Multiplex PCR (polymerase chain reaction) primer combination and sequencing analysis method for identifying species of Alocasia mali

Abstract

The invention provides a multiplex PCR (polymerase chain reaction) primer combination and a sequencing analysis method for identifying a species of Schizothorax mali, and belongs to the technical field of molecular detection. According to the multiple PCR primer combination disclosed by the invention, a plurality of primer pairs are designed according to multiple key gene SNP locus areas of mitochondrial genomes (imagoes, larvae and pupae) of different forms and different populations and sibling species of the mitochondrial genomes, so that a plurality of target fragments can be synchronously amplified by single PCR reaction; the misjudgment caused by single gene variation or sequencing errors is effectively avoided, and the sibling species and the cryptic species with highly similar forms can be accurately distinguished. The invention further provides a multiple PCR and sequencing analysis method for identifying the species of the malodinia mali, targeted high-throughput sequencing is conducted on multiple PCR amplification products, sequence information of all key sites is obtained, the similarity of the sites to be detected can be obtained through comparison, and decisive data support is provided for high-throughput accurate identification of the species level of the malodinia mali.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of molecular detection, and particularly relates to a multiplex PCR primer combination and sequencing analysis method for identifying apple borer species. BACKGROUND

[0002] Apple borer (such as Agrilus mali ) is an important borer pest of Rosaceae fruit trees such as apple and pear. The larvae drill into the trunk and cause damage, leading to tree decline and even whole plant death, posing a major threat to orchard economic benefits. Traditionally, species identification of apple borer mainly relies on morphological characteristics such as body color, wing vein structure, or larval tunneling pattern. However, apple borer individuals are small (usually a few millimeters in length), inter-specific morphological differences are not significant, and there are cryptic species that are difficult to distinguish morphologically, making morphological identification prone to misjudgment. Especially when incomplete samples are collected in the field, incomplete stages are operated by non-classification experts, the accuracy of the identification results is further reduced. In addition, the wide distribution of the pest and the complex geographical population variation also increase the difficulty of morphological identification. The above limitations often lead to improper prevention and control strategies, such as misuse of pesticides and delayed prevention and control, which in turn exacerbates the risk of pest resistance development and spread. Therefore, developing a rapid and accurate molecular identification method has become an urgent need to make up for the deficiencies of morphological identification of apple borer and improve the efficiency of integrated management.

[0003] Currently, most molecular identification techniques for apple borer are based on single target detection, which cannot meet the actual needs of rapid identification of mixed populations in the field. SUMMARY

[0004] The present application aims to provide a multiplex PCR primer combination and sequencing analysis method for identifying apple borer species, which can target multiple key gene regions of apple borer and its close relatives, and meet the actual needs of rapid identification of mixed populations in the field.

[0005] The application provides a multiplex PCR primer combination for identifying Agelopsis species, comprising a first primer pair, a second primer pair, a third primer pair, a fourth primer pair, a fifth primer pair, a sixth primer pair, a seventh primer pair, an eighth primer pair, a ninth primer pair, a tenth primer pair and an internal reference primer pair; the first primer pair comprises an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 1 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 2; the second primer pair comprises an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 3 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 4; the third primer pair comprises an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 5 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 6; the fourth primer pair comprises an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 7 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 8; the fifth primer pair comprises an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 9 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 10; the sixth primer pair comprises an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 11 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 12; the seventh primer pair comprises an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 13 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 14; the eighth primer pair comprises an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 15 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 16; the ninth primer pair comprises an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 17 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 18; the tenth primer pair comprises an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 19 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 20; and the internal reference primer pair comprises an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 21 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 22.

[0006] The application also provides a kit for identifying Agelopsis species, comprising the multiplex PCR primer combination described in the above scheme.

[0007] Preferably, the kit further comprises 5x multiplex amplification enzyme, 2x HiFi Mix, reagents for library construction and 96 indexes.

[0008] The application also provides the use of the multiplex PCR primer combination described in the above scheme or the kit described in the application in identifying Agelopsis species.

[0009] The application further provides a method for identifying apple sawfly species by multiplex PCR and sequencing analysis, comprising the following steps: extracting genomic DNA of a sample to be tested; using the genomic DNA as a template, performing multiplex PCR amplification by using the multiplex PCR primer combination in the above-mentioned scheme to obtain a multiplex PCR amplification product; performing high-throughput sequencing on the multiplex PCR amplification product to obtain sequencing data; performing quality control and alignment on the sequencing data, counting reads to evaluate the relative content of each species, and completing sample identification.

