Primer group and kit for identifying variety of cabbage mustard and application of primer group and kit
By using PCR amplification and sequencing with 500 primer pairs, combined with DNA fingerprint database comparison, the problems of accuracy and efficiency in Chinese kale variety identification were solved, achieving high-resolution and rapid variety identification.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- 深圳市农产品质量安全检验检测中心(深圳市动植物疫病预防控制中心)
- Filing Date
- 2025-12-15
- Publication Date
- 2026-04-14
AI Technical Summary
Existing technologies are insufficient to accurately distinguish Chinese kale varieties. SRAP and SSR methods suffer from unstable amplification and insufficient polymorphism, which affect the accuracy of typing and make it difficult to meet the requirements for high-accuracy and high-throughput variety identification.
PCR amplification and sequencing were performed using 500 primer pairs to obtain DNA fingerprint data. By comparing the DNA fingerprint data with that of known varieties, high-resolution and high-accuracy variety identification was achieved, and a DNA fingerprint database was constructed for rapid identification.
It has significantly improved the accuracy and efficiency of Chinese kale variety identification, reaching the single-base level, enabling rapid and high-throughput identification of Chinese kale varieties, and improving resolution and digitization.
Smart Images
Figure CN121852580A_ABST
Abstract
Description
Technical Field
[0001] This application relates to the field of Chinese kale identification technology, and in particular to a primer set, reagent kit, and application for identifying Chinese kale varieties. Background Technology
[0002] Chinese kale (Brassica oleracea var. alboglabra Bailey), belonging to the Brassicaceae family and the Brassica genus, is a variety of cabbage and is widely enjoyed both domestically and internationally as an important vegetable and economic crop. In recent years, to meet market demand, many domestic research institutions have strengthened the utilization of hybrid vigor and the breeding of new varieties of Chinese kale, leading to a surge in the number of hybrid varieties on the market. Simultaneously, the introduction of numerous different Chinese kale varieties, many of which share similar traits, makes them difficult to distinguish visually. Therefore, research into the genetic background of Chinese kale and the strengthening of new variety management have become particularly important.
[0003] Currently, there is a lack of standard methods for identifying Chinese kale varieties. Only a few publications report studies on the genetic background and diversity of Chinese kale based on molecular marker technologies, such as SRAP (Sequence-Related Amplified Polymorphism) and SSR (Simple Sequence Repeat, also known as Microsatellite DNA). SRAP technology mainly utilizes the differences in GC and AT base content within exons and introns of a species to design unique primers for specific amplification of open reading frames. However, primers that are too long or too short can affect the experiment. Primers that are too long will affect autoradiography observation, while primers that are too short will easily produce multiple bands with poor reproducibility. In addition, gel electrophoresis has limited resolution, making this method unsuitable for variety identification where high accuracy is required.
[0004] SSR technology, based on the high conservation of SSR flanking sequences, designs specific primers for PCR (polymerase chain reaction) amplification and agarose gel electrophoresis, and then performs polymorphism analysis based on the electrophoretic bands. However, this method suffers from limited throughput and insufficient polymorphism, making it difficult to distinguish all varieties and meet the needs of large-scale sample testing. Furthermore, during the PCR reaction, DNA polymerase slippage upon binding to the template easily leads to non-specific amplification. This results in multiple bands appearing during electrophoresis, making it difficult to distinguish between the true genotype band and the slipped genotype band, severely impacting the accuracy of genotyping.
[0005] Therefore, developing detection technologies that can accurately identify Chinese kale varieties has become an urgent technical problem to be solved in the field of Chinese kale identification technology. Summary of the Invention
[0006] The purpose of this application is to provide a new primer set, kit, and application for identifying Chinese kale varieties.
[0007] The following technical solution is adopted in this application:
[0008] The first aspect of this application discloses a primer set for identifying Chinese kale varieties, comprising primer pairs 1 to 500, each primer pair including a forward primer and a reverse primer. The forward primers of primer pairs 1 to 500 are shown in SEQ ID NO.1 to SEQ ID NO.500, and the reverse primers of primer pairs 1 to 500 are shown in SEQ ID NO.501 to SEQ ID NO.1000, respectively. That is, the forward and reverse primers of primer pair 1 are shown in SEQ ID NO.1 and SEQ ID NO.501, respectively; the forward and reverse primers of primer pair 2 are shown in SEQ ID NO.2 and SEQ ID NO.502, respectively; and so on, with the forward and reverse primers of primer pair 500 being shown in SEQ ID NO.500 and SEQ ID NO.1000, respectively.
