A SNP site combination, a KASP primer combination, a kit and application thereof in identifying carrot varieties

Abstract

The application discloses a SNP site combination, a KASP primer combination, a kit and application thereof in identifying carrot varieties. The application is based on whole genome resequencing of 244 carrot germplasm resources with wide genetic diversity, 7300 candidate SNP sites are screened from the carrot germplasm resources, and finally, 30 SNP sites covering all 9 chromosomes of the carrot and corresponding KASP primers are developed, which can be used for accurate genetic background identification and material identification.

Description

Technical Field

[0001] This invention belongs to the field of agricultural technology, specifically relating to an SNP site combination, a KASP primer combination, a reagent kit, and their application in identifying carrot varieties. Background Technology

[0002] In crop hybridization breeding, especially backcrossing, the efficiency of breeding selection can be improved by quickly and accurately screening individuals whose genetic background is as consistent as possible with superior parents. Furthermore, for newly introduced or collected breeding resources or varieties, clarifying their genetic background allows for targeted breeding improvements and utilization. Carrots are biennial root vegetable crops with long breeding cycles, and the accuracy of traditional phenotypic identification is easily affected by environmental factors. Therefore, accurate and efficient "background selection" technology plays a significant role in the breeding of carrot hybrid varieties.

[0003] Currently, carrot breeding selection mainly relies on field phenotypic observations, while a limited number of traditional molecular marker technologies have been developed, such as simple sequence repeats (SSR, or microsatellite markers) and random amplified polymorphic DNA (RAPD) markers. Breeders use these limited markers to perform genotyping on hybrid offspring populations, and by analyzing the inheritance patterns of the markers, they can indirectly infer the degree of restoration of an individual's genetic background, thus assisting in the selection of target individuals.

[0004] However, traditional empirical breeding has many uncertainties, is easily affected by environmental factors, has low selection efficiency and poor reproducibility, and is difficult to accurately identify breeding materials. Traditional marker technologies, represented by SSR, have a limited number of markers, are mostly concentrated in gene-rich regions, have low background selection resolution, and rely on gel electrophoresis or capillary electrophoresis, which are cumbersome, time-consuming, have low automation, and high identification costs.

[0005] Overcoming the above technical bottlenecks and establishing an efficient carrot background selection technology presents several key challenges: ① Ideal background selection requires tens of thousands of markers evenly distributed across the genome; however, developing a massive number of markers and ensuring their stable and accurate detection is a challenge both technically and practically. ② Traditional markers struggle to evenly cover the entire genome, resulting in low accuracy, cumbersome processes, and inconsistent detection standards. ③ While high-throughput sequencing technology can provide whole-genome information, its high cost and complex data analysis process make it difficult to apply on a large scale and in multiple rounds in conventional breeding. Therefore, a cost-effective technical solution that provides accurate genetic information is urgently needed. Summary of the Invention

[0006] In view of this, the present invention proposes an SNP locus combination, a KASP primer combination, a kit, and their application in identifying carrot varieties. The SNP locus combination screened by the present invention is evenly distributed in the genome, avoiding the problem of traditional markers being concentrated in gene-rich regions, and significantly improving the resolution and accuracy of genetic background identification. It can be used for precise genetic background identification and material identification.

[0007] The technical solution of this invention is implemented as follows:

[0008] In a first aspect, the present invention provides a combination of SNP loci for identifying carrot varieties, the combination of SNP loci consisting of 30 SNP loci, the information of the 30 SNP loci including:

[0009] SNP1 is located at position 3378909 on chromosome 1, and its allelic variation is T / G.

[0010] SNP2 is located at position 36213768 on chromosome 1, and its allelic variation is T / C.

[0011] SNP3 is located at position 49750001 on chromosome 1, and its allelic variation is T / C.

[0012] SNP4 is located at position 12004076 on chromosome 2, and its allelic variation is T / C.

[0013] SNP5 is located at position 32694778 on chromosome 2, and its allelic variation is A / T.

[0014] SNP6 is located at position 54566859 on chromosome 2, and its allelic variation is C / T.

[0015] SNP7 is located at position 2139316 on chromosome 3, and its allelic variation is C / T.

