Method for detecting emamectin benzoate resistant molecules of megalustris megalustris

Abstract

The invention discloses a megalurothrips megalurothrips methylamino abamectin benzoate resistance molecule detection method which comprises the following steps: (1) collecting megalurothrips megalurothrips female adults in a plurality of regions, and extracting DNA (deoxyribonucleic acid) in a single region in a mixed manner; (2) designing glutamic acid gated chloride ion channel gene specific primers of the megalurothrips megalurothrips, respectively adding specific tags at the front ends of the primers in all regions, and carrying out PCR (Polymerase Chain Reaction) amplification; mixing the PCR products of each region to obtain a library; (3) performing second-generation amplicon sequencing on the library to obtain off-machine data; and (4) analyzing offline data, merging the data in the same region, calculating the mutation frequency of each region by taking the ratio of the mutation (G232S) of the target sequence as an index, and judging the resistance of the megalurothrips usitatus to the emamectin benzoate. The method can be used for rapidly and massively monitoring the emamectin benzoate resistance condition of the field megalurothrips megalurothrips, and an important basis is provided for high-level emamectin benzoate resistance early warning and megalurothrips megalurothrips chemical treatment.

Description

Technical Field

[0001] This invention relates to the field of pesticide resistance detection technology, specifically to a molecular detection method for emamectin benzoate resistance in soybean thrips. Background Technology

[0002] Bean thrips ( Megalurothrips usitatus The common thrips, also known as the common thrips, belongs to the superfamily Thripsoidea in the order Thysanoptera and is one of the most important pests of crops in Hainan. This insect is small in size and is characterized by its strong concealment and rapid reproduction rate. Thrips occur year-round, but are particularly active in Hainan's tropical climate, with multiple generations per year. The development from egg to adult takes only about 14 days, allowing the thrips population to grow rapidly in a short period and easily become a pest infestation.

[0003] Avermectin is a macrolide antibiotic insecticide produced by Streptomyces fermentation. It is a natural product with high efficacy, low toxicity, and low residue, and is widely used in agricultural pest control. Emamectin benzoate (abamectin benzoate for short) is a derivative of avermectin, a semi-synthetic compound obtained through chemical modification of the avermectin molecule. Compared to avermectin, it has higher insecticidal activity and lower mammalian toxicity. The target of avermectin insecticides is the glutamate-gated chloride channel (GluCl) in insects. Based on existing research reports, only a small portion of studies suggest that insect resistance to emamectin benzoate may be related to metabolism; most research results support a target-based resistance mechanism.

[0004] Currently, resistance testing for agricultural pests such as soybean thrips is mainly divided into biological detection and molecular detection, but both have limitations in practical applications. Biological detection requires collecting field test insects and breeding them to a certain scale in the laboratory, resulting in a long experimental cycle and allowing only a single test to assess resistance levels in a single region. Furthermore, this method is susceptible to sampling errors and variations in the developmental state and health of individual test insects, leading to insufficient result stability. In addition, biological detection is costly in terms of manpower and resources—constructing a standardized toxicity curve requires at least 200-300 standard test insects, making it difficult to meet the needs of large-scale, multi-regional resistance monitoring. Molecular detection commonly uses techniques including conventional PCR and Sanger sequencing. Conventional PCR, represented by CN112342283A, typically uses specific primers to amplify and identify a single known mutation site (such as the sodium ion channel I904S mutation), resulting in low detection efficiency. Although Sanger sequencing technology is mature, it is difficult to apply it directly to the detection of resistance in soybean thrips: the pest is small and has a complex genetic background. To achieve accurate identification of resistance alleles in a single insect, a lot of manpower and sequencing costs are required, making it difficult to carry out large-scale monitoring under limited funding and manpower conditions. Summary of the Invention

[0005] The purpose of this invention is to provide a molecular detection method for emamectin benzoate resistance in soybean thrips, in order to overcome the shortcomings of the prior art.

[0006] The present invention adopts the following technical solution: A molecular detection method for emamectin benzoate resistance in soybean thrips includes the following steps: (1) Collect female adult bean thrips from multiple regions, and extract whole genome DNA after mixing female adult bean thrips from a single region; (2) Design specific primers for the glutamate-gated chloride channel gene of soybean thrips. The upstream primer sequence is shown in SEQ ID NO.1 and the downstream primer sequence is shown in SEQ ID NO.2. Add specific tags to the front end of the upstream and downstream primers for each region to obtain specific tag primers, and perform PCR amplification. Mix the PCR products from each region to obtain a library. (3) Perform second-generation amplicon sequencing on the library to obtain the data. (4) Analyze the data from the machine and merge the data from the same region, i.e. the same population. Calculate the mutation frequency of each region based on the proportion of mutations in the target sequence. Determine the resistance of soybean thrips to emamectin benzoate based on the mutation frequency. The mutation in the target sequence is that the base composition at position 232 of the soybean thrips glutamate-gated chloride channel gene changes from GGC to AGC, and the amino acid changes from glycine to serine, i.e., G232S.

