A method for identifying the purity of carrot hybrids and the InDel primer combination used therein.

Abstract

This invention discloses a method for identifying the purity of carrot hybrids and the InDel primer set used therein, belonging to the field of hybrid purity identification. The InDel primer set provided by this invention consists of 10 primer sets; each primer amplifies one of ten InDel loci to perform genotyping or determine homozygosity or heterozygosity at each InDel locus. This primer set can be used to identify the purity of carrot hybrids, and can be used for early identification at the seed or seedling stage, thereby ensuring the purity of hybrids and effectively protecting the legitimate rights and interests of producers and breeders.

Description

Technical Field

[0001] This invention belongs to the field of purity identification of vegetable hybrids, specifically involving a method for identifying the purity of carrot hybrids and the InDel primer combination used therein. Background Technology

[0002] Carrots, one of my country's major vegetable varieties, rank among the world's top in both planting scale and yield. In carrot production, hybrid varieties have become the dominant force due to their superior genetic characteristics and production performance. However, hybrid varieties must undergo rigorous purity testing before entering the market. According to the "Regulations for Seed Inspection of Agricultural Crops" (GB / T 3543.1-3543.7-1995), the purity requirement for carrot hybrid varieties is over 98%. Currently, traditional field purity testing methods are costly and time-consuming, failing to meet the needs of the rapidly developing modern seed industry. Therefore, DNA-based molecular detection technology has emerged, quickly becoming the preferred method for identifying the purity of carrot hybrid varieties due to its low cost and high efficiency. By screening suitable combinations of molecular markers for DNA detection technology, purity identification can be completed more accurately and quickly. Therefore, screening molecular marker combinations based on DNA detection technology for identifying the purity of carrot hybrid varieties is particularly crucial.

[0003] InDel (Insertion-Deletion) markers are insertion / deletion markers that indicate a certain number of nucleotide insertions or deletions in the genome of other samples relative to a reference genome. InDel variants are widely distributed throughout the genome, second only to SNP variants in number. Specific InDel marker primers can be designed based on the sequences flanking the insertion / deletion site. Compared to SNP and SSR markers, InDel markers have a wider range of applications, can be detected using ordinary agarose gel electrophoresis platforms, and have lower requirements for sample DNA quality. Their banding stability and interpretation performance are also superior to SSRs. They are characterized by good stability, wide distribution, high polymorphism, strong versatility, simple typing systems, and low cost.

[0004] Based on this, the present invention is proposed. Summary of the Invention

[0005] In view of the shortcomings of the prior art, one of the objectives of this invention is to provide an InDel primer combination for identifying the purity of carrot hybrids.

[0006] A second objective of this invention is to provide a kit containing the above-described primer combination.

[0007] The third objective of this invention is to provide applications of the above-mentioned primer combinations or kits.

[0008] The fourth objective of this invention is to provide a method for identifying the purity of a carrot hybrid to be tested.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] The first aspect of this invention provides an InDel primer set, comprising ten primer sets, each corresponding to amplify ten InDel loci (or InDel markers) to perform genotyping or homozygous / heterozygous determination for each InDel locus, wherein:

[0011] The InDel site includes:

[0012] IndHB10 site (first site): located at position 62600640 on chromosome 3, is nucleotide A or the nucleotide sequence shown in SEQ ID NO.21;

[0013] IndHB12 site (second site): located at position 24462630 on chromosome 4, is nucleotide A or the nucleotide sequence shown in SEQ ID NO.22;

[0014] IndHB15 site (third site): located at position 25653852 on chromosome 5, is nucleotide A or the nucleotide sequence shown in SEQ ID NO.23;

[0015] IndHB22 site (fourth site): located at position 37949132 on chromosome 7, is nucleotide T or the nucleotide sequence shown in SEQ ID NO.24;

[0016] IndHB27 (the fifth site): located at position 34173400 on chromosome 9, is nucleotide A or the nucleotide sequence shown in SEQ ID NO.25;

[0017] IndHB32 (sixth site): located at position 62564623 on chromosome 3, is nucleotide G or the nucleotide sequence shown in SEQ ID NO.26;

