Method for identifying coconut moth
By using specific primer combinations in the AFLP amplification reaction, the problem of difficult to effectively identify and track coconut moths in the prior art is solved, and the precise traceability and prevention of coconut moths is achieved.
Patent Information
- Application Number
- CN202411856680.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-17
- Publication Date
- 2025-02-14
AI Technical Summary
The existing technology has not yet effectively utilized AFLP molecular marker technology for identification and tracking of coconut coconut moth, which has led to difficulties in traceability and prevention of coconut coconut moth.
By designing a specific primer combination for AFLP amplification reaction, the identification efficiency of coconut coconut moth is improved and the precise traceability of coconut coconut moth is achieved.
It improves the sensitivity of AFLP molecular markers, can effectively identify and trace the origin of coconut coconut moth, and provides a scientific basis for preventing and controlling the invasion of coconut coconut moth.
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Figure CN119433053A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of molecular markers and relates to a method for identifying coconut palm moths, and more specifically to a method for identifying the sources of coconut palm moths by utilizing AFLP molecular marker technology. Background Art
[0002] Coconut moth (Agonoxena sp.) belongs to the genus Agonoxena of the family Agonoxenidae.
[0003] AFLP molecular marker technology, namely amplified fragment length polymorphism, is a molecular detection and identification technology based on DNA level, which has the advantages of small amount of DNA required, high detection efficiency, high polymorphism, good reliability and high repeatability. Although AFLP molecular marker technology has been involved in the fields of zoology, botany, medicine, etc., there has been no research on its use in identifying and tracking coconut moths. Summary of the invention
[0004] The purpose of the present invention is to provide a method for identifying the coconut palm moth, and the efficiency of identifying the coconut palm moth is improved by using a specific primer combination, so the present invention can realize the accurate tracing of the coconut palm moth.
[0005] The present invention provides a primer combination for AFLP amplification reaction, the primer combination comprising: a pre-amplification reaction primer pair and a selective amplification primer pair;
[0006] The pre-amplification reaction primer pair includes: E00 and M00; the nucleotide sequence of E00 is shown in SEQ ID NO.5; the nucleotide sequence of M00 is shown in SEQ ID NO.6;
[0007] The selective amplification primer pair includes: a primer pair consisting of primer E and primer M;
[0008] The primer E includes: E42, E46, E83, E31, E32, E38, E41, E43, E47 or E62; the primer M includes: M55, M60, M66, M83, M85, M48, M51, M58, M61 or M70; the nucleotide sequence of E42 is shown in SEQ ID NO.19; the nucleotide sequence of M55 is shown in SEQ ID NO.20; the nucleotide sequence of E46 is shown in SEQ ID NO.25; the nucleotide sequence of M60 is shown in SEQ ID NO.26; the nucleotide sequence of E47 is shown in SEQ ID NO.27; the nucleotide sequence of M61 is shown in SEQ ID NO.28; the nucleotide sequence of E62 is shown in SEQ ID NO.37; the nucleotide sequence of M66 is shown in SEQ ID NO.38; the nucleotide sequence of E83 is shown in SEQ ID NO.47; the nucleotide sequence of M83 is shown in SEQ ID NO. NO.48; the nucleotide sequence of E31 is shown in SEQ ID NO.55; the nucleotide sequence of M85 is shown in SEQ ID NO.52; the nucleotide sequence of E32 is shown in SEQ ID NO.7; the nucleotide sequence of M48 is shown in SEQ ID NO.10; the nucleotide sequence of E38 is shown in SEQ ID NO.11; the nucleotide sequence of M51 is shown in SEQ ID NO.14; the nucleotide sequence of E41 is shown in SEQ ID NO.17; the nucleotide sequence of E43 is shown in SEQ ID NO.21; the nucleotide sequence of M58 is shown in SEQ ID NO.24; the nucleotide sequence of M61 is shown in SEQ ID NO.28; the nucleotide sequence of M70 is shown in SEQ ID NO.40.
