A method for identifying the purity of brassica rapa var. pekinensis hybrids and an in del primer combination used thereby

Abstract

The application discloses a method for identifying purity of Chinese cabbage hybrid and an InDel primer combination used in the method. The InDel primer combination provided by the application is composed of 10 primer groups; each primer group is composed of two primer sequences, and is used for amplifying an InDel site; and the nucleotide sequences of the primers are sequentially shown in SEQ ID NO:1 to SEQ ID NO:20. The InDel primer combination is suitable for purity identification of most Chinese cabbage varieties, and can be used for early identification in the seed or seedling stage of the Chinese cabbage hybrid, so as to ensure the purity of the hybrid, protect the rights and interests of producers and breeders, and provide technical support for seed quality management of the Chinese cabbage varieties. The method provided by the application has the advantages of accuracy, low cost, simple operation, clear results, saving of manpower and material resources, and the like, and has a very wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of hybrid purity identification, specifically relating to a method for identifying the purity of non-heading Chinese cabbage hybrids and the InDel primer combination used therein. Background Technology

[0002] Non-heading Chinese cabbage, a crucial vegetable crop belonging to the Brassicaceae family, also known as bok choy, rapeseed, or Chinese cabbage, has a cultivation history of thousands of years in southern China. Its rich variety and adaptability make it a key player in China's vegetable industry. In 2022, its national planting area reached 4.5 million hectares, with an annual output of approximately 18 million tons, making it one of the main varieties for ensuring year-round vegetable supply during emergencies. Modern breeding techniques, through the utilization of hybrid vigor, have significantly improved the disease resistance, environmental adaptability, and commercial quality of non-heading Chinese cabbage. However, technical challenges in the breeding process cannot be ignored, including issues such as inadequate parental isolation, insufficient self-compatibility, and incomplete field weeding, all of which can reduce seed purity, thereby affecting field performance and commercial value. Therefore, seed purity identification is of great significance for improving the seed quality of non-heading Chinese cabbage varieties, as well as seed production and sales. Currently, traditional field phenotypic identification is easily affected by the external environment, has a long identification cycle, and low reproducibility. The industry has gradually shifted towards more precise DNA molecular identification technology, but this is only applicable to the purity identification of a few non-heading cabbage varieties. According to statistics from the China Seed Industry Big Data Platform website, more than 1,000 non-heading cabbage varieties have applied for protection. How to quickly screen stable mutation sites in non-heading cabbage and establish a DNA molecular detection technology suitable for the purity identification of most varieties is an urgent problem to be solved.

[0003] SSR and SNP are two commonly used molecular marker types. However, SSR has abundant variations, making results difficult to interpret, and the amplified products show little difference, requiring detection by polyacrylamide gel electrophoresis or capillary electrophoresis. SNP markers are based on the fluorescence signal of the amplified products for genotyping, but the equipment used is expensive. InDel (Insertion-Deletion) markers represent the insertion or deletion of a nucleotide in the genome. Specific InDel marker primers can be designed based on the sequences flanking the insertion / deletion site. Compared to SNP and SSR markers, InDel markers are second only to SNP variants in terms of the number of variants in the genome. They contain only two variant types, and their banding stability and interpretation are superior to SSR. Furthermore, long InDel variants can be detected using ordinary agarose gel electrophoresis platforms, and the requirements for sample DNA quality are not high, making it suitable for most companies to conduct rapid identification of hybrid variety purity.

[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 that can be used to identify the purity of non-heading Chinese cabbage 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 non-heading Chinese cabbage hybrid.

[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 a first primer set to an eighth primer set, corresponding to the amplification of ten InDel markers for genotyping (or determination of homozygosity or heterozygosity) of each InDel marker, wherein:

[0011] The ten InDel markers include:

[0012] The marker IndBrX05 is located at position 12957778 on chromosome 2 and is either nucleotide G or the nucleotide sequence shown in SEQ ID NO.21; or it can be described as being located at position 12957778 on chromosome 2 and being either nucleotide G or the nucleotide at that position being replaced by the nucleotide sequence shown in SEQ ID NO.21.