[0016] SNP8 is located at position 59862819 on chromosome 3, and its allelic variation is T / C.

[0017] SNP9 is located at position 15000238 on chromosome 4, and its allelic variation is G / T;

[0018] SNP10 is located at position 31185658 on chromosome 4, and its allelic variation is G / A.

[0019] SNP11 is located at position 33289495 on chromosome 4, and its allelic variation is C / T.

[0020] SNP12 is located at position 48827130 on chromosome 4, and its allelic variation is G / A.

[0021] SNP13 is located at position 2674035 on chromosome 5, and its allelic variation is A / C.

[0022] SNP14 is located at position 30110137 on chromosome 5, and its allelic variation is T / C.

[0023] SNP15 is located at position 42967130 on chromosome 5, and its allelic variation is T / C.

[0024] SNP16 is located at position 25653180 on chromosome 6, and its allelic variant base is T / G.

[0025] SNP17 is located at position 27564723 on chromosome 6, and its allelic variation is G / A.

[0026] SNP18 is located at position 38383474 on chromosome 6, and its allelic variation is T / C.

[0027] SNP19 is located at position 4019817 on chromosome 7, and its allelic variation is A / G.

[0028] SNP20 is located at position 26295659 on chromosome 7, and its allelic variation is A / T.

[0029] SNP21 is located at position 33609659 on chromosome 7, and its allelic variation is T / A.

[0030] SNP22 is located at position 35540647 on chromosome 7, and its allelic variation is C / T.

[0031] SNP23 is located at position 10,158,974 on chromosome 8, and its allelic variation is T / C.

[0032] SNP24 is located at position 23348460 on chromosome 8, and its allelic variation is A / G.

[0033] SNP25 is located at position 31837294 on chromosome 8, and its allelic variation is A / T.

[0034] SNP26 is located at position 1061607 on chromosome 9, and its allelic variation is A / G.

[0035] SNP27 is located at position 2609626 on chromosome 9, and its allelic variation is C / T.

[0036] SNP28 is located at position 12172120 on chromosome 9, and its allelic variation is C / T.

[0037] SNP29 is located at position 22280031 on chromosome 9, and its allelic variant base is G / T.

[0038] SNP30 is located at position 32876062 on chromosome 9, and its allelic variation is A / G.

[0039] This invention utilizes whole-genome resequencing of 244 carrot germplasm resources with broad genetic diversity to screen for 7300 candidate SNP loci, ultimately developing 30 SNP loci covering all nine carrot chromosomes. These SNP loci are evenly distributed across the genome, avoiding the problem of traditional markers being concentrated in gene-rich regions, and significantly improving the resolution and accuracy of genetic background identification.

[0040] Secondly, the present invention provides a KASP primer combination for detecting the 30 SNP sites, wherein the KASP primer combination corresponding to each SNP site includes a forward primer 1, a forward primer 2 and a common reverse primer;

[0041] Forward primer 1, forward primer 2, and common reverse primer used to detect SNP1 are shown in SEQ ID NO. 1~3;

[0042] Forward primer 1, forward primer 2, and common reverse primer used to detect SNP2 are shown in SEQ ID NO. 4~6;

[0043] Forward primer 1, forward primer 2, and common reverse primer used to detect SNP3 are shown in SEQ ID NO. 7~9;

[0044] Forward primer 1, forward primer 2, and common reverse primer used to detect SNP4 are shown in SEQ ID NO. 10~12;

[0045] Forward primer 1, forward primer 2, and common reverse primer used to detect SNP5 are shown in SEQ ID NO. 13~15;

[0046] Forward primer 1, forward primer 2 and common reverse primer used to detect SNP6 are shown in SEQ ID NO.16~18;

[0047] The forward primer 1, forward primer 2 and common reverse primer used to detect SNP7 are shown in SEQ ID NO.19~21;

[0048] The forward primer 1, forward primer 2 and common reverse primer used to detect SNP8 are shown in SEQ ID NO. 22~24;

[0049] Forward primer 1, forward primer 2 and common reverse primer used to detect SNP9 are shown in SEQ ID NO. 25~27;

[0050] The forward primer 1, forward primer 2 and common reverse primer used to detect SNP10 are shown in SEQ ID NO.28~30;

