A method for identifying cauliflower varieties

Abstract

This application relates to a method for cauliflower variety identification. Based on the results of resequencing 820 advanced cauliflower inbred lines, the method ultimately screened 41 SNP loci and corresponding KASP primer sets for genotyping cauliflower germplasm resources and varieties, establishing the first cauliflower DNA fingerprint, making genotyping more efficient, stable, accurate, and cost-effective. The SNP markers described in this application can be widely used for authenticity authentication of cauliflower varieties, genetic diversity analysis of germplasm resources, molecular marker-assisted breeding of cauliflower, and purity identification of cauliflower varieties.

Description

Technical Field

[0001] This application relates to a method for identifying cauliflower varieties, and belongs to the field of biotechnology. This application claims priority from CN202210624694.0, and incorporates the entire contents of CN202210624694.0 into this application. Background Art

[0002] Cauliflower (Brassica oleracea L.var. botrytis), also known as cauliflower, is a variety of the Brassica oleracea species in the Brassicaceae family. Its delicious flavor, sweet and crisp texture, rich nutrition, and cancer-fighting and health-promoting properties have made it a popular choice among consumers. Cauliflower plays a vital role in my country's vegetable production. With the development of my country's cauliflower industry, counterfeit seeds have appeared on the market, with some selling inferior products as genuine ones. Some people even illegally obtain breeding material for seed propagation, seriously impacting the interests of breeding companies and the healthy development of the cauliflower seed market.

[0003] DNA fingerprinting technology offers numerous advantages, including speed and accuracy, making it a powerful tool for detecting seed authenticity and purity. It has been widely used in analyzing genetic diversity and identifying plant varieties. SNP markers, as the most advanced third-generation molecular markers, offer low mutation rates and excellent genetic stability. However, they present two challenges: First, extensive screening is required to obtain valid, reliable SNP markers, leading to an urgent need for SNP markers that are broadly applicable to a wide range of cauliflower resources. Second, with hundreds or even thousands of cauliflower samples, how can the minimum number of SNP markers be used to detect the greatest number of samples? Therefore, it is necessary to identify the simplest combination of SNP markers or primer combinations corresponding to these SNP markers. Summary of the Invention

[0004] To solve the above problems, this application provides SNP markers and primers for constructing a cauliflower DNA map and for identifying cauliflower varieties, and selects 41 real and effective SNP markers and corresponding primer sets, some or all of which can solve the above problems. The information of the 41 SNP markers is shown in Table 1 and Figure 6 The information of the corresponding primer sets is shown in Table 2 and Figure 7-Figure 8 The combinations of 25 primer sets and / or 25 SNP markers that can distinguish all 153 core germplasm resources of cauliflower are shown in Table 3 (corresponding to Figure 9) as shown in Tables 4 and 5, along with other exemplary primer combinations. Therefore, researchers only need to select one or more marker combinations from among the 41 markers for cauliflower breeding, variety identification, and other applications, eliminating the extensive initial screening effort and enabling testing of a maximum number of cauliflower samples using a minimal number of SNP markers, greatly improving efficiency.

[0005] In some embodiments, the number of primer sets is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or 41.

[0006] The present application also provides the use of the above-mentioned primer sets and SNP sites in identifying the authenticity of cauliflower varieties or in detecting the purity of cauliflower varieties. For example, the present application provides a method for identifying the purity or authenticity of cauliflower germplasm, comprising the step of detecting the SNP site genotype of the genomic DNA of a cauliflower sample, wherein the SNP site is any one or any combination of the SNP sites in Table 1 or Tables 3-5. In some embodiments, the detection utilizes any one or any multiple primer sets mentioned above, preferably, the primer set and the SNP site have the same number. The present application also provides the use of the above-mentioned primer sets and SNP sites in constructing a cauliflower fingerprint map. The present application also provides the use of the above-mentioned primer sets and SNP sites in cauliflower breeding. The present application also provides the use of the above-mentioned primer sets and SNP sites in analyzing or evaluating the genetic diversity of cauliflower. The present application also provides a method for constructing a SNP fingerprint of cauliflower, comprising: (1) obtaining a corresponding primer (or primer set) according to any of the above-mentioned SNP sites, or using any of the above-mentioned primer sets; (2) amplifying cauliflower DNA with the primer set in (1), and constructing a SNP fingerprint of cauliflower based on the amplification results.

[0007] Table 1 41 SNP markers in cauliflower

[0008]

[0009]

[0010] Table 2 Primer set information for 41 cauliflower SNP markers

[0011]

[0012]

[0013]

[0014] Table 3 Preferred combinations of primer sets and SNP marker combinations (1)

[0015]

[0016]

[0017] Table 4 Preferred combinations of primer sets and SNP marker combinations (2)

[0018]

[0019]

[0020] Table 5 Preferred combinations of primer sets and SNP marker combinations (3)

[0021]

[0022] BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram of the distribution of 41 SNP markers on chromosomes.

[0024] Figure 2 It is a polymorphism indicator of 41 SNP markers.

[0025] Figure 3 Fluorescence scanning results of amplification products were obtained for 41 primer sets.

[0026] Figure 4 The result of constructing a phylogenetic tree for 820 cauliflower inbred lines using two sets of core SNP markers.

[0027] Figure 5 The results of population genetic analysis of 329 cauliflower varieties using 41 SNP markers. Figure 5 A is the result of principal component analysis, Figure 5 B is the result diagram of the constructed developmental tree. Figure 5 C is the result of population structure analysis using ADMIXTURE software.

[0028] Figure 6 Detailed information of 41 SNP markers is provided.

