Method for detecting resistance of polypogon fugax to ACCase inhibitor herbicide

Abstract

The invention discloses a method for detecting the resistance of polypogon fugax to ACCase inhibitor herbicides, which comprises the following steps: detecting the resistance of polypogon fugax to ACCase inhibitor herbicides, extracting the genome DNA of a polypogon fugax plant to be detected, carrying out PCR amplification on the genome DNA by using a KASP primer group, detecting the fluorescence signal of the PCR product, and determining the genotype of the 1781st nucleotide of the ACCase gene. And if the nucleotide at the site is T, judging that the polypogon fugax plant has resistance to ACCase inhibitor herbicides. According to the method, the ACCase inhibitor herbicide resistance of the polypogon fugax is accurately judged, the problems that a traditional whole plant bioassay method is time-consuming and labor-consuming, and the drug resistance mechanism is difficult to directly determine are effectively solved, a powerful technical support is provided for early-stage resistance monitoring and accurate prevention and control of farmland weeds, and the method is suitable for popularization and application. Grain loss caused by drug resistance of weeds can be reduced, and agricultural production efficiency and sustainable development are guaranteed.

Description

Technical Field

[0001] The present invention belongs to the field of agricultural biotechnology and relates to a KASP typing marker method and application for the ACCase gene 1781 site mutation associated with herbicide resistance in the weed Polypogon fugax Neesex Steud. The method is mainly used for rapidly detecting the resistance of Polypogon fugax Neesex Steud to ACCase inhibitor herbicides, providing technical support for the precise prevention and control of weeds in farmland. Background Art

[0002] Farmland weeds compete with crops for resources such as light, water, fertilizer, and space, and spread pests and diseases, significantly reducing crop yield and quality, increasing agricultural management costs, and posing a serious threat to agricultural production. Polypogon fugax Nees ex Steud., an annual or biennial grass weed, has a wide range of adaptability and strong competitive advantages, easily becoming a dominant species in its growing environment. Its growth cycle closely matches that of summer crops such as wheat and rapeseed, making it a major weed pest in the fields of these summer crops. Because Polypogon fugax Nees ex Steud. occurs in large numbers in fields and grows slender plants, manual weeding is inefficient and ineffective. Chemical control, owing to its labor-saving, time-saving, and highly effective weed control, has become the preferred method.

[0003] Currently, the most commonly used foliar treatments for controlling grass weeds in wheat and rapeseed fields are acetyl-CoA carboxylase (ACCase) inhibitors. However, the long-term use of a single weed control technique or agent has led to increasing resistance (or tolerance) in gramineous weeds. Numerous studies, both domestically and internationally, have found that the mechanisms of weed resistance to herbicides are primarily categorized as target-site resistance (TSR) and non-target-site resistance (NTSR). Target-site resistance primarily results from mutations in the target enzyme gene within the weed, resulting in decreased sensitivity to the herbicide or overexpression of the target enzyme. Non-target resistance, on the other hand, results from the regulation of physiological and biochemical metabolic processes by multiple genes, leading to weed resistance.

[0004] Currently, the commonly used whole-plant bioassay method can be used to determine insecticide resistance in cloverleaf weed. However, this method is difficult to directly determine the mechanism of resistance, is time-consuming, and can easily miss the optimal period for weed control, leading to significant losses in agricultural production. Therefore, a method that can quickly determine the resistance status of cloverleaf weed in the same season is urgently needed to enable timely and accurate chemical control and reduce food losses caused by weeds.

[0005] During the development of this invention, it was discovered that KASP (Kompetitive allele specific PCR) typing technology has attracted considerable attention due to its high specificity, low cost, and operability. This technology achieves high-throughput genotyping by competitively binding specific primers to SNP (single nucleotide polymorphism) sites, combined with universal fluorescent probes. Specific matching of 3'-end bases enables precise typing with a low error rate. The use of universal fluorescent probes eliminates the need for customized specific probes for each SNP, significantly reducing reagent costs. Consequently, KASP technology has been widely used in fields such as medicine and agricultural mutation analysis, providing a new technical approach for detecting resistance to clover. Summary of the Invention

