KASP labeled primer for detecting resistance mutation of tetranychus urticae to carbamate insecticides and application of KASP labeled primer

Abstract

The invention discloses a KASP labeled primer for detecting the resistance of tetranychus urticae to carbamate insecticides 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 TurtG119S, TurtF331W, TurtF331Y, TurtA201S, TurtT280A and TurtG328A of the carbamate insecticide. The KASP labeled primer group disclosed by the invention comprises a primer combination which is used for detecting the resistance mutations of TurtG119S, TurtF331W, TurtF331Y, TurtA201S, TurtT280A and TurtG328A. The group of KASP labeled primers has good specificity and high sensitivity, and can accurately detect the resistance mutation frequency of the tetranychus urticae to carbamate insecticides.

Description

Technical Field

[0001] The present invention belongs to the field of agricultural biotechnology and relates to the rapid molecular detection of mutations related to carbamate resistance in Tetranychus urticae Koch. Background Art

[0002] Tetranychus urticae Koch is a worldwide agricultural pest mite that can damage more than 1,100 host plants in 140 families, causing huge economic losses to agricultural production. Tetranychus urticae Koch has developed resistance to most acaricides with different modes of action, resulting in serious problems of blind use of pesticides in production. Timely and rapid high-throughput detection of acaricide resistance in Tetranychus urticae Koch is a key link in effectively controlling this pest mite.

[0003] Carbamate acaricides include isoprocarb, thiodicarb, and carbaryl, which have contact and stomach poisoning effects. At the same time, isoprocarb also has plant systemic conductivity, and all of them mainly inhibit acetylcholinesterase (AChE) as the main insecticidal and acaricidal mechanism. Currently, relevant studies have found that some Tetranychus urticae Koch populations have developed resistance to carbamate acaricides.

[0004] For the detection of acaricide resistance in Tetranychus urticae Koch, the traditional method is mainly the bioassay method. Although the results of this method are reliable, it requires live detection, has high requirements for the processes of sample collection, transportation, and feeding, and has a slow detection speed and a large workload, and is not suitable for simultaneously detecting multiple populations. By performing PCR amplification, sequencing, and sequence comparison on target genes to determine whether there are resistance mutations, this method does not require live detection and can simultaneously detect multiple populations, but there are still problems such as a long detection period and high costs. KASP (Kompetitive Allele-Specific PCR) technology uses fluorescence labeling to accurately detect SNP (Single Nucleotide Polymorphism) sites in sample DNA. Compared with the methods of PCR amplification and sequencing, KASP can achieve high-throughput and low-cost detection of resistance mutations. However, there are currently no KASP detection primers for carbamate acaricide resistance. Therefore, it is necessary to develop a KASP primer and method for detecting resistance mutation sites G119S, F331W, F331Y, A201S, T280A, and G328A on the acetylcholinesterase (AChE) gene of Tetranychus urticae Koch, so as to monitor the resistance mutation sites of field Tetranychus urticae Koch to carbamate acaricides. Summary of the Invention

[0005] The main object of the present invention is to achieve high-throughput and rapid molecular detection of carbamate insecticide resistance in Tetranychus urticae Koch.

[0006] To achieve the object of the present invention, a KASP primer combination for 6 resistance mutation sites was first designed, which can be used to detect SNP variation sites related to carbamate resistance on the acetylcholinesterase (AChE) gene of Tetranychus urticae Koch.

[0007] Furthermore, the detection primers include primer combinations for detecting the mutation Turt_G119S at position 119 of the acetylcholinesterase (AChE) protein sequence of Tetranychus urticae; primer combinations for detecting the mutations Turt_F331W and Turt_F331Y at position 331; primer combinations for detecting the mutation Turt_A201S at position 201; primer combinations for detecting the mutation Turt_T280A at position 280; and primer combinations for detecting the mutation Turt_G328A at position 328.

[0008] Furthermore, the two upstream primers included in the primer combination Turt_G119S have sequences shown in SEQ ID NO.: 1 and SEQ ID NO.: 2 respectively, and the downstream primer has a sequence shown in SEQ ID NO.: 3; the two upstream primers included in the primer combination Turt_F331W have sequences shown in SEQ ID NO.: 4 and SEQ ID NO.: 5 respectively, and the downstream primer has a sequence shown in SEQ ID NO.: 6; the two upstream primers included in the primer combination Turt_F331Y have sequences shown in SEQ ID NO.: 7 and SEQ ID NO.: 8 respectively, and the downstream primer has a sequence shown in SEQ ID NO.: 9; the two upstream primers included in the primer combination Turt_A201S have sequences shown in SEQ ID NO.: 10 and SEQ ID NO.: 11 respectively, and the downstream primer has a sequence shown in SEQ ID NO.: 12; the two upstream primers included in the primer combination Turt_T280A have sequences shown in SEQ ID NO.: 13 and SEQ ID NO.: 14 respectively, and the downstream primer has a sequence shown in SEQ ID NO.: 15; the two upstream primers included in the primer combination Turt_G328A have sequences shown in SEQ ID NO.: 16 and SEQ ID NO.: 17 respectively, and the downstream primer has a sequence shown in SEQ ID NO.: 18.

