Weak seed dormancy (SD) is prone to pre-harvest sprouting (PHS), which reduces cereal yield and quality. Here, through map-based analysis, we identify TaCNGC-2A, encoding a cyclic nucleotide-gated channel protein, as a negative regulator of wheat SD. Knocking out of TaCNGC-2A enhances SD and PHS resistance, with no yield penalty. Two transcription factors, TaMYB-5B and TaMYB-5D, directly bind to the T/A mutation site of TaCNGC-2A promoter to synergistically repress its expression. The calmodulin TaCaM-3A interacts with TaCNGC-2A to jointly modulate SD and PHS resistance through influencing calcium and multiple hormonal signaling pathways. Knocking out of TaCaM-3A not only enhances SD and PHS resistance, but also increases grain weight and per-plant yield. Finally, we identify allele combinations of TaCNGC-2A and other known dormancy genes associated with strong SD. This study uncovers a regulatory mechanism underlying SD and PHS resistance and provides gene targets for breeding wheat varieties with PHS resistance.

A novel major locus (Qgi.245.ahau-4B.3) controlling seed dormancy, and its candidate genes (TaF-box-B1 and TaF-box-B2), were identified by integrating association and linkage mapping with expression and sequence variation analyses. Moderate seed dormancy is essential for reducing pre-harvest sprouting (PHS) and ensuring uniform germination in cereal crops. In this study, seed dormancy was evaluated in 245 wheat varieties with diverse genetic backgrounds across seven environments, and genotypes were obtained using the Wheat 90 K SNP array. A genome-wide association study (GWAS) identified 55 loci associated with seed dormancy, including a novel major locus (Qgi.245.ahau-4B.3) on chromosome 4B. This locus was validated by integrating molecular marker development, re-GWAS, linkage mapping, and expression analysis. Two candidate F-box protein-encoding genes underlying this locus were identified: TraesCS4B03G0269800 (TaF-box-B1) and TraesCS4B03G0270500 (TaF-box-B2). The expression levels of TaF-box-B1 and TaF-box-B2 were significantly lower in the moderate dormancy wheat variety Annong 1124 (AN1124) than in weak dormancy variety Annong 8455 (AN8455). Sequence and haplotype analyses showed that variations in TaF-box-B1 and TaF-box-B2 were completely linked, forming two haplotypes: TaF-box-Hap1 for strong dormancy and TaF-box-Hap2 for weak dormancy. Frequency analysis further revealed that the favorable haplotype TaF-box-Hap1 was predominantly distributed in the Middle and Lower Yangtze River winter wheat region, characterized by relatively high rainfall and humidity. These findings establish a robust foundation for molecular marker-assisted breeding of wheat varieties with enhanced climate resilience and stable PHS resistance, thereby contributing substantively to global food security.

Abstract In wheat, weak seed dormancy (SD) is related to an increased tendency for pre-harvest sprouting (PHS), which reduces yield and quality. However, the molecular mechanism underlying SD remains elusive. Here, we identified a wheat R2R3-MYB transcription factor ( TaMYB83-7B ) related to SD. Expression analysis showed that TaMYB83-7B was highly expressed in wheat seeds, and was more highly expressed in strong-dormancy varieties than in weak-dormancy varieties. Sequence and association analysis indicated that T/C mutations at −907 bp and −1133 bp in the TaMYB83-7B promoter were significantly associated with wheat SD, with C at both sites related to strong dormancy. Dual-luciferase reporter assays demonstrated that the transcriptional activity of the TaMYB83-7B promoter was significantly higher in strong-dormancy varieties than in weak-dormancy varieties. Further analyses indicated that TaMYB83-7B functions as a transcriptional inhibitor. Germination experiments revealed that overexpression of TaMYB83-7B significantly enhanced SD, while its loss-of-function reduced SD. Finally, TaMYB83-7B was found to regulate SD by influencing the balance between abscisic acid (ABA) and gibberellin (GA) in wheat seeds. Overall, the results of this study enhance our understanding of the complex regulatory mechanism underlying SD, and provide gene targets and molecular markers for the genetic improvement of PHS resistance in wheat.

