KASP labeled primer for detecting resistance mutation of chilo suppressalis to chlorantraniliprole insecticide and application of KASP labeled primer

Abstract

The invention discloses a KASP labeled primer for detecting resistance of chilo suppressalis to chlorantraniliprole insecticide and application of the KASP labeled primer, and relates to the technical field of agricultural biology. The KASP labeled primer group disclosed by the invention comprises a primer combination which is used for detecting the resistance mutations of the chlorantraniliprole insecticide, such as CsupI4758M, CsupY4667C, CsupY4667D, CsupY4894F and CsupG4915E. The group of KASP labeled primers is good in specificity and high in sensitivity, and can accurately detect the resistance mutation frequency of the chilo suppressalis to the chlorantraniliprole insecticide.

Description

Technical Field

[0001] The invention belongs to the field of agricultural biotechnology and relates to rapid molecular detection of mutations related to drug resistance of Chilo suppressalis. Background Art

[0002] The Chilo suppressalis (Chipioides suppressalis) is one of the most serious and common pests affecting rice cultivation. It is found in most rice-growing areas of my country. In recent years, due to climate warming, the expansion of the insect's source area, and the accumulation of insect populations, the damage caused by the Chilo suppressalis has become increasingly severe, causing significant economic losses to agricultural production. O-carbamidobenzamide insecticides have stomach toxicity and contact activity, while isoprocarb also has plant systemic activity. Chlorantraniliprole, among them, is a key chemical control agent for the Chilo suppressalis (Chipioides suppressalis). Due to long-term use of chlorantraniliprole, Chilo suppressalis (Chipioides suppressalis) has developed significant resistance to the agent in some areas. These insecticides primarily target the ryanodine receptor (RyR). Multiple mutations in the RyR have been reported as the molecular mechanism of resistance to chlorantraniliprole in Chilo suppressalis. Detecting the frequency of these mutations in populations can help quickly determine the level of resistance in Chilo suppressalis and provide a basis for the scientific and rational use of chlorantraniliprole. KASP (competitive allele-specific PCR) utilizes fluorescent labeling to accurately detect SNPs (single nucleotide polymorphisms) in sample DNA. Compared to PCR amplification and sequencing, KASP allows for high-throughput, low-cost detection of resistance mutations. However, KASP primers specifically targeting chlorantraniliprole resistance are currently unavailable. Therefore, it is necessary to develop KASP primers and methods to detect resistance mutations I4758M, Y4667C, Y4667D, Y4894F, and G4915E in the ryanodine receptor (RyR) gene of the Chilo suppressalis (C. suppressalis) to monitor chlorantraniliprole resistance mutations in field plants. Summary of the Invention

[0003] The main purpose of the present invention is to achieve high-throughput rapid molecular detection of the resistance of Chilo suppressalis to the insecticide chlorantraniliprole.

[0004] To achieve the purpose of the present invention, a KASP primer combination of five resistance mutation sites was first designed, which can be used to detect drug resistance-related SNP variation sites on the ryanodine receptor (RyR) gene of the Chilo suppressalis.

[0005] Furthermore, the detection primers include a primer combination for detecting the 4758th mutation Csup_I4758M of the ryanodine receptor (RyR) protein sequence of the striped stem borer; a primer combination for detecting the 4667th mutations Csup_Y4667C and Csup_Y4667D; a primer combination for detecting the 4894th mutation Csup_Y4894F; and a primer combination for detecting the 4915th mutation Csup_G4915E.

[0006] Furthermore, the sequences of the two upstream primers included in the primer combination Csup_I4758M are shown in SEQ ID NO.: 1 and SEQ ID NO.: 2, respectively, and the sequence of the downstream primer is shown in SEQ ID NO.: 3; the sequences of the two upstream primers included in the primer combination Csup_Y4667C are shown in SEQ ID NO.: 4 and SEQ ID NO.: 5, respectively, and the sequence of the downstream primer is shown in SEQ ID NO.: 6; the sequences of the two upstream primers included in the primer combination Csup_Y4667D are shown in SEQ ID NO.: 7 and SEQ ID NO.: 8, respectively, and the sequence of the downstream primer is shown in SEQ ID NO.: 9; the sequences of the two upstream primers included in the primer combination Csup_Y4894F are shown in SEQ ID NO.: 10 and SEQ ID NO.: 11, respectively, and the sequence of the downstream primer is shown in SEQ ID NO.: 12 The sequences of the two upstream primers contained in the primer combination Csup_G4915E are shown in SEQ ID NO.: 13 and SEQ ID NO.: 14, respectively, and the sequence of the downstream primer is shown in SEQ ID NO.: 15;

[0007] Furthermore, the sequences of the two upstream primers of each primer combination are respectively connected to different fluorescent modified tags, which are used to distinguish different alleles during KASP typing;

