":"Deng","suffix":""},{"id":590859528,"identity":"4262ccc8-8e1a-4ef8-aaba-98392522428c","order_by":1,"name":"Juanyu Zhang","email":"","orcid":"","institution":"Chinese Academy of 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\u003c/strong\u003eindicated comparison of PH, SL, SNS, GL, GW, TGW, TN and GNS between \u003cem\u003ewph3\u003c/em\u003eand K1, respectively.\u003c/p\u003e","description":"","filename":"Fig.1Phenotypiccomparisonofwph3andK1.png","url":"https://assets-eu.researchsquare.com/files/rs-8634203/v1/974fa059ba0852b395927365.png"},{"id":102928843,"identity":"751d848c-5d02-4d5f-b5f0-ed0ce437c6d1","added_by":"auto","created_at":"2026-02-18 14:27:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1244552,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of relationship between GA and dwarf genotype of \u003cem\u003ewph3\u003c/em\u003e. \u003cstrong\u003ea \u003c/strong\u003eand \u003cstrong\u003eb\u003c/strong\u003e, The response of PH to exogenously applied GA\u003csub\u003e3\u003c/sub\u003e. \u003cstrong\u003ec\u003c/strong\u003e, Quantification of endogenous GA levels of the peduncle. The contents of GAs (ng / g fresh weight) were integrated with the sketch map of GA biosynthesis pathway (Adapted from Phillips et al., 2025).\u003c/p\u003e\n\u003cp\u003e*, **, and *** indicated \u003cem\u003ep\u003c/em\u003e\u0026lt;=0.05, 0.01, and 0.001, respectively. n.s. indicated no significance. ND means not detectable.\u003c/p\u003e","description":"","filename":"Fig.2AnalysisofrelationshipbetweenGAanddwarfgenotypeofwph3.png","url":"https://assets-eu.researchsquare.com/files/rs-8634203/v1/0a4a287ac50b05486b4de107.png"},{"id":102928714,"identity":"d845015f-c321-4e7b-870a-11fc64dc5382","added_by":"auto","created_at":"2026-02-18 14:26:50","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4206869,"visible":true,"origin":"","legend":"\u003cp\u003eGenetic analysis and molecular mapping of \u003cem\u003eRht29\u003c/em\u003e. \u003cstrong\u003ea \u003c/strong\u003eand \u003cstrong\u003eb\u003c/strong\u003e, Phenotypic comparison between parental lines and F\u003csub\u003e1\u003c/sub\u003e hybrids. \u003cstrong\u003ec\u003c/strong\u003e, Plotting of frequency distribution of plant height in the RK6-F\u003csub\u003e2\u003c/sub\u003e population. \u003cstrong\u003ed\u003c/strong\u003e, BSE based on plotting of \u003cem\u003eG’\u003c/em\u003e value on 21 chromosomes of wheat. \u003cstrong\u003ee\u003c/strong\u003e, Genetic map of \u003cem\u003eRht29\u003c/em\u003e. \u003cstrong\u003ef\u003c/strong\u003e, Fine mapping of \u003cem\u003eRht29\u003c/em\u003e. *** indicated \u003cem\u003ep\u003c/em\u003e\u0026lt;=0.001.\u003c/p\u003e","description":"","filename":"Fig.3GeneticanalysisandmolecularmappingofRht29.png","url":"https://assets-eu.researchsquare.com/files/rs-8634203/v1/22d55ce99be15d926584f2b8.png"},{"id":102928763,"identity":"bdd0689a-2cce-4d31-8825-94cff5981989","added_by":"auto","created_at":"2026-02-18 14:27:15","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2700726,"visible":true,"origin":"","legend":"\u003cp\u003eTranscriptome analysis of the elongating peduncle. \u003cstrong\u003ea\u003c/strong\u003e, Principal Component Analysis (PCA) based on DEGs. \u003cstrong\u003eb\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003eVolcano plotting of DEGs. \u003cstrong\u003ec\u003c/strong\u003e, The expression of candidate gene \u003cem\u003eTraesCS2B03G1527800\u003c/em\u003e in K1 and \u003cem\u003ewph3\u003c/em\u003e. \u003cstrong\u003ed\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003eComparison of expression levels of DEGs encoding key enzymes catalyzing bioactive GA biosynthesis and metabolism. FDR, False discovery rate; FC, fold change.\u003c/p\u003e","description":"","filename":"Fig.4Transcriptomeanalysisoftheelongatingpeduncle.png","url":"https://assets-eu.researchsquare.com/files/rs-8634203/v1/038ea49040f80b9a15478c58.png"},{"id":102928717,"identity":"7604db35-912d-4dc3-b769-0a735d57aeaa","added_by":"auto","created_at":"2026-02-18 14:26:51","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":4627474,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of the genetic effects of the mutant gene in the RK6-F\u003csub\u003e2:3\u003c/sub\u003e population. \u003cstrong\u003ea-h,\u003c/strong\u003e corresponded to PH, SL, SNS, TN, GL, GW, TGW, and GNS, respectively. \u003cstrong\u003ei,\u003c/strong\u003e indicated the interaction between \u003cem\u003eRht-B1 \u003c/em\u003eand \u003cem\u003eRht-29\u003c/em\u003e (\u003cem\u003eRht-B29\u003c/em\u003e). + or - for \u003cem\u003eRht-B1\u003c/em\u003e indicated \u003cem\u003eRht-B1b\u003c/em\u003e or \u003cem\u003eRht-B1i\u003c/em\u003e, and for \u003cem\u003eRht-29\u003c/em\u003e indicated WT\u003cem\u003e \u003c/em\u003e(\u003cem\u003eRht-B29a\u003c/em\u003e)\u003cem\u003e \u003c/em\u003eor mutant type (\u003cem\u003eRht-B29b\u003c/em\u003e), respectively. *** indicated \u003cem\u003ep\u003c/em\u003e\u0026lt;=0.001, n.s. indicated no significant difference.\u003c/p\u003e","description":"","filename":"Fig.5AnalysisofthegeneticeffectsofthemutantgeneintheRK6F23population.png","url":"https://assets-eu.researchsquare.com/files/rs-8634203/v1/0ab0879ecd1fe41410707ad5.png"},{"id":102928857,"identity":"0900dbff-4d39-4a39-9e60-fef222109a48","added_by":"auto","created_at":"2026-02-18 14:27:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":18679961,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8634203/v1/dcfdfadd-db06-4b44-99a8-5ee63237731d.pdf"},{"id":102928759,"identity":"b98b18aa-7a6c-475f-b472-b0175c56ec82","added_by":"auto","created_at":"2026-02-18 14:27:07","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":10171,"visible":true,"origin":"","legend":"","description":"","filename":"Table1CandidateSNPsinthetargetinterval.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8634203/v1/d04f0d73e14623765dbe5a95.xlsx"},{"id":102928754,"identity":"62fc78f4-d85e-44ba-85fc-f6a2dfdd24c8","added_by":"auto","created_at":"2026-02-18 14:27:06","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":38247,"visible":true,"origin":"","legend":"","description":"","filename":"Supportingmaterial.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8634203/v1/c71f91e0beacf287f2cb945c.