[0006] In view of this, the object of the present invention is to provide a method for detecting the resistance of Codonopsis pilosula to ACCase inhibitor herbicides, a KASP primer set, a kit for detecting resistant Codonopsis pilosula, the application of the KASP primer set or the kit for detecting resistant Codonopsis pilosula, and a method for identifying resistant Codonopsis pilosula populations, so as to address the shortcomings of the existing methods for detecting Codonopsis pilosula resistance, achieve rapid and accurate detection of Codonopsis pilosula resistance, promote the precise identification of Codonopsis pilosula resistant populations, and provide a new approach for cultivating herbicide-resistant plants, thereby providing strong technical support for the efficient prevention and control of farmland weeds.

[0007] Through long-term exploration and experimentation, as well as numerous experiments and efforts, the inventors have continuously innovated and reformed to solve the above technical problems. The present invention provides a method for detecting the resistance of Coleus chinensis to ACCase inhibitor herbicides, comprising the following steps: (a) extracting genomic DNA from the tested Herba Corydalis plants; (b) performing fluorescent PCR amplification on the genomic DNA using a KASP primer set, wherein the KASP primer set comprises: KASP-1F: sequence shown in SEQ ID NO: 3; KASP-2F: sequence shown in SEQ ID NO: 4; KASP-R: sequence shown in SEQ ID NO: 5; (c) detecting the fluorescent signal of the PCR product to determine the genotype of nucleotide 1781 of the ACCase gene; (d) If the nucleotide at position 1781 is T, the Echinops chinensis plant is determined to be resistant to the ACCase inhibitor herbicide.

[0008] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a faster, more accurate and high-throughput detection method. By designing a specific KASP primer set targeting the 1781st nucleotide mutation site of the ACCase gene and combining it with fluorescence signal detection technology, it can accurately judge the resistance of Codonopsis pilosula to ACCase inhibitor herbicides. This effectively solves the problem that traditional whole-plant bioassay methods are time-consuming and labor-intensive and difficult to directly determine the resistance mechanism. It provides strong technical support for early resistance monitoring and precise prevention and control of farmland weeds, helps reduce food losses caused by weed resistance, and ensures agricultural production efficiency and sustainable development.

[0009] On the basis of the above technical solution, the present invention can also be improved as follows: Furthermore, the ACCase inhibitor herbicide is selected from at least one of clodinafop-butyl, quizalofop-ethyl and fenoxaprop-butyl.

[0010] Compared with the prior art, the beneficial effects of adopting the above further technical solution are: The resistance of the grass with the ACCase gene 1781 mutation site to three herbicides with the same mechanism of action was verified. The resistance can be expanded to more types of herbicides of the same type, making the detection method more widely applicable and better meeting practical application needs, further enhancing the effectiveness and reliability of the present invention in guiding farmland weed control.

[0011] On the basis of the above technical solution, the present invention can also be improved as follows: Furthermore, the 1781st nucleotide mutation causes the ACCase gene to change from ATA to TTA, and the 126th amino acid to change from isoleucine to leucine.

[0012] Compared with the prior art, the beneficial effects of adopting the above further technical solution are: The present invention clarifies the molecular mechanism of resistance, provides a more in-depth theoretical basis for the precise detection and study of the resistance of Codonopsis pilosula to ACCase inhibitor herbicides, and helps to formulate targeted weed management strategies.

[0013] On the basis of the above technical solution, the present invention can also be improved as follows: Furthermore: the PCR amplification procedure includes: pre-denaturation at 94°C for 15 minutes; Denature at 94°C for 15 seconds. Annealing at 61°C for 1 minute, decreasing the temperature by 0.6°C each cycle for 10 cycles; denaturation at 94°C for 20 seconds and annealing at 57°C for 1 minute for 40 cycles; Extension at 72°C for 7 minutes.

[0014] Compared with the prior art, the beneficial effects of adopting the above further technical solution are: By optimizing the PCR amplification procedure, the specificity and accuracy of the amplification reaction are improved, the reliability of the detection results is ensured, and at the same time it helps to improve the detection efficiency and repeatability, further enhancing the feasibility and stability of the present invention in practical applications.

