InDel marker primer for identifying authenticity or seed purity of cauliflower variety Jinjiang 81 and identification method of InDel marker primer

Abstract

The invention discloses an InDel marker primer for identifying the authenticity or seed purity of a cauliflower variety Jinjiang 81 and an identification method of the InDel marker primer. According to the invention, re-sequencing data of male and female parents of a cauliflower variety 'Jinjiang 81' is analyzed in a whole genome range, specific InDel loci are screened and designed, five pairs of InDel labeled primer pairs are selected from the specific InDel loci, and nucleotide sequences of the five pairs of InDel labeled primer pairs are respectively shown as SEQ ID NO.1-SEQ ID NO.10; the identification method established by applying the five pairs of labeled primers can quickly and accurately complete the identification of the authenticity of the cauliflower variety 'Jinjiang 81' and the purity of the hybrid seeds of the cauliflower variety 'Jinjiang 81', solves the problems of long period, high cost, low accuracy and the like of the traditional identification method, has the advantages of high identification accuracy, simplicity, convenience and rapidness in operation, low demand quantity of detection samples and the like, and has a wide application prospect. The identification efficiency and accuracy are greatly improved, and the seed production quality of the cauliflower variety 'Jinjiang 81' is effectively guaranteed.

Description

Technical Field

[0001] The invention relates to molecular markers for cauliflower variety authenticity or seed purity identification, in particular to InDel marker primers and an identification method for cauliflower variety 'Jinpin 81' authenticity or seed purity identification, belonging to the field of molecular markers for cauliflower variety authenticity or seed purity identification. Background Art

[0002] Cauliflower (Brassica oleracea var. Botrytis) is one of the main vegetable varieties in the world, and is widely popular for its rich nutritional value and unique flavor. Cauliflower is rich in vitamin C and anti-cancer substances such as glucosinolates, and represents a high-quality and healthy vegetable variety, which meets the current demand for nutritious and healthy diets.

[0003] In recent years, the number of cauliflower varieties has continued to increase, but some parents have been frequently used, resulting in reduced genetic differences between varieties and increased difficulty in identification. It is particularly important to develop a rapid genome-based identification method. At present, traditional identification methods for cauliflower varieties mostly rely on morphological markers, which are complex and easily affected by environmental and human factors. Therefore, it is crucial to establish an efficient molecular marker identification method.

[0004] Molecular marker technology can directly detect genetic diversity at the DNA molecular level, is not limited by growth stage, tissue type or environmental conditions, and has high polymorphism and genetic stability. Insertion-Deletion Length Polymorphism (InDel) is one of the third-generation molecular marker types, which is caused by the insertion or deletion of nucleotides in alleles and is developed based on whole genome sequencing technology. InDel markers have a higher density than SSR (Simple Sequence Repeat) markers, and have the characteristics of strong specificity, good stability, simple and economical detection methods. They have been successfully applied in the identification of crop varieties such as rice, soybeans and cotton, avoiding the uncertainty of identification based solely on morphological characteristics.

[0005] 'Jinpin 81' is a mid-to-late autumn maturing cauliflower variety (the cauliflower variety 'Jinpin 81' is sold by Tianjin Kerun Vegetable Research Institute). It has good growth potential and disease resistance, a semi-erect plant shape, white and firm flower heads, and a single head weighs about 1.9 kilograms. The flower heads need to be harvested in time after maturity; however, to date there is a lack of specific molecular markers and identification methods suitable for the authenticity of the cauliflower variety 'Jinpin 81' or the purity of its seeds. Summary of the invention

[0006] One of the purposes of the present invention is to provide an InDel molecular marker primer pair set for identifying the authenticity of the cauliflower variety 'Jinpin 81' or the purity of its seeds;

[0007] The second object of the present invention is to provide a PCR detection kit for identifying the authenticity of the cauliflower variety 'Jinpin 81' or identifying the purity of the seeds of the cauliflower hybrid 'Jinpin 81';

[0008] The third object of the present invention is to apply the InDel marker primer pair or the PCR detection kit containing the InDel marker primer pair to identify the authenticity of the cauliflower variety 'Jinpin 81' or the purity of its seeds.

