Integrated Genomic and Transcriptomic Analysis Reveals Candidate Genes Underlying Herbicide Resistance in Sorghum

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Results: In this study, 356 sorghum accessions were screened for herbicide tolerance at the seedling stage using gradient herbicide treatments. Under application of the ACCase inhibitor 10% feproxydim, five accessions showed reduced phytotoxicity: IS1219 displayed the highest resistance, while IS10867, SJ72, SJ85, and PI61 exhibited moderate tolerance. For the ALS inhibitor mesosulfuron-methyl, only two accessions, SJ304 and PI47, showed visible tolerance. To elucidate the molecular basis of resistance, a bulked segregant analysis sequencing (BSA-Seq) approach was applied to resistant and susceptible gene pools constructed from the IS1219 × RTx430 F₂ population. The analysis identified a major quantitative trait locus (QTL) for herbicide resistance located on chromosome 1.Transcriptome (RNA-Seq) data of leaf tissues collected after feproxydim treatment revealed five co-expressed candidate genes within the mapped interval. Among them, Sobic.001G431500 , encoding a carboxylesterase 17 (α/β-hydrolase), plays a pivotal role in feproxydim resistance. This gene was markedly upregulated in the resistant line (IS1219) but not in the susceptible line (RTx430), indicating that enhanced hydrolytic or metabolic activity may be a major resistance mechanism. Protein sequence comparison also showed that the IS1219 allele carries a missense and deletion mutation at and after position 300 (V300A and P301_P303del). Conclusions: These findings clarify the physiological and molecular mechanisms underlying herbicide resistance in sorghum and provide valuable genetic resources for breeding herbicide-tolerant varieties. Sorghum Herbicide RNA-Seq BSA-Seq Candidate genes Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Sorghum [ Sorghum bicolor (L.) Moench] is a globally important grain, forage, and bioenergy crop with remarkable adaptability to harsh environments such as drought, heat, and nutrient-poor soils [ 1 – 3 ] . However, its production is often severely constrained by weed infestation [ 4 ] . Weeds compete with sorghum for essential growth resources such as light, water, and nutrients, while also reducing canopy ventilation and light penetration, which in turn increases the incidence of diseases and insect pests. These effects ultimately cause substantial yield losses and deterioration of grain quality [ 5 ] . At present, chemical herbicide application remains the predominant method for weed control in sorghum production. Nevertheless, chemical weeding is frequently associated with issues such as phytotoxicity, the evolution of herbicide-resistant weed populations, and environmental contamination of farmland ecosystems [ 6 ] . Alternative strategies, including agronomic and biological control, generally fail to achieve effective and sustainable weed management [ 7 ] . Therefore, the identification and utilization of genetic resources and germplasm materials with herbicide resistance have become critical priorities for sorghum improvement [ 8 ] . In recent years, herbicide-resistant germplasms have been developed through approaches such as mutation breeding, natural population screening, and molecular marker-assisted selection, providing key genetic resources for the breeding of herbicide-tolerant sorghum varieties. The herbicide resistance mechanisms identified to date can be broadly classified into two categories: target-site resistance (TSR) and non-target-site resistance (NTSR) [ 9 ] . TSR results from mutations in herbicide target genes that reduce the affinity between the herbicide molecule and its target protein, thereby conferring tolerance [ 10 ] . For example, Zhang et al. [ 11 ] identified a novel OsEPSPS allele in rice harboring an Asp-213-Asn substitution within the predicted glyphosate-binding domain, which conferred tolerance to glyphosate at four times the recommended field concentration. In sorghum, two allelic variants of the SbALS gene— sbals-1 (A93T) and sbals-2 (S624N)—have been demonstrated to confer strong resistance to imidazolinone herbicides, enabling plants to survive treatments up to 16-fold the standard application rate [ 12 ] . In contrast, NTSR operates through physiological and biochemical mechanisms that minimize the effective herbicide concentration at its site of action. These mechanisms include reduced herbicide absorption or translocation, enhanced metabolic detoxification, and active efflux [ 13 ] . The major molecular systems underlying NTSR involve cytochrome P450 monooxygenases, glutathione S-transferases (GSTs), and ATP-binding cassette (ABC) transporters [ 14 ] . For instance, CYP81A6 , a cytochrome P450 gene in rice, has been shown to confer broad-spectrum tolerance to bentazone and metsulfuron-methyl—two herbicides widely used in rice and wheat cultivation [ 15 ] . In this study, we aimed to address the challenge of weed control in sorghum by identifying and characterizing herbicide-resistant germplasms. Herbicide tolerance was evaluated at the seedling stage among diverse sorghum accessions, and the identified resistant and susceptible genotypes were used to construct an F₂ segregating population. Whole-genome resequencing of resistant and susceptible bulks via BSA-Seq identified genomic regions associated with herbicide resistance. By integrating transcriptomic data from resistant and susceptible parents under herbicide stress, key candidate genes were identified. These findings provide valuable insights and technical support for the molecular breeding of herbicide-resistant sorghum. 2. Materials and Methods 2.1. Plant Materials A total of 356 sorghum germplasm accessions were used in this study (Table S1), all provided by the Anhui Provincial Key Laboratory of Forage Breeding and Utilization. Among them, the herbicide-resistant accession IS1219 and the susceptible accession RTx430 were selected for further analysis. An F₁ hybrid population was generated by crossing IS1219 (♀) with RTx430 (♂). The harvested F₁ plants were self-pollinated to produce an F₂ segregating population. 2.2. Screening of Herbicide-Resistant Sorghum at the Seedling Stage and Evaluation of Resistance Levels Seeds were surface-sterilized with 75% ethanol for 5 min and rinsed 2–3 times with sterile distilled water. The sterilized seeds were placed in Petri dishes and incubated at 28°C for germination. Germinated seeds with visible radicles were transplanted into seedling trays filled with nutrient soil (hole size: 4.8 × 4.8 × 4.8 cm). The trays were maintained in a greenhouse under controlled conditions and watered from the bottom to ensure adequate moisture. At the five-leaf stage, seedlings were sprayed with either 10% feproxydim (200 mL/667 m²) or mesosulfuron-methyl (160 mL/667 m²). Phytotoxicity symptoms and plant mortality were recorded at 3, 5, 7, 9, 11, and 14 days after treatment. Phytotoxicity levels were evaluated according to the five-grade classification method specified in the Agricultural Industry Standards of the People’s Republic of China (Table S2). At the five-leaf stage, seedlings were exposed to a gradient of herbicide concentrations (Table S3), including the recommended field rate (1×), a control (0×, sprayed with water), and higher dosages. Two types of herbicides—an ALS inhibitor (mesosulfuron-methyl) and an ACCase inhibitor (10% feproxydim)—were tested. Twenty-one days after spraying, plant mortality was recorded, and the GR₅₀ (herbicide dose causing 50% growth reduction) and resistance index (RI) were calculated. 2.3. RNA-Seq Analysis Resistant sorghum line IS1219 and susceptible line RTx430 were treated with 10% feproxydim at the recommended field concentration when plants reached the five-leaf stage. Leaf samples were collected from both genotypes 48 h after herbicide application, with three biological replicates per accession. Total RNA was extracted and sent to BGI-Wuhan (Wuhan, China) for transcriptome sequencing. Sequencing reads were aligned to the Sorghum bicolor reference genome (version 5.1) for downstream analysis. Raw reads were quality-checked using FastQC, and clean reads were aligned to the reference genome. Gene expression levels were normalized using the TMM (Trimmed Mean of M-values) method with FeatureCounts. Differentially expressed genes (DEGs) were identified with DESeq2 under the criteria of |log₂FoldChange| ≥ 1 and FDR < 0.05. Functional enrichment analysis of DEGs was performed for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) terms in the R environment, with p < 0.05 considered significant. 2.4. Bulked Segregant Analysis Sequencing (BSA-Seq) The F₂ population was grown in pots under greenhouse conditions. At the five-leaf stage, leaf samples from each individual were collected for DNA extraction. Subsequently, the plants were treated with 10% feproxydim at the recommended field concentration. Two weeks after herbicide application, resistant and susceptible individuals were identified based on their phenotypic responses. According to phenotypic classification, equal numbers of extremely resistant and susceptible plants were selected to construct two bulks for bulked segregant analysis (BSA). High-quality genomic DNA was extracted from individual plants using the DNAsecure Plant Genomic DNA Extraction Kit (Tiangen, Beijing, China). DNA concentrations were quantified using a Qubit 4 Fluorometer (Thermo Fisher Scientific, USA) and diluted to 20 ng/µL. Equal amounts of DNA from individuals within each bulk were pooled to form the resistant and susceptible bulks. Sequencing libraries were prepared and sequenced on the DNBSEQ-T7 platform (MGI, Wuhan, China) with an average depth of 30× per bulk. Raw sequencing reads were quality-checked using FastQC, and high-quality reads were aligned to the Sorghum bicolor reference genome (v5.1) using BWA-MEM. Single nucleotide polymorphisms (SNPs) were identified with samtools (v1.10) under default parameters. SNP index values for each bulk were calculated using QTL-seq, and candidate genomic regions associated with herbicide resistance were identified based on the 95% and 99% confidence thresholds. 