[0051] Forward primer 1, forward primer 2 and common reverse primer used to detect SNP11 are shown in SEQ ID NO. 31~33;

[0052] The forward primer 1, forward primer 2 and common reverse primer used to detect SNP12 are shown in SEQ ID NO. 34~36;

[0053] The forward primer 1, forward primer 2 and common reverse primer used to detect SNP13 are shown in SEQ ID NO. 36~39;

[0054] Forward primer 1, forward primer 2 and common reverse primer used to detect SNP14 are shown in SEQ ID NO.40~42;

[0055] The forward primer 1, forward primer 2 and common reverse primer used to detect SNP15 are shown in SEQ ID NO.43~45;

[0056] Forward primer 1, forward primer 2 and common reverse primer used to detect SNP16 are shown in SEQ ID NO.46~48;

[0057] The forward primer 1, forward primer 2 and common reverse primer used to detect SNP17 are shown in SEQ ID NO.49~51;

[0058] Forward primer 1, forward primer 2 and common reverse primer used to detect SNP18 are shown in SEQ ID NO. 52~54;

[0059] Forward primer 1, forward primer 2 and common reverse primer used to detect SNP19 are shown in SEQ ID NO. 55~57;

[0060] The forward primer 1, forward primer 2 and common reverse primer used to detect SNP20 are shown in SEQ ID NO.58~60;

[0061] The forward primer 1, forward primer 2 and common reverse primer used to detect SNP21 are shown in SEQ ID NO. 61~63;

[0062] The forward primer 1, forward primer 2 and common reverse primer used to detect SNP22 are shown in SEQ ID NO. 64~66;

[0063] The forward primer 1, forward primer 2 and common reverse primer used to detect SNP23 are shown in SEQ ID NO. 67~69;

[0064] The forward primer 1, forward primer 2 and common reverse primer used to detect SNP24 are shown in SEQ ID NO. 70~72;

[0065] Forward primer 1, forward primer 2 and common reverse primer used to detect SNP25 are shown in SEQ ID NO.73~75;

[0066] The forward primer 1, forward primer 2 and common reverse primer used to detect SNP26 are shown in SEQ ID NO.76~78;

[0067] The forward primer 1, forward primer 2 and common reverse primer used to detect SNP27 are shown in SEQ ID NO.79~81;

[0068] The forward primer 1, forward primer 2 and common reverse primer used to detect SNP28 are shown in SEQ ID NO. 82~84;

[0069] The forward primer 1, forward primer 2 and common reverse primer used to detect SNP29 are shown in SEQ ID NO. 85~87;

[0070] The forward primer 1, forward primer 2, and common reverse primer used to detect SNP30 are shown in SEQ ID NO. 88~90.

[0071] Thirdly, the present invention provides a method for genotyping carrots using the KASP primers, comprising the following steps: S1, mixing carrot genomic DNA with a reaction system containing the KASP primer combination, performing PCR amplification, and obtaining PCR amplification products;

[0072] S2. Genetically type carrots based on the fluorescence signal of the PCR amplification product.

[0073] Fourthly, the present invention provides a kit comprising the aforementioned KASP primer combination.

[0074] Fifthly, the present invention provides the application of the SNP site combination, KASP primer combination or kit in any of the following: (1) identification of the genetic background of carrot breeding materials;

[0075] (2) Analysis of the phylogenetic relationships of carrot varieties, strains, or germplasm resources;

[0076] (3) Background selection of carrot hybrid offspring population.

[0077] Compared with the prior art, the present invention has the following beneficial effects:

[0078] (1) This invention is based on whole-genome resequencing of 244 carrot germplasm resources with broad genetic diversity, from which 7300 candidate SNP loci were screened, and finally 30 SNP loci covering all 9 chromosomes of carrot and their corresponding KASP primers were developed. These SNP loci are evenly distributed in the genome, avoiding the problem of traditional markers being concentrated in gene-rich regions, and significantly improving the resolution and accuracy of genetic background identification.