[0009] To achieve the above-mentioned purpose, the main technical solutions adopted by the present invention include:

[0010] In one aspect, the present invention provides InDel molecular marker primers for identifying the authenticity of cauliflower variety 'Jinpin 81' or identifying the seed purity of cauliflower variety 'Jinpin 81', wherein the InDel molecular marker primers are selected from any one of the primer pairs or a primer set consisting of more than one primer pair described in (1) to (5) below:

[0011] (1) Primer pair 1: Primer pair 1 consisting of a forward primer shown in SEQ ID NO. 1 and a reverse primer shown in SEQ ID NO. 2;

[0012] (2) Primer pair 2: Primer pair 2 consisting of a forward primer shown in SEQ ID NO. 3 and a reverse primer shown in SEQ ID NO. 4;

[0013] (3) Primer pair 3: Primer pair 3 consisting of the forward primer shown in SEQ ID NO. 5 and the reverse primer shown in SEQ ID NO. 6;

[0014] (4) Primer pair 4: Primer pair 4 consisting of a forward primer shown in SEQ ID NO. 7 and a reverse primer shown in SEQ ID NO. 8;

[0015] (5) Primer pair 5: Primer pair 5 consisting of the forward primer shown in SEQ ID NO. 9 and the reverse primer shown in SEQ ID NO. 10.

[0016] The present invention screens and designs specific InDel sites by performing genome-wide analysis on the resequencing data of the parents of the cauliflower variety 'Jinpin 81', and screens out 5 pairs of nucleotide sequence-specific InDel marker primer pairs. The 5 pairs of InDel marker primer pairs can be used to quickly and accurately identify the authenticity of the cauliflower variety 'Jinpin 81' or the purity of hybrid seeds.

[0017] Another aspect of the present invention is to provide a PCR detection kit for identifying the authenticity of cauliflower variety 'Jinpin 81' or identifying the seed purity of cauliflower variety 'Jinpin 81', the PCR detection kit comprising: dNTPs, Taq enzyme, MgCl2, a PCR primer pair consisting of a forward primer and a reverse primer, an amplification buffer and sterile water; wherein the PCR primer pair is selected from a primer set consisting of any one or more primer pairs among the following five pairs of InDel labeled primer pairs:

[0018] (1) Primer pair 1: Primer pair 1 consisting of a forward primer shown in SEQ ID NO.1 and a reverse primer shown in SEQ ID NO.2; (2) Primer pair 2: Primer pair 2 consisting of a forward primer shown in SEQ ID NO.3 and a reverse primer shown in SEQ ID NO.4; (3) Primer pair 3: Primer pair 3 consisting of a forward primer shown in SEQ ID NO.5 and a reverse primer shown in SEQ ID NO.6; (4) Primer pair 4: Primer pair 4 consisting of a forward primer shown in SEQ ID NO.7 and a reverse primer shown in SEQ ID NO.8; (5) Primer pair 5: Primer pair 5 consisting of a forward primer shown in SEQ ID NO.9 and a reverse primer shown in SEQ ID NO.10.

[0019] Another aspect of the present invention is to apply the InDel molecular marker primer or a PCR detection kit containing the InDel molecular marker primer to the authenticity identification of the cauliflower variety 'Jinpin 81' or the seed purity identification, including: using the DNA extracted from the sample to be detected as a template, using the InDel molecular marker primer as a PCR primer to establish a PCR amplification system, and achieving the purpose of identification by detecting the polymorphism of the InDel site between different individuals.

[0020] Specifically, the present invention provides a method for authenticating the authenticity of a cauliflower variety 'Jinpin 81', comprising:

[0021] (1) extracting genomic DNA from a cauliflower plant sample to be tested;

[0022] (2) using the extracted sample genomic DNA as a template and using the five pairs of InDel molecular marker primers as forward and reverse primers to establish a PCR amplification system and perform PCR amplification respectively;

[0023] (3) If the banding patterns of the five amplified products all have the banding patterns of the parent strain of the cauliflower variety 'Jinpin 81', the cauliflower variety to be tested is the cauliflower variety 'Jinpin 81'; if the banding patterns of only one of the five amplified products do not all have the banding patterns of the parent strain of the cauliflower variety 'Jinpin 81', the cauliflower variety to be tested is not the cauliflower variety 'Jinpin 81'.

