Spodoptera frugiperda SNP (Single Nucleotide Polymorphism) molecular marker combination, chip and application thereof
By using liquid-phase chip and liquid-phase probe capture sequencing genotyping technology for fall armyworm SNPs, the problems of low throughput and high cost in fall armyworm SNP detection have been solved. This technology enables efficient and accurate detection of fall armyworm biotypes and resistance gene frequencies, supporting the scientific control of fall armyworm.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- AGRICULTURAL GENOMICS INSTITUTE AT SHENZHEN CHINESE ACADEMY OF AGRICULTURAL SCIENCES (SHENZHEN BRANCH GUANGDONG LABORATORY FOR LINGNAN MODERN AGRICULTURE)
- Filing Date
- 2026-01-09
- Publication Date
- 2026-04-10
AI Technical Summary
Existing SNP detection methods for fall armyworm suffer from low throughput, high cost, and cumbersome operation, making it difficult to achieve efficient and accurate detection of a large number of samples and numerous SNP sites. Furthermore, there is a lack of effective means to detect the mutation frequency of resistance genes in fall armyworm.
We developed a molecular marker combinatorial system for SNPs in the fall armyworm, including 27,006 SNPs. We designed a liquid-phase chip for the fall armyworm SNPs and used liquid-phase probe capture sequencing genotyping technology, combined with whole-genome resequencing and population structure analysis, to screen SNP sites linked to biotype and resistance-related genes in the fall armyworm, achieving efficient and accurate detection.
It enables rapid and accurate detection of fall armyworm biotypes and resistance gene frequencies, reduces detection costs, increases detection throughput, and can simultaneously cover nearly a thousand materials, supporting the analysis of resistance evolution patterns and resistance management.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology, specifically relating to a combination of SNP molecular markers for the fall armyworm, a chip, and their applications. Background Technology
[0002] fall armyworm Spodoptera frugiperda The fall armyworm (JE Smith), also known as the fall armyworm, belongs to the family Noctuidae in the order Lepidoptera. Native to tropical and subtropical regions of the Americas, it possesses an extremely strong migratory ability. Since its invasion of West Africa in 2016, it has spread to more than 80 countries and regions in the Eastern Hemisphere, including Australia, New Zealand, and parts of Europe, becoming one of the world's most significant invasive species and drawing widespread international attention. In its native habitat, the fall armyworm exhibits different biotypes ("maize type" and "rice type") depending on the host plant. Furthermore, field-resistant populations of the fall armyworm to chemical pesticides and Bt toxin have been detected in its native habitat, demonstrating a strong ability to evolve resistance, posing a serious challenge to effective control.
[0003] Currently, control methods for fall armyworm mainly include biological control, chemical control, and ecological regulation. Although chemical pesticides have advantages such as speed and efficiency in pest control, the long-term selective action of different insecticides has led to mutations in the resistance genes of fall armyworm, potentially further increasing its resistance level. While fall armyworm is currently relatively sensitive to Bt toxins, the evolution of resistant populations in its native habitat indicates the potential for resistance mutations, posing a threat to the future commercialization of Bt crops. Therefore, preventative testing for resistance-related variations (gene mutations) and analysis of their mutation frequency and patterns will help establish resistance management and integrated control for fall armyworm.
[0004] Single nucleotide polymorphisms (SNPs) are DNA sequence polymorphisms caused by single-base mutations at the genomic level. They are characterized by their wide distribution, high stability, and ease of automated analysis, making them ideal tools for studying genetic variation, molecular breeding, and germplasm resource assessment. SNP molecular markers theoretically offer broad prospects for research on the fall armyworm, especially in areas such as biotype identification based on SNP variation, population structure analysis, and monitoring of drug resistance mutations. However, in practical applications, traditional SNP detection methods, such as PCR-RFLP and Sanger sequencing, suffer from drawbacks such as low throughput, high cost, and cumbersome operation, making it difficult to achieve efficient and accurate detection of large numbers of samples and numerous SNP loci. Liquid-phase chip technology effectively avoids these problems, offering advantages such as high detection accuracy and high throughput. Currently, no liquid-phase chips have been developed for the fall armyworm.