[0007] Further, in step (1), the multiple regions are 3-24 regions, and 30-100 adult female thrips of the bean thrips are collected in each region.

[0008] Furthermore, in step (1), 50 female adult bean thrips were collected from each region.

[0009] Furthermore, in step (1), whole-genome DNA is extracted using the CTAB method.

[0010] Furthermore, in step (2), each region adds a specific tag to the front end of the upstream and downstream primers respectively. The specific tags on the front end of the upstream and downstream primers in the same region are the same, while the specific tags on the front end of the upstream and downstream primers in different regions are different.

[0011] Furthermore, in step (2), each region adds a 6-8 BP specific tag to the front end of the upstream and downstream primers, respectively.

[0012] Furthermore, in step (2), each region adds a 6BP specific tag to the front end of the upstream and downstream primers, respectively.

[0013] Further, in step (2), the PCR products from each region are mixed according to the same DNA mass.

[0014] Furthermore, in step (4), the offline data is analyzed, and the analysis steps are as follows: (a) Use the Trimmomatic tool to remove sequencing adapters from the analyte data and perform data quality control; (b) Use the Seqtk_demultiplex tool to separate the data for each region using specific labels; (c) Use the PANDAseq tool to merge the two-end data; (d) Use BWA & Python tools to compare and remove non-target sequences; (e) Merge duplicate sequences using the FASTX toolkit; (f) Use Python tools to filter sequencing errors using a threshold of 1 / (2n-1), where n is the number of individuals in the pool; (g) Calculate allele frequencies based on haplotype frequencies.

[0015] Furthermore, in step (4), the mutation frequency cutoff point is 95%.

[0016] The beneficial effects of this invention are: 1. This invention proposes for the first time to determine the resistance of soybean thrips to abamectin by detecting whether the base composition at position 232 of the glutamate-gated chloride channel gene in soybean thrips changes from GGC to AGC, resulting in a mutation of the encoded glycine (G) to serine (S), i.e., G232S. Soybean thrips exhibiting this point mutation are resistant to abamectin, while those without the mutation are not. This invention also designs and screens highly specific primer pairs that can accurately amplify key fragments of the glutamate-gated chloride channel gene targeting abamectin from the complex soybean thrips genome, avoiding non-specific amplification and demonstrating high sensitivity and specificity in practical applications. This invention allows for rapid and large-scale monitoring of abamectin resistance in soybean thrips in the field, providing important evidence for early warning of high-level abamectin resistance and chemical control of soybean thrips.

[0017] 2. The detection method of this invention can simultaneously monitor the resistance of soybean thrips field populations to the insecticide abamectin in multiple regions.

[0018] 3. The detection method of the present invention does not require the extraction of DNA or RNA from each individual, which can solve the difficulty of preparing DNA from a single sample when the number of samples is large or the size of the test insect is small.

[0019] 4. The detection method of this invention does not require raising test insects or conducting cumbersome drug resistance tests. The experimental cycle is short and the cost and manpower consumption are low.

[0020] 5. The detection method of this invention does not rely on insect rearing and bioassay, and the results are stable and accurate. Detailed Implementation

[0021] The present invention will be further explained below with reference to the embodiments. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0022] Example 1

[0023] Abamectin insecticides work by directly activating glutamate-gated chloride channels (GluCls) in pests and regulating other cysteine ​​cyclic ion channels. This invention provides a molecular detection method for the resistance of soybean thrips to abamectin. The method determines the resistance of the soybean thrips to abamectin by detecting whether the base composition at position 232 of the glutamate-gated chloride channel (GenBank: KAJ1526384.1) in soybean thrips changes from GGC to AGC, resulting in a mutation from glycine (G) to serine (S), i.e., G232S. Soybean thrips exhibiting this point mutation are resistant to abamectin, while those without the point mutation are not.

