CN113621732B - InDel labeled primer set and its application in purity identification of cauliflower ‘CB-30’ variety or seeds - Google Patents

Abstract

The invention discloses an InDel marker primer group and application thereof in identifying the purity of cauliflower CB-30 variety or seeds. According to the invention, the parental resequencing data of the broccoli variety CB-30' is utilized to screen different InDel loci in the whole genome range, and a specific primer is designed, 8 pairs of InDel marker primers are obtained through screening, and the nucleotide sequences of the primers are respectively shown as SEQ ID NO.1-SEQ ID NO. 16. Based on PCR amplification reaction, the 8 pairs of InDel molecular marker primers can be used for rapidly and accurately identifying the purity of the broccoli variety and the hybrid seed in a short time, the required detection sample amount is small, the period is short, the method is simple and rapid, the identification result is accurate, the seed production quality of the hybrid seed 'CB-30' can be controlled, and the problems of long period, large labor investment, inaccurate identification and the like in the conventional broccoli variety identification are solved.

Description

InDel marker primer set and its purity identification in cauliflower ‘CB-30’ variety or seeds Applications in

Technical field

The present invention relates to a cauliflower molecular marker primer set and its application, in particular to a cauliflower InDel marker primer set and its application in cauliflower 'CB-30' variety or seed purity identification, and belongs to cauliflower 'CB-30' variety or seed purity molecules. identification field.

Background technique

Cauliflower (Brassica oleracea var.Botrytis) is one of the most important vegetable varieties in the world. It is deeply loved by Chinese and Western consumers because of its nutritious and delicious flower bulbs. China is the country with the largest cultivation and production of cauliflower in the world. In 2018, China's cauliflower production reached 10.26 million tons, accounting for 40.67% of the world's total production (FAO, 2019). As a representative of high-quality healthy vegetables, the curds composed of fleshy stems and inflorescence meristems are rich in vitamin C and glucosinolate anti-cancer substances, which meets the current Chinese people's demand for healthy vegetables.

As the number of cauliflower varieties increases year by year, and a small number of backbone parents are used intensively, the genetic differences between varieties become smaller and smaller, making it difficult to effectively identify. Effectively managing and screening cauliflower varieties with similar genetic backgrounds from the source is a major problem that needs to be solved. . At present, the identification of cauliflower varieties is mainly through traditional methods such as morphological marker research, which takes a long time, and the conclusions obtained are often incomplete, and it is difficult to eliminate the influence of environmental and human factors. Therefore, it is necessary to establish a rapid identification method based on genotype.

Molecular markers are markers at the gene level that directly detect genetic variation at the DNA molecular level. They are not affected by tissue and organ types, developmental stages, habitat conditions and many other factors. They are highly polymorphic and genetically stable. Insertion-Deletion Length Polymorphism (InDel) marker is a length polymorphism variation caused by the insertion or deletion of a certain number of nucleotides at the allelic site. It is a third-generation molecular marker and is mainly Developed based on whole-genome sequencing, it has a higher marker density than SSR (Simple Sequence Repeat) in the genome. It has the advantages of high marker specificity, good stability, simple and economical detection methods, and has been successfully used in rice, soybean, cotton and other varieties. In terms of identification, it overcomes the uncertainty of identifying new varieties based only on morphological characteristics.

Cauliflower variety 'CB-30' is a high-yielding, high-quality cauliflower hybrid variety cultivated by the Tianjin Academy of Agricultural Sciences. It is selected from the cauliflower cytoplasmic sterile line 'GT-42' and the excellent inbred line 'GS-57' Early maturing hybrids. 'CB-30' is a spring cultivar with a maturity period of 55 to 60 days, a compact plant shape, dark green leaves, and excellent ball protection. The flower bulbs are round, firm, white, tender and flat. The weight of a single ball is 1.21kg, the planting density is 37500 plants/hm ² , and the average yield is 45290kg/hm ² . So far, there is no molecular identification marker suitable for cauliflower variety 'CB-30'. The development of molecular identification markers suitable for cauliflower variety 'CB-30' will help standardize cauliflower variety registration, strengthen market supervision and strengthen intellectual property protection.