[0024] A preferred specific embodiment of the present invention is to use the extracted sample genomic DNA as a template, and use the primer pair 1 described in claim 1 as a forward and reverse primer to establish a PCR amplification system for PCR amplification, and the length of the paternal band type of the amplified product is 247bp, and the length of the maternal band type of the amplified product is 399bp; use the extracted sample genomic DNA as a template, and use the primer pair 2 described in claim 1 as a forward and reverse primer to establish a PCR amplification system for PCR amplification, and the length of the paternal band type of the amplified product is 370bp, and the length of the maternal band type of the amplified product is 462bp; use the extracted sample genomic DNA as a template, and use the primer pair 3 described in claim 1 as a forward and reverse primer to establish a P The CR amplification system performs PCR amplification respectively, and the length of the paternal band type of the amplified product is 314bp, and the length of the maternal band type of the amplified product is 354bp; using the extracted sample genomic DNA as a template, the primer pair 4 described in claim 1 is used as the forward and reverse primers to establish a PCR amplification system for PCR amplification respectively, and the length of the paternal band type of the amplified product is 169bp, and the length of the maternal band type of the amplified product is 211bp; using the extracted sample genomic DNA as a template, the primer pair 5 described in claim 1 is used as the forward and reverse primers to establish a PCR amplification system for PCR amplification respectively, and the length of the paternal band type of the amplified product is 379bp, and the length of the maternal band type of the amplified product is 440bp.

[0025] The PCR amplification system described in step (2) is preferably: 10× buffer 1.0 μL, 25 mM MgCL2 1.0 μL, 2 mM dNTPs 1.0 μL, 10 μM forward primer and reverse primer 1.0 μL each, 0.5 units of Taq DNA polymerase 0.2 μL, 50 ng of sample template to be detected, and ddH2O added to 10 μL;

[0026] The PCR amplification procedure is preferably: 95°C for 3 min; 95°C for 15 s, 58°C for 15 s, 72°C for 20 s, 35 cycles; 72°C for 5 min.

[0027] The present invention further provides a method for identifying the purity of seeds of the cauliflower variety 'Jinpin 81', the method comprising:

[0028] (1) extracting genomic DNA from a cauliflower seed sample to be tested;

[0029] (2) Using the extracted genomic DNA of the seed samples as a template, PCR amplification systems were established using five pairs of InDel molecular marker primers as forward and reverse primers to perform PCR amplification respectively;

[0030] (3) If the band patterns of the five amplified products all have the band patterns of the parent strain of the cauliflower variety 'Jinpin 81', the cauliflower seeds to be tested are seeds of the cauliflower variety 'Jinpin 81'; if the band patterns of only one of the five amplified products do not all have the band patterns of the parent strain of the cauliflower variety 'Jinpin 81', the cauliflower seeds to be tested are not seeds of the cauliflower variety 'Jinpin 81';

[0031] (4) Calculate the ratio of the number of true 'Jinpin 81' cauliflower varieties to the total number of tested seeds to obtain the seed purity of the 'Jinpin 81' cauliflower variety sample to be tested.

[0032] A preferred specific embodiment of the present invention is to use the extracted sample genomic DNA as a template, and use the primer pair 1 described in claim 1 as a forward and reverse primer to establish a PCR amplification system for PCR amplification, and the length of the paternal band type of the amplified product is 247bp, and the length of the maternal band type of the amplified product is 399bp; use the extracted sample genomic DNA as a template, and use the primer pair 2 described in claim 1 as a forward and reverse primer to establish a PCR amplification system for PCR amplification, and the length of the paternal band type of the amplified product is 370bp, and the length of the maternal band type of the amplified product is 462bp; use the extracted sample genomic DNA as a template, and use the primer pair 3 ... P The CR amplification system performs PCR amplification respectively, and the length of the paternal band type of the amplified product is 314bp, and the length of the maternal band type of the amplified product is 354bp; using the extracted sample genomic DNA as a template, the primer pair 4 described in claim 1 is used as the forward and reverse primers to establish a PCR amplification system for PCR amplification respectively, and the length of the paternal band type of the amplified product is 169bp, and the length of the maternal band type of the amplified product is 211bp; using the extracted sample genomic DNA as a template, the primer pair 5 described in claim 1 is used as the forward and reverse primers to establish a PCR amplification system for PCR amplification respectively, and the length of the paternal band type of the amplified product is 379bp, and the length of the maternal band type of the amplified product is 440bp.