[0009] It should be noted that this application is the first to develop DNA fingerprint data for Chinese kale. By using the primer set of this application for PCR amplification and sequencing of the sample, the DNA fingerprint data of the sample can be obtained. By comparing this DNA fingerprint data with the DNA fingerprint data of known Chinese kale varieties, the variety identification result of the sample can be obtained. The DNA fingerprint data of this application consists of multiple MNP-labeled base sequences obtained after sequencing the PCR amplification products. The resolution reaches the single-base level, and the accuracy and digitization are both high. Hundreds or thousands of samples can be compared at once using sequence analysis software, providing rapid and high-throughput variety identification results, significantly improving the accuracy and efficiency of Chinese kale variety identification. The primer set of this application has good application value in Chinese kale variety identification and DNA fingerprint database construction, providing technical support for the protection of Chinese kale varieties and thus promoting the healthy development of the industry.
[0010] In this application, the DNA fingerprint data refers to the sequencing results obtained by PCR amplification of the DNA of Chinese kale using the primer set of this application and sequencing the PCR amplification products, namely the sequencing results of the 500 primer pairs amplification fragments of this application.
[0011] The second aspect of this application discloses a kit for identifying Chinese kale varieties, the kit including the primer set of this application.
[0012] In one implementation of this application, the kit further includes at least one of PCR amplification reagents and positive standards.
[0013] It should be noted that the key to this kit is the primer set specified in this application. Other reagents can be incorporated into the kit as needed, or purchased separately. The positive standard in this application can be a nucleic acid sample from any known Chinese kale variety to verify the accuracy of the entire experimental procedure.
[0014] The third aspect of this application discloses a database for identifying Chinese kale varieties, which contains MNP molecular marker information for several known Chinese kale varieties; the MNP molecular marker information is obtained by amplifying and sequencing the nucleic acids of known Chinese kale varieties using the primer set of this application.
[0015] It should be noted that the database used in this application for identifying Chinese kale varieties is actually a DNA fingerprint database of known Chinese kale varieties obtained by amplifying and sequencing known varieties using the primer set described in this application. The identification result of the sample to be tested can be obtained by comparing the sequencing results with this database. If no matching result meeting the genetic similarity coefficient is obtained in this database, the sample to be tested is determined to be a new Chinese kale variety. After identification using traditional methods, the DNA fingerprint data of this sample can be added to this database to expand the range of Chinese kale varieties in this application's database.
[0016] The fourth aspect of this application discloses the application of the primer set, reagent kit, or database of this application in the identification of Chinese kale varieties.
[0017] The fifth aspect of this application discloses a method for identifying Chinese kale varieties, comprising the following steps:
[0018] The primer set of this application was used to perform PCR amplification on the DNA of the sample to be tested;
[0019] Sequencing of PCR amplification products;
[0020] The sequencing results were compared with the database of this application to obtain the genetic similarity coefficient between the sample to be tested and each known Chinese kale variety in the database;
[0021] Variety identification results of the test samples are obtained based on the genetic similarity coefficient.
[0022] The sixth aspect of this application discloses an apparatus for identifying Chinese kale varieties, comprising an input module, a comparison module, and an output module; wherein, the input module includes a sequencing module for obtaining the sequencing results of PCR amplification and sequencing of the DNA of the sample to be tested using the primer set of this application; the comparison module includes a module for comparing the sequencing results with the database of this application to obtain the genetic similarity coefficient between the sample to be tested and each known Chinese kale variety in the database; and the output module includes a module for obtaining the variety identification result of the sample to be tested based on the genetic similarity coefficient.