[0013] The marker IndBrX07 is located at positions 709237-709294 on chromosome 3 and is either the nucleotide sequence shown in SEQ ID NO.22 or the nucleotide sequence with positions 709238-709294 deleted, which is nucleotide C.

[0014] The marker IndBrX12 is located on chromosome 4 at positions 10620027-10620080, and is either the nucleotide sequence shown in SEQ ID NO.23 or the nucleotide sequence missing from positions 10620028-10620080, which is nucleotide C.

[0015] The marker IndBrX28 is located at position 7888084 on chromosome 5 and is either nucleotide T or the nucleotide sequence shown in SEQ ID NO.24; or it can be described as being located at position 7888084 on chromosome 5 and being either nucleotide T or the nucleotide at that position being replaced by the nucleotide sequence shown in SEQ ID NO.24.

[0016] The marker IndBrX34 is located on chromosome 6 at positions 9167633-9167690 and is either the nucleotide sequence shown in SEQ ID NO.25 or the nucleotide sequence missing from positions 9167634-9167690, which is nucleotide G.

[0017] The marker IndBrX35 is located at position 15513284 on chromosome 6 and is either nucleotide A or the nucleotide sequence shown in SEQ ID NO.26; or it can be described as being located at position 15513284 on chromosome 6 and being either nucleotide A or the nucleotide at that position being replaced by the nucleotide sequence shown in SEQ ID NO.26.

[0018] The marker IndBrX15 is located at position 1493949 on chromosome 8 and is either nucleotide G or the nucleotide sequence shown in SEQ ID NO.27; or it can be described as being located at position 1493949 on chromosome 8 and being either nucleotide G or the nucleotide at that position being replaced by the nucleotide sequence shown in SEQ ID NO.27.

[0019] The marker IndBrX39 is located at position 24696172 on chromosome 9 and is either nucleotide T or the nucleotide sequence shown in SEQ ID NO.28; or it can be described as being located at position 24696172 on chromosome 9 and being either nucleotide T or the nucleotide at that position being replaced by the nucleotide sequence shown in SEQ ID NO.28.

[0020] The marker IndBrX18 is located on chromosome 9 at positions 37489568-37489627, and is either the nucleotide sequence shown in SEQ ID NO.29 or the nucleotide sequence missing from positions 37489569-37489627, which is nucleotide C.

[0021] The marker IndBrX30 is located on chromosome 10 at positions 735120-735176 and is either the nucleotide sequence shown in SEQ ID NO.30 or the nucleotide sequence with positions 735121-735176 omitted, which is nucleotide A.

[0022] The positions of the ten InDel markers on the chromosome were determined based on sequence alignment with the CHIIFU_V1.5 reference genome of non-heading Chinese cabbage;

[0023] The first primer set was used to amplify the InDel label IndBrX05; the second primer set was used to amplify the InDel label IndBrX07; the third primer set was used to amplify the InDel label IndBrX12; the fourth primer set was used to amplify the InDel label IndBrX28; the fifth primer set was used to amplify the InDel label IndBrX34; the sixth primer set was used to amplify the InDel label IndBrX35; the seventh primer set was used to amplify the InDel label IndBrX15; the eighth primer set was used to amplify the InDel label IndBrX39; the ninth primer set was used to amplify the InDel label IndBrX18; and the tenth primer set was used to amplify the InDel label IndBrX30.

[0024] The InDel primer set provided by this invention can be used to identify the purity of non-heading Chinese cabbage hybrids. If the PCR product of the primer set used to identify the purity of a certain non-heading Chinese cabbage hybrid is a heterozygous band, it means that the non-heading Chinese cabbage variety being tested is a hybrid. If it is a single band, it means that the non-heading Chinese cabbage variety being tested is not a hybrid, but may be one of the parents, that is, the parents and the hybrid are mixed.

[0025] The non-heading cabbage of this invention includes rapeseed, bok choy, rapeseed, and other similar varieties.

[0026] The hybrid species mentioned in this invention refer to first-generation hybrids, which may contain a mixture of parental lines.