2.5 Data analysis For the dose–response assay, plants were treated with a series of herbicide concentrations, and plant mortality was recorded after treatment. Dose–response curves were constructed using GraphPad Prism (v9.0; GraphPad Software, San Diego, CA, USA) to assess the herbicide sensitivity of each genotype and to estimate the effective concentration causing 50% growth inhibition (EC₅₀). For the F₂ segregation population, the numbers of resistant and susceptible plants were recorded based on phenotypic evaluation. A chi-square (χ²) test was performed in Microsoft Excel 2016 (Microsoft Corporation, Redmond, WA, USA) to assess the goodness of fit between the observed segregation ratio and the expected Mendelian ratio. 3 Results 3.1. Screening of Herbicide-resistant Sorghum Materials at the Seedling Stage As shown in Table 1 , among the 356 sorghum accessions screened, only a few exhibited noticeable tolerance to herbicide treatment. Under application of the ACCase inhibitor 10% feproxydim, five accessions (1.40% of the total) showed reduced phytotoxicity: IS1219 (grade 2) displayed the highest resistance, while IS10867, SJ72, SJ85, and PI61 (all grade 4) exhibited moderate tolerance. For the ALS inhibitor mesosulfuron-methyl, only two accessions—SJ304 (grade 2) and PI47 (grade 4)—showed visible tolerance, accounting for 0.56% of all materials tested. These results indicate that herbicide resistance is rare among the tested germplasm, and that IS1219 is the most promising resistant materials for subsequent dose–response and molecular analyses. Table 1 Identification of phytotoxicity grades of 356 sorghum accessions treated with two herbicides during seedling-stage screening. Herbicide (Target enzyme) Phytotoxicity Grade Accession Number Accessions Percentage (%) 10% Feproxydim(ACCase) 2 1 IS 1219 0.28 4 4 IS 10867、SJ72、SJ85、PI61 1.12 Mesosulfuron-methyl(ALS) 2 1 SJ304 0.28 4 1 PI47 0.28 3.2 Analysis of Resistance Levels of Sorghum Accessions at the Seedling Stage under Different Herbicide Concentrations Table 2 Resistance Levels of Sorghum Resistant Accessions to Different Herbicides Herbicide Name Sorghum Material(R/S) Regression Equation (y=) Correlation Coefficient r GR50Value (95%CL) Resistance Index (RI) 10% Feproxydim(ACCase) IS 1219 y = − 1.624 + 102.824/(1 + 10 − 2.465×(2.627−x) ) 0.9962 423.7(352.2 ~ 554.9) 4.65 RTx430 y = 2.674 + 99.426/(1 + 10 − 4.007*(1.960− x) ) 0.9889 91.19(72.80 ~ 120.4) Mesosulfuron-methyl(ALS) SJ304 y = − 4.428 + 104.208/(1 + 10 − 2.268×(2.173−x) ) 0.9990 148.9(135.1 ~ 166.2) 3.15 RTx430 y = − 1.616 + 96.666/(1 + 10 − 2.854×(1.674−x) ) 0.9913 47.21(37.57 ~ 58.31) As shown in Table 2 , significant differences in herbicide sensitivity were observed among the tested sorghum genotypes. For the ACCase inhibitor feproxydim, the resistant line IS1219 exhibited markedly higher tolerance than the susceptible control RTx430. The R₅₀ value of IS1219 (423.7 mL/667 m²) was approximately 4.65 times greater than that of RTx430 (91.19 mL/667m²), indicating a high level of resistance to feproxydim. The corresponding regression model showed an excellent fit (r = 0.9962), confirming the reliability of the dose–response relationship. For the ALS inhibitor mesosulfuron-methyl, the resistant accession SJ304 displayed moderate resistance relative to RTx430, with an R₅₀ value of 148.9 mL/667 m² and a resistance index (RI) of 3.15. The correlation coefficients (r = 0.9913–0.9990) further demonstrated that the fitted regression models were robust and statistically sound. Overall, these results reveal that sorghum germplasm exhibits distinct resistance levels to herbicides targeting different enzymatic pathways. The strong resistance of IS1219 to feproxydim may be attributed to mutations in the ACCase target site and/or enhanced metabolic detoxification, while the moderate resistance of SJ304 to mesosulfuron-methyl likely involves ALS gene variation or non–target-site metabolic resistance mechanisms. 3.3 Genetic Analysis of Herbicide Resistance and BSA-Seq of the F₂ Population To identify the genetic basis underlying herbicide resistance, an F₂ population was developed by crossing the resistant sorghum line IS1219 with the susceptible line RTx430. Figure 1 shows the phenotypic differences between RTx430 (sensitive control) and IS1219 (resistant accession) 14 days after treatment with 2× dose of 10% feproxydim. RTx430 exhibited severe phytotoxicity (e.g., plant dwarfing, leaf yellowing and withering), while IS1219 showed no obvious phytotoxicity and maintained good growth status. Herbicide treatments were applied to both the F₁ plants and the F₂ population to evaluate resistance segregation. The F₁ plants exhibited resistance similar to the resistant parent, suggesting that the resistance trait is dominant. A chi-square (χ²) test of the F₂ population indicated that the segregation of resistant and susceptible phenotypes fit a 3:1 Mendelian ratio (Table S4), consistent with inheritance controlled by a single dominant gene. These results suggest that herbicide resistance in IS1219 is primarily governed by a major allele conferring dominant resistance. To identify the genomic region associated with feproxydim resistance, a bulked segregant analysis sequencing (BSA-Seq) approach was performed using F 2 population. As shown in Fig. 2 A, most regions exhibited Δ(SNP-index) values fluctuating around zero, indicating random allele distribution. However, a distinct peak exceeding the 95% confidence threshold was detected on chromosome 1 (Chr01) ( Fig. 2 B). Within this significant region (74.47–75.37 Mb), the Δ(SNP-index) reached its maximum value, suggesting strong association with the herbicide resistance trait. Gene annotation of this 0.9-Mb interval revealed 117 predicted genes ( Fig. 2 C, Table S5). 3.4. RNA-Seq Analysis of Sorghum Response to Feproxydim Treatment Six RNA-Seq libraries were constructed from the resistant line IS1219 and the susceptible line RTx430, sampled 48 hours after treatment with 10% feproxydim at the recommended field dosage. Quality assessment confirmed that all sequencing datasets were of high quality and suitable for downstream analysis (Table S6). Cluster analysis showed that the three biological replicates of each genotype grouped closely together, indicating good experimental reproducibility ( Fig. 3 A). A total of 2,578 differentially expressed genes (DEGs) were identified between IS1219 and RTx430, including 1,319 upregulated and 1,259 downregulated genes ( Fig. 3 B). Gene Ontology (GO) enrichment analysis revealed that, under herbicide stress, upregulated DEGs were significantly enriched in terms such as intrinsic component of plasma membrane, integral component of plasma membrane, intermediate filament-based process, intermediate filament cytoskeleton organization, and 2-oxoglutarate-dependent dioxygenase activity ( Fig. 3 C; Table S7). KEGG pathway enrichment analysis further showed that upregulated DEGs were predominantly involved in flavonoid biosynthesis and stilbenoid, diarylheptanoid and gingerol biosynthesis, suggesting that enhanced secondary metabolism contributes to detoxification and antioxidant defense in resistant plants. In contrast, downregulated DEGs were enriched in zeatin biosynthesis, MAPK signaling, FoxO signaling, and plant–pathogen interaction pathways ( Fig. 3 D; Table S8), indicating that growth- and defense-related processes are suppressed under herbicide stress. Collectively, these results suggest that feproxydim resistance in sorghum involves a complex regulatory network integrating hormone signaling, membrane-associated transport, defense responses, and secondary-metabolite biosynthesis. These transcriptional adjustments likely enable the resistant line IS1219 to mitigate herbicide-induced oxidative damage and maintain cellular homeostasis. 