[0015] The present invention also provides a KASP primer set for detecting mutations at site 1781 of the ACCase gene of the tricholoma serrata, comprising: KASP-1F: sequence shown in SEQ ID NO: 3; KASP-2F: sequence shown in SEQ ID NO: 4; KASP-R: The sequence is shown in SEQ ID NO: 5.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a specific KASP primer set targeting the mutation at site 1781 of the ACCase gene of the herbicide Herba Eupatorii, thereby achieving high-specificity, high-throughput, and rapid detection of the herbicide resistance of the herbicide Herba Eupatorii to ACCase inhibitors, providing a highly practical and cost-effective tool for monitoring and precise prevention and control of weed resistance in farmland.

[0017] On the basis of the above technical solution, the present invention can also be improved as follows: Furthermore, the 5' end of the KASP-1F is connected to a FAM fluorescent group, and the 5' end of the KASP-2F is connected to a HEX fluorescent group.

[0018] Compared with the prior art, the beneficial effects of adopting the above further technical solution are: By connecting FAM and HEX fluorescent groups to the 5' end of the KASP primer, specific labeling and real-time monitoring of different alleles can be achieved, significantly improving the accuracy and detection efficiency of genotyping, facilitating the intuitive distinction of mutation types at site 1781 of the ACCase gene in the sedge grass, and further enhancing the reliability and stability of the test results.

[0019] The present invention also provides a kit for detecting resistant Echinops serrata, comprising the KASP primer set, a Master Mix required for PCR reaction, a DNA extraction reagent and a positive control sample.

[0020] Compared with the prior art, the present invention has the following beneficial effects: The kit for detecting resistant Echinops spp. provided by the present invention integrates a specific KASP primer set, reagents required for PCR reaction, DNA extraction reagents and positive control samples, making the detection process more convenient, efficient and standardized, reducing operational difficulty and error risks, providing a complete solution for rapid on-site detection and wide application, and significantly improving the feasibility and reliability of detection.

[0021] The present invention also provides a method for identifying a resistant Echinops truncatus population. The KASP typing method of the Echinops truncatus ACCase gene 1781 resistance mutation site is used to detect plants with the T allele at the ACCase gene 1781 site, and confirm that the plants are resistant populations.

[0022] Compared with the prior art, the present invention has the following beneficial effects: The present invention directly identifies resistant populations associated with mutations at site 1781 of the ACCase gene of A. chinensis through molecular detection technology, overcoming the shortcomings of traditional methods that are time-consuming and labor-intensive and difficult to directly determine the resistance mechanism. It achieves rapid and accurate identification and provides a strong basis for monitoring farmland weed resistance and formulating prevention and control strategies. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0024] Figure 1 This is the dose-response curve of the Echinops chinensis population to different herbicides.

[0025] Figure 2 This is the result of PCR amplification of the ACCase gene of the Herba Eupatorii. Figure 2 In the figure, M represents Marker D2000, and the unmarked lanes represent ACCase gene clones from other Echinops species.

[0026] Figure 3 This is a schematic diagram of the ACCase gene sequence alignment and mutation sites.

[0027] Figure 4 This is the result of monoclonal sequencing.

[0028] Figure 5 This is the KASP typing result diagram. DETAILED DESCRIPTION

[0029] The following describes the details in conjunction with specific embodiments.

[0030] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention.

[0031] In the present invention, unless otherwise specified, all equipment and raw materials can be purchased from the market or are commonly used in the industry. The methods in the following embodiments, unless otherwise specified, are all conventional methods in the art.

[0032] Example 1 This example describes the complete process for resistance detection in C. truncatum, including DNA extraction, KASP amplification, and fluorescence detection. The effectiveness of the primer set and kit was verified, and they can be used for resistance detection and accurately identify resistant populations. Through genotyping, a tool is provided for resistance research and management. This example provides strong support for related patent applications and provides a comprehensive solution for resistance detection and management in C. truncatum.