[0009] Preferably, the selective amplification primer pair includes: a primer pair consisting of primer E42 and primer M55, a primer pair consisting of primer E46 and primer M60, a primer pair consisting of primer E47 and primer M61, a primer pair consisting of primer E62 and primer M66, a primer pair consisting of primer E83 and primer M83, a primer pair consisting of primer E31 and primer M85, a primer pair consisting of primer E32 and primer M48, a primer pair consisting of primer E38 and primer M51, a primer pair consisting of primer E41 and primer M55, a primer pair consisting of primer E43 and primer M58, a primer pair consisting of primer E46 and primer M61, or a primer pair consisting of primer E62 and primer M70.
[0010] The present invention provides the application of the primer combination described in the above technical scheme in identifying the coconut palm moth and / or tracing the coconut palm moth.
[0011] The invention provides a method for identifying coconut palm moth, comprising the following steps:
[0012] DNA is extracted from the sample to be tested to obtain genomic DNA; AFLP amplification is performed on the genomic DNA to obtain AFLP amplification products; the AFLP amplification sequentially includes enzyme cutting-ligation reaction, pre-amplification reaction using the primer combination described in the above technical solution, and selective amplification reaction; genetic diversity analysis and / or kinship analysis are performed on the AFLP amplification products to identify the source of the coconut moth.
[0013] Preferably, the enzyme digestion-ligation reaction comprises: double digestion of genomic DNA with EcoRI and MseI, and adding EcoRI adapter and MseI adapter to the double digestion product for ligation;
[0014] The EcoRI linker is composed of EcoRI1 and EcoRI2; the nucleotide sequence of EcoRI1 is shown in SEQ ID NO.1; the nucleotide sequence of EcoRI2 is shown in SEQ ID NO.3;
[0015] The MseⅠ linker is composed of MseⅠ1 and MseⅠ2; the nucleotide sequence of MseⅠ1 is shown in SEQ ID NO.2; the nucleotide sequence of MseⅠ2 is shown in SEQ ID NO.4.
[0016] Preferably, the pre-amplification reaction uses the enzyme cleavage-ligation product as template DNA;
[0017] The system of the pre-amplification reaction includes: 2×PCR Mix 10 μL, 20 μM E00 1 μL, 20 μM E00 1 μL, template DNA 4 μL, and ddH2O added to 20 μL;
[0018] The procedure of the pre-amplification reaction is: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s; annealing at 50°C for 30 s, extension at 72°C for 1 min, and 30 cycles.
[0019] Preferably, the selective amplification uses the product of the pre-amplification reaction as template DNA;
[0020] The selective amplification reaction system includes: 2×PCR Mix 10 μL, 20 μM primer E 1 μL, 20 μM primer M 1 μL, template DNA 5 μL, and ddH2O added to 20 μL;
[0021] The selective amplification reaction procedure is: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 35 s, annealing at 65°C for 35 s, extension at 72°C for 1 min, 12 cycles; denaturation at 94°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 1 min, 23 cycles.
[0022] Beneficial effects:
[0023] The invention provides a primer combination for AFLP amplification reaction, which comprises a pre-amplification reaction primer and a selective amplification primer. The sensitivity of AFLP molecular markers is increased by using specific primers to carry out AFLP amplification reaction.
[0024] Based on the above technical advantages, the present invention also provides the use of the primer combination in identifying coconut moth and / or tracing the coconut moth. By sequentially performing DNA extraction, AFLP amplification and capillary electrophoresis analysis on the sample to be tested, the source of the foreign coconut moth can be accurately traced, laying a scientific foundation for preventing and controlling the invasion of coconut moth. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.
[0026] Figure 1 The electrophoresis diagram of the sample DNA provided by the present invention;
[0027] Figure 2 The electrophoresis diagram of DNA after double enzyme digestion provided by the present invention;
[0028] Figure 3 3730xl test result diagram provided by the present invention;
[0029] Figure 4 The best primer result diagram provided by the present invention;
[0030] Figure 5 A readable summary diagram of the fragments provided by the present invention;
[0031] Figure 6 This is a cluster analysis diagram based on the genetic distance UPGMA method provided by the present invention. DETAILED DESCRIPTION
[0032] In the present invention, unless otherwise specified, the reagents and methods used are conventional.
[0033] In the present invention, the coconut moth comes from Pingtan, Fujian; there is no requirement for the sample size of the coconut moth, subject to meeting the statistical requirements.
[0034] The primer sequences used in the experimental process of the present invention are shown in Table 1.