[0024] In this embodiment, bioassays of abamectin resistance were performed on indoor susceptible strains of *Thrips spp.* and five populations collected from fields in Sanya, Lingshui, Ledong, Danzhou, and Haikou, Hainan Province. The detection method of this invention was used to test the resistance of indoor susceptible strains and various populations of *Thrips spp.* to abamectin. The five populations were collected from cowpea hosts in Sanya, Lingshui, Ledong, Danzhou, and Haikou between April and December 2024. The bioassay data for abamectin resistance (determining the lethal median concentration of the pest by setting different concentration gradients of the insecticide abamectin) for the above populations are shown in Table 1.

[0025] Table 1

[0026] Note: The susceptible strain (i.e., the non-mutated strain, with GGC at position 232 (corresponding to glycine) and no mutation) is a susceptible material that has been housed indoors for many years. Its median lethal concentration (LC50) for emamectin benzoate is... 50 The value was used as a baseline in this experiment to determine the resistance levels of the other five populations.

[0027] The detection method of the present invention includes the following steps: 1. Collect 30-100 female adult soybean thrips from multiple regions (3-24 regions, 5 regions in this example: Sanya, Ledong, Lingshui, Danzhou, and Haikou, respectively), and extract whole-genome DNA using the CTAB method. Specific method: (1) Place 50 female adult insects from each of the five regions in order (Sanya, Ledong, Lingshui, Danzhou, Haikou) into five 2.0ml EP tubes, named GS1, GS2, GS3, GS4, and GS5 respectively. Add steel balls, 700ul CTAB lysis buffer, and grind in a grinder (60 times per second) for 5 minutes.

[0028] (2) Centrifuge the EP tube at 14000g for 10min in a centrifuge, add 700ul of the mixture (phenol: chloroform: isoamyl alcohol volume ratio = 25: 24: 1, hereinafter referred to as the mixture) to the EP tube, shake vigorously, let stand for 5min, and centrifuge at 14000g for 10min.

[0029] (3) Pipette 600ul of supernatant into a 1.5ml EP tube, add 600ul of mixed solution, mix gently and let stand for 5min, then centrifuge at 14000g for 10min.

[0030] (4) Pipette 500ul of supernatant into a new 1.5ml EP tube, add 50ul of 7.5M sodium acetate solution, fill the tube to 1.5ml with anhydrous ethanol, and store it in a -20℃ refrigerator (1h, or overnight). The EP tube should be labeled with the final label.

[0031] (5) After the sample is taken out, the centrifuge is pre-cooled to 4°C and centrifuged at 14000g for 10min.

[0032] (6) After centrifugation, a large amount of white precipitate should be seen at the bottom of the EP tube (if there is no precipitate, it does not mean that the extraction was unsuccessful, and the subsequent steps should be continued). Discard the supernatant, add 500ul of 70v / v% ethanol solution to wash the DNA, and centrifuge at 4℃ and 14000g for 5min.

[0033] (7) After pouring out most of the ethanol solution in the EP tube, place it in a centrifuge and start the rapid centrifugation mode, i.e., centrifuge at 1000g for 10s. Use a 200ul pipette to remove the remaining ethanol solution (avoid touching the precipitate), and let it air dry at room temperature (about 10-15min).

[0034] (8) Add 30 μL of DNA elution buffer, dissolve by pipetting, and store properly (store at -20°C).

[0035] 2. In this study, five sets of primers with specific tags were designed to ensure that samples from different regions were labeled with different specific tags after the PCR tagging step.

[0036] (1) Design specific primers for the glutamate-gated chloride channel gene of soybean thrips (GenBank: KAJ1526384.1). The upstream primer F sequence is: 5'-AACGTTAACAACCCTGCGG-3' (as shown in SEQ ID NO.1), and the downstream primer R sequence is: 5'-GCGCTGCTTCTTCATGTTC-3' (as shown in SEQ ID NO.2).