[0033] The PCR amplification system in step (2) is preferably: 10× buffer 1.0 μL, 25 mM MgCL2 1.0 μL, 2 mM dNTPs 1.0 μL, 10 μM forward primer and reverse primer 1.0 μL each, 0.5 unit Taq DNA polymerase 0.2 μL, template 50 ng, add ddH2O to 10 μL;

[0034] The PCR amplification procedure is preferably: 95°C for 3 min; 95°C for 15 s, 58°C for 15 s, 72°C for 20 s, 35 cycles; 72°C for 5 min.

[0035] Detailed description of the overall technical solution of the present invention

[0036] The present invention extracts the genomic DNA of the maternal and paternal parents of the cauliflower variety 'Jinpin 81', performs Illumina double-end sequencing on the genomic DNA of each sterile line and inbred line, removes the adapter sequence and data quality control from the obtained raw data, and compares the effective sequencing data with the cauliflower reference genome through BWA software, and removes duplications from the comparison results through SAMTOOLS. The GATK HaplotypeCaller module is used to detect and analyze the InDel variation in the sample; based on the above-mentioned Illumina high-throughput sequencing, the present invention further adopts differential InDel site screening and specific primer design: the InDel sites in the parental genome of the cauliflower variety 'Jinpin 81' are screened using a Perl language script, 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 are located on the genome, and the nucleotide sequences of 200bp upstream and downstream of the InDel marker site are extracted. Primers are designed in batches using Primer3.0 and self-programmed Perl language scripts. Using the designed and synthesized primers, PCR amplification was carried out on the cauliflower variety 'Jinpin 81' and its parents, and 5 pairs of InDel marker primers were screened; using the screened 5 pairs of InDel marker primers, the authenticity of the cauliflower variety 'Jinpin 81' or the purity of its seeds can be identified quickly, efficiently, stably and accurately, solving the problems of long identification cycle and high labor input in the existing cauliflower 'Jinpin 81' variety.

[0037] Main beneficial effects of the present invention

[0038] 1. The present invention utilizes the resequencing data of the parents of the cauliflower variety 'Jinpin 81' to screen differential InDel sites in the whole genome and design specific primers, with high accuracy and stable and reliable results.

[0039] 2. The present invention utilizes agarose gel electrophoresis for detection, which has the advantages of simple operation, low cost, high speed, wide application range, etc. compared with polyacrylamide gel electrophoresis.

[0040] 3. The present invention is based on PCR amplification reaction and utilizes InDel molecular markers at 5 loci to quickly and accurately complete the purity identification of 'Jinpin 81' cauliflower variety and hybrid seeds in a short time. The method is simple and fast, the detection cycle is short, and labor costs are saved, and it has a good application and promotion prospect.

[0041] 4. In addition to identifying the cauliflower variety 'Jinpin 81' according to the band pattern amplified by the primer set, the present invention can also identify different varieties including 'Zhe Nong Songhua 85', 'Zhe Nong Songhua 90', 'Shang Mei 65', 'Jinpin 70', 'Jinpin 69', 'Fugui 80', 'B11', 'BX110', 'BX120', 'CB-30', 'CY17', 'DX-108', 'FUZiYAMA', 'H2194', 'JN152', 'JN157', '1522', 'ID3', 'ID23', '21JN74', '2 It is possible to distinguish cauliflower varieties such as '1JN79', '21JN81', 'KB18', 'ID51', 'ID53', 'ID62', 'PK4', 'PK5', 'PK10', 'PK12', 'ID24', '20CP1', '20CP3', '20CP9', '21NY19', '19QK42', '19QK67', '19QK68', 'ID46', '16SY55', '16SY64', '16SY69' or '16SY96', which can effectively regulate variety registration, strengthen market supervision and enhance intellectual property protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 The figure shows the electrophoresis of the PCR amplification products of the parents of the cauliflower variety 'Jinpin 81' and 5 pairs of InDel-labeled primer pairs of 'Jinpin 81'; lanes 1, 2, and 3 in the figure are the amplification results of the male parent of 'Jinpin 81', the female parent of 'Jinpin 81', and the mixed samples of 5 individual plants of 'Jinpin 81' respectively.