3.5. Integrated Analysis of BSA-Seq and RNA-Seq To identify key genes associated with herbicide resistance, an integrated analysis combining BSA-Seq and RNA-Seq data was performed. Genes located within the candidate interval identified by BSA-Seq (Chr01: 74.47–75.37 Mb) were intersected with differentially expressed genes (DEGs) between the resistant line IS1219 and the susceptible line RTx430 collected 48 hours after treatment with 10% feproxydim at the recommended field dosage. This integrative analysis identified five genes responsive to feproxydim stress within the candidate region (Table 3 ). Cluster analysis revealed distinct expression patterns ( Fig. 3 A), dividing the genes into two groups: one upregulated gene and four downregulated genes. The upregulated gene, Sobic.001G431500 , encodes an α/β-hydrolase fold enzyme/carboxylesterase 17, which may be involved in herbicide metabolism or detoxification. The four downregulated genes included Sobic.001G425500 (encoding an HSP20-like chaperone involved in protein processing in the endoplasmic reticulum), Sobic.001G430500 (a dynamin-related GTPase effector), and two genes ( Sobic.001G432500 and Sobic.001G432600 ) containing PGG domains, which may participate in cellular stress response and membrane remodeling. Together, these results highlight Sobic.001G431500 as the most likely candidate gene conferring feproxydim resistance in IS1219, while the co-regulated downregulated genes may function in associated stress or transport pathways contributing to the overall resistance phenotype. Table 3 Upregulated and downregulated genes identified by integrated BSA-Seq and RNA-Seq analysis. Gene Number Chromosomal Location (Chr01) Function Annotation Expression direction Sobic.001G430500 Chr01:75097727..75100237 Dynamin GTPase effector down Sobic.001G431500 Chr01:75194400..75196997 Alpha/Beta hydrolase fold up Sobic.001G425500 Chr01:74680740..74681565 HSP20-like chaperone down Sobic.001G432500 Chr01:75266077..75267585 PGG domain down Sobic.001G432600 Chr01:75270555..75271610 PGG domain down 3.6 Sobic.001G431500 comparison between IS1219 and RTx430 To further explore how the gene confers resistance in IS1219, we compared its protein sequence with RTx430 and found that compared with the RTx430 allele, the IS1219 allele contained a missense mutation (V300A) and a deletion of three amino acids (P301_P303del) ( Fig. 4 ). It is possible that these changes explain the resistance in IS1219. 4. Discussion Herbicide-resistant accessions derived from natural populations represent valuable resources for herbicide-resistance breeding and genetic research. Such populations harbor abundant natural genetic variation and preserve resistance-associated loci that have adapted to local ecological conditions, while maintaining favorable agronomic traits through long-term natural selection. For example, Bao et al. [ 16 ] screened 854 maize inbred lines using 1 g/L glufosinate at the three-leaf stage and identified the line L336R, a naturally derived variant of L336 that displayed over twice the tolerance of the parental line. Similarly, in the present study, herbicide-resistant sorghum accessions were screened from a natural population. By applying 10% feproxydim at more than twice the recommended field dosage at the four- to five -leaf stage—consistent with the seedling treatment stage used by Tang et al. [ 12 ] , the survival rate of IS1219 reached 69%, exceeding the maximum population survival rate of 51.9% reported by Rizwan et al. [ 17 ] using an Atlantis-type herbicide on lentil populations. Compared with earlier growth stages, sorghum seedlings at the four- to five -leaf stage exhibit stronger physiological metabolism and stress tolerance [ 18 – 19 ] , thereby improving the precision and reliability of herbicide resistance evaluation. TSR, one of the most common herbicide resistance mechanisms, arises from point mutations or overexpression of herbicide target enzymes. Nine single-amino-acid substitutions in the ALS gene (Ala122, Pro197, Ala205, Phe206, Asp376, Arg377, Trp574, Ser653, and Gly654) have been confirmed to confer resistance to ALS-inhibiting herbicides [ 20 – 22 ] . In contrast, NTSR involves detoxification and sequestration processes mediated by plant metabolic systems. Two major pathways underlie NTSR: (i) cytochrome P450 monooxygenases (P450s) and glutathione S-transferases (GSTs) detoxify herbicide molecules through oxidative or conjugative metabolism, and (ii) ATP-binding cassette (ABC) transporters reduce cellular herbicide concentrations by compartmentalization and transport. These systems function synergistically to mitigate herbicide toxicity [ 10 ] . In this study, five genes were identified within the candidate interval on chromosome 1 through RNA-seq, showing herbicide-responsive expression patterns. Integration of transcriptomic profiling, functional annotation, and physiological validation suggests that Sobic.001G431500 , encoding carboxylesterase, plays a pivotal role in feproxydim resistance. This gene was markedly upregulated in the resistant line (IS1219) but not in the susceptible line (RTx430), indicating that enhanced hydrolytic or metabolic activity may be a major resistance mechanism. The α/β-hydrolase family is widely known for mediating Phase I detoxification reactions, in which active herbicide molecules are transformed into less toxic or more conjugation-ready intermediates through hydrolysis, deesterification, or oxidation. Carboxylesterases (CXEs/CarEs), a key subgroup of α/β-hydrolases [ 23 ] , hydrolyze carboxylate esters, thioesters, and amide bonds, facilitating the detoxification of xenobiotics and herbicides [ 24 ] . Many herbicides—such as 2,4-D methyl ester and aryloxyphenoxypropionate (AOPP) herbicides (e.g., diclofop-methyl, clodinafop-propargyl, fenoxaprop-ethyl, fenthioprop-ethyl)—are applied as inactive ester forms that readily penetrate plant cuticles. Once inside, carboxylesterases catalyze their hydrolysis into active carboxylic acid forms, conferring both herbicidal selectivity and plant tolerance [ 25 – 27 ] . In resistant sorghum, the elevated expression of Sobic.001G431500 , likely functioning as carboxylesterase or related hydrolase, may accelerate the Phase I degradation of herbicides such as fepropydim and topramezone, thereby lowering their intracellular accumulation and preventing the inhibition of key biosynthetic pathways (e.g., fatty acid and carotenoid synthesis). It is possible that the missense and deletion mutation in IS1219 allele ( Fig. 4 ) enhances this process. The detoxification intermediates produced can subsequently undergo Phase II conjugation—catalyzed by UDP-glycosyltransferases (UGTs) and GSTs—and Phase III sequestration, where conjugates are transported into vacuoles by ABC transporters [ 28 – 30 ] . Together, these findings support the presence of a three-phase metabolic detoxification network in resistant sorghum, involving coordinated actions of hydrolytic enzymes, conjugative transferases, and transport proteins. This network effectively reduces the cellular concentration of active herbicides and represents a typical NTSR mechanism. The identification of Sobic.001G431500 as a key α/β-hydrolase/carboxylesterase gene provides novel insights into metabolic resistance mechanisms in sorghum and offers a promising molecular target for breeding herbicide-tolerant forage and grain sorghum varieties. 5. Conclusions In this study, 356 sorghum accessions were screened for herbicide tolerance at the seedling stage using gradient herbicide treatments. Under application of the ACCase inhibitor 10% feproxydim, five accessions showed reduced phytotoxicity: IS1219 displayed the highest resistance, while IS10867, SJ72, SJ85, and PI61 exhibited moderate tolerance. For the ALS inhibitor mesosulfuron-methyl, only two accessions, SJ304 and PI47, showed visible tolerance. To elucidate the molecular basis of resistance, a bulked segregant analysis sequencing (BSA-Seq) approach was applied to resistant and susceptible gene pools constructed from the IS1219 × RTx430 F₂ population. The analysis identified a major quantitative trait locus (QTL) for herbicide resistance located on chromosome 1. Transcriptome (RNA-Seq) data of leaf tissues collected after feproxydim treatment revealed five co-expressed candidate genes within the mapped interval. Among them, Sobic.001G431500, encoding a carboxylesterase 17 (α/β-hydrolase), plays a pivotal role in feproxydim resistance. This gene was markedly upregulated in the resistant line (IS1219) but not in the susceptible line (RTx430), indicating that enhanced hydrolytic or metabolic activity may be a major resistance mechanism. Protein sequence comparison also showed that the IS1219 allele carries a missense and deletion mutation at and after position 300 (V300A and P301_P303del). These findings clarify the physiological and molecular mechanisms underlying herbicide resistance in sorghum and provide valuable genetic resources for breeding herbicide-tolerant varieties. Declarations Author Contribution Xing Zhichao: Investigation, Writing – original draft, Writing – review & editing. Cheng Zhengxiao: Writing – original draft, Investigation. Yang Xiaochun: Writing– original draft. Hu Lu: Investigation. Wang Kai: Writing– original draft. Wang Yongfei: Writing – original draft. Hu Die: Investigation. Wang Yi-Hong: Writing– review & editing. Du Junli: Investigation. Wang Lihua: Conceptualization, Funding acquisition, Investigation. Li Jieqin: Investigation, Supervision, Writing – review & editing. Funding The study was supported by the National Natural Science Foundation of China (32372134), Chuzhou "Star of Innovation and Entrepreneurship" Industrial Innovation Team. Data availability No data was used for the research described in the article. 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Cite Share Download PDF Status: Published Journal Publication published 01 Apr, 2026 Read the published version in BMC Plant Biology → Version 1 posted Editorial decision: Revision requested 20 Jan, 2026 Reviews received at journal 10 Jan, 2026 Reviewers agreed at journal 08 Jan, 2026 Reviewers agreed at journal 05 Jan, 2026 Reviews received at journal 05 Jan, 2026 Reviewers agreed at journal 05 Jan, 2026 Reviewers invited by journal 02 Jan, 2026 Editor invited by journal 31 Dec, 2025 Editor assigned by journal 31 Dec, 2025 Submission checks completed at journal 31 Dec, 2025 First submitted to journal 29 Dec, 2025 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. 