Contents of the invention

One of the objects of the present invention is to provide an InDel marker primer pair for identifying the authenticity or seed purity of the cauliflower variety 'CB-30';

The second object of the present invention is to provide a PCR detection kit for identifying the authenticity of cauliflower variety ‘CB-30’ or identifying the purity of cauliflower hybrid ‘CB-30’ seeds;

The third object of the present invention is to apply the InDel marker primer pair set to identify the authenticity or seed purity of cauliflower variety 'CB-30'.

The above objects of the present invention are achieved through the following technical solutions:

On the one hand, the present invention provides a primer pair set for identifying cauliflower variety 'CB-30' based on InDel markers. The primer pair set consists of the following primer pair 1 - primer pair 8, a total of 8 pairs of primers. The 8 pairs of primers The nucleotide sequences are as follows:

Primer pair 1: consists of the forward primer shown in SEQ ID NO.1 and the reverse primer shown in SEQ ID NO.2;

Primer pair 2: consists of the forward primer shown in SEQ ID NO.3 and the reverse primer shown in SEQ ID NO.4;

Primer pair 3: consists of the forward primer shown in SEQ ID NO.5 and the reverse primer shown in SEQ ID NO.6;

Primer pair 4: consists of the forward primer shown in SEQ ID NO.7 and the reverse primer shown in SEQ ID NO.8;

Primer pair 5: consists of the forward primer shown in SEQ ID NO.9 and the reverse primer shown in SEQ ID NO.10;

Primer pair 6: consists of the forward primer shown in SEQ ID NO.11 and the reverse primer shown in SEQ ID NO.12;

Primer pair 7: consists of the forward primer shown in SEQ ID NO.13 and the reverse primer shown in SEQ ID NO.14;

Primer pair 8: consists of the forward primer shown in SEQ ID NO. 15 and the reverse primer shown in SEQ ID NO. 16.

Another aspect of the present invention is to provide a PCR detection kit for identifying the authenticity of cauliflower variety 'CB-30' or identifying the purity of cauliflower hybrid 'CB-30' seeds. The PCR detection kit includes: dNTPs, Taq enzyme, MgCl ₂ , a PCR primer pair consisting of a forward primer and a reverse primer, an amplification buffer and sterilized water; wherein the PCR primer pair is the InDel labeled primer pair set (the above primer pair 1- Any pair of primers 8).

Another aspect of the present invention is to provide a method for identifying the authenticity or seed purity of cauliflower variety 'CB-30': applying the InDel-based marker primer pair, amplifying by PCR, and detecting by agarose gel electrophoresis, The polymorphism of the InDel site among different individuals can be displayed to achieve the purpose of identification.

Specifically, the present invention provides an application of the InDel marker primer pair set in the authenticity identification of cauliflower hybrid 'CB-30' variety, including:

(1) Extract the genomic DNA of the cauliflower plant sample to be tested;

(2) Using the extracted sample genomic DNA as a template, establish a PCR amplification system using 8 pairs of primer pairs as forward and reverse primers to perform PCR amplification respectively;

(3) If the banding pattern of each of the 8 amplification products has the banding pattern of the parents of the cauliflower hybrid 'CB-30', then the cauliflower variety to be tested is the cauliflower hybrid 'CB-30' '; If the banding pattern of any one of the 8 amplification products does not have the banding pattern of the parents of the cauliflower hybrid 'CB-30', then the cauliflower variety to be tested is not the cauliflower hybrid 'CB-30' '.

Among them, the PCR amplification system described in step (2) is preferably: 10× buffer 1.0 μL, 25mM MgCL ₂ 1.0 μL, 2mM dNTPs 1.0 μL, 10 μM forward primer and reverse primer 1.0 μL each, 0.5 units of Taq DNA polymerase 0.2μL, template 50ng, add ddH ₂ O to 10μL;

The PCR amplification program is: 95°C for 3 minutes; 95°C for 15 seconds, 58°C for 15 seconds, 72°C for 20 seconds, 35 cycles; 72°C for 5 minutes.