[0018] IndHB36 (seventh site): located at position 40686460 on chromosome 7, is nucleotide T or the nucleotide sequence shown in SEQ ID NO.27;

[0019] IndHB44 (eighth site): located at position 6247289 on chromosome 9, is nucleotide T or the nucleotide sequence shown in SEQ ID NO.28;

[0020] IndHB47 (ninth site): located at position 39038965 on chromosome 1, is nucleotide T or the nucleotide sequence shown in SEQ ID NO.29;

[0021] IndHB50 (tenth position): located at position 4341982 on chromosome 3, is nucleotide T or the nucleotide sequence shown in SEQ ID NO.30;

[0022] The positions of the ten InDel sites on the chromosome were determined based on sequence alignment with the carrot reference genome Reference DH1V3.

[0023] The first primer set (primer set 1) is used to amplify the IndHB10 site; the second primer set (primer set 2) is used to amplify the IndHB12 site; the third primer set (primer set 3) is used to amplify the IndHB15 site; the fourth primer set (primer set 4) is used to amplify the IndHB22 site; the fifth primer set (primer set 5) is used to amplify the IndHB27 site; the sixth primer set (primer set 6) is used to amplify the IndHB32 site; the seventh primer set (primer set 7) is used to amplify the IndHB36 site; the eighth primer set (primer set 8) is used to amplify the IndHB44 site; the ninth primer set (primer set 9) is used to amplify the IndHB47 site; and the tenth primer set (primer set 10) is used to amplify the IndHB50 site.

[0024] The InDel primer combination mentioned above can be used to identify the purity of carrot hybrids. Based on the InDel primer combination, a primer set suitable for identifying the carrot variety to be tested is selected. PCR is performed using the selected primer set. If the PCR product is a heterozygous band, it means that the carrot variety to be tested is a hybrid. If it is a single band, it means that the carrot variety to be tested is not a hybrid, but may be one of the parents, such as a mixture of parents and hybrids.

[0025] The hybrids described in this invention refer to first-generation hybrids, the purity of which may be affected by the mixing or contamination of parental lines.

[0026] Furthermore, in the InDel primer combination,

[0027] The first primer set consists of the forward primer F1 shown in SEQ ID NO.1 and the reverse primer R1 shown in SEQ ID NO.2;

[0028] The second primer set consists of the forward primer F2 shown in SEQ ID NO.3 and the reverse primer R2 shown in SEQ ID NO.4.

[0029] The third primer set consists of the forward primer F3 shown in SEQ ID NO.5 and the reverse primer R3 shown in SEQ ID NO.6.

[0030] The fourth primer set consists of the forward primer F4 shown in SEQ ID NO.7 and the reverse primer R4 shown in SEQ ID NO.8.

[0031] The fifth primer set consists of the forward primer F5 shown in SEQ ID NO.9 and the reverse primer R5 shown in SEQ ID NO.10.

[0032] The sixth primer set consists of the forward primer F6 shown in SEQ ID NO.11 and the reverse primer R6 shown in SEQ ID NO.12.

[0033] The seventh primer set consists of the forward primer F7 shown in SEQ ID NO.13 and the reverse primer R7 shown in SEQ ID NO.14.

[0034] The eighth primer set consists of the forward primer F8 shown in SEQ ID NO.15 and the reverse primer R8 shown in SEQ ID NO.16.

[0035] The ninth primer set consists of the forward primer F9 shown in SEQ ID NO.17 and the reverse primer R9 shown in SEQ ID NO.18.

[0036] The tenth primer set consists of the forward primer F10 shown in SEQ ID NO.19 and the reverse primer R10 shown in SEQ ID NO.20.

[0037] Furthermore, the InDel primer set consists of primer set 1, primer set 2, primer set 3, primer set 4, primer set 5, primer set 6, primer set 7, primer set 8, primer set 9 and primer set 10.

[0038] A second aspect of the present invention provides a kit comprising the InDel primer combination described in the first aspect.

[0039] In addition to primer combinations, the kit may also include PCR auxiliary reagents such as ultrapure water, PCR buffer, dNTPs, and DNA polymerase, as well as standard experimental equipment for PCR.

[0040] In the primer set mentioned above, the molar ratio of primers containing "F" in their names to primers containing "R" in their names can be 1:1 during PCR.