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Molecular mapping of a novel non-canonical gibberellin-sensitive dwarfing gene Rht29 in wheat (Triticum aestivum)","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCommon wheat (\u003cem\u003eTriticum aestivum\u003c/em\u003e L.) is one of the most vital staple crops in the world, sustaining over one-third of the global population. Its yield and quality are thus directly linked to global food security. Plant height (PH) is a core but complex agronomic trait governed by both multiple quantitative trait loci (QTLs) and major genes. It critically determines lodging resistance and is intimately associated with harvest index, biomass partitioning, and yield potential (Song et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Dwarfing alleles such as \u003cem\u003eRht-B1b\u003c/em\u003e and \u003cem\u003eRht-D1b\u003c/em\u003e successfully reduced wheat plant height, significantly improved lodging resistance and harvest index, thereby revolutionized wheat productivity during the Green Revolution in the 1960s (Mohan et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Chai et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe primary dwarfing genes discovered in wheat were categorized into gibberellin (GA) signal transduction pathway genes (GA-insensitive type) and GA biosynthesis or deactivation pathway genes (GA-sensitive type). \u003cem\u003eRht-B1b\u003c/em\u003e and \u003cem\u003eRht-D1b\u003c/em\u003e belong to GA-insensitive type and encode N-terminally truncated DELLA proteins that conferred resistance to GA-dependent degradation, thereby suppressed stem elongation persistently. Conversely, there are some genes belonging to the GA-sensitive type. For example, \u003cem\u003eRht12\u003c/em\u003e (Sun et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and \u003cem\u003eRht18\u003c/em\u003e (Grant et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) encode GA2-oxidase (GA2ox) A13 and GA2oxA9, respectively. The up-regulated expression of \u003cem\u003eRht12\u003c/em\u003e and \u003cem\u003eRht18\u003c/em\u003e significantly reduced endogenous GA levels, thereby reducing plant height (Ford et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, Buss et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eApart from genes directly participating in the GA biosynthesis and signaling pathways, dwarfing genes indirectly influencing these pathways have also been identified. The GA-sensitive gene \u003cem\u003eRht8\u003c/em\u003e encoding a ribonuclease (RNase) H-like protein modulated bioactive GA ratios through regulating the expressions of GA biosynthetic enzyme genes. The \u003cem\u003eRht8\u003c/em\u003e mutations effectively reduced plant height (Chai et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Xiong et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). \u003cem\u003eRht-13\u003c/em\u003e is a \u003cem\u003enucleotide-binding site/leucine-rich repeat\u003c/em\u003e (\u003cem\u003eNB-LRR\u003c/em\u003e) gene and is GA-sensitive. A point mutation in the semi-dwarfing \u003cem\u003eRht-B13b\u003c/em\u003e alle resulted in autoactivation of \u003cem\u003eNB-LRR\u003c/em\u003e genes and up-regulation of pathogenesis-related genes, thereby enhancing cell wall cross-linking and ultimately inhibiting cell elongation (Borrill et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). \u003cem\u003eRht22\u003c/em\u003e reduced plant height by disrupting cell proliferation and decreasing the number of internode cells, thereby shortening internodes(Wang et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). A recent study has revealed that GA-sensitive dwarfing gene \u003cem\u003eRht25\u003c/em\u003e encodes a PLATZ transcription factor regulating wheat plant height through directly interacting with DELLA. Consequently, loss of function mutations in \u003cem\u003ePLATZ-A1\u003c/em\u003e reduced plant height (Zhang et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDespite the remarkable success of these dwarfing genes, their widespread deployment has also revealed limitations and potential drawbacks, including reduced coleoptile length, diminished early growth vigor, decreased grain size, and potentially increased susceptibility to certain diseases (Guo et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Furthermore, modern breeding's heavy reliance on this limited set of major loci has narrowed genetic diversity. However, less attention has been paid to plant height of wheat in recent years due to the widespread perception that the lodging seemed solved by the Green Revolution. In the face of intensifying global climate change, increased frequency of extreme weather events, and escalating threats from pests and diseases, as well as the unknown weakness of the existing repertoire of dwarfing genes, exploring new genes regulating plant height is still necessary.\u003c/p\u003e \u003cp\u003eIn this study, a plant height-reduced mutant was identified from an EMS-mutagenized population of an elite wheat variety Kechengmai 1 (K1), designated as \u003cem\u003ewheat plant height 3\u003c/em\u003e (\u003cem\u003ewph3\u003c/em\u003e). Phenotypical and genetic characterization of \u003cem\u003ewph3\u003c/em\u003e were performed. The Exome Capture Sequencing based Bulked Segregant Analysis (BSE) and subsequent molecular mapping were conducted to localize causal gene for \u003cem\u003ewph3\u003c/em\u003e in an approximately 3.9 Mb interval at the end of the long arm of chromosome 2B, representing a genetically novel dwarfing gene, designed as \u003cem\u003eRht29.\u003c/em\u003e We further analyzed its response to exogenous GA\u003csub\u003e3\u003c/sub\u003e application and endogenous GA content. Candidate gene and its potential mechanism in controlling plant height were also inferred and discussed by combining variant and transcriptome data. This research provided a novel dwarfing gene with breeding potential and laid the foundation for future cloning and functional characterization of \u003cem\u003eRht29\u003c/em\u003e.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant Materials\u003c/h2\u003e \u003cp\u003eTwo elite wheat varieties, K1 and Kechengmai 6 (K6), were both developed and conserved by our lab. The dwarf mutant \u003cem\u003ewph3\u003c/em\u003e was generated by EMS mutagenesis of K1(Zhang et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). For genetic analysis, reciprocal crosses were performed between \u003cem\u003ewph3\u003c/em\u003e and K1, as well as between \u003cem\u003ewph3\u003c/em\u003e and K6. The hybrid seeds were collected and sown for summer propagation in Maerkang, Sichuan, China (31\u0026deg;54\u0026rsquo;25.99''N, 102\u0026deg;10'34.72''E, alt. 3025 m). Seeds of all F\u003csub\u003e1\u003c/sub\u003e of \u003cem\u003ewph3\u003c/em\u003e \u0026times; K6 and K6 \u0026times; \u003cem\u003ewph3\u003c/em\u003e were harvested and planted in Shifang, Sichuan, China (31\u0026deg;06'37.07''N, 104\u0026deg;09'09.70''E, alt. 521 m) to generate the F\u003csub\u003e2\u003c/sub\u003e genetic population RK6-F\u003csub\u003e2\u003c/sub\u003e for inheritance investigation and molecular mapping.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMeasurements of agronomic traits\u003c/h3\u003e\n\u003cp\u003eAt the jointing stage, ten randomly selected plants of K1, K6, and \u003cem\u003ewph3\u003c/em\u003e, and all individuals of the population were phenotyped in the field for plant height (PH), spike length (SL), spikelet number per spike (SNS), and tiller number (TN). After seed harvest, thousand grain weight (TGW), grain length (GL), grain width (GW), and grain number per spike (GNS) were measured by using SC-G software (WSeen, Hangzhou, Zhejiang, China). All data were analyzed by Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) or ANOVA.