The female parent of the cauliflower hybrid 'CB-30' is GT-42, and the male parent of the cauliflower hybrid 'CB-30' is GS-57.

Specifically, the present invention provides a method for identifying the purity of cauliflower hybrid 'CB-30' seeds using the InDel marker primer pair set. The identification method includes:

(1) Extract the genomic DNA of the cauliflower seed sample to be tested;

(2) Using the extracted genomic DNA of the seed sample as a template, establish a PCR amplification system using 8 pairs of primer pairs as forward and reverse primers to perform PCR amplification respectively;

(3) If the banding pattern of each of the eight amplification products has the banding pattern of the parents of the cauliflower hybrid 'CB-30', then the cauliflower seed to be tested is the cauliflower hybrid 'CB-30' 'Seeds; if the banding pattern of any one of the eight amplification products does not have the banding pattern of the parents of the cauliflower hybrid 'CB-30', then the cauliflower seeds to be tested are not the cauliflower hybrid 'CB- 30';

(4) Calculate the ratio of the true number of 'CB-30' cauliflower hybrids to the total number of tested seeds, and obtain the seed purity of 'CB-30' cauliflower hybrids.

Among them, the PCR amplification system described in step (2) is preferably: 10× buffer 1.0 μL, 25mM MgCL ₂ 1.0 μL, 2mM dNTPs 1.0 μL, 10 μM forward primer and reverse primer 1.0 μL each, 0.5 units of Taq DNA polymerase 0.2μL, template 50ng, add ddH ₂ O to 10μL;

The PCR amplification program is: 95°C for 3 minutes; 95°C for 15 seconds, 58°C for 15 seconds, 72°C for 20 seconds, 35 cycles; 72°C for 5 minutes.

The female parent of the cauliflower hybrid 'CB-30' is 'GT-42', and the male parent of the cauliflower hybrid 'CB-30' is GS-57.

The present invention uses a Perl language script to screen the unique InDel sites in the parental genome of the cauliflower variety 'CB-30'. The InDel size is set to 20-500 bp, and the sequencing depth is greater than 4. Based on the reference genome information, the screened sites were positioned on the genome, and the 200 bp upstream and downstream nucleotide sequences of the InDel marker site were extracted. Primer3.0 and self-programmed Perl language scripts were used to design primers in batches. The designed and synthesized primers were used to conduct PCR amplification of the cauliflower 'CB-30' variety and its parents. 8 pairs of InDel marker primers were screened and the 8 screened primers were used. InDel labeled primers can quickly and accurately complete the purity identification of 'CB-30' cauliflower varieties and hybrid seeds in a short time, solving the existing problems of long cycle and high labor investment in the identification of existing cauliflower 'CB-30' varieties. The cauliflower variety 'CB-30' was detected quickly, efficiently, stably and accurately based on InDel molecular markers.

Compared with the prior art, the present invention mainly has the following beneficial effects:

1. The present invention uses the resequencing data of the parents of the cauliflower variety 'CB-30' to screen differential InDel sites across the entire genome and design specific primers, with high accuracy and stable and reliable results.

2. The present invention uses agarose gel electrophoresis for detection. Compared with polyacrylamide gel electrophoresis, it has the advantages of simple operation, low cost, fast speed, and wide application range.

3. This invention is based on PCR amplification reaction and uses InDel molecular markers at 8 sites to quickly and accurately complete the purity identification of 'CB-30' cauliflower varieties and hybrid seeds in a short time. The method is simple and fast, and the detection cycle is short. It saves labor costs and has good application and promotion prospects.

4. In addition to identifying the cauliflower variety 'CB-30' based on the band pattern amplified by the primer set, the present invention can also identify the different band patterns among different cauliflower varieties based on the primer set. Different varieties include 'Jinpin 74', 'Jinpin 79' ', 'Jinpin 80', 'Chunxue 30', 'BX-110', 'BX-120' or 'SX-60' and other cauliflower varieties to distinguish.

