Molecular markers and methods for identifying the resistance of Bactrocera dorsalis to spinosad

Abstract

The invention discloses a molecular marker and method for identifying the resistance of citrus fruit fly to spinosad, and belongs to the field of biotechnology. The invention provides an application of a substance for detecting a SNP site in the genome of citrus fruit fly in identifying or assisting in identifying the resistance of citrus fruit fly genes to spinosad, wherein the SNP site is the first nucleotide of the codon encoding the 345th amino acid of the basic acetylcholine receptor α9 subunit in the genome of citrus fruit fly, and the nucleotide type is A or G. By using the molecular marker information provided by the invention, the resistance of a population to spinosad can be determined efficiently and quickly, thereby providing data reference for guiding the early monitoring and early warning of the resistance of citrus fruit fly and comprehensive management.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a molecular marker and a method for identifying the resistance of Bactrocera dorsalis to spinosad. Background Art

[0002] The citrus fruit fly (Bactrocera dorsalis Hendel) is an important quarantine pest for imported plants in my country. It has high reproductive capacity, strong adaptability, and a wide host range. It can harm more than 250 species of fruit and vegetable crops from more than 40 families. The citrus fruit fly lays eggs in fruits and vegetables, directly damaging fruit and vegetable commodities and causing serious economic impacts on the fruit and vegetable industry. Although my country advocates the use of integrated control technology for the citrus fruit fly, field control of the citrus fruit fly mainly relies on chemical pesticides. However, problems such as continuous chemical control and abuse of pesticides have led to the rapid development of citrus fruit fly resistance to multiple pesticides, which not only increases the difficulty of control, but also affects environmental safety and human health.

[0003] Spinosad is a macrolide insecticide developed by Dow AgroSciences in the United States in the 1990s. It is produced by aerobic fermentation of soil actinomycetes - Saccharopolyspora spinosa. The insecticide has the characteristics of low toxicity, low residue and high insecticidal activity. It can be used for pest control of fruits, vegetables, rice, flowers and other crops. It can effectively control a variety of pests such as Lepidoptera, Diptera, Thysanoptera, etc. It is a commonly used insecticide in chemical control of citrus fruit fly. Although no significant resistance to spinosad has been monitored in the field population of citrus fruit fly, there have been reports that citrus fruit fly has a certain risk of resistance to spinosad. Under the condition of large-scale and continuous use of spinosad for control, the possibility of citrus fruit fly developing resistance to spinosad is very high, and the situation of resistance control of citrus fruit fly may be more severe in the future. Spinosad mainly acts on nicotinic acetylcholine receptors (nAChR) and gamma-aminobutyric acid receptors (GABAR). nAChR mutation is the main cause of spinosad target resistance. Among them, the nAChRα6 subunit has been widely studied. Truncation of the subunit transcript will significantly reduce the sensitivity of the citrus fruit pest to spinosad (Hsu, JC, Feng, HT, Wu, WJ, Geib, SM, Mao, CH, & Vontas, J. (2012). Truncated transcripts of nicotinicacetylcholine subunit gene Bdα6are associated with spinosad resistance in Bactrocera dorsalis. Insect Biochemistry and Molecular Biology, 2012. Biology, 42(10):806-815. https: / / doi.org / https: / / doi.org / 10.1016 / j.ibmb.2012.07.010). Currently, the research on the resistance of Bactrocera dorsalis to spinosad is limited to this target, and no related research involves other potential targets.

[0004] In field control, monitoring of the resistance of orange fruit flies to spinosad helps to grasp the development trend of its resistance in real time, so it is urgent to develop a fast and accurate insecticide resistance detection technology. At present, the spinosad resistance of orange fruit flies is mainly monitored by bioassays, but this method has a long cycle and requires a large number of test insects. In addition, it is difficult to monitor early resistance or low-frequency resistance alleles by bioassays, and it cannot meet the requirements of fast and accurate detection. In addition, a rapid molecular detection method for the target resistance of spinosad caused by nAChR mutations in orange fruit flies has not yet been established in China. Among other pests, the resistance molecular detection technology based on insecticide target genes mainly relies on PCR amplification technology, such as CN 105713959A, which uses specific primers designed according to the gene sequence of the glutamate chloride ion channel of the diamondback moth, and uses the genomic DNA of the single-headed diamondback moth as a template to perform PCR amplification of the TM2 and TM3 regions of the glutamate chloride ion channel gene. By analyzing the direct sequencing chromatogram of the purified target fragment product, the genotype of the individual diamondback moth can be distinguished, and the resistance allele frequency of the population sample can be detected. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a molecular marker and method for identifying spinosad resistance of Bactrocera dorsalis. The technical problem to be solved is not limited to the described technical subject matter, and those skilled in the art can clearly understand other technical subjects not mentioned in this article through the following description.