[0029] Figure 7 Information of 41 SNP markers and corresponding 41 primer sets.

[0030] Figure 8Figure 3 is a diagram of the optimization process of 41 SNP markers and primer sets, among which the SNP markers marked in yellow and the corresponding primer sets were unstable and were eventually screened out.

[0031] Figure 9 Information for 2 groups of core SNP markers.

[0032] Figure 10 Example 1 of SNP markers for distinguishing PK_5 and PK_9.

[0033] Figure 11 Example 2 of SNP markers for distinguishing PK_5 and PK_9.

[0034] Figure 12 A map of 41 SNP markers distinguishing 329 cauliflower varieties. DETAILED DESCRIPTION

[0035] In order to specifically illustrate the universal design concept of this application, specific experimental parameters are used as an example below, but this should not be used as a reason to limit the scope of protection of this application.

[0036] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. Unless otherwise specified, the materials, reagents, etc. used in the following examples are all commercially available.

[0037] This application provides 41 SNP sites and corresponding primer sets. Each primer set in the primer set can be used individually for separate PCR amplifications or together for multiplex PCR. When each primer pair is used together, the molar ratio of each primer can be arbitrary, for example, the same, that is, the molar ratio of the three primers in each primer set can be 1:1:1.

[0038] In some embodiments of the methods for authenticating cauliflower varieties, authenticating cauliflower seed purity, or constructing a cauliflower SNP fingerprint, the PCR products can be analyzed using software provided by an instrument, such as the software provided by an AB-Q6Flex fluorescence quantitative PCR instrument or the ArrayTape platform, to determine the genotypes of multiple SNP sites in the genomic DNA of the cauliflower sample to be tested. The genotypes of multiple SNP sites in the genomic DNA of the cauliflower sample to be tested can be determined by a method comprising the following steps: PCR amplifying the genomic DNA of the cauliflower sample to be tested using the KASP primer set to obtain PCR products, and detecting the PCR products to determine the genotypes of multiple SNP sites in the genomic DNA of the cauliflower sample to be tested.

[0039] When identifying seed purity, one strategy is to first use the 41 SNP sites to test the sample to be tested and select SNP sites that are beneficial to identifying the seed purity of the sample to be tested. The selected SNP sites can be 1-41 (i.e., 41≥n≥1, n is a natural number), and then use the selected SNP sites for further testing.

[0040] In some embodiments, the method for constructing a SNP fingerprint map of all cauliflower germplasm includes comparing the genotypes of 41 SNP sites in the genomic DNA of the cauliflower sample to be tested, and constructing a SNP fingerprint map of the cauliflower sample to be tested based on the genotypes of the 41 SNP sites; the 41 SNP sites are the SNP sites numbered 01-41 as shown in Table 1.

[0041] The present application also provides any of the following applications: X1) application of the KASP primer set in preparing a product for identifying or assisting in identifying the authenticity of a cauliflower variety; X2) application of the KASP primer set in preparing a product for identifying or assisting in identifying the purity of a cauliflower seed; X3) application of the KASP primer set in identifying or assisting in identifying the authenticity of a cauliflower variety; X4) application of the KASP primer set in identifying or assisting in identifying the purity of a cauliflower seed; X5) application of the KASP primer set in cauliflower breeding; X6) application of the method for constructing a cauliflower SNP fingerprint in identifying or assisting in identifying the authenticity of a cauliflower variety; X7) application of the method for constructing a cauliflower SNP fingerprint in identifying or assisting in identifying the purity of a cauliflower seed; X8) application of the method for constructing a cauliflower SNP fingerprint in cauliflower breeding; X9) application of the SNP marker in preparing a product for identifying or assisting in identifying a product for identifying or assisting in identifying a product for identifying a product for identifying a product for identifying a product for identifying a product for identifying a product for identifying a product for identifying a product for identifying a product for identifying a product for identifying a product for identifying a product for identifying a product for identifying a product for identifying a product for identifying a product for identifying a product for identifying a product for identifying a product for identifying a product for identifying a product for identifying a product for identifying a product for identifying a product for identifying a product for identifying a product for Application of the SNP marker in products for identifying the authenticity of cauliflower varieties; X10) Application of the SNP marker in the preparation of products for identifying or assisting in identifying the purity of cauliflower seeds; X11) Application of the SNP marker in identifying or assisting in identifying the authenticity of cauliflower varieties; X12) Application of the SNP marker in identifying or assisting in identifying the purity of cauliflower seeds; X13) Application of the SNP marker in cauliflower breeding; X14) Application of a substance for detecting nucleotides at SNP sites in the preparation of products for identifying the authenticity of cauliflower varieties; X15) Application of a substance for detecting nucleotides at SNP sites in identifying the authenticity of cauliflower varieties; X16) Application of a substance for detecting nucleotides at SNP sites in the preparation of products for identifying the purity of cauliflower seeds; X17) Application of a substance for detecting nucleotides at SNP sites in identifying the purity of cauliflower seeds; X18) Application of a substance for detecting nucleotides at SNP sites in cauliflower breeding.

[0042] Example 1 Development of SNP markers

[0043] 1.1 Construction of cauliflower SNP information database

[0044] 820 high-generation inbred lines of cauliflower (covering nearly all cauliflower germplasm) were planted, DNA extracted, and resequenced using second-generation Illumina Hi-Seq sequencing technology. The raw sequencing data for each line was at least 15x. GATK and other tools were used to identify SNP / InDel variants found in the inbred lines. Bioinformatics methods were used to analyze these variants and establish a database containing SNP / InDel molecular marker information.