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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-8473307","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":569079147,"identity":"6d4ccc88-1980-43f6-b242-bb4bc21642b3","order_by":0,"name":"Zhichao Xing","email":"","orcid":"","institution":"Anhui Science and Technology University","correspondingAuthor":false,"prefix":"","firstName":"Zhichao","middleName":"","lastName":"Xing","suffix":""},{"id":569079148,"identity":"47e58a59-29b1-4424-a667-7ccbaea3506f","order_by":1,"name":"Zhengxiao Cheng","email":"","orcid":"","institution":"Anhui Science and Technology University","correspondingAuthor":false,"prefix":"","firstName":"Zhengxiao","middleName":"","lastName":"Cheng","suffix":""},{"id":569079151,"identity":"c3b1d17a-a949-479b-8b9b-f5861b48a0af","order_by":2,"name":"Xiaochun Yang","email":"","orcid":"","institution":"Anhui Science and Technology University","correspondingAuthor":false,"prefix":"","firstName":"Xiaochun","middleName":"","lastName":"Yang","suffix":""},{"id":569079152,"identity":"e2b9e4d3-1d1c-418a-9973-3cc643f44149","order_by":3,"name":"Lu Hu","email":"","orcid":"","institution":"Anhui Science and Technology University","correspondingAuthor":false,"prefix":"","firstName":"Lu","middleName":"","lastName":"Hu","suffix":""},{"id":569079153,"identity":"ebbad9f2-834d-4f74-af80-9b71f98462eb","order_by":4,"name":"Kai Wang","email":"","orcid":"","institution":"Anhui Science and Technology 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11:07:40","extension":"xml","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":98850,"visible":true,"origin":"","legend":"","description":"","filename":"5691c3f6374b45208c0a245d6480da0c1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8473307/v1/602403db67c283d1c1f66aed.xml"},{"id":99521191,"identity":"2bd639e6-6fd5-4043-9277-d2832dd1bb4f","added_by":"auto","created_at":"2026-01-05 11:07:39","extension":"html","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":108170,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8473307/v1/61aad4047e7ca6ee55f86bc7.html"},{"id":99521175,"identity":"ed88aeed-9da7-4854-838d-5cffd5c0b2c2","added_by":"auto","created_at":"2026-01-05 11:07:39","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":177285,"visible":true,"origin":"","legend":"\u003cp\u003ePhenotypes of RTx430 (sensitive control) and IS1219 (resistant accession) 14 days after treatment with 2× dose of 10% feproxydim.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8473307/v1/e034c7ded083c1df1479251f.png"},{"id":99521176,"identity":"2d5a9d90-f152-4c96-b990-c1dad852fd0b","added_by":"auto","created_at":"2026-01-05 11:07:39","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":245517,"visible":true,"origin":"","legend":"\u003cp\u003eBSA-Seq analysis for mapping feproxydim resistance loci in the F₂ population. (A) Genome-wide Δ(SNP-index) plot showing the distribution of SNP-index differences between resistant and susceptible bulks across all 10 sorghum chromosomes. The red line indicates the smoothed trend of Δ(SNP-index), and the green dashed lines represent the 95% confidence interval (\u003cem\u003ep\u003c/em\u003e = 0.05). (B) Localized Δ(SNP-index) plot of chromosome 1 (Chr01), revealing a prominent candidate region associated with herbicide resistance located between 74.47 Mb and 75.37 Mb. (C) 117 genes annotated in the candidate gene region.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8473307/v1/40bc0a99120f595b8afdeb5a.png"},{"id":99792040,"identity":"8d54805f-88a9-4b28-98b3-f9aa94343807","added_by":"auto","created_at":"2026-01-08 13:12:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":283113,"visible":true,"origin":"","legend":"\u003cp\u003eTranscriptomic analysis of sorghum response to feproxydim treatment.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8473307/v1/92d302937266917c52ae97ca.png"},{"id":99790854,"identity":"767e771b-6040-4f4b-9e6b-8d0d7c959d95","added_by":"auto","created_at":"2026-01-08 12:58:46","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":955814,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u0026nbsp;\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8473307/v1/d893807886783d846af4715f.png"},{"id":106344280,"identity":"f56dd1a3-1b3f-462c-b2a4-de930f83e56f","added_by":"auto","created_at":"2026-04-07 16:13:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2632678,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8473307/v1/23c9fe9d-2587-421a-8e46-31e3ae34d83a.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Integrated Genomic and Transcriptomic Analysis Reveals Candidate Genes Underlying Herbicide Resistance in Sorghum","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eSorghum [\u003cem\u003eSorghum bicolor\u003c/em\u003e (L.) Moench] is a globally important grain, forage, and bioenergy crop with remarkable adaptability to harsh environments such as drought, heat, and nutrient-poor soils \u003csup\u003e[\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. However, its production is often severely constrained by weed infestation \u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Weeds compete with sorghum for essential growth resources such as light, water, and nutrients, while also reducing canopy ventilation and light penetration, which in turn increases the incidence of diseases and insect pests. These effects ultimately cause substantial yield losses and deterioration of grain quality \u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAt present, chemical herbicide application remains the predominant method for weed control in sorghum production. Nevertheless, chemical weeding is frequently associated with issues such as phytotoxicity, the evolution of herbicide-resistant weed populations, and environmental contamination of farmland ecosystems \u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. Alternative strategies, including agronomic and biological control, generally fail to achieve effective and sustainable weed management \u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. Therefore, the identification and utilization of genetic resources and germplasm materials with herbicide resistance have become critical priorities for sorghum improvement \u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn recent years, herbicide-resistant germplasms have been developed through approaches such as mutation breeding, natural population screening, and molecular marker-assisted selection, providing key genetic resources for the breeding of herbicide-tolerant sorghum varieties. The herbicide resistance mechanisms identified to date can be broadly classified into two categories: target-site resistance (TSR) and non-target-site resistance (NTSR) \u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. TSR results from mutations in herbicide target genes that reduce the affinity between the herbicide molecule and its target protein, thereby conferring tolerance \u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. For example, Zhang et al. \u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e identified a novel \u003cem\u003eOsEPSPS\u003c/em\u003e allele in rice harboring an Asp-213-Asn substitution within the predicted glyphosate-binding domain, which conferred tolerance to glyphosate at four times the recommended field concentration. In sorghum, two allelic variants of the \u003cem\u003eSbALS\u003c/em\u003e gene\u0026mdash;\u003cem\u003esbals-1\u003c/em\u003e (A93T) and \u003cem\u003esbals-2\u003c/em\u003e (S624N)\u0026mdash;have been demonstrated to confer strong resistance to imidazolinone herbicides, enabling plants to survive treatments up to 16-fold the standard application rate \u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. In contrast, NTSR operates through physiological and biochemical mechanisms that minimize the effective herbicide concentration at its site of action. These mechanisms include reduced herbicide absorption or translocation, enhanced metabolic detoxification, and active efflux \u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. The major molecular systems underlying NTSR involve cytochrome P450 monooxygenases, glutathione S-transferases (GSTs), and ATP-binding cassette (ABC) transporters \u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. For instance, \u003cem\u003eCYP81A6\u003c/em\u003e, a cytochrome P450 gene in rice, has been shown to confer broad-spectrum tolerance to bentazone and metsulfuron-methyl\u0026mdash;two herbicides widely used in rice and wheat cultivation \u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this study, we aimed to address the challenge of weed control in sorghum by identifying and characterizing herbicide-resistant germplasms. Herbicide tolerance was evaluated at the seedling stage among diverse sorghum accessions, and the identified resistant and susceptible genotypes were used to construct an F₂ segregating population. Whole-genome resequencing of resistant and susceptible bulks via BSA-Seq identified genomic regions associated with herbicide resistance. By integrating transcriptomic data from resistant and susceptible parents under herbicide stress, key candidate genes were identified. These findings provide valuable insights and technical support for the molecular breeding of herbicide-resistant sorghum.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Plant Materials\u003c/h2\u003e \u003cp\u003eA total of 356 sorghum germplasm accessions were used in this study (Table S1), all provided by the Anhui Provincial Key Laboratory of Forage Breeding and Utilization. Among them, the herbicide-resistant accession IS1219 and the susceptible accession RTx430 were selected for further analysis. An F₁ hybrid population was generated by crossing IS1219 (♀) with RTx430 (♂). The harvested F₁ plants were self-pollinated to produce an F₂ segregating population.