A method and product suitable for on-site rapid detection of plant variety nucleic acid

Abstract

The application discloses a method and product suitable for on-site rapid detection of plant variety nucleic acids. The core of the application is to construct a general set of plant variety DNA on-site rapid detection technology. By designing a set of dominant primers and supporting detection technology, it is converted into a "dominant state / recessive state" interpretation mode suitable for on-site rapid detection, and a mobile, integrated and localized instant variety identification molecular rapid detection technology is developed to realize on-site sampling and rapid detection, quickly provide results, meet the rapid identification needs in the scenes of variety rights protection, seed supervision, enterprise self-checking and the like, and improve the variety identification efficiency.

Description

Technical Field

[0001] This invention belongs to the field of plant detection and breeding, specifically relating to a method and product for rapid on-site detection of nucleic acids in plant varieties. Background Technology

[0002] The seed industry is the core foundation of food security and agricultural modernization. Variety management is an important institutional guarantee for promoting agricultural modernization. As a key technology for maintaining agricultural production security and regulating the seed market order, variety identification, including the detection of variety authenticity and purity, is of great significance for protecting variety rights, improving seed management, and stimulating innovation in the seed industry.

[0003] Current plant variety identification techniques mainly rely on centralized molecular testing in laboratories, which suffers from problems such as long testing cycles, difficulties in sample return, and limited application at the grassroots level. Particularly in areas such as seed regulation, variety rights protection, and corporate rights enforcement, there is a lack of rapid, convenient, efficient, and universal molecular rapid testing techniques. These techniques suffer from low efficiency and poor adaptability, failing to meet the urgent needs of the current seed industry market for rapid, portable, and on-site plant variety identification.

[0004] Therefore, providing a rapid, portable, and on-site method for plant variety identification is of paramount importance in this field. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, this invention provides a method and product suitable for rapid on-site detection of plant varieties.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: The first aspect of the present invention provides a primer design method for rapid detection of nucleic acids in plant varieties. The method designs dominant primers based on the InDel site. The dominant primer design method is as follows: the 3' end of one dominant primer is designed within the InDel insertion sequence or at the deletion junction.

[0007] Furthermore, the dominant primer can be based on either strand of the double-stranded DNA as a template.

[0008] Furthermore, if the variant sequence at the InDel site is different from the adjacent downstream sequence, a dominant primer is designed using the template strand or the complementary strand as a template; if the variant sequence at the InDel site is similar to or the same as the adjacent downstream sequence, a dominant primer is designed using the complementary strand as a template.

[0009] Furthermore, InDel sites were screened using MAF, heterozygosity, deletion rate, size of the variant interval, and chromosome location as indicators.

[0010] Furthermore, dynamic programming algorithms were used to screen InDel sites.

[0011] Furthermore, we further screened InDel sites that were consistent with the genotyping results of co-dominant primers and dominant primers.

[0012] Furthermore, the plant in question is a monocotyledonous plant.

[0013] Furthermore, the monocotyledonous plant is a member of the Poaceae family.

[0014] Furthermore, the grass species mentioned is maize.

[0015] In some embodiments, the dominant primers are dominant-associated primers designed for the InDel site, and the genotype can be determined by the presence or absence of the amplification product.

[0016] In some embodiments, the codominant primers are primers designed with codominance associated with the InDel site, capable of distinguishing between homozygotes and heterozygotes.

[0017] In some embodiments, the plants include, but are not limited to, monocotyledons, dicotyledons, gymnosperms, ferns, and algae.

[0018] In some embodiments, the monocotyledonous plants include, but are not limited to, grasses (including but not limited to corn, rice, wheat, barley, sorghum, sugarcane, and bamboo), orchids (including but not limited to phalaenopsis, dendrobium, slipper orchid, and cattleya), lilies (including but not limited to lilies, tulips, onions, garlic, and aloe), palms (including but not limited to coconuts, oil palms, areca nuts, fan palms, and date palms), araceae (including but not limited to taro, taro, pothos, monstera, and calla lilies), sedges (including but not limited to water chestnuts, cyperus, onions, and sedges), irises (including but not limited to irises, gladioli, and saffron), and amaryllis (including but not limited to daffodils, clivia, amaryllis, zephyranthes, and lycoris).

[0019] In some embodiments, the dicotyledonous plants include, but are not limited to, legumes (including but not limited to soybeans, peanuts, and peas), cruciferous plants (including but not limited to rapeseed, cabbage, radish, and Arabidopsis), Rosaceae plants (including but not limited to apples, peaches, pears, roses, and strawberries), Solanaceae plants (including but not limited to potatoes, tomatoes, peppers, and tobacco), Cucurbitaceae plants (including but not limited to cucumbers, watermelons, pumpkins, and loofahs), Asteraceae plants (including but not limited to sunflowers, chrysanthemums, and lettuce), Apiaceae plants (including but not limited to carrots, celery, and fennel), Malvaceae plants (including but not limited to cotton, hibiscus, and okra), and Euphorbiaceae plants (including but not limited to rubber trees, cassava, and castor beans).

[0020] A second aspect of the present invention provides a method for screening InDel sites for plant variety detection, the method comprising: screening InDel sites using MAF, heterozygosity, deletion rate, size of variation interval and chromosome position as indicators.

[0021] Furthermore, the screening indexes meet any one or more of the following conditions: MAF≥0.3, heterozygosity≤0.05, deletion rate<0.05, variation interval size is 3-20bp, and they are evenly distributed on the chromosome.

[0022] Furthermore, the method also includes further screening InDel sites using a dynamic programming algorithm.

[0023] Furthermore, the method also includes further screening for InDel sites that are consistent with the typing results of the same DNA co-dominant primer and dominant primer.

[0024] Furthermore, the plant in question is a monocotyledonous plant.

[0025] Furthermore, the monocotyledonous plant is a member of the Poaceae family.

[0026] Furthermore, the grass species mentioned is maize.

[0027] Furthermore, the screening criteria for maize InDel sites must meet one or more of the following conditions: MAF > 0.4, heterozygosity < 0.02, deletion rate < 0.015, and variation size of 3-10 bp.

[0028] The third aspect of the invention provides any of the following tools: (1) A primer design system for plant variety detection, the system comprising: Primer design module: Design dominant primers based on template DNA, with the 3' end of one dominant primer designed within the InDel insertion sequence or at the deletion junction.

[0029] Furthermore, the system also includes: Template selection module: If the variant sequence at the InDel site is different from the adjacent downstream sequence, a dominant primer is designed using the template strand or the complementary strand as the template; if the variant sequence at the InDel site is similar to or the same as the adjacent downstream sequence, a dominant primer is designed using the complementary strand as the template.

[0030] Furthermore, the system also includes: InDel site screening module, the InDel site screening module includes: Indicator screening submodule: Screening InDel sites using MAF, heterozygosity, deletion rate, size of the variant interval, and chromosome location as indicators; Algorithm filtering submodule: Filters InDel sites based on dynamic programming algorithm; Primer screening submodule: Screening InDel sites based on the amplification results of co-dominant primers and dominant primers to obtain InDel sites with consistent genotyping results between co-dominant primers and dominant primers; (2) An InDel site screening system for plant variety detection, the system comprising: Indicator screening module: InDel sites are screened using MAF, heterozygosity, deletion rate, size of the variation interval, and chromosome location as indicators; Algorithm filtering module: Filters InDel sites based on dynamic programming algorithm; Primer screening module: Screens InDel sites based on the amplification results of co-dominant primers and dominant primers, and obtains InDel sites that are consistent with the genotyping results of co-dominant primers and dominant primers; (3) An apparatus comprising a memory and a processor, the memory being used to store program instructions; the processor being used to invoke the program instructions, which, when executed, implement the method described in the first or second aspect of the present invention; (4) A computer-readable storage medium having a computer program thereon, which, when executed by a processor, implements the method described in the first or second aspect of the present invention; (5) A computer program product comprising a computer program that, when executed by a processor, implements the method described in the first or second aspect of the present invention.