[0006] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0007] The present invention provides the use of a substance for detecting SNP sites in the genome of the citrus fruit fly in any of the following:

[0008] C1) Identify or assist in identifying the resistance of Bactrocera dorsalis to spinosad;

[0009] C2) preparing products for identifying or assisting in identifying the resistance of Bactrocera dorsalis to spinosad;

[0010] C3) Screening or breeding varieties or strains of Bactrocera dorsalis for resistance to spinosad;

[0011] C4) preparing products for screening or breeding varieties or strains of Bactrocera dorsalis for resistance to spinosad;

[0012] C5) Screening or breeding varieties or strains of Bactrocera dorsalis susceptible to spinosad;

[0013] C6) preparing products for screening or breeding species or strains of Bactrocera dorsalis susceptible to spinosad;

[0014] The SNP site is the first nucleotide of the codon encoding the 345th amino acid of the basic acetylcholine receptor α9 subunit in the genome of the oriental fruit fly, and the nucleotide type is A or G.

[0015] The 345th amino acid of the basic acetylcholine receptor α9 subunit is the 345th amino acid of SEQ ID No. 1. The SNP site may specifically be the 1033rd nucleotide of SEQ ID No. 2.

[0016] In the above application, the substance is any one of the following:

[0017] D1) the substance is a primer composition for amplifying a genomic DNA fragment of the citrus fruit fly including the SNP site;

[0018] D2) the substance is a PCR reagent containing the primer combination described in D1);

[0019] D3) The substance is a kit containing the primer composition described in D1) or the PCR reagent described in D2).

[0020] In the above application, the primer composition is composed of the single-stranded DNA shown in SEQ ID No.3 and the single-stranded DNA shown in SEQ ID No.4, or can be composed of any upstream primer and downstream primer that can amplify the above-mentioned SNP site, the upstream primer refers to the primer located at the 5' end relative to the SNP site on the genome according to the gene transcription direction, and the downstream primer refers to the primer located at the 3' end relative to the SNP site on the genome.

[0021] The present invention also provides a product for detecting the polymorphism or genotype of the SNP site in the genome of the aforementioned Bactrocera dorsalis, wherein the product is the aforementioned substance, and the product is any one of the following:

[0022] A1) Products for detecting single nucleotide polymorphisms or genotypes associated with the resistance of Bactrocera dorsalis to spinosad;

[0023] A2) Products for identifying or assisting in identifying the resistance of Bactrocera dorsalis to spinosad;

[0024] A3) Screening or breeding products for Bactrocera dorsalis resistant to spinosad;

[0025] A4) Products for screening or breeding Bactrocera dorsalis susceptible to spinosad.

[0026] In the above applications and methods, the substance for detecting the polymorphism or genotype of SNP can be used to determine the nucleotide type of the SN site in the above-mentioned Bactrocera dorsalis genome by at least one of the following methods: DNA sequencing, restriction fragment length polymorphism, single-strand conformation polymorphism, denaturing high performance liquid chromatography and SNP chip. Among them, SNP chips include chips based on nucleic acid hybridization reactions, chips based on single base extension reactions, chips based on allele-specific primer extension reactions, chips based on "one-step" reactions, chips based on primer ligation reactions, chips based on restriction endonuclease reactions, chips based on protein DNA binding reactions, and chips based on fluorescent molecule DNA binding reactions.

[0027] In the above products, the above substances are any of the following:

[0028] D1) the substance is a primer composition for amplifying a genomic DNA fragment of the citrus fruit fly including the SNP site;

[0029] D2) the substance is a PCR reagent containing the primer combination described in D1);

[0030] D3) The substance is a kit containing the primer composition described in D1) or the PCR reagent described in D2).