[0045] 1.2 SNP site screening based on resequencing

[0046] Resequencing data were aligned with the known cauliflower 'C-8' reference genome to identify SNPs. The identified SNPs were screened according to the following criteria: ① quadruple degenerate SNPs located within a gene and biallelic; ② heterozygosity ≤ 0.2, deletion rate < 0.05, and minimum allele frequency ≥ 0.3; ③ no other SNPs or indels within 100 bp upstream or downstream; and ④ uniform distribution across the chromosome. Plink and PowerMarker software were used to calculate SNP variant type, polymorphism information content (PIC), and heterozygosity. These SNPs were then used for KASP primer design.

[0047] 1.3 Computer simulation of optimal SNP combinations

[0048] Randomly select 1-100 marker combinations and simulate their typing ability using a self-developed computer program. Simulate 5000 times for each marker number, and select the optimal combination. The distance between markers must be no less than 0.6 times the genome length divided by the number of markers to ensure even distribution of marker combinations across the genome.

[0049] 1.4KASP primer design and detection

[0050] For each identified SNP, the 50 bp sequence before and after the markers was truncated. Genomic copy number, GC content, and repetitive sequence content of the sequences near these markers were evaluated. High-quality KASP primers were designed and their efficiency verified. DNA from over 300 cauliflower hybrids was extracted and purified using the CTAB method. The DNA samples were then added to a 96-well plate for PCR amplification. The PCR reaction system consisted of 5 μL of KASP Master mix, 0.14 μL of KASP Primer mix, and 5 μL of 20 ng μL template DNA. The PCR reaction conditions were: a first cycle at 94°C for 15 min; 10 cycles of 94°C for 20 s, 61-55°C for 60 s, and touchdown; and a second cycle at 94°C for 20 s, 55°C for 60 s, for 26 cycles. PCR products were detected using a fluorescence microplate reader, and data were read using SNPviewer software.

[0051] 1.5 KASP detection data processing and fingerprint map construction

[0052] Based on the typing results, the allele frequency of each marker was calculated, and the PIC for each marker was calculated using the software PIC_Calc 0.6. Based on the dimorphic nature of SNP markers, the typing data were converted into binary encoding data, with the wild type (consistent with the reference genome) represented as (1,0), the mutant as (0,1), the heterozygous genotype as (1,1), and missing base sites as (999,999). Genotyping statistics were performed for each accession. The KASP testing results for over 300 cauliflower accessions were summarized, and missing genotypes were imputed using Beagle software. Fingerprints were constructed for different cauliflower accessions based on the KASP typing results.

[0053] 1.6 Genetic analysis of germplasm resource populations

[0054] FastTree and Figtree were used to cluster and construct a molecular evolutionary tree for over 300 cauliflower hybrids. Principal component analysis of SNP data was performed using GCTA software, and population structure analysis was performed using Admixture software.

[0055] result:

[0056] SNP calling was performed on the resequencing gene data of 820 cauliflower inbred lines, generating 17.9 million SNP site data. These SNP sites were screened according to the screening criteria, and 1662 SNPs were obtained. The most common mutation types are A / G and C / T, which belong to transition and transversion types, respectively, accounting for 27.02% and 25.51%, with a ratio of 1.11. The PIC of 1662 SNPs ranged from 0.363 to 0.504, with an average of 0.389; the minor allele frequency MAF ranged from 0.343 to 0.475, with an average of 0.419; the heterozygosity ranged from 0.029 to 0.193, with an average of 0.075; and the genetic diversity ranged from 0.473 to 0.591, with an average of 0.506. The PIC values ​​of 52% of the SNPs were between 0.38 and 0.39, and the PIC values ​​of 484 SNPs were between 0.39 and 0.504, indicating that the screened SNPs have good polymorphism.

[0057] A total of 41 SNP markers were finally selected. The average polymorphism index (PIC) and heterozygosity of 90% of the KASP markers were between 0.3 and 0.6; the diversity of 85% of the markers was between 0.4 and 0.6; and the minimum allele frequency (MAF) of 63% of the markers was between 0.4 and 0.5, showing good polymorphism and uniform distribution on the genome ( Figure 1 ). Figure 2 The polymorphism indices of 41 markers were listed. Figure 3 The results of reading the fluorescence scanning data of the amplified products using representative KASP markers are listed.

[0058] To test the representativeness of these 41 loci among the 1662 SNPs, phylogenetic trees were constructed for 820 cauliflower inbred lines using the two groups of SNP markers listed in Table 3. All 820 cauliflower germplasms could be divided into three groups. The two results showed the same differentiation trend and degree, indicating that the discrimination ability and representativeness of the 41 KASPs were reliable ( Figure 4 ) can all be used to construct a fingerprint for cauliflower. Techniques for constructing SNP fingerprints are known in the art. Therefore, the 41 SNP and KASP primer sets can fully distinguish all 820 cauliflower germplasms. Furthermore, the 41 SNP and KASP primer sets can also distinguish the 153 core cauliflower germplasms constructed by the inventors' team.

[0059] Example 2 Utilization of SNP markers

[0060] To further validate the discriminatory effects of one or more of the 41 SNP markers or KASP primer sets in cauliflower varieties, population genetic analysis was performed on 329 purchased cauliflower cultivars.

[0061] Results: The constructed phylogenetic tree showed that the 329 materials could be divided into three groups, and small subgroups were found between each group ( Figure 5 B). The results of principal component analysis (PCA) are consistent with the developmental tree, showing three parts ( Figure 5 A). Then, ADMIXTURE software was used to analyze the population structure, and the results showed that the optimal number of clusters was 9 ( Figure 5 C) This result is consistent with the subgroups shown in the developmental tree (taking into account the phenomenon of different names for the same species).