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Screening of Herbicide-Resistant Sorghum at the Seedling Stage and Evaluation of Resistance Levels\u003c/h2\u003e \u003cp\u003eSeeds were surface-sterilized with 75% ethanol for 5 min and rinsed 2\u0026ndash;3 times with sterile distilled water. The sterilized seeds were placed in Petri dishes and incubated at 28\u0026deg;C for germination. Germinated seeds with visible radicles were transplanted into seedling trays filled with nutrient soil (hole size: 4.8 \u0026times; 4.8 \u0026times; 4.8 cm). The trays were maintained in a greenhouse under controlled conditions and watered from the bottom to ensure adequate moisture. At the five-leaf stage, seedlings were sprayed with either 10% feproxydim (200 mL/667 m\u0026sup2;) or mesosulfuron-methyl (160 mL/667 m\u0026sup2;). Phytotoxicity symptoms and plant mortality were recorded at 3, 5, 7, 9, 11, and 14 days after treatment. Phytotoxicity levels were evaluated according to the five-grade classification method specified in the Agricultural Industry Standards of the People\u0026rsquo;s Republic of China (Table S2).\u003c/p\u003e \u003cp\u003eAt the five-leaf stage, seedlings were exposed to a gradient of herbicide concentrations (Table S3), including the recommended field rate (1\u0026times;), a control (0\u0026times;, sprayed with water), and higher dosages. Two types of herbicides\u0026mdash;an ALS inhibitor (mesosulfuron-methyl) and an ACCase inhibitor (10% feproxydim)\u0026mdash;were tested. Twenty-one days after spraying, plant mortality was recorded, and the GR₅₀ (herbicide dose causing 50% growth reduction) and resistance index (RI) were calculated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. RNA-Seq Analysis\u003c/h2\u003e \u003cp\u003eResistant sorghum line IS1219 and susceptible line RTx430 were treated with 10% feproxydim at the recommended field concentration when plants reached the five-leaf stage. Leaf samples were collected from both genotypes 48 h after herbicide application, with three biological replicates per accession. Total RNA was extracted and sent to BGI-Wuhan (Wuhan, China) for transcriptome sequencing. Sequencing reads were aligned to the \u003cem\u003eSorghum bicolor\u003c/em\u003e reference genome (version 5.1) for downstream analysis. Raw reads were quality-checked using FastQC, and clean reads were aligned to the reference genome. Gene expression levels were normalized using the TMM (Trimmed Mean of M-values) method with FeatureCounts. Differentially expressed genes (DEGs) were identified with DESeq2 under the criteria of |log₂FoldChange| \u0026ge; 1 and FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Functional enrichment analysis of DEGs was performed for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) terms in the R environment, with \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 considered significant.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Bulked Segregant Analysis Sequencing (BSA-Seq)\u003c/h2\u003e \u003cp\u003eThe F₂ population was grown in pots under greenhouse conditions. At the five-leaf stage, leaf samples from each individual were collected for DNA extraction. Subsequently, the plants were treated with 10% feproxydim at the recommended field concentration. Two weeks after herbicide application, resistant and susceptible individuals were identified based on their phenotypic responses. According to phenotypic classification, equal numbers of extremely resistant and susceptible plants were selected to construct two bulks for bulked segregant analysis (BSA). High-quality genomic DNA was extracted from individual plants using the DNAsecure Plant Genomic DNA Extraction Kit (Tiangen, Beijing, China). DNA concentrations were quantified using a Qubit 4 Fluorometer (Thermo Fisher Scientific, USA) and diluted to 20 ng/\u0026micro;L. Equal amounts of DNA from individuals within each bulk were pooled to form the resistant and susceptible bulks. Sequencing libraries were prepared and sequenced on the DNBSEQ-T7 platform (MGI, Wuhan, China) with an average depth of 30\u0026times; per bulk.\u003c/p\u003e \u003cp\u003eRaw sequencing reads were quality-checked using FastQC, and high-quality reads were aligned to the \u003cem\u003eSorghum bicolor\u003c/em\u003e reference genome (v5.1) using BWA-MEM. Single nucleotide polymorphisms (SNPs) were identified with samtools (v1.10) under default parameters. SNP index values for each bulk were calculated using QTL-seq, and candidate genomic regions associated with herbicide resistance were identified based on the 95% and 99% confidence thresholds.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Data analysis\u003c/h2\u003e \u003cp\u003eFor the dose\u0026ndash;response assay, plants were treated with a series of herbicide concentrations, and plant mortality was recorded after treatment. Dose\u0026ndash;response curves were constructed using GraphPad Prism (v9.0; GraphPad Software, San Diego, CA, USA) to assess the herbicide sensitivity of each genotype and to estimate the effective concentration causing 50% growth inhibition (EC₅₀). For the F₂ segregation population, the numbers of resistant and susceptible plants were recorded based on phenotypic evaluation. A chi-square (χ\u0026sup2;) test was performed in Microsoft Excel 2016 (Microsoft Corporation, Redmond, WA, USA) to assess the goodness of fit between the observed segregation ratio and the expected Mendelian ratio.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Screening of Herbicide-resistant Sorghum Materials at the Seedling Stage\u003c/h2\u003e \u003cp\u003eAs shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, among the 356 sorghum accessions screened, only a few exhibited noticeable tolerance to herbicide treatment. Under application of the ACCase inhibitor 10% feproxydim, five accessions (1.40% of the total) showed reduced phytotoxicity: IS1219 (grade 2) displayed the highest resistance, while IS10867, SJ72, SJ85, and PI61 (all grade 4) exhibited moderate tolerance. For the ALS inhibitor mesosulfuron-methyl, only two accessions\u0026mdash;SJ304 (grade 2) and PI47 (grade 4)\u0026mdash;showed visible tolerance, accounting for 0.56% of all materials tested. These results indicate that herbicide resistance is rare among the tested germplasm, and that IS1219 is the most promising resistant materials for subsequent dose\u0026ndash;response and molecular analyses.\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\u003eIdentification of phytotoxicity grades of 356 sorghum accessions treated with two herbicides during seedling-stage screening.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" 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=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHerbicide\u003c/p\u003e \u003cp\u003e(Target enzyme)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePhytotoxicity Grade\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAccession Number\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAccessions\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePercentage (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e10% Feproxydim(ACCase)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIS 1219\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.28\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIS 10867、SJ72、SJ85、PI61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eMesosulfuron-methyl(ALS)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSJ304\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.28\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePI47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.28\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=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Analysis of Resistance Levels of Sorghum Accessions at the Seedling Stage under Different Herbicide Concentrations\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eResistance Levels of Sorghum Resistant Accessions to Different Herbicides\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=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHerbicide Name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSorghum Material(R/S)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRegression\u003c/p\u003e \u003cp\u003eEquation (y=)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCorrelation Coefficient r\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eGR50Value\u003c/p\u003e \u003cp\u003e(95%CL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eResistance Index (RI)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e10% Feproxydim(ACCase)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIS 1219\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ey\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;1.624\u0026thinsp;+\u0026thinsp;102.824/(1\u0026thinsp;+\u0026thinsp;10\u003csup\u003e\u0026minus;\u0026thinsp;2.465\u0026times;(2.627\u0026minus;x)\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.9962\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e423.7(352.2\u0026thinsp;~\u0026thinsp;554.