[0023] The seventh aspect of this application discloses an apparatus for identifying Chinese kale varieties, the apparatus comprising a memory and a processor; the memory for storing a program; and the processor for executing the program stored in the memory to implement the following method:
[0024] Step 1: Obtain the DNA of the sample to be tested by PCR amplification and sequencing using the primer set of this application;
[0025] Step 2: Compare the sequencing results with the database of this application to obtain the genetic similarity coefficient between the sample to be tested and each known Chinese kale variety in the database;
[0026] Step 3: Obtain the variety identification results of the sample to be tested based on the genetic similarity coefficient.
[0027] The eighth aspect of this application discloses a computer-readable storage medium that includes the database of this application.
[0028] In one implementation of this application, the computer-readable storage medium further includes a program that can be executed by a processor to implement the following method:
[0029] Step 1: Obtain the DNA of the sample to be tested by PCR amplification and sequencing using the primer set described in claim 1;
[0030] Step 2: Compare the sequencing results with the database described in claim 4 to obtain the genetic similarity coefficient between the sample to be tested and each known Chinese kale variety in the database;
[0031] Step 3: Obtain the variety identification results of the sample to be tested based on the genetic similarity coefficient.
[0032] Due to the adoption of the above technical solutions, the beneficial effects of this application are as follows:
[0033] The primer set of this application allows for PCR amplification and sequencing of the test sample to obtain its DNA fingerprint data. By comparing this DNA fingerprint data with the DNA fingerprint data of known Chinese kale varieties, the variety identification result of the test sample can be obtained. Using the primer set of this application for Chinese kale variety identification achieves a resolution down to the single-base level, with high accuracy and digitization, enabling rapid and high-throughput Chinese kale variety identification, significantly improving the accuracy and efficiency of Chinese kale variety identification. Attached Figure Description
[0034] Figure 1 This is a distribution map of the number of MNP marker detection sites for different Chinese kale varieties in the embodiments of this application, where the horizontal axis represents the sample to be tested and the vertical axis represents the number of MNP markers detected.
[0035] Figure 2 This is a distribution diagram showing the difference in the proportion of MNP marker sites among different Chinese kale varieties in the embodiments of this application. Detailed Implementation
[0036] To address the issue of Chinese kale variety identification, this application pioneered the development of primer sets capable of amplifying and obtaining Chinese kale DNA fingerprint data, namely primer pairs 1 to 500.
[0037] Based on the primer set of this application, this application further develops a method for identifying Chinese kale varieties, specifically including: performing PCR amplification on the DNA of the sample to be tested using the primer set of this application; sequencing the PCR amplification product; comparing the sequencing results with the database of this application to obtain the genetic similarity coefficient between the sample to be tested and each known Chinese kale variety in the database; and obtaining the variety identification result of the sample to be tested based on the genetic similarity coefficient.
[0038] Those skilled in the art will understand that all or part of the functions of the above methods can be implemented in hardware or by computer programs. When all or part of the functions in the above embodiments are implemented by computer programs, the program can be stored in a computer-readable storage medium, which may include: read-only memory, random access memory, disk, optical disk, hard disk, etc., and the above functions are implemented by executing the program with a computer. For example, the program can be stored in the memory of a device, and when the program in the memory is executed by the processor, all or part of the above functions can be implemented. In addition, when all or part of the functions in the above embodiments are implemented by computer programs, the program can also be stored in a storage medium such as a server, another computer, disk, optical disk, flash drive, or portable hard drive, and can be downloaded or copied to the memory of a local device, or the system of the local device can be updated. When the program in the memory is executed by the processor, all or part of the functions in the above embodiments can be implemented.
[0039] Therefore, this application further develops a device for identifying Chinese kale varieties, including an input module, a comparison module, and an output module; wherein, the input module includes a sequencing result for obtaining the DNA of the sample to be tested by PCR amplification and sequencing using the primer set of this application; the comparison module includes a device for comparing the sequencing result with the database of this application to obtain the genetic similarity coefficient between the sample to be tested and each known Chinese kale variety in the database; the output module includes a device for obtaining the variety identification result of the sample to be tested based on the genetic similarity coefficient.