Description of the drawings

Figure 1 is the electrophoresis pattern of PCR amplification products of 8 primer pairs of cauliflower variety 'CB-30' female parent GT-42, cauliflower variety 'CB-30' male parent GS-57, and cauliflower variety 'CB-30'; Figure M in the middle is Marker, and each pair of primers has 9 lanes. From left to right, there are 3 individual plants each of the female parent, male parent and 'CB-30' of the cauliflower variety 'CB-30'.

Figure 2 shows the cauliflower variety 'CB-30' and Jinpin 74', 'Jinpin 79', 'Jinpin 80', 'Chunxue 30', 'BX-110', 'BX-120' or 'SX-60' Electrophoresis diagram of PCR amplification products of 8 pairs of primers; M in the picture is Marker, and lanes 01 to 08 are CB30-36, CB30-46, CB30-48, CB30-57, CB30-63, CB30-67, and CB30 respectively. -98, CB30-99 and other 8 pairs of primers, each pair of primers corresponds to 1 lane, and each is a mixed sample of 5 individual plants. .

Figure 3 is a partial electrophoresis diagram of using primers CB30-36, CB30-48, and CB30-57 to detect the seed purity of cauliflower variety 'CB-30'; M in the figure is the Marker, and lane 01 is the mother of cauliflower variety 'CB-30' In this example, lane 02 is the male parent of cauliflower variety 'CB-30', and lanes 03-24 are the 22 individual plants of the sample to be tested.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, these embodiments are only exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that the details and forms of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and substitutions all fall within the protection scope of the present invention.

Test Example 1: Screen differential InDel sites across the entire genome and design screening-specific primers

1Test method

1.1 Illumina high-throughput sequencing

Extract the maternal cytoplasmic sterile line 'GT-42' of cauliflower variety 'CB-30', the male superior inbred line 'GS-57', and other PN-601, PN-602, PN-603, PN- Genomic DNA of 7 excellent inbred lines, including 604, PN-643, PN-644, and PN-737, was analyzed. Illumina paired-end sequencing was performed on the genomic DNA of each sterile line and inbred line. The obtained raw data removed the adapter sequences. After data quality control, the effective sequencing data was compared with the cauliflower reference genome through BWA software, and the comparison results were removed by SAMTOOLS to remove duplicates. Use the GATK HaplotypeCaller module to detect and analyze InDel mutations in samples;

1.2 Differential InDel site screening and specific primer design

Use a Perl language script to screen the unique InDel sites in the parental genome of cauliflower variety 'CB-30'. The InDel size is set to 20-500bp, and the sequencing depth is greater than 4. Based on the reference genome information, the screened sites were positioned on the genome, and the 200 bp upstream and downstream nucleotide sequences of the InDel marker site were extracted. Use Primer3.0 and self-programmed Perl language script to design primers. Set the parameters as T _m value is (58±3)℃, primer length is (20±2)bp, the expected length of PCR product is 100~400bp, and other parameters are default .

2 test results

Based on the predicted InDel sites, a total of 125 InDel sites and their specific primers were obtained. After PCR testing, it was found that 125 pairs of primers could amplify bands in 'GT-42', 'GS-57' and F1'CB-30', and the size of the amplified product was consistent with the predicted size, and 8 pairs of InDel The primers in 'CB-30' can simultaneously amplify the characteristic bands of both parents, which can be used to identify the cauliflower variety 'CB-30' and its seed purity (Figure 1).