\u003c/p\u003e\n\u003ch3\u003eExogenous GA treatment and endogenous GA content measurement\u003c/h3\u003e\n\u003cp\u003eGA\u003csub\u003e3\u003c/sub\u003e (MeilunBio\u003csup\u003e\u0026reg;\u003c/sup\u003e) was initially dissolved in a small amount of ethanol to formulate the 100 mM stock solution and then stored at 4℃. Thirty plants each of \u003cem\u003ewph3\u003c/em\u003e and K1 were grown in the greenhouse under a 16-h light/8-h dark photoperiod, with the temperature maintained at 24 ℃. At the initiation of the jointing stage, GA\u003csub\u003e3\u003c/sub\u003e stock solution was diluted 1000-fold then evenly sprayed onto the leaves and stems of both \u003cem\u003ewph3\u003c/em\u003e and K1. The control group was sprayed with the solution containing 0.1% ethanol. Both treatments were performed in three replicates, with each replicate consisting of five plants.\u003c/p\u003e \u003cp\u003eFor endogenous GA content measurement, thirty plants each of \u003cem\u003ewph3\u003c/em\u003e and K1 were planted in the greenhouse. The peduncle tissues of \u003cem\u003ewph3\u003c/em\u003e and K1 were collected during the jointing stage when the peduncles were about 2-3cm. Three biological replicates were performed, with each replicate consisting of samples from at least five uniformly growing plants. Fresh plant samples were immediately frozen in liquid nitrogen, ground into powder, and stored at -80 ℃ until needed. 50 mg sample from each replicate was weighed and transferred into a 2 mL microcentrifuge tube. GAs was extracted with 1 mL methanol/H\u003csub\u003e2\u003c/sub\u003eO/formic acid (15:4:1, v/v/v) and quantified by MetWare (Wuhan, Hubei, China) based on the AB Sciex QTRAP\u0026reg;6500\u0026thinsp;+\u0026thinsp;LC-MS/MS platform.\u003c/p\u003e\n\u003ch3\u003eExome capture sequencing based bulked segregant analysis (BSE)\u003c/h3\u003e\n\u003cp\u003eThe BSE was employed to identify key loci responsible for reduced plant height in \u003cem\u003ewph3\u003c/em\u003e. At the flowering stage, leaves of the 30 individuals in RK6-F\u003csub\u003e2\u003c/sub\u003e population with extreme plant height were collected in equal amounts to construct two extreme phenotype bulks: a wild-type (WT) bulk and a dwarf bulk. Simultaneously, two parental pools were constructed using equal quantities leaf tissue from 30 individuals each of \u003cem\u003ewph3\u003c/em\u003e and K6.\u003c/p\u003e \u003cp\u003eLibrary preparation and exome capture sequencing were performed by Tcuni Technology Co., Ltd. (Chengdu, Sichuan, China). The SpeedSeq pipeline was utilized for reads mapping and variant calling (Chiang et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Bulk segregant analysis was conducted using the Gene Mapping module of the Tcuni platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.wheatgmap.org\u003c/span\u003e\u003cspan address=\"https://www.wheatgmap.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eMolecular marker development and gene mapping\u003c/h3\u003e\n\u003cp\u003eSingle nucleotide polymorphisms (SNPs) within the target region were selected to design molecular markers, including kompetitive allele-specific PCR (KASP) markers and Sanger sequencing markers for genotyping. Marker design and application followed the procedures described previously (Pu et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). KASP markers were subsequently used to genotype a subset of the F\u003csub\u003e2\u003c/sub\u003e population to construct a genetic linkage map. Once a primary genetic interval was determined, the fine mapping was performed by designing more markers within this interval to genotype more individuals in the mapping population. The key recombinants were validated by performing genotyping and phenotyping on their progeny.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eRNA-seq analysis\u003c/h2\u003e \u003cp\u003eThe peduncle tissues of K1 and \u003cem\u003ewph3\u003c/em\u003e were sampled when they elongated to 2\u0026ndash;3 cm. The whole peduncles dissected from the plant was immediately flash-frozen in liquid nitrogen and subsequently stored at -80\u0026deg;C until further processing. To minimize experimental error, all samples were collected at the same time point. Three biological replicates were collected with each replicate consisting of samples from the main tiller of at least 10 individuals with similar growth status(Zhang et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRNA extraction, library preparation, RNA-seq and subsequent analysis were performed by Biomarker Technologies Co., Ltd. (Beijing, China). Paired-end sequencing was conducted on the Illumina NovaSeq platform. HISAT2 (Kim et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) was used to align the clean reads to the Chinese Spring reference genome (IWGSC RefSeq v2.1) (Zhu et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Gene expression levels were quantified as Fragments Per Kilobase of transcript per Million mapped reads (FPKM). Differentially expressed genes (DEGs) between \u003cem\u003ewph3\u003c/em\u003e and K1 were identified using DESeq2 (Love et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) with thresholds of false discovery rate (FDR)\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and absolute fold change\u0026thinsp;\u0026ge;\u0026thinsp;1.5. Finally, DEGs were functionally annotated via homology search against the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (Kanehisa et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2004\u003c/span\u003e) using DIAMOND (Buchfink et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003ePhenotypic Characterization of\u003c/b\u003e \u003cb\u003ewph3\u003c/b\u003e\u003c/p\u003e \u003cp\u003eA dwarf mutant, designated \u003cem\u003ewph3\u003c/em\u003e, was derived from EMS-induced mutagenesis of K1. From the appearance, the plant height and spike length were significantly decreased in \u003cem\u003ewph3\u003c/em\u003e compared to K1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, b). The length of all stem internodes of \u003cem\u003ewph3\u003c/em\u003e was reduced and resulted in the 38.41% decrease in plant height (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, d). The uppermost internode (peduncle) contributed the most. Measurement of agronomic traits indicated that compared to the WT, the SL of the \u003cem\u003ewph3\u003c/em\u003e decreased by 19.30% (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee), while there was no significant change in the SNS, GL and GW (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh). Additionally, the TGW, TN and GNS significantly decreased in \u003cem\u003ewph3\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ek). These results indicated that the mutation in \u003cem\u003ewph3\u003c/em\u003e showed pleiotropic effects on agronomic traits.