[0062] The final differentiation results of 329 cauliflower varieties using 41 SNP markers or KASP primer sets are shown in Figure 12 , where the vertical direction is 41 SNP markers and the horizontal direction is 329 cauliflower materials. Figure 12 It can also be found that using a specific combination of 25 markers or primer combinations (as shown in Table 3) is sufficient to achieve the same differentiation effect as 41 markers. Therefore, for these 329 cauliflower varieties, only 25 of the 41 markers are needed to distinguish them all.

[0063] Example 3 Cauliflower Variety Authenticity and Purity Identification

[0064] The authenticity and purity of cauliflower varieties PK-5 and PK-9 were identified using the marker or primer sets in Table 4 (e.g., PK-5 and PK-9 were mixed in a designed ratio). It can be seen that the set3 or set4 marker or primer set in Table 4 can identify and distinguish PK-5 and PK-9 varieties with high quality (see Figure 10-11 ).

[0065] from Figure 10-11 It can also be seen that when using the set3 or set4 marker or primer set for detection, the two varieties have many identical sites (redundant sites). Therefore, in order to detect as many cauliflower varieties as possible using as few primer sets as possible, any combination of one or more markers in Table 5 (such as set5 or set6) can be used to accurately identify and differentiate cauliflower PK-5 and PK-9 varieties without redundancy ( Figure 10-11 medium gray columns).

[0066] For other cauliflower varieties, researchers can simply use the cauliflower fingerprint (such as Figure 12 ), one or more marker combinations or primer sets can be selected from the above 41 markers or primer sets for use in breeding and variety identification of various cauliflowers, avoiding the extensive work of initial screening and enabling the detection of as many cauliflower samples as possible with a minimum number of SNP markers, thus greatly saving time.

[0067] In summary, this application, based on the resequencing of 820 high-generation inbred lines of cauliflower, constructed a database of tens of millions of SNP molecular marker information, developed KASP markers through computer simulation screening of SNP sites, and finally obtained 41 SNP sites and KASP primer sets through experimental screening to realize genotyping detection of cauliflower germplasm resources and varieties. The first cauliflower DNA fingerprint map was constructed, making genotyping more efficient, stable, accurate and low-cost. The SNP markers of this application can be widely used in the authenticity identification of cauliflower varieties, genetic diversity analysis of germplasm resources, molecular marker-assisted breeding and purity identification of cauliflower varieties.

[0068] Those skilled in the art may make various changes and modifications to the present application without departing from the spirit and scope of the present application. Thus, if these modifications and variations of the present application fall within the scope of the claims of the present application and their equivalents, the present application is intended to include such modifications and variations.

