KASP primers for detecting drug resistance mutation of bemisia tabaci and application of KASP primers

Abstract

The invention discloses a group of KASP primers for detecting drug resistance mutation of bemisia tabaci and application of the KASP primers, and relates to the technical field of agricultural biology. The KASP primer disclosed by the invention comprises a primer combination which is used for detecting resistance mutations of deltamethrin, spirodiclofen and chlorpyrifos, such as BtabM918V (BtabM918V), BtabL925I (BtabL925I), BtabT929V (BtabA2083V) and BtabF392W (BtabF392W). One or more primer combinations are used for detecting the drug resistance mutation of the bemisia tabaci. The group of KASP labeled primers has good specificity and high sensitivity, and can accurately detect the resistance mutation frequency of bemisia tabaci to insecticides.

Description

Technical Field

[0001] The invention belongs to the field of agricultural biotechnology and relates to rapid molecular detection of pesticide-resistance-related mutations in Bemisia tabaci. Background Art

[0002] Bemisia tabaci Bemisiatabaci The whitefly is a worldwide agricultural pest that harms over 400 plant species, including those in the Cruciferae, Solanaceae, Cucurbitaceae, and Leguminosae families. It is a significant pest of numerous crops, including tomatoes, eggplants, peppers, cucumbers, and cowpeas, causing significant economic losses to agricultural production. Small and fast-reproducing, it not only feeds directly on crop sap, impacting growth, but also excretes honeydew, causing sooty mold and spreading plant viral diseases. Tomato viral diseases spread by whiteflies often result in the complete loss of tomato harvests, making it one of the most serious pests in agricultural production. Spraying chemical insecticides is a key control measure for Bemisia whiteflies. Commonly used agents include deltamethrin, spirodiclofen, chlorpyrifos, and abamectin. Deltamethrin targets the sodium ion channels (KDRs) that conduct excitation in nerve cells, prolonging their open time and causing sustained depolarization of the cell membrane. This leads to symptoms of poisoning in insects, including movement disorders, paralysis, and death. Spirodiclofen, a tetronix acid insecticide with the active ingredient tetronix tetronix, inhibits fat synthesis in the insects, disrupting energy metabolism and ultimately killing them. Chlorpyrifos is a neurotoxin that inhibits acetylcholinesterase, causing high levels of acetylcholine to accumulate at synapses, causing prolonged excitation of nerve fibers and blocking normal nerve conduction, leading to poisoning and death in the insects. Due to long-term use of chemical pesticides, some Bemisia whitefly populations have developed resistance to commonly used agents such as deltamethrin, spirodiclofen, and chlorpyrifos. Detecting the resistance level of whitefly populations to different pesticides is the basis for scientific and rational selection of control agents. It is of great significance for improving the effectiveness of chemical control, reducing the use of chemical pesticides, and improving the safety of agricultural products.

[0003] Traditional methods for detecting insecticide resistance in whiteflies rely primarily on bioassays. While reliable, these methods require live specimens, placing high demands on sample collection, transportation, and rearing. Furthermore, they are slow and labor-intensive, making them unsuitable for testing multiple populations simultaneously. Methods that identify resistance mutations by amplifying target genes through PCR, sequencing, and sequence comparison do not require live specimens and can test multiple populations simultaneously, but they still present challenges such as long testing cycles and high costs. KASP (competitive allele-specific PCR) utilizes fluorescent markers to accurately detect SNPs (single nucleotide polymorphisms) in sample DNA. Compared to PCR amplification and sequencing, KASP enables high-throughput, low-cost detection of resistance mutations. However, KASP primers for detecting resistance to deltamethrin, spirodiclofen, and chlorpyrifos are currently unavailable. Therefore, it is necessary to develop a KASP primer and method to detect the resistance mutation sites M918V, L925I and T929V on the sodium ion channel (kdr) gene of Bemisia tabaci, the resistance mutation site A2083V on the acetyl-CoA carboxylase (ACC) gene, and the resistance mutation site F392W on the acetylcholinesterase (ace1) gene, so as to monitor the frequency of insecticide resistance mutations in Bemisia tabaci in the field. Summary of the Invention

[0004] The main purpose of the present invention is to achieve high-throughput rapid molecular detection of pesticide resistance of Bemisia tabaci.