Moderate seed dormancy is essential for reducing pre-harvest sprouting (PHS) and ensuring uniform germination in cereal crops. In this study, seed dormancy was evaluated in 245 wheat varieties with diverse genetic backgrounds across seven environments, and genotypes were obtained using the Wheat 90K SNP array. Genome-wide association analysis identified 55 loci associated with seed dormancy, including a novel major locus, Qgi.245.ahau-4B.3 , on chromosome 4B. By integrating molecular marker development, re-GWAS, linkage mapping, and expression analysis, this locus was validated and two candidate genes underlying Qgi.245.ahau-4B.3 were identified: TraesCS4B02G118000 ( TaF-box-B1 ) and TraesCS4B02G118200 ( TaF-box-B2 ), both encoding F-box proteins. The expression levels of TaF-box-B1 and TaF-box-B2 were significantly lower in the moderate dormancy wheat variety Annong 1124 (AN1124) than in weak dormancy variety Annong 8455 (AN8455). Sequence and haplotype analyses showed that variations in TaF-box-B1 and TaF-box-B2 were completely linked, forming two haplotypes: TaF-box-Hap1 for strong dormancy and TaF-box-Hap2 for weak dormancy. Frequency analysis further revealed that the favorable haplotype TaF-box-Hap1 was predominantly distributed in the Middle and Lower Yangtze River winter wheat region, characterized by relatively high rainfall and humidity. These findings establish a robust foundation for molecular marker-assisted breeding of wheat varieties with enhanced climate resilience and stable PHS resistance, thereby contributing substantively to global food security.

Mixograph properties represent important quantitative traits that are controlled by multiple genes and influenced by environmental factors. In this study, we conducted quantitative trait locus (QTL) mapping for key Mixograph paraments using a recombinant inbred line (RIL) population derived from a cross between Yangxiaomai and Zhongyou 9507. Based on a high-density genetic map, six stable QTLs were identified on chromosomes 1A, 1B, and 1D across four environments, with individual phenotypic variation explained, ranging from 2.26 to 28.70%. Among these, QTh.ahau-1A, QMt/QPa.ahau-1B, and QTw.ahau-1D.1 are potentially novel loci. Furthermore, four functional Kompetitive Allele-Specific PCR (KASP) markers were developed based on tightly linked SNPs and validated in 110 advanced breeding lines, confirming their significant association with the target traits and utility for marker-assisted selection (MAS). Additionally, six candidate genes were predicted, which encoded proteins such as a hydroxyproline-rich glycoprotein, a CCCH-type zinc finger protein, protease, kinase, a phosphoglucan water dikinase, and a TRP-like family protein. Collectively, these findings provide valuable genetic loci, functional molecular markers, and candidate gene resources for improving wheat processing quality through MAS-based breeding.

Seed dormancy (SD) is the primary genetic determinant of pre-harvest sprouting (PHS) resistance. However, the molecular mechanisms underlying SD remain incompletely understood. Here, we identified a wheat cytochrome P450 gene, TaCYP94-A1, that is expressed at significantly higher levels in weak-dormancy varieties than in strong-dormancy varieties. TaCYP94-A1 expression increased during SD release and decreased during dormancy establishment. Knockout of TaCYP94-A1 markedly enhanced SD and PHS resistance without adversely affecting yield-related traits. Two key single-nucleotide polymorphisms (T/C at -1,895 bp and T/C at -1,225 bp) in the TaCYP94-A1 promoter were significantly associated with SD variation, with the TaCYP94-A11,895C and TaCYP94-A11,225C allele combination (haplotype Hap4) strongly associated with enhanced dormancy. Two transcription factors, TaABI4 and TaNAC-A1, bind directly to the 5'-ACCGC-3' (C, -1,895 bp) and 5'-GACTTC-3' (C, -1,225 bp) motifs in the TaCYP94-A1 promoter, respectively, and regulate its transcription through antagonistic protein-protein interactions in the nucleus. Physiological, biochemical, and gene expression analyses revealed that the TaABI4/TaNAC-A1-TaCYP94-A1 module regulates SD through crosstalk with the gibberellic acid, abscisic acid, and jasmonic acid pathways. Together, these findings uncover a previously uncharacterized regulatory module controlling SD and provide valuable genetic resources and molecular markers for developing PHS-resistant wheat cultivars through molecular design breeding.