[0035] Table 1 Primer sequence information
[0036]
[0037]
[0038] Example 1
[0039] A method for identifying coconut palm moth, comprising the following steps:
[0040] 1. Sample preparation: 30 test insects were collected from the coconut moth occurrence area in Pingtan, Fujian, and DNA was extracted from the 30 test insects using the CTAB method. The extracted DNA samples were numbered 1-30 in sequence; the extraction results of the 30 DNA samples were detected using agarose with a mass concentration of 1.5%, as shown in the figure. Figure 1 (exist Figure 1 In the figure, the first 16 strips in the first row represent the strips of samples numbered 1-16, and the last one is MK; the first 14 strips in the second row represent the strips of samples numbered 17-30, and the last one is MK; MK represents DL2000)
[0041] Depend on Figure 1 It can be seen that DNA samples were successfully extracted from the test insects numbered 1-30.
[0042] 2. AFLP test
[0043] (1) Enzyme digestion-ligation: The 30 DNA samples extracted above were double-digested with EcoRⅠ and MseⅠ, and the enzyme digestion and ligation reactions were carried out simultaneously. The enzyme digestion-ligation system was calculated as 20 μL, specifically including: 2 μL of 10×AFLP digest-ligation Buffer, 1.8 μL of AFLP digest-ligation Enzyme Mix; 1 μL of 10 μM EcoR I Adaptor, 1 μL of 10 μM Mse I Adaptor, 5 μL of DNA sample, and supplemented with ddH2O to 20 μL;
[0044] Among them, the sequence of the EcoRⅠ linker consists of EcoRⅠ1 and EcoRⅠ2, the sequence of EcoRⅠ1 is shown in SEQ ID NO.1, and the sequence of EcoRⅠ2 is shown in SEQ ID NO.3; the sequence of the MseⅠ linker consists of MseⅠ1 and MseⅠ2, the sequence of MseⅠ1 is shown in SEQ ID NO.2, and the sequence of MseⅠ2 is shown in SEQ ID NO.4.
[0045] The conditions for the enzyme digestion-ligation reaction were: 25°C for 5 h. After the reaction, the enzyme digestion-ligation template DNA was obtained. The electrophoresis of each enzyme digestion-ligation template DNA product was performed using agarose with a mass concentration of 1.5%. The results are shown in Figure 2 .
[0046] Depend on Figure 2 It can be seen that there are no obvious stripes after the DNA sample is double digested with EcoRⅠ and MseⅠ.
[0047] (2) Pre-amplification: taking the above-mentioned enzyme-cut-ligated template DNA for pre-amplification, and then obtaining DNA pre-amplification products respectively;
[0048] The pre-amplification reaction system is 20 μL, specifically including: 10 μL of 2×PCR Mix, 1 μL of 20 μM E00, 1 μL of 20 μM E00, 4 μL of enzyme-cut-ligated template DNA, and ddH2O to 20 μL.
[0049] The pre-amplification reaction program was as follows: 94°C pre-denaturation for 3 min; 94°C denaturation for 30 s; 50°C annealing for 30 s, 72°C extension for 1 min, 30 cycles;
[0050] (3) Selective amplification: Take the above DNA pre-amplification products and dilute them 20 times as template DNA for selective amplification to obtain selective PCR amplification products;
[0051] The selective amplification reaction system is 20 μL, specifically including: 10 μL of 2×PCR Mix, 1 μL of 20 μM primer E, 1 μL of 20 μM primer M, 5 μL of template DNA, and ddH2O added to 20 μL.
[0052] The selective amplification reaction program was as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 35 s, annealing at 65°C for 35 s (0.7°C reduction per cycle), extension at 72°C for 1 min, 12 cycles; denaturation at 94°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 1 min, 23 cycles;
[0053] Among them, primer E and primer M are the selective amplification primers in Table 1, such as E32, M47, etc., and any primer E and primer M are selected as a pair of primers (for example, E84 and M32 are a pair of primers, E32 and M47 are a pair of primers, etc.), which can be directly applied to the selective amplification reaction system.
[0054] 3. Capillary electrophoresis method
[0055] After formamide and molecular weight internal standard were mixed at a volume ratio of 10:1, 9uL was taken and added to the sample plate, and then 1uL of the selective PCR amplification product diluted 10 times was added. Then, capillary electrophoresis was performed using a 3730XL sequencer. The reduction electrophoresis diagram of samples 1-30 after amplification with primers E81 and M55 is shown in Figure 3 .