[0040] In another implementation of this application, a device for identifying Chinese kale varieties has also been developed. This device includes a memory and a processor. The memory stores a program. The processor executes the program stored in the memory to implement the following method: Step 1, obtaining the sequencing results of PCR amplification and sequencing of the DNA of the sample to be tested using the primer set of this application; Step 2, comparing the sequencing results with the database of this application to obtain the genetic similarity coefficient between the sample to be tested and each known Chinese kale variety in the database; Step 3, obtaining the variety identification result of the sample to be tested based on the genetic similarity coefficient.
[0041] In another implementation of this application, a computer-readable storage medium is also developed, which stores the database of this application. This database can be read by a processor for comparison with the DNA fingerprint data of the sample to be tested.
[0042] Furthermore, the computer-readable storage medium of this application also contains a program that can be executed by a processor to implement the following method: Step 1, obtaining the sequencing results of PCR amplification and sequencing of the DNA of the sample to be tested using the primer set of this application; Step 2, comparing the sequencing results with the database of this application to obtain the genetic similarity coefficient between the sample to be tested and each known Chinese kale variety in the database; Step 3, obtaining the variety identification result of the sample to be tested based on the genetic similarity coefficient.
[0043] The present application will be further described in detail below through specific embodiments. The following embodiments are only for further illustration of the present application and should not be construed as limiting the present application.
[0044] Example
[0045] In this example, primer sets capable of amplifying and obtaining DNA fingerprint data of Chinese kale were developed based on the genomic DNA of Chinese kale, namely primer pairs 1 to 500. Each primer pair includes a forward primer and a reverse primer. The forward primers of primer pairs 1 to 500 are shown in SEQ ID NO.1 to SEQ ID NO.500, and the reverse primers of primer pairs 1 to 500 are shown in SEQ ID NO.501 to SEQ ID NO.1000, respectively. That is, the forward and reverse primers of primer pair 1 are SEQ ID NO.1 and SEQ ID NO.501, respectively; the forward and reverse primers of primer pair 2 are SEQ ID NO.2 and SEQ ID NO.502, respectively; and so on, with the forward and reverse primers of primer pair 500 being SEQ ID NO.500 and SEQ ID NO.1000, respectively. The primer sequences are shown in Table 1. All primers were synthesized by Shanghai Sangon Biotech.
[0046] Table 1
[0047]
[0048] Continued from Table 1
[0049]
[0050] Continued from Table 1
[0051]
[0052] Continued from Table 1
[0053]
[0054] Continued from Table 1
[0055]
[0056] Continued from Table 1
[0057]
[0058] Continued from Table 1
[0059]
[0060] Continued from Table 1
[0061]
[0062] The primers in the above primer set do not interfere with each other, ensuring that each primer pair can react normally in the same amplification reaction and specifically amplify the target gene sequence.
[0063] This example uses the above primer set to identify Chinese kale varieties, specifically including:
[0064] The DNA of the test sample was amplified by multiplex PCR using the above primer set to obtain multiplex PCR amplification products;
[0065] Purify multiplex PCR amplification products;
[0066] High-throughput sequencing libraries were constructed using purified multiplex PCR amplification products to obtain high-throughput libraries of the samples to be tested.
[0067] Purifying high-throughput libraries of test samples;
[0068] Sequencing data was obtained by sequencing the high-throughput library of the sample to be tested.
[0069] Analyze sequencing data to obtain DNA fingerprint data;
[0070] The genetic similarity coefficient is obtained by comparing DNA fingerprint data with control samples;
[0071] Based on the genetic similarity coefficient, the variety identification conclusion between the test sample and the control variety is obtained.
[0072] Specifically, based on the genetic similarity coefficient, the variety identification conclusion between the test sample and the control variety is obtained, including: when the genetic similarity coefficient is greater than or equal to 99%, the test sample and the control sample are determined to be extremely similar varieties or the same varieties.
[0073] In this embodiment, the samples to be tested were the Chinese kale varieties collected by the applicant. Ten representative varieties were selected for the experiment. The ten Chinese kale varieties were: Chinese kale, Hong Kong Chinese medium-flowered kale, four-season thick-striped kale, Dasun yellow-flowered kale (401), hybrid dwarf large-bone kale, red kale, Yangmei yellow-flowered kale, Taiwan Dixian kale king, Xinghui kale, and American sweet and crisp white-flowered chicken leg kale.