Strong seed dormancy is crucial for preventing pre-harvest sprouting (PHS) in cereal crops. However, the underlying molecular mechanism in wheat remains unclear. Here, we identified a gibberellin (GA)-stimulated regulator gene, TaGASR25, which negatively modulates wheat seed dormancy. Further analyses showed that TaC3HC4, a member of the C3HC4-type zinc finger family, enhances TaGASR25 transcription and interacts with TaGASR25 in the nucleus to negatively regulate wheat seed dormancy through crosstalk with the GA and abscisic acid (ABA) pathways. Marker-trait association analyses revealed that the A/G (-1317 bp) and CGG/GA- (-1645 bp) mutations in the TaGASR25 promoter were significantly associated with differences in seed dormancy among wheat varieties, with A and CGG associated with strong dormancy. Collectively, our findings uncover a novel TaC3HC4-TaGASR25 module regulating seed dormancy and provide promising targets and molecular markers for the molecular breeding of PHS-resistant wheat varieties.

Understanding the regulatory mechanisms underlying wheat plant architecture is essential for yield improvement and molecular breeding. The miR156-SPL module plays a pivotal role in controlling plant architecture and agronomic traits, however, its regulatory functions in wheat remain incompletely understood. In this study, we first report the miR156h-TaSPL4-TaPIN18 module regulates plant architecture and grain size by modulating auxin transport in wheat. Overexpression of miR156h resulted in reducing the size of leaf, spikelet and grain, accompanied by increased tillering and a more compact plant architecture. And miR156h directly cleaves and suppresses the expression of TaSPL4. CRISPR/Cas9-mediated knockout of TaSPL4 leads to increased tiller number, compact growth, and significant reductions in grain width and thousand-grain weight. In contrast, overexpression of TaSPL4 reduced tillering, increased tiller angle, and significantly enhanced grain length, grain width, and thousand-grain weight, highlighting its critical role in regulating plant architecture and grain size. Furthermore, we identified TaPIN18 as a novel downstream gene regulated by TaSPL4 and demonstrated that TaSPL4 positively regulates TaPIN18 expression and mediates auxin transport and distribution. Taken together, our findings reveal a previously uncharacterised miR156h-TaSPL4-TaPIN18 regulatory module that modulates wheat plant architecture and grain size, and provides potential molecular targets for yield improvement.

In wheat, exposure to low temperatures (LTs) during the middle and late stages of seed development induces dormancy release; however, the underlying regulatory mechanism remains unclear. Here, using whole-transcriptome sequencing, we identified a novel microRNA (miR1832) that is downregulated by LT and located at a key node of the associated regulatory network. Germination assays showed that overexpression of miR1832 enhanced seed dormancy, whereas its silencing reduced seed dormancy. Sequence variation and association analyses further indicated that an A/G mutation at -670 bp in the miR1832 promoter is significantly associated with phenotypic variation in seed dormancy among wheat varieties, with the A allele correlated with strong dormancy. Through yeast one-hybrid, electrophoretic mobility shift assay, and dual-luciferase (LUC) reporter assays, we found that the LT-responsive Dof transcription factor TaDof-2D directly binds to the A site in the miR1832 promoter and inhibits its transcription. Subsequent expression analysis, dual-LUC assays, and 5' rapid amplification of cDNA ends confirmed that miR1832 targets the cytochrome P450 gene TaP450-7A, which is upregulated by LT and negatively regulates seed dormancy. Finally, physiological and biochemical analyses further demonstrated that the TaDof-2D-miR1832-TaP450-7A module participates in LT-induced dormancy release by modulating α-amylase activity and the abscisic acid and gibberellin pathways. These findings uncover a previously uncharacterized regulatory mechanism underlying LT-induced dormancy release and provide promising genetic resources and molecular markers for breeding wheat varieties with optimal dormancy levels.