[0005] Therefore, by utilizing efficient modern molecular techniques, the development of the first liquid-phase chip related to biotype identification and resistance gene mutation detection of fall armyworm can fill the current technological gap. It can quickly and accurately detect the frequency of resistance gene mutations in fall armyworm in different regions, monitor the dynamic development of resistance in fall armyworm in different regions in a timely manner, accelerate the analysis of the resistance evolution law of fall armyworm, and guide the scientific prevention and control of fall armyworm. Summary of the Invention
[0006] This invention aims to address at least one of the technical problems existing in the prior art. To this end, this invention proposes a combination of molecular markers for detecting the biotype of the fall armyworm.
[0007] This invention also proposes a combination of SNP molecular markers for the fall armyworm containing the above-mentioned molecular marker combination.
[0008] The present invention also proposes a primer set and / or probe for detecting the above-mentioned SNP molecular marker combination of fall armyworm.
[0009] The present invention also proposes a chip.
[0010] The present invention also proposes a reagent kit.
[0011] This invention also proposes a method for screening the above-mentioned SNP molecular marker combinations of fall armyworm.
[0012] This invention also proposes the application of the above-mentioned molecular marker combination, the fall armyworm SNP molecular marker combination, primer set and / or probe, chip or kit.
[0013] This invention also proposes a method for identifying the biotype of the fall armyworm.
[0014] According to one aspect of the present invention, a molecular marker combination for detecting fall armyworm biotypes is provided, the molecular marker combination comprising SNP1, SNP2, SNP3, SNP4, SNP5 and SNP6; The SNP1 is located at nucleotide 8307876 on chromosome 1 of the fall armyworm reference genome GCA_012979215.2, or at the corresponding site on a homologous genomic fragment between species, and the nucleotide bases at this site are A / G. The SNP2 is located at nucleotide 8307894 on chromosome 1 of the fall armyworm reference genome GCA_012979215.2, or at the corresponding site on a homologous genomic fragment between species, and the nucleotide base of this site is G / A. The SNP3 is located at nucleotide 8307915 on chromosome 1 of the fall armyworm reference genome GCA_012979215.2, or at the corresponding site on a homologous genomic fragment between species, and the nucleotide base of this site is T / C. The SNP4 is located at nucleotide 6588069 on chromosome 1 of the fall armyworm reference genome GCA_012979215.2, or at the corresponding site on a homologous genomic fragment between species, and the nucleotide bases at this site are A / C. The SNP5 is located at nucleotide 6588070 on chromosome 1 of the fall armyworm reference genome GCA_012979215.2, or at the corresponding site on a homologous genomic fragment between species, and the nucleotide bases at this site are A / C. The SNP6 is located at nucleotide 6588071 on chromosome 1 of the fall armyworm reference genome GCA_012979215.2, or at the corresponding site on a homologous genomic fragment between species, and the nucleotide bases at this site are G / C.
[0015] According to a second aspect of the present invention, a fall armyworm SNP molecular marker combination is provided, the fall armyworm SNP molecular marker combination comprising the above-mentioned molecular marker combination.
[0016] In some embodiments of the present invention, the fall armyworm SNP molecular marker combination includes 27,006 SNP molecular markers. The physical locations of the 27,006 SNP molecular markers are determined by sequence alignment based on the fall armyworm reference genome GCA_012979215.2. The specific site information is shown in Table 1 below.
[0017] Table 1 ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ;
[0019] In some embodiments of the present invention, the location information of the marker is represented in the form of chromosome number: physical location.
[0020] In some embodiments of the present invention, the Alt value being "." indicates that it has multiple mutation possibilities.
[0021] In some embodiments of the present invention, when ref is A, Alt is “.”, indicating that the mutation can be T, G, C or N; When ref is T, Alt is “.”, indicating that the mutation can be A, G, C, or N; When ref is G, Alt is “.”, indicating that the mutation can be T, A, C, or N; When ref is C, Alt is “.”, indicating that the mutation can be T, A, G, or N, where N is deletion.