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eExogenous GA treatment and endogenous GA levels analysis\u003c/h3\u003e\n\u003cp\u003eGiven that most dwarfing genes participate in GA biosynthesis or signaling pathways, we exogenously applied GA\u003csub\u003e3\u003c/sub\u003e to \u003cem\u003ewph3\u003c/em\u003e and the WT plants during the jointing stage. Our results indicated that both \u003cem\u003ewph3\u003c/em\u003e and the WT were responsive to GA\u003csub\u003e3\u003c/sub\u003e, and the dwarf phenotype of \u003cem\u003ewph3\u003c/em\u003e could be completely rescued by GA\u003csub\u003e3\u003c/sub\u003e treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, b), showing that \u003cem\u003ewph3\u003c/em\u003e is a GA-sensitive dwarf mutant. This result suggested potential endogenous bioactive GA deficiency in \u003cem\u003ewph3\u003c/em\u003e. Therefore, we further quantified the levels of endogenous bioactive GAs, along with precursors, intermediates and catabolites in the elongating peduncle. Compared to the WT, levels of bioactive GA\u003csub\u003e1\u003c/sub\u003e and GA\u003csub\u003e3\u003c/sub\u003e, synthesized through the early-13-hydroxylation (13-OH) pathway, were dramatically reduced by 68.71% and 28.22% in the \u003cem\u003ewph3\u003c/em\u003e, respectively. (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Meanwhile, the intermediates of GA\u003csub\u003e1\u003c/sub\u003e and GA\u003csub\u003e3,\u003c/sub\u003e including GA\u003csub\u003e20\u003c/sub\u003e, GA\u003csub\u003e19\u003c/sub\u003e, GA\u003csub\u003e44\u003c/sub\u003e, and GA\u003csub\u003e53\u003c/sub\u003e in the 13-OH pathway were all decreased in \u003cem\u003ewph3\u003c/em\u003e, while GA\u003csub\u003e5\u003c/sub\u003e showed a slight increase. Consistently, the level of GA\u003csub\u003e8\u003c/sub\u003e, the catabolite of GA\u003csub\u003e1\u003c/sub\u003e, was lower in \u003cem\u003ewph3\u003c/em\u003e than in K1.\u003c/p\u003e \u003cp\u003eFurthermore, the levels of bioactive GA\u003csub\u003e4\u003c/sub\u003e and its intermediates GA\u003csub\u003e15\u003c/sub\u003e and GA\u003csub\u003e24\u003c/sub\u003e and catabolite GA\u003csub\u003e34\u003c/sub\u003e in the non-13-hydroxylation (13-H) pathway were not detectable or decreased in \u003cem\u003ewph3\u003c/em\u003e, whereas the GA\u003csub\u003e9\u003c/sub\u003e slightly increased in \u003cem\u003ewph3\u003c/em\u003e. GA\u003csub\u003e12\u003c/sub\u003e, the precursor for bioactive GAs was not detectable in both \u003cem\u003ewhp3\u003c/em\u003e and K1. These results suggested significant suppression of GA biosynthesis in \u003cem\u003ewph3\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e*, **, and *** indicated \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;=\u0026thinsp;0.05, 0.01, and 0.001, respectively. n.s. indicated no significance. ND means not detectable.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGenetic Analysis and molecular mapping of reduced plant height phenotype in\u003c/b\u003e \u003cb\u003ewph3\u003c/b\u003e\u003c/p\u003e \u003cp\u003eReciprocal crosses were performed between \u003cem\u003ewph3\u003c/em\u003e and each of two elite wheat varieties K1 and K6. All F\u003csub\u003e1\u003c/sub\u003e progeny from all crosses exhibited plant height comparable to that of the WT parents (K1 or K6) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, b). Meanwhile, the PH in the RK6-F\u003csub\u003e2\u003c/sub\u003e population was continuously distributed within the range of 40 to 110 cm, with most values concentrated between 60 and 90 cm (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Therefore, we hypothesize that multiple genetic loci regulating plant height may be present in the RK6-F\u003csub\u003e2\u003c/sub\u003e population.\u003c/p\u003e \u003cp\u003eTo identify the mutant gene in \u003cem\u003ewph3\u003c/em\u003e, we used the RK6-F\u003csub\u003e2\u003c/sub\u003e as the mapping population and performed BSE analysis with QTL-seq software (Takagi et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). A sharp peak of \u003cem\u003eG\u003c/em\u003e' value was identified on chromosome 2B, indicating that a candidate chromosome region of approximately 750.0-817.0 Mb was responsible for the dwarf phenotype (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). We developed five KASP markers (\u003cem\u003eM1\u003c/em\u003e-\u003cem\u003eM5\u003c/em\u003e, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) to genotype a total of 200 F\u003csub\u003e2\u003c/sub\u003e individuals (Table S2) from the population, and constructed a genetic linkage map (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). This map localized the target to an interval between markers \u003cem\u003eM3\u003c/em\u003e and \u003cem\u003eM5\u003c/em\u003e, corresponding to 799.5-807.3 Mb of chromosome 2B. Additional 562 F\u003csub\u003e2\u003c/sub\u003e individuals were further genotyped using markers \u003cem\u003eM3\u003c/em\u003e and \u003cem\u003eM5\u003c/em\u003e. As a result, 172 recombination events between the two markers were identified (Table S3). Five markers (\u003cem\u003eM6\u003c/em\u003e-\u003cem\u003eM10\u003c/em\u003e, Table S4) were further developed to genotype the recombinants and their F\u003csub\u003e2:3\u003c/sub\u003e progeny, thereby further narrowing the candidate region. Finally, the candidate gene was mapped to a 3.9 Mb segment (799.5-803.4 Mb) on chromosome 2B, flanked by markers \u003cem\u003eM8\u003c/em\u003e and \u003cem\u003eM10\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef). The plants carrying genotypes of the two markers derived from \u003cem\u003ewph3\u003c/em\u003e exhibited a dwarf phenotype, indicating that the identified locus was derived from \u003cem\u003ewph3\u003c/em\u003e. No GA-sensitive dwarf genes or their homologues had been reported in this region previously. Therefore, we proposed that it is a novel locus regulating plant height, designated \u003cem\u003eReduced height 29\u003c/em\u003e (\u003cem\u003eRht29\u003c/em\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCandidate gene mining\u003c/h2\u003e \u003cp\u003eBased on the Chinese Spring reference genome annotation (IWGSC RefSeq Annotation v2.1) and BSE results, a total of 26 high-confidence genes carrying high or moderate variants were identified within the target region (Table S5). Given that \u003cem\u003ewph3\u003c/em\u003e is an EMS-induced mutant of K1, the causal variant in \u003cem\u003ewph3\u003c/em\u003e should be unique, and not present in Chinese Spring, K6 or K1. Therefore, whole-genome resequencing data of K1 was integrated with the reference genome (Li et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) to filter out the non-causal SNPs in \u003cem\u003ewph3\u003c/em\u003e. Meanwhile, we focused on the typical EMS-induced guanine (G) \u0026rarr; adenine (A) or cytosine (C) \u0026rarr; thymine (T) transition. This integrated analysis resulted in only two candidate SNPs in two genes \u003cem\u003eTraesCS2B03G1539200\u003c/em\u003e and \u003cem\u003eTraesCS2B03G1527800\u003c/em\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). \u003cem\u003eTraesCS2B03G1539200\u003c/em\u003e encodes a peroxidase and \u003cem\u003eTraesCS2B03G1527800\u003c/em\u003e encodes the SKP1-interacting partner 15 (SKIP15) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The candidate SNPs on these two genes resulted in amino acid changes or splice region variants. Thus, they were predicted as the potential causal genes for \u003cem\u003eRht29\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCandidate SNPs in the target interval.