[0027] Furthermore, in the InDel primer combination,

[0028] 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;

[0029] 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;

[0030] 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;

[0031] 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;

[0032] 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;

[0033] 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;

[0034] 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;

[0035] 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;

[0036] 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;

[0037] 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.

[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 contain PCR auxiliary reagents, such as ultrapure water, PCR buffer, dNTPs, and DNA polymerase.

[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 non-heading Chinese cabbage hybrids.

[0042] Furthermore, the non-heading Chinese cabbage hybrid varieties are selected from 19qing47, 23qing2080, 23qing1390, 23qing735, Qing1262, 22qing106, 20qing49, 20qing138, 23qing1644, 23qing604, 21qing515, 15qing104, 19qing764, Yingqing349, 20qing110, 21qing124, 22qing1385, 23qing112, 23qing96, Q2337, 23qing891, 23qing1389, 23qing1980, 22 Qing1067, 23 Qing1703, 23 Qing2000, Jinpin Yixia, 21 Qing647, 23 Qing2623, 23 Qing1698, Chang'an Feng, 23 Qing246, Jingguan No.4, 23 Qing482, 23 Qing533, Jingguan No.2, Jingguan No.5, Naibai No.1, Jingyan Heiye, 22 Qing1262, Waltz Meili, 21 Qing110, 16 Qing1022, 24 Qing1313, 24 Qing166, 24 Qing1331, 23 Qing2634, 24 Qing29, 24 Qing147, 22 Qing1400.

[0043] All of the above-mentioned non-heading Chinese cabbage hybrids are commercially available varieties. Among these non-heading Chinese cabbage hybrids, at least one of the ten InDel markers mentioned in the first aspect of this invention is heterozygous. There are 4 non-heading Chinese cabbage hybrids with one heterozygous locus among the ten InDel markers, 8 non-heading Chinese cabbage hybrids with two heterozygous loci, 10 non-heading Chinese cabbage hybrids with three heterozygous loci, 7 non-heading Chinese cabbage hybrids with four heterozygous loci, 14 non-heading Chinese cabbage hybrids with five heterozygous loci, 5 non-heading Chinese cabbage hybrids with six heterozygous loci, 1 non-heading Chinese cabbage hybrid with seven heterozygous loci, and 1 non-heading Chinese cabbage hybrid with eight heterozygous loci. Therefore, the InDel primer combination provided by this invention can be used for purity identification of the above 50 non-heading Chinese cabbage hybrids.

[0044] A fourth aspect of the present invention provides a method for identifying the purity of a non-heading Chinese cabbage hybrid, comprising the following steps:

[0045] (1) Randomly select N non-heading Chinese cabbage hybrids to be tested and obtain their genomic DNA;

[0046] (2) Using the genomic DNA of at least 10 (e.g., 10, 15 or 20) non-heading Chinese cabbage hybrids selected from the N non-heading Chinese cabbage hybrids obtained in step (1) as templates, PCR amplification was 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 were obtained.

[0047] (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 bands as the target primer set.

[0048] (4) Using the genomic DNA of the N non-heading Chinese cabbage hybrids 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 non-heading Chinese cabbage hybrid under different target primer sets.

[0049] (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. Among them, the non-heading Chinese cabbage lines to be tested with heterozygous bands are hybrids. The purity of the non-heading Chinese cabbage hybrids to be tested is obtained according to the number of plants with heterozygous bands amplified by each target primer group.

[0050] 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 non-heading Chinese cabbage hybrid to be tested, specifically:

[0051] 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 non-heading Chinese cabbage hybrids obtained from each target primer set was calculated, and then the average value was calculated.

[0052] The purity of a target primer set = number of strains showing heterozygous bands with the target primer set / (N - number of strains without bands with the target primer set) × 100%.