Table 1 Primer pairs for identification of InDel-labeled cauliflower hybrid ‘CB-30’

serial number Primer pair name Forward primer sequence (5‘-3’) Reverse primer sequence (5‘-3’) 1 CB30-36 TGGGGATAGAGAGAGACCCA(SEQ ID No.1) AAATCCCAGTGTCCGAACGA(SEQ ID No.2) 2 CB30-46 ACCATTGTGCTCCCAGTTCC(SEQ ID No.3) GCTTTGTTTCAGACCAGTGTGT(SEQ ID No.4) 3 CB30-48 ATACTGACAAGCCACTGCCC(SEQ ID No.5) AATGGACAATCAGGGCAGGG(SEQ ID No.6) 4 CB30-57 GCGGATGCTACTGTAGAGAAAC(SEQ ID No.7) AGCGGCTTCTCATTCCCAAT(SEQ ID No.8) 5 CB30-63 ACGCTGAGTCGTTTCTGGAG(SEQ ID No.9) CGGAAGGTTTCAGTTTGCCG(SEQ ID No.10) 6 CB30-67 CGGTCTATCAGGTCACAGGC(SEQ ID No.11) CTGTGCAACTTCCAACGTGG(SEQ ID No.12) 7 CB30-98 TCTCCGCATGTTCGTGTGAA(SEQ ID No.13) TTTGCAGGCAAACCAATCCG(SEQ ID No.14) 8 CB30-99 ACGGTCCGGCAAAGAATCTT(SEQ ID No.15) CGTATCAATGCCACCGTCCT(SEQ ID No.16)

The eight pairs of InDel marker primers used to identify the purity of ‘CB-30’ cauliflower variety or ‘CB-30’ cauliflower hybrid seeds in the following Test Examples 1 and 2 are shown in Table 1.

Test Example 2 ‘CB-30’ Cauliflower Variety Rapid Identification Test

1 identification method

1.1 Extraction of genomic DNA from leaves

Cauliflower hybrid 'CB-30' and its female parent GT-42 and male parent GS-57, 'Jinpin 74', 'Jinpin 79', 'Jinpin 80', 'Chunxue 30', and 'BX-110' , 'BX-120' or 'SX-60' cauliflower varieties, mixed DNA samples of five individual plants each.

1.2 PCR amplification

Use the genomic DNA of each cauliflower variety described in 1.1 as the template, and use CB30-36, CB30-46, CB30-48, CB30-57, CB30-63, CB30-67, CB30-98 and CB30-99 as primers, respectively. Perform PCR amplification;

The PCR amplification system is: 10× buffer 1.0μL, 25mM MgCL ₂ 1.0μL, 2mM dNTPs 1.0μL, 10μM forward primer and reverse primer 1.0μL each, 0.5 unit Taq DNA polymerase 0.2μL, template 50ng, add ddH ₂ O to 10 μL;

The PCR amplification program was: 95°C for 3 min, 35 cycles of (95°C for 15 s, 58°C for 15 s, 72°C for 20 s), and 72°C for 5 min.

1.3 Detection and analysis of PCR amplification products

Add 1 μL of 10× Loding Buffer to the amplified product described in 1.2, conduct 2% agarose gel electrophoresis for 90 min at a constant voltage of 120 V/cM, Gelred staining, and take pictures with a gel imaging system. Identification of varieties based on electrophoresis band patterns. If the band patterns of the eight InDel sites also have the band patterns of the parents of the cauliflower hybrid ‘CB-30’, the cauliflower variety to be tested is determined to be the cauliflower hybrid ‘CB-30’.

2 identification results

Use the above method to test the cauliflower hybrid cauliflower variety 'CB-30' and 'Jinpin 74', 'Jinpin 79', 'Jinpin 80', 'Chunxue 30', 'BX-110', 'BX-120' or 'SX-60' was used for variety identification. The 8 pairs of primers were verified to be polymorphic in cauliflower varieties, with 3 banding types. For intuitive representation, the amplification map of the same primer pair is generally based on the molecular weight of the smallest amplification product. Classify by size. When the molecular weight of the smallest product is the same, it is classified by the second molecular weight, and so on. The type of low molecular weight band is defined as 1, the type of high molecular weight band is defined as 2, the type of hybrid co-banding is defined as 3, and the type of no band is defined as 0. Convert the corresponding map type into a unique code like an ID number, and use the code to distinguish the differences in varieties.