[0031] In the above product, the primer composition consists of the single-stranded DNA shown in SEQ ID No.3 and the single-stranded DNA shown in SEQ ID No.4.

[0032] In the above-mentioned applications and methods, the PCR primer may be labeled or not labeled with a marker. The marker refers to any atom or molecule that can be used to provide a detectable effect and can be attached to a nucleic acid. Markers include, but are not limited to, dyes; radioactive labels, such as 32P; binding moieties, such as biotin; haptens, such as digoxin (DIG); luminescent, phosphorescent or fluorescent moieties; and fluorescent dyes alone or in combination with moieties that can inhibit or shift the emission spectrum by fluorescence resonance energy transfer (FRET). The marker can provide a signal that can be detected by fluorescence, radioactivity, colorimetry, weight determination, X-ray diffraction or absorption, magnetism, enzyme activity, etc. The marker can be a charged moiety (positive or negative charge) or, alternatively, can be charge neutral. The marker can include a nucleic acid or protein sequence or a combination thereof, as long as the sequence containing the marker is detectable. In some embodiments, the nucleic acid is directly detected (e.g., directly reading the sequence) without a marker. In the above applications and methods, the product may be a reagent or a kit or a system, and the system may include a combination product of a reagent or a kit, an instrument and an analysis software, such as a product consisting of PCR primers, PARMS master mix reagents, an ELISA reader and an online software SNP decoder (http: / / www.snpway.com / snpdecoder01 / ), and a combination product consisting of PCR primers, PARMS master mix reagents, an online software SNP decoder and a fluorescence quantitative PCR instrument. The product may include the above-mentioned substance for detecting the polymorphism or genotype of the SNP site in the genome of the citrus fruit fly.

[0033] The present invention also provides a method for identifying or assisting in identifying citrus fruit fly resistant to spinosad, the method comprising detecting the genotype of a SNP site in the genome of the citrus fruit fly to be tested, the SNP site being the first nucleotide of the codon encoding the 345th amino acid of the basic acetylcholine receptor α9 subunit in the citrus fruit fly genome, and the nucleotide type thereof is A or G.

[0034] In the above method, the nucleotide sequence of the gene encoding the basic acetylcholine receptor α9 subunit is SEQ ID No.2.

[0035] In the above method, according to the genotype identification or auxiliary identification of the resistance of the citrus fruit fly to spinosad, the genotype of the SNP site is AA, and the resistance to spinosad is lower than that of the citrus fruit fly to be tested with a genotype of AG or GG. The genotype of the SNP site is AA, which is a homozygous type of the SNP1 site is A, the genotype of the SNP site is AG, which is a heterozygous type of the SNP1 site is A and G, and the genotype of the SNP site is AA, which is a homozygous type of the SNP1 site is G.

[0036] The method comprises the following steps:

[0037] (1) using the genomic DNA of the fruit fly to be identified as a template, performing PCR amplification using the aforementioned PCR primers to obtain a PCR product;

[0038] (2) Identifying the resistance of Bactrocera dorsalis to spinosad according to the type of SEQ ID No. 2 contained in the PCR product.

[0039] The types of SEQ ID No. 2 include that the 1033rd nucleotide of SEQ ID No. 2 is A or the 1033rd nucleotide is G.

[0040] The above substances or primer compositions also fall within the protection scope of the present invention.

[0041] The present invention also provides any of the following applications of DNA molecules:

[0042] C1) Identify or assist in identifying the resistance of Bactrocera dorsalis to spinosad;

[0043] C2) preparing products for identifying or assisting in identifying the resistance of Bactrocera dorsalis to spinosad;

[0044] C3) Screening or breeding varieties or strains of Bactrocera dorsalis for resistance to spinosad;

[0045] C4) preparing products for screening or breeding varieties or strains of Bactrocera dorsalis for resistance to spinosad;

[0046] C5) Screening or breeding varieties or strains of Bactrocera dorsalis susceptible to spinosad;

[0047] C6) preparing products for screening or breeding species or strains of Bactrocera dorsalis susceptible to spinosad;

[0048] The DNA molecule is a DNA molecule whose nucleotide sequence is SEQ ID No.2.

[0049] Identification of the resistance of the orange fruit fly to spinosad: the resistance multiple of the orange fruit fly with genotype AA was significantly lower than that of the orange fruit fly with genotype AG or GG, that is, the effect of using spinosad to kill the orange fruit fly with genotype AA is better than that with genotype AG or GG.