[0031] In some embodiments, the system, device, computer-readable storage medium, or computer program product may perform preliminary screening using the method described in the first or second aspect of the present invention. The system, device, computer-readable storage medium, or computer program product may display preliminary design results (candidate primers, parameters, preliminary validation data) to the user, who can manually adjust primer sequences and optimize parameters according to the detailed requirements of the customer's design method. The program provides real-time feedback on performance changes after adjustments, assisting the user in completing the optimal design.

[0032] A fourth aspect of the present invention provides an InDel site for plant variety detection, wherein the InDel site is obtained by screening using the method described in the second aspect of the present invention or the tool described in the third aspect of the present invention.

[0033] Furthermore, the plant in question is a monocotyledonous plant.

[0034] Furthermore, the monocotyledonous plant is a member of the Poaceae family.

[0035] Furthermore, the grass species mentioned is maize.

[0036] Furthermore, the InDel sites of the maize include any one or more of MKT01-MKT16, wherein the mutation initiation position of MKT01 is at position 278,976,599 on chromosome 1, the mutation initiation position of MKT02 is at position 24,653,642 on chromosome 2, the mutation initiation position of MKT03 is at position 88,426,353 on chromosome 2, the mutation initiation position of MKT04 is at position 172,717,920 on chromosome 3, the mutation initiation position of MKT05 is at position 124,035,057 on chromosome 3, the mutation initiation position of MKT06 is at position 192,970,579 on chromosome 4, the mutation initiation position of MKT07 is at position 14,612,889 on chromosome 5, and the mutation initiation position of MKT08 is at position 5. The mutations of MKT09 and MKT010 originate from chromosome 6 at position 15,209,041. MKT09 originates from chromosome 6 at position 159,829,453. MKT010 originates from chromosome 6 at position 3,240,073. MKT011 originates from chromosome 6 at position 138,858,575. MKT012 originates from chromosome 6 at position 132,187,454. MKT013 originates from chromosome 7 at position 151,285,049. MKT014 originates from chromosome 8 at position 159,528,845. MKT015 originates from chromosome 9 at position 113,082,501. MKT016 originates from chromosome 10 at position 145,370,739.

[0037] Furthermore, the sequence of the InDel site in maize is shown in any one or more of SEQ ID NO:1-16.

[0038] The fifth aspect of the present invention provides a primer set for plant variety detection, the primer set comprising primers designed by the method described in the first aspect of the present invention, primers for amplifying the InDel site obtained by the method described in the second aspect of the present invention, primers designed by the tool described in the third aspect of the present invention, primers for amplifying the InDel site obtained by the tool described in the third aspect of the present invention, or primers for amplifying the InDel site described in the fourth aspect of the present invention.

[0039] Furthermore, the plant in question is a monocotyledonous plant.

[0040] Furthermore, the monocotyledonous plant is a member of the Poaceae family.

[0041] Furthermore, the grass species mentioned is maize.

[0042] Furthermore, the primer set for the corn includes primers capable of amplifying the corn InDel site described in the fourth aspect of the present invention.

[0043] Furthermore, the primer sequence of the corn is shown as any one or more of SEQ ID NO:17-48 or SEQ ID NO:49-80.

[0044] In some embodiments, the primer set further includes primers having at least 90% sequence identity with the sequences shown in SEQ ID NO:17-48 or SEQ ID NO:49-80, preferably at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. These sequence identity ranges do not significantly affect the primers' ability to function, and the sequence identity ranges provided above are for guidance only; primers outside these ranges may also be used.

[0045] In some embodiments, the primer set further includes a degenerate sequence of the primer set's nucleotide sequence. The degenerate sequence encodes the same protein as the nucleotide sequence shown in SEQ ID NO:17-48 or SEQ ID NO:49-80, but differs from the nucleotide sequence shown in SEQ ID NO:17-48 or SEQ ID NO:49-80 due to the degeneracy of the genetic code. The degenerate position of the degenerate sequence includes R, W, M, Y, K, B, D, H, N, S, or V, wherein R includes A or G, W includes A or T, M includes A or C, Y includes C or T, K includes G or T, B includes A, G, or T, D includes A, G, or T, H includes A, C, or T, N includes A, G, C, or T, S includes G or C, and V includes A, C, or G.

[0046] A sixth aspect of the present invention provides a product for plant variety detection, the product comprising primers designed by the method described in the first aspect of the present invention, primers for amplifying the InDel site obtained by the method described in the second aspect of the present invention, primers designed by the tool described in the third aspect of the present invention, primers for amplifying the InDel site obtained by the tool described in the third aspect of the present invention, primers for amplifying the InDel site described in the fourth aspect of the present invention, or a primer set described in the fifth aspect of the present invention.

[0047] Furthermore, the primer set includes individually packaged single primers, mixed-package primer mixtures, and primer premixes premixed with buffer solution.

[0048] Furthermore, the primer set includes lyophilized primers, liquid primers, and solid primers.

[0049] Furthermore, the solid primers are adsorbed onto the carrier.

[0050] Furthermore, the carrier includes a filter membrane, microspheres, a chip, and a test strip.

[0051] Furthermore, the product includes reagent kits, chips, and test strips.

[0052] Furthermore, the product also includes amplification reaction reagents.

[0053] Furthermore, the amplification reaction reagents include buffer, enzyme, and dNTPs.

[0054] Furthermore, the buffer solution includes Tris-HCl, KCl, betaine, and BSA.

[0055] Furthermore, the enzyme includes DNA polymerase.

[0056] Furthermore, the DNA polymerase includes Taq, Vent, Pfu, Klenow, Phi29, and Bst.

[0057] Furthermore, the product also includes extraction reagents, washing reagents, and reference standards.

[0058] Furthermore, the extraction reagents include Tris-HCl, NaCl, EDTA, and SDS.

[0059] Furthermore, the washing reagent includes Tween 20, Tris-HCl, isopropanol, and ethanol.

[0060] Furthermore, the product also includes a sample processing component.

[0061] Furthermore, the sample processing component includes a punch and a grinding device.

[0062] Furthermore, the product also includes an instruction manual.

[0063] Furthermore, the product also includes detectable reagents.

[0064] Furthermore, the detectable reagents include fluorescent dyes and enzymes.

[0065] In some embodiments, the instruction manual describes sample processing methods, product testing procedures, PCR program settings (such as real-time quantitative PCR program settings), Ct value determination threshold rules, and result determination principles.

[0066] In some embodiments, the fluorescent dyes include, but are not limited to, fluorescein isothiocyanate (FITC), carboxyfluorescein (FAM), tetramethylrhodamine (TAMRA), cyanine dyes (Cy3, Cy5, Cy7), indocyanine green (ICG), and dansyl chloride (Dansyl); the enzymes include, but are not limited to, alkaline phosphatase, horseradish peroxidase, fluorescent reagents used in the SYBR Green protocol (such as SYBR Green I), and TaqMan probes (reporter groups: such as VIC, HEX, TET, JOE, ROX, NED, Texas Red; quencher groups: such as TAMRA, BHQ series, MGB).