[0079] (2) Through actual verification on 96 carrot breeding materials, the 30 pairs of KASP primers corresponding to the 30 SNP sites in this invention can clearly distinguish materials of different color types and different genetic origins, and achieve highly consistent cluster analysis on multiple single plants of the same material, proving that it has high accuracy and reliability in assessing genetic background restoration and identifying materials.

[0080] (3) The KASP primer combination used in this invention does not require gel electrophoresis or capillary electrophoresis. High-throughput and automated genotyping can be achieved through fluorescence signals, which greatly simplifies the operation process, shortens the detection time, reduces human error, and is suitable for large-scale, multi-round screening of breeding materials.

[0081] (4) This invention provides a powerful molecular tool for carrot hybridization breeding, backcrossing and conversion breeding and germplasm resource identification, which helps to realize the transformation from "experience breeding" to "precision breeding", significantly improves breeding efficiency, shortens the breeding cycle and accelerates the selection and promotion of superior varieties. Attached Figure Description

[0082] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0083] Figure 1 Results of genotyping using some KASP primers;

[0084] Figure 2 The results of phylogenetic analysis of 96 materials using 30 pairs of KASP primers are presented. Detailed Implementation

[0085] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0086] Unless otherwise specified, the experimental methods used in the following specific embodiments are conventional experimental methods, and the reagents used are conventional commercially available reagents.

[0087] Example 1: SNP site mining and KASP primer development

[0088] 1. Natural population whole-genome resequencing

[0089] To comprehensively cover the genetic diversity of carrots, 244 broadly representative carrot accessions were selected, including 182 carrot germplasm resources preserved in the National Vegetable Germplasm Resource Interim Bank, 28 breeding lines from our team, and 34 carrot germplasm resources with published whole-genome resequencing data from abroad (as shown in Tables 1-3). The materials cover major domestic and international carrot cultivation types (such as Chanette, Kuroda, Nantes, Danfoss, etc.), local varieties, and wild relatives, ensuring the broad applicability of the SNP markers subsequently developed.

[0090] After high-quality genomic DNA extraction and quality testing of each sample, whole-genome resequencing with a depth of at least 10X was performed.

[0091] Table 1 28 breeding line materials

[0092]

[0093] Table 2 182 carrot germplasm resources

[0094]

[0095] Table 3 34 foreign germplasm accessions

[0096]

[0097] 2. Bioinformatics Analysis and Mining of Highly Variable SNP Sites

[0098] The raw sequencing data from the resequencing were evaluated for quality. The median Phred quality score (Q-score) for each position in the sequencing sequence should be ≥30 (i.e., error rate ≤0.1%), and the content of adapter sequences should be less than 1%.

[0099] Filter and trim the Raw Data based on the evaluation results to generate high-quality valid data (CleanData): Remove low-quality bases (using a 4-bp sliding window to scan from the 5' end to the 3' end of the sequence. When the average quality value within the window drops below <Q20, truncate the sequence from the starting position of the window), excise the ends of the sequences (excise the last few bases with generally low quality from the 3' end of each sequence), and perform overall read length filtering (remove sequences with a length less than 50% of the original read length) to obtain high-quality Clean Data.

[0100] Subsequently, align the valid data of all materials to the carrot reference genome (DH1 v3.0). Through genome alignment analysis, initial SNP sites are identified across the entire genome. Based on the principle of high polymorphism, SNPs located in high-variation regions of the carrot genome are preferentially selected. These regions generally have a stronger ability to distinguish different genetic backgrounds. At the same time, sites located in repetitive sequences or complex regions are excluded to ensure the success rate and specificity of subsequent primer design. A total of 7300 candidate SNP sites are screened for the subsequent development of SNP sites and KASP primers.

[0101] 3. Development of KASP primers

[0102] Use 15 carrot breeding materials from different sources to develop and validate SNP sites and KASP primers (as shown in Table 4).

[0103] Include the following steps:

[0104] (1) Sample DNA preparation: Collect young leaves of 15 carrot plants, extract DNA using the CTAB method, and detect the purity and integrity of DNA using a micro nucleic acid protein analyzer or agarose gel electrophoresis. Dilute the DNA concentrations of all qualified samples to a working concentration of 50 ng / μL uniformly.