[0033] 1. Test materials 1.1 Collection of the Cistanche population: A suspected resistant population of R. truncatula (R, MY) was collected from a wheat field in Zhongtai Town, Santai County, Mianyang City, Sichuan Province (31.358074°N, 105.019238°E). The sampling area exceeded 2 hectares, and more than 200 plants were sampled. The seeds were mixed and used in the experiment.

[0034] Susceptible Echinops chinensis populations (S, DY) were collected from wheat fields in Xinan Town, Mianzhu City, Deyang City, Sichuan Province (31.334331°N, 104.160411°E). Seeds harvested through single-plant multiplication were used as the sensitive population.

[0035] The collected seeds were air-dried and stored at 4°C for future use.

[0036] 1.2 Main reagents and materials: 15% clodinafop-propargyl wettable powder (Syngenta Crop Protection GmbH, Switzerland).

[0037] 10% Quizalofop-P-ethyl emulsifiable concentrate (Jiangsu Ruibang Agrochemical Co., Ltd.).

[0038] 69g / L Fenoxaprop-P-ethyl emulsion in water (Bayer CropScience (China) Co., Ltd.).

[0039] High-efficiency plant genome extraction kit, DNA gel recovery kit, I-5™ 2× High-Fidelity Master Mix, high-purity low-electrosmotic agarose, and pClone007 Blunt Vector Kit (Beijing Qingke Biotechnology Co., Ltd.)

[0040] 2 Test methods 2.1 Determination of resistance level Sowing of grass seeds: The seeds of the two populations of MY and DY were sown in pots with a diameter of 13 cm, with 54 pots sown for each population.

[0041] When the grass plants grow to the 3-4 leaf stage, plant them in a pot to 20 plants.

[0042] Herbicide treatment: Use a handheld sprayer to spray herbicides according to the dosage and concentration listed in Table 1. Each treatment was repeated three times.

[0043] Table 1: Types and dosages of herbicides used in different treatments Data Analysis: 21 days after the application of the medicine, the aboveground part of each pot of grass was cut and the fresh weight was weighed.

[0044] SigmaPlot v12.5 software was used to fit the nonlinear curve according to formula (1) and calculate the GR50 value.

[0045] The RI value was calculated using formula (2).

[0046] Y = C + (D - C) / [1 + (X / GR50)b] (1), RI=GR50(resistant population) / GR50(sensitive population) (2).

[0047] The results of whole-plant dose-response assays showed that DY was a sensitive population, with GR50 values ​​for the three herbicides being 1 / 5 of the respective recommended field doses.

[0048] MY showed high resistance to two herbicides, clodinafop-butyl and quizalofop-p-butyl (RI values ​​were 23.54 and 14.26, respectively), and moderate resistance to fenoxaprop-butyl (RI=5.83) ( Figure 1 ).

[0049] Figure 1Dose-response curves of C. chinensis to three different herbicides (Clodinafop-propargyl, Quizalofop-P-ethyl, Fenoxaprop-P-ethyl) are shown. Figure 1 The fresh weight percentages (relative to the control) of the sensitive population (S) and the resistant population (R) at different herbicide doses are plotted respectively. The horizontal axis represents the amount of active ingredient of the herbicide (ga.i.ha⁻¹), and the vertical axis represents the fresh weight (as a percentage of the control).

[0050] Clodinafop-propargyl: GR of sensitive populations (S) 50 is 10.78 g aiha⁻¹; GR of resistant population (R) 50 is 253.78 g aiha⁻¹.

[0051] Quizalofop-P-ethyl: GR of sensitive populations (S) 50 is 10.54 g aiha⁻¹; GR of resistant population (R) 50 It is 150.33 g aiha⁻¹.

[0052] Fenoxaprop-P-ethyl: GR of sensitive populations (S) 50 is 9.26 g aiha⁻¹; GR of resistant population (R) 50 It is 53.95 g aiha⁻¹.

[0053] These curves and data show that the tolerance dose of the resistant population (R) to these three herbicides is significantly higher than that of the sensitive population (S), indicating that R. truncatula has developed resistance to these ACCase inhibitor herbicides.

