A molecular identification reagent for bothibos and end of the large chrysanthemum thrips and its use

Abstract

The application belongs to the technical field of biological identification, and provides a molecular identification reagent for Olotanussus and Endothrips spp., which comprises a pair of primers for detecting COI gene sequence of the thrips: olotCOI-F: 5-ATATTAGGAGCTCCAGATATAGC-3; olotCOI-R: 5-GGGTGACCAAAAAATCAAAATAA-3; and further comprises a BclI enzyme cutting site designed in the sequence of the Olotanussus. According to the sequence difference of the mitochondrial cytochrome C oxidase subunit I gene between the thrips species, a species-specific variation site of the Olotanussus and the Endothrips spp. is found, a molecular identification system for the thrips species is established, and finally the rapid, accurate and efficient identification of the thrips and the closely related species is realized.

Description

Technical Field

[0001] This invention belongs to the field of biological identification technology, and in particular relates to a molecular identification reagent and method of using *Thrips horn-toothed* and *Thrips endoglossus*. Background Technology

[0002] Herbivorous thrips are important agricultural pests. Thrips are generally 0.5–2.0 mm in length, and most are indistinguishable to the naked eye, often causing species confusion. Currently, thrips identification is mainly based on morphological characteristics, such as body color, number of antennal segments, and the quantity and morphology of bristles. *Odontothripsloti* and *Megalurothrips distalis* both belong to the subfamily Thripsinae and are morphologically similar. They are brown with 8 antennal segments, segment I having a pair of dorsal apical bristles, dark yellow foreleg tibiae, and dark gray forewings with the basal portion (about 1 / 7) being colorless and transparent. The abdominal tergites VIII have posterior margin combs on both sides, and the posterior margin bristles in sternite VII are located anterior to the posterior margin. The difference lies in that *Megalurothrips distalis* has antennal segments matching its body color and 3 bristles on the forewing anterior vein; *Odontothripsloti* has yellow antennal segment III and 2 bristles on the forewing anterior vein. The above characteristics can only be distinguished by experienced workers using a microscope. Slight carelessness can lead to confusion, increasing the difficulty of field collection and differentiation.

[0003] The mitochondrial cytochrome C oxidase subunit I gene (COI gene) is a conserved gene widely present in animals. Due to its highly conserved sequence and sufficient interspecific variation, it is widely used in species identification and evolutionary analysis. Molecular identification methods have been applied to the identification of Thysanoptera insects. However, methods such as sequencing and genetic distance determination are time-consuming, expensive, and technically challenging. Currently, there is no rapid and convenient molecular marker method for identifying thrips, which are easily contaminated in the field. Therefore, how to quickly and accurately identify morphologically similar, concomitant thrips populations has become an urgent problem to be solved. Summary of the Invention

[0004] The purpose of this invention is to provide a molecular identification reagent and method for using *Thrips horn-toothed* and *Thrips telsonii*, in order to solve the problems mentioned in the background art.

[0005] This invention is implemented as follows: a molecular identification reagent for *Thrips horn-toothed* and *Thrips endornifax* includes a pair of primers designed to detect the COⅠ gene sequence of thrips:

[0006] olotCOⅠ-F:5-ATATTAGGAGCTCCAGATATAGC-3

[0007] olotCOⅠ-R: 5-GGGTGACCAAAAAATCAAAATAA-3;

[0008] It also includes the BclⅠ restriction site designed in the horn-toothed thrips sequence.

[0009] In a further technical solution, the molecular identification reagent further includes a homogenate prepared by a mixture of solution A: solution B: solution C = 8:1:1, wherein solution A comprises 0.05 mol / L Tris base, 0.1 mol / L NaCl, and 0.1 mol / L EDTA, the pH of solution A is 7.6, solution B is 5% SDS, and solution C is 2 mg / mL proteinase K.