[0037] (2) The upstream and downstream primers of the five regions of Sanya, Ledong, Lingshui, Danzhou and Haikou were respectively given a 6BP specific tag. The 6BP specific tags of the five regions are ACTTGA, AGTCAA, AGTTCC, ATCACG and ATCGAC. After adding the 6BP specific tag, the primers for the five regions are as follows: Sanya: upstream primer F sequence: 5'-ACTTGAAACGTTAACAACCCTGCGG-3' (as shown in SEQ ID NO.3), downstream primer R sequence: 5'-ACTTGAGCGCTGCTTCTTCATGTTC-3' (as shown in SEQ ID NO.4); Ledong: upstream primer F sequence: 5'-AGTCAAAACGTTAACAACCCTGCGG-3' (as shown in SEQ ID NO.5), downstream primer R sequence: 5'-AGTCAAGCGCTGCTTCTTCATGTTC-3' (as shown in SEQ ID NO.6); Lingshui: upstream primer F sequence: 5'-AGTTCCAACGTTAACAACCCTGCGG-3' (as shown in SEQ ID NO.7), downstream primer R sequence: 5'-AGTTCCGCGCTGCTTCTTCATGTTC-3' (as shown in SEQ ID NO.7). As shown in SEQ ID NO. 8), in Danzhou: the upstream primer F sequence is 5'-ATCACGAACGTTAACAACCCTGCGG-3' (as shown in SEQ ID NO. 9), and the downstream primer R sequence is 5'-ATCACGGCGCTGCTTCTTCATGTTC-3' (as shown in SEQ ID NO. 10). In Haikou: the upstream primer F sequence is 5'-ATCGACAACGTTAACAACCCTGCGG-3' (as shown in SEQ ID NO. 11), and the downstream primer R sequence is 5'-ATCGACGCGCTGCTTCTTCATGTTC-3' (as shown in SEQ ID NO. 12). Primer synthesis was completed by the Hainan Branch of Beijing Qingke Biotechnology Co., Ltd.

[0038] (3) Take out 5 0.2ml PCR tubes and name them GS1, GS2, GS3, GS4 and GS5 respectively, corresponding to the population order of Sanya, Ledong, Lingshui, Danzhou and Haikou respectively. Complete the amplification of the target fragment with a total reaction volume of 50ul (Table 2) (the Genomic DNA template in Table 2 is the DNA sample extracted in step 1 above, and the DNA sample concentration is about 600ng / ul). The primers of the five regions are added to GS1, GS2, GS3, GS4 and GS5 in the corresponding order.

[0039] Table 2

[0040] The PCR reaction procedure was as follows: first, pre-denaturation at 94℃ for 3 min; then 35 cycles were performed: denaturation at 94℃ for 30 s, annealing at 60℃ for 30 s, extension at 72℃ for 30 s; and finally extension at 72℃ for 10 min.

[0041] The PCR products were subjected to agarose gel electrophoresis, and the specific steps are as follows: (a) To prepare a 1 m / v% gel, weigh 0.25 g of agarose powder and pour it into an Erlenmeyer flask. Add 25 mL of 1x TAE buffer to the Erlenmeyer flask and gently shake to disperse the agarose powder.

[0042] (b) Microwave heating until it is completely transparent and melted, then add 2ul of nucleic acid dye after it cools slightly, shake well and pour it into a mold to cool.

[0043] (c) After placing the sample into the electrophoresis apparatus, add PCR samples of GS1, GS2, GS3, GS4 and GS5 to the second to sixth cells respectively, and add 2000 DNA marker to the first cell. The program is set to 144V, 20mA and 25min.

[0044] (d) Under UV light, cut the gel strip from each well with a knife and place it into the corresponding 2ml centrifuge tubes of GS1, GS2, GS3, GS4, and GS5. Purify and recover the gel strip using the gel recovery kit from Nanjing Novizan Biotechnology Co., Ltd. The collected gel recovery products were named GS1, GS2, GS3, GS4, and GS5 in sequence. Mix the DNA from the five regions in the same proportion (each region has the same DNA mass, 1000ug DNA per region, for a total of 5000ug DNA) to obtain the library.

[0045] 3. Submit the library obtained in step 2 to Guangzhou Novogene Medical Laboratory Co., Ltd. for next-generation amplicon sequencing.

[0046] 4. Analyze the data from the second-generation amplicon sequencing (i.e., the sum of various fragments from five regions) obtained by Guangzhou Novogene Medical Laboratory Co., Ltd. The analysis steps are as follows: (1) Remove sequencing adapters from the data after sequencing and perform data quality control (using Trimmomatic).

[0047] (2) Separate the data of each region by using specific labels (the tool is Seqtk_demultiplex).

[0048] (3) Merge the two-end data (using PANDAseq).

[0049] (4) Align and remove non-target sequences (tools: BWA & Python).

[0050] (5) Merge repeating sequences (using FASTX toolkit).

[0051] (6) Filter sequencing errors by a threshold of 1 / (2n-1) (n is the number of individuals in the pool) (tool: Python).

[0052] (7) Calculate allele frequencies based on haplotype frequencies.

[0053] After analyzing the data above, the data of the same population were merged, and the mutation frequency of each region was calculated using the mutation ratio of the target sequence (mutation ratio: the ratio of a single base mutation in a certain region to the total number of samples (50), i.e. the proportion of cases where the 232 site is AGC (corresponding to serine)) as the indicator.