[0041] The third aspect of this invention provides the application of the InDel primer combination of the first aspect or the kit of the second aspect in identifying the purity of carrot hybrids.

[0042] A fourth aspect of this invention provides a method for identifying the purity of a carrot hybrid to be tested, comprising the following steps:

[0043] (1) Randomly select N carrot hybrids to be tested and obtain their genomic DNA;

[0044] (2) Using the genomic DNA of at least 8 (e.g., 10, 12, 15 or 20) carrot hybrids selected from the N carrot hybrids to be tested obtained in step (1) as templates, PCR amplification is performed using the first to tenth primer sets in the InDel primer combination provided in the first aspect above, and the corresponding PCR amplification products are obtained.

[0045] (3) Perform agarose gel electrophoresis on the PCR amplification products obtained in step (2), and count the number of heterozygous bands in the first to tenth primer sets according to the electrophoresis results; then select the primer set with a relatively large number of heterozygous bands (e.g., more than 5, the second largest number of bands, the largest number of bands) and clear as the target primer set.

[0046] (4) Using the genomic DNA of the N carrot hybrids to be tested obtained in step (1) as templates, PCR amplification was performed using the target primer sets obtained in step (3) to obtain the PCR amplification products of each carrot hybrid to be tested under different target primer sets.

[0047] (5) Perform agarose gel electrophoresis on the PCR amplification products obtained in step (4). According to the electrophoresis results, count the number of plants with heterozygous bands in the amplification products of each target primer group. The carrot lines to be tested with heterozygous bands are hybrids. The purity of the carrot hybrids to be tested is obtained according to the number of plants with heterozygous bands amplified by each target primer group.

[0048] In the fourth aspect of the method of the present invention, the target primer set determined by step (3) can be one, two or more. When there are multiple target primer sets, the purity of the hybrid obtained from multiple target primer sets can be used to determine the final purity of the carrot hybrid to be tested, specifically:

[0049] The number of plants showing heterozygous bands and the number of plants without bands for each target primer set were counted. The purity of the carrot hybrids obtained from each target primer set was calculated, and then the average value was calculated.

[0050] The purity of a target primer set = number of plants showing heterozygous bands with the target primer set / (N - number of plants without bands with the target primer set) × 100%.

[0051] As an optional implementation of the above method, the PCR amplification reaction system may specifically consist of: 5 µl template DNA (50 ng / µl), 10 µl 2×Mix, 1 µl forward primer and 1 µl reverse primer (20 µmol / L), and 3 µl ultrapure water. The PCR amplification reaction program may specifically consist of: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s; 58℃ annealing for 30 s; 72℃ extension for 1 min, 35 cycles; and 72℃ extension for 10 min.

[0052] In the method described above, the larger the value of N, the higher the accuracy of identifying the purity of the carrot hybrid to be tested, such as N≥80, 100, 120, 150 or 200.

[0053] Furthermore, the carrot hybrids to be tested in the fourth aspect of the present invention and the carrot hybrids in the third aspect are both selected from Hongdu 107, Naaisi, Dali, Zixia, Zilong carrot, Hongdu 345, Black Nebula carrot, Ziyu 199, 23-864, Red Core Seven-Inch Ginseng, Jingyuan, Carlos, H1502, Zhongyu 2048, Universe Purple Carrot, Zhongyu 1182, Beauty Finger Carrot, Mingzhu Zhenpin, Shinongchun 95, Asahi Five-Inch Ginseng, Early Spring 95, Lijiajimei, Marite, Shaanxi Touxinhong, Zhongyu 1504, Zhongyu 1877, 23-1094, Fruit Carrot, Hama, Dennis, Early Red Crown, Zhuang 2203, Mini cxpress, H20181, Red Rainbow 5-inch, All Red 191-3, Betarichi, Zhongyu 181, Shinashi 5-inch 2, Huangdi 9-inch, Extremely Early 3-inch Ginseng, Shinashi 5-inch 1, Sichuan Red Heart Carrot, Huangjian No. 1 Carrot, Slender Finger No. 1 Carrot, Qitou Yellow Carrot, Hongsheng No. 4, Royal Red, Hongtianxia, ​​Jinhong Daigen, All Red 14, All Red 785, Xifeng No. 2, Jinmei, Resistant to Smoking Japan, CA-86, CA-137, Improved Kuroda Ginseng, Leichuan Red, 22-621, Rainbow, Japan Kuroda 5-inch Ginseng, Xiaoding 8-inch Ginseng, Fumei Red, Purple Ginseng, Mendel, Zhongyu 1729, Zhongyu 1749, Ziyu 212, Jingyu 189, 23-1096, Japanese Improved 7-inch Ginseng, Hongsheng No. 9, WC-1803, 904, Huili, Jixiang, Ruyi, Zhuang 2201, Yangzhongbao, Kuroda 5-inch Ginseng, Zhongyu 1870, Huayu 388, Oufang No. 1 Carrot, 23-881, Shenghong 601, Hongsheng No. 5, WC-2302, Huangshi, New Kuroda 5-inch, Xiangyang No. 2, Hongyu 7-inch, 22-779, Beka.