[0010] Another objective of this invention is to provide a method for using a molecular identification reagent for *Thrips hornbillii* and *Thrips endoglossus*, comprising the following steps:

[0011] Step 1: Extraction of total DNA from thrips:

[0012] Take a single thrips and place it in a 1.5 ml centrifuge tube. Add 200 μl of freshly prepared molecular identification reagent and grind thoroughly. Add another 400 μl of molecular identification reagent and mix well to make a final volume of 600 μl. Heat in a 45°C water bath for 1 h until the tissue is completely digested. Add 600 μl of Tris-saturated phenol and gently shake for 10 min to mix. Centrifuge at 7000 r / min at room temperature for 10 min. Transfer the supernatant to a new centrifuge tube. Add 550 μl of chloroform and isoamyl alcohol (24:1) and mix. Mix the DNA precipitate by gently shaking for 10 minutes, centrifuge at 8000 rpm for 10 minutes at room temperature, and transfer the supernatant to a new centrifuge tube. Add 1 ml of pre-chilled anhydrous ethanol solution, centrifuge at 12000 rpm for 10 minutes at room temperature, and discard the supernatant. Add 500 μl of pre-chilled 70% anhydrous ethanol solution, gently tap the bottom of the centrifuge tube to suspend the DNA precipitate, centrifuge at 12000 rpm for 10 minutes at room temperature, and discard the supernatant. Allow to air dry, dissolve in an appropriate amount of ddH2O, and store at -20℃ for later use.

[0013] Step 2, PCR amplification:

[0014] Primers were designed based on the COⅠ gene sequence of the genus *Thrips* (accession number MG521378.1) using DNA as a template.

[0015] olotCOⅠ-F:5-ATATTAGGAGCTCCAGATATAGC-3

[0016] olotCOⅠ-R: 5-GGGTGACCAAAAAATCAAAATAA-3;

[0017] The reaction system is constructed as follows:

[0018]

[0019] Electrophoresis was performed on a 1% agarose gel at 120V for 20 minutes. The electrophoretic bands were observed using a gel imaging system, and the PCR products were sequenced.

[0020] Step 3, Enzyme digestion analysis:

[0021] Sequencing results showed that the *Thrips hornbillae* sequence was 427 bp long, and the *Thrips telsonii* sequence was 431 bp long. A suitable Bcl-I restriction enzyme site was designed in the *Thrips hornbillae* sequence using DNAMAN V6. If the obtained enzyme digestion products contained bands of 167 bp and 260 bp, the sample was identified as *Thrips hornbillae*; otherwise, it was identified as *Thrips telsonii*.

[0022] Incubate at 37°C for 30 minutes. Use a 1% agarose gel for electrophoresis at 120V for 20 minutes, and observe the electrophoretic bands using a gel imaging system.

[0023] In a further technical solution, the PCR reaction procedure in step 2 is as follows:

[0024]

[0025] A further technical solution, in step 3, involves constructing the following enzyme digestion system:

[0026]

[0027] This invention provides a molecular identification reagent and method for *Thrips horn-toothed* and *Thrips endornifi*. Based on the differences in mitochondrial cytochrome C oxidase subunit I gene sequences among thrips genera and species, it identifies species-specific variation sites in different thrips species, establishes a molecular identification system for these thrips species, and ultimately achieves rapid, accurate, and efficient identification of thrips and their closely related species. It also possesses the following beneficial effects:

[0028] (1) The testing time is short, requiring only 1 day from sample collection to completion of testing;

[0029] (2) It is easy to operate and highly accurate, and even non-insect classification professionals can complete the test;

[0030] (3) Low cost, detection can be completed with a single thrips sample. Attached Figure Description

[0031] Figure 1 This is an electrophoresis image of the bands after PCR amplification;

[0032] Figure 2 This is an electrophoretic banding image after enzyme digestion. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0034] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0035] One embodiment of the present invention provides a molecular identification reagent for *Thrips hornbillii* and *Thrips endornifi*, the molecular identification reagent comprising a pair of primers designed to detect the COⅠ gene sequence of thrips:

[0036] olotCOⅠ-F:5-ATATTAGGAGCTCCAGATATAGC-3

[0037] olotCOⅠ-R: 5-GGGTGACCAAAAAATCAAAATAA-3;

[0038] It also includes the BclⅠ restriction site designed in the *Thrips hornbillii* sequence. The molecular identification reagent is prepared from a homogenate of solution A: solution B: solution C = 8:1:1. Solution A comprises 0.05 mol / L Trisbase, 0.1 mol / L NaCl, and 0.1 mol / L EDTA, with a pH of 7.6. Solution B contains 5% SDS, and solution C contains 2 mg / mL proteinase K.