[0050] The advantage of the present invention is that the bioassay requires the collection of Bactrocera dorsalis and the reproduction to a certain scale before the detection can be carried out, which takes at least 2 weeks. The present invention can significantly shorten the detection time by detecting the type of nucleotides at the SNP site. In addition, the bioassay is a relatively rough method for detecting the resistance level, which will be affected by many factors and the uncertainty of the results is high. The present detection technology sequences the nucleotides, which meets the accuracy requirements while ensuring the rapidity of the results. In addition, the present invention significantly reduces the material requirements compared to the bioassay. In the bioassay technology, the LC of a certain population to a certain insecticide is determined. 50 The value requires at least about 300 orange fruit flies. However, the present invention only needs about 30 flies to detect a population, and the mutation frequency of alleles involved in the resistance to spinosad in the population can be obtained, providing data reference for early monitoring, early warning and comprehensive management of the resistance of orange fruit flies. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 These are the chromatograms of three genotypes of the I345 mutation of the oriental fruit fly, and the mutation is marked with a triangle in the chromatogram.

[0052] Figure 2 The linear regression relationship between the resistance multiple and the I345V mutant allele frequency. Each point represents a different population. In addition, r is marked in the figure. 2 value and significance. DETAILED DESCRIPTION

[0053] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0054] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0055] SPSS Statistics 26 statistical software was used to process the data in the following examples, and univariate linear regression analysis was performed. P<0.05 (*) indicated a significant difference.

[0056] Example 1. Method establishment

[0057] The inventors' research shows that the 1033rd nucleotide of the nicotinic acetylcholine receptor α9 subunit gene (SEQ ID No. 2) sequence of some individuals in the field population of the citrus fruit fly mutates from A to G, and the protein it encodes mutates from I to V. The mutation frequency is related to the resistance of the population to spinosad, that is, the mutation leads to a high level of target resistance of the citrus fruit fly to spinosad. The amino acid sequence of the nicotinic acetylcholine receptor α9 subunit is SEQ ID No. 1.

[0058] 1. Primer design

[0059] Primers were designed for the α9 subunit of the nicotinic acetylcholine receptor of Bactrocera dorsalis (I345V resistance mutation), and the nucleotide sequence of the α9 subunit of the nicotinic acetylcholine receptor of Bactrocera dorsalis (I345V resistance mutation) is SEQ ID No. 2. The details are as follows:

[0060] Bdα9-F (SEQ ID No. 3): 5'-GTTGTACCTTTGTTACCGTA-3';

[0061] Bdα9-R (SEQ ID No. 4): 5'-TCTCCATAAAGCTCGCCAA-3'.

[0062] The above primers were synthesized by Beijing Qingke Company.

[0063] 2. RNA extraction from single Bactrocera dorsalis

[0064] Ambion's Trizol reagent was used to extract RNA from a single-headed Bactrocera dorsalis. The specific steps are as follows:

[0065] (1) Grind the adult insect tissue in liquid nitrogen using an electric grinder until the sample appears as a white powder;

[0066] (2) Add 1000 μL Trizol solution, vortex gently for 1 min to mix evenly, and let stand at room temperature for 5 min;

[0067] (3) Centrifuge at 12000 rpm for 5 min at 4°C and transfer 1000 μL of the supernatant to a new tube;

[0068] (4) Add 200 μL of chloroform, invert several times, and let stand for 5 min;

[0069] (5) Centrifuge at 12,000 rpm for 15 min at 4°C. The sample is separated into three layers: organic phase, middle layer, and aqueous phase. The desired RNA is mainly present in the aqueous phase. Take 350 μL of the supernatant and transfer it to a new tube.

[0070] (6) Add 500 μL of isopropanol, invert several times, and let stand for 10 min;

[0071] (7) Centrifuge at 12,000 rpm for 10 min at 4°C, remove the supernatant and retain the precipitate;

[0072] (8) Add 1000 μL of 75% ethanol solution to the sample, gently suspend the precipitate, and invert several times;

[0073] (9) Centrifuge at 12,000 rpm for 5 min at 4°C, remove the supernatant and retain the precipitate;

[0074] (10) Repeat steps (8) and (9);

[0075] (11) Place the centrifuge tube upside down on the filter paper and blow dry with a gentle breeze for about 20 minutes.