[0054] 2.2 Extraction of DNA from Echinops chinensis and amplification and sequencing of the plastid ACCase gene DNA extraction: Use a high-efficiency plant genome extraction kit to extract the DNA of the grass, and refer to the instructions for the steps.

[0055] DNA quality was checked using agarose gel electrophoresis.

[0056] PCR amplification: Synthesize a pair of primers based on the primer sequences of Zhao et al. (2019): F:5'-TTTCCCAGCGGCAGACAGAT-3', R:5'-TCCCTGGAGTCTTGCTTTCA-3'.

[0057] Reaction system: 30 μL, including 15 μL of I-5™ 2× High-Fidelity Master Mix, 1 μL each of primers F / R and DNA template, and 12 μL of ddH2O.

[0058] Amplification program: pre-denaturation at 98°C for 3 min; denaturation at 98°C for 10 s, annealing at 53°C for 15 s, extension at 72°C for 15 s, 37 cycles; extension at 72°C for 5 min, and storage at 4°C.

[0059] The target fragments were recovered after electrophoresis of the PCR products, purified, and sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing.

[0060] Using the DNA extracted from two populations of Echinops truncatula (DY and MY) as templates, PCR amplification yielded 1437 bp gene fragments (SEQ ID NO. 1 and SEQ ID NO. 2). Figure 2 shown.

[0061] Comparison of the ACCase gene between different biotypes revealed that all resistant plants carried a mutation from ATA to TTA ( Figure 3 A mutation in the ACCase gene (Figure A) results in the replacement of isoleucine (Ile) with leucine (Leu) at codon 178 (corresponding to amino acid 126). This mutation is a key molecular mechanism by which cloverleaf grass develops resistance to ACCase inhibitor herbicides.

[0062] In the spectrum of direct sequencing of PCR products, the position of the MY mutant base often shows a double peak, while DY shows a single peak ( Figure 3 Middle B).

[0063] 2.3 TA cloning and sequencing of the plastid ACCase gene of Rhizoma Corydalis TA clone: Ligate the target gene fragment from the sequencing results in 1.2.2 into the pClone007 Blunt Vector and transform into Trelief™ 5α competent cells.

[0064] Single clones that were positive for colony PCR were selected and sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing again. The number of clones in each population was ≥15.

[0065] The sequencing results were analyzed using SnapGene 6.2.2.

[0066] The results of monoclonal sequencing showed that there were two ACCase gene sequences in the sensitive Echinops truncatus population (DY) and four ACCase gene sequences in the resistant Echinops truncatus population (MY). Figure 4 ).

[0067] 2.4 KASP marker development and validation KASP primer design: To detect the differences between the 1781 mutation site (A / T) of the plastid ACCase gene of Rhizoctonia foetida, two forward primers and one reverse primer were designed.

[0068] Fluorescent tag sequences (FAM and HEX) were added upstream of the two forward primers for modification (Table 2).

[0069] Table 2 KASP primers and fluorescent tag sequences Note: Underlined bases represent FAM or HEX fluorophores.

[0070] Perform fluorescent PCR amplification on the diluted DNA: Reaction system: 10 μL, including 5 μL of qPCR 2× Master Mix, 1.4 μL of primer mixture, 1 μL of DNA template, and 2.6 μL of ddH2O.

[0071] Reaction program: 94°C for 15 min; 94°C for 15 sec, 61°C (decrease 0.6°C each cycle) for 1 min, 10 cycles; 94°C for 20 sec, 57°C for 1 min, 40 cycles; 72°C for 7 min.

[0072] The samples were detected and analyzed using a CFX96 real-time system (Applied Biopsystems, USA).

[0073] Based on the SNP site differences between the 1781 mutant and the non-mutant, genotyping can be performed efficiently using KASP markers.

[0074] like Figure 5 As shown, the green ▲ indicates that the MY ACCase gene of the grass is a mutation type at position 1781 and is a heterozygote; the blue ■ indicates that the DY ACCase gene of the grass is a wild type.