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePosition\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSNP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eType\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGene\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eVariant DNA\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eVariant Protein\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e800072182\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC/T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003emissense_variant\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTraesCS2B03G1527800\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eG1214A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eGly405Asp\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e803273626\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC/T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003emissense_variant \u0026amp;\u003c/p\u003e \u003cp\u003esplice_region_variant\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTraesCS2B03G1539200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eG215A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eGly72Asp\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eTranscriptome analysis\u003c/h2\u003e \u003cp\u003eTo further mine the genes and transcription network responsible for the dwarf phenotype of the \u003cem\u003ewph3\u003c/em\u003e, we collected peduncle samples of \u003cem\u003ewph3\u003c/em\u003e and K1 during the jointing stage for RNA sequencing (RNA-seq). Principal Component Analysis (PCA) revealed that the K1 and \u003cem\u003ewph3\u003c/em\u003e groups showed a clear clustering pattern in the 3D PCA space. Sample points within each group were relatively tightly clustered, indicating good consistency in transcriptome expression patterns among biological replicates of the same group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Quantification of gene expression level using FPKM values was performed. From this dataset, we found that the candidate gene \u003cem\u003eTraesCS2B03G1539200\u003c/em\u003e was not expressed in either K1 or \u003cem\u003ewph3\u003c/em\u003e, while \u003cem\u003eTraesCS2B03G1527800\u003c/em\u003e exhibited no significant expression difference between \u003cem\u003ewph3\u003c/em\u003e and K1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). This result indicated that \u003cem\u003eTraesCS2B03G1539200\u003c/em\u003e could be excluded from candidate genes.\u003c/p\u003e \u003cp\u003eBased on the threshold of FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and fold change\u0026thinsp;\u0026ge;\u0026thinsp;1.5, a total of 3479 DEGs, including 2228 up-regulated and 1251 down-regulated, were identified (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Since exogenous GA application could restore the PH phenotype of \u003cem\u003ewph3\u003c/em\u003e and \u003cem\u003ewph3\u003c/em\u003e exhibited apparent deficiency in bioactive GAs, as well as their intermediates and catabolites, we focused on DEGs related to the GA biosynthesis pathway. Several key enzymes, such as GA 13-oxidase (GA13ox), GA 20-oxidase (GA20ox) and GA3-oxidase (GA3ox), participate in converting the common precursor GA\u003csub\u003e12\u003c/sub\u003e to several intermediates and the bioactive GAs through 13-OH pathway (GA\u003csub\u003e1\u003c/sub\u003e and GA\u003csub\u003e3\u003c/sub\u003e) and the 13-H pathway (GA\u003csub\u003e4\u003c/sub\u003e and GA\u003csub\u003e7\u003c/sub\u003e) (Shani et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The bioactive GAs could also be further deactivated by GA2-oxidase (GA2ox). According to our transcriptome data, seven genes encoding key enzymes of GA biosynthesis were detected to express in the peduncle and exhibit differential expression levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). One DEG encoding GA20ox2 was up-regulated in \u003cem\u003ewph3\u003c/em\u003e, and four \u003cem\u003eGA2oxs\u003c/em\u003e were identified showing down-regulated expression in \u003cem\u003ewph3\u003c/em\u003e compared to K1. Interestingly, two genes, annotated as \u003cem\u003eGA3ox2-2\u003c/em\u003e and \u003cem\u003eGA3ox2-3\u003c/em\u003e, encoding GA3ox, which catalyzes the key step for bioactive GA biosynthesis, exhibited significantly down-regulated expression in \u003cem\u003ewph3\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of the\u003c/b\u003e \u003cb\u003eRht29\u003c/b\u003e \u003cb\u003eon agronomic traits\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo evaluate effects of \u003cem\u003eRht29\u003c/em\u003e on agronomic traits in different genetic background, we genotyped RK6-F\u003csub\u003e2:3\u003c/sub\u003e population using a molecular marker \u003cem\u003eM4\u003c/em\u003e tightly linked with \u003cem\u003eRht29\u003c/em\u003e and randomly selected 84 homozygous K6-type individuals and 73 mutant individuals for phenotyping. Compared to K6-type, \u003cem\u003ewph3\u003c/em\u003e-type showed average 34.13% decrease in PH, 15.35% decrease in SL, but similar SNS and TN (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-d). For the grain-related traits, GL, GW, and TGW decreased by 5.99%, 6.58%, and 11.51% in \u003cem\u003ewph3\u003c/em\u003e, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee-g). Similar to that in the genetic background of K1, the 30.41% decrease of GNS was found in the \u003cem\u003ewph3\u003c/em\u003e-type plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eh).\u003c/p\u003e \u003cp\u003ePH segregation in the RK6-F\u003csub\u003e2\u003c/sub\u003e population did not show monogenic inheritance, suggesting potential additional dwarf gene(s) besides \u003cem\u003eRht29\u003c/em\u003e between K6 and \u003cem\u003ewph3\u003c/em\u003e. Based on our exome sequencing data, we found a premature termination variant in K6 and a missense variant in \u003cem\u003ewph3\u003c/em\u003e of \u003cem\u003eRht-B1\u003c/em\u003e, corresponding to alleles \u003cem\u003eRht-B1b\u003c/em\u003e and \u003cem\u003eRht-B1i\u003c/em\u003e, respectively (Table S6). We therefore analyzed interaction between \u003cem\u003eRht-B1\u003c/em\u003e and \u003cem\u003eRht29\u003c/em\u003e. We used molecular marker \u003cem\u003eKASP_Rht-B1\u003c/em\u003e linked with \u003cem\u003eRht-B1\u003c/em\u003e (Li et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and those linked with \u003cem\u003eRht29\u003c/em\u003e to identify F\u003csub\u003e2\u003c/sub\u003e individuals homozygous at both loci. Under the background of \u003cem\u003eRht-B1b\u003c/em\u003e or \u003cem\u003eRht-B1i\u003c/em\u003e, mutant allele of \u003cem\u003eRht-29\u003c/em\u003e (\u003cem\u003eRht-B29b\u003c/em\u003e) caused approximately 23% PH decrease. A greater reduction (32.67%) in PH could be observed when comparing the combination of \u003cem\u003eRht-B1b\u003c/em\u003e and \u003cem\u003eRht-B29b\u003c/em\u003e to the combination of \u003cem\u003eRht-B1i\u003c/em\u003e and WT \u003cem\u003eRht-29\u003c/em\u003e (\u003cem\u003eRht-B29a\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ei).