[0076] (12) After the sample is dry, add 40-60 μL of DEPC-treated water to dissolve it, and continue to pump it;

[0077] (13) 1 μL of each sample was taken for quality assessment, and the rest was frozen at -80°C.

[0078] 3. Reverse transcription to synthesize cDNA

[0079] Using the RNA extracted in the above steps as a template, reverse transcription was performed using TaKaRa's PrimeScriptTM RT reagent Kit with gDNAEraser to obtain cDNA. The following steps must be performed on ice, as follows:

[0080] (1) The first step reaction system (Table 1) was established in a 200 μL centrifuge tube. A maximum of 1 mg RNA was added to the system. The required RNA volume was calculated based on the measured RNA concentration. The RNA volume was less than 7 μL. The reaction conditions were: 42°C metal bath for 2 min and storage at 4°C.

[0081] Table 1 Reverse transcription first step reaction system

[0082] Components Volume (μL) 5×gDNAEraserBuffer 2 gDNAEraser 1 Total RNA * <![CDATA[RNaseFreeddH20]]> Upto10 Total 10

[0083] (2) The second step reaction system (Table 2) was established. The reaction conditions were: 37°C metal bath for 15 min, 85°C for 5 s, and storage at 4°C.

[0084] Table 2 Reverse transcription second step reaction system

[0085] Components Volume (μL) The reaction solution in step (1) 10 5×PrimeScriptBuffer2(forRealTime) 4 PrimeScriptRTEnzymeMixI 1 RTPrimeMix 1 <![CDATA[RNaseFreeddH2O]]> 4 Total 20

[0086] 4. PCR amplification

[0087] The specific primers used for PCR amplification were SEQ ID No.3 and SEQ ID No.4. The reaction system was shown in Table 3. The reaction conditions were: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at the corresponding temperature for 30 s, extension at 72°C for 1 min / 1 kb, for a total of 38 cycles, and finally extension at 72°C for 10 min and storage at 4°C.

[0088] Table 3 PCR amplification reaction system

[0089]

[0090] 5. PCR product sequencing

[0091] The PCR amplification product was sent to the company for sequencing, and the sequencing primers were Bdα9-F and Bdα9-R. The chromatogram of the sequencing results was analyzed using Chromas software. The 1033rd base of the Bdα9 gene (SEQ ID No.1) was a single peak chromatogram of A, indicating that the individual was a sensitive homozygote without a nicotinic acetylcholine receptor mutation, and the genotype of this type of orange fruit fly was named AA; the 1033rd base was an A / G double peak chromatogram, indicating that the individual was a heterozygote with an amino acid mutation at position 345, and the genotype of this type of orange fruit fly was named AG; the 1033rd base was a single peak chromatogram of G, indicating that the individual was a homozygote with an amino acid mutation at position 345, and the genotype of this type of orange fruit fly was named GG. The calculation method of the Bdα9I345V resistance allele frequency is as follows: resistance allele frequency (RF) = (number of resistant homozygote individuals × 2 + number of resistant heterozygotes) / (total number of detected individuals × 2).

[0092] Example 2: Determination of the genotype of Bdα9 encoding amino acid 345 in different geographical populations of Bactrocera dorsalis using the method provided in Example 1

[0093] 1. Population Information

[0094] LAB population: a control population sensitive to insecticides that has been raised in the inventor's laboratory for many years, which is recorded in the non-patent document "Zhao Yan, Duan Yongheng, Zhao Zihua, Wang Yuning, Wu Qin, Ding Jianyun, Li Zhihong (2019). Study on wintering of citrus fruit fly experimental population in Beijing area. China Plant Protection Guide, 39(10): 68-71." The public can obtain it from China Agricultural University. This biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes. This population is named LAB population.