[0039] An embodiment of the present invention provides a method for using a molecular identification reagent for *Thrips hornbillii* and *Thrips telsonii*, comprising the following steps:

[0040] Step 1: Extraction of total DNA from thrips:

[0041] Take a single thrips and place it in a 1.5 ml centrifuge tube. Add 200 μl of freshly prepared molecular identification reagent and grind thoroughly. Add another 400 μl of molecular identification reagent and mix well to make a final volume of 600 μl. Heat in a 45°C water bath for 1 h until the tissue is completely digested. Add 600 μl of Tris-saturated phenol and gently shake for 10 min to mix. Centrifuge at 7000 r / min at room temperature for 10 min. Transfer the supernatant to a new centrifuge tube. Add 550 μl of chloroform and isoamyl alcohol (24:1) and mix. Mix the DNA precipitate by gently shaking for 10 minutes, centrifuge at 8000 rpm for 10 minutes at room temperature, and transfer the supernatant to a new centrifuge tube. Add 1 ml of pre-chilled anhydrous ethanol solution, centrifuge at 12000 rpm for 10 minutes at room temperature, and discard the supernatant. Add 500 μl of pre-chilled 70% anhydrous ethanol solution, gently tap the bottom of the centrifuge tube to suspend the DNA precipitate, centrifuge at 12000 rpm for 10 minutes at room temperature, and discard the supernatant. Allow to air dry, dissolve in an appropriate amount of ddH2O, and store at -20℃ for later use.

[0042] Step 2, PCR amplification:

[0043] Primers were designed using DNA as a template based on the COⅠ gene sequence of the genus *Thrips* (accession number MG521378.1):

[0044] olotCOⅠ-F:5-ATATTAGGAGCTCCAGATATAGC-3

[0045] olotCOⅠ-R: 5-GGGTGACCAAAAAATCAAAATAA-3;

[0046] The reaction system is constructed as follows:

[0047]

[0048] PCR reaction procedure:

[0049]

[0050] Electrophoresis was performed on a 1% agarose gel at 120V for 20 minutes. The electrophoretic bands were observed using a gel imaging system. The PCR products were sequenced, and the sequencing results are shown in the sequence listing.

[0051] Step 3, Enzyme digestion analysis:

[0052] Sequencing results showed that the *Thrips hornbillii* sequence was 427 bp long, and the *Thrips teliophora* sequence was 431 bp long. A suitable Bcl-I restriction enzyme site was designed in the *Thrips hornbillii* sequence using DNAMAN V6. If the resulting digestion product contained bands of 167 bp and 260 bp, the sample was identified as *Thrips hornbillii*; otherwise, it was identified as *Thrips teliophora*. The enzyme digestion system was constructed as follows:

[0053]

[0054] Incubate at 37°C for 30 minutes. Use a 1% agarose gel for electrophoresis at 120V for 20 minutes, and observe the electrophoretic bands using a gel imaging system.

[0055] Experimental results:

[0056] After PCR amplification, such as Figure 1 As shown, it can be seen that 1 is the DL2000 DNAMaker, with band lengths of 2000bp, 1000bp, 750bp, 500bp, 250bp, and 100bp from top to bottom; 2 is the PCR product of *Thrips hornbillii*; and 3 is the PCR product of *Thrips telsonii*. The size of the two thrips PCR products is consistent with the sequencing results.

[0057] After enzymatic digestion, such as Figure 2 As shown, 1 represents DL2000 DNAMaker, with bands ranging from 2000bp, 1000bp, 750bp, 500bp, 250bp, and 100bp in length from top to bottom; 2 represents the enzyme digestion product of *Thrips horn-toothed*; and 3 represents the enzyme digestion product of *Thrips teliophora*. Thrips 2 was digested by Bcl-I into two bands of 167bp and 260bp, while thrips 3 was not digested, indicating that thrips 2 is *Thrips horn-toothed* and thrips teliophora is *Thrips teliophora*.

[0058] The PCR products of the two types of thrips were sent for sequencing, and the results are shown in the sequence listing. When compared with GenBank, sequence 1 is the partial COI sequence of *Thrips telsonii*, and sequence 2 is the partial COI sequence of *Thrips hornbillii*. This detection result is consistent with the detection result of the present invention, proving that the method of the present invention is correct.