[0005] To achieve the purpose of the present invention, three KASP primer combinations were designed for detecting SNP variant sites associated with deltamethrin resistance in the sodium ion channel (kdr) gene of Bemisia tabaci; one KASP primer combination was designed for detecting SNP variant sites associated with spirodiclofen resistance in the acetyl-CoA carboxylase (ACC) gene of Bemisia tabaci; and one KASP primer combination was designed for detecting SNP variant sites associated with chlorpyrifos resistance in the acetylcholinesterase (ace1) gene of Bemisia tabaci.

[0006] Furthermore, the detection primers include a primer combination for detecting the 918th mutation Btab_M918V, the 925th mutation Btab_L925I, and the 929th mutation Btab_T929V of the sodium ion channel (kdr) protein sequence of the whitefly; a primer combination for detecting the 2083rd mutation Btab_A2083V of the acetyl-CoA carboxylase (ACC) protein sequence; and a primer combination for detecting the 392nd mutation Btab_F392W of the acetylcholinesterase (ace1) protein of the whitefly;

[0007] Furthermore, the sequences of the two upstream primers included in the primer combination Btab_M918V 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 Btab_L925I 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 Btab_T929V 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 Btab_A2083V 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 Btab_F392W 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;

[0008] 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;

[0009] The present invention also establishes a high-throughput rapid molecular detection method for pesticide resistance of Bemisia tabaci, the specific steps of which are as follows:

[0010] (1) KASP primer combination Btab_M918V was used to perform KASP reaction and SNP typing using the DNA of Bemisia tabaci 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 Bemisia tabaci population to deltamethrin.

[0011] (2) KASP primer combination Btab_L925I was used to perform KASP reaction and SNP typing using the DNA of B. tabaci 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 T:T, 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 A in the tested population was counted to infer the resistance level of the B. tabaci population to cypermethrin.

[0012] (3) KASP primer combination Btab_T929V was used to perform KASP reaction and SNP typing using the DNA of B. tabaci 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 B. tabaci population to cypermethrin.

[0013] (4) KASP primer combination Btab_A2083V was used to perform KASP reaction and SNP typing using the DNA of whitefly 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 C:C, it was a homozygous sensitive individual; if the fluorescence signal data was typed as C:T, it was a heterozygous individual. The frequency of the resistance allele T in the tested population was counted to infer the resistance level of the whitefly population to spiroclofen.

[0014] (5) KASP primer combination Btab_F392W was used to perform KASP reaction and SNP typing using the DNA of B. tabaci 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 to infer the level of resistance of the B. tabaci population to chlorpyrifos.

[0015] The beneficial effect of this invention lies in the design of a KASP detection primer set for identifying pesticide-resistant individuals, addressing the urgent need for rapid detection of pesticide resistance in whiteflies. This primer set enables rapid, accurate, low-cost, and high-throughput detection of the frequency of pesticide-resistant mutations in whiteflies. This has broad application prospects in the rational and precise selection of pesticides. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 KASP genotype diagram for detecting insecticide-resistant mutations in Bemisia tabaci. DETAILED DESCRIPTION

[0017] The following describes in detail the method for detecting insecticide resistance of Bemisia tabaci using KASP primers designed in the present invention with reference to specific examples. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, features and embodiments of the present invention.

[0018] Example 1 Screening of KASP Primers for Mutation Sites Related to Insecticide Resistance in Bemisia tabaci

[0019] Genomic DNA sequences within 500 base pairs (bp) of five resistance mutation sites in Bemisia tabaci to deltamethrin, spirodiclofen, and chlorpyrifos were extracted and used to design KASP primers using Primer 3. Their sequences are shown in Table 1. KASP reactions and SNP typing were performed using Bemisia tabaci DNA as a template to verify that each KASP primer set produced clear typing and a success rate greater than 90%. For each mutation site, a preferred combination was identified, as shown in Table 1.