[0054] All the aforementioned carrot hybrids or the carrot hybrids to be tested are commercially available varieties. In the aforementioned carrot hybrid varieties, at least one of the ten InDel loci mentioned in the first aspect of this invention is heterozygous, and in some cases, up to eight InDel loci are heterozygous. The InDel primer combination provided by this invention can be used for purity identification of the aforementioned carrot hybrid varieties. Of course, this invention does not limit the variety of carrot hybrid to be tested.

[0055] It should be noted that this invention focuses on ensuring the purity of carrot hybrids, specifically referring to first-generation hybrids. The core challenge lies in avoiding parental mixing rather than mechanical mixing, that is, not simply mixing multiple carrot hybrids.

[0056] Compared with the prior art, the present invention has the following beneficial effects:

[0057] The InDel primer combination and purity identification method provided by this invention enable early identification of existing representative carrot hybrids at the seed or seedling stage, simplifying the identification cycle and improving efficiency. While ensuring hybrid purity, it also effectively protects the legitimate rights and interests of producers and breeders, providing technical support for the market and quality management of carrot hybrids. The method provided by this invention has advantages such as high throughput, accuracy, low cost, simple operation, and saving manpower and resources, and has a very broad application prospect. Attached Figure Description

[0058] Figure 1 The InDel typing effect of 10 primer sets in some tested carrot hybrids was studied.

[0059] Figure 2 The distribution of 10 InDel sites as heterozygous sites in 94 carrot varieties.

[0060] Figure 3 The results show the purity of primer group 5 in the Huayu 388 heterozygote. Detailed Implementation

[0061] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0062] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0063] Example 1: Obtaining the InDel primer combination for identifying the purity of carrot hybrids

[0064] I. Discovery of 10 InDel sites

[0065] This invention develops whole-genome InDel markers for carrots based on resequencing data from 380 representative carrot resources, and uses them for varietal purity identification, obtaining 10 InDel loci. These 380 carrot resources are diverse in type, covering early cultivated varieties, improved cultivated varieties, local varieties, and wild varieties, basically encompassing the main ecological types and agronomic traits of carrots, reflecting germplasm representativeness as much as possible, and possessing high genetic diversity.

[0066] Specifically, the screening criteria for InDel loci were as follows: First, InDel loci belonging to the dimorphic type were screened from the entire genome chromosome. InDel loci with a conserved 200 bp margin and >30 bp were selected. Then, InDel loci with Miss <0.2, MAF >0.2, and He <0.05 were screened. Subsequently, InDel loci with MAF >0.2 from both early-cultivated and improved-cultivated populations were screened. Finally, BLAST-specific analysis was performed, and 52 pairs of specific primers were designed. These primers were used to screen the 94 tested carrot hybrids listed in Table 3. During the screening process, through careful comparison and analysis, primers exhibiting high heterozygosity and polymorphism were ultimately selected. After rigorous screening and evaluation, 10 pairs of primers were finally retained.

[0067] Basic information on the 10 InDel sites is detailed in Table 1. The positions of the InDel sites on the chromosome were determined based on sequence alignment with the carrot reference genome Reference DH1 V3 (URL: https: / / www.ncbi.nlm.nih.gov / datasets / genome / GCA_001625215.2 / ).