The stripe rust resistance molecular module ‘1347B’, comprising Yr9-1RS/1BL , Yr30-3BS , YrZH22-4BL , and YrZH84-7BL , could provide stable and durable stripe rust resistance without a yield penalty in wheat breeding program. Wheat stripe rust is a significant epidemiological disease that severely impacts wheat production. The aggregation of multiple stripe rust resistance genes constitutes a pivotal breeding strategy for reducing pathogen evolution of virulence and enhancing the resistance level in wheat. Investigating the resistance conferred by various combinations of stripe rust resistance genes and assessing their impact on wheat yield are essential for understanding the synergistic regulatory mechanisms of these genes on host immunity and yield. In this study, 254 F7 recombinant inbred lines (RILs) derived from the cross ZK331/ZK32 were evaluated for stripe rust severity at the adult-plant stage and thousand-kernel weight (TKW) across six environments over a two-year period. Through QTL mapping and molecular marker detection, four stripe rust resistance genes, Yr9 (1RS/1BL), Yr30 (3BS), YrZH22 (4BL), and YrZH84 (7BL) were identified in the RIL population. The results demonstrated that pyramiding these genes conferred a high level of adult plant stage resistance to stripe rust. Analysis of agronomic traits, including TKW, plant height, spike length, and kernel number per spike, in RIL population with different combinations of these genes revealed no adverse effects under rust-free conditions and a highly significant positive effect under stripe rust infection. The molecular module ‘1347B’, comprising Yr9-1RS/1BL , Yr30-3BS , YrZH22-4BL , and YrZH84-7BL , provided stripe rust resistance across multiple growth stages without a yield penalty. This study elucidates the resistance efficacy and yield stability provided by the ‘1347B’ module, providing a valuable genetic resource and theoretical foundation for designing high-yielding wheat varieties with enhanced stripe rust resistance.

The plant hormone gibberellin (GA) plays a key role in breaking seed dormancy, but the underlying regulatory mechanism is not fully understood. Here, we reported TraesCS3B02G166100 (named TaGA2ox2-3B), encoding a GA metabolism enzyme GA2ox family member, to be differentially expressed in strong- and weak-dormancy wheat seeds during germination. We confirmed that the ERF transcription factor TaERF-2 A directly bound to the TaGA2ox2-3B promoter and enhanced its transcription. Germination tests indicated that TaERF-2 A positively regulated seed dormancy in wheat. Additionally, 12 mutations were identified within the promoter and coding regions of TaGA2ox2-3B when comparing strong- and weak-dormancy wheat varieties. Six molecular markers were developed to verify correlations between these mutations and seed dormancy. Transgenic experiments verified the potential of the TaGA2ox2-3B+2246-A allele to enhance seed dormancy. Physiological and biochemical analyses indicated that the TaERF-2 A-TaGA2ox2-3B module modulated seed dormancy by influencing GA metabolism and signaling pathways. Collectively, this study revealed the molecular mechanism of GA regulating seed dormancy, and identified genetic resources and molecular markers to breed wheat varieties with preharvest sprouting resistance.