[0074] DNA extraction from the test samples: DNA was extracted from the leaves of the above 10 Chinese kale varieties using the Plant Genomic DNA Extraction Kit (catalog number: DP320) manufactured by Tiangen Biotech (Beijing) Co., Ltd. Detailed operating procedures are described in the kit's instruction manual. DNA from 10 test samples was obtained. Then, 1 μL of DNA from each test sample was taken and the DNA concentration of the test sample was determined using a Qubit fluorescence quantitative quantitation instrument. In this embodiment, the DNA concentration of the test samples was measured to be between 20 ng / μL and 50 ng / μL, which meets the requirements for subsequent experiments.
[0075] The primer set designed in this example was used to perform multiplex PCR amplification on the DNA of the test sample to obtain multiplex PCR amplification products.
[0076] Specifically, each amplification reaction system included: 4 μL primer set mixture, 4 μL DNA from the sample to be tested, 10 μL LenoPlexs 3×T Master Mix (manufacturer: Shijiazhuang Borui Biotechnology Co., Ltd.), and 12 μL water. The mixture was vortexed and mixed thoroughly to obtain a multiplex PCR amplification system. The multiplex PCR amplification program was: 95℃ for 3 min; (95℃ for 20 sec, 60℃ for 4 min) × 17 cycles; 72℃ for 4 min to obtain the multiplex PCR amplification product.
[0077] The primer set mixture is composed of equal amounts of forward and reverse primers from 500 primer pairs.
[0078] Purification of multiplex PCR amplification products:
[0079] Specifically, the amplification products after the reaction were then purified using DNA purification magnetic beads (manufacturer: Nanjing Novizan Biotechnology Co., Ltd.). For detailed operating procedures, please refer to the product's instruction manual.
[0080] High-throughput sequencing libraries were constructed using purified multiplex PCR amplification products to obtain high-throughput libraries of the samples to be tested.
[0081] Specifically, 10 μL of GenoPlexs 3×T Master Mix, 2 μL of 5 μM Illumina sequencing adapter primers (manufactured by Borui Biotechnology Co., Ltd.), and 16 μL of water were added to the purified multiplex PCR amplification product, and the PCR amplification reaction was performed according to the following program: 95℃ for 3 min; (95℃ for 15 s, 58℃ for 15 s, 70℃ for 30 s) × 8 cycles; a final extension at 72℃ for 5 min, and the reaction was terminated at 16℃. After the reaction, a high-throughput sequencing library of the sample to be tested was obtained.
[0082] Purifying the high-throughput library of the sample to be tested: Specifically, use DNA purification magnetic beads (VAHTS DNA CleanBeads, catalog number N411-01) to purify the high-throughput sequencing library to obtain the purified high-throughput sequencing library. For specific purification methods, refer to the product instructions of the DNA purification magnetic beads.
[0083] The high-throughput library of the sample to be tested was sequenced to obtain sequencing data. Specifically, the high-throughput sequencing library was sequenced using an Illumina NextSeq1000 sequencer to obtain sequencing data of the sample to be tested. For detailed sequencing steps, please refer to the instruction manual of the sequencer.
[0084] Sequencing data was analyzed to obtain DNA fingerprint data. Specifically, Bowtie2 (version 2.1.0) was used for sequence analysis to obtain the MNP marker for each Chinese kale variety, i.e., the DNA fingerprint data of each Chinese kale.
[0085] Detection rate of MNP markers:
[0086] Multiplex PCR amplification and sequencing library construction were performed using the primer set provided in this embodiment. Multiplex amplification, next-generation high-throughput sequencing and data analysis were performed on these 10 Chinese kale DNA samples. The 500 markers were detected in all 10 samples to be tested. The specific detection information is shown in Table 2.