Claims

1. A KASP primer set, comprising the following 41 groups: Group 01: forward primer CTTCTCCGGCGACTTCC, forward primer GCTCTTCTCCCGGCGACTTCG, universal reverse primer GGACACGCTCACAATCTGGCCAA; Group 02: forward primer GGTCCGAGAAAAGCGGTTCC, forward primer AGGTCCGAGAAAAGCGGTTCT, universal reverse primer CACATCCGTAAAGACGAAATCCGCAT; Group 03: forward primer GAAGACCTAGAGTGGCTATCC, forward primer GGAAGACCTAGAGTGGCTATCA, universal reverse primer GGTTTAACCGGAACGTACTGCCTTT; Group 04: forward primer CATGCCTTCTAAGATGACCAAGTCT, forward primer CATGCCTTCTAAGATGACCAAGTCA, universal reverse primer AGAGGTTGCCTACCTTTCAACCGAT; Group 05: forward primer GAACAGAACTAGACCATGGTGTG, forward primer GGAACAGAACTAGACCATGGTGTA, universal reverse primer GATCCCTTTGATGAACCGTACCCAA; Group 06: forward primer AGCCTGAAACTTAGTCCATCTCTTG, forward primer CAGCCTGAAACTTAGTCCATCTCTTT, universal reverse primer GAGGAGTTGAGATGGAACGAGACTT; Group 07: forward primer TCTCGGAGTAGGTGATGAAGAAT, forward primer CTCGGAGTAGGTGATGAAGAAG, universal reverse primer TCTCTACTCGCTGTCCTCTTCCTAT; Group 08: forward primer GATATGTGTGATGAGTCTGTCTGCA, forward primer GATATGTGTGATGAGTCTGTCTGCT, universal reverse primer TGTGCTCAGAGTTGTGATCAGGGAA; Group 09: forward primer ATGGAGCTAGCTCCAGGGGTT, forward primer GGAGCTAGCTCCAGGGGTC, universal reverse primer TACGGCAACTCATCTAACCCTGGAT; Group 10: forward primer ACATCTCTGTTTATCATCAGGTGTC, forward primer CACATCTCTGTTTATCATCAGGTGTT, universal reverse primer CCATGTGGGCTAAAGCTTTGGGATT; Group 11: forward primer GTGTCAACAATGTTGGCAAAGGCT, forward primer GTGTCAACAATGTTGGCAAAGGCA, universal reverse primer CATCCGCGTCCTTCCTTATAACCAA; Group 12: forward primer GGGTAACTTGATGATAAAAGAAACCG, forward primer GGGGTAACTTGATGATAAAAGAAACCA, universal reverse primer CGAATGTGTTGGCACTTCTTGGCAA; Group 13: forward primer ACGATGAAGGGAGAGACTGACA, forward primer CGATGAAGGGAGAGACTGACT, universal reverse primer CGATATCCAAGCTCCACCTACTCTT; Group 14: forward primer CCTGGCTGCATCTTCTAAATTGGTA, forward primer CTGGCTGCATCTTCTAAATTGGTG, universal reverse primer GTTGTTGGAGACTTGATGACCCCTT; Group 15: forward primer GTTGTATCATTAACCCAACGAATA, forward primer CTGTTGTATCATTAACCCAACGAATG, universal reverse primer AGGACTACTGATCTGGAGAGGCTTT; Group 16: forward primer CCACCATTGTTTCCTCATGGAAG, forward primer GTCCACCATTGTTTCCTCATGGAAA, universal reverse primer AAGATTCCGAGCCTGAGAAGACCAA; Group 17: forward primer GATATCAAACTTGGAAGAGCCATCC, forward primer GATATCAAACTTGGAAGAGCCATCG, universal reverse primer CCCTCTTTGGAGGTGATGCATTGAA; Group 18: forward primer AATTTTGGGTGTTGTGGTGGCG, forward primer GAATAATTTGGGTGTTGTGGTGGCA, universal reverse primer GAAGCTGTGGCCAACTCACAAGAAT; Group 19: forward primer GTTGAACGTGAAAATCAAGAGGG, forward primer ATGTTGAACGTGAAAATCAAGAGGA, universal reverse primer CTCTTCGCCAGAGGAAGGAATTGTT; Group 20: forward primer CGGTCGGAGTCACTTCGGTG, forward primer CGGTCGGAGTCACTTCGGTC, universal reverse primer CTAACTTGTAGACCGGTTCGTGCAT; Group 21: forward primer CTGAGTCCAGCTACAGAGGAC, forward primer CTGAGTCCAGCTACAGAGGAG, universal reverse primer ACCGTGAAGATATCGTCTCAGGGAA; Group 22: forward primer AATGTCCGACGAAGCAAAACCG, universal reverse primer AATGTCCGACGAAGCAAAACCC, universal reverse primer AATGCAGTGATCGAGCTGCTTCGTA; Group 23: forward primer AGGTACGAATGTTGGGTTCAGGT, forward primer GGTACGAATGTTGGGTTCAGGC, universal reverse primer CATCAATGATTCCTGGGATGGGCAT; Group 24: forward primer GCACCATACCAGAACTTCAAGAAC, forward primer AATGCACCATACCAGAACTTCAAGAAT, universal reverse primer TGGGAGGAACTACGTTGCTCTTCAT; Group 25: forward primer CCAGTATCTGTTGAAATAACTTATGGC, forward primer CCAGTATCTGTTGAAATAACTTATGGG, universal reverse primer TGAAGCAACATGAGGATGCGCTCAA; Group 26: forward primer GAAGGCCTTTTCCAACATTGGC, forward primer TTGAAGGCCTTTTCCAACATTGGA, universal reverse primer CTGACCTTGAGACAGTAAGACGCTA; Group 27: forward primer CGGCGCCGGCACATTTTAGG, forward primer CCGGCGCCGGCACATTTTAGA, universal reverse primer AACGGTAACTGCGTCACAGGAGATT; Group 28: forward primer TATAGGTGGAGACAATGGAGGC, forward primer GTTATAGGTGGAGACAATGGAGGT, universal reverse primer GATTCCACCAACGGTTTCTCCACAA; Group 29: forward primer CCCCGCTCATTAGCACAGGT, forward primer CCCGCTCATTAGCACAGGG, universal reverse primer GCTCAGAGGGCCCTTAAGAAGATTA; Group 30: forward primer ACAGCTTTTTGTTCCCGAGCTCG, forward primer ACAGCTTTTTGTTCCCGAGCTCC, universal reverse primer TGGACCGGAATCACCTGCAACAAAT; Group 31: forward primer TTTCTTCTCAGGAGCAGCGACA, forward primer CTTCTCAGGAGCAGCGACG, universal reverse primer CGAATATGATCACCAGCACCAGCAA; Group 32: forward primer GTCTAGATTAATCTGGACTGTGTTG, forward primer CGTCTAGATTAATCTGGACTGTGTTA, universal reverse primer CTACCGAGACACTTGATGTGGGTTA; Group 33: forward primer CAAGCAGCCTACAAGCAACTTGT, forward primer AAGCAGCCTACAAGCAACTTGC, universal reverse primer CCTTGGATGACCCTGAGAGAAGTT; Group 34: forward primer CCGTGCACTAACATCGACTCGA, forward primer CCGTGCACTAACATCGACTCGT, universal reverse primer CGCTGTTCTCATACGACGATGGATA; Group 35: forward primer AGGTTTAGATACGAAGGTCTTGTC, forward primer AGGTTTAGATACGAAGGTCTTGTG, universal reverse primer TTGCTATGAAGAGCGACACAGCCAA; Group 36: forward primer CCGACAAAACCTAACTGAGCC, forward primer CTCCGACAAAACCTAACTGAGCT, universal reverse primer CTCACTGCACGACCGGTTCCTAA; Group 37: forward primer CCCAACCCAAGCCACGACCA, forward primer CCAACCCAAGCCACGACCG, universal reverse primer GGTTAACCCTCCGCTTGAGAATGAT; Group 38: forward primer AGATATGGTTCCCTTCTGAAGGC, forward primer GAGATATGGTTCCCTTCTGAAGGA, universal reverse primer GGCGCACTCCTGTAAATTTGTGGAT; Group 39: forward primer ATGGTCAACGGTCTCATCGGC, forward primer CATGGTCAACGGTCTCATCGGA, universal reverse primer CAGTTCTGAGCACTGCACACACAAA; Group 40: forward primer TTTGAAATGGATGGCGAGGAGCTT, forward primer GAAATGGATGGCGAGGAGCTC, universal reverse primer GCTCTCGGATAAGCACAGCTTTCTT; Group 41: forward primer GAAGACGAGGAGCGGAATGTG, forward primer GGAAGACGAGGAGCGGAATGTA, universal reverse primer CGACACCGACCAAAGAGTGCAAAAT.