[0010] Preferably, the amplification system of the multiplex PCR amplification comprises the following components in 20 μL: 4 μL of 5x multiplex amplification enzyme, 2 μL of the multiplex PCR primer combination, 1-2 μL of a template, and the balance of water; and the final concentration of each primer in the multiplex PCR primer combination is 0.1-0.3 μM.

[0011] Preferably, the amplification procedure of the multiplex PCR amplification comprises 95℃, 2 min; 95℃, 10 s, 56℃, 30 s, 72℃, 30 s, 17 cycles; and 72℃, 5 min.

[0012] Preferably, the purification of the multiplex PCR amplification product comprises purifying the multiplex PCR amplification product by magnetic beads to remove primer dimers and non-specific fragments and retain target DNA fragments.

[0013] Preferably, the high-throughput sequencing comprises DNBSEQ-T7 sequencing; the high-throughput sequencing of the multiplex PCR amplification product comprises: purifying the multiplex PCR amplification product to obtain a purified product; adding adapters to the purified product to obtain a sequencing library; and performing high-throughput sequencing on the sequencing library.

[0014] Preferably, the alignment comprises aligning the sequencing fragments after quality control to COI, COII, and CytB gene sequences; and the counting of reads to evaluate the relative content of each species comprises calculating the number of sequencing fragments and the alignment rate on the alignment, and determining the species by the size of the index data.

[0015] The application provides a multiplex PCR primer combination for identifying Agrilus sinuatus species, comprising a first primer pair, a second primer pair, a third primer pair, a fourth primer pair, a fifth primer pair, a sixth primer pair, a seventh primer pair, an eighth primer pair, a ninth primer pair, a tenth primer pair and an internal reference primer pair. The multiplex PCR primer combination of the application targets Agrilus sinuatus, and can simultaneously amplify multiple target fragments in a single PCR reaction through SNP difference information of multiple key gene regions (such as COI, COII, CytB, etc.) of different morphologies and different populations and their close species; and simultaneously obtains specific recognition sites of COI, COII, CytB and other multiple gene regions, effectively avoids misjudgment caused by single gene variation or sequencing error through multi-site sequence alignment and cross verification; secondly, based on the SNP site sequence information of different populations and different developmental morphologies, the multiplex PCR primer combination of the application can be used for accurately distinguishing close species and cryptic species with highly similar morphologies, and the identification accuracy is significantly better than that of traditional methods. In addition, the application also introduces a universal primer for all Curculionidae insects as an internal reference control in the multiplex PCR primer combination, which is used for monitoring the DNA extraction quality and the effectiveness of the PCR reaction; if the internal reference is not amplified, it is determined that the experiment is invalid, so as to eliminate false negative results caused by operation errors or invalid reagents, and improve the reliability of the detection results. Compared with the traditional single PCR combined with sequencing technology, the application realizes the technical leap from "single point in series" to "multiple points in parallel". The traditional method can only detect one target site per reaction, and if it is necessary to distinguish mixed populations or multiple close species, multiple independent experiments are required, the process takes several days, and the cost of reagents and manpower increases significantly. The application simultaneously amplifies multiple target gene fragments in a single tube reaction through the multiplex PCR technology, greatly shortens the overall detection time, and truly realizes efficient and high-throughput variety identification.

[0016] The application also provides a multiplex PCR and sequencing analysis method for identifying Agrilus sinuatus species, which targets high-throughput sequencing of the multiplex PCR amplification product to obtain key SNP sequence information of all target genes, and obtains genetic information of the sample to be detected through sequencing, thereby providing decisive data support for accurate identification of Agrilus sinuatus. BRIEF DESCRIPTION OF DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings in the following description are only some embodiments of the application, and other drawings can be obtained by those skilled in the art without creative labor.

[0018] Figure 1 The application provides a multiplex PCR and sequencing analysis method for identifying Agrilus sinuatus species. DETAILED DESCRIPTION

[0019] The application provides a multiplex PCR primer combination for identifying agilus specie, which is characterized in that the combination comprises a first primer pair, a second primer pair, a third primer pair, a fourth primer pair, a fifth primer pair, a sixth primer pair, a seventh primer pair, an eighth primer pair, a ninth primer pair, a tenth primer pair and an internal reference primer pair; the first primer pair comprises an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 1 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 2; the second primer pair comprises an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 3 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 4; the third primer pair comprises an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 5 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 6; the fourth primer pair comprises an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 7 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 8; the fifth primer pair comprises an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 9 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 10; the sixth primer pair comprises an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 11 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 12; the seventh primer pair comprises an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 13 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 14; the eighth primer pair comprises an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 15 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 16; the ninth primer pair comprises an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 17 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 18; the tenth primer pair comprises an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 19 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 20; and the internal reference primer pair comprises an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 21 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 22.