[0095] The specific screening method for the RES population of Bactrocera dorsalis is as follows: 3-5 day-old adults of the LAB population are used as the target, and each generation is treated with the drug film method, and the drug concentration is maintained at the 24h LC of the previous bioassay. 50About. Measure 100mL of liquid medicine (spinosad, purchased from Hunan Nonghai Haite Agrochemical Co., Ltd., pesticide registration number PD20160704) in a large beaker, fully rotate the beaker to form a uniform drug film on the wall of the cup, and then pour out the liquid medicine. After the residual liquid in the beaker is completely evaporated, put a cotton ball soaked in 10% sucrose water, and then induce the adult citrus fruit fly to enter, seal the cup mouth with breathable gauze and tighten it with a rubber band. After 24 hours of treatment, the surviving individuals continue to be raised and reproduced. The 24h LC of the antagonistic population is determined by the drug film method every 5 generations 50 , monitor resistance in time and adjust the concentration of screening agent.

[0096] The specific collection information of the field populations of Bactrocera dorsalis BJ, HN, and SD is shown in Table 4, and all of them have been identified as Bactrocera dorsalis.

[0097] Table 4 Collection information of three field populations of Bactrocera dorsalis

[0098]

[0099] Results of identification of Bactrocera dorsalis BJ field population (COI gene):

[0100] 5’-ATATGTTTTATCTTCGGAGCCTGAGCAGGAATAGTAGGAACATCCCTTAGA ATTTTAGTCCGAGCTGAACTCGGTCACCCAGGAGCTTTAATTGGTGACGATCAAATTTATAATGTAATTGTAACAGCTCATGCTTTCGTAATAATTTTCTTTATAGTTATACCAATTATAATTGGTGGATTTGGAAATTGACTTGTTCCTTTAATATTAGGAGCTCCCGATATAGCATTTCCACGAATGAATAATATAAGATTTTGATTATTACCTCCTTCCCTTACATTACTATTAGTAAGAAGTATAGTAGAAAACGGAGCTGGTACAGGTTGAACAGTTTACCCACCCCTATCATCTGTTATTGCACACGGAGGAGCTTCAGTTGACCTAGCTATTTTTTCACTTCACTTAGCGGGTATTTCCTCAATTTTAGGAGCAGTAAATTTCATTACAACAGTAATTAATATACGATCGACAGGAATCACCTTTGATCGAATACCTCTATTCGTTTGAGCAGTTGTATTAACAGCTTTATTACTTTTATTATCATTACCAGTTTTAGCAGGGGCTATTACTATATTACTAACAGACCGAAACTTAAATACTTCCTTTTTTGACCCTGCCGGAGGAGGAGATCCTATTCTTTACCAACATTTATTTTGATTTTTTGGCACCCGGGAAAAGTTAAAAAG-3’

[0101] Identification results of the Bactrocera dorsalis HN field population (COI gene):

[0102] 5’-GGGCTTTTTATTTTATCTTCGGAGCCTGAGCAGGAATAGTAGGAACATCCC TTAGAATTTTAGTCCGAGCTGAACTCGGTCACCCAGGAGCTTTAATTGGTGACGATCAAATTTATAATGTAATTGTAACAGCTCATGCTTTCGTAATAATTTTCTTTATAGTTATACCAATTATAATTGGTGGATTTGGAAATTGACTTGTTCCTTTAATATTAGGAGCTCCCGATATAGCATTTCCACGAATGAATAATATAAGATTTTGATTATTACCTCCTTCCCTTACATTACTATTAGTAAGAAGTATAGTAGAAAACGGAGCTGGTACAGGTTGAACAGTTTACCCACCCCTATCATCTGTTATTGCACACGGAGGAGCTTCAGTTGACCTAGCTATTTTTTCACTTCACTTAGCGGGTATTTCCTCAATTTTAGGAGCAGTAAATTTCATTACAACAGTAATTAATATACGATCGACAGGAATCACCTTTGATCGAATACCTCTATTCGTTTGAGCAGTTGTATTAACAGCTTTATTACTTTTATTATCATTACCAGTTTTAGCAGGGGCTATTACTATATTACTAACAGACCGAAACTTAAATACTTCCTTTTTTGACCCTGCCGGAGGAGGAGATCCTATTCTTTACCAACATTTATTTTGATTTTTTGGTCACCTTGGAAAGTTTAAA-3’

[0103] Identification results of the Bactrocera dorsalis SD field population (COI gene):

[0104] 5'-CATTAAATTTTTCTTCGGAGCTGAGCAGGAATAGTAGGAACATCCCTTAG -3'

[0105] 2. Statistics of resistance to spinosad and allele frequencies in different populations