[0067] In some embodiments, as additional equipment required in the product, examples include pipettes, pipette tips, PCR tubes, and other equipment widely used in molecular biology experiments; as devices, examples include PCR instruments (such as real-time quantitative PCR instruments), clean benches, mixers, centrifuges, and other instruments widely used in molecular biology experiments.

[0068] The seventh aspect of the present invention provides a method for detecting plant varieties, wherein the method uses primers designed by the method described in the first aspect of the present invention, primers for amplifying the InDel site obtained by the method described in the second aspect of the present invention, primers designed by the tool described in the third aspect of the present invention, primers for amplifying the InDel site obtained by the tool described in the third aspect of the present invention, primers for amplifying the InDel site described in the fourth aspect of the present invention, primer sets described in the fifth aspect of the present invention, or products described in the sixth aspect of the present invention for detection.

[0069] Furthermore, the method also includes visual interpretation of the detection results.

[0070] Furthermore, the visualization interpretation involves determining the dominant / recessive state of the detection results.

[0071] Furthermore, plant variety detection can be achieved through the determination of dominant / recessive forms.

[0072] Furthermore, primer amplification techniques include thermal cycling amplification and isothermal amplification.

[0073] Furthermore, the thermal cycling amplification technique includes PCR.

[0074] Furthermore, the PCR includes qPCR and dPCR.

[0075] Furthermore, the isothermal amplification techniques include RPA, LAMP, RCA, SDA, and HDA.

[0076] Furthermore, the primer set detection platform includes electrophoresis, gene chip, KASP platform, and sequencing.

[0077] Furthermore, the electrophoresis includes gel electrophoresis and capillary electrophoresis.

[0078] Furthermore, the gene chip includes microbead chips, laser micro-etching chips, and magnetic bead chips.

[0079] Furthermore, the sequencing includes site-directed sequencing.

[0080] Furthermore, the plant in question is a monocotyledonous plant.

[0081] Furthermore, the monocotyledonous plant is a member of the Poaceae family.

[0082] Furthermore, the grass species mentioned is maize.

[0083] Furthermore, the range threshold Ct0 is calculated based on the following formula for visual interpretation of maize varieties:

[0084] Among them, Ct I-MAX The maximum Ct value of the dominant amplification curve; Ct D-MIN This represents the minimum Ct value of the recessive state amplification curve.

[0085] Furthermore, the conversion rules for visualization interpretation are as follows: Ct < 28 is converted to 1, Ct > 30 is converted to 0, and 28 ≤ Ct ≤ 30 is converted to 9, generating a DNA fingerprint database for on-site detection of maize variety authenticity consisting of 16 digits.

[0086] Furthermore, the endpoint fluorescence detection method was used to visually identify maize varieties.

[0087] In some implementations, fluorescence signals can be collected at any suitable cycle, such as cycles 31, 32, 33, 34, and 35. The fluorescence signal values ​​for different cycles depend on the instrument used, therefore, fluorescence signal conversion is required using a reference sample with a known genotype. Taking a fluorescence quantitative PCR instrument collecting fluorescence signals in cycle 31 as an example, a fluorescence signal value < 500,000 is considered a recessive genotype, and the result is converted to 0; a fluorescence signal value > 1,000,000 is considered a dominant genotype, and the result is converted to 1. The relevant threshold conversion is consistent with the co-dominance detection result of the reference sample. Finally, a 16-digit DNA fingerprint of maize variety authenticity for on-site detection is formed based on the fluorescence signal conversion under this cycle.

[0088] In some implementations, thermal cycling amplification technology utilizes a thermal cycler (PCR instrument) to control temperature and achieve exponential DNA amplification through a cycle of high-temperature denaturation (unwinding of double strands), low-temperature annealing (primer binding), and medium-temperature extension (synthesis of new strands). This thermal cycling amplification technology includes PCR, which includes, but is not limited to, qPCR (qPCR, real-time quantitative PCR) and dPCR (digital PCR).

[0089] In some implementations, the primer sets shown in any one of SEQ ID NO:17-48, combined with real-time quantitative PCR technology, can enable rapid on-site detection of maize varieties.

[0090] In some implementations, the primer set shown in any one of SEQ ID NO:49-80 is used in conjunction with capillary electrophoresis for detection, and a fingerprint database is established to enable rapid on-site detection of maize varieties using the fingerprint database.

[0091] In some implementations, isothermal amplification technology utilizes specialized enzyme systems (such as strand displacement enzymes, recombinases, etc.) to achieve DNA melting and synthesis at a single constant temperature (typically 37°C-65°C), without the need for a thermal cycler. These isothermal amplification technologies include, but are not limited to, RPA (Recombinase Polymerase Amplification), LAMP (Loop-mediated Isothermal Amplification), RCA (Rolling Circle Amplification), SDA (Strand Displacement Amplification), and HDA (Helicase-dependent Isothermal Amplification).

[0092] In some implementations, isothermal amplification technology can be combined with CRISPR-Cas protein detection technology. CRISPR-Cas protein detection technology utilizes Cas proteins (such as Cas12, Cas13, Cas9, or Cas14) to specifically recognize and bind to target DNA or RNA, guided by guide RNA (gRNA).

[0093] In some implementations, the method for rapid on-site detection of plant varieties based on dominant InDel markers specifically includes: (1) Collect plant samples to be tested and extract their nucleic acids on site quickly.

[0094] (2) The nucleic acid of the sample to be tested was amplified and detected on-site using the designed dominant primers to obtain the amplification signal of each InDel site.

[0095] (3) Based on the amplification signal, each InDel site is converted into a dominant state, a recessive state, or an undeterminable state to form a digital feature code of the sample to be tested.

[0096] (4) Compare the digital feature code of the sample to be tested with the feature code of the reference sample or the reference feature library, and output the on-site detection results of the plant variety.

[0097] The eighth aspect of the present invention provides any of the following tools: (1) A plant variety detection system, the system comprising: Data receiving module: receives primers designed by the method described in the first aspect of the present invention, primers for amplifying the InDel site obtained by the method described in the second aspect of the present invention, primers designed by the tool described in the third aspect of the present invention, primers for amplifying the InDel site obtained by the tool described in the third aspect of the present invention, primers for amplifying the InDel site described in the fourth aspect of the present invention, primer sets described in the fifth aspect of the present invention, or product amplification data obtained by the sixth aspect of the present invention. The amplified data is compared with a preset plant variety characteristic database; Output plant variety identification results; (2) An apparatus comprising a memory and a processor, the memory being used to store program instructions; the processor being used to invoke the program instructions, which, when executed, implement the method described in the seventh aspect of the present invention; (3) A computer-readable storage medium having a computer program thereon, which, when executed by a processor, implements the method described in the seventh aspect of the present invention; (4) A computer program product comprising a computer program that, when executed by a processor, implements the method described in the seventh aspect of the present invention.

[0098] Furthermore, the plant in question is a monocotyledonous plant.

[0099] Furthermore, the monocotyledonous plant is a member of the Poaceae family.

[0100] Furthermore, the grass species mentioned is maize.