[0068] Table 1. Basic information of 10 InDel sites

[0069] II. Obtaining the InDel primer combination for identifying the purity of carrot hybrids

[0070] Based on the 10 InDel sites discovered in step one, the inventors of this invention developed a combination of InDel primers with high polymorphism for identifying the purity of carrot hybrids.

[0071] The InDel primer set consists of 10 primer sets, the name of which is shown in column 2 of Table 2. Each primer set consists of 2 primer sequences used to amplify the corresponding InDel site. The nucleotide sequences of each primer in the 10 primer sets are shown in column 4 of Table 2.

[0072] Table 2. Ten pairs of Indel primer sequences suitable for carrot purity identification.

[0073] Example 2: Validation of the InDel primer combinations developed in Example 1

[0074] The basic information of the 94 tested carrot hybrids in this embodiment is shown in Table 3. All 94 tested carrot hybrids are common superior hybrids or imported hybrids, and can be obtained through commercial channels.

[0075] Table 3. Basic information of 94 tested carrot hybrids

[0076] 1. Obtaining genomic DNA from the tested carrot hybrid (DNA extraction using magnetic beads).

[0077] A suitable amount of carrot leaf samples were collected in centrifuge tubes, steel beads were added, and the samples were frozen in liquid nitrogen. Then, the samples were ground using an ultra-high throughput grinder. After grinding, 800 µl of SDS extraction buffer was added to each sample. The samples were dried in a 60°C oven for 30-40 minutes, inverting and mixing three times during this period. 240 µl of 3M potassium acetate solution (0.3 times the volume of the SDS extraction buffer) was added to each sample, and the samples were thoroughly inverted and mixed five times. The samples were then placed in a 4°C refrigerator and allowed to stand for 20 minutes. The samples were then centrifuged at 4000 rpm for 10 minutes. New centrifuge tubes or deep-well plates were prepared in advance, and 600 µl of magnetic bead isopropanol was added (ensuring the volume was equal to the supernatant). Note that the order of operations is to add the magnetic bead isopropanol first, followed by the supernatant, and the volume of the supernatant aspirated should not exceed a certain limit. Fill the container to more than 2 / 3 of its total volume; invert and mix thoroughly, let stand at -20℃ for 30 minutes until the magnetic beads precipitate, invert and mix again, adsorb using a magnetic rack, discard the supernatant, and invert onto absorbent paper to absorb residual liquid; add 200µl of 75% ethanol solution to the sample, invert and mix thoroughly, and let stand until the magnetic beads are completely precipitated; invert and mix again, adsorb using a magnetic rack, discard the supernatant, and invert onto absorbent paper to absorb residual liquid; after the magnetic beads are completely air-dried, add 100µl of purified water, invert and mix thoroughly to ensure complete DNA dissolution; use a Nanodrop (micro-spectrophotometer) to take 2µl of the DNA extract for detection, measure its OD260 / 280 and OD260 / 230 ratios (ideal range for both is 1.8-2.0), and record the DNA concentration. Subsequently, adjust the DNA concentration to 50ng / µl for subsequent use.

[0078] 2. Using genomic DNA from 94 tested carrot hybrids as templates, PCR amplification was performed using the 10 primer sets given in Table 2 to obtain the corresponding PCR amplification products. In each PCR reaction system, the concentration ratio of primers containing "F" in their names to primers containing "R" in their names was 1:1.

[0079] The PCR amplification reaction system consisted of: 5 µl template DNA, 10 µl 2×Mix, 1 µl forward primer and 1 µl reverse primer (20 µmol / L), and 3 µl ultrapure water.

[0080] The reaction program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s; 58℃ annealing for 30 s; 72℃ extension for 1 min, 35 cycles; 72℃ extension for 10 min.

[0081] 3. After completing step 2, the PCR amplification products were subjected to agarose gel electrophoresis, and the results were photographed and read using a gel imaging system. The genotypes of the 94 tested carrot hybrids based on each InDel locus were determined by the banding pattern. The specific determination principles are as follows: if a tested carrot hybrid shows a heterozygous band at a certain InDel locus, then the genotype of the tested carrot hybrid based on that InDel locus is heterozygous; if a tested carrot hybrid shows a single band at a certain InDel locus, then the genotype of the tested carrot hybrid based on that InDel locus is homozygous.