[0106] 1. Resistance multiple determination

[0107] The median lethal concentration (LC) of spinosad was measured by the LAB population. 50) as the sensitive baseline, and the resistance level of other populations to the spinosad insecticide spinosad (provided by Hunan Nonghai Special Agrochemical Co., Ltd., pesticide registration certificate number: PD20160704) was determined. The specific operation was as follows: First, the test solution was prepared, and spinosad was diluted into 5 concentration gradients with 0.1% Triton. 10mL of the solution was measured in a glass cup for each concentration gradient, and the glass cup was fully rotated to form a uniform drug film on the wall of the cup, and then the solution was poured out. After the residual liquid in the glass cup was completely evaporated, a cotton ball soaked in 10% sucrose water was placed, and then different populations of citrus fruit flies were induced to enter, and the cup mouth was sealed with breathable gauze and tightened with a rubber band. Finally, it was placed upside down in an incubator with a temperature of 26-27°C, a humidity of 60%-70%, and a light cycle of L / D=14h / 10h. A 0.1% Triton without spinosad was set as a blank control, and 3 replicates were set for each concentration, with 20 adult Bactrocera dorsalis in each replicate. After the cup mouth was sealed with gauze, the death of Bactrocera dorsalis in each cup was observed for 24 hours. If there was no reaction within 30 seconds when the test adult was touched with a brush, it was judged that the insect was dead. The number of dead adults in each cup was recorded and the mortality rate was calculated. The mortality rate of the control group was less than 20% as valid measurement data. Finally, after the data were processed with Excel, the slope and standard error of the toxicity regression equation of the test agent, LC 50 The test results are compared with the toxicity test results of the relatively sensitive population in the room to obtain the resistance multiple, which is the LC of the test population. 50 / LC of laboratory population 50 .

[0108] The results are shown in Table 5. The LC of RES population 50 (158.686 mg / L) was significantly higher than the LC of LAB population 50 (10.289 mg / L), and was also significantly higher than that of BJ, HN and SD populations.

[0109] Table 5 The resistance multiples of five Bactrocera dorsalis populations to spinosad

[0110]

[0111]

[0112] 2. Allele frequency determination

[0113] The fruit flies from five fruit fly populations LAB, RES, BJ, HN and SD were collected, and 29 were randomly selected from LAB, 29 from RES, 15 from BJ, 18 from HN and 10 from SD. RNA of the fruit flies was extracted according to the method shown in Example 1, and the RNA was reverse transcribed to synthesize a cDNA template, and the cDNA template was used to perform PCR amplification of the target fragment of the Bdα9 gene, and the obtained PCR amplification product was sent to Beijing Qingke Company for direct sequencing using specific primers Bdα9-F / Bdα9-R.

[0114] The results are as follows Figure 1 As shown in the figure, according to the peak of the 1033rd base of the Bdα9 gene, it is determined whether the individual has a gene mutation. The statistical results are shown in Table 6. All 29 orange fruit flies in the LAB population were of AA genotype, and 16 of the 29 orange fruit flies in the RES population were of AG genotype and 3 were of GG genotype, indicating that the increase in the frequency of AG and GG genotypes led to a significant increase in the resistance of orange fruit flies to spinosad.

[0115] Table 6 The frequency of I345V genotypes in five Bactrocera dorsalis populations

[0116]

[0117] Note: AA represents the frequency of gene A in the homozygotes whose 1033rd position of SEQ ID No.2 of Bdα9 gene of Bactrocera dorsalis is A in the population; AG represents the frequency of gene A in the heterozygotes whose 1033rd position of SEQ ID No.2 of Bdα9 gene of Bactrocera dorsalis is G and A in the population; GG represents the frequency of gene A in the homozygotes whose 1033rd position of SEQ ID No.2 of Bactrocera dorsalis is G in the population.

[0118] Table 7 Phenotypes and resistance multiples of different genotypes of Bactrocera dorsalis

[0119]

[0120]

[0121] As shown in Table 7, the LC of Bactrocera dorsalis with genotype AA 50 It was significantly lower than that of Bactrocera dorsalis with genotype AA or genotype GG (P<0.05).

[0122] 3. Correlation analysis between the frequency of I345V genotypes and the multiplicity of spinosad resistance in five Bactrocera dorsalis populations

[0123] The resistance multiples of various populations to spinosad obtained from the above bioassay were correlated with the mutation frequency of the 1033 allele of the Bdα9 gene detected by the present invention.