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cb\u003eRht29\u003c/b\u003e \u003cb\u003eis a novel wheat dwarfing gene\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn this study, a dwarfing gene was identified within the physical interval of 799.5-803.4 Mb on the long arm of chromosome 2B of wheat. Several \u003cem\u003eRht\u003c/em\u003e genes have been cloned or localized on the specific chromosomes. For example, \u003cem\u003eRht1\u003c/em\u003e and \u003cem\u003eRht2\u003c/em\u003e were located on chromosomes 4B and 4D, respectively (Peng et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1999\u003c/span\u003e); \u003cem\u003eRht5\u003c/em\u003e was on chromosome 3B (Cui et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2022\u003c/span\u003e); \u003cem\u003eRht14\u003c/em\u003e, \u003cem\u003eRht18\u003c/em\u003e, \u003cem\u003eRht24\u003c/em\u003e, and \u003cem\u003eRht25\u003c/em\u003e were all located on chromosome 6A (Haque et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, Ford et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, Tian et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Zhang et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Several \u003cem\u003eRht\u003c/em\u003e genes have been reported to be derived from the group 2 homoeologous chromosomes. \u003cem\u003eRht8\u003c/em\u003e was derived from chromosome 2D and its homologous locus \u003cem\u003eRht8-2B\u003c/em\u003e was located on the short arm of chromosome 2B (Chai et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). \u003cem\u003eRht28\u003c/em\u003e(Liu et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) was located at 558.90-562.08Mb on chromosome 2A, which was not collinear with the interval we defined for \u003cem\u003eRht29\u003c/em\u003e. The homologues of the predicted candidate genes of \u003cem\u003eRht28\u003c/em\u003e on chromosome 2B, \u003cem\u003eTraesCS2B03G0888600\u003c/em\u003e (495,354,127\u0026ndash;495,360,583) and \u003cem\u003eTraesCS2B03G0889100\u003c/em\u003e (496,131,191\u0026ndash;496,141,611), were far apart from \u003cem\u003eRht29\u003c/em\u003e. \u003cem\u003eRht4\u003c/em\u003e was the dwarfing gene closest to \u003cem\u003eRht29\u003c/em\u003e on chromosome 2B (770\u0026ndash;773 Mb) (Qiao \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, it still did not overlap with the \u003cem\u003eRht29\u003c/em\u003e interval. Its candidate gene, \u003cem\u003eTraesCS2B03G1429200\u003c/em\u003e, exhibited no variant and expression in K6 and \u003cem\u003ewph3\u003c/em\u003e. Additionally, other known genes associated with plant height on chromosome 2B, such as \u003cem\u003eTaAP1-3-2B\u003c/em\u003e, \u003cem\u003eTaAP1-2-2B\u003c/em\u003e, and \u003cem\u003eTaARF12-2B\u003c/em\u003e (Li et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Li et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), also fall outside the \u003cem\u003eRht29\u003c/em\u003e region. Therefore, \u003cem\u003eRht29\u003c/em\u003e is a novel dwarfing gene.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRht29\u003c/b\u003e \u003cb\u003eis potentially a non-canonical GA-sensitive dwarfing gene\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn this study, \u003cem\u003eRht29\u003c/em\u003e was characterized as a GA-sensitive dwarfing gene due to the two aspects: First, the PH of \u003cem\u003ewph3\u003c/em\u003e is sensitive to exogenous GA\u003csub\u003e3\u003c/sub\u003e application, which can rescue the dwarf phenotype of \u003cem\u003ewph3.\u003c/em\u003e Second, \u003cem\u003ewph3\u003c/em\u003e showed apparent GA biosynthesis deficiency (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Quantitative analysis of GAs revealed that both the 13-OH and 13-H pathways were suppressed in the \u003cem\u003ewph3\u003c/em\u003e. The intermediates of the 13-OH pathway (e.g. GA\u003csub\u003e53\u003c/sub\u003e, GA\u003csub\u003e44\u003c/sub\u003e, GA\u003csub\u003e19\u003c/sub\u003e, GA\u003csub\u003e20\u003c/sub\u003e) exhibited a more pronounced reduction compared to those of the 13-H pathway. This observation was consistent with previous study showing that GA biosynthesis in wheat mainly relies on the 13-OH pathway, with GA\u003csub\u003e1\u003c/sub\u003e as the major bioactive GA. In line with this, 13-hydroxylated GAs, such as GA\u003csub\u003e1\u003c/sub\u003e, GA\u003csub\u003e20\u003c/sub\u003e, and GA\u003csub\u003e19\u003c/sub\u003e, predominantly accumulate in vegetative tissues of wheat, whereas GA\u003csub\u003e4\u003c/sub\u003e from the 13-H pathway is present at very low levels in these tissues (Appleford et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTypical GA-sensitive dwarfing genes usually encode key enzymes for bioactive GA synthesis or deactivation. For example, rice \u0026lsquo;green revolution\u0026rsquo; gene \u003cem\u003esd1\u003c/em\u003e encodes a GA20ox (Sasaki et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). In wheat, \u003cem\u003eRht12\u003c/em\u003e (Sun et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and \u003cem\u003eRht18\u003c/em\u003e (Grant et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) encode GA2oxA13 and GA2oxA9, respectively. However, some non-canonical GA-sensitive dwarfing genes have also been identified. \u003cem\u003eRht8\u003c/em\u003e encodes an RNase H-like protein which can modulate bioactive GA ratios by regulating the expressions of \u003cem\u003eGA13ox\u003c/em\u003e and \u003cem\u003eGA20ox\u003c/em\u003e (Xiong et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). GA-sensitive \u003cem\u003eRht13\u003c/em\u003e encodes an NB-LRR protein (Borrill et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and \u003cem\u003eRht25\u003c/em\u003e encodes a PLATZ transcription factor, which regulated wheat PH via interacting with DELLA (Zhang et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTwo high-confidence genes, \u003cem\u003eTraesCS2B03G1527800\u003c/em\u003e and \u003cem\u003eTraesCS2B03G1539200\u003c/em\u003e, carrying \u003cem\u003ewph3\u003c/em\u003e-specific SNPs were identified within the \u003cem\u003eRht29\u003c/em\u003e interval. Our transcriptome data showed that \u003cem\u003eTraesCS2B03G1539200\u003c/em\u003e was not expressed either in \u003cem\u003ewph3\u003c/em\u003e or in K1(Table S7). We also queried the spatiotemporal gene expression dataset of Chinese Spring (Consortium \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) and found that it did not express in the stem. Thus, \u003cem\u003eTraesCS2B03G1527800\u003c/em\u003e is considered to be the most possible candidate gene for \u003cem\u003eRht29.\u003c/em\u003e However, it did not encode the known key enzymes for GA synthesis or metabolism, suggesting that \u003cem\u003eRht29\u003c/em\u003e might be a non-canonical GA-sensitive dwarfing gene.