[0053] Further, the non-heading Chinese cabbage hybrids to be tested were selected from 19qing47, 23qing2080, 23qing1390, 23qing735, Qing1262, 22qing106, 20qing49, 20qing138, 23qing1644, 23qing604, 21qing515, 15qing104, 19qing764, Yingqing349, 20qing110, 21qing124, 22qing1385, 23qing112, 23qing96, Q2337, 23qing891, 23qing1389, 23qing1980, and 2 2qing1067, 23qing1703, 23qing2000, Jinpin Yixia, 21qing647, 23qing2623, 23qing1698, Chang'an Feng, 23qing246, Jingguan No.4, 23qing482, 23qing533, Jingguan No.2, Jingguan No.5, Naibai No.1, Jingyan Heiye, 22qing1262, Waltz Meili, 21qing110, 16qing1022, 24qing1313, 24qing166, 24qing1331, 23qing2634, 24qing29, 24qing147, 22qing1400.

[0054] 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.

[0055] In the method described above, the larger the value of N, the higher the accuracy of identifying the purity of the non-heading Chinese cabbage hybrid to be tested.

[0056] It should be noted that this invention focuses on ensuring the purity of non-heading Chinese cabbage 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 non-heading Chinese cabbage hybrids.

[0057] Compared with the prior art, the present invention has the following beneficial effects:

[0058] The InDel primer combination provided by this invention enables early identification of existing representative non-heading Chinese cabbage hybrids at the seed or seedling stage, thereby ensuring the purity of hybrids, effectively protecting the legitimate rights and interests of producers and breeders, and providing technical support for seed quality management of non-heading Chinese cabbage varieties. 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

[0059] Figure 1The distribution of 10 InDel primer sets on the genome.

[0060] Figure 2 The typing effects of 10 InDel primer sets in some tested non-heading Chinese cabbage hybrids were investigated.

[0061] Figure 3 The distribution of 10 InDel primer sets at heterozygous sites in 50 non-heading Chinese cabbage varieties.

[0062] Figure 4 The results show the purity identification of primer group 9 in the Shangnongqing No. 1 hybrid. Detailed Implementation

[0063] 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.

[0064] 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.

[0065] Example 1: Obtaining the InDel primer combination for identifying the purity of non-heading Chinese cabbage hybrids.

[0066] I. Discovery of 10 InDel sites

[0067] This invention, based on resequencing data from 30 representative non-heading Chinese cabbage resources, developed whole-genome InDel markers for non-heading Chinese cabbage and used them for varietal purity identification, obtaining 10 InDel loci. These 30 non-heading Chinese cabbage resources are diverse, covering types such as green-stemmed, white-stemmed, dwarf yellow, black-tumbled, and dark-tumbled, basically encompassing the main ecological types and agronomic traits of non-heading Chinese cabbage. Specifically, the screening criteria for InDel loci are as follows: First, InDel loci belonging to the bimorphic variation type were screened on the whole genome chromosome, with a variation length >30 bp, 300 conserved limbs with no other variations. Further screening was conducted for InDel loci with a deletion rate <0.2, a minimum allele frequency >0.2, and a heterozygosity <0.05. Finally, BLAST-specific analysis was performed, and 40 pairs of specific primers were designed. These primers were used to screen tested non-heading Chinese cabbage hybrids. During the screening process, through careful comparison and analysis, primers exhibiting high heterozygosity and polymorphism were ultimately selected. After rigorous screening and evaluation, 10 InDel sites that are evenly distributed on the chromosome were ultimately retained. Figure 1).

[0068] 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 the alignment of the Chinese cabbage Chiifu-401-42 reference genome sequence, version V1.5 (downloadable from: http: / / 39.100.233.196:82 / download_genome / Brassica_Genome_data / Brara_Chiifu_V1.5 / Brapa_sequence_v1.5.fa.gz).

[0069] Table 1. Basic information of 10 InDel sites

[0070]

[0071]

[0072] II. Obtaining the InDel primer combination for identifying the purity of non-heading Chinese cabbage hybrids

[0073] Based on the 10 InDel sites discovered in step one, the inventors of this invention developed an InDel primer combination with high polymorphism and heterozygosity for identifying the purity of non-heading Chinese cabbage hybrids.