Wheat (Triticum aestivum L.) is a crucial global cereal crop, but its yield is severely affected by Leaf rust (LR) caused by Puccinia triticina. LR results in reddish-brown lesions on wheat leaves, which disrupt photosynthesis and reduce crop productivity. This study identifies three novel quantitative trait loci (QTLs) associated with LR resistance in wheat and evaluates optimal cross combinations within a segregating F2 population. Yield-related traits including 1000-grain weight, peduncle length, spike length, and grain yield per plant were evaluated over three replications. The parental genotypes, AN179, AN1687, PR123, and PR127, demonstrated high potential for both yield and reduced LR infection rates. Among the crosses, AN179 x PR127 was the most effective, exhibiting healthier grains, higher grain counts per spike, longer peduncles for better photosynthate translocation, and adult plant resistance to slow rusting. Disease severity ranged from 2.9 % to 29.1 % in the parental genotypes and from 4.7 % to 51.1 % in the crosses. Parents PR123, PR127, AN179, and AN1687 showed resistance to field disease severity. The crosses AN176 x PR127, AN1687 x PR123, and AN179 x PR123 exhibited high resistance, while AN179 x PR127 showed the lowest field disease severity at 4.7 %. A linkage map of the F2 population revealed three novel LR resistance QTLs on chromosomes 2AS, 1BL, and 6B in the cross AN179 x PR127. Microsatellite markers Xgwm273, Xgwm610, and Xgwm493 were closely linked to these QTLs. These novel QTLs can be utilized in wheat hybridization programs for marker-assisted selection and pyramiding of all-stage LR genes to enhance resistance.

The comprehensive annotation of regulatory elements in linear genomes is needed to elucidate the molecular mechanisms underlying chromatin loop formation in plants. Here, we characterized a novel family of conserved noncoding sequences (CNSs) in the rice (Oryza sativa) genome. These sequences, known as AT-rich pincer-like elements (APEs), are composed of 13-bp repeat unit arrays in a reverse-forward configuration. Our findings revealed that there are 611 APE copies across the japonica genome. Deletion of single APEs disrupted the long-range chromatin loops anchoring target-APE regions and moderately remodeled the profile of A/B compartments, topologically associating domains (TADs), and chromatin loops, thereby rewiring the expression of looped gene(s) including those controlling important agronomic traits. Thus, APEs function as hub motifs directly mediating chromatin looping and maintaining 3D genome integrity and stability at the levels of compartments, TADs, and loops. Moreover, neighboring genomic regions harboring numerous paired non-APE (NA) CNSs were more likely to interact with each other. This finding suggests that NA CNS pairs might play a helper role in determining loop frequency in a dose-dependent manner, likely by ensuring the pairing selectivity of anchor sites. Our study highlights the importance of APEs and NA CNSs in maintaining 3D genome structure, thereby providing the framework required to link many noncoding repetitive elements to their molecular functions in plants.

Global warming is primarily characterized by asymmetric temperature increases, with higher temperature rises in winter/spring and at night compared to summer/autumn and daytime. We investigated the impact of winter night warming on wheat leaves using the spring wheat cultivar Yangmai 18 and the semi-winter wheat cultivar Yannong 19 during the 2020–2021 growing season. This study aimed to examine the effect of winter night warming on the top expanded leaf of wheat plants. The results showed that the night mean temperature in the treatment group increased by 1.27°C compared to the ambient temperature and winter night warming increased the yield of both wheat cultivars, the activities of sucrose synthase and sucrose phosphate synthase after anthesis, and the biosynthesis of sucrose and soluble sugars. The differentially expressed genes were identified using P-value<0.05 and fold change>2, and subjected to Gene Ontology annotation and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses. The genes differentially expressed in wheat leaves treated with night warming were primarily associated with starch and sucrose metabolism, amino acid biosynthesis, carbon metabolism, plant hormone signal transduction, and amino sugar and nucleotide sugar metabolism. Comparison between groups identified 14 differentially expressed genes related to temperature. These results highlight the effects of winter night warming on wheat development from various perspectives. Our results provide new insights into the molecular mechanisms of the wheat response to winter night warming and the candidate genes involved in this process.