[0022] In a third aspect of the invention, primer sets and / or probes are provided for detecting the above-described molecular marker combinations or the fall armyworm SNP molecular marker combinations.
[0023] In a fourth aspect of the invention, a chip is provided that includes the aforementioned primer set and / or probes.
[0024] In some embodiments of the present invention, the chip is a liquid phase chip.
[0025] In some embodiments of the present invention, the chip is a resistance gene liquid phase chip.
[0026] In a fifth aspect of the invention, a kit is provided comprising the primer set and / or probes described above.
[0027] In a sixth aspect of the present invention, a method for screening the above-mentioned SNP molecular marker combinations of fall armyworm is proposed, the method comprising the following steps: (1) Whole genome resequencing and population structure analysis were performed on fall armyworm samples to screen out SNP sites that are stably associated with the biotype of fall armyworm; (2) Based on whole-genome sequencing data, screen for SNPs linked to resistance-related genes; the resistance-related genes include CHS2 , ABCC3 , ABCC2 , Myb , VipR , VipP , AChE , VGSC , RyR Gene; (3) Integrate the SNP sites that are stably associated with the biotype of fall armyworm obtained in (1) and the SNPs that are linked to resistance-related genes obtained in (2) to obtain the molecular marker combination of fall armyworm SNPs.
[0028] In a seventh aspect of the invention, the use of the above-described molecular marker combinations, fall armyworm SNP molecular marker combinations, primer sets and / or probes, chips, or kits in any of the following is proposed: 1) Identify the biotype of the fall armyworm; 2) Prepare products for identifying the biotype of fall armyworm; 3) Detection of resistance gene frequencies; 4) Identify new targets for fall armyworm control and assess resistance evolution; 5) Genotyping detection of fall armyworm; 6) Prepare products for controlling fall armyworm.
[0029] In some embodiments of the present invention, the fall armyworm biotypes include "corn-type" fall armyworm and "rice-type" fall armyworm.
[0030] In an eighth aspect of the present invention, a method for identifying the biotype of fall armyworm is provided, comprising the following steps: detecting the DNA of the sample to be tested using one of the above-mentioned molecular marker combination, fall armyworm SNP molecular marker combination, primer set and / or probe, chip and kit, and determining the biotype of fall armyworm based on the detection results.
[0031] In some embodiments of the present invention, the fall armyworm biotypes include "corn-type" fall armyworm and "rice-type" fall armyworm.
[0032] In some embodiments of the present invention, when the nucleotide base corresponding to SNP1 is G, the nucleotide base corresponding to SNP2 is G, the nucleotide base corresponding to SNP3 is C, the nucleotide base corresponding to SNP4 is A, the nucleotide base corresponding to SNP5 is A, and the nucleotide base corresponding to SNP6 is G, the fall armyworm biotype is maize type; when the nucleotide base corresponding to SNP1 is A, the nucleotide base corresponding to SNP2 is A, the nucleotide base corresponding to SNP3 is C, the nucleotide base corresponding to SNP4 is C, the nucleotide base corresponding to SNP5 is C, and the nucleotide base corresponding to SNP6 is C, the fall armyworm biotype is maize type.
[0033] In some embodiments of the present invention, the detection is performed based on liquid-phase probe capture sequencing genotyping technology.
[0034] The present invention has at least the following beneficial effects: The fall armyworm SNP molecular marker combinatorial provided by this invention is designed based on key resistance genes of fall armyworm. It has the characteristics of good representativeness, high specificity and high polymorphism. It can be applied to detect the frequency of resistance genes of fall armyworm in different regions and provide technical support for carrying out resistance evolution assessment.
[0035] This invention provides the first SNP liquid phase chip for the fall armyworm based on the SNP molecular marker combination of the fall armyworm. This chip effectively reduces the cost of scientific research analysis of the fall armyworm and can be applied to biotype classification and resistance gene frequency detection of the fall armyworm in different regions. It provides technical support for resistance evolution assessment, accelerates the functional verification of resistance genes, and helps to cultivate new insect-resistant crop varieties.