[0105] (2) KASP primer design: Randomly select 5 SNP sites from the candidate SNP sites on each chromosome from the 7300 candidate SNP sites obtained above to get 45 SNP sites, as shown in Table 5.

[0106] Use professional primer design software (Primer6) to design specific PCR amplification primers for each site and add allele-specific fluorescent primers.

[0107] (3) Preparation of KASP reaction system (10 μL): KASP Master Mix (2X) 5 μL, template genomic DNA 2 μL, primer mixture 0.14 μL (0.07 μM for each allele-specific primer (forward primer 1, forward primer 2), 0.18 μM for common reverse primer), double-distilled water 0.16 μL.

[0108] (4) PCR amplification and fluorescence detection: The PCR reaction program is as follows: 94℃ hot start for 10 min; first stage annealing extension at 94℃ for 20 s denaturation, 58-65℃ for 60 s annealing, with the annealing temperature decreasing by 0.6-0.8℃ for each cycle, for 10 cycles; second stage standard amplification at 94℃ for 20 s denaturation, 56℃ for 60 s annealing, for 30 cycles; endpoint fluorescence scanning.

[0109] (5) Fluorescence signal reading and genotype analysis: Fluorescence signal data were read using SNPviewer software, and genotype (AA, AB, BB) data of 15 samples at 45 SNP loci were exported.

[0110] Genotyping results of some KASP primers are as follows: Figure 1 As shown in Tables 6 and 7. Based on the obtained genotyping results, 15 SNP sites with no polymorphism or low signal intensity were removed from the 45 SNP sites, and finally 30 SNP sites covering 9 chromosomes and their corresponding 30 KASP primer combinations were screened out.

[0111] Table 4. Carrot breeding materials from 15 different sources

[0112]

[0113] Table 5 45 SNP sites

[0114]

[0115] Table 6 30 SNP sites

[0116]

[0117] Table 7 30 pairs of KASP primers

[0118]

[0119] Example 2: Genotyping of carrots using the aforementioned KASP primer combination.

[0120] Using the 30 pairs of KASP primers in Table 7, genotyping was performed on 96 carrot breeding materials (as shown in Table 8), including 12 orange fleshy root materials, 1 purple fleshy root material and 83 red fleshy root materials.

[0121] Includes the following steps:

[0122] (1) Sample DNA preparation: Young leaves of the carrot plants to be tested were collected, and DNA was extracted using the CTAB method. The purity and integrity of the DNA were detected using a micro-nucleic acid protein analyzer or agarose gel electrophoresis. The DNA concentration of all qualified samples was uniformly diluted to a working concentration of 50 ng / μL, and the diluted genomic DNA samples were aliquoted into 96-well PCR plates.

[0123] (2) Preparation of KASP reaction system (10 μL): KASP Master Mix (2X) 5 μL, template genomic DNA 2 μL, primer mixture 0.14 μL (0.07 μM for each allele-specific primer (forward primer 1, forward primer 2), and 0.18 μM for the common reverse primer), double-distilled water 0.16 μL.

[0124] (3) PCR amplification and fluorescence detection: The PCR reaction program is as follows: 94℃ hot start for 10 min; first stage annealing extension at 94℃ for 20 s denaturation, 58-65℃ for 60 s annealing, with the annealing temperature decreasing by 0.6-0.8℃ for each cycle, for 10 cycles; second stage standard amplification at 94℃ for 20 s denaturation, 56℃ for 60 s annealing, for 30 cycles; endpoint fluorescence scanning.

[0125] (4) Fluorescence signal reading and genotype analysis: Fluorescence signal data were read using SNPviewer software, and genotype data (AA, AB, BB) of 96 samples at 30 SNP loci were exported.

[0126] (5) Cluster analysis: Genotypes were converted into numerical matrices (A coded as 0, AB as 1, BB as 2), and matrix analysis was performed using TASSEL software. Cluster analysis was conducted using the unweighted group average method (UPGMA) to generate a phylogenetic tree. The results are as follows: Figure 2 As shown.

[0127] Depend on Figure 2 It can be seen that these 30 pairs of KASP primers can clearly and stably identify and distinguish 96 materials without any confusion.