[0087] Table 2
[0088] Serial Number serial number Variety Name Number of detected sites Detection rate 1 JL240517004 Chinese kale 475 95.00% 2 JL240517011 Chinese Hong Kong Chinese kale 494 98.80% 3 JL240517017 Four Seasons Thick-stemmed Chinese Mustard Greens 487 97.40% 4 JL240517022 Large bamboo shoots and yellow-flowered kale (401) 486 97.20% 5 JL240517029 Hybrid dwarf kale 495 99.00% 6 JL240517047 Red mustard 485 97.00% 7 JL240517059 Yangmei Yellow Chinese Mustard 474 94.80% 8 JL240517014 Taiwan Emperor Immortal Mustard King 487 97.40% 9 JL240517038 Starry Chinese Mustard 480 96.00% 10 JL240517039 American Sweet and Crispy White Chicken Legs and Kale 496 99.20%
[0089] Table 2 shows that the highest number of detected markers was 496 for the Chinese kale variety, with an average of 485.9 MNP markers detected per variety, resulting in an average detection rate of 97.18%. The distribution of MNP marker detection sites in the tested samples is shown below. Figure 1 As shown.
[0090] Accuracy analysis:
[0091] To verify the accuracy of the primer pairs, a reproducibility experiment was conducted on the 10 Chinese kale varieties. This included two independent experiments performed by different personnel, using different batches of reagents, and different instruments. The data from the two experiments for each sample were compared and analyzed. The accuracy of the genotyping was calculated using the formula: accuracy = 1 - (1 - reproducibility) / 2. Reproducibility refers to the proportion of MNP marker sites with consistent genotyping results in the two experiments out of all MNP marker sites. The statistical results are shown in Table 3.
[0092] Table 3
[0093]
[0094] The serial numbers in Table 3 correspond to those in Table 2. Library 1 and Library 2 with the same serial number, such as 1-11 and 1-21, refer to two libraries of Chinese mustard green “JL240517004”, that is, two libraries from two independent experiments conducted by different personnel, with different batches of reagents and different instruments. “Number of comparisons” refers to the number of marker sites compared between the two libraries. “Number of differences” refers to the number of marker sites with different genotypes in the two libraries being compared. “Number of reproducibility” refers to the number of reproducible marker sites in the two libraries, that is, marker sites that are present in both libraries.
[0095] As shown in Table 3, a total of 4819 MNP markers were compared, with 5 non-repeating sites, resulting in a typing accuracy of 99.95%. High marker accuracy indicates that the identification results are not affected by different personnel, different batches of reagents, or different instruments, providing technical support for the sharing of DNA fingerprint data.
[0096] Distinguishing features of MNP-marked varieties:
[0097] All detected MNP marker genotypes from the 10 Chinese kale samples were compared pairwise. Based on the principle that a difference of at least one SNP at the same MNP marker locus across different varieties is considered significant, the number of differentially expressed MNP markers in each pairwise comparison of the 10 Chinese kale samples was counted, resulting in 45 pairs of comparisons. The average difference per sample was 186.64 marker loci, with an average difference rate of 40.65%. The distribution of the difference rate is shown below. Figure 2 As shown. By Figure 2 It can be seen that the selected MNP markers have high polymorphism and can significantly distinguish any Chinese kale variety.
[0098] Chinese kale variety identification:
[0099] DNA fingerprint data is compared with control samples to obtain a genetic similarity coefficient. Based on the genetic similarity coefficient, the variety of the test sample is identified. A variety identification conclusion is obtained between the test sample and the control variety based on the genetic similarity coefficient, including: when the genetic similarity coefficient is greater than or equal to 99%, the test sample and the control sample are determined to be very similar varieties or the same variety. The control sample can be amplified and sequenced together with the test sample to obtain DNA fingerprint data, or it can be amplified and sequenced beforehand and then compared with the test sample, depending on the actual situation.
[0100] Specifically, in this example, another sample of Chinese kale (named DC1) was tested according to the above experimental procedure. The DNA fingerprint data was then compared with that of the 10 samples to be tested to obtain the genetic similarity coefficient. Referring to the existing national standard "MNP Marker Method for Plant Variety Identification," the genetic similarity coefficient was used as the basis for determining the variety identification conclusion. When the genetic similarity coefficient (GS) was greater than or equal to 99%, the sample to be tested and the control sample were determined to be "very similar varieties or the same variety." The results are shown in Table 4.