2. A KASP primer set, characterized in that: The primer set includes 25 primer sets, specifically: ①Set1: Group 01: forward primer CTTCTCCGGCGACTTCC, forward primer GCTCTTCTCCCGGCGACTTCG, universal reverse primer GGACACGCTCACAATCTGGCCAA; Group 03: forward primer GAAGACCTAGAGTGGCTATCC, forward primer GGAAGACCTAGAGTGGCTATCA, universal reverse primer GGTTTAACCGGAACGTACTGCCTTT; Group 04: forward primer CATGCCTTCTAAGATGACCAAGTCT, forward primer CATGCCTTCTAAGATGACCAAGTCA, universal reverse primer AGAGGTTGCCTACCTTTCAACCGAT; Group 06: forward primer AGCCTGAAACTTAGTCCATCTCTTG, forward primer CAGCCTGAAACTTAGTCCATCTCTTT, universal reverse primer GAGGAGTTGAGATGGAACGAGACTT; Group 08: forward primer GATATGTGTGATGAGTCTGTCTGCA, forward primer GATATGTGTGATGAGTCTGTCTGCT, universal reverse primer TGTGCTCAGAGTTGTGATCAGGGAA; Group 10: forward primer ACATCTCTGTTTATCATCAGGTGTC, forward primer CACATCTCTGTTTATCATCAGGTGTT, universal reverse primer CCATGTGGGCTAAAGCTTTGGGATT; Group 12: forward primer GGGTAACTTGATGATAAAAGAAACCG, forward primer GGGGTAACTTGATGATAAAAGAAACCA, universal reverse primer CGAATGTGTTGGCACTTCTTGGCAA; Group 14: forward primer CCTGGCTGCATCTTCTAAATTGGTA, forward primer CTGGCTGCATCTTCTAAATTGGTG, universal reverse primer GTTGTTGGAGACTTGATGACCCCTT; Group 17: forward primer GATATCAAACTTGGAAGAGCCATCC, forward primer GATATCAAACTTGGAAGAGCCATCG, universal reverse primer CCCTCTTTGGAGGTGATGCATTGAA; Group 18: forward primer AATTTTGGGTGTTGTGGTGGCG, forward primer GAATAATTTGGGTGTTGTGGTGGCA, universal reverse primer GAAGCTGTGGCCAACTCACAAGAAT; Group 22: forward primer AATGTCCGACGAAGCAAAACCG, universal reverse primer AATGTCCGACGAAGCAAAACCC, universal reverse primer AATGCAGTGATCGAGCTGCTTCGTA; Group 23: forward primer AGGTACGAATGTTGGGTTCAGGT, forward primer GGTACGAATGTTGGGTTCAGGC, universal reverse primer CATCAATGATTCCTGGGATGGGCAT; Group 25: forward primer CCAGTATCTGTTGAAATAACTTATGGC, forward primer CCAGTATCTGTTGAAATAACTTATGGG, universal reverse primer TGAAGCAACATGAGGATGCGCTCAA; Group 26: forward primer GAAGGCCTTTTCCAACATTGGC, forward primer TTGAAGGCCTTTTCCAACATTGGA, universal reverse primer CTGACCTTGAGACAGTAAGACGCTA; Group 28: forward primer TATAGGTGGAGACAATGGAGGC, forward primer GTTATAGGTGGAGACAATGGAGGT, universal reverse primer GATTCCACCAACGGTTTCTCCACAA; Group 29: forward primer CCCCGCTCATTAGCACAGGT, forward primer CCCGCTCATTAGCACAGGG, universal reverse primer GCTCAGAGGGCCCTTAAGAAGATTA; Group 30: forward primer ACAGCTTTTTGTTCCCGAGCTCG, forward primer ACAGCTTTTTGTTCCCGAGCTCC, universal reverse primer TGGACCGGAATCACCTGCAACAAAT; Group 32: forward primer GTCTAGATTAATCTGGACTGTGTTG, forward primer CGTCTAGATTAATCTGGACTGTGTTA, universal reverse primer CTACCGAGACACTTGATGTGGGTTA; Group 33: forward primer CAAGCAGCCTACAAGCAACTTGT, forward primer AAGCAGCCTACAAGCAACTTGC, universal reverse primer CCTTGGATGACCCTGAGAGAAGTT; Group 34: forward primer CCGTGCACTAACATCGACTCGA, forward primer CCGTGCACTAACATCGACTCGT, universal reverse primer CGCTGTTCTCATACGACGATGGATA; Group 35: forward primer AGGTTTAGATACGAAGGTCTTGTC, forward primer AGGTTTAGATACGAAGGTCTTGTG, universal reverse primer TTGCTATGAAGAGCGACACAGCCAA; Group 36: forward primer CCGACAAAACCTAACTGAGCC, forward primer CTCCGACAAAACCTAACTGAGCT, universal reverse primer CTCACTGCACGACCGGTTCCTAA; Group 38: forward primer AGATATGGTTCCCTTCTGAAGGC, forward primer GAGATATGGTTCCCTTCTGAAGGA, universal reverse primer GGCGCACTCCTGTAAATTTGTGGAT; Group 40: forward primer TTTGAAATGGATGGCGAGGAGCTT, forward primer GAAATGGATGGCGAGGAGCTC, universal reverse primer GCTCTCGGATAAGCACAGCTTTCTT; Group 41: forward primer GAAGACGAGGAGCGGAATGTG, forward primer GGAAGACGAGGAGCGGAATGTA, universal