[0075] Figure 5The results of allele discrimination at the 1781 locus of the ACCase gene in the cloverleaf grass were shown. Using the KASP labeling technology, the genotypes of the resistant population (MY) and the sensitive population (DY) were successfully distinguished. The horizontal and vertical axes represent the relative fluorescence units (RFU) of two fluorescent signals (such as FAM and HEX), respectively. The green triangles represent the sensitive population (wild type), and the blue squares represent the resistant population (mutant). Figure 5 It can be clearly seen that the clustering areas of the two types of data points are different, indicating that the typing is successful and can effectively identify mutations.

[0076] In the description of the present invention, it should be understood that "-" and "~" represent a range between two values, and the range includes the endpoints. For example, "AB" represents a range greater than or equal to A and less than or equal to B. "A~B" represents a range greater than or equal to A and less than or equal to B.

[0077] In the description of the present invention, the term "and / or" is merely a description of the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B may represent three situations: A exists alone, A and B exist at the same time, and B exists alone.

[0078] In the description of the invention, the numerical values ​​of time, temperature, ratio and mass involved may be based on actual measurements, standard parameters of equipment, simplified rounding results, or within an acceptable error range, ensuring the practicality and repeatability of the invention.

[0079] In the description of the present invention, the term "about" or "approximately" is used to express the approximate value of a numerical value or range, allowing a certain error to ensure the flexibility and practicality of the description while remaining within an acceptable error range, with the maximum error range not exceeding 10% of the corresponding numerical value or numerical range.

[0080] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be construed as limiting the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. Persons skilled in the art will appreciate that improvements and modifications may be made without departing from the spirit and scope of the present invention, and such improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for detecting resistance of Coleus chinensis to ACCase inhibitor herbicides, characterized in that: The following steps are involved: (a) extracting genomic DNA from the tested Herba Corydalis plants; (b) performing fluorescent PCR amplification on the genomic DNA using a KASP primer set, wherein the KASP primer set comprises: KASP-1F: sequence shown in SEQ ID NO: 3; KASP-2F: sequence shown in SEQ ID NO: 4; KASP-R: sequence shown in SEQ ID NO: 5; (c) detecting the fluorescence signal of the PCR product to determine the genotype of nucleotide 1781 of the ACCase gene; (d) If the nucleotide at position 1781 is T, the Echinops chinensis plant is determined to be resistant to the ACCase inhibitor herbicide.

2. The method according to claim 1, characterized in that The ACCase inhibitor herbicide is selected from at least one of clodinafop-butyl, quizalofop-ethyl and fenoxaprop-butyl.

3. The method according to claim 1, characterized in that The 1781st nucleotide mutation causes the 178th codon of the ACCase gene to change from ATA to TTA, and causes the 126th amino acid to change from isoleucine to leucine.

4. The method according to claim 1, wherein The PCR amplification procedure includes: pre-denaturation at 94°C for 15 minutes; Denature at 94°C for 15 seconds. Annealing at 61°C for 1 minute, decreasing the temperature by 0.6°C each cycle for 10 cycles; denaturation at 94°C for 20 seconds and annealing at 57°C for 1 minute for 40 cycles; Extension at 72°C for 7 minutes.

5. A KASP primer set for detecting mutations at site 1781 of the ACCase gene of the tricholoma serrata, characterized in that: include: KASP-1F: sequence shown in SEQ ID NO: 3; KASP-2F: sequence shown in SEQ ID NO: 4; KASP-R: The sequence is shown in SEQ ID NO:

5.

6. The KASP primer set according to claim 5, characterized in that The 5' end of the KASP-1F is connected to a FAM fluorescent group, and the 5' end of the KASP-2F is connected to a HEX fluorescent group.

7. A kit for detecting resistant Codonopsis pilosula, characterized in that: The method comprises the KASP primer set according to claim 4, a Master Mix required for PCR reaction, a DNA extraction reagent and a positive control sample.

8. Use of the primer set according to claim 5 or the kit according to claim 7 in detecting resistance of Echinops chinensis to ACCase inhibitor herbicides.

9. A method for identifying resistant Codonopsis pilosula populations, characterized in that: The plants having the T allele at the ACCase gene 1781 site are detected by the method of claim 1 and confirmed to be a resistant population.

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