[0082] Some results can be found Figure 1 The results showed that each primer set could achieve good genotyping results in the tested carrot hybrids.

[0083] 4. Distribution of heterozygous loci and efficiency evaluation

[0084] (1) Based on the genotypes of 94 tested carrot hybrids at 10 InDel loci, the number of heterozygous loci for each tested carrot hybrid was counted.

[0085] The distribution of heterozygous loci in 94 tested carrot hybrids established on 10 primer sets is shown in the figure. Figure 2 The results showed that the 10 primer sets could ensure that each tested carrot hybrid had at least one heterozygous site.

[0086] (2) Multiplex PCR can be used to reduce the workload for hybrid purity identification.

[0087] The results showed that multiplex PCR (10 primer sets) achieved 100% coverage of heterozygous sites in 94 tested carrot hybrids.

[0088] Therefore, it can be seen that the InDel primer combination developed in Example 1 can be applied to the purity identification of carrot hybrids.

[0089] Example 3: Detection of the purity of Huayu 388 hybrid using the InDel primer combination developed in Example 1.

[0090] 1. Obtaining genomic DNA from the Huayu 388 hybrid

[0091] (1) Plant 200 seeds of Huayu 388 hybrid seed to obtain Huayu 388 hybrid seedlings.

[0092] (2) Leaves or roots of 96 Huayu 388 hybrid seedlings were randomly selected and genomic DNA was extracted by magnetic bead method to obtain 96 Huayu 388 hybrid genomic DNA samples.

[0093] 2. Primer set screening

[0094] (1) From the genomic DNA of 96 Huayu 388 hybrids obtained, the genomic DNA of 8 hybrid seedlings was randomly selected as templates, and PCR amplification was performed using 10 primer sets from the InDel primer combination developed in Example 1 to obtain the corresponding PCR amplification products. In each PCR reaction system, the concentration ratio of primers containing "F" in their names to primers containing "R" in their names was 1:1.

[0095] The specific reaction system for PCR amplification can be: 5 µl template DNA (50 ng / µl), 10 µl 2×Mix, 1 µl forward primer and 1 µl reverse primer (20 µmol / L), and 3 µl ultrapure water.

[0096] The specific reaction procedure for PCR amplification can be as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s; 58℃ annealing for 30 s; 72℃ extension for 1 min, 35 cycles; 72℃ extension for 10 min.

[0097] (2) After completing step (1), perform agarose gel electrophoresis and use a gel imaging system to photograph and record the electrophoresis results. Compare the number of heterozygous sites in the 10 primer sets. The primer set with the most heterozygous sites is the target primer set selected.

[0098] The results showed that primer set 5 had the highest number of heterozygous sites. Therefore, primer set 5 was selected as the primer set for subsequent experiments.

[0099] 3. Obtain the purity of Huayu 388 hybrid.

[0100] (1) Using the genomic DNA of the 96 Huayu 388 hybrids obtained in step 1 as templates, PCR amplification was performed using primer set 5 from the InDel primer combination developed in Example 1 to obtain the corresponding PCR amplification products. In each PCR reaction system, the concentration ratio of primers containing "F" in their names to primers containing "R" in their names was 1:1.

[0101] The specific reaction system for PCR amplification can be: 5 µl template DNA (50 ng / µl), 10 µl 2×Mix, 1 µl forward primer and 1 µl reverse primer (20 µmol / L), and 3 µl ultrapure water.

[0102] The specific reaction procedure for PCR amplification can be as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s; 58℃ annealing for 30 s; 72℃ extension for 1 min, 35 cycles; 72℃ extension for 10 min.

[0103] (2) After completing step (1), perform agarose gel electrophoresis and use a gel imaging system to photograph and record the electrophoresis results.

[0104] InDel typing results are shown below Figure 3 .

[0105] (3) After completing step (2), count the number of plants with heterozygous sites and the number of plants without heterozygous sites in primer group 5; calculate the purity of Huayu 388 hybrid according to the following formula.