[0056] In addition, 12 pairs of primers with good amplification were screened out through AFLP test on samples No. 1 and 2 for batch test. For details, see the primer screening results. Figure 4 .
[0057] Depend on Figure 4 It can be seen that the primer combinations E42 and M55, E46 and M60, E47 and M61, E62 and M66, E83 and M83, E31 and M85, E32 and M48, E38 and M51, E41 and M55, E43 and M58, E46 and M61, and E62 and M70 have better amplification results.
[0058] 4. Data Analysis
[0059] GeneMarker2.2 software was used to analyze the raw data obtained by the sequencer of the amplified products of 30 samples and 12 primer combinations (E42 and M55, E46 and M60, E47 and M61, E62 and M66, E83 and M83, E31 and M85, E32 and M48, E38 and M51, E41 and M55, E43 and M58, E46 and M61, E62 and M70). The position of the molecular weight internal standard in each lane was compared with the position of the peak of each sample to obtain the fragment size. Then it was converted into a 0, 1 data matrix according to the absence and presence of bands. The individual similarity coefficient was calculated using the NTSYSpc 2.11 software package and the UPGMA method was used for cluster analysis. Popgene and NTSYS software were used to analyze the genetic diversity of the samples.
[0060] The fragments read using primers E31 and M85 as primer pair are summarized in Figure 5 shown.
[0061] The results of genetic diversity analysis are as follows:
[0062] Genetic diversity data analysis was performed on samples numbered 1-30, and the results are shown in Table 2 (in Table 2, na represents the number of observed alleles; ne represents the number of effective alleles; I represents the Shannon information index; P represents the percentage of polymorphic sites; and h represents the Shannon diversity index).
[0063] Table 2 Genetic diversity data analysis
[0064]
[0065]
[0066] The genetic parameters generated by AFLP analysis of samples numbered 1-30 are shown in Table 3 (in Table 3, PIC stands for polymorphic information content; EMR stands for effective multiple ratio; RP stands for resolution power; MI stands for marker index).
[0067] Table 3 Genetic parameters generated by AFLP analysis
[0068]
[0069] The results of kinship analysis are as follows:
[0070] The genetic similarity coefficients between samples numbered 1-30 were summarized, and the results are shown in Tables 4 and 5 (Table 5 is a supplement to the data in Table 4).
[0071] Table 4 Genetic similarity coefficients between different samples
[0072]
[0073] Table 5 Genetic similarity coefficients between different samples (supplement)
[0074]
[0075] The genetic distances between samples numbered 1-30 are summarized, and the results are shown in Tables 6 and 7 (Table 7 is a supplement to the data in Table 6). The cluster analysis based on the genetic distance UPGMA method is shown in Figure 6 .
[0076] Table 6 Genetic distance between samples
[0077]
[0078] Table 7 Genetic distance between samples (supplement)
[0079]
[0080] It can be seen from Tables 6 and 7 that after adopting the technical solution of the present invention, the genetic distances of different samples can be statistically analyzed. When the genetic distance parameter between two samples is small, it means that the genetic relationship between the two samples is relatively close, and the introduced coconut moth can be identified.
[0081] In summary, it can be seen that the technical solution provided by the present invention can not only determine and identify the coconut palm moth, but also track and trace it.
[0082] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.