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e4.65\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRTx430\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ey\u0026thinsp;=\u0026thinsp;2.674\u0026thinsp;+\u0026thinsp;99.426/(1\u0026thinsp;+\u0026thinsp;10\u003csup\u003e\u0026minus;\u0026thinsp;4.007*(1.960\u0026minus; x)\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.9889\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e91.19(72.80\u0026thinsp;~\u0026thinsp;120.4)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eMesosulfuron-methyl(ALS)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSJ304\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ey\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;4.428\u0026thinsp;+\u0026thinsp;104.208/(1\u0026thinsp;+\u0026thinsp;10\u003csup\u003e\u0026minus;\u0026thinsp;2.268\u0026times;(2.173\u0026minus;x)\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.9990\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e148.9(135.1\u0026thinsp;~\u0026thinsp;166.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e3.15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRTx430\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ey\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;1.616\u0026thinsp;+\u0026thinsp;96.666/(1\u0026thinsp;+\u0026thinsp;10\u003csup\u003e\u0026minus;\u0026thinsp;2.854\u0026times;(1.674\u0026minus;x)\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.9913\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e47.21(37.57\u0026thinsp;~\u0026thinsp;58.31)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAs shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, significant differences in herbicide sensitivity were observed among the tested sorghum genotypes. For the ACCase inhibitor feproxydim, the resistant line IS1219 exhibited markedly higher tolerance than the susceptible control RTx430. The R₅₀ value of IS1219 (423.7 mL/667 m\u0026sup2;) was approximately 4.65 times greater than that of RTx430 (91.19 mL/667m\u0026sup2;), indicating a high level of resistance to feproxydim. The corresponding regression model showed an excellent fit (r\u0026thinsp;=\u0026thinsp;0.9962), confirming the reliability of the dose\u0026ndash;response relationship. For the ALS inhibitor mesosulfuron-methyl, the resistant accession SJ304 displayed moderate resistance relative to RTx430, with an R₅₀ value of 148.9 mL/667 m\u0026sup2; and a resistance index (RI) of 3.15. The correlation coefficients (r\u0026thinsp;=\u0026thinsp;0.9913\u0026ndash;0.9990) further demonstrated that the fitted regression models were robust and statistically sound.\u003c/p\u003e \u003cp\u003eOverall, these results reveal that sorghum germplasm exhibits distinct resistance levels to herbicides targeting different enzymatic pathways. The strong resistance of IS1219 to feproxydim may be attributed to mutations in the ACCase target site and/or enhanced metabolic detoxification, while the moderate resistance of SJ304 to mesosulfuron-methyl likely involves ALS gene variation or non\u0026ndash;target-site metabolic resistance mechanisms.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Genetic Analysis of Herbicide Resistance and BSA-Seq of the F₂ Population\u003c/h2\u003e \u003cp\u003eTo identify the genetic basis underlying herbicide resistance, an F₂ population was developed by crossing the resistant sorghum line IS1219 with the susceptible line RTx430. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows the phenotypic differences between RTx430 (sensitive control) and IS1219 (resistant accession) 14 days after treatment with 2\u0026times; dose of 10% feproxydim. RTx430 exhibited severe phytotoxicity (e.g., plant dwarfing, leaf yellowing and withering), while IS1219 showed no obvious phytotoxicity and maintained good growth status. Herbicide treatments were applied to both the F₁ plants and the F₂ population to evaluate resistance segregation. The F₁ plants exhibited resistance similar to the resistant parent, suggesting that the resistance trait is dominant. A chi-square (χ\u0026sup2;) test of the F₂ population indicated that the segregation of resistant and susceptible phenotypes fit a 3:1 Mendelian ratio (Table S4), consistent with inheritance controlled by a single dominant gene. These results suggest that herbicide resistance in IS1219 is primarily governed by a major allele conferring dominant resistance.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo identify the genomic region associated with feproxydim resistance, a bulked segregant analysis sequencing (BSA-Seq) approach was performed using F\u003csub\u003e2\u003c/sub\u003e population. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, most regions exhibited Δ(SNP-index) values fluctuating around zero, indicating random allele distribution. However, a distinct peak exceeding the 95% confidence threshold was detected on chromosome 1 (Chr01) ( Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Within this significant region (74.47\u0026ndash;75.37 Mb), the Δ(SNP-index) reached its maximum value, suggesting strong association with the herbicide resistance trait. Gene annotation of this 0.9-Mb interval revealed 117 predicted genes ( Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, Table S5).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.4. RNA-Seq Analysis of Sorghum Response to Feproxydim Treatment\u003c/h2\u003e \u003cp\u003eSix RNA-Seq libraries were constructed from the resistant line IS1219 and the susceptible line RTx430, sampled 48 hours after treatment with 10% feproxydim at the recommended field dosage. Quality assessment confirmed that all sequencing datasets were of high quality and suitable for downstream analysis (Table S6). Cluster analysis showed that the three biological replicates of each genotype grouped closely together, indicating good experimental reproducibility ( Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). A total of 2,578 differentially expressed genes (DEGs) were identified between IS1219 and RTx430, including 1,319 upregulated and 1,259 downregulated genes ( Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Gene Ontology (GO) enrichment analysis revealed that, under herbicide stress, upregulated DEGs were significantly enriched in terms such as intrinsic component of plasma membrane, integral component of plasma membrane, intermediate filament-based process, intermediate filament cytoskeleton organization, and 2-oxoglutarate-dependent dioxygenase activity ( Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC; Table S7).\u003c/p\u003e \u003cp\u003eKEGG pathway enrichment analysis further showed that upregulated DEGs were predominantly involved in flavonoid biosynthesis and stilbenoid, diarylheptanoid and gingerol biosynthesis, suggesting that enhanced secondary metabolism contributes to detoxification and antioxidant defense in resistant plants. In contrast, downregulated DEGs were enriched in zeatin biosynthesis, MAPK signaling, FoxO signaling, and plant\u0026ndash;pathogen interaction pathways ( Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD; Table S8), indicating that growth- and defense-related processes are suppressed under herbicide stress. Collectively, these results suggest that feproxydim resistance in sorghum involves a complex regulatory network integrating hormone signaling, membrane-associated transport, defense responses, and secondary-metabolite biosynthesis. These transcriptional adjustments likely enable the resistant line IS1219 to mitigate herbicide-induced oxidative damage and maintain cellular homeostasis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Integrated Analysis of BSA-Seq and RNA-Seq\u003c/h2\u003e \u003cp\u003eTo identify key genes associated with herbicide resistance, an integrated analysis combining BSA-Seq and RNA-Seq data was performed. Genes located within the candidate interval identified by BSA-Seq (Chr01: 74.47\u0026ndash;75.37 Mb) were intersected with differentially expressed genes (DEGs) between the resistant line IS1219 and the susceptible line RTx430 collected 48 hours after treatment with 10% feproxydim at the recommended field dosage.\u003c/p\u003e \u003cp\u003eThis integrative analysis identified five genes responsive to feproxydim stress within the candidate region (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Cluster analysis revealed distinct expression patterns ( Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), dividing the genes into two groups: one upregulated gene and four downregulated genes. The upregulated gene, \u003cem\u003eSobic.001G431500\u003c/em\u003e, encodes an α/β-hydrolase fold enzyme/carboxylesterase 17, which may be involved in herbicide metabolism or detoxification. The four downregulated genes included \u003cem\u003eSobic.001G425500\u003c/em\u003e (encoding an HSP20-like chaperone involved in protein processing in the endoplasmic reticulum), \u003cem\u003eSobic.001G430500\u003c/em\u003e (a dynamin-related GTPase effector), and two genes (\u003cem\u003eSobic.001G432500\u003c/em\u003e and \u003cem\u003eSobic.001G432600\u003c/em\u003e) containing PGG domains, which may participate in cellular stress response and membrane remodeling.\u003c/p\u003e \u003cp\u003eTogether, these results highlight \u003cem\u003eSobic.001G431500\u003c/em\u003e as the most likely candidate gene conferring feproxydim resistance in IS1219, while the co-regulated downregulated genes may function in associated stress or transport pathways contributing to the overall resistance phenotype.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eUpregulated and downregulated genes identified by integrated BSA-Seq and RNA-Seq analysis.