[0020] The application successfully screens specific variation sites on three conservative genes COI, COII and CytB in a mitochondrial genome, the sites have high interspecific polymorphism and can effectively distinguish agilus from main close species, thereby providing a theoretical basis and target region for subsequent primer design.

[0021] In the application, the main close species include agilus; the agilus includes agilus cinctus and / or agilus planipennis.

[0022] The multiplex PCR primer combination of the present application targets SNP sites in multiple key gene regions (such as COI, COII, CytB, etc.) of the apple small weevil, can accurately distinguish its closely related species, there is no cross reaction between the primers, and the length difference of the amplification products is obvious, which is convenient for detection, can realize the synchronous amplification of multiple target fragments in a single PCR reaction, breaks through the bottleneck of low throughput of traditional single PCR, supports one-time synchronous identification of mixed populations or multiple closely related species, and significantly improves the detection efficiency and coverage. Moreover, the multiplex PCR primer combination of the present application has no cross reaction to the main closely related species (such as the genus of narrow weevil) of the apple small weevil, simultaneously realizes the wide compatibility of different insect states (adults, larvae) and incomplete samples, effectively makes up for the dependence of morphological identification on sample integrity, significantly reduces the risk of misjudgment of cryptic species, and provides reliable basis at the molecular level for pest tracing, population monitoring and accurate prevention and control.

[0023] The design principles of the multiplex PCR primer combination designed for the three conserved genes of mitochondrial COI, COII and CytB include: high specificity, balance between non-specific amplification and target region coverage (SNP≥99%, STR≥98%); strict control of primer dimer (no continuous 4 base complementary); balance of amplification uniformity, primer length 17-35 bp, Tm value uniformity 61°C (no modification); amplicon length 160-260 bp (adapted to PE150 sequencing), STR region preferentially short amplicon and single-end measurement, and attention to haplotype region to avoid missing expansion. The present application is a primer probe development kit designed by finding out all SNPs of the three fragments of the bar code from the mitochondrial genome sequence, which is more accurate than a single fragment (only a single mitochondrial fragment COI before). The SNPs are mainly compared with the closely related species of the apple small weevil, and the primer probe is developed.

[0024] The present application introduces an internal reference control system to monitor the whole process quality from DNA extraction to PCR amplification in real time, eliminates false negative results, and improves data reliability.

[0025] The present application also provides a kit for identifying apple small weevil species, which comprises the multiplex PCR primer combination described in the above scheme.

[0026] As an implementation manner, the kit further comprises 5x multiplex amplification enzyme, 2x HiFi Mix, reagents for library construction, and 96 indexes.

[0027] As an implementation manner, the 5x multiplex amplification enzyme is purchased from Yisen Biological, and the item number is 12948ES24.

[0028] The application also provides application of the multiplex PCR primer combination or the kit in identifying Agelopsis species.

[0029] The application further provides a method for identifying Agelopsis species by multiplex PCR and sequencing analysis, comprising the following steps: extracting genomic DNA of a sample to be tested; performing multiplex PCR amplification on the genomic DNA as a template by using the multiplex PCR primer combination to obtain a multiplex PCR amplification product; sequencing the multiplex PCR amplification product at high throughput to obtain sequencing data; performing quality control and alignment on the sequencing data, counting reads to evaluate the relative content of each species, and completing sample identification.

[0030] The application first extracts genomic DNA of a sample to be tested.

[0031] The method for extracting genomic DNA of a sample to be tested is not particularly limited in the application, and a conventional method in the art can be used.

[0032] After obtaining the genomic DNA of a sample to be tested, the application performs multiplex PCR amplification on the genomic DNA as a template by using the multiplex PCR primer combination to obtain a multiplex PCR amplification product.

[0033] As an embodiment, the amplification system of the multiplex PCR amplification comprises the following components in 20 μL: 4 μL of 5× multiplex amplification enzyme, 2 μL of the multiplex PCR primer combination, 1-2 μL of a template, and the rest of water; the final concentration of each primer in the multiplex PCR primer combination is 0.1-0.3 μM. As an embodiment, the amplification procedure of the multiplex PCR amplification comprises 95℃, 2 min; 95℃, 10 s, 56℃, 30 s, 72℃, 30 s, 17 cycles; 72℃, 5 min.

[0034] In the application, the multiplex PCR amplification can effectively avoid cross-reaction and non-specific amplification between primers, and ensure that the DNA template is in a trace amount (as low as 10 ng).

[0035] The innovation of the present application is that a high-efficiency and reliable multiplex PCR amplification system is developed for the first time for Agelopsis ursi, filling the gap in the rapid high-throughput identification technology at the molecular level for this species. Moreover, by reasonably setting the reaction conditions, the present application realizes the balance of multi-target amplification, effectively avoids the cross-reaction and non-specific amplification between primers, avoids primer dimers, and ensures high sensitivity to micro DNA templates (as low as 10 ng). The method flow of the present application is simplified, the dependence on high-cost equipment is reduced, and it is suitable for rapid detection in the field of grass-plant protection units.