Pre-harvest sprouting (PHS) reduces grain yield and quality and poses a serious threat to global wheat production. Seed dormancy and germination are closely related to PHS resistance. However, the intricate regulatory processes governing seed dormancy and germination remain largely unknown. Here, we reported that a NAC transcription factor gene (TaNAC018-7D) is highly expressed in weak-dormancy wheat cultivar Jing 411 compared with a strong-dormancy landrace Hongmangchun 21 during seed germination. Germination tests revealed that TaNAC018-7D negatively regulates seed dormancy and positively mediates germination in transgenic lines of Arabidopsis and rice, and wheat mutants induced by ethyl methane sulfonate in the Jing 411 background. Subcellular localization analysis indicated that TaNAC018-7D is located in the nucleus and cytoplasm. Physiological, biochemical, and molecular experiments indicated that TaNAC018-7D interacts with the promoter of the GA biosynthesis gene TaGA7ox-A1 and activates its expression, thereby shortening seed dormancy and promoting germination. In summary, this study provides a new target gene for improving wheat PHS resistance, and improves the understanding of the complex regulatory network of PHS resistance.

The bHLH transcription factor SD6 regulates rice seed dormancy. However, sequence variants of wheat SD6 homologs and their roles in seed dormancy remain unknown. Here, we cloned three wheat homologous genes of SD6 (named TaSD6-7A, TaSD6-7B, and TaSD6-7D), and found that TaSD6-7A had abundant sequence variations, while TaSD6-7B and TaSD6-7D had no variation. Based on sequence variations in the promoter and coding regions of TaSD6-7A, we developed three molecular markers and verified their associations with seed dormancy in 160 wheat varieties. We further confirmed that the TaSD6-7 A + 1307-T allele positively regulated seed dormancy, but had no adverse effects on other important agronomic traits. Resequencing and phenotypic data indicated that the favorable haplotype TaSD6-7AHapII associated with high dormancy level was more common in landraces than in modern varieties. Additionally, we demonstrated the pyramid effects of TaSD6-7A with known dormancy-related genes in enhancing seed dormancy. Overall, this study provides novel genetic resources and molecular markers for improving PHS resistance of modern wheat varieties.

ABSTRACT Leaf rust (LR) poses a global threat to wheat crops and can lead to severe yield losses if environmental conditions favour its spread. Using resistant wheat cultivars offers a sustainable approach to managing LR. This study aimed to identify promising wheat lines for LR‐resistance breeding using classical analytical methods to screen for LR tolerance. We evaluated 10 parental lines, comprising 6 lines and 4 testers, crossed into 24 combinations using a line × tester mating design. These germplasm were grown in a triplicate RCB design under both optimal and LR‐stress conditions. We recorded data on various morphological, physiochemical, yield and component traits at key growth stages. The analysis of combining ability indicated significant variations among genotypes, with non‐additive gene action influencing most traits. Four promising parents (AN179, AN1687, PR123 and PR127) and two crosses (AN179 × PR127 and AN179 × PR123) showed high combining ability for yield traits under LR‐stress. Cluster analysis revealed divergent groups among the genotypes, with shifting clustering under LR‐stress suggesting varied genotypic responses. Factor analysis identified genotypes that performed consistently well under LR‐stress. These genotypes are suitable for use in LR‐resistance breeding programs. We also recommend peduncle length and tillers per plant as phenotypic markers for wheat selection and breeding due to their positive correlation with grain yield. The findings of this study can contribute valuable insights to sustainable wheat breeding research.