[0043] Figure 2The cauliflower varieties are 'Jinpin 81', 'Zhe Nong Songhua 85', 'Zhe Nong Songhua 90', 'Shang Mei 65', 'Jinpin 70', 'Jinpin 69', 'Fugui 80', 'B11', 'BX110', 'BX120', 'CB-30', 'CY17', 'DX-108', 'FUZiYAMA', 'H2194', 'JN152', 'JN157', '1522', 'ID3', 'ID23', '21JN74', '21JN79', '21JN81', 'KB18', 'ID51', 'ID53', 'ID62', 'PK4', 'PK5', 'PK10', Electrophoresis of PCR amplification products of 5 pairs of InDel-labeled primer pairs: 'PK12', 'ID24', '20CP1', '20CP3', '20CP9', '21NY19', '19QK42', '19QK67', '19QK68', 'ID46', '16SY55', '16SY64', '16SY69' or '16SY96'; in the figure, M is Marker, and lanes 01 to 05 are the amplification products of 5 pairs of InDel-labeled primers: InD-JP81-1, InD-JP81-5, InD-JP81-6, InD-JP81-9, InD-JP81-13, respectively.

[0044] Figure 3 This is an electrophoresis diagram of the seed purity of cauliflower variety 'Jinpin 81' detected using primer InD-JP81-1; in the figure, M is a marker, lane 01 is the amplification product of the male parent of cauliflower variety 'Jinpin 81', lane 02 is the amplification product of the female parent of cauliflower variety 'Jinpin 81', and lanes 03-96 are the amplification products of 94 individual plants of the sample to be tested.

[0045] Figure 4 This is an electrophoresis diagram of the seed purity of the cauliflower variety 'Jinpin 81' detected using primer InD-JP81-5; in the figure, M is a marker, lane 01 is the amplification product of the male parent of the cauliflower variety 'Jinpin 81', lane 02 is the amplification product of the female parent of the cauliflower variety 'Jinpin 81', and lanes 03-96 are the amplification products of 94 individual plants of the sample to be tested.

[0046] Figure 5 This is an electrophoresis diagram of the seed purity of the cauliflower variety 'Jinpin 81' detected using primer InD-JP81-6; in the figure, M is a marker, lane 01 is the amplification product of the male parent of the cauliflower variety 'Jinpin 81', lane 02 is the amplification product of the female parent of the cauliflower variety 'Jinpin 81', and lanes 03-96 are the amplification products of 94 individual plants of the sample to be tested.

[0047] Figure 6This is an electrophoresis diagram of the seed purity of the cauliflower variety 'Jinpin 81' detected using primer InD-JP81-9; in the figure, M is a marker, lane 01 is the amplification product of the male parent of the cauliflower variety 'Jinpin 81', lane 02 is the amplification product of the female parent of the cauliflower variety 'Jinpin 81', and lanes 03-96 are the amplification products of 94 individual plants of the sample to be tested.

[0048] Figure 7 This is an electrophoresis diagram of the seed purity of cauliflower variety 'Jinpin 81' detected using primer InD-JP81-13; in the figure, M is a marker, lane 01 is the amplification product of the male parent of cauliflower variety 'Jinpin 81', lane 02 is the amplification product of the female parent of cauliflower variety 'Jinpin 81', and lanes 03-96 are the amplification products of 94 individual plants of the sample to be tested. DETAILED DESCRIPTION

[0049] 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 as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0050] The five pairs of InDel marker primer pairs screened in Experimental Example 1 for identifying the purity of the 'Jinpin 81' cauliflower variety or the 'Jinpin 81' cauliflower hybrid seeds and the five pairs of InDel marker primer pairs used to identify the authenticity of the 'Jinpin 81' cauliflower variety or the purity of the 'Jinpin 81' cauliflower hybrid seeds in Experimental Examples 2 and 3 are shown in Table 1.