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGene Number\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChromosomal Location (Chr01)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFunction Annotation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eExpression direction\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSobic.001G430500\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChr01:75097727..75100237\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDynamin GTPase effector\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003edown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSobic.001G431500\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChr01:75194400..75196997\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAlpha/Beta hydrolase fold\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eup\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSobic.001G425500\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChr01:74680740..74681565\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHSP20-like chaperone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003edown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSobic.001G432500\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChr01:75266077..75267585\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePGG domain\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003edown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSobic.001G432600\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChr01:75270555..75271610\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePGG domain\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003edown\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=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.6 \u003cem\u003eSobic.001G431500\u003c/em\u003e comparison between IS1219 and RTx430\u003c/h2\u003e \u003cp\u003eTo further explore how the gene confers resistance in IS1219, we compared its protein sequence with RTx430 and found that compared with the RTx430 allele, the IS1219 allele contained a missense mutation (V300A) and a deletion of three amino acids (P301_P303del) ( Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). It is possible that these changes explain the resistance in IS1219.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eHerbicide-resistant accessions derived from natural populations represent valuable resources for herbicide-resistance breeding and genetic research. Such populations harbor abundant natural genetic variation and preserve resistance-associated loci that have adapted to local ecological conditions, while maintaining favorable agronomic traits through long-term natural selection. For example, Bao et al. \u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e screened 854 maize inbred lines using 1 g/L glufosinate at the three-leaf stage and identified the line L336R, a naturally derived variant of L336 that displayed over twice the tolerance of the parental line. Similarly, in the present study, herbicide-resistant sorghum accessions were screened from a natural population. By applying 10% feproxydim at more than twice the recommended field dosage at the four- to five -leaf stage\u0026mdash;consistent with the seedling treatment stage used by Tang et al. \u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e, the survival rate of IS1219 reached 69%, exceeding the maximum population survival rate of 51.9% reported by Rizwan et al. \u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e using an Atlantis-type herbicide on lentil populations. Compared with earlier growth stages, sorghum seedlings at the four- to five -leaf stage exhibit stronger physiological metabolism and stress tolerance \u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e, thereby improving the precision and reliability of herbicide resistance evaluation.\u003c/p\u003e \u003cp\u003eTSR, one of the most common herbicide resistance mechanisms, arises from point mutations or overexpression of herbicide target enzymes. Nine single-amino-acid substitutions in the ALS gene (Ala122, Pro197, Ala205, Phe206, Asp376, Arg377, Trp574, Ser653, and Gly654) have been confirmed to confer resistance to ALS-inhibiting herbicides \u003csup\u003e[\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. In contrast, NTSR involves detoxification and sequestration processes mediated by plant metabolic systems. Two major pathways underlie NTSR: (i) cytochrome P450 monooxygenases (P450s) and glutathione S-transferases (GSTs) detoxify herbicide molecules through oxidative or conjugative metabolism, and (ii) ATP-binding cassette (ABC) transporters reduce cellular herbicide concentrations by compartmentalization and transport. These systems function synergistically to mitigate herbicide toxicity \u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. In this study, five genes were identified within the candidate interval on chromosome 1 through RNA-seq, showing herbicide-responsive expression patterns. Integration of transcriptomic profiling, functional annotation, and physiological validation suggests that \u003cem\u003eSobic.001G431500\u003c/em\u003e, encoding carboxylesterase, plays a pivotal role in feproxydim resistance. This gene was markedly upregulated in the resistant line (IS1219) but not in the susceptible line (RTx430), indicating that enhanced hydrolytic or metabolic activity may be a major resistance mechanism.\u003c/p\u003e \u003cp\u003eThe α/β-hydrolase family is widely known for mediating Phase I detoxification reactions, in which active herbicide molecules are transformed into less toxic or more conjugation-ready intermediates through hydrolysis, deesterification, or oxidation. Carboxylesterases (CXEs/CarEs), a key subgroup of α/β-hydrolases \u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e, hydrolyze carboxylate esters, thioesters, and amide bonds, facilitating the detoxification of xenobiotics and herbicides \u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. Many herbicides\u0026mdash;such as 2,4-D methyl ester and aryloxyphenoxypropionate (AOPP) herbicides (e.g., diclofop-methyl, clodinafop-propargyl, fenoxaprop-ethyl, fenthioprop-ethyl)\u0026mdash;are applied as inactive ester forms that readily penetrate plant cuticles. Once inside, carboxylesterases catalyze their hydrolysis into active carboxylic acid forms, conferring both herbicidal selectivity and plant tolerance \u003csup\u003e[\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. In resistant sorghum, the elevated expression of \u003cem\u003eSobic.001G431500\u003c/em\u003e, likely functioning as carboxylesterase or related hydrolase, may accelerate the Phase I degradation of herbicides such as fepropydim and topramezone, thereby lowering their intracellular accumulation and preventing the inhibition of key biosynthetic pathways (e.g., fatty acid and carotenoid synthesis). It is possible that the missense and deletion mutation in IS1219 allele ( Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) enhances this process. The detoxification intermediates produced can subsequently undergo Phase II conjugation\u0026mdash;catalyzed by UDP-glycosyltransferases (UGTs) and GSTs\u0026mdash;and Phase III sequestration, where conjugates are transported into vacuoles by ABC transporters \u003csup\u003e[\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTogether, these findings support the presence of a three-phase metabolic detoxification network in resistant sorghum, involving coordinated actions of hydrolytic enzymes, conjugative transferases, and transport proteins. This network effectively reduces the cellular concentration of active herbicides and represents a typical NTSR mechanism. The identification of \u003cem\u003eSobic.001G431500\u003c/em\u003e as a key α/β-hydrolase/carboxylesterase gene provides novel insights into metabolic resistance mechanisms in sorghum and offers a promising molecular target for breeding herbicide-tolerant forage and grain sorghum varieties.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eIn this study, 356 sorghum accessions were screened for herbicide tolerance at the seedling stage using gradient herbicide treatments. Under application of the ACCase inhibitor 10% feproxydim, five accessions showed reduced phytotoxicity: IS1219 displayed the highest resistance, while IS10867, SJ72, SJ85, and PI61 exhibited moderate tolerance. For the ALS inhibitor mesosulfuron-methyl, only two accessions, SJ304 and PI47, showed visible tolerance. To elucidate the molecular basis of resistance, a bulked segregant analysis sequencing (BSA-Seq) approach was applied to resistant and susceptible gene pools constructed from the IS1219 \u0026times; RTx430 F₂ population. The analysis identified a major quantitative trait locus (QTL) for herbicide resistance located on chromosome 1. Transcriptome (RNA-Seq) data of leaf tissues collected after feproxydim treatment revealed five co-expressed candidate genes within the mapped interval. Among them, Sobic.001G431500, encoding a carboxylesterase 17 (α/β-hydrolase), plays a pivotal role in feproxydim resistance. This gene was markedly upregulated in the resistant line (IS1219) but not in the susceptible line (RTx430), indicating that enhanced hydrolytic or metabolic activity may be a major resistance mechanism. Protein sequence comparison also showed that the IS1219 allele carries a missense and deletion mutation at and after position 300 (V300A and P301_P303del). These findings clarify the physiological and molecular mechanisms underlying herbicide resistance in sorghum and provide valuable genetic resources for breeding herbicide-tolerant varieties.