[0036] After obtaining the multiplex PCR amplification product, the present application performs high-throughput sequencing on the multiplex PCR amplification product to obtain sequencing data.

[0037] The present application can realize rapid detection and accurate interpretation of the multiplex PCR amplification product through high-throughput sequencing. Moreover, by combining the multiplex PCR technology with the high-throughput capability of the second-generation sequencing platform, the present application improves the detection throughput by about 10 times, greatly shortens the overall detection time, and truly realizes efficient and high-throughput species identification.

[0038] As an embodiment, the high-throughput sequencing includes DNBSEQ-T7 sequencing; the high-throughput sequencing on the multiplex PCR amplification product includes: purifying the multiplex PCR amplification product to obtain a purified product; adding adapters to the purified product to obtain a sequencing library; and performing high-throughput sequencing on the sequencing library. As an embodiment, the purification of the multiplex PCR amplification product includes purifying the multiplex PCR amplification product by magnetic beads to remove primer dimers and non-specific fragments and retain target DNA fragments; the method for purifying the magnetic beads is not particularly limited in the present application, and a conventional method in the art can be used; the adapter contains a sequencing primer binding site; the method for adding adapters to the purified product is not particularly limited in the present application, and a conventional method in the art can be used.

[0039] As an embodiment, the DNBSEQ-T7 sequencing includes: linear amplification of the library, circularization to generate single-stranded circular DNA templates, and then rolling circle amplification to prepare DNA nanoballs (DNB); the DNB is loaded to a sequencing chip (strictly controlling temperature and humidity to ensure uniformity and stability) by an automatic device, sequenced by using combined probe anchor polymerization (cPAS) technology, and the raw data is standardized to obtain off-machine data.

[0040] As an embodiment, the sequencing chip is purchased from Tianjin Baoao Huaxing Gene Technology Co., Ltd.

[0041] After obtaining the sequencing data, the present application performs quality control and alignment on the sequencing data, counts the reads to evaluate the relative content of each species, and completes sample identification.

[0042] As an implementation, the present application uses fastp / bwa / samtools for quality control and alignment; the alignment includes aligning the quality-controlled sequencing fragments to COI, COII, CytB gene sequences; and the read count evaluation of the relative content of each species includes calculating the number of aligned sequencing fragments and the alignment rate, and determining the species by the size of the index data.

[0043] In the present application, when the alignment rate is greater than or equal to 95%, the sample to be tested is determined to be the apple small tit beetle, and further, when the alignment rate is greater than or equal to 97%, the sample to be tested is determined to be the apple small tit beetle; when the alignment rate is less than 95%, the sample to be tested is determined to be a non-apple small tit beetle. The alignment rates of other species except the apple small tit beetle and the apple small tit beetle are not in the same order of magnitude (it is verified by experiments that the alignment rates of the closely related species of the same genus are less than 5%).

[0044] The present application establishes a bioinformatics analysis process for the quarantine pest apple small tit beetle, aligns the sequencing sequences obtained with the target gene sequences of the apple small tit beetle and its closely related species, and distinguishes or identifies the species based on the quantitative results, the alignment depth, the alignment rate and even the variation sites. This method can effectively distinguish closely related species and cryptic species with similar morphology, and improve the accuracy and resolution of the identification results.

[0045] The present application is aimed at the rapid molecular identification needs of the apple small tit beetle ( Agrilus mali ) and its main closely related species (such as Agrilus sinensis , etc.), and establishes a high-efficiency identification method that can simultaneously identify the apple small tit beetle and its closely related species in a single reaction, realizes the one-time targeted identification of the apple small tit beetle in field samples, effectively solves the problem of cryptic species that are difficult to distinguish by morphological identification, and has the advantages of simple operation, reasonable cost and accurate results. This method breaks through the limitations of traditional methods in terms of throughput and efficiency, realizes the simultaneous identification of multiple target samples, and significantly improves the detection efficiency and accuracy. The implementation of the present application fills the gap in the field of multiple molecular identification of the apple small tit beetle, and provides important technical support for the accurate monitoring and scientific prevention and control of this type of pest.

[0046] The method of the present application fully considers the actual application needs in the field, and maintains good detection effect for samples in different insect stages such as adults, larvae and residual limbs, and under different storage conditions, solving the dependence of morphological identification on sample integrity. Moreover, only conventional PCR instruments and other equipment are needed to complete the detection, reducing the hardware threshold for technology popularization. In addition, the method has early warning capability, and still maintains high sensitivity for low-age larvae or trace DNA samples, which is beneficial to early detection and timely prevention and control. Furthermore, the present application has a significant cost advantage, with reduced reagent consumption, shortened experimental time and reduced labor cost.