[0036] The first SNP liquid phase chip for fall armyworm provided by this invention is based on 11 key genes of fall armyworm obtained in previous research. By designing specific probes, it can simultaneously capture and genotype analyze multiple key resistance genes. It can efficiently integrate tens of thousands of SNP sites in a single detection, overcoming the problem of limited number of traditional PCR markers.
[0037] Compared with traditional solid-phase chips, the first SNP liquid-phase chip for fall armyworm provided by this invention is more flexible and can add marker sites at any time according to the application scenario. At the same time, the liquid-phase chip relies on the second-generation sequencing platform, which has lower typing cost, high detection throughput, and large data output at one time, and can simultaneously cover the detection of nearly a thousand materials, providing a technical means for large-scale typing.
[0038] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. Attached Figure Description
[0039] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which: Figure 1 This is a flowchart of the experiment for genotyping of fall armyworm SNP liquid phase chip in an embodiment of the present invention. Figure 2 This is a graph showing the genotype consistency rate detection results of replicate samples using the fall armyworm technique in an embodiment of the present invention. Figure 3 This is a diagram showing the biotype detection results of the fall armyworm in an embodiment of the present invention. Detailed Implementation
[0040] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.
[0041] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.
[0042] Example 1 This embodiment provides a fall armyworm SNP molecular marker combinatorial system and the resulting fall armyworm SNP liquid-phase chip. The fall armyworm SNP molecular marker combinatorial system includes 27,006 SNP sites. The physical locations of these 27,006 SNP sites were determined based on sequence alignment from the fall armyworm genome (GenBank: GCA_012979215.2). Specific information about these 27,006 SNP sites is shown in Table 1 of the specification.
[0043] 1. The specific process for obtaining the SNP molecular marker combination screening of fall armyworm is as follows: (1) By performing whole-genome resequencing and population structure analysis on the fall armyworm, the fall armyworm was divided into "rice type" and "maize type" genetic populations. By identifying SNP marker sites closely related to the population structure, two candidate genes were located: OR13 Genes (fall armyworm sex pheromone receptors) and Tpi The gene (triose phosphate isomerase) was further screened to identify 6 SNP sites on this candidate gene that are stably associated with the biotype, which can accurately reflect the biotype characteristics of the fall armyworm.
[0044] (2) Integrate the previously reported and obtained 9 resistance-related genes of fall armyworm, including 6 Bt resistance-related genes ( CHS2 , ABCC3 , ABCC2 , Myb , VipR , VipP ), and 3 pesticide resistance-related genes ( AChE , VGSC , RyR Gene annotations were extracted from the reference genome annotation file, and probes were designed by selecting exon regions, ultimately obtaining 27,000 SNP sites on 9 genes.
[0045] All the sites (1)-(2) above were integrated to form a liquid phase chip of fall armyworm SNP. The final number of sites was 27,006, involving 11 genes. Site information is shown in Table 1 of the instruction manual. Key resistance gene information of fall armyworm is shown in Table 2.
[0046] Table 2. Key resistance genes of fall armyworm
[0047] 2. Preparation of SNP liquid phase chip for fall armyworm The 27,006 loci obtained from the above screening were then used by Huazhi Biotechnology's independently developed liquid-phase probe precise localization sequencing and genotyping technology (Genotyping by Pinpoint Sequencing of liquid). c aptured target, c GPS was used to develop a liquid phase chip for the fall armyworm SNP. c GPS is based on an optimized thermodynamic stability algorithm model. It designs specific probes for target region sequences, then uses these synthesized specific probes to capture and enrich multiple different target sequences located at different genomic positions through liquid-phase hybridization. Finally, it constructs sequencing libraries and performs high-throughput sequencing on the captured and enriched target genomic sequences to obtain the genotypes of all SNP / InDel loci within the target region. The specific process is as follows: Figure 1 As shown.