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contribution\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXing Zhichao: Investigation, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing. Cheng Zhengxiao:\u0026nbsp;Writing \u0026ndash; original draft, Investigation. Yang Xiaochun: Writing\u0026ndash; original draft. Hu Lu: Investigation. Wang Kai: Writing\u0026ndash; original draft. Wang Yongfei: Writing \u0026ndash; original draft. Hu Die: Investigation. Wang Yi-Hong: Writing\u0026ndash; review \u0026amp; editing. Du Junli: Investigation. Wang Lihua: Conceptualization, Funding acquisition, Investigation. Li Jieqin: Investigation, Supervision, Writing \u0026ndash; review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was supported by the National Natural Science Foundation of China (32372134), Chuzhou \u0026quot;Star of Innovation and Entrepreneurship\u0026quot; Industrial Innovation Team.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo data was used for the research described in the article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePaterson AH, Bowers JE, Bruggmann R, Dubchak I, Grimwood J, Gundlach H, Haberer G, Hellsten U, Mitros T, Poliakov A, Schmutz J, Spannagl M, Tang H, Wang X, Wicker T, Bharti AK, Chapman J, Feltus FA, Gowik U, Grigoriev IV, Lyons E, Maher CA, Martis M, Narechania A, Otillar RP, Penning BW, Salamov AA, Wang Y, Zhang L, Carpita NC, Freeling M, Gingle AR, Hash CT, Keller B, Klein P, Kresovich S, McCann MC, Ming R, Peterson DG, Mehboob-ur-Rahman, Ware, Westhoff D, Mayer P, Messing KF, Rokhsar J. 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Weed Technol. 2020;34(4):624\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePeerzada AM, Ali HH, Chauhan BS. Weed management in sorghum [Sorghum bicolor (L.) Moench] using crop competition: A review. Crop Prot. 2017;95:74\u0026ndash;80.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBeckie HJ. Herbicide resistance in plants. Plants. 2020;9(4):435.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHasan M, Ahmad-Hamdani MS, Rosli AM, Hamdan H. Bioherbicides: An eco-friendly tool for sustainable weed management. Plants. 2021;10(6):1212.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOuyang C, Jin X, Zhao H, Chen S, Zhao G, Li D, Liu W, He X, Wu Y, Yang J, An B. Generating Broad-Spectrum Resistance to ALS-Inhibiting Herbicides in Rice by CRISPR/Cas9-Mediated NHEJ. Rice. 2025;18(1):86.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDeng W, Yang Q, Chen Y, Yang M, Xia Z, Zhu J, Chen Y, Cai J, Yuan S. Cyhalofop-butyl and glyphosate multiple-herbicide resistance evolved in an Eleusine indica population collected in Chinese direct-seeding rice. J Agric Food Chem. 2020;68(9):2623\u0026ndash;30.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eD\u0026eacute;lye C, Duhoux A, Pernin F, Riggins CW, Tranel PJ. Molecular mechanisms of herbicide resistance. Weed Sci. 2015;63(SP1):91\u0026ndash;115.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang C, Zhong X, Li S, Yan L, Li J, He Y, Lin Y, Zhang Y, Xia L. Artificial evolution of OsEPSPS through an improved dual cytosine and adenine base editor generated a novel allele conferring rice glyphosate tolerance. J Integr Plant Biol. 2023;65(9):2194\u0026ndash;203.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTang S, Shi J, Li X, Yang M, Li C, Zhang D, Yang S, Mei C, Luo Z, Zhang L, Zhang W. Development and Breeding of Herbicide-Resistant Sorghum for Effective Cereal‐Legume Intercropping. Adv Sci. 2025;12(27):e2503083.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSuzukawa AK, Bobadilla LK, Mallory-Smith C, Brunharo CA. Non-target-site resistance in Lolium spp. globally: a review. Front Plant Sci. 2021;11:609209.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGaines TA, Duke SO, Morran S, Rigon CA, Tranel PJ, K\u0026uuml;pper A, Dayan FE. Mechanisms of evolved herbicide resistance. J Biol Chem. 2020;295(30):10307\u0026ndash;30.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLu HP, Edwards M, Wang QZ, Zhao HJ, Fu HW, Huang JZ, Gatehouse A, Shu QY. Expression of cytochrome P450 CYP81A6 in rice: tissue specificity, protein subcellular localization, and response to herbicide application. J Zhejiang Univ-SCI B. 2015;16(2):113\u0026ndash;22.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBao J, Gao Y, Li Y, Wu S, Li J, Dong Z, Wan X. 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Afr J Agric Res. 2013;8(8):660\u0026ndash;70.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhan Y, Liu H, Cao Z, Qi J, Bai L, Pan L. Target-site and non-target-site resistance mechanisms confer mesosulfuron-methyl resistance in Alopecurus aequalis. Plant Physiol Biochem. 2024;210:108597.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang J, Cao W, Guo Q, Yang Y, Bai L, Pan L. Resistance to mesosulfuron-methyl in Beckmannia syzigachne may involve ROS burst and non-target-site resistance mechanisms. Ecotoxicol Environ Saf. 2022;229:113072.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao N, Yan Y, Luo Y, Zou N, Liu W, Wang J. Unravelling mesosulfuron-methyl phytotoxicity and metabolism-based herbicide resistance in Alopecurus aequalis: Insight into regulatory mechanisms using proteomics. 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A wheat ABC transporter contributes to both grain formation and mycotoxin tolerance. J Exp Bot. 2015;66(9):2583\u0026ndash;93.\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":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Sorghum, Herbicide, RNA-Seq, BSA-Seq, Candidate genes","lastPublishedDoi":"10.21203/rs.3.rs-8473307/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8473307/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBackground: Herbicide-resistant germplasms provide critical genetic resources for improving weed control and understanding resistance mechanisms in crops.\u003c/p\u003e\n\u003cp\u003eResults: In this study, 356 sorghum accessions were screened for herbicide tolerance at the seedling stage using gradient herbicide treatments. Under application of the ACCase inhibitor 10% feproxydim, five accessions showed reduced phytotoxicity: IS1219 displayed the highest resistance, while IS10867, SJ72, SJ85, and PI61 exhibited moderate tolerance. For the ALS inhibitor mesosulfuron-methyl, only two accessions, SJ304 and PI47, showed visible tolerance. To elucidate the molecular basis of resistance, a bulked segregant analysis sequencing (BSA-Seq) approach was applied to resistant and susceptible gene pools constructed from the IS1219 × RTx430 F₂ population. The analysis identified a major quantitative trait locus (QTL) for herbicide resistance located on chromosome 1.Transcriptome (RNA-Seq) data of leaf tissues collected after feproxydim treatment revealed five co-expressed candidate genes within the mapped interval. Among them, \u003cem\u003eSobic.001G431500\u003c/em\u003e, encoding a carboxylesterase 17 (α/β-hydrolase), plays a pivotal role in feproxydim resistance. This gene was markedly upregulated in the resistant line (IS1219) but not in the susceptible line (RTx430), indicating that enhanced hydrolytic or metabolic activity may be a major resistance mechanism. Protein sequence comparison also showed that the IS1219 allele carries a missense and deletion mutation at and after position 300 (V300A and P301_P303del).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConclusions: These findings clarify the physiological and molecular mechanisms underlying herbicide resistance in sorghum and provide valuable genetic resources for breeding herbicide-tolerant varieties.\u003c/p\u003e","manuscriptTitle":"Integrated Genomic and Transcriptomic Analysis Reveals Candidate Genes Underlying Herbicide Resistance in Sorghum","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-05 11:07:35","doi":"10.21203/rs.3.rs-8473307/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-20T06:36:51+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-10T22:58:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"305536824278588015899233657087244152146","date":"2026-01-08T09:59:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"134761304131829774580902836064406895272","date":"2026-01-05T10:05:39+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-05T06:31:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"97331238959720755258717076803964234909","date":"2026-01-05T05:58:10+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-02T13:46:05+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-12-31T09:47:05+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-31T09:27:37+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-31T09:25:52+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2025-12-29T12:52:49+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"78b4b51f-8d0b-453a-8d93-09c96b6181c5","owner":[],"postedDate":"January 5th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-04-07T16:06:27+00:00","versionOfRecord":{"articleIdentity":"rs-8473307","link":"https://doi.org/10.1186/s12870-026-08624-5","journal":{"identity":"bmc-plant-biology","isVorOnly":false,"title":"BMC Plant Biology"},"publishedOn":"2026-04-01 15:59:26","publishedOnDateReadable":"April 1st, 2026"},"versionCreatedAt":"2026-01-05 11:07:35","video":"","vorDoi":"10.1186/s12870-026-08624-5","vorDoiUrl":"https://doi.org/10.1186/s12870-026-08624-5","workflowStages":[]},"version":"v1","identity":"rs-8473307","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8473307","identity":"rs-8473307","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}