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePotential functions of\u003c/b\u003e \u003cb\u003eRht29\u003c/b\u003e \u003cb\u003ein GA-dependent PH modulation\u003c/b\u003e\u003c/p\u003e \u003cp\u003ePrevious studies have shown that expression of \u003cem\u003eGA20ox\u003c/em\u003e and \u003cem\u003eGA3ox\u003c/em\u003e genes is under negative feedback control of GA, whereas that of \u003cem\u003eGA2ox\u003c/em\u003e genes is under positive feedforward control (Hedden and Phillips \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2000\u003c/span\u003e, Olszewski et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Consistent with these reports, a \u003cem\u003eGA20ox\u003c/em\u003e gene and four \u003cem\u003eGA2ox\u003c/em\u003e genes of \u003cem\u003ewph3\u003c/em\u003e showed up-regulated and down-regulated expression in responsive to GA deficiency, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). However, the expression of two detectable \u003cem\u003eGA3ox\u003c/em\u003e genes in \u003cem\u003ewph3\u003c/em\u003e was significantly suppressed, which contradicted the negative feedback regulation pattern of GA. Therefore, we speculated that \u003cem\u003eRht29\u003c/em\u003e may function in bioactive GA biosynthesis through regulating \u003cem\u003eGA3ox\u003c/em\u003e expression.\u003c/p\u003e \u003cp\u003eThe candidate gene \u003cem\u003eTraesCS2B03G1527800\u003c/em\u003e is homologous to \u003cem\u003eBAF1\u003c/em\u003e of \u003cem\u003eArabidopsis\u003c/em\u003e, which encodes an F-box family E3 ubiquitin ligase. BAF1 interacts with BRI1-EMS-SUPPRESSOR1 (BES1), the core transcription factor of the brassinosteroid signaling pathway, and mediates its ubiquitination and degradation (Wang et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Notably, BES1 plays a key role in the crosstalk between Brassinosteroids (BRs) and GA biosynthesis pathways. It has been reported that BES1 was able to interact with the promoter of \u003cem\u003eGA3ox1D\u003c/em\u003e and activate its expression to promote fiber cell elongation in cotton (Hou et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Therefore, it is possible that the mutation in \u003cem\u003eBAF1\u003c/em\u003e of \u003cem\u003ewph3\u003c/em\u003e may enhance the degradation of BES1, thereby preventing the activation of \u003cem\u003eGA3ox\u003c/em\u003e expression. This model proposed the functional relevance between \u003cem\u003eTraesCS2B03G1527800\u003c/em\u003e and the dwarf phenotype of \u003cem\u003ewph3\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRht29\u003c/b\u003e \u003cb\u003ehas pleiotropic effects on agronomic traits\u003c/b\u003e\u003c/p\u003e \u003cp\u003eBy comparing with WT and analyzing its effects in RK6 population, \u003cem\u003eRht29\u003c/em\u003e was found to cause more than 30% PH and more than 15% SL decrease (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). This combined effect was consistent with the GA-sensitive \u003cem\u003eRht8\u003c/em\u003e (Chai et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and \u003cem\u003eRht25\u003c/em\u003e (Zhang et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), as well as GA-insensitive \u003cem\u003eRht-B1\u003c/em\u003e (Song et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), indicating that this is a common feature of GA-related dwarfing genes. \u003cem\u003eRht29\u003c/em\u003e showed relatively large degree of PH reduction under semi-dwarf genetic background and exhibited additive effects with different \u003cem\u003eRht-B1\u003c/em\u003e alleles. Such effect may originate from the significant suppression of \u003cem\u003eGA3ox\u003c/em\u003e and the consequent extensive inhibition of synthesis of bioactive GA\u003csub\u003e1\u003c/sub\u003e, GA\u003csub\u003e3\u003c/sub\u003e, and GA\u003csub\u003e4\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This also led to a significant decrease in biomass, which may contribute to the decrease of the grain weight. In contrast, \u003cem\u003eRht8\u003c/em\u003e functions in modulating the ratio of GA\u003csub\u003e3\u003c/sub\u003e and GA\u003csub\u003e4\u003c/sub\u003e by regulating the expression of \u003cem\u003eGA20ox\u003c/em\u003e and \u003cem\u003eGA13ox.\u003c/em\u003e Such a relatively mild change in GA synthesis generated\u0026thinsp;~\u0026thinsp;20% PH decrease and had no obvious negative impact on grain yield (Xiong et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAdditionally, GA is closely associated with TN and inflorescence development. For example, the wheat \u003cem\u003ega3ox2\u003c/em\u003e mutant showed approximately 3-fold lower concentration of the bioactive GA\u003csub\u003e1\u003c/sub\u003e compared to the WT, and exhibited significantly more TN, fewer spikelets, and complete sterility (Phillips et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). In this study, \u003cem\u003ewph3\u003c/em\u003e showed significantly down-regulated expression of \u003cem\u003eGA3ox\u003c/em\u003e genes and a similar reduction of GA\u003csub\u003e1\u003c/sub\u003e, but exhibited different performance on tiller and inflorescence development compared to the \u003cem\u003ega3ox2\u003c/em\u003e mutant. We observed\u0026thinsp;~\u0026thinsp;30% TN reduction in \u003cem\u003ewph3\u003c/em\u003e compared with that in K1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ek). However, this negative impact was undetectable in RK6 population, indicating that the influence of \u003cem\u003eRht29\u003c/em\u003e on TN may be genetic background-dependent. Additionally, \u003cem\u003ewph3\u003c/em\u003e showed no difference in SNS under different backgrounds (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg and Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec) and significantly decreased GNS (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ek and Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eh), suggesting that \u003cem\u003eRht29\u003c/em\u003e does not affect differentiation of spikelet. Although it remains unclear that whether \u003cem\u003eRht29\u003c/em\u003e affects GNS by regulating the floret number or floret fertility, our results indicated that the pleiotropy of \u003cem\u003eRht29\u003c/em\u003e on agronomic traits is not equivalent to that of the \u003cem\u003eGA3ox\u003c/em\u003e mutant. Thus, further causal gene cloning, functional validation, and molecular mechanism dissection are worth conducting, which will facilitate gaining deeper insights into the mechanisms underlying the transcriptional regulation of \u003cem\u003eGA3ox2\u003c/em\u003e genes and the maintenance of bioactive GA homeostasis, as well as their pleiotropic effects on agronomic traits. This will lay the foundation for breaking the linkage between \u003cem\u003eRht29\u003c/em\u003e and unfavorable traits and expanding wheat yield potential through fine tuning plant height in breeding.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAuthor contribution statement\u003c/b\u003e \u003c/p\u003e \u003cp\u003eGD and HL conceived and designed the study. YT developed mapping population, FD, YT, FH and GD undertook the field and greenhouse trials, sampling, phenotyping and genotyping, JZ, TL, JW, JL and HZ assisted in genetic mapping, FD, JZ, and HL analyzed data, drafted and revised the manuscript, and all authors reviewed the manuscript.