[0101] In some implementations, the system may be implemented manually, automatically, or in combination thereof to perform or complete the selected tasks. Furthermore, the actual instruments and equipment used in the implementations of the methods and / or systems of this application may implement multiple selected tasks via hardware, software, firmware, or a combination thereof using an operating system.

[0102] In some embodiments, the memory includes one or more of the following: a non-transitory computer-readable medium; a magnetic disk or other magnetic storage medium; an optical disk or other optical storage medium; random access memory (RAM), read-only memory (ROM) or other electronic memory devices or a collection of chips or operatively interconnected chips. The processor includes one or more of a microprocessor, microcontroller, graphics processing unit (GPU), application-specific integrated circuit (ASIC), and field-programmable gate array (FPGA).

[0103] In some embodiments, computer-readable storage media include, but are not limited to, electrical, magnetic, optical, electromagnetic, infrared, or semiconductor devices or any combination thereof. Specifically, computer-readable storage media include: electrical connections having one or more wires, portable computer disks, hard disks, random access memory (RAM), read-only memory (ROM), erasable programmable read-only memory (EPROM or flash memory), optical fibers, portable compact disk read-only memory (CD-ROM), optical storage devices, magnetic storage devices, or any suitable combination thereof. In this application, a computer-readable storage medium can be any tangible medium containing or storing a program that can be used by or in conjunction with an instruction execution system, device, or apparatus.

[0104] In some implementations, the data receiving module in the system processes the plant variety amplification result data (such as signal peak analysis, threshold comparison, etc.) and generates a detection report (including sample information, detection parameters, result judgment, signal spectrum, etc.).

[0105] In some implementations, amplification results, comparison results, or plant variety identification results can be output via system printing or data export (electrical transmission to terminal devices).

[0106] In some embodiments, the system or device includes equipment used in thermal cycling amplification technology and isothermal amplification technology; computer-readable storage media and computer program products are connectable to the equipment used in thermal cycling amplification technology and isothermal amplification technology. The equipment used in thermal cycling amplification technology includes PCR instruments (including but not limited to qPCR instruments and dPCR instruments), and the equipment used in isothermal amplification technology includes isothermal amplification instruments, isothermal fluorescence detectors, etc.

[0107] In some embodiments, the system, device, computer-readable storage medium, and computer program product have a built-in computer program / processor. This computer program / processor has built-in algorithms (such as signal thresholding algorithms and specificity comparison algorithms) to analyze the collected electrical signal data, compare the signal spectra of positive and negative controls, determine whether the sample contains the target plant variety, and simultaneously verify the detection data (such as repeatability analysis and error calculation). The system automatically stores the detection results, detection parameters, signal spectra, sample information, and other data in a database (data backup can be achieved via an electrical network). Users can view the detection results through the program interface and can also instruct the program to print reports and export data, completing the entire detection process.

[0108] In some embodiments, the computer-readable storage medium and the result terminal of the computer program product can be a computer, mobile phone, etc. Taking corn as an example, the primer set shown in any one of SEQ ID NO:17-48 can be used in conjunction with real-time fluorescence quantitative PCR technology to achieve rapid on-site detection of corn varieties; alternatively, the primer set shown in any one of SEQ ID NO:49-80 can be used in conjunction with capillary electrophoresis for detection, and a fingerprint database can be established. The fingerprint database can then be used to achieve rapid on-site detection of corn varieties, and the detection results can be obtained on terminal devices such as computers and mobile phones.

[0109] In some implementations, the steps for plant variety detection using the above-described tools include: 1) Sample preparation: Collect plant samples (leaves, seeds, fruits, tissues, etc.) to be tested, perform simple pretreatment according to conventional methods (such as crushing and grinding) to obtain sample homogenate for later use.

[0110] 2) Primer and reagent preparation: Prepare the reaction system by mixing the primer set (lyophilized powder / solution form) with the auxiliary reagents required for nucleic acid amplification (buffer, dNTPs, DNA polymerase, etc.) in the specified proportions.

[0111] 3) Amplification and data transmission: Input the amplification parameters of the appropriate primer set (such as temperature gradient, reaction time, number of cycles), the computer program / processor sends electrical control instructions to execute the amplification program; after amplification is completed, the computer program / processor sends instructions to start the fluorescence detector (if necessary), the detector collects the fluorescence signal of the reaction system, converts the signal into an electrical signal, transmits it to the computer program / processor, and automatically records the signal data and plots the signal spectrum.

[0112] 4) Result determination: The computer program / processor analyzes the collected electrical signal data through built-in algorithms (such as signal threshold algorithm and specificity comparison algorithm), compares the signal spectra of positive and negative controls, determines whether the sample to be tested contains the target plant species, and verifies the detection data.

[0113] In some embodiments, the methods, systems, devices, computer-readable storage media, or computer program products of this application may be implemented in the form of a completely hardware embodiment, a completely software embodiment, or an embodiment combining software and hardware aspects. The embodiments described above are merely illustrative. For example, the division of modules is only a logical functional division; in actual implementation, there may be other division methods. For instance, multiple modules or components may be combined or integrated into another system, or some features may be ignored or not executed. Furthermore, the couplings or direct couplings or communication connections shown or discussed may be indirect couplings or communication connections through some interfaces, devices, or modules, and may be electrical, mechanical, or other forms.

[0114] The ninth aspect of the present invention provides a method for constructing a plant variety fingerprint database. The method includes amplifying the InDel site obtained by the method described in the first aspect of the present invention, amplifying the InDel site obtained by the method described in the second aspect of the present invention, amplifying the InDel site obtained by the tool described in the third aspect of the present invention, amplifying the InDel site obtained by the tool described in the third aspect of the present invention, amplifying the InDel site described in the fourth aspect of the present invention, a primer set described in the fifth aspect of the present invention, or a product described in the sixth aspect of the present invention to obtain a plant variety fingerprint database.

[0115] Furthermore, the plant in question is a monocotyledonous plant.

[0116] Furthermore, the monocotyledonous plant is a member of the Poaceae family.

[0117] Furthermore, the grass species mentioned is maize.

[0118] In some embodiments, the construction method uses the visualization and conversion rules described in the seventh aspect of the present invention to construct a plant variety fingerprint database.

[0119] The tenth aspect of the present invention provides a plant variety fingerprint database, which is constructed by the method described in the ninth aspect of the present invention.

[0120] Furthermore, the plant in question is a monocotyledonous plant.

[0121] Furthermore, the monocotyledonous plant is a member of the Poaceae family.

[0122] Furthermore, the grass species mentioned is maize.

[0123] The eleventh aspect of the present invention provides the application of primers designed by the method described in the first aspect of the present invention, primers for amplifying the InDel site obtained by the method described in the second aspect of the present invention, primers designed by the tool described in the third aspect of the present invention, primers for amplifying the InDel site obtained by the tool described in the third aspect of the present invention, primers for amplifying the InDel site described in the fourth aspect of the present invention, primer sets described in the fifth aspect of the present invention, or products described in the sixth aspect of the present invention in plant variety detection.

[0124] Furthermore, the plant variety testing includes plant variety identification, plant genetic breeding, and plant germplasm resource analysis.

[0125] Furthermore, the plant variety identification includes plant variety authenticity identification and plant purity identification.

[0126] Furthermore, the identification of the authenticity of the plant varieties includes plant pair comparison and database screening comparison.

[0127] Furthermore, the plant genetic breeding includes plant genetic analysis.

[0128] Furthermore, the plant in question is a monocotyledonous plant.