[0048] Example 2 This embodiment provides a method for genotyping fall armyworm samples using the SNP liquid phase chip prepared in Example 1. The steps are as follows: 1. Extraction and quality control of fall armyworm genomic DNA DNA was extracted from fall armyworm samples using a magnetic bead method, and the DNA samples underwent quality testing. Quality testing included determining DNA concentration using a Qubit real-time fluorescence analyzer and assessing DNA integrity using 1% agarose gel electrophoresis. Samples that passed quality control were used for library preparation.
[0049] 2. c GPS Experiment and Analysis Process (1) Take 200 ng of qualified genomic DNA, digest the DNA sample with fragmentation enzyme, repair the enzyme ends and add an A base to the 3' end, and detect the fragment size by agarose gel electrophoresis. (2) Use T4 ligase to ligate the adapter fragments to both ends of the DNA, and purify the ligation product using fragment sorting magnetic beads. Detect the concentration of the purified product using a Qubit fluorescence quantitative instrument and detect the fragment size by agarose gel electrophoresis. (3) Perform PCR amplification on the purified ligation product, and screen the amplified product using magnetic beads. Detect the concentration of the screened product using a Qubit fluorescence quantitative instrument and detect the fragment size by agarose gel electrophoresis. (4) Place the qualified library, blocking reagent, RNase inhibitor, and probe on a PCR instrument for hybridization reaction, and incubate at 55°C for 16-24 hours. (5) Capture the hybridization product using streptavidin, amplify and enrich the captured library, and perform PE150 sequencing using the BGI sequencing DNBSEQ-T7 platform.
[0050] (6) The raw data after high-throughput sequencing were processed through quality control filtering, and adapter fragments and low-quality reads were removed using FASTP software to obtain high-quality Clean Reads. The obtained Clean Reads were compared with the reference genome using BWA software, and the positions were sorted to obtain the sorted BAM file. The sequencing results were analyzed for variant sites using GATK software to obtain the genotyping results of the target loci. c The principles and processes for constructing a GPS library are detailed in [link / documentation]. Figure 1 .
[0051] Example 3: Evaluation of the genotyping effect of SNP liquid phase chip in fall armyworm To verify the genotyping effect of the fall armyworm SNP liquid phase chip, the fall armyworm SNP liquid phase chip obtained in Example 1 was used to perform genotyping detection on 21 fall armyworm samples (including 2 duplicate samples). The specific operation method is described in Example 2.
[0052] Table 3
[0053] The results of sequencing and data analysis are shown in Table 3 and Figure 2As shown in Table 3, the detection rate of loci in the 23 samples ranged from 99.33% to 99.91%, with an average detection rate of 99.70%. The genotypic consistency between the two replicate samples ranged from 99.90% to 99.95%, with an average consistency rate of 99.93%. Figure 2 The genotyping effect of the SNP liquid phase chip on fall armyworm showed that the chip has good detection stability, high detection rate of target sites, and accurate and reliable genotyping results.
[0054] Example 4: SNP liquid phase chip biotype identification of fall armyworm Genotyping of 91 fall armyworm samples (with known genotypes) was performed using the fall armyworm SNP liquid phase chip prepared in Example 1 (see Example 2 for specific operation method). OR13 and Tpi Genotyping results of SNPs on two marker genes (Table 4) that are stably associated with biotypes were used to identify the biotype of fall armyworm.
[0055] Table 4
[0056] The results are as follows Figure 3 As shown in the figure, the test materials were all corn-type, and the results are consistent with the actual results. This indicates that the SNP liquid phase chip for fall armyworm can accurately identify the biotype characteristics of fall armyworm.
[0057] The embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.