[0051] Table 1 Five pairs of InDel marker primers used for identification of cauliflower 'Jinpin 81' variety

[0052]

[0053]

[0054] Experimental Example 1 Screening of InDel marker primer pairs for identifying the purity of 'Jinpin 81' cauliflower variety or 'Jinpin 81' cauliflower hybrid seeds

[0055] 1 Test method

[0056] 1.1Illumina high-throughput sequencing

[0057] Extract genomic DNA from the parents of the cauliflower variety 'Jinpin 81' (the seeds of the cauliflower variety 'Jinpin 81' are sold by Tianjin Kerun Vegetable Research Institute; in addition, Tianjin Kerun Vegetable Research Institute sells seeds of the male and female parents of the cauliflower variety 'Jinpin 81') and perform Illumina double-end sequencing on the genomic DNA. After removing the adapter sequence and data quality control from the obtained raw data, the effective sequencing data is compared with the cauliflower reference genome using BWA software, and the comparison results are deduplicated using SAMTOOLS. Use the GATK HaplotypeCaller module to detect and analyze InDel variations in the samples;

[0058] 1.2 Screening of differential InDel sites and design of specific primers

[0059] The InDel sites in the genome of the parents of the cauliflower variety 'Jinpin 81' were screened using a Perl language script. The InDel size was set to 20-500bp, and the sequencing depth was greater than 4. Based on the reference genome information, the screened sites were located on the genome, and the nucleotide sequences of 200bp upstream and downstream of the InDel marker site were extracted. Primers were designed in batches using Primer3.0 and a self-programmed Perl language script. PCR amplification was performed on the cauliflower variety 'Jinpin 81' and its parents using the designed and synthesized primers.

[0060] 2 Test results

[0061] Figure 1 The electrophoresis diagram of the PCR amplification products of the cauliflower variety 'Jinpin 81' parents and 5 pairs of InDel-labeled primer pairs; Figure 1 It can be seen that the five pairs of InDel marker primers screened can simultaneously amplify the characteristic bands of the parents in the cauliflower variety 'Jinpin 81', and can be used to identify the cauliflower variety 'Jinpin 81' and its seed purity ( Figure 1 ) Experimental Example 2: Authenticity test of cauliflower variety 'Jinpin 81' using the five selected InDel marker primer pairs

[0062] 1 Test method

[0063] 1.1 Extraction of leaf genomic DNA

[0064] The cauliflower hybrid 'Jinpin 81' and its parents, 'Zhe Nong Songhua 85', 'Zhe Nong Songhua 90', 'Shang Mei 65', 'Jinpin 70', 'Jinpin 69', 'Fugui 80', 'B11', 'BX110', 'BX120', 'CB-30', 'CY17', 'DX-108', 'FUZiYAMA', 'H2194', 'JN152', 'JN157', '1522', 'ID3', 'ID23', '21JN74', '21JN79', ' The DNA samples were mixed from 5 individual plants of cauliflower varieties including '21JN81', 'KB18', 'ID51', 'ID53', 'ID62', 'PK4', 'PK5', 'PK10', 'PK12', 'ID24', '20CP1', '20CP3', '20CP9', '21NY19', '19QK42', '19QK67', '19QK68', 'ID46', '16SY55', '16SY64', '16SY69' or '16SY96'.

[0065] 1.2 PCR amplification

[0066] Using the genomic DNA of each cauliflower variety extracted in 1.1 as a template, and using InDel-JP81-1, InDel-JP81-5, InDel-JP81-6, InDel-JP81-9 and InDel-JP81-13 as primers, PCR amplification was performed respectively;

[0067] The PCR amplification system was: 10× buffer 1.0 μL, 25 mM MgCL2 1.0 μL, 2 mM dNTPs 1.0 μL, 10 μM forward primer and reverse primer 1.0 μL each, 0.5 units of Taq DNA polymerase 0.2 μL, template 50 ng, add ddH2O to 10 μL;

[0068] The PCR amplification program was as follows: 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.

[0069] 1.3 Detection and analysis of PCR amplification products

[0070] Add 1 μL 10× Loding Buffer to the amplified product described in 1.2, and perform 2% agarose gel electrophoresis for 90 min at a constant voltage of 120 V / cM, then stain with Gelred and photograph using a gel imaging system. Identify the variety based on the electrophoresis band pattern. If the band patterns of the five InDel sites have the band patterns of the parents of the cauliflower variety 'Jinpin 81' at the same time, the cauliflower variety to be tested is determined to be the cauliflower variety 'Jinpin 81'.