[0129] Furthermore, the monocotyledonous plant is a member of the Poaceae family.

[0130] Furthermore, the grass species mentioned is maize.

[0131] Advantages and beneficial effects of the present invention: 1. High method versatility: The universal Point-of-Variety Identification (POVID) technique developed in this application is applicable to rapid on-site detection of plant variety DNA. It can be extended to crops such as rice and wheat simply by changing the core InDel primers for the target plant. Different primers and systems can be designed to meet different needs such as variety authenticity, purity, and transformant purity, achieving a leap from laboratory-dependent identification to on-site, portable detection.

[0132] 2. Extremely fast and efficient process: The entire process can be completed within 1 hour, which is an order of magnitude more efficient than traditional methods, meeting the rapid screening needs of fields, seed production bases and other scenarios.

[0133] 3. Digitalized results and prevention of contamination: Closed-tube testing avoids electrophoresis with the cap open, significantly reducing the risk of contamination; the output result is a binary digital barcode (DNA fingerprint database), which facilitates cross-platform comparison.

[0134] 4. This application further provides a more specific and optimized Maize Point-of-Genuineness Identification (M-POG) kit for on-site detection of authentic maize inbred lines. Based on a premixed reaction system optimized for 16 core InDel sites, combined with a portable real-time quantitative PCR platform, the entire process takes only about 42 minutes. It exhibits high consistency with the national standard SSR method, providing reliable results. It comes with ready-to-use extraction components and standardized consumables, requiring no laboratory equipment or professional training, and is suitable for non-laboratory environments such as fields and seed production bases. It can quickly screen for samples with significant differences, effectively reducing the number of samples submitted for testing and significantly lowering time and economic costs. It is suitable for on-site enforcement of seed quality supervision and variety rights protection. Attached Figure Description

[0135] Figure 1 These are primer design diagrams, where 1A is a flanking primer design diagram, 1B is a competitive primer design diagram, 1C is a dominant primer design diagram, and 1D is a detailed diagram of a dominant primer design. Figure 2 These are M-POG candidate locus feature maps, where 2A is a map showing the distribution of candidate loci on maize chromosomes, 2B is a map showing the proportion of different variation sizes of candidate loci, and 2C is a statistical map of MAF of candidate loci. Figure 3 These are the amplification results of dominant primers. Among them, 3A and 3B are images showing clear distinction between dominant and recessive states in the test samples when the dominant primers (C-KT09 and C-KT24) were successfully designed. 3C is an image showing no recessive state genotyping in the test samples when the dominant primers were designed incorrectly. 3D is an image showing no recessive state genotyping or no amplification in the test samples when the dominant primers were designed incorrectly. 3E is an image showing unclear distinction between dominant and recessive states in the test samples when the dominant primers were designed incorrectly. 3F is an image showing fluorescence signals and obvious primer dimers in the blank control when the dominant primers were designed incorrectly. Figure 4 This is a diagram showing the M-POG barcode conversion rules and judgment thresholds. In this diagram, 4A is a conversion diagram of the Ct value of the core primer amplification and the result, and 4B is a comparison diagram of the number of differences in the core site. Figure 5 This is a schematic diagram of RPA primer and crRNA design; Figure 6 This is a visualization of the amplification results; Figure 7 These are schematic diagrams of DNA extraction procedures and qPCR results. 7A and 7B are schematic diagrams of DNA extraction procedures, and 7C is a schematic diagram of qPCR results. Figure 8 This is the capillary electrophoretic fingerprint of the codominant primers eMKT13 (left) and eMKT14 (right); Figure 9 This is a schematic diagram comparing the results of the M-POG method with the standard method; Figure 10 This is a comparison chart of the average time taken per sample for the M-POG method and the standard method. 10A is a schematic diagram of the kit operation process, and 10B is a comparison chart of the time taken for the two identification methods. Detailed Implementation

[0136] The invention is further illustrated below with reference to specific embodiments. It should be understood that the specific embodiments described herein are by way of example and are not intended to limit the invention. The main features of the invention can be used in various embodiments without departing from the scope of the invention.

[0137] Example 1: Dominant primer design scheme suitable for field identification of varieties Existing technologies, such as conventional flanking primers suitable for electrophoresis platforms and competitive primers suitable for high-throughput detection platforms, cannot achieve on-site identification of varieties. The dominant primer design in this application is a short single-stranded primer that binds complementary to the template DNA strand, providing a 3'-OH end as the starting point for DNA synthesis, extending to synthesize the complementary strand of the template DNA. The 3' end of one of the short single strands is designed within an InDel insertion sequence or a deletion junction. The dominant primer design differs from the design strategies of flanking and competitive primers. As shown in Table 1, flanking primers require polymorphism identification, making conventional electrophoresis equipment unsuitable for on-site detection. Competitive primer designs require three primers, while the dominant primer design only requires one upstream and downstream primer pair, resulting in lower requirements for the reaction system. The dominant primer is compatible with real-time quantitative PCR or visual detection schemes, achieving the goal of immediate detection.

[0138] When designing InDel-labeled dominant primers, if the variant sequence is highly similar to or identical to the immediately adjacent downstream sequence, the complementary strand should be used as a template, and a reverse design scheme should be employed to design the primers. Figure 1 Finally, the success of primer design was qualitatively determined using a real-time quantitative PCR platform, with the following criteria: Figure 3 As shown.

[0139] Table 1. Comparison of InDel marker primer designs suitable for variety identification

[0140] Example 2: POVID General Technical Process The main process includes the following three steps: crude sample extraction --- rapid / isothermal amplification reaction --- fingerprint conversion and result interpretation.

[0141] a. Crude extraction of samples: A rapid DNA extraction protocol for plant leaves / seeds was established, using a two-step grinding method that requires no laboratory equipment and can obtain DNA within 3 minutes.

[0142] b. Rapid / Isothermal Amplification Reaction: Add crude DNA to the reaction system and run a rapid or isothermal program on a portable qPCR instrument or isothermal device. The entire process takes about 20-40 minutes.

[0143] c. Fingerprint Conversion and Result Interpretation: If qPCR is used for interpretation, the amplified Ct result is converted into a 16-bit rapid detection fingerprint based on a preset Ct threshold. If isothermal amplification reactions such as RPA / LAMP are used, the presence / absence of the locus is determined based on the test strip reaction, colorimetric reaction, or precipitation reaction, and then converted into a rapid detection fingerprint. The rapid detection fingerprint of the sample is compared with the control sample / control sample library, or a rapid purity analysis is performed.

[0144] The selected InDel loci should possess the following characteristics: (a) abundant polymorphism, (b) good reproducibility, (c) uniform distribution throughout the genome, and (d) neutral loci. The method includes screening InDel loci based on indicators such as an MAF of not less than 0.3, a heterozygosity of not more than 0.05, a deletion rate of less than 0.05, a variation range of 3-20 bp, and uniform distribution on the chromosome.

[0145] Example 3: Screening of core loci for rapid detection of maize authenticity and evaluation of dominant primers (1) Locus Assessment and Screening: Using a database of 5000 loci from 2270 maize inbred lines based on the Maize IDP50k chip, the selection criteria included minimum allele frequency (MAF), heterozygosity, deletion rate, variation range size, and chromosome location. Dynamic programming was used for optimization to select a set of superior InDel candidate loci. Specifically, MAF > 0.4, heterozygosity < 0.02, deletion rate < 0.015, and variation size of 3-10 bp were used as selection criteria to obtain 506 M-POG candidate InDel loci from the 5000 superior loci in the Maize IDP50k chip. For example... Figure 2 As shown in Figure A, the above-mentioned sites are relatively evenly distributed across the maize genome, with sites having a variation size of 3-4 bp accounting for 48% ( Figure 2 B), its average MAF is 0.45-0.46 ( Figure 2 C).