[0008] The present invention also establishes a high-throughput rapid molecular detection method for the resistance of Chilo suppressalis to the insecticide chlorantraniliprole, and the specific steps are as follows:

[0009] (1) KASP primer combination Csup_I4758M was used to perform KASP reaction and SNP typing using the DNA of Chilo suppressalis as a template. If the fluorescence signal data of the amplified product was typed as G:G, it was a homozygous resistant individual; if the fluorescence signal data was typed as C:C, it was a homozygous sensitive individual; if the fluorescence signal data was typed as G:C, it was a heterozygous individual. The frequency of the resistance allele G in the tested population was counted to infer the resistance level of the Chilo suppressalis population to chlorantraniliprole.

[0010] (2) KASP primer combination Csup_Y4667C was used to perform KASP reaction and SNP typing using the DNA of Chilo suppressalis as a template. If the fluorescence signal data of the amplified product was typed as G:G, it was a homozygous resistant individual; if the fluorescence signal data was typed as A:A, it was a homozygous sensitive individual; if the fluorescence signal data was typed as G:A, it was a heterozygous individual. The frequency of the resistance allele G in the tested population was counted to infer the resistance level of the Chilo suppressalis population to chlorantraniliprole.

[0011] (3) KASP primer combination Csup_Y4667D was used to perform KASP reaction and SNP typing using the DNA of the Chilo suppressalis as a template. If the fluorescence signal data of the amplified product was typed as G:G, it was a homozygous resistant individual; if the fluorescence signal data was typed as T:T, it was a homozygous sensitive individual; if the fluorescence signal data was typed as G:T, it was a heterozygous individual. The frequency of the resistance allele G in the tested population was counted, and the resistance level of the Chilo suppressalis population to chlorantraniliprole could be inferred.

[0012] (4) KASP primer combination Csup_Y4894F was used to perform KASP reaction and SNP typing using the DNA of the Chilo suppressalis as a template. If the fluorescence signal data of the amplified product was typed as T:T, it was a homozygous resistant individual; if the fluorescence signal data was typed as A:A, it was a homozygous sensitive individual; if the fluorescence signal data was typed as A:T, it was a heterozygous individual. The frequency of the resistance allele T in the tested population was counted, and the resistance level of the Chilo suppressalis population to chlorantraniliprole could be inferred.

[0013] (5) KASP primer combination Csup_G4915E was used to perform KASP reaction and SNP typing using the DNA of the Chilo suppressalis as a template. If the fluorescence signal data of the amplified product was typed as A:A, it was a homozygous resistant individual; if the fluorescence signal data was typed as G:G, it was a homozygous sensitive individual; if the fluorescence signal data was typed as G:A, it was a heterozygous individual. The frequency of the resistance allele A in the tested population was counted, and the resistance level of the Chilo suppressalis population to chlorantraniliprole could be inferred.

[0014] The present invention has the beneficial effect of designing a KASP detection primer set for identifying resistant individuals, addressing the urgent need for rapid detection of chlorantraniliprole resistance in Chilo suppressalis. This primer set enables rapid, accurate, low-cost, and high-throughput detection of chlorantraniliprole resistance in Chilo suppressalis. This has broad application prospects in the rational and precise selection of pesticides. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1This is the KASP genotype diagram of the Chilo suppressalis resistance to the insecticide chlorantraniliprole. DETAILED DESCRIPTION

[0016] The following describes in detail the method for detecting chlorantraniliprole resistance in Chilo suppressalis using KASP primers designed in the present invention with reference to specific examples. This detailed description should not be considered a limitation of the present invention, but rather a more detailed description of certain aspects, features, and embodiments of the present invention.

[0017] Example 1 Screening of KASP Primers for Mutation Sites Related to Chilo suppressalis Resistance to Chlorantraniliprole

[0018] Using Primer 3, we extracted 500 base pairs of genomic DNA sequences from five chlorantraniliprole-resistant mutations in the Chilo suppressalis (C. suppressalis) and designed KASP primers for each site. KASP reactions and SNP typing were performed using Chilo suppressalis DNA as a template. Each KASP primer set was tested for clear typing and a success rate exceeding 90%. For each mutation site, a preferred combination was identified, as shown in Table 1.

[0019] Table 1 Preferred combinations of KASP primers obtained by screening

[0020]

[0021] Example 2 Application of KASP Primers in Detecting Chilo suppressalis Resistance to Chlorantraniliprole (O-formylaminobenzamide) Insecticide

[0022] (1) Sample collection

[0023] Chilo suppressalis samples were collected in 2022 from field populations in a rice orchard in Hannan District, Hubei Province, and from greenhouses in a rice orchard in Jiangxia District, Hubei Province. Both populations fed on rice leaves. The Hannan population was labeled CsA, and the Jiangxia population was labeled CsB. Live insects were collected, soaked in 100% alcohol, and stored at -20°C.