[0047] The application organically combines multiplex PCR, targeted sequencing and bioinformatics analysis to form a complete technical closed loop. The technical framework can be flexibly adjusted to facilitate the addition of target species or adaptation to different detection needs. In summary, the application is significantly superior to the prior art in terms of detection efficiency, accuracy, cost-effectiveness, practical application value and innovation, and provides reliable technical support for scientific monitoring and precise prevention and control of the apple small jewel beetle.

[0048] The method of the application has significant universality and extensibility in application level, and its core value is not limited to the identification of the apple small jewel beetle, but also shows important potential in the comprehensive management of agricultural pests, biodiversity protection and commercial services, etc. In the field of agriculture, the technology can be used for field pest population dynamic monitoring, and a population distribution and change database can be established through large-scale screening to provide data support for prevention and control decision-making; combined with target gene mutation detection, the development of pest resistance can be monitored in real time to guide scientific use of pesticides; at the same time, the control effect of natural enemy insects on target pests can also be accurately evaluated to promote the application of green prevention and control strategies. In the field of ecological research and biodiversity protection, the application helps to reveal the genetic structure and gene exchange mode of different geographical populations, and provides molecular technical support for the protection of rare and endangered jewel beetle species. In addition, in the commercial expansion, the system can also provide professional technical services and data interpretation for agricultural departments, scientific research institutions, etc., and has good industrialization prospects.

[0049] The application provided not only a preferred technical solution for identifying the apple small jewel beetle, but also a highly adaptable and expandable technical platform. Through flexible adjustment of target genes, detection methods and analysis processes, the system can respond to the needs of various application scenarios, and has wide applicability in the fields of port quarantine, ecological monitoring, population genetic research, etc. This multi-dimensional and multi-level extensibility not only highlights the technical value and development potential of the application, but also lays a solid foundation for its actual promotion and continuous optimization in the future.

[0050] In order to further illustrate the application, a method for identifying the apple small jewel beetle species using a multiplex PCR primer combination and sequencing analysis is described in detail below in conjunction with the accompanying drawings and examples, but they should not be understood as limiting the scope of protection of the application.

[0051] Example 1: A method for identifying the apple small jewel beetle species using multiplex PCR and sequencing analysis The method development scheme flowchart of the application is shown in Figure 1 .

[0052] The detailed content is as follows: The application successfully screens specific variation sites on three conservative genes COI, COII and CytB in the mitochondrial genome, which have high interspecific polymorphism and can effectively distinguish the apple small weevil from its main relative species (such as white wax narrow weevil, rainbow narrow weevil, etc.), providing a theoretical basis and target region for subsequent primer design. The variation sites of the target region are shown in Table 1.

[0053] Table 1 variation sites of the target region

[0054] 1. Primer design For the three conservative gene sequences COI, COII and CytB in the mitochondrion, the primer design follows the following basic principles: a) The specificity of the primer needs to be high: the more the number of primer binding sites in the genome, the higher the failure rate of amplification; at the same time, the risk of non-specific amplification needs to be balanced with the coverage rate of the target region, wherein the SNP coverage rate generally needs to be ≥ 99%, and the STR coverage rate generally needs to be ≥ 98%. b) Strictly control the formation of primer dimers: there should be no continuous 4-base complementary pairing between primers to avoid affecting the amplification efficiency due to dimer competition. c) The amplification uniformity of different regions of the target gene needs to be balanced to ensure that the amount of each fragment amplification product is less different. d) The length of the primer is controlled at 17-35 bp, the first step is to extend the primer, and the sequencing primer + target segment primer; the second step is to introduce a 96-well plate sample tag primer. The sequencing primer is shown in Table 2 in bold. The sample tag primer is shown in the index table; the annealing temperature needs to be basically the same, and the Tm value of all primers is set to 61℃, and no chemical modification is performed. e) The length of the amplicon is generally controlled at 160-260 bp (adapted to PE150 sequencing strategy) to ensure the integrity and accuracy of the sequencing data. f) For the STR region: the priority of short amplicon design is higher, and single-end measurement is required to ensure complete capture of STR site information. g) The haplotype region in the target gene needs to be paid attention to, and the amplification bias or omission caused by the sequence characteristics of the region needs to be avoided.