[0146] (2) Dominant primer design and testing: Dominant and co-dominant primers were designed for the above loci. Further screening of candidate loci with high amplification efficiency and consistent results was conducted. Loci exhibiting uniform amplification efficiency, clear distinction between dominant and recessive genotypes, and good reproducibility in the 12 backbone inbred lines were considered as successfully designed dominant primers. Figure 3 A, Figure 3 B), no genotyping or amplification failure in the tested material ( Figure 3 C, Figure 3 D) Indistinct subtypes ( Figure 3 E) or the presence of obvious primer dimers ( Figure 3 Sites with issues such as F) were identified as failures in dominant primer design. Further analysis was conducted on sites where dominant and co-dominant results were consistent.

[0147] Figure 3 The number at the peak position represents the Ct value. There is a clear boundary between the two genotypes. The Ct values ​​of the dominant genotype are all less than 27, and the Ct values ​​of the recessive genotype are all greater than 31 or there is no curve (considered as a Ct value of 35). The amplification curves of the two types are closely aligned, indicating that the amplification efficiency is uniform and the reproducibility is good. Taking the three loci in Table 2 as examples, the Ct values ​​of the dominant genotypes of the dominant primers were all less than 27, while the Ct values ​​of the recessive genotypes of the dominant primers were all greater than 31. The co-dominant primer results corresponding to the dominant genotypes contained larger fragments containing the InDel variant region. For example, at the C-KT12 locus, the fragment size of the co-dominant primer amplification product of Huangzao 4 was 338 / 338 containing a 4bp variant, and its dominant primer genotyping result was a dominant genotype with a Ct value of 25.1. In contrast, the fragment size of the co-dominant primer amplification product of Jing 724 was 334 / 334 without the 4bp variant, and its dominant primer genotyping result was a recessive genotype with a Ct value of 35.0. These results indicate that the dominant primer and co-dominant results are consistent. After screening out the loci with consistent results, the 16 core loci with the highest variety identification rate were further obtained.

[0148] Table 2. Statistical analysis of amplification results using co-dominant and dominant primers.

[0149] Example 4: Fingerprint Conversion and Result Interpretation To achieve rapid and standardized comparison of test results, this study systematically analyzed qPCR amplification data of the InDel core site, established the Ct threshold range for genotype determination and the rules for constructing digital barcodes.

[0150] The range threshold Ct0 is calculated according to the following inequality, where “1” represents the dominant state, “0” represents the recessive state, and “9” represents the state that cannot be determined.

[0151]

[0152] Note: Ct I-MAX : Maximum Ct value of the dominant amplification curve; Ct D-MIN Minimum Ct value of recessive amplification curve The Ct values ​​of the dominant and recessive genotypes at the core locus of the 12 tested materials showed significant differences. The Ct value for the dominant genotype ranged from 22.49 to 26.91, while that for the recessive genotype ranged from 31.92 to 35.00 (the recessive genotype that did not show an amplification curve after exceeding the set number of cycles was recorded as having a Ct value of 35.00). The Ct values ​​of the two genotyping schemes differed significantly. The calculation results showed that the threshold range between the two was 28 ≤ Ct0 ≤ 30. Figure 4 Based on this, the fingerprint conversion rules are defined as follows: referring to Ct0, homozygous dominant genotype II and heterozygous dominant genotype ID with Ct < 28 are determined as dominant and converted to 1; homozygous recessive genotype DD with Ct > 30 and data missing are determined as recessive and converted to 0; results with 28 ≤ Ct ≤ 30 are determined as undeterminable and converted to 9, thereby generating a 16-digit DNA barcode for the on-site detection of maize varieties.

[0153] Example 5: RPA primer development using maize as an example and visualization detection using CRISPR / Cas12a technology. Using the Chinese maize backbone inbred lines LX9801, Zheng 58, and Chang 7-2 as materials, a superior InDel site RC001 (Table 3) obtained through the above screening method was selected for primer design. Figure 5 As shown, two pairs of RPA primers and two crRNAs were designed and synthesized. Amplification was performed according to the TwistAmp kit instructions. Maize IDP50k microarray fingerprint data for this site was used as a control. Figure 6 As shown, the detection results were obtained under ultraviolet light irradiation. From left to right, they are LX9801, Zheng 58, Chang 7-2, and blank control. Green fluorescence indicates that the sample is in the dominant state at the RC001 site, while no fluorescence indicates that the sample is in the recessive state at the RC001 site.

[0154] Table 3 RC001 site information

[0155] Example 6: Primer set for maize variety authenticity identification (M-POG) Detailed information on the M-POG core site is shown in Table 4. Detailed information on the core site primers based on the real-time quantitative PCR platform is shown in Table 5. Detailed information on the core site primers based on the fluorescence capillary electrophoresis platform is shown in Table 6.

[0156] Table 4. Information on M-POG core sites

[0157] Table 5. Information on core site primers designed based on the real-time quantitative PCR platform.

[0158] Table 6. Information on core site primers designed based on the fluorescence capillary electrophoresis platform.

[0159] Twelve core inbred lines of Chinese maize were selected as test materials. The sample names of these 12 core inbred lines are: Jing 92, Jing 724, LX9801, Huangzao 4, Zheng 58, Zong 3, Qi 319, Ye 478, Chang 7-2, Zheng 58 (approximate sample A), Huangzao 4 (approximate sample B), and Qi 319 (approximate sample C). Seeds were cultured in moist sand under light for 7 days. DNA extraction was performed using a two-step grinding method. A schematic diagram of the grinding operation is shown below. Figure 7 As shown in A and 7B. The extraction buffer consisted of 15-25 mmol / L Tris-HCl, 20-30 mmol / L NaCl, 0.3-0.6 mmol / L EDTA, and 0.02%-1.08% SDS. The washing buffer consisted of 0.08%-0.12% Tween 20 and 8-12 mmol / L Tris-HCl. Simultaneously, control DNA was extracted from 12 samples using the Tiangen Biotech DNA Extraction Kit.

[0160] The following reaction system and program were used for nucleic acid amplification using the real-time quantitative PCR (qPCR) platform. The reaction system consisted of 5 μL DNA, 0.4 μmol / L each of 2×TB Green Premix Ex Taq II (Tli RNaseH Plus) and forward and reverse primers, 0.4 μL ROX Reference Dye, and ddH2O to a final volume of 20 μL. The reaction program was 95 °C for 30 s; 95 °C for 5 s, 60 °C for 30 s, for 35 cycles. Fluorescence signals were collected at the end of each cycle. The amplification results are shown in the diagram below. Figure 7 As shown in C.

[0161] The amplification reaction on the fluorescent capillary electrophoresis platform was performed using a system and procedure based on the universal tailed sequence (UTS). The amplified products were detected by fluorescent capillary electrophoresis using a DNA fragment analyzer (Applied Biosystem, 3730XL). Raw data were collected using Date Collection V1.0 software and analyzed using SSRAnalyser (V1.2.6). The capillary electrophoresis results were analyzed using the codominant primer eMKT13 (… Figure 8Left), eMKT14 ( Figure 8 Taking the right as an example, the recessive genotype of eMKT13 is 140 / 140, and the dominant genotype is 145 / 145. The recessive genotype of eMKT14 is 148 / 148, and the dominant genotype is 155 / 155.