[0024] (2) DNA extraction

[0025] The genomic DNA was extracted from 46 individuals of different populations of Chilo suppressalis using the DNA lysis crude extraction method.

[0026] (3) KASP amplification, typing, and drug resistance prediction

[0027] The reagents used were 2× KASP master mixture (LGC). A 3 μL reaction system included 1.5 ng of dry powdered genomic DNA, 0.14 μL of primer mix (upstream primer concentration: 12 μmol / L, downstream primer concentration: 30 μmol / L), and 3 μL of 2× KASP master mixture.

[0028] The touchdown reaction procedure included: initial denaturation at 94°C for 15 min; followed by 10 cycles of denaturation at 94°C for 20 s and annealing at 61–55°C for 1 min, with a decrease of 0.6°C per cycle; and 40 cycles of denaturation at 94°C for 20 s and annealing at 55°C for 1 min.

[0029] Finally, check the typing status on the typing detector. Figure 1 The statistical genotype frequencies are shown in Table 2. The statistical allele frequencies are shown in Table 3.

[0030] Table 2 Genotype frequencies at five resistance mutation sites in two Chilo suppressalis populations

[0031]

[0032] Table 3 Allele frequencies at five resistance mutation sites in two Chilo suppressalis populations

[0033]

Claims

1. A KASP primer combination for detecting five mutation sites of Chilo suppressalis resistance to chlorantraniliprole, characterized in that: The invention comprises a primer combination for detecting the mutation Csup_I4758M at position 4758 of the ryanodine receptor (RyR) protein sequence of the striped stem borer, a primer combination for detecting the mutations Csup_Y4667C and Csup_Y4667D at position 4667, a primer combination Csup_Y4894F for detecting the mutation at position 4894, and a primer combination for detecting the mutation Csup_G4915E at position 4915; The sequences of the two upstream primers and one downstream primer included in the primer combination for detecting Csup_I4758M are shown in SEQ ID NOs.: 1-3, respectively; the sequences of the two upstream primers and one downstream primer included in the primer combination for detecting Csup_Y4667C are shown in SEQ ID NOs.: 4-6, respectively; the sequences of the two upstream primers and one downstream primer included in the primer combination for detecting Csup_Y4667D are shown in SEQ ID NOs.: 7-9, respectively; the sequences of the two upstream primers and one downstream primer included in the primer combination for detecting Csup_Y4894F are shown in SEQ ID NOs.: 10-12, respectively; and the sequences of the two upstream primers and one downstream primer included in the primer combination for detecting Csup_G4915E are shown in SEQ ID NOs.: 13-15, respectively.

2. The KASP primer combination of five resistance mutation sites according to claim 1, characterized in that: The sequences of the two upstream primers of each primer combination are respectively connected to different fluorescent modification tags.

3. A method for detecting a chlorantraniliprole-resistant mutation in Chilo suppressalis, characterized in that: The KASP primer combination according to claim 1 is used to detect whether there is a drug resistance-related mutation in the ryanodine receptor (RyR) gene of the chilo suppressalis using the DNA of the chilo suppressalis as a template.

4. The method for detecting a chlorantraniliprole-resistant mutation in Chilo suppressalis according to claim 3, wherein If the KASP primer combination Csup_I4758M is used, the sample with the SNP genotype of G:G is a homozygous individual for resistance, the sample with the genotype of C:C is a homozygous individual for sensitivity, and the sample with the genotype of G:C is a heterozygous individual; if the KASP primer combination Csup_Y4667C is used, the sample with the SNP genotype of G:G is a homozygous individual for resistance, the sample with the genotype of A:A is a homozygous individual for sensitivity, and the sample with the genotype of G:A is a heterozygous individual; if the KASP primer combination Csup_Y4667D is used, the sample with the SNP genotype of G:G is a homozygous individual for resistance, the sample with the genotype of A:A is a homozygous individual for sensitivity, and the sample with the genotype of G:A is a heterozygous individual The samples with SNP genotype of T:T are homozygous individuals for resistance, those with genotype of G:T are heterozygous individuals for sensitivity; if the KASP primer combination Csup_Y4894F is used, the samples with SNP genotype of T:T are homozygous individuals for resistance, those with genotype of A:A are homozygous individuals for sensitivity, and those with genotype of A:T are heterozygous individuals; if the KASP primer combination Csup_G4915E is used, the samples with SNP genotype of A:A are homozygous individuals for resistance, those with genotype of G:G are homozygous individuals for sensitivity, and those with genotype of G:A are heterozygous individuals.

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