Claims
1. A primer combination for AFLP amplification reaction, characterized in that: The primer combination includes: a pre-amplification reaction primer pair and a selective amplification primer pair; The pre-amplification reaction primer pair includes: E00 and M00; The nucleotide sequence of E00 is shown in SEQ ID NO.5; the nucleotide sequence of M00 is shown in SEQ ID NO.6; The selective amplification primer pair includes: a primer pair consisting of primer E and primer M; The primer E includes: E42, E46, E83, E31, E32, E38, E41, E43, E47 or E62; The primer M includes: M55, M60, M66, M83, M85, M48, M51, M58, M61 or M70; The nucleotide sequence of E42 is shown in SEQ ID NO.19; The nucleotide sequence of M55 is shown in SEQ ID NO.20; The nucleotide sequence of E46 is shown in SEQ ID NO.25; The nucleotide sequence of M60 is shown in SEQ ID NO.26; The nucleotide sequence of E47 is shown in SEQ ID NO.27; The nucleotide sequence of M61 is shown in SEQ ID NO.28; The nucleotide sequence of E62 is shown in SEQ ID NO.37; The nucleotide sequence of M66 is shown in SEQ ID NO.38; The nucleotide sequence of E83 is shown in SEQ ID NO.47; The nucleotide sequence of M83 is shown in SEQ ID NO.48; The nucleotide sequence of E31 is shown in SEQ ID NO.55; The nucleotide sequence of M85 is shown in SEQ ID NO.52; The nucleotide sequence of E32 is shown in SEQ ID NO.7; The nucleotide sequence of M48 is shown in SEQ ID NO.10; The nucleotide sequence of E38 is shown in SEQ ID NO.11; The nucleotide sequence of M51 is shown in SEQ ID NO.14; The nucleotide sequence of E41 is shown in SEQ ID NO.17; The nucleotide sequence of E43 is shown in SEQ ID NO.21; The nucleotide sequence of M58 is shown in SEQ ID NO.24; The nucleotide sequence of M61 is shown in SEQ ID NO.28; The nucleotide sequence of M70 is shown in SEQ ID NO.
40.
2. The primer combination according to claim 1, characterized in that The selective amplification primer pairs include: a primer pair consisting of primer E42 and primer M55, a primer pair consisting of primer E46 and primer M60, a primer pair consisting of primer E47 and primer M61, a primer pair consisting of primer E62 and primer M66, a primer pair consisting of primer E83 and primer M83, a primer pair consisting of primer E31 and primer M85, a primer pair consisting of primer E32 and primer M48, a primer pair consisting of primer E38 and primer M51, a primer pair consisting of primer E41 and primer M55, a primer pair consisting of primer E43 and primer M58, a primer pair consisting of primer E46 and primer M61, or a primer pair consisting of primer E62 and primer M70.
3. Use of the primer combination described in claim 1 or 2 in identifying the coconut palm moth and / or tracing the coconut palm moth.
4. A method for identifying the coconut palm moth, characterized in that: The steps include: Extract DNA from the sample to be tested to obtain genomic DNA; Performing AFLP amplification on the genomic DNA to obtain an AFLP amplification product; The AFLP amplification sequentially comprises an enzyme cutting-ligation reaction, a pre-amplification reaction using the primer combination of claim 1 or 2, and a selective amplification reaction; The AFLP amplification products are subjected to genetic diversity analysis and / or phylogenetic analysis to identify the source of the coconut moth.
5. The method according to claim 4, characterized in that The enzyme digestion-ligation reaction comprises: performing double digestion of genomic DNA with EcoRI and MseI, and adding EcoRI adapter and MseI adapter to the double digestion product for ligation; The EcoRI linker is composed of EcoRI1 and EcoRI2; the nucleotide sequence of EcoRI1 is shown in SEQ ID NO.1; the nucleotide sequence of EcoRI2 is shown in SEQ ID NO.3; The MseⅠ linker is composed of MseⅠ1 and MseⅠ2; the nucleotide sequence of MseⅠ1 is shown in SEQ ID NO.2; the nucleotide sequence of MseⅠ2 is shown in SEQ ID NO.
4.
6. The method according to claim 4, characterized in that The pre-amplification reaction uses the enzyme-cut-ligation product as template DNA; The system of the pre-amplification reaction includes: 10 μL of 2×PCR Mix, 1 μL of 20 μM E00, 1 μL of 20 μM E00, 4 μL of enzyme-cut-ligated template DNA, and ddH2O added to 20 μL; The procedure of the pre-amplification reaction is: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s; annealing at 50°C for 30 s, extension at 72°C for 1 min, and 30 cycles.
7. The method according to claim 4, characterized in that The selective amplification uses the product of the pre-amplification reaction as template DNA; The selective amplification reaction system includes: 2×PCR Mix 10 μL, 20 μM Primer E 1 μL, 20 μM Primer M 1 μL, template DNA 5 μL, and ddH2O added to 20 μL; The selective amplification reaction procedure is: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 35 s, annealing at 65°C for 35 s, extension at 72°C for 1 min, 12 cycles; denaturation at 94°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 1 min, 23 cycles.