[0074] 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 and is used to amplify one InDel site. The nucleotide sequences of each primer in the 10 primer sets are shown in column 4 of Table 2.

[0075] Table 2. Ten pairs of Indel primer sequences applicable to purity identification of non-heading Chinese cabbage.

[0076]

[0077]

[0078] Example 2: Validation of the InDel primer combinations developed in Example 1

[0079] The basic information of the 50 tested non-heading Chinese cabbage hybrids in this embodiment is shown in Table 3. All 50 tested non-heading Chinese cabbage hybrids are common and excellent hybrids that can be obtained through commercial channels.

[0080] Table 3. Basic information of 50 tested non-heading Chinese cabbage hybrids

[0081]

[0082]

[0083] 1. Obtaining genomic DNA from the tested non-heading Chinese cabbage hybrid (DNA extraction using magnetic beads)

[0084] A suitable amount of leaf samples from each non-heading Chinese cabbage material 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℃ 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℃ 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 the volume of the supernatant. The volume should exceed 2 / 3 of the total volume; invert and mix well, 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 200 μl 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 100 μl of purified water, invert and mix thoroughly to ensure complete DNA dissolution; use a Nanodrop (micro-spectrophotometer) to take 2 μl 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 50 ng / μl for subsequent use.

[0085] 2. Using genomic DNA from 50 tested non-heading Chinese cabbage hybrids as templates, PCR amplification was performed using the 10 primer sets given in Table 2, and the corresponding PCR amplification products were obtained. 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.

[0086] 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.

[0087] 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.

[0088] 3. After completing step 2, perform agarose gel electrophoresis on the PCR amplification products, and take pictures and read the results using a gel imaging system. Determine the genotype of each InDel locus for the 50 tested non-heading Chinese cabbage hybrids based on the banding pattern. The specific determination principle is as follows: if a tested non-heading Chinese cabbage hybrid shows a heterozygous band at a certain InDel locus, then the genotype of the tested non-heading Chinese cabbage hybrid based on that InDel locus is heterozygous; if a tested non-heading Chinese cabbage hybrid shows a single band at a certain InDel locus, then the genotype of the tested non-heading Chinese cabbage hybrid based on that InDel locus is homozygous.

[0089] Some results can be found in Figure 2 The results showed that each primer set could achieve good typing results in the tested non-heading Chinese cabbage hybrids.

[0090] 4. Distribution of heterozygous loci and efficiency evaluation

[0091] (1) Based on the genotypes of 50 tested non-heading Chinese cabbage hybrids at 10 InDel loci, the number of heterozygous loci for each tested non-heading Chinese cabbage hybrid was counted.

[0092] The distribution of heterozygous loci in 50 tested non-heading Chinese cabbage hybrids established on 10 primer sets is shown in the figure. Figure 3 The results showed that the 10 primer sets ensured that each tested non-heading Chinese cabbage hybrid had at least one heterozygous site.

[0093] (2) Multiplex PCR can be used to reduce the workload for hybrid purity identification.

[0094] The results showed that multiplex PCR (10 primer sets) achieved 100% coverage of heterozygous sites in 50 tested non-heading Chinese cabbage hybrids.

[0095] Therefore, it can be seen that the InDel primer combination developed in Example 1 can be applied to the purity identification of non-heading Chinese cabbage hybrids.

[0096] Example 3: Detection of the purity of Shangnongqing No. 1 hybrid (commercially available variety) using the InDel primer combination developed in Example 1.

[0097] I. Detection of the purity of Shangnongqing No. 1 hybrid using the InDel primer combination developed in Example 1

[0098] 1. Obtaining genomic DNA from the Shangnongqing No. 1 hybrid

[0099] (1) Plant 200 seeds of Shangnongqing No. 1 hybrid seedlings to obtain Shangnongqing No. 1 hybrid seedlings.

[0100] (2) Leaves or roots of 96 Shangnongqing No. 1 hybrid seedlings were randomly selected and genomic DNA was extracted by magnetic bead method to obtain 96 genomic DNA samples of Shangnongqing No. 1 hybrid.