[0059] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A molecular identification reagent for Aphis craccivora and Aphis spiraecola, characterized by comprising a polynucleotide having a nucleotide sequence represented by SEQ ID NO: 1 or 2. The primers for detecting the sequence of the COI gene of the thrips include: olot COI-F: 5-ATATTAGGAGCTCCAGATATAGC-3 olot COI-R: 5-GGGTGACCAAAAAATCAAAATAA-3; The primers are used to amplify the fragment including a specific variation site in the COI gene of the Odontothrips loti; The enzyme cutting site BclI which can recognize the variation site and is used to distinguish the Odontothrips loti from the Thrips parellus is also included; When the PCR product is cut by the BclI, electrophoresis is performed in the 1% agarose gel, if two bands of 167bp and 260bp appear in the electrophoretogram, it is determined that the sample is the Odontothrips loti, otherwise it is the Thrips parellus; The molecular identification reagent further includes the homogenate prepared by mixing A liquid, B liquid and C liquid in the ratio of 8:1:1, the A liquid includes 0.05mol / L Tris base, 0.1mol / L NaCl and 0.1mol / L EDTA, the pH of the A liquid is 7.6, the B liquid is 5% SDS and the C liquid is 2mg / mL proteinase K.

2. A method of using a molecular identification reagent for A. cowpea and A. terminalis as claimed in claim 1, characterized in that, The method includes the following steps: Step 1, extraction of the total DNA of the thrips: Step 2, PCR amplification: The primers are designed according to the COI gene sequence of the Odontothrips loti with the sequence number of MG521378.1: olot COI-F: 5-ATATTAGGAGCTCCAGATATAGC-3 olot COI-R: 5-GGGTGACCAAAAAATCAAAATAA-3; The PCR product is sequenced by using the 1% agarose gel, electrophoresis for 20min under the voltage of 120V and observing the electrophoretic bands in the gel imaging system; Step 3, enzyme cutting analysis: The enzyme cutting site BclI is designed in the sequence of the Odontothrips loti by using the DNA MAN V6; The 1% agarose gel is used for electrophoresis for 20min under the voltage of 120V, and the electrophoretic bands are observed in the gel imaging system after 37℃ water bath for 30min.

3. The method of using the molecular identification reagent of the Aphis craccivora and the Aphis gossypii according to claim 2, characterized in that, The specific steps of the step 1 are as follows: Respectively take single head chrysanthemum thrips in 1.5ml centrifugal tube, add the molecular identification reagent 200ul prepared, grind thoroughly, add 400ul of molecular identification reagent again, mix well, make the final volume 600ul; 45℃ water bath heating 1h to complete the tissue digestion, add 600ul of Tris saturated phenol, shake 10min, 7000r / min room temperature centrifugation 10min, take supernatant to new centrifugal tube; Add 550ul of mixed solution, shake 10min, 8000r / min room temperature centrifugation 10min, take supernatant to new centrifugal tube; Add 1ml pre-cooled anhydrous ethanol solution, 12000r / min room temperature centrifugation 10min, discard supernatant; Add 500ul pre-cooled 70% anhydrous ethanol solution, light bounce centrifugal tube bottom makes DNA precipitate suspension, 12000r / min room temperature centrifugation 10min, discard supernatant; Natural drying, add appropriate amount of ddH2O dissolution, save in-20℃ for standby.

4. The method of claim 3, wherein the method comprises the steps of: a) obtaining a sample from a plant suspected of being infested with A. maidiradicis or A. maidireducens; b) contacting the sample with the molecular identification reagent; and c) determining the presence or absence of a positive reaction between the sample and the molecular identification reagent. In the step 1, chloroform: isoamyl alcohol = 24:

1.

5. The method of using the molecular identification reagent for *Thrips horn-toothed* and *Thrips terminalis* according to claim 2, characterized in that... In the step 2, the PCR construction reaction system is as follows:

6. The method of using the molecular identification reagent of the Aphis craccivora and the Aphis gossypii according to claim 2, characterized in that, In the step 2, the PCR reaction program is as follows:

7. The method of claim 2, wherein the method comprises the steps of: a) obtaining a sample from a plant suspected of being infested with the thrips; b) contacting the sample with the molecular identification reagent; and c) determining the presence or absence of the thrips in the sample based on the color change of the molecular identification reagent. In the step 3, the constructed enzyme cutting system is as follows:

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