[0162] Twelve maize samples were identified using the M-POG protocol (real-time fluorescence PCR) and the national standard detection method (GB / T 39914-2021), respectively. The percentage of different sites between pairs of samples was statistically analyzed for each method.

[0163]

[0164] A scatter plot was drawn based on the proportion of differing sites from the two methods. For example... Figure 9 As shown, based on the judgment thresholds of 16 core sites and 40 SSR sites of M-POG, the following will be used: Figure 9 The samples were divided into four regions, with all results falling within regions a, b, and c. 90.91% (60 groups) of the samples were identified as having differences using both methods (region b), indicating a high degree of reliability. This allows for rapid on-site screening of samples with significant differences, serving as a direct exclusion criterion in the field or seed production base, thus significantly reducing the number of samples requiring laboratory testing. Three groups of samples—Zheng 58 and its similar sample A, Huangzao 4 and its similar sample B, and Qi 319 and its similar sample C—had results below the threshold using both methods (region c), demonstrating high accuracy when detecting identical or very similar samples. Three other groups of samples fell below the on-site detection threshold but could be identified using SSR markers (region a). No comparison results fell into region d, indicating no missed detections where the kit detected differences but SSR detection showed no differences. These results effectively demonstrate the reliability and accuracy of the M-POG detection's "difference exists" result, significantly reducing the number of samples requiring laboratory testing and effectively lowering time and economic costs. For the purpose of protecting the rights of inbred lines, samples with fewer differential sites than the determination threshold (i.e., regions a and c) should be sent to a professional testing institution for further identification.

[0165] Twelve maize samples were identified using both the M-POG protocol and the national standard test method (GB / T 39914-2021). The average time per sample for M-POG kit testing and the national standard laboratory test for variety authenticity identification were statistically analyzed and compared. Figure 10This kit significantly reduces the time required for each step, from DNA extraction and system preparation to amplification detection and result reading. The entire detection process takes only 42 minutes, which is 1 / 4 of the total detection time of the standard method (excluding sampling and transportation time), significantly improving detection efficiency. The actual efficiency improvement is even more significant when sample shipping time is taken into account.

[0166] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. A primer design method for rapid detection of nucleic acids in plant varieties, characterized in that, The method is based on the design of dominant primers at the InDel site. The design method of the dominant primer is as follows: the 3' end of one of the dominant primers is designed in the InDel insertion sequence or at the deletion junction. Preferably, the dominant primer can be based on either strand of double-stranded DNA as a template; Preferably, if the variant sequence at the InDel site is different from the adjacent downstream sequence, a dominant primer is designed using the template strand or the complementary strand as a template; if the variant sequence at the InDel site is similar to or the same as the adjacent downstream sequence, a dominant primer is designed using the complementary strand as a template. Preferably, InDel sites are screened using MAF, heterozygosity, deletion rate, size of the variant interval, and chromosome location as indicators; Preferably, a dynamic programming algorithm is further used to screen InDel sites; Preferably, the InDel sites that are consistent with the genotyping results of co-dominant primers and dominant primers are further screened; Preferably, the plant is a monocotyledonous plant; Preferably, the monocotyledonous plant is a grass (Poaceae). Preferably, the grass plant is maize.

2. A method for screening InDel sites for plant variety detection, characterized in that, The method includes screening InDel sites using MAF, heterozygosity, deletion rate, size of the variant interval, and chromosome location as indicators; Preferably, the screening index meets any one or more of the following conditions: MAF≥0.3, heterozygosity ≤0.05, deletion rate <0.05, variation interval size of 3-20bp, and uniform distribution on chromosome; Preferably, the method further includes using a dynamic programming algorithm to screen InDel sites; Preferably, the method further includes screening for InDel sites that are consistent with the typing results of the same DNA co-dominant primer and dominant primer; Preferably, the plant is a monocotyledonous plant; Preferably, the monocotyledonous plant is a grass (Poaceae). Preferably, the grass is maize; Preferably, the screening index for maize InDel sites meets any one or more of the following conditions: MAF>0.4, heterozygosity <0.02, deletion rate <0.015, and variation size of 3-10 bp.

3. Any one of the following tools: (1) A primer design system for plant variety detection, characterized in that, The system includes: Primer design module: Design dominant primers based on template DNA, with the 3' end of one dominant primer designed inside the InDel insertion sequence or at the deletion junction; Preferably, the system further includes: Template selection module: If the variant sequence where the InDel site is located is different from the immediately adjacent downstream sequence, then the template strand or the complementary strand is used as the template to design a dominant primer; if the variant sequence where the InDel site is located is similar to or the same as the immediately adjacent downstream sequence, then the complementary strand is used as the template to design a dominant primer. Preferably, the system further includes: InDel site screening module, the InDel site screening module includes: Indicator screening submodule: Screening InDel sites using MAF, heterozygosity, deletion rate, size of the variant interval, and chromosome location as indicators; Algorithm filtering submodule: Filters InDel sites based on dynamic programming algorithm; Primer screening submodule: Screening InDel sites based on the amplification results of co-dominant primers and dominant primers to obtain InDel sites with consistent genotyping results between co-dominant primers and dominant primers; (2) An InDel site screening system for plant variety detection, characterized in that the system comprises: Indicator screening module: InDel sites are screened using MAF, heterozygosity, deletion rate, size of the variant interval, and chromosome location as indicators; Algorithm filtering module: Filters InDel sites based on dynamic programming algorithm; Primer screening module: Screens InDel sites based on the amplification results of co-dominant primers and dominant primers, and obtains InDel sites that are consistent with the genotyping results of co-dominant primers and dominant primers; (3) An apparatus, characterized in that the apparatus includes a memory and a processor, the memory being used to store program instructions; the processor being used to invoke the program instructions, which, when executed, implement the method of claim 1 or 2; (4) A computer-readable storage medium having a computer program thereon, characterized in that the computer program, when executed by a processor, implements the method of claim 1 or 2; (5) A computer program product, characterized in that it includes a computer program that, when executed by a processor, implements the method of claim 1 or 2.

4. An InDel site for plant variety detection, characterized in that, The InDel sites are obtained by screening using the method of claim 2 or the tool of claim 3; Preferably, the plant is a monocotyledonous plant; Preferably, the monocotyledonous plant is a grass (Poaceae). Preferably, the grass is maize; Preferably, the InDel sites of the maize include any one or more of MKT01-MKT16, wherein the mutation initiation position of MKT01 is at position 278,976,599 on chromosome 1, the mutation initiation position of MKT02 is at position 24,653,642 on chromosome 2, the mutation initiation position of MKT03 is at position 88,426,353 on chromosome 2, the mutation initiation position of MKT04 is at position 172,717,920 on chromosome 3, the mutation initiation position of MKT05 is at position 124,035,057 on chromosome 3, the mutation initiation position of MKT06 is at position 192,970,579 on chromosome 4, the mutation initiation position of MKT07 is at position 14,612,889 on chromosome 5, and the mutation initiation position of MKT08 is at position 5. The mutations of MKT09 and MKT010 originate from chromosome 6 at position 15,209,041; MKT011 originates from chromosome 6 at position 138,858,575; MKT012 originates from chromosome 6 at position 132,187,454; MKT013 originates from chromosome 7 at position 151,285,049; MKT014 originates from chromosome 8 at position 159,528,845; MKT015 originates from chromosome 9 at position 113,082,501; and MKT016 originates from chromosome 10 at position 145,370,739. Preferably, the sequence of the InDel site in maize is as shown in any one or more of SEQ ID NO:1-16.