[0106] Purity = Number of strains with heterozygous sites shown in primer set / (96 - Number of strains with no band in primer set) × 100%.

[0107] The results showed that primer set 5 showed 94 plants with heterozygous bands and 2 plants with homozygous bands, with a purity of 94 / 96×100%=97.9%.

[0108] Four heteromorphic plants were obtained from 200 hybrid plants in the field. The purity of the hybrid of this variety is 98%, which shows that the identification method of the present invention has high accuracy.

[0109] Finally, it should be noted that the above specific embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. The InDel primer set consists of primer sets 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. The first primer set consists of the forward primer shown in SEQ ID NO.1 and the reverse primer shown in SEQ ID NO.2; The second primer set consists of the forward primer shown in SEQ ID NO.3 and the reverse primer shown in SEQ ID NO.4; The third primer set consists of the forward primer shown in SEQ ID NO.5 and the reverse primer shown in SEQ ID NO.6; The fourth primer set consists of the forward primer shown in SEQ ID NO.7 and the reverse primer shown in SEQ ID NO.8; The fifth primer set consists of the forward primer shown in SEQ ID NO.9 and the reverse primer shown in SEQ ID NO.10; The sixth primer set consists of the forward primer shown in SEQ ID NO.11 and the reverse primer shown in SEQ ID NO.12; The seventh primer set consists of the forward primer shown in SEQ ID NO.13 and the reverse primer shown in SEQ ID NO.14; The eighth primer set consists of the forward primer shown in SEQ ID NO.15 and the reverse primer shown in SEQ ID NO.16; The ninth primer set consists of the forward primer shown in SEQ ID NO.17 and the reverse primer shown in SEQ ID NO.18; The tenth primer set consists of the forward primer shown in SEQ ID NO.19 and the reverse primer shown in SEQ ID NO.

20.

2. A kit comprising the InDel primer combination of claim 1.

3. The application of the InDel primer combination of claim 1 or the kit of claim 2 in identifying the purity of carrot hybrids.

4. The application according to claim 3, characterized in that, The carrot hybrids mentioned are selected from Hongdu 107, Naaisi, Dali, Zixia, Zilong Carrot, Hongdu 345, Black Nebula Carrot, Ziyu 199, 23-864, Red Core Seven-Inch Ginseng, Jingyuan, Carlos, H1502, Zhongyu 2048, Universe Purple Carrot, Zhongyu 1182, Beauty Finger Carrot, Mingzhu Zhenpin, Shinongchun 95, Asahi Five-Inch Ginseng, Early Spring 95, Lijiajimei, Marite, Shaanxi Touxinhong, Zhongyu 1504, Zhongyu 1877, 23-1094, Fruit Carrot, Hama, Dennis, Early Red Crown, Zhuang 2203, and Mini. cxpress, H20181, Red Rainbow 5-inch, All Red 191-3, Betarichi, Zhongyu 181, Shinashi 5-inch 2, Huangdi 9-inch, Extremely Early 3-inch Ginseng, Shinashi 5-inch 1, Sichuan Red Heart Carrot, Huangjian No. 1 Carrot, Slender Finger No. 1 Carrot, Qitou Yellow Carrot, Hongsheng No. 4, Royal Red, Hongtianxia, ​​Jinhong Daigen, All Red 14, All Red 785, Xifeng No. 2, Jinmei, Resistant to Smoking Japan, CA-86, CA-137, Improved Kuroda Ginseng, Leichuan Red, 22-621, Rainbow, Japan Kuroda 5-inch Ginseng, Xiaoding 8-inch Ginseng, Fumei Red, Purple Ginseng, Mendel, Zhongyu 1729, Zhongyu 1749, Ziyu 212, Jingyu 189, 23-1096, Japanese Improved 7-inch Ginseng, Hongsheng No. 9, WC-1803, 904, Huili, Jixiang, Ruyi, Zhuang 2201, Yangzhongbao, Kuroda 5-inch Ginseng, Zhongyu 1870, Huayu 388, Oufang No. 1 Carrot, 23-881, Shenghong 601, Hongsheng No. 5, WC-2302, Huangshi, New Kuroda 5-inch, Xiangyang No. 2, Hongyu 7-inch, 22-779, Beka.

