Integrated Genomic and Transcriptomic Analysis Reveals Candidate Genes Underlying Herbicide Resistance in Sorghum
Zhichao
Xing
2
Zhengxiao
Cheng
2
Xiaochun
Yang
2
Lu
Hu
2
Kai
Wang
2
Yongfei
Wang
2
Die
Hu
2
Yi-Hong
Wang
3
Junli
Du
2
Lihua
Wang
2
Jieqin
Li
2✉
Emailwlhljq@163.com
1
College of Agriculture
Anhui Science and Technology University
233100
Fengyang
Anhui
China
2
International Cooperative Research Center for Forage Biological Breeding in Anhui Province
233100
Chuzhou
China
3
Department of Biology
University of Louisiana at Lafayette
70504
Lafayette
LA
United States
Zhichao Xing1,2, Zhengxiao Cheng1,2, Xiaochun Yang1,2, Lu Hu1,2, Kai Wang1,2, Yongfei Wang1,2, Die Hu1,2, Yi-Hong Wang3, Junli Du1,2, Lihua Wang1,2, Jieqin Li1,2*
1*College of Agriculture, Anhui Science and Technology University, Fengyang, Anhui, 233100, China.
2 International Cooperative Research Center for Forage Biological Breeding in Anhui Province, Chuzhou,233100, China.
3 Department of Biology, University of Louisiana at Lafayette, Lafayette, LA 70504, United States.
*Corresponding author(s). E-mail(s): wlhljq@163.com
Abstract
Background
Herbicide-resistant germplasms provide critical genetic resources for improving weed control and understanding resistance mechanisms in crops.
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.
Keywords:
Sorghum
Herbicide
RNA-Seq
BSA-Seq
Candidate genes
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].
A
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
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
A
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).A
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)
A
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. A
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.
|
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
|
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
A
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.Fig. 1
Phenotypes of RTx430 (sensitive control) and IS1219 (resistant accession) 14 days after treatment with 2× dose of 10% feproxydim.
A
To identify the genomic region associated with feproxydim resistance, a bulked segregant analysis sequencing (BSA-Seq) approach was performed using F2 population. As shown in Fig. 2A, 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. 2B). 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. 2C, Table S5).Fig. 2
BSA-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 (p = 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.
3.4. RNA-Seq Analysis of Sorghum Response to Feproxydim Treatment
A
A
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. 3A). 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. 3B). 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. 3C; Table S7).A
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. 3D; 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.Fig. 3
Transcriptomic analysis of sorghum response to feproxydim treatment.
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. 3A), 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.
|
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.
Fig. 4
Protein sequence alignment between IS1219 and RTx430 (SbiRTX430.01G453700) alleles of Sobic.001G431500. “.” Indicates missense and “---” deletion mutations. Sequence color coding: Red-Small amino acids (AVFPMILW); Blue-Acidic (DE); Magenta-Basic (RHK); Green-Hydroxly/sulfhydryl/amine (STYHCNGQ).
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.
A
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.
A
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.
Declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The 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.
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