Cauliflower hybrid varieties 'CB-30' and 'Jinpin 74', 'Jinpin 79', 'Jinpin 80', 'Spring Snow 30', 'BX-110', 'BX-120' or 'SX-60' The agarose gel electrophoresis diagram amplified by 8 pairs of primers is shown in Figure 2. In Figure 2, M is Marker, and lanes 01 to 08 are respectively the amplification results of eight pairs of primers: CB30-36, CB30-46, CB30-48, CB30-57, CB30-63, CB30-67, CB30-98 and CB30-99. Increased product; its coding table is shown in Table 2.

Table 2 Fingerprint codes of the parents of cauliflower variety ‘CB-30’, hybrid ‘CB-30’ and 7 cauliflower hybrid varieties

According to the test results, it can be seen that the banding patterns of the eight InDel sites of the cauliflower hybrid 'CB-30' also have the banding patterns of the parents of the cauliflower hybrid 'CB-30', 'Jinpin 74', 'Jinpin 79', The banding patterns of the eight InDel loci of 'Jinpin 80', 'Chunxue 30', 'BX-110', 'BX-120' and 'SX-60' are different and they all have the parental parent of the cauliflower hybrid 'CB-30'. The band pattern; according to the identification results, it can be seen that the cauliflower hybrid 'CB-30' and other cauliflower varieties can be accurately identified using the primer set designed in the present invention.

Test Example 3 Identification of Purity of ‘CB-30’ Cauliflower Hybrid Seeds

1. Identification method

1.1 Extraction of genomic DNA from leaves

94 random hybrid sample seeds from a batch of ‘CB-30’ cauliflower hybrid seeds.

Seeds from the parent parent (female parent GT-42 and male parent GS-57) and sample seeds were sown in a 72-hole hole tray at the same time, and watered regularly until two true leaves grew; 5 plants from each parent were planted separately. Mixed sampling was conducted, and the leaves of each of the 94 hybrid sample seeds were sampled from individual plants. Genomic DNA was extracted separately according to the conventional CTAB method and stored at -20°C for later use.

1.2 PCR amplification

The same PCR amplification conditions as in Test Example 2.

1.3 Detection and analysis of PCR amplification products

The methods for detecting PCR amplification products and identifying hybrid varieties are the same as those in Experimental Example 1.

2. Identification results

Partial results of agarose gel electrophoresis for seed purity identification of cauliflower hybrid ‘CB-30’ using the above method are shown in Figure 3. In the figure, M is the marker, lane 01 is the female parent of the cauliflower hybrid variety 'CB-30', lane 02 is the male parent of the cauliflower hybrid variety 'CB-30', and lanes 03-24 are the 22 individual plants of the sample to be tested.

Among the 22 sample seeds, 22 are true hybrids, so the purity of this batch of ‘CB-30’ hybrid seeds is 100%, in line with national standards and consistent with field survey results.

SEQUENCE LISTING

<110> Tianjin Academy of Agricultural Sciences

<120> InDel labeled primer set and its application in purity identification of cauliflower 'CB-30' variety or seeds

<130> TJ-2002-210704A

<160> 16

<170> PatentIn version 3.5

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Claims (9)