Sequence replacement is the most direct and powerful genome editing strategy. However, it remains currently an urgent need to develop protospacer adjacent motif (PAM)-less, off-target-free and simple tools for precise sequence replacement. To address this challenge, we fused a rice-derived AT-rich pincer-like elements (APE), which is composed of unique repeat-spacer array, to donor template against target sequence, thus forming guider and donor template (gdt). The donor template harbors multiple sites of DNA fragment insertion/deletion (MsDFID) which function as donor sites. APE plays as MsDFID gdt scaffold to repurpose Cas3 or Cas9 to mediate transposition of DNA fragment insertion/deletion from MsDFID donor template into genome target in E . coli , thus realizing seamless sequence replacement. These results established putative gdt/Cas3 or gdt/Cas9 ribonucleoprotein as compact genome editors which feature PAM-lessness, no observable off-target, and simplicity based on the dual role of MsDFID gdt per se as both guider and donor template. This strategy provides significant potential for precise sequence replacement in both animals and plants.

Leaf rust (LR) epidemics present a persistent threat to global wheat production, despite the presence of resistance (Lr) genes in wheat. The evolving pathogen Puccinia triticina continually challenges these resistance mechanisms. This study assessed 10 wheat lines for relative resistance index (RRI) and screened them for Lr genes or quantitative trait loci (QTLs) using microsatellite markers. The lines were classified into three groups: Ssusceptible (< 5; 4.32 +/- 0.68), moderate (5-7; 6.05 +/- 0.67) and resistant (> 7; 8.50 +/- 0.22) (p < 0.001). Genetic analysis with 12 polymorphic markers revealed 186 alleles with varying allelic diversity. Markers Xbarc124 and Xgwm512 showed greater diversity, and resistance-related alleles were linked to markers Xgwm512 and Xgwm493, associated with the Lr34 gene. Moderate associations were found with Lr37 (Xbarc1138 and Xgwm400) and Lr24 (Xgwm273), while Lr26 (Xwmc407) was linked to susceptibility. Parental line crosses resulted in higher RRI, indicating beneficial recombination. Structure analysis revealed genetic diversity among resistance groups, with susceptible groups showing distinct clustering. Lines AN179 and PR127 clustered together, showing key resistance alleles, particularly in crosses with resistant PR123. The findings highlight novel pathogen races contributing to resistance breakdown and suggest combining all-stage resistance genes (Lr9, Lr24, Lr37) with adult plant resistance (APR) genes (Lr48, Lr22a, Lr34, Lr46) for durable LR resistance. The identified alleles offer valuable insights for marker-assisted breeding to enhance wheat resistance to LR.

A new stripe rust resistance gene YrAn1589 in Chinese wheat Annong1589 was mapped to a 160.9-166.6 kb interval on chromosome arm 3BL and co-segregated with a marker CAPS9 developed from candidate gene TraesCS3B03G1054600. Stripe rust, caused by Puccinia. striiformis f. sp. tritici (Pst), is a devastating fungal disease that can significantly reduce wheat yield. The Chinese wheat cultivar Annong1589 demonstrates high resistance against the predominant Pst races in the Huang-Huai valley wheat region. The present study aimed to identify the stripe rust resistance gene in Annong1589. Genetic analysis indicated that the resistance in Annong1589 was conferred by a single dominant gene, provisionally designated YrAn1589. Using Wheat660K SNP array, bulked segregant RNA sequencing and new molecular markers developed, the resistance gene was mapped to a 160.9–166.6 kb region between CAPS8 and CAPS10 on chromosome 3BL based on IWGSC CS RefSeq v2.1 and eight other reference genome sequences, including eight high-confidence annotated genes. Transcriptome and qRT-PCR analyses revealed significantly upregulated expression of TraesCS3B03G1054600 in resistant plants following CYR32 inoculation, suggesting it is a potential candidate gene for YrAn1589. A functional marker CAPS9 developed from a A/G polymorphic SNP in the candidate co-segregated with YrAn1589 in the F2 population. Subcellular localization experiments showed that TraesCS3B03G1054600 protein was localized in the cytoplasm and nucleus, implying its role in immune response and resistance. Our findings establish YrAn1589 as a new stripe rust resistance gene, providing valuable gene resource and molecular markers for improvement of stripe rust resistance in wheat.