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of interest\u003c/h2\u003e \u003cp\u003eAll authors declare that they have no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work is supported National Natural Science Foundation of China (32272125, 32301790), the National Key R\u0026amp;D Program of China (2024YFD1201200), Sichuan Science and Technology Program, China (2022ZDZX0014), and Sichuan Provincial Agricultural Department Innovative Research Team (SCCXTD-2024-11).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eGD and HL conceived and designed the study. YT developed mapping population, FD, YT, FH and GD undertook the field and greenhouse trials, sampling, phenotyping and genotyping, JZ, TL, JW, JL and HZ assisted in genetic mapping, FD, JZ, and HL analyzed data, drafted and revised the manuscript, and all authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe BSA data and transcriptome data generated and used in this study have been deposited in The Genome Sequence Archive (GSA) (Accession No. XXXXXXXXX, under checked), Beijing Institute of Genomics, Chinese Academy of Sciences, and are publicly available.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAppleford, N. E. J., D. J. Evans, J. R. Lenton, P. Gaskin, S. J. Croker, K. M. Devos, A. L. Phillips and P. Hedden (2006). \"Function and transcript analysis of gibberellin-biosynthetic enzymes in wheat.\" Planta 223(3): 568\u0026ndash;582.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBorrill, P., R. Mago, T. Xu, B. Ford, S. J. Williams, A. Derkx, W. D. Bovill, J. Hyles, D. Bhatt, X. Xia, C. MacMillan, R. White, W. Buss, I. Molnar, S. Walkowiak, O.-A. Olsen, J. Dolezel, C. J. Pozniak and W. Spielmeyer (2022). \"An autoactive \u003cem\u003eNB-LRR\u003c/em\u003e gene causes \u003cem\u003eRht13\u003c/em\u003e dwarfism in wheat.\" Proc Natl Acad Sci U S A 119(48).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBuchfink, B., C. Xie and D. H. Huson (2015). \"Fast and sensitive protein alignment using DIAMOND.\" Nature Methods 12(1): 59\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBuss, W., B. A. Ford, E. Foo, W. Schnippenkoetter, P. Borrill, B. Brooks, A. R. Ashton, P. M. Chandler and W. 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Pu, X. Qiu, J. Wang, T. Li, Z. Yang, Y. Zhou, Y. Chang, J. Liang, H. Zhang, G. Deng and H. Long (2022). \"Genetic and transcriptomic dissection of an artificially induced paired spikelets mutant of wheat (\u003cem\u003eTriticum aestivum\u003c/em\u003e L.).\" Theor Appl Genet 135(7): 2543\u0026ndash;2554.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhu, T., L. Wang, H. Rimbert, J. C. Rodriguez, K. R. Deal, R. De Oliveira, F. Choulet, G. Keeble-Gagnere, J. Tibbits, J. Rogers, K. Eversole, R. Appels, Y. Q. Gu, M. Mascher, J. Dvorak and M.-C. Luo (2021). \"Optical maps refine the bread wheat \u003cem\u003eTriticum aestivum\u003c/em\u003e cv. Chinese Spring genome assembly.\" Plant J 107(1): 303\u0026ndash;314.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"theoretical-and-applied-genetics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"taag","sideBox":"Learn more about [Theoretical and Applied Genetics](https://www.springer.com/journal/122)","snPcode":"122","submissionUrl":"https://submission.nature.com/new-submission/122/3","title":"Theoretical and Applied Genetics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Triticum aestivum, Plant height, Rht genes, Gibberellin, GA3oxidase","lastPublishedDoi":"10.21203/rs.3.rs-8634203/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8634203/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBreeding semi-dwarf cultivars has long been a major objective for wheat improvement due to the inseparable association between plant height (PH) and grain yield. Although the utilization of \u003cem\u003eRht-B1b\u003c/em\u003e and \u003cem\u003eRht-D1b\u003c/em\u003e genes successfully achieved semi-dwarfism in the 1960s, these genes were associated with undesirable traits. The current wheat breeding urgently requires continuously exploring PH-controlling genes and their regulatory mechanisms, which will expand the genetic diversity of the PH gene pool to achieve precise PH regulation while maintaining or even increasing the grain yield potential. In this study, we identified a gibberellin (GA) - sensitive dwarf mutant, designated \u003cem\u003ewph3\u003c/em\u003e (\u003cem\u003ewheat plant height 3\u003c/em\u003e). It showed GA biosynthesis deficiency and had pleiotropic effects on PH, spike length, grain weight, and grain number per spike. Using Exome Capture Sequencing for Bulked Segregant Analysis and molecular marker mapping, a novel recessive nuclear dwarfing gene was identified and localized into a\u0026thinsp;~\u0026thinsp;3.9 Mb physical interval on chromosome 2B, designated \u003cem\u003eRht29\u003c/em\u003e (\u003cem\u003eReduced height 29\u003c/em\u003e). Transcriptome analysis and candidate gene mining indicated that \u003cem\u003eRht29\u003c/em\u003e may not encode a canonical key enzyme for GA biosynthesis, but participate in the GA biosynthesis by regulating the expression level of \u003cem\u003eGA3ox\u003c/em\u003e. This study enriches the genetic resources available for wheat dwarfing breeding and establishes a foundation for further molecular characterization of phenotypic regulation by \u003cem\u003eRht29.\u003c/em\u003e\u003c/p\u003e","manuscriptTitle":"Molecular mapping of a novel non-canonical gibberellin-sensitive dwarfing gene Rht29 in wheat (Triticum aestivum)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-18 14:24:57","doi":"10.21203/rs.3.rs-8634203/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-06T17:28:00+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-21T05:56:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"110319030163317198815603094030192433763","date":"2026-02-13T11:38:00+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-13T00:34:07+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-22T22:37:26+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-19T17:02:36+00:00","index":"","fulltext":""},{"type":"submitted","content":"Theoretical and Applied Genetics","date":"2026-01-19T02:00:25+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"theoretical-and-applied-genetics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"taag","sideBox":"Learn more about [Theoretical and Applied Genetics](https://www.springer.com/journal/122)","snPcode":"122","submissionUrl":"https://submission.nature.com/new-submission/122/3","title":"Theoretical and Applied Genetics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"96d4a908-772c-453d-992f-49b859fc62b6","owner":[],"postedDate":"February 18th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-06T17:28:00+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-05-06T17:38:43+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-18 14:24:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8634203","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8634203","identity":"rs-8634203","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}