[0020] Table 1 Preferred combination KASP primer sequences obtained by screening

[0021]

[0022] Example 2 Application of KASP Primers in Detecting the Frequency of Pesticide-Resistant Mutations in Bemisia tabaci

[0023] (1) Sample collection

[0024] Bemisia tabaci samples were collected in 2022 from field populations in a plantation in Miyun District, Beijing, and from greenhouses in a plantation in Shunyi District, Beijing. Both populations fed on cucumber leaves. The Miyun District field population was labeled BtMY, and the Shunyi District population was labeled BtSY. Live insects were collected, soaked in 100% alcohol, and stored at -20°C.

[0025] (2) DNA extraction

[0026] Genomic DNA was extracted from 22 individuals of different populations of Bemisia tabaci using the DNA lysis crude extraction method.

[0027] (3) KASP amplification, typing, and drug resistance prediction

[0028] 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.

[0029] 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.

[0030] 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.

[0031] Table 2 Genotype frequencies at five insecticide-resistant mutation sites in two Bemisia tabaci populations

[0032]

[0033] Table 3 Allele frequencies at five insecticide-resistant mutation sites in two Bemisia tabaci populations

[0034]

Claims

1. A KASP primer combination for detecting insecticide-resistant mutations in Bemisia tabaci, characterized in that The invention comprises KASP primers for detecting three mutation sites of resistance of Bemisia tabaci to deltamethrin; KASP primers for detecting one mutation site of resistance of Bemisia tabaci to spiroclofen; and KASP primers for detecting one mutation site of resistance of Bemisia tabaci to chlorpyrifos.

2. The KASP primers for detecting three mutation sites of Bemisia tabaci resistance to deltamethrin according to claim 1, characterized in that A primer combination comprising Btab_M918V, Btab_L925I and Btab_T929V; a KASP primer for detecting a mutation site of Bemisia tabaci resistance to spiroclofen, characterized by comprising a primer combination of Btab_A2083V; a KASP primer for detecting a mutation site of Bemisia tabaci resistance to chlorpyrifos, characterized by comprising a primer combination of Btab_F392W.

3. According to claim 2, the sequences of the two upstream primers and one downstream primer contained in the primer combination for detecting Btab_M918V are shown in SEQ ID NOs.: 1-3, respectively; the sequences of the two upstream primers and one downstream primer contained in the primer combination for detecting Btab_L925I are shown in SEQ ID NOs.: 4-6, respectively; the sequences of the two upstream primers and one downstream primer contained in the primer combination for detecting Btab_T929V are shown in SEQ ID NOs.: 7-9, respectively; the sequences of the two upstream primers and one downstream primer contained in the primer combination for detecting Btab_A2083V are shown in SEQ ID NOs.: 10-12, respectively; the sequences of the two upstream primers and one downstream primer contained in the primer combination for detecting Btab_F392W are shown in SEQ ID NOs.: 13-15, respectively.

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

5. A method for detecting insecticide-resistant mutations in Bemisia tabaci, characterized in that: The KASP primer combination according to claim 2 is used to detect whether there is a drug resistance-related mutation using the DNA of Bemisia tabaci as a template.

6. The method for detecting insecticide-resistant mutations in Bemisia tabaci according to claim 5, characterized in that: If the KASP primer combination Btab_M918V 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 Btab_L925I is used, the sample with the SNP genotype of A:A is a homozygous individual for resistance, the sample with the genotype of T:T is a homozygous individual for sensitivity, and the sample with the genotype of A:T is a heterozygous individual; if the KASP primer combination Btab_T929V is used, the sample with the SNP genotype of G:G is a homozygous individual for resistance If the KASP primer combination Btab_A2083V is used, the samples with SNP genotypes of T:T are homozygous for resistance, C:C are homozygous for sensitivity, and C:T are heterozygous for heterozygous. If the KASP primer combination Btab_F392W is used, the samples with SNP genotypes of G:G are homozygous for resistance, T:T are homozygous for sensitivity, and G:T are heterozygous.

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