[0101] 2. Primer set screening

[0102] (1) Select the genomic DNA of 10 hybrid seedlings obtained in step 1 (2), and use them as templates to perform PCR amplification using 10 primer sets from the InDel primer combination developed in Example 1, and 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 is 1:1.

[0103] 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.

[0104] The specific reaction program 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.

[0105] (2) After completing step (1), perform agarose gel electrophoresis and record the electrophoresis results using a gel imaging system. Compare the number of heterozygous sites in the 10 primer sets, and the primer set with the most heterozygous sites is the selected primer set.

[0106] The results showed that primer set 9 had the highest number of heterozygous sites. Therefore, primer set 9 was selected as the primer set for subsequent experiments.

[0107] 3. Obtain the purity of Shangnongqing No. 1 hybrid.

[0108] (1) Using genomic DNA from 96 Shangnongqing No. 1 hybrids as templates, PCR amplification was performed using the selected primer set 9 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.

[0109] 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.

[0110] The specific reaction program 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.

[0111] (2) After completing step (1), perform agarose gel electrophoresis and use a gel imaging system to photograph and record the electrophoresis results.

[0112] InDel typing results are shown below Figure 4 .

[0113] (3) After completing step (2), count the number of plants with heterozygous sites and the number of plants without heterozygous sites shown by primer group 9; calculate the purity of Shangnongqing No. 1 hybrid according to the following formula.

[0114] Purity = Number of strains with heterozygous sites shown in primer set / (96 - Number of strains with no band in primer set) × 100%.

[0115] The results showed that primer set 9 showed 95 plants with heterozygous bands and 1 plant with homozygous bands, with a purity of 95 / 96 × 100% = 98.9%.

[0116] The purity of the hybrids obtained from field identification of 200 hybrid plants was 98.5%, which demonstrates the high accuracy of the identification method of this invention.

[0117] 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. InDel primer sets, including primer sets 1 through 10, amplify ten InDel markers to perform genotyping on each InDel marker, wherein: The ten InDel markers include: The marker IndBrX05 is located at position 12957778 on chromosome 2 and is either nucleotide G or the nucleotide sequence shown in SEQ ID NO.

21. The marker IndBrX07 is located on chromosome 3 at positions 709237-709294, and is either the nucleotide sequence shown in SEQ ID NO.22 or the nucleotide sequence at positions 709238-709294 that is missing. The marker IndBrX12 is located on chromosome 4 at positions 10620027-10620080, and is either the nucleotide sequence shown in SEQ ID NO.23 or the nucleotide sequence at positions 10620028-10620080 is missing. The marker IndBrX28 is located at position 7888084 on chromosome 5 and is either nucleotide T or the nucleotide sequence shown in SEQ ID NO.

24. The marker IndBrX34 is located on chromosome 6 at positions 9167633-9167690 and is either the nucleotide sequence shown in SEQ ID NO.25 or the nucleotide sequence at positions 9167634-9167690 that is missing. The marker IndBrX35 is located at position 15513284 on chromosome 6 and is either nucleotide A or the nucleotide sequence shown in SEQ ID NO.

26. The marker IndBrX15 is located at position 1493949 on chromosome 8 and is either nucleotide G or the nucleotide sequence shown in SEQ ID NO.

27. The marker IndBrX39 is located at position 24696172 on chromosome 9 and is either nucleotide T or the nucleotide sequence shown in SEQ ID NO.

28. The marker IndBrX18 is located on chromosome 9 at positions 37489568-37489627, and is either the nucleotide sequence shown in SEQ ID NO.29 or the nucleotide sequence at positions 37489569-37489627 is missing. The marker IndBrX30 is located on chromosome 10 at positions 735120-735176 and is either the nucleotide sequence shown in SEQ ID NO.30 or the nucleotide sequence at positions 735121-735176 that is missing. The positions of the ten InDel markers on the chromosome were determined based on sequence alignment with the CHIIFU_V1.5 reference genome of non-heading Chinese cabbage; The first primer set was used to amplify the InDel label IndBrX05; the second primer set was used to amplify the InDel label IndBrX07; the third primer set was used to amplify the InDel label IndBrX12; the fourth primer set was used to amplify the InDel label IndBrX28; the fifth primer set was used to amplify the InDel label IndBrX34; the sixth primer set was used to amplify the InDel label IndBrX35; the seventh primer set was used to amplify the InDel label IndBrX15; the eighth primer set was used to amplify the InDel label IndBrX39; the ninth primer set was used to amplify the InDel label IndBrX18; and the tenth primer set was used to amplify the InDel label IndBrX30.