5. A primer set for plant variety detection, characterized in that, The primer set includes primers designed by the method of claim 1, primers for amplifying the InDel site obtained by the method of claim 2, primers designed by the tool of claim 3, primers for amplifying the InDel site obtained by the tool of claim 3, or primers for amplifying the InDel site of claim 4. Preferably, the plant is a monocotyledonous plant; Preferably, the monocotyledonous plant is a grass (Poaceae). Preferably, the grass is maize; Preferably, the primer set for the maize includes primers capable of amplifying the maize InDel site as described in claim 4; Preferably, the primer sequence of the corn is as shown in any one or more of SEQ ID NO:17-48 or SEQ ID NO:49-80.

6. A product for plant variety detection, characterized in that, The product includes primers designed by the method of claim 1, primers for amplifying the InDel site obtained by the method of claim 2, primers designed by the tool of claim 3, primers for amplifying the InDel site obtained by the tool of claim 3, primers for amplifying the InDel site of claim 4, or the primer set of claim 5. Preferably, the primer set includes individually packaged single primers, mixed-package primer mixtures, and primer premixes premixed with buffer solution; Preferably, the primer set includes lyophilized primers, liquid primers, and solid primers. Preferably, the solid primer is adsorbed onto the carrier; Preferably, the carrier includes a filter membrane, microspheres, a chip, or a test strip; Preferably, the product includes a reagent kit, a chip, and a test strip; Preferably, the product further includes amplification reaction reagents; Preferably, the amplification reaction reagents include buffer, enzyme, and dNTPs; Preferably, the buffer solution comprises Tris-HCl, KCl, betaine, and BSA; Preferably, the enzyme includes DNA polymerase; Preferably, the DNA polymerase includes Taq, Vent, Pfu, Klenow, Phi29, and Bst; Preferably, the product further includes extraction reagents, washing reagents, and reference standards; Preferably, the extraction reagent includes Tris-HCl, NaCl, EDTA, and SDS; Preferably, the washing reagent includes Tween 20, Tris-HCl, isopropanol, and ethanol; Preferably, the product further includes a sample processing component; Preferably, the sample processing component includes a punch and a grinding device; Preferably, the product also includes an instruction manual; Preferably, the product further includes a detection reagent; Preferably, the detectable reagent includes fluorescent dyes and enzymes.

7. A method for detecting plant varieties, characterized in that, The method uses primers designed by the method of claim 1, primers for amplifying the InDel site obtained by the method of claim 2, primers designed by the tool of claim 3, primers for amplifying the InDel site obtained by the tool of claim 3, primers for amplifying the InDel site of claim 4, primer sets of claim 5, or the product of claim 6 for detection. Preferably, the method further includes visual interpretation of the detection results; Preferably, the visualization interpretation involves determining the dominant / recessive state of the detection results; Preferably, plant variety detection is achieved through the determination of dominant / recessive forms; Preferably, the primer set amplification techniques include thermal cycling amplification and isothermal amplification. Preferably, the thermal cycling amplification technique includes PCR; Preferably, the PCR includes qPCR and dPCR; Preferably, the isothermal amplification techniques include RPA, LAMP, RCA, SDA, and HDA; Preferably, the primer set detection platform includes electrophoresis, gene chip, KASP platform, and sequencing; Preferably, the electrophoresis includes gel electrophoresis and capillary electrophoresis; Preferably, the gene chip includes a microbead chip, a laser micro-etched chip, and a magnetic bead chip; Preferably, the sequencing includes site-directed sequencing; Preferably, the plant is a monocotyledonous plant; Preferably, the monocotyledonous plant is a grass (Poaceae). Preferably, the grass is maize; Preferably, the range threshold Ct0 is calculated based on the following formula for visual interpretation of maize varieties: Among them, Ct I-MAX The maximum Ct value of the dominant amplification curve; Ct D-MIN The minimum Ct value for the recessive state amplification curve; Preferably, the conversion rules for visualization interpretation are as follows: convert Ct<28 to 1, convert Ct>30 to 0, and convert 28≤Ct≤30 to 9, thereby generating a DNA fingerprint database for on-site detection of maize variety authenticity consisting of 16 digits; Preferably, the visual interpretation of maize varieties is based on endpoint fluorescence detection.

8. Any one of the following tools: (1) A plant variety detection system, characterized in that, The system includes: Data receiving module: receives primers designed by the method of claim 1, primers for amplifying the InDel site obtained by the method of claim 2, primers designed by the tool of claim 3, primers for amplifying the InDel site obtained by the tool of claim 3, primers for amplifying the InDel site of claim 4, primer sets of claim 5, or plant variety amplification data obtained by product amplification of claim 6. The amplified data is compared with a preset plant variety characteristic database; Output plant variety identification results; (2) An apparatus, characterized in that the apparatus includes a memory and a processor, the memory being used to store program instructions; the processor being used to invoke the program instructions, and when the program instructions are executed, to implement the method of claim 7; (3) A computer-readable storage medium having a computer program thereon, characterized in that the computer program, when executed by a processor, implements the method of claim 7; (4) A computer program product, characterized in that it includes a computer program that, when executed by a processor, implements the method of claim 7; Preferably, the plant is a monocotyledonous plant; Preferably, the monocotyledonous plant is a grass (Poaceae). Preferably, the grass plant is maize.

9. A method for constructing a plant variety fingerprint database, characterized in that, The method includes amplifying the InDel site obtained by the method of claim 1 using primers designed according to claim 1, primers for amplifying the InDel site obtained by the method of claim 2, primers designed according to the tool of claim 3, primers for amplifying the InDel site obtained by the tool of claim 3, primers for amplifying the InDel site of claim 4, primer set according to claim 5, or product according to claim 6, to obtain a plant variety fingerprint database. Preferably, the plant is a monocotyledonous plant; Preferably, the monocotyledonous plant is a grass (Poaceae). Preferably, the grass plant is maize.

10. A plant variety fingerprint database, characterized in that, The plant variety fingerprint database is constructed by the method described in claim 9; Preferably, the plant is a monocotyledonous plant; Preferably, the monocotyledonous plant is a grass (Poaceae). Preferably, the grass plant is maize.

11. The application of the primers designed by the method of claim 1, the primers for amplifying the InDel site obtained by the method of claim 2, the primers designed by the tool of claim 3, the primers for amplifying the InDel site obtained by the tool of claim 3, the primers for amplifying the InDel site of claim 4, the primer set of claim 5, or the product of claim 6 in plant variety detection; Preferably, the plant variety testing includes plant variety identification, plant genetic breeding, and plant germplasm resource analysis; Preferably, the plant variety identification includes plant variety authenticity identification and purity identification; Preferably, the identification of plant variety authenticity includes plant composition comparison and database screening comparison; Preferably, the plant genetic breeding includes plant genetic analysis; Preferably, the plant is a monocotyledonous plant; Preferably, the monocotyledonous plant is a grass (Poaceae). Preferably, the grass plant is maize.

Images