1. The InDel labeling primer pair set of cauliflower is characterized in that the InDel labeling primer pair set consists of the following primer pair 1-primer pair 8: Primer pair 1: consists of the forward primer shown in SEQ ID NO.1 and the reverse primer shown in SEQ ID NO.2; Primer pair 2: consists of the forward primer shown in SEQ ID NO.3 and the reverse primer shown in SEQ ID NO.4; Primer pair 3: consists of the forward primer shown in SEQ ID NO.5 and the reverse primer shown in SEQ ID NO.6; Primer pair 4: consists of the forward primer shown in SEQ ID NO.7 and the reverse primer shown in SEQ ID NO.8; Primer pair 5: consists of the forward primer shown in SEQ ID NO.9 and the reverse primer shown in SEQ ID NO.10; Primer pair 6: consists of the forward primer shown in SEQ ID NO.11 and the reverse primer shown in SEQ ID NO.12; Primer pair 7: consists of the forward primer shown in SEQ ID NO.13 and the reverse primer shown in SEQ ID NO.14; Primer pair 8: consists of the forward primer shown in SEQ ID NO. 15 and the reverse primer shown in SEQ ID NO. 16. 2. A PCR detection kit used to identify the authenticity of cauliflower variety 'CB-30' or to identify the purity of cauliflower hybrid 'CB-30' seeds. The PCR detection kit includes: dNTPs, Taq enzyme, MgCl ₂ , from Forward A PCR primer pair consisting of a primer and a reverse primer, an amplification buffer and sterilized water; characterized in that the PCR primer pair is primer pair 1-primer pair 8 in claim 1. 3. Application of the InDel labeled primer pair set according to claim 1 in the authenticity identification of cauliflower hybrid 'CB-30' variety. 4. The application according to claim 3, characterized in that it includes: (1) Extract the genomic DNA of the cauliflower plant sample to be tested; (2) Using the extracted genomic DNA of the sample as a template, establish a PCR amplification system using the 8 pairs of primers of claim 1 as forward and reverse primers to perform PCR amplification respectively; (3) If the banding pattern of each of the 8 amplification products has the banding pattern of the parents of the cauliflower hybrid 'CB-30', then the cauliflower variety to be detected is the cauliflower hybrid 'CB-30' '; If the banding pattern of any one of the eight amplification products does not have the banding pattern of the parents of the cauliflower hybrid 'CB-30', then the cauliflower variety to be detected is not the cauliflower hybrid 'CB-30' '. 5. The application according to claim 4, characterized in that the PCR amplification system described in step (2) is: 10×buffer 1.0 μL, 25mM MgCL ₂ 1.0 μL, 2mM dNTPs 1.0 μL, 10 μM forward primer and 1.0 μL of each reverse primer, 0.2 μL of 0.5 units of Taq DNA polymerase, 50 ng of template, and add ddH ₂ O to 10 μL; The PCR amplification program is: 95°C for 3 minutes; 95°C for 15 seconds, 58°C for 15 seconds, 72°C for 20 seconds, 35 cycles; 72°C for 5 minutes. 6. Application according to claim 4, characterized in that the female parent of the cauliflower hybrid 'CB-30' is GT-42, and the male parent of the cauliflower hybrid 'CB-30' is GS-57. . 7. Application of the InDel labeled primer pair set according to claim 1 in identifying the purity of cauliflower hybrid 'CB-30' seeds. 8. The application according to claim 7, characterized in that it includes: (1) Extract the genomic DNA of the cauliflower seed sample to be tested; (2) Using the extracted genomic DNA of the seed sample as a template, establish a PCR amplification system using the 8 pairs of primers of claim 1 as forward and reverse primers to perform PCR amplification respectively; (3) If the banding pattern of each of the eight amplification products has the banding pattern of the parents of the cauliflower hybrid 'CB-30', then the cauliflower seed to be tested is the cauliflower hybrid 'CB-30' 'Seeds; if the banding pattern of any one of the 8 amplification products does not have the banding pattern of the parents of the cauliflower hybrid 'CB-30', then the cauliflower seeds to be detected are not the cauliflower hybrid 'CB- 30'; (4) Calculate the ratio of the true number of 'CB-30' cauliflower hybrids to the total number of tested seeds, and obtain the seed purity of 'CB-30' cauliflower hybrids. 9. The application according to claim 8, characterized in that the PCR amplification system described in step (2) is: 10×buffer 1.0 μL, 25mM MgCL ₂ 1.0 μL, 2mM dNTPs 1.0 μL, 10 μM forward primer and 1.0 μL of each reverse primer, 0.2 μL of 0.5 units of Taq DNA polymerase, 50 ng of template, and add ddH ₂ O to 10 μL; The PCR amplification program is: 95°C for 3 minutes; 95°C for 15 seconds, 58°C for 15 seconds, 72°C for 20 seconds, 35 cycles; 72°C for 5 minutes.