[0101] Table 4
[0102]
[0103] As shown in Table 4, the number of varietal differences between the DC1 Chinese kale and the 10 tested samples of Chinese kale was 1, and the GS value reached 99.79%, thus it was determined to be a very similar variety or the same variety. However, the number of significantly different varietal differences (235-329 loci) between the DC1 and other kale samples, and the GS value was extremely low (less than or equal to 49.68%), were determined to be different varieties. Therefore, the primer set provided in this embodiment can be used to compare and analyze the DNA fingerprint data of the collected samples with existing data, thereby accurately identifying kale varieties.
[0104] The above description, in conjunction with specific embodiments, provides a further detailed explanation of this application and should not be construed as limiting the specific implementation of this application to these descriptions. Those skilled in the art to which this application pertains can make several simple deductions or substitutions without departing from the concept of this application.
Claims
1. A primer set for identifying Chinese kale varieties, characterized in that: The primers include primer pairs 1 through 500, each primer pair consisting of a forward primer and a reverse primer. The forward primers of primer pairs 1 through 500 are shown in SEQ ID NO.1 through SEQ ID NO.500, and the reverse primers of primer pairs 1 through 500 are shown in SEQ ID NO.501 through SEQ ID NO.1000, respectively. That is, the forward and reverse primers of primer pair 1 are shown in SEQ ID NO.1 and SEQ ID NO.501, respectively; the forward and reverse primers of primer pair 2 are shown in SEQ ID NO.2 and SEQ ID NO.502, respectively; and so on, with the forward and reverse primers of primer pair 500 being shown in SEQ ID NO.500 and SEQ ID NO.1000, respectively.
2. A kit for identifying Chinese kale varieties, characterized in that: Includes the primer set as described in claim 1.
3. The reagent kit according to claim 2, characterized in that: It also includes at least one of PCR amplification reagents and positive standards.
4. A database for identifying Chinese kale varieties, characterized in that: The database contains MNP molecular marker information for several known Chinese kale varieties; The MNP molecular marker information is obtained by amplifying and sequencing the nucleic acids of a known Chinese kale variety using the primer set described in claim 1.
5. The application of the primer set of claim 1, the kit of claim 2 or 3, or the database of claim 4 in the identification of Chinese kale varieties.
6. A method for identifying varieties of Chinese kale, characterized in that: Includes the following steps, The DNA of the sample to be tested was amplified by PCR using the primer set described in claim 1; Sequencing of PCR amplification products; The sequencing results are compared with the database described in claim 4 to obtain the genetic similarity coefficient between the sample to be tested and each known Chinese kale variety in the database; Variety identification results of the test samples are obtained based on the genetic similarity coefficient.
7. An apparatus for identifying varieties of Chinese kale, characterized in that: It includes an input module, a comparison module, and an output module; The input module includes a sequencing result for obtaining PCR amplification and sequencing of the DNA sample to be tested using the primer set described in claim 1. The comparison module includes a function to compare the sequencing results with the database of claim 4 to obtain the genetic similarity coefficient between the sample to be tested and each known Chinese kale variety in the database. The output module includes a variety identification result for obtaining the test sample based on the genetic similarity coefficient.
8. An apparatus for identifying varieties of Chinese kale, characterized in that: The device includes a memory and a processor; The memory is used to store programs; The processor is configured to implement the following method by executing a program stored in the memory; Step 1: Obtain the DNA of the sample to be tested by PCR amplification and sequencing using the primer set described in claim 1; Step 2: Compare the sequencing results with the database described in claim 4 to obtain the genetic similarity coefficient between the sample to be tested and each known Chinese kale variety in the database; Step 3: Obtain the variety identification results of the sample to be tested based on the genetic similarity coefficient.
9. A computer-readable storage medium, characterized in that: Includes the database described in claim 4.
10. The computer-readable storage medium according to claim 9, characterized in that: It also includes a program that can be executed by a processor to implement the following methods; Step 1: Obtain the DNA of the sample to be tested by PCR amplification and sequencing using the primer set described in claim 1; Step 2: Compare the sequencing results with the database described in claim 4 to obtain the genetic similarity coefficient between the sample to be tested and each known Chinese kale variety in the database; Step 3: Obtain the variety identification results of the sample to be tested based on the genetic similarity coefficient.