[0054] Sensitive strains can be established separately following the steps described above, except that primers do not need to be labeled with specific tags.

[0055] The results are shown in Table 3.

[0056] Table 3

[0057] Table 1 shows the bioassay results, indicating that the lethal median concentration of the susceptible soybean thrips strain is extremely low, at 0.91. Correspondingly, the lowest resistance multiple of the five soybean thrips field populations is over 30 times, all indicating medium-to-high resistance levels. Table 3 shows the results of the detection method of this invention, indicating that the mutation frequency of the susceptible soybean thrips strain is 0%, while the mutation frequency of the five soybean thrips field populations is all above 95%, essentially indicating complete mutation. This suggests that all five soybean thrips field populations have high resistance to abamectin, corresponding to the high resistance multiples in Table 1. This verifies that the detection method of this invention can rapidly and extensively detect the resistance of soybean thrips field populations to abamectin, enabling rapid and large-scale monitoring of soybean thrips resistance in the field, and timely informing farmers of their pesticide application strategies, including whether to increase or decrease the use of abamectin in controlling soybean thrips.

Claims

1. A molecular detection method for emamectin benzoate resistance in soybean thrips, characterized in that, Includes the following steps: (1) Collect female adult bean thrips from multiple regions, and extract whole genome DNA after mixing female adult bean thrips from a single region; (2) Design specific primers for the glutamate-gated chloride channel gene of soybean thrips. The upstream primer sequence is shown in SEQ ID NO.1 and the downstream primer sequence is shown in SEQ ID NO.

2. Add specific tags to the front end of the upstream and downstream primers for each region to obtain specific tag primers, and perform PCR amplification. Mix the PCR products from each region to obtain a library. (3) Perform second-generation amplicon sequencing on the library to obtain the data; (4) Analyze the data from the machine and merge the data from the same region, i.e. the same population. Calculate the mutation frequency of each region based on the proportion of mutations in the target sequence. Determine the resistance of soybean thrips to emamectin benzoate based on the mutation frequency. The mutation in the target sequence is that the base composition at position 232 of the soybean thrips glutamate-gated chloride channel gene changes from GGC to AGC, and the amino acid changes from glycine to serine, i.e., G232S.

2. The molecular detection method for emamectin benzoate resistance in soybean thrips according to claim 1, characterized in that, In step (1), the multiple regions are 3-24 regions, and 30-100 adult female thrips of the bean thrips are collected in each region.

3. The molecular detection method for emamectin benzoate resistance in soybean thrips according to claim 2, characterized in that, In step (1), 50 adult female bean thrips were collected from each region.

4. The molecular detection method for emamectin benzoate resistance in soybean thrips according to claim 1, characterized in that, In step (1), whole genome DNA is extracted using the CTAB method.

5. The molecular detection method for emamectin benzoate resistance in soybean thrips according to claim 1, characterized in that, In step (2), each region adds a specific tag to the front end of the upstream and downstream primers respectively. The specific tags on the front end of the upstream and downstream primers in the same region are the same, while the specific tags on the front end of the upstream and downstream primers in different regions are different.

6. The molecular detection method for emamectin benzoate resistance in soybean thrips according to claim 5, characterized in that, In step (2), each region adds a 6-8 BP specific tag to the front end of the upstream and downstream primers, respectively.

7. The molecular detection method for emamectin benzoate resistance in soybean thrips according to claim 6, characterized in that, In step (2), each region adds a 6BP specific tag to the front end of the upstream and downstream primers respectively.

8. The molecular detection method for emamectin benzoate resistance in soybean thrips according to claim 1, characterized in that, In step (2), the PCR products from different regions are mixed according to the same DNA mass.

9. The molecular detection method for emamectin benzoate resistance in soybean thrips according to claim 1, characterized in that, In step (4), the offline data is analyzed. The analysis steps are as follows: (a) Use the Trimmomatic tool to remove sequencing adapters from the analyte data and perform data quality control; (b) Use the Seqtk_demultiplex tool to separate the data for each region using specific labels; (c) Use the PANDAseq tool to merge the two-end data; (d) Use BWA & Python tools to compare and remove non-target sequences; (e) Merge duplicate sequences using the FASTX toolkit; (f) Use Python tools to filter sequencing errors using a threshold of 1 / (2n-1), where n is the number of individuals in the pool; (g) Calculate allele frequencies based on haplotype frequencies.

10. The molecular detection method for emamectin benzoate resistance in soybean thrips according to claim 1, characterized in that, In step (4), the mutation frequency cutoff point is 95%.

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