2. The InDel primer combination as described in claim 1, characterized in that: 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; 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; 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; 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; 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; 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; 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; 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; 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; 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.

3. A kit comprising the InDel primer combination as described in claim 1 or 2.

4. The application of the InDel primer combination as described in claim 1 or 2, or the kit as described in claim 3, in identifying the purity of non-heading Chinese cabbage hybrids.

5. The application according to claim 4, characterized in that, The non-heading Chinese cabbage hybrids are selected from 19qing47, 23qing2080, 23qing1390, 23qing735, Qing1262, 22qing106, 20qing49, 20qing138, 23qing1644, 23qing604, 21qing515, 15qing104, 19qing764, Yingqing349, 20qing110, 21qing124, 22qing1385, 23qing112, 23qing96, Q2337, 23qing891, 23qing1389, 23qing1980, and 22qing10. 67, 23qing1703, 23qing2000, Jinpin Yixia, 21qing647, 23qing2623, 23qing1698, Chang'an Feng, 23qing246, Jingguan No.4, 23qing482, 23qing533, Jingguan No.2, Jingguan No.5, Naibai No.1, Jingyan Heiye, 22qing1262, Waltz Meili, 21qing110, 16qing1022, 24qing1313, 24qing166, 24qing1331, 23qing2634, 24qing29, 24qing147, 22qing1400.

6. A method for identifying the purity of a non-heading Chinese cabbage hybrid, characterized in that, Includes the following steps: (1) Randomly select N non-heading Chinese cabbage hybrids to be tested and obtain their genomic DNA; (2) Using the genomic DNA of at least 10 non-heading Chinese cabbage hybrids selected from the N non-heading Chinese cabbage hybrids 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 or 2 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 non-heading Chinese cabbage hybrids 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 non-heading Chinese cabbage hybrid 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. Among them, the non-heading Chinese cabbage lines to be tested with heterozygous bands are hybrids. The purity of the non-heading Chinese cabbage hybrids to be tested is obtained according to the number of plants with heterozygous bands amplified by each target primer group.

7. The method as described in claim 6, characterized in that, The method for "obtaining the purity of the non-heading Chinese cabbage hybrid seedlings 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 showing heterozygous bands and the number of plants without bands for each target primer set, calculate the purity of the non-heading Chinese cabbage hybrid seedlings to be tested obtained by each target primer set, and then calculate the average value. Purity = Number of strains showing heterozygous bands with a specific primer set / (N - Number of strains without bands with that primer set) × 100%.

8. The method as described in claim 6 or 7, characterized in that, The non-heading Chinese cabbage hybrids to be tested were selected from 19qing47, 23qing2080, 23qing1390, 23qing735, Qing1262, 22qing106, 20qing49, 20qing138, 23qing1644, 23qing604, 21qing515, 15qing104, 19qing764, Yingqing349, 20qing110, 21qing124, 22qing1385, 23qing112, 23qing96, Q2337, 23qing891, 23qing1389, 23qing1980, and 22qing1 067, 23qing1703, 23qing2000, Jinpin Yixia, 21qing647, 23qing2623, 23qing1698, Chang'an Feng, 23qing246, Jingguan No.4, 23qing482, 23qing533, Jingguan No.2, Jingguan No.5, Naibai No.1, Jingyan Heiye, 22qing1262, Waltz Meili, 21qing110, 16qing1022, 24qing1313, 24qing166, 24qing1331, 23qing2634, 24qing29, 24qing147, 22qing1400.

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