[0071] 2 Identification results

[0072] The above method was used to test the hybrid cauliflower varieties 'Jinpin 81' and 'Zhe Nong Songhua 85', 'Zhe Nong Songhua 90', 'Shang Mei 65', 'Jinpin 70', 'Jinpin 69', 'Fugui 80', 'B11', 'BX110', 'BX120', 'CB-30', 'CY17', 'DX-108', 'FUZiYAMA', 'H2194', 'JN152', 'JN157', '1522', 'ID3', 'ID23', '21JN74', '21JN79', '21JN81', 'KB18', 'ID51', 'ID53', 'ID62', 'PK4', 'PK54', 'PK63', 'PK70', 'PK71', 'PK81', 'PK90', 'PK91', 'PK11', 'PK120', 'CB-30', 'CY17', 'DX-108', 'FUZiYAMA', 'H2194', 'JN152', 'JN157', '1522', 'ID3', 'ID23', '21JN74', '21JN79', '21JN81', 'KB18', 'ID51', 'ID53', 'ID62', 'PK4 'PK5', 'PK10', 'PK12', 'ID24', '20CP1', '20CP3', '20CP9', '21NY19', '19QK42', '19QK67', '19QK68', 'ID46', '16SY55', '16SY64', '16SY69' or '16SY96' for variety identification; 5 pairs of InDel marker primer pairs were verified to be polymorphic in cauliflower varieties, all with 3 band types. For intuitive representation, the amplification map of the same primer pair is generally classified according to the molecular weight of the minimum amplification product. When the molecular weight of the minimum product is the same, it is classified according to 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, and the type of heterozygous co-dominant band is defined as 3. Convert the corresponding map type into a unique code like an ID number, and use the code to distinguish the differences between varieties. Cauliflower hybrids Cauliflower varieties 'Jinpin 81' and 'Zhe Nong Songhua 85', 'Zhe Nong Songhua 90', 'Shang Mei 65', 'Jinpin 70', 'Jinpin 69', 'Fugui 80', 'B11',

[0073] 'BX110', 'BX120', 'CB-30', 'CY17', 'DX-108', 'FUZiYAMA',

[0074] 'H2194', 'JN152', 'JN157', '1522', 'ID3', 'ID23',

[0075] '21JN74', '21JN79', '21JN81', 'KB18', 'ID51', 'ID53',

[0076] 'ID62', 'PK4', 'PK5', 'PK10', 'PK12', 'ID24', '20CP1',

[0077] '20CP3', '20CP9', '21NY19', '19QK42', '19QK67', '19QK68',

[0078] Agarose gel electrophoresis of the five pairs of InDel-labeled primers amplified with 'ID46', '16SY55', '16SY64', '16SY69' or '16SY96' is shown in Figure 2 shown. Figure 2 In the figure, M is a marker, and lanes 1 to 5 are the amplification results of InDel-JP81-1, InDel-JP81-5, InDel-JP81-6, InDel-JP81-9 and InDel-JP81-13, respectively. The coding table is shown in Table 2.

[0079] Table 2 Fingerprint codes of cauliflower variety 'Jinpin 81' and 43 cauliflower hybrid varieties

[0080]

[0081]

[0082] according to Figure 2 As can be seen from the test results in Table 2, the banding patterns of the five InDel loci of the cauliflower hybrid 'Jinpin 81' also have the banding patterns of the parents of the cauliflower hybrid 'Jinpin 81'; 'Zhe Nong Songhua 85', 'Zhe Nong Songhua 90', 'Shang Mei 65', 'Jinpin 70', 'Jinpin 69', 'Fugui 80', 'B11', 'BX110', 'BX120', 'CB-30', 'CY17', 'DX-108', 'FUZiYAMA', 'H2194', 'JN152', 'JN157', '1522', 'ID3', 'ID23', '21JN74', '21JN79', '21JN81', 'KB18', 'I The banding patterns of the five InDel loci of 'D51', 'ID53', 'ID62', 'PK4', 'PK5', 'PK10', 'PK12', 'ID24', '20CP1', '20CP3', '20CP9