[0128] First, the 12 orange materials and 83 red materials were clearly distinguishable, forming two large branches, and the purple material 733-37 was also clustered separately. Among the 12 orange materials, the two individual plants (-1 and -10) of material 318 and the six sister lines P52A-P100B were identified as having high homology.

[0129] The 83 red materials were further clustered into 8 sub-branches, matching the 8 genotype sources of these materials. For example, 10 materials with the same domestication source (407-15~450-23) were clustered in the same branch.

[0130] At the same time, different individual plants of the same material were also accurately identified, such as the four individual plants of material 491 (-37, -39, -41, -43), the five individual plants of material 561, and the four individual plants of material 594 (-38, -40, -42, -44), and the phylogenetic trees also showed consistent high homology.

[0131] The above results fully demonstrate that the 30 SNP loci and their corresponding 30 pairs of KASP primers provided by this invention can be used for accurate genetic background identification and material identification.

[0132] Table 8 96 carrot breeding materials

[0133]

[0134] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of a SNP locus combination in identifying carrot varieties, characterized in that, The SNP locus combination consists of 30 SNP loci in the carrot reference genome DH1v3.0, and the information of the 30 SNP loci includes: SNP1 is located at position 3378909 on chromosome 1, and its allelic variation is T / G. SNP2 is located at position 36213768 on chromosome 1, and its allelic variation is T / C. SNP3 is located at position 49750001 on chromosome 1, and its allelic variation is T / C. SNP4 is located at position 12004076 on chromosome 2, and its allelic variation is T / C. SNP5 is located at position 32694778 on chromosome 2, and its allelic variation is A / T. SNP6 is located at position 54566859 on chromosome 2, and its allelic variation is C / T. SNP7 is located at position 2139316 on chromosome 3, and its allelic variation is C / T. SNP8 is located at position 59862819 on chromosome 3, and its allelic variation is T / C. SNP9 is located at position 15000238 on chromosome 4, and its allelic variation is G / T; SNP10 is located at position 31185658 on chromosome 4, and its allelic variation is G / A. SNP11 is located at position 33289495 on chromosome 4, and its allelic variation is C / T. SNP12 is located at position 48827130 on chromosome 4, and its allelic variation is G / A. SNP13 is located at position 2674035 on chromosome 5, and its allelic variation is A / C. SNP14 is located at position 30110137 on chromosome 5, and its allelic variation is T / C. SNP15 is located at position 42967130 on chromosome 5, and its allelic variation is T / C. SNP16 is located at position 25653180 on chromosome 6, and its allelic variant base is T / G. SNP17 is located at position 27564723 on chromosome 6, and its allelic variation is G / A. SNP18 is located at position 38383474 on chromosome 6, and its allelic variation is T / C. SNP19 is located at position 4019817 on chromosome 7, and its allelic variation is A / G. SNP20 is located at position 26295659 on chromosome 7, and its allelic variation is A / T. SNP21 is located at position 33609659 on chromosome 7, and its allelic variation is T / A. SNP22 is located at position 35540647 on chromosome 7, and its allelic variation is C / T. SNP23 is located at position 10,158,974 on chromosome 8, and its allelic variation is T / C. SNP24 is located at position 23348460 on chromosome 8, and its allelic variation is A / G. SNP25 is located at position 31837294 on chromosome 8, and its allelic variation is A / T. SNP26 is located at position 1061607 on chromosome 9, and its allelic variation is A / G. SNP27 is located at position 2609626 on chromosome 9, and its allelic variation is C / T. SNP28 is located at position 12172120 on chromosome 9, and its allelic variation is C / T. SNP29 is located at position 22280031 on chromosome 9, and its allelic variant base is G / T. SNP30 is located at position 32876062 on chromosome 9, and its allelic variation is A / G.