[0009] Furthermore, the sequences of the two upstream primers of each primer combination are respectively linked to different fluorescent modification tags for differentiating different alleles during KASP genotyping.

[0010] The present invention also establishes a high-throughput and rapid molecular detection method for the resistance of Tetranychus urticae to carbamate insecticides, and the specific steps are as follows:

[0011] (1) Use the KASP primer combination Turt_G119S to perform KASP reaction and SNP genotyping with the DNA of Tetranychus urticae as the template; if the fluorescence signal data of the amplification product is genotyped as A:A, it is a resistant homozygous individual; if the fluorescence signal data is genotyped as G:G, it is a sensitive homozygous individual; if the fluorescence signal data is genotyped as G:A, it is a heterozygous individual; count the frequency of the resistant allele A in the detected population to infer the resistance level of the Tetranychus urticae population to carbamate acaricides.

[0012] (2) Use the KASP primer combination Turt_F331W to perform KASP reaction and SNP genotyping with the DNA of Tetranychus urticae as the template; if the fluorescence signal data of the amplification product is genotyped as G:G, it is a resistant homozygous individual; if the fluorescence signal data is genotyped as T:T, it is a sensitive homozygous individual; if the fluorescence signal data is genotyped as G:T, it is a heterozygous individual; count the frequency of the resistant allele G in the detected population to infer the resistance level of the Tetranychus urticae population to carbamate acaricides.

[0013] (3) Use the KASP primer combination Turt_F331Y to perform KASP reaction and SNP genotyping with the DNA of Tetranychus urticae as the template; if the fluorescence signal data of the amplification product is genotyped as A:A, it is a resistant homozygous individual; if the fluorescence signal data is genotyped as T:T, it is a sensitive homozygous individual; if the fluorescence signal data is genotyped as T:A, it is a heterozygous individual; count the frequency of the resistant allele A in the detected population to infer the resistance level of the Tetranychus urticae population to carbamate acaricides.

[0014] (4) Use the KASP primer combination Turt_A201S to perform KASP reaction and SNP genotyping with the DNA of Tetranychus urticae as the template; if the fluorescence signal data of the amplification product is genotyped as T:T, it is a resistant homozygous individual; if the fluorescence signal data is genotyped as G:G, it is a sensitive homozygous individual; if the fluorescence signal data is genotyped as G:T, it is a heterozygous individual; count the frequency of the resistant allele T in the detected population to infer the resistance level of the Tetranychus urticae population to carbamate acaricides.

[0015] (5) Use the KASP primer combination Turt_T280A to perform KASP reaction and SNP genotyping with the DNA of Tetranychus urticae as the template; if the fluorescence signal data of the amplification product is genotyped as G:G, it is a resistant homozygous individual; if the fluorescence signal data is genotyped as A:A, it is a sensitive homozygous individual; if the fluorescence signal data is genotyped as G:A, it is a heterozygous individual; count the frequency of the resistant allele G in the detected population to infer the resistance level of the Tetranychus urticae population to carbamate acaricides.

[0016] (6) KASP primer combination Turt_G328A was used to perform KASP reaction and SNP typing with DNA of two-spotted spider mite as template; if the fluorescence signal data of the amplified product was typed as C:C, it was a homozygous individual for resistance; if the fluorescence signal data was typed as G:G, it was a homozygous individual for sensitivity; if the fluorescence signal data was typed as G:C, it was a heterozygous individual; the frequency of the resistance allele C in the tested population was statistically analyzed to infer the resistance level of the two-spotted spider mite population to carbamate acaricides.

[0017] The beneficial effect of the present invention is that, in view of the actual demand for rapid detection of the resistance of two-spotted spider mites to carbamate acaricides in production, a KASP detection primer set for identifying resistant individuals is designed. The primer set can be used to detect the resistance of two-spotted spider mites to carbamate acaricides quickly, accurately, at low cost and with high throughput. It has a wide application prospect in the rational and accurate selection of pesticides. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the KASP genotype diagram of the resistance of two-spotted spider mites to carbamate pesticides. Implementation

[0019] The following is a detailed description of the method for detecting the resistance of Tetranychus urticae to carbamate acaricides using KASP primers designed by 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.

[0020] Example 1 Screening of KASP primers for mutation sites associated with resistance of Tetranychus urticae to carbamate acaricides

[0021] The genomic DNA sequences of 500 bp above and below the six mutation sites of resistance to carbamate acaricides of Tetranychus urticae were intercepted, and two sets of KASP primers were designed using Primer 3, and their sequences are shown in Table 1. The DNA of Tetranychus urticae was used as a template for KASP reaction and SNP typing to test whether each set of KASP primers had clear typing and a success rate greater than 90%. For each mutation site, a preferred combination was screened, as indicated in Table 1.

[0022] Table 1 KASP primer sequences used for screening

[0023]

[0024] Example 2 Application of KASP primers in detecting resistance of Tetranychus urticae to carbamate acaricides

[0025] (1) Sample collection

[0026] The Tetranychus urticae samples were collected from the field populations in a planting park in Changping District, Beijing and in a greenhouse in a planting park in Daxing District, Beijing in 2022. The host plants were all strawberry leaves. The Changping field population was labeled TUWD, and the Daxing population was labeled TUQM. Live insects were collected and immersed in 100% alcohol and stored at -20°C.