reverse primer CGACACCGACCAAAGAGTGCAAAAT; Or ②Set2: Group 01: forward primer CTTCTCCGGCGACTTCC, forward primer GCTCTTCTCCCGGCGACTTCG, universal reverse primer GGACACGCTCACAATCTGGCCAA; Group 03: forward primer GAAGACCTAGAGTGGCTATCC, forward primer GGAAGACCTAGAGTGGCTATCA, universal reverse primer GGTTTAACCGGAACGTACTGCCTTT; Group 04: forward primer CATGCCTTCTAAGATGACCAAGTCT, forward primer CATGCCTTCTAAGATGACCAAGTCA, universal reverse primer AGAGGTTGCCTACCTTTCAACCGAT; Group 05: forward primer GAACAGAACTAGACCATGGTGTG, forward primer GGAACAGAACTAGACCATGGTGTA, universal reverse primer GATCCCTTTGATGAACCGTACCCAA; Group 08: forward primer GATATGTGTGATGAGTCTGTCTGCA, forward primer GATATGTGTGATGAGTCTGTCTGCT, universal reverse primer TGTGCTCAGAGTTGTGATCAGGGAA; Group 09: forward primer ATGGAGCTAGCTCCAGGGGTT, forward primer GGAGCTAGCTCCAGGGGTC, universal reverse primer TACGGCAACTCATCTAACCCTGGAT; Group 10: forward primer ACATCTCTGTTTATCATCAGGTGTC, forward primer CACATCTCTGTTTATCATCAGGTGTT, universal reverse primer CCATGTGGGCTAAAGCTTTGGGATT; Group 11: forward primer GTGTCAACAATGTTGGCAAAGGCT, forward primer GTGTCAACAATGTTGGCAAAGGCA, universal reverse primer CATCCGCGTCCTTCCTTATAACCAA; Group 12: forward primer GGGTAACTTGATGATAAAAGAAACCG, forward primer GGGGTAACTTGATGATAAAAGAAACCA, universal reverse primer CGAATGTGTTGGCACTTCTTGGCAA; Group 14: forward primer CCTGGCTGCATCTTCTAAATTGGTA, forward primer CTGGCTGCATCTTCTAAATTGGTG, universal reverse primer GTTGTTGGAGACTTGATGACCCCTT; Group 17: forward primer GATATCAAACTTGGAAGAGCCATCC, forward primer GATATCAAACTTGGAAGAGCCATCG, universal reverse primer CCCTCTTTGGAGGTGATGCATTGAA; Group 18: forward primer AATTTTGGGTGTTGTGGTGGCG, forward primer GAATAATTTGGGTGTTGTGGTGGCA, universal reverse primer GAAGCTGTGGCCAACTCACAAGAAT; Group 19: forward primer GTTGAACGTGAAAATCAAGAGGG, forward primer ATGTTGAACGTGAAAATCAAGAGGA, universal reverse primer CTCTTCGCCAGAGGAAGGAATTGTT; Group 21: forward primer CTGAGTCCAGCTACAGAGGAC, forward primer CTGAGTCCAGCTACAGAGGAG, universal reverse primer ACCGTGAAGATATCGTCTCAGGGAA; Group 22: forward primer AATGTCCGACGAAGCAAAACCG, universal reverse primer AATGTCCGACGAAGCAAAACCC, universal reverse primer AATGCAGTGATCGAGCTGCTTCGTA; Group 26: forward primer GAAGGCCTTTTCCAACATTGGC, forward primer TTGAAGGCCTTTTCCAACATTGGA, universal reverse primer CTGACCTTGAGACAGTAAGACGCTA; Group 28: forward primer TATAGGTGGAGACAATGGAGGC, forward primer GTTATAGGTGGAGACAATGGAGGT, universal reverse primer GATTCCACCAACGGTTTCTCCACAA; Group 29: forward primer CCCCGCTCATTAGCACAGGT, forward primer CCCGCTCATTAGCACAGGG, universal reverse primer GCTCAGAGGGCCCTTAAGAAGATTA; Group 30: forward primer ACAGCTTTTTGTTCCCGAGCTCG, forward primer ACAGCTTTTTGTTCCCGAGCTCC, universal reverse primer TGGACCGGAATCACCTGCAACAAAT; Group 32: forward primer GTCTAGATTAATCTGGACTGTGTTG, forward primer CGTCTAGATTAATCTGGACTGTGTTA, universal reverse primer CTACCGAGACACTTGATGTGGGTTA; Group 34: forward primer CCGTGCACTAACATCGACTCGA, forward primer CCGTGCACTAACATCGACTCGT, universal reverse primer CGCTGTTCTCATACGACGATGGATA; Group 35: forward primer AGGTTTAGATACGAAGGTCTTGTC, forward primer AGGTTTAGATACGAAGGTCTTGTG, universal reverse primer TTGCTATGAAGAGCGACACAGCCAA; Group 38: forward primer AGATATGGTTCCCTTCTGAAGGC, forward primer GAGATATGGTTCCCTTCTGAAGGA, universal reverse primer GGCGCACTCCTGTAAATTTGTGGAT; Group 40: forward primer TTTGAAATGGATGGCGAGGAGCTT, forward primer GAAATGGATGGCGAGGAGCTC, universal reverse primer GCTCTCGGATAAGCACAGCTTTCTT; Group 41: forward primer GAAGACGAGGAGCGGAATGTG, forward primer GGAAGACGAGGAGCGGAATGTA, universal reverse primer CGACACCGACCAAAGAGTGCAAAAT.