2. A KASP primer combination, characterized in that, The KASP primer combination is used to detect the 30 SNP sites in claim 1, and the KASP primers corresponding to each SNP site include forward primer 1, forward primer 2 and common reverse primer; Forward primer 1, forward primer 2, and common reverse primer used to detect SNP1 are shown in SEQ ID NO. 1~3; Forward primer 1, forward primer 2, and common reverse primer used to detect SNP2 are shown in SEQ ID NO. 4~6; Forward primer 1, forward primer 2, and common reverse primer used to detect SNP3 are shown in SEQ ID NO. 7~9; Forward primer 1, forward primer 2, and common reverse primer used to detect SNP4 are shown in SEQ ID NO. 10~12; Forward primer 1, forward primer 2, and common reverse primer used to detect SNP5 are shown in SEQ ID NO. 13~15; Forward primer 1, forward primer 2 and common reverse primer used to detect SNP6 are shown in SEQ ID NO.16~18; The forward primer 1, forward primer 2 and common reverse primer used to detect SNP7 are shown in SEQ ID NO.19~21; The forward primer 1, forward primer 2 and common reverse primer used to detect SNP8 are shown in SEQ ID NO. 22~24; Forward primer 1, forward primer 2 and common reverse primer used to detect SNP9 are shown in SEQ ID NO. 25~27; The forward primer 1, forward primer 2 and common reverse primer used to detect SNP10 are shown in SEQ ID NO.28~30; Forward primer 1, forward primer 2 and common reverse primer used to detect SNP11 are shown in SEQ ID NO. 31~33; The forward primer 1, forward primer 2 and common reverse primer used to detect SNP12 are shown in SEQ ID NO. 34~36; The forward primer 1, forward primer 2 and common reverse primer used to detect SNP13 are shown in SEQ ID NO. 36~39; Forward primer 1, forward primer 2 and common reverse primer used to detect SNP14 are shown in SEQ ID NO.40~42; The forward primer 1, forward primer 2 and common reverse primer used to detect SNP15 are shown in SEQ ID NO.43~45; Forward primer 1, forward primer 2 and common reverse primer used to detect SNP16 are shown in SEQ ID NO.46~48; The forward primer 1, forward primer 2 and common reverse primer used to detect SNP17 are shown in SEQ ID NO.49~51; Forward primer 1, forward primer 2 and common reverse primer used to detect SNP18 are shown in SEQ ID NO. 52~54; Forward primer 1, forward primer 2 and common reverse primer used to detect SNP19 are shown in SEQ ID NO. 55~57; The forward primer 1, forward primer 2 and common reverse primer used to detect SNP20 are shown in SEQ ID NO.58~60; The forward primer 1, forward primer 2 and common reverse primer used to detect SNP21 are shown in SEQ ID NO. 61~63; The forward primer 1, forward primer 2 and common reverse primer used to detect SNP22 are shown in SEQ ID NO. 64~66; The forward primer 1, forward primer 2 and common reverse primer used to detect SNP23 are shown in SEQ ID NO. 67~69; The forward primer 1, forward primer 2 and common reverse primer used to detect SNP24 are shown in SEQ ID NO. 70~72; Forward primer 1, forward primer 2 and common reverse primer used to detect SNP25 are shown in SEQ ID NO.73~75; The forward primer 1, forward primer 2 and common reverse primer used to detect SNP26 are shown in SEQ ID NO.76~78; The forward primer 1, forward primer 2 and common reverse primer used to detect SNP27 are shown in SEQ ID NO.79~81; The forward primer 1, forward primer 2 and common reverse primer used to detect SNP28 are shown in SEQ ID NO. 82~84; The forward primer 1, forward primer 2 and common reverse primer used to detect SNP29 are shown in SEQ ID NO. 85~87; The forward primer 1, forward primer 2, and common reverse primer used to detect SNP30 are shown in SEQ ID NO. 88~90.

3. A method for genotyping carrots using the KASP primer combination as described in claim 2, characterized in that, The process includes the following steps: S1, mixing carrot genomic DNA with a reaction system containing the KASP primer combination described in claim 2, performing PCR amplification, and obtaining PCR amplification products; S2. Genetically type carrots based on the fluorescence signal of the PCR amplification product.

4. A reagent kit, characterized in that, It includes the KASP primer combination as described in claim 2.

5. The use of the SNP site combination of claim 1, the KASP primer combination of claim 2, or the kit of claim 4 in any of the following: (1) Identification of the genetic background of carrot breeding materials; (2) Analysis of the phylogenetic relationships of carrot varieties, strains, or germplasm resources; (3) Background selection of carrot hybrid offspring population.

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