[0027] (2)DNA Extraction

[0028] The genomic DNA of 47 individuals from different populations of Tetranychus urticae was extracted using the crude DNA lysis method.

[0029] (3)KASP Amplification, Genotyping, and Resistance Prediction

[0030] The reagent used was 2 × KASP master mixture (LGC). The reaction system was 3 μL, including 1.5 ng of dry powder genomic DNA and 0.14 μL of primer mixture (in the primer mixture system, the concentration of the upstream and downstream primers was 12 μmol / L and 30 μmol / L respectively), and 3 μL of 2 × KASP master mixture.

[0031] The touchdown reaction program included: pre-denaturation at 94°C for 15 min; then 10 cycles, each cycle including denaturation at 94°C for 20 s, annealing at 61 - 55°C for 1 min, with a decrease of 0.6°C for each cycle; then another 40 cycles, each cycle including denaturation at 94°C for 20 s and annealing at 55°C for 1 min.

[0032] Finally, the genotyping results were viewed on a genotyping detector, and the results are shown in Figure 1 . The genotype frequencies were statistically analyzed, as shown in Table 2. The allele frequencies were statistically analyzed, as shown in Table 3.

[0033] Table 2 Genotype Frequencies of Two Tetranychus urticae Populations at 6 Resistance Mutation Loci

[0034]

[0035] Table 3 Allele Frequencies of Two Tetranychus urticae Populations at 6 Resistance Mutation Loci

[0036]

Claims

1. A KASP primer combination for detecting 6 mutation sites of Tetranychus urticae Koch's resistance to carbamate insecticides, characterized in that, A primer combination for detecting the mutation Turt_G119S at position 119 of the acetylcholinesterase (AChE) protein sequence of Tetranychus urticae, a primer combination for detecting the mutations Turt_F331W and Turt_F331Y at position 331, a primer combination Turt_A201S for detecting the mutation at position 201, a primer combination for detecting the mutation Turt_T280A at position 280, and a primer combination for detecting the mutation Turt_G328A at position 328; The sequences of the two upstream primers and one downstream primer included in the primer combination for detecting Turt_G119S are shown in SEQ ID NO.: 1 - 3 respectively; the sequences of the two upstream primers and one downstream primer included in the primer combination for detecting Turt_F331W are shown in SEQ ID NO.: 4 - 6 respectively; the sequences of the two upstream primers and one downstream primer included in the primer combination for detecting Turt_F331Y are shown in SEQ ID NO.: 7 - 9 respectively; the sequences of the two upstream primers and one downstream primer included in the primer combination for detecting Turt_A201S are shown in SEQ ID NO.: 10 - 12 respectively; the sequences of the two upstream primers and one downstream primer included in the primer combination for detecting Turt_T280A are shown in SEQ ID NO.: 13 - 15 respectively; the sequences of the two upstream primers and one downstream primer included in the primer combination for detecting Turt_G328A are shown in SEQ ID NO.: 16 - 18 respectively.

2. The KASP primer combination for 6 resistance mutation sites according to claim 1, characterized in that, The sequences of the two upstream primers of each primer combination are respectively linked with different fluorescent modification tags.

3. A method for detecting resistance mutations of Tetranychus urticae to carbamate insecticides, characterized in that, Using the KASP primer combination according to Claim 1, with the DNA of Tetranychus urticae as a template, to detect whether there are drug resistance-related mutations on the acetylcholinesterase (AChE) gene of Tetranychus urticae.

4. The method for detecting carbamate insecticide resistance mutations in Tetranychus urticae according to claim 3, characterized in that, If the KASP primer combination Turt_G119S is used, samples with the SNP genotype A:A are resistant homozygous individuals, those with the genotype G:G are sensitive homozygous individuals, and those with the genotype G:A are heterozygous individuals; if the KASP primer combination Turt_F331W is used, samples with the SNP genotype G:G are resistant homozygous individuals, those with the genotype T:T are sensitive homozygous individuals, and those with the genotype G:T are heterozygous individuals; if the KASP primer combination Turt_F331Y is used, samples with the SNP genotype A:A are resistant homozygous individuals, those with the genotype T:T are sensitive homozygous individuals, and those with the genotype T:A are heterozygous individuals; if the KASP primer combination Turt_A201S is used, samples with the SNP genotype T:T are resistant homozygous individuals, those with the genotype G:G are sensitive homozygous individuals, and those with the genotype G:T are heterozygous individuals; if the KASP primer combination Turt_T280A is used, samples with the SNP genotype G:G are resistant homozygous individuals, those with the genotype A:A are sensitive homozygous individuals, and those with the genotype G:A are heterozygous individuals; if the KASP primer combination Turt_G328A is used, samples with the SNP genotype C:C are resistant homozygous individuals, those with the genotype G:G are sensitive homozygous individuals, and those with the genotype G:C are heterozygous individuals.

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