3. A method for identifying the purity or authenticity of cauliflower germplasm or a method for constructing a cauliflower DNA fingerprint, comprising the step of detecting the genotype of a single nucleotide polymorphism (SNP) site in genomic DNA of a cauliflower sample, wherein the SNP site comprises the following 41 SNPs: Cauliflower C-8 was used as the reference genome; The SNP numbered 01 is located at position 860653 on chromosome 1, and its deoxynucleotide is C or G; SNP numbered 02 is located at position 975765 on chromosome 1, and its deoxynucleotide is C or T; SNP numbered 03 is located at position 1581568 on chromosome 1, and its deoxynucleotide is C or A; SNP numbered 04 is located at position 2022083 on chromosome 1, and its deoxynucleotide is A or T; SNP numbered 05 is located at position 2022902 on chromosome 1, and its deoxynucleotide is C or T; SNP numbered 06 is located at position 9494597 on chromosome 1, and its deoxynucleotide is G or T; SNP numbered 07 is located at position 16591554 on chromosome 1, and its deoxynucleotide is A or C; SNP numbered 08 is located at position 45449565 on chromosome 1, and its deoxynucleotide is T or A; SNP numbered 09 is located at position 3174530 on chromosome 2, and its deoxynucleotide is A or G; SNP numbered 10 is located at position 14297682 on chromosome 2, and its deoxynucleotide is G or A; SNP numbered 11 is located at position 39230790 on chromosome 2, and its deoxynucleotide is T or A; SNP numbered 12 is located at position 65005201 on chromosome 2, and its deoxynucleotide is C or T; SNP numbered 13 is located at position 66045832 on chromosome 2, and its deoxynucleotide is A or T; SNP numbered 14 is located at position 6005226 on chromosome 3, and its deoxynucleotide is T or C; SNP numbered 15 is located at position 73929470 on chromosome 3, and its deoxynucleotide is T or C; SNP numbered 16 is located at position 75221240 on chromosome 3, and its deoxynucleotide is T or C; SNP numbered 17 is located at position 14063327 on chromosome 4, and its deoxynucleotide is C or G; SNP numbered 18 is located at position 52102326 on chromosome 4, and its deoxynucleotide is G or A; SNP numbered 19 is located at position 311091 on chromosome 5, and its deoxynucleotide is C or T; SNP numbered 20 is located at position 326763 on chromosome 5, and its deoxynucleotide is G or C; SNP numbered 21 is located at position 4246754 on chromosome 5, and its deoxynucleotide is G or C; SNP numbered 22 is located at position 42917099 on chromosome 5, and its deoxynucleotide is G or C; SNP numbered 23 is located at position 42919228 on chromosome 5, and its deoxynucleotide is T or C; SNP numbered 24 is located at position 1872141 on chromosome 6, and its deoxynucleotide is G or A; SNP numbered 25 is located at position 34,494,441 on chromosome 6, and its deoxynucleotide is G or C; SNP numbered 26 is located at position 40582776 on chromosome 6, and its deoxynucleotide is G or T; SNP numbered 27 is located at position 46214109 on chromosome 6, and its deoxynucleotide is C or T; SNP numbered 28 is located at position 6266029 on chromosome 7, and its deoxynucleotide is G or A; SNP numbered 29 is located at position 7108476 on chromosome 7, and its deoxynucleotide is T or G; SNP numbered 30 is located at position 18611643 on chromosome 7, and its deoxynucleotide is C or G; SNP numbered 31 is located at position 26326101 on chromosome 7, and its deoxynucleotide is G or A; SNP numbered 32 is located at position 49710841 on chromosome 7, and its deoxynucleotide is C or T; SNP numbered 33 is located at position 685258 on chromosome 8, and its deoxynucleotide is A or G; SNP numbered 34 is located at position 7993815 on chromosome 8, and its deoxynucleotide is A or T; SNP numbered 35 is located at position 10333745 on chromosome 8, and its deoxynucleotide is C or G; SNP numbered 36 is located at position 10995531 on chromosome 8, and its deoxynucleotide is G or A; SNP numbered 37 is located at position 16285895 on chromosome 8, and its deoxynucleotide is A or G; SNP numbered 38 is located at position 23662826 on chromosome 8, and its deoxynucleotide is C or A; SNP numbered 39 is located at position 24753546 on chromosome 8, and its deoxynucleotide is C or A; SNP numbered 40 is located at position 51980792 on chromosome 8, and its deoxynucleotide is T or C; SNP numbered 41 is located at position 2367375 on chromosome 9, and its deoxynucleotide is C or T.

4. The method according to claim 3, wherein include: (1) obtaining an amplification primer set corresponding to the SNP site according to claim 3, or using the primer set according to claim 1; (2) amplifying cauliflower DNA with the primer set in (1), and identifying the purity or authenticity of the cauliflower germplasm based on the amplification results, or constructing a cauliflower DNA fingerprint.

5. Use of the primer set according to claim 1 or the SNP site according to claim 3 in identifying the authenticity of a cauliflower variety or detecting the purity of a cauliflower variety, or in constructing a DNA fingerprint of a cauliflower.

6. Use of the primer set according to claim 1 or the SNP site according to claim 3 in analyzing or evaluating the genetic diversity of a cauliflower population.

7. Use of the primer set according to claim 2 in identifying the authenticity of cauliflower varieties or detecting the purity of cauliflower varieties.

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