Claims
1. A molecular marker combination for detecting the biotype of fall armyworm, characterized in that, The molecular marker combination includes SNP1, SNP2, SNP3, SNP4, SNP5 and SNP6; The SNP1 is located at nucleotide 8307876 on chromosome 1 of the fall armyworm reference genome GCA_012979215.2, or at the corresponding site on a homologous genomic fragment between species, and the nucleotide bases at this site are A / G. The SNP2 is located at nucleotide 8307894 on chromosome 1 of the fall armyworm reference genome GCA_012979215.2, or at the corresponding site on a homologous genomic fragment between species, and the nucleotide base of this site is G / A. The SNP3 is located at nucleotide 8307915 on chromosome 1 of the fall armyworm reference genome GCA_012979215.2, or at the corresponding site on a homologous genomic fragment between species, and the nucleotide base of this site is T / C. The SNP4 is located at nucleotide 6588069 on chromosome 1 of the fall armyworm reference genome GCA_012979215.2, or at the corresponding site on a homologous genomic fragment between species, and the nucleotide bases at this site are A / C. The SNP5 is located at nucleotide 6588070 on chromosome 1 of the fall armyworm reference genome GCA_012979215.2, or at the corresponding site on a homologous genomic segment among different species, and the nucleotide bases at this site are A / C. The SNP6 is located at nucleotide 6588071 on chromosome 1 of the fall armyworm reference genome GCA_012979215.2, or at the corresponding site on a homologous genomic fragment between species, and the nucleotide bases at this site are G / C.
2. A molecular marker combinatorial system for the fall armyworm SNP, characterized in that, The fall armyworm SNP molecular marker combination includes the molecular marker combination as described in claim 1.
3. The fall armyworm SNP molecular marker combination according to claim 2, characterized in that, The fall armyworm SNP molecular marker assemblage includes 27,006 SNP molecular markers. The physical locations of these 27,006 SNP molecular markers were determined by sequence alignment based on the fall armyworm reference genome GCA_012979215.
2. The specific site information is shown in Table 1 of the specification.
4. Primer sets and / or probes for detecting the molecular marker combination as described in claim 1 or the fall armyworm SNP molecular marker combination as described in any one of claims 2-3.
5. A chip, characterized in that, The chip includes the primer set and / or probe as described in claim 4; Preferably, the chip comprises a liquid phase chip.
6. A reagent kit, characterized in that, The kit contains the primer set and / or probe as described in claim 4.
7. A method for screening SNP molecular marker combinations of fall armyworm as described in any one of claims 2-3, characterized in that, The method includes the following steps: (1) Whole genome resequencing and population structure analysis were performed on fall armyworm samples to screen out SNP sites that are stably associated with the biotype of fall armyworm; (2) Based on whole-genome sequencing data, screen for SNPs linked to resistance-related genes; the resistance-related genes include CHS2 , ABCC3 , ABCC2 , Myb , VipR , VipP , AChE , VGSC , RyR Gene; (3) Integrate the SNP sites that are stably associated with the biotype of fall armyworm obtained in (1) and the SNPs that are linked to resistance-related genes obtained in (2) to obtain the molecular marker combination of fall armyworm SNPs.
8. The use of the molecular marker combination of claim 1, the fall armyworm SNP molecular marker combination of any one of claims 2-3, the primer set and / or probe of claim 4, the chip of claim 5, or the kit of claim 6 in any one of the following: 1) Identify the biotype of the fall armyworm; 2) Prepare products for identifying the biotype of fall armyworm; 3) Detection of resistance gene frequencies; 4) Identify new targets for fall armyworm control and assess resistance evolution; 5) Genotyping detection of fall armyworm; 6) Prepare products for controlling fall armyworm.
9. The application according to claim 8, characterized in that, The fall armyworm biotypes include the "corn-type" fall armyworm and the "rice-type" fall armyworm.
10. A method for identifying the biotype of the fall armyworm, characterized in that, The process includes the following steps: using the molecular marker combination as described in claim 1, the fall armyworm SNP molecular marker combination as described in any one of claims 2-3, the primer set and / or probe as described in claim 4, the chip as described in claim 5, or the kit as described in claim 6 to detect the DNA of the sample to be tested, and determining the fall armyworm biotype based on the detection results; Preferably, the fall armyworm biotypes include the "corn-type" fall armyworm and the "rice-type" fall armyworm; Preferably, the detection is performed based on liquid-phase probe capture sequencing genotyping technology.