[0124] The results are as follows Figure 2 As shown in Figure 2, the mutation frequency of the Bdα9 allele was found to be highly positively correlated with spinosad resistance (r 2 =0.912, df=42.61, P=0.004).

[0125] The molecular detection technology of the present invention can quickly determine the genotype of Bdα9 encoding amino acid 1033 in different geographical populations of Bactrocera dorsalis, and the above calculation formula can be used to obtain the mutation frequency of the Bdα9 resistance allele in the population. In addition, the data of this example verified through correlation analysis that the mutation frequency of the Bdα9I345V allele of Bactrocera dorsalis was significantly correlated with spinosad resistance.

[0126] The inventors' research has found for the first time that the I345V mutation of the α9 subunit of the nicotinic acetylcholine receptor of the fruit fly is associated with spinosad resistance, and different frequencies of allele mutations can be detected in multiple field populations in my country. Based on the above findings, the present invention has established a molecular detection method for the target of the resistance of the fruit fly to spinosad.

[0127] SEQ ID No.1

[0128] MKCLKFTSWSALSSFYLLLFSIVITAESKFYSASVARQSLITDLFVNYNALVKPTGEGQRIDVHTNLVLEHFDFRESDGSVHFVGLLNVLWQDPKLGWNPRDYNNL TRIPVRQKLIWVPDLEVYNNAPDKFLRMHHHGTVILAHTGMVLWSDNVDMNVFCSTNMNNWPHDKHECKLNLGSWTYDGFELDFKNYTNPNDSMSFEDKYMASMKYK VTDFSVERVATVYSCCAEPYIVMEYHLSFERRCTFVTVFRALGSTVILLSMLTLCFETSHHSKICLNGLNLIIITFVLLYFAQNVGKFTRTTPYIVKFFSCSYILVT FQQLLTVFSIFATHASYRGKLHPTISNLLRHSFVSYITPKRKMRLAQPETCDVNAQFEDVTLQEFSNLISADETPYEWMQLASFMESFAVIIFSIIYLILATVCFV.

[0129] SEQ ID No.2

[0130]

[0131] The above R is A or G.

[0132] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. Application, characterized in that, Application of a substance for detecting SNP sites in the genome of Bactrocera dorsalis in any of the following: C1) Identify or assist in identifying the resistance of Bactrocera dorsalis to spinosad; C2) preparing products for identifying or assisting in identifying the resistance of Bactrocera dorsalis to spinosad; C3) Screening or breeding varieties or strains of Bactrocera dorsalis for resistance to spinosad; C4) preparing products for screening or breeding varieties or strains of Bactrocera dorsalis for resistance to spinosad; C5) Screening or breeding varieties or strains of Bactrocera dorsalis susceptible to spinosad; C6) preparing products for screening or breeding species or strains of Bactrocera dorsalis susceptible to spinosad; The SNP site is the first nucleotide of the codon encoding the 345th amino acid of the α9 subunit of the nicotinic acetylcholine receptor in the genome of the oriental fruit fly, and the nucleotide type is A or G.

2. The use according to claim 1, characterized in that: The substance is any of the following: D1) the substance is a primer composition for amplifying a genomic DNA fragment of the citrus fruit fly including the SNP site; D2) the substance is a PCR reagent containing the primer combination described in D1); D3) The substance is a kit containing the primer composition described in D1) or the PCR reagent described in D2).

3. The use according to claim 2, characterized in that: The primer composition consists of the single-stranded DNA shown in SEQ ID No.3 and the single-stranded DNA shown in SEQ ID No.

4.

4. A method for identifying or assisting in identifying the resistance of Bactrocera dorsalis to spinosad, characterized in that: The method comprises detecting the genotype of a SNP site in the genome of the to-be-tested fruit fly, wherein the SNP site is the first nucleotide of the codon encoding the 345th amino acid of the alpha 9 subunit of the nicotinic acetylcholine receptor in the genome of the fruit fly, and the nucleotide type thereof is A or G.

5. The method according to claim 4, characterized in that The nucleotide sequence of the gene encoding the nicotinic acetylcholine receptor α9 subunit is SEQ ID No.2.

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