5. A method for identifying the purity of a carrot hybrid to be tested, characterized in that, Includes the following steps: (1) Randomly select N carrot hybrids to be tested and obtain their genomic DNA; (2) Using the genomic DNA of at least 8 carrot hybrids selected from the N carrot hybrids to be tested obtained in step (1) as templates, PCR amplification was performed using the first to tenth primer sets in the InDel primer combination described in claim 1 to obtain the corresponding PCR amplification products. (3) Perform agarose gel electrophoresis on the PCR amplification products obtained in step (2), and count the number of heterozygous bands in the first to tenth primer sets according to the electrophoresis results; then select the primer set with a relatively large number of heterozygous bands and clear bands as the target primer set. (4) Using the genomic DNA of the N carrot hybrids to be tested obtained in step (1) as templates, PCR amplification was performed using the target primer sets obtained in step (3) to obtain the PCR amplification products of each carrot hybrid to be tested under different target primer sets. (5) Perform agarose gel electrophoresis on the PCR amplification products obtained in step (4). According to the electrophoresis results, count the number of plants with heterozygous bands in the amplification products of each target primer group. The carrot lines to be tested with heterozygous bands are hybrids. The purity of the carrot hybrids to be tested is obtained according to the number of plants with heterozygous bands amplified by each target primer group.

6. The identification method as described in claim 5, characterized in that, The target primer set consists of two or more primers.

7. The identification method as described in claim 5, characterized in that, The method for "obtaining the purity of the carrot hybrid to be tested based on the number of plants with heterozygous bands amplified by each target primer set" is as follows: count the number of plants that show heterozygous bands and the number of plants that do not show bands for each target primer set, calculate the purity of the carrot hybrid to be tested obtained by each target primer set, and then calculate the average value. Purity = Number of strains showing heterozygous bands with a certain target primer set / (N - Number of strains without bands with that target primer set) × 100%.

8. The identification method as described in claim 5, characterized in that, The value of N is ≥100.

9. The identification method according to any one of claims 5-8, characterized in that, The tested carrot hybrids were selected from Hongdu 107, Naaisi, Dali, Zixia, Zilong Carrot, Hongdu 345, Black Nebula Carrot, Ziyu 199, 23-864, Red Core Seven-Inch Ginseng, Jingyuan, Carlos, H1502, Zhongyu 2048, Universe Purple Carrot, Zhongyu 1182, Beauty Finger Carrot, Mingzhu Zhenpin, Shinongchun 95, Asahi Five-Inch Ginseng, Early Spring 95, Lijiajimei, Marite, Shaanxi Touxinhong, Zhongyu 1504, Zhongyu 1877, 23-1094, Fruit Carrot, Hama, Dennis, Early Red Crown, Zhuang 2203, and Mini. cxpress, H20181, Red Rainbow 5-inch, All Red 191-3, Betarichi, Zhongyu 181, Shinashi 5-inch 2, Huangdi 9-inch, Extremely Early 3-inch Ginseng, Shinashi 5-inch 1, Sichuan Red Heart Carrot, Huangjian No. 1 Carrot, Slender Finger No. 1 Carrot, Qitou Yellow Carrot, Hongsheng No. 4, Royal Red, Hongtianxia, ​​Jinhong Daigen, All Red 14, All Red 785, Xifeng No. 2, Jinmei, Resistant to Smoking Japan, CA-86, CA-137, Improved Kuroda Ginseng, Leichuan Red, 22-621, Rainbow, Japan Kuroda 5-inch Ginseng, Xiaoding 8-inch Ginseng, Fumei Red, Purple Ginseng, Mendel, Zhongyu 1729, Zhongyu 1749, Ziyu 212, Jingyu 189, 23-1096, Japanese Improved 7-inch Ginseng, Hongsheng No. 9, WC-1803, 904, Huili, Jixiang, Ruyi, Zhuang 2201, Yangzhongbao, Kuroda 5-inch Ginseng, Zhongyu 1870, Huayu 388, Oufang No. 1 Carrot, 23-881, Shenghong 601, Hongsheng No. 5, WC-2302, Huangshi, New Kuroda 5-inch, Xiangyang No. 2, Hongyu 7-inch, 22-779, Beka.

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