[0055] Around the above three gene regions, the application designs 10 groups of specific primers, and establishes a set of efficient and stable multiplex PCR amplification system after repeated optimization and screening. Through standardized experimental operation, the simultaneous amplification of the sequences of the three target genes is successfully realized, the amplification product band is clear, and the specificity is good, and there is no non-specific amplification or primer dimer phenomenon, indicating that the primer design is reasonable, and the reaction system is stable and reliable. The primer information and the sequence of the amplification region are shown in Table 2.

[0056] Table 2 primer information and amplification region

[0057] 2. Multiplex PCR experimental procedure The experiment was performed using a 1-tube amplification multiplex PCR kit (Table 3) purchased from Bionovogene. The experimental method is as follows: Table 3 1 Multiplex PCR kit kit composition

[0058] Reagents, consumables and instruments to be prepared: 1) nucleic acid extraction kit: nucleic acid extraction kit from Tian Gen; 2) nucleic acid quantification kit: A. Quantus dsDNA / RNA HS Assay Kit from Promega, Catalog No.: E2670 / E3310; B. Qubit dsDNA / RNA HS Assay Kit from Thermo Fisher Scientific, Catalog No.: Q32854 / Q32852.

[0059] 3) nucleic acid purification kit (magnetic bead method): A. Agencourt AMPure XP Kit from Beckman Coulter, Catalog No.: A63880 / A63881 / A63882; B. CleanNGS purification magnetic beads from Suzhou Fuduo Biotech, Catalog No.: CNGS0005 / CNGS 0050 / CNGS0500.

[0060] The purified magnetic beads need to be equilibrated to room temperature before use.

[0061] 4) DNase and RNase-free purified water, Low TE solution (10mM Tris, 0.1mM EDTA, pH 8.0).

[0062] 5) Anhydrous ethanol (analytical pure).

[0063] 6) fluorometer: A. Quantus™ Fluorometer from Promega, Catalog No.: E6150; B. Qubit 4.0 fluorometer from Thermo Fisher Scientific, Catalog No.: Q33226.

[0064] 7) vortex mixer and microcentrifuge.

[0065] 8) magnetic stand: A, DynaMag™-2 Magnet, Thermo Fisher Scientific, Cat# 12321D; B, DynaMag™-96 Side Magnet, Thermo Fisher Scientific, Cat# 12331D.

[0066] 9) Pipette filter tips gun without DNase and RNase.

[0067] 3, Experimental operation process Library construction: Applied Biosystems™ 2720 Thermal Cycler, ABI MiniAmp A37028 PCR instrument, Bio-Rad T100 PCR instrument or equivalent PCR instrument.

[0068] 3.1 Preparation before experiment 3.1.1 Thaw the reagents on ice before the experiment starts; 3.1.2 Before use, take the purified magnetic beads out of the 4°C refrigerator 30 minutes in advance and balance to room temperature; 3.1.3 Freshly prepare 80% ethanol for purification; 3.2 1 st PCR 3.2.1 The sample input is 1~50ng, and the PCR program is set as shown in Table 4.

[0069] Table 41 st PCR program

[0070] 3.2.2 1 st Prepare the PCR amplification system according to Table 5.

[0071] Table 51 st PCR reaction system

[0072] 3.2.3 After the system is prepared, mix well, centrifuge briefly, place in the PCR instrument, and start amplification.

[0073] 3.3 1 st Amplification and purification 3.3.1 Add 20μL purified magnetic beads to the above PCR tube, mix well by blowing, and stand at room temperature for 5 minutes, then place it on the magnetic stand for 3~5 minutes until the liquid is clear; 3.3.2 Carefully aspirate the liquid (be careful not to aspirate the magnetic beads), add 200μL of freshly prepared 80% ethanol, and stand for 30 seconds; 3.3.3 Repeat Step 3.3.2 once, for a total of two washes; 3.3.4 Aspirate the ethanol and let the tube sit at room temperature for 3-5 min to allow the residual ethanol to evaporate. The beads should be dull and not shiny. 3.3.5 Remove the tube from the magnet and add 12 μL of purified water to resuspend the beads. Let the tube sit at room temperature for 5 min. 3.3.6 Place the tube back on the magnet and let it sit for 3-5 min until the liquid is clear. Carefully open the tube and aspirate 10 μL of the eluted product into a 200 μL PCR tube.

[0074] 3.4 2 nd PCR 3.4.1 Set up the PCR reactions as shown in Table 6 nd PCR program: Table 6 2 nd PCR program

[0075] Set up the PCR reactions as shown in Table 7: Table 6 2 nd PCR reactions Note: The Index information for each sample must be recorded in detail and must not be confused.

[0076] 3.5 2 nd Post-amplification purification 3.5.1 Add 20 μL of purified magnetic beads to each of the PCR tubes, mix by pipetting, and let the tubes sit at room temperature for 5 min. Then place the tubes on the magnet and let them sit for 3-5 min until the liquid is clear.

[0077] 3.5.2 Carefully aspirate the liquid (be careful not to aspirate the magnetic beads), add 200 μL of freshly prepared 80% ethanol, and let the tubes sit for 30 s.

[0078] 3.5.3 Repeat Step 3.5.2 once.

[0079] 3.5.4 Aspirate the ethanol and let the tube sit at room temperature for 3-5 min to allow the residual ethanol to evaporate. The beads should be dull and not shiny.

[0080] 3.5.5 Remove the tube from the magnet and add 32 μL of purified water to resuspend the beads. Let the tube sit at room temperature for 5 min.

[0081] 3.5.6 Place the tube back on the magnet and let it sit for 3-5 min until the liquid is clear.

[0082] 3.5.7 Carefully open the tube cap, and pipette 30 μL elution product into a 200 μL PCR tube, which is the desired library to be tested.

[0083] 3.5.8 Quantus / Qubit detects library concentration.

[0084] The kit contains library construction reagents, index, etc. See Table 8 for details.

[0085] Table 8 Library construction index

[0086] 3. DNBSEQ-T7 sequencing The multiplex PCR amplification product needs to be purified first to effectively remove primer dimers and non-specific amplification products; usually magnetic bead purification method is used for fragment screening, and specific DNA fragments of target size are reserved.

[0087] The purified PCR product needs to be further subjected to adapter ligation reaction to introduce the necessary sequence elements (including sequencing primer binding sites and index sequence barcode) for sequencing, and finally form a complete sequencing library. Subsequently, the purified sequencing library is subjected to linear amplification and circularization reaction to generate single-stranded circular DNA template, and then in a special buffer system, DNA nanoballs (DNB) are prepared by rolling circle amplification technology. The prepared DNB is loaded into the DNBSEQ-T7 special sequencing chip through automatic equipment, forming a high-density and regular arrangement of sequencing array. The chip loading process needs to be strictly controlled in terms of temperature and humidity to ensure the uniformity and stability of DNB distribution. The DNBSEQ-T7 platform uses combined probe anchor polymerization (cPAS) sequencing technology. After sequencing, the raw data is standardized to obtain the off-machine data.

[0088] 4. Species identification results After preprocessing the sequencing data, professional bioinformatics processes are used for analysis, including sequence alignment, variant site identification and species discrimination analysis, verifying the smooth connection of the whole process from wet experiments to dry analysis and the credibility of the results. Specifically, first, use fastp software to quality control the off-machine data, calculate sample Q20, Q30, GC content and other indicators. After filtering out low-quality reads, use bwa software to align the reads to COI, COII, CytB gene sequences, calculate the number of aligned reads and alignment rate, and determine the species through the size of the index data to obtain the final identification results.

[0089] In the present application, when the matching rate is ≥95%, the sample to be tested is determined to be the apple borer, and further, when the matching rate is ≥97%, the sample to be tested is determined to be the apple borer; when the matching rate is <95%, the sample to be tested is determined to be a non-apple borer.

[0090] Four typical samples were selected for testing in this step, including two positive samples E11 and E24 (known as apple borers) and two negative samples E3 (Iridesma prasina) and EAB (Paratimia laevicollis) (closely related species, from the morphology, it is more difficult to distinguish between apple borers and Iridesma prasina; from the genome, Paratimia laevicollis is more closely related to Iridesma prasina and apple borers), wherein the positive samples are from Beijing Wulumuqi County street trees and Yili fruit forest, and the samples are identified by experts. The negative samples are provided by insect classification experts. The experimental results are shown in Table 9.

[0091] Table 9: Experimental results of typical samples

[0092] The experimental results are summarized in Table 9, and the identification results of all samples are completely consistent with the known species information. Specifically: the alignment depths of positive samples E11 and E24 are significantly higher, and the target sequence matching rates are both greater than 95%, and they are accurately identified as apple borers; the alignment depths of negative samples E3 (Iridesma prasina) and EAB (Paratimia laevicollis) are lower, and the matching rates are both lower than 95%, and they are correctly determined to be non-apple borers.

[0093] Table 9: Experimental results

[0094] In this verification, the identification results of all samples are consistent with the actual species identity, and there is no false positive or false negative misjudgment, which shows that the method has 100% accuracy in species identification. The results fully prove that the method of the present application has high reliability and practical value in the identification of apple borers.

[0095] Although the above embodiment has made a detailed description of the present application, it is only a part of the embodiments of the present application, not all the embodiments, and other embodiments can be obtained according to the present embodiment without creativity, which all belong to the protection scope of the present application.

Claims

1. A multiplex PCR primer set for identifying species of *Acer palmatum*, characterized in that, It includes the first primer pair, the second primer pair, the third primer pair, the fourth primer pair, the fifth primer pair, the sixth primer pair, the seventh primer pair, the eighth primer pair, the ninth primer pair, the tenth primer pair, and the internal reference primer pair; The first primer pair includes an upstream primer with a nucleotide sequence as described in SEQ ID NO.1 and a downstream primer with a nucleotide sequence as described in SEQ ID NO.2; The second primer pair includes an upstream primer with a nucleotide sequence as described in SEQ ID NO.3 and a downstream primer with a nucleotide sequence as described in SEQ ID NO.4; The third primer pair includes an upstream primer with a nucleotide sequence as described in SEQ ID NO. 5 and a downstream primer with a nucleotide sequence as described in SEQ ID NO. 6; The fourth primer pair includes an upstream primer with a nucleotide sequence as described in SEQ ID NO. 7 and a downstream primer with a nucleotide sequence as described in SEQ ID NO. 8; The fifth primer pair includes an upstream primer with a nucleotide sequence as described in SEQ ID NO. 9 and a downstream primer with a nucleotide sequence as described in SEQ ID NO. 10; The sixth primer pair includes an upstream primer with a nucleotide sequence as described in SEQ ID NO. 11 and a downstream primer with a nucleotide sequence as described in SEQ ID NO. 12; The seventh primer pair includes an upstream primer with a nucleotide sequence as described in SEQ ID NO. 13 and a downstream primer with a nucleotide sequence as described in SEQ ID NO. 14; The eighth primer pair includes an upstream primer with a nucleotide sequence as described in SEQ ID NO. 15 and a downstream primer with a nucleotide sequence as described in SEQ ID NO. 16; The ninth primer pair includes an upstream primer with a nucleotide sequence as described in SEQ ID NO. 17 and a downstream primer with a nucleotide sequence as described in SEQ ID NO. 18; The tenth primer pair includes an upstream primer with a nucleotide sequence as described in SEQ ID NO. 19 and a downstream primer with a nucleotide sequence as described in SEQ ID NO. 20; The internal reference primer pair includes an upstream primer with a nucleotide sequence as described in SEQ ID NO. 21 and a downstream primer with a nucleotide sequence as described in SEQ ID NO.

22.

2. A kit for identifying species of *Acer palmatum*, characterized in that, Includes the multiplex PCR primer combination as described in claim 1.

3. The reagent kit according to claim 2, characterized in that, It also includes 5× multiplex amplification enzyme, 2× HiFi Mix, library construction reagents, and 96 indexes.

4. The use of the multiplex PCR primer combination of claim 1 or the kit of claim 2 or 3 in the identification of species of *Acer palmatum*.

5. A method for identifying species of *Acer palmatum* using multiplex PCR and sequencing analysis, characterized in that... Includes the following steps: Extract genomic DNA from the sample to be tested; Using the genomic DNA as a template, multiplex PCR amplification was performed using the multiplex PCR primer combination described in claim 1 to obtain multiplex PCR amplification products; The multiplex PCR amplification products were subjected to high-throughput sequencing to obtain sequencing data. The sequencing data were subjected to quality control and comparison, and the relative abundance of each species was assessed by counting the reads to complete sample identification.

6. The method according to claim 5, characterized in that, The amplification system for the multiplex PCR amplification, in 20 μL increments, comprises the following components: 4 μL of 5× multiplex amplification enzyme, 2 μL of the multiplex PCR primer combination, 1-2 μL of template, and the remainder being water; the final concentration of each primer in the multiplex PCR primer combination is 0.1-0.3 μM.

7. The method according to claim 5, characterized in that, The amplification program for the multiplex PCR amplification includes: 95℃ for 2 min; 95℃ for 10 s, 56℃ for 30 s, 72℃ for 30 s, 17 cycles; 72℃ for 5 min.

8. The method according to claim 5, characterized in that, Purification of the multiplex PCR amplification product includes purifying the multiplex PCR amplification product with magnetic beads, removing primer dimers and non-specific fragments, and retaining the target DNA fragment.

9. The method according to claim 5, characterized in that, The high-throughput sequencing includes DNBSEQ-T7 sequencing; High-throughput sequencing of the multiplex PCR amplification products includes: purifying the multiplex PCR amplification products to obtain purified products; adding adapters to the purified products to obtain sequencing libraries; and performing high-throughput sequencing on the sequencing libraries.

10. The method according to claim 5, characterized in that, The alignment includes aligning the quality-controlled sequencing fragments to the COI, COII, and CytB gene sequences; the reading count assessment of the relative abundance of each species includes calculating the number of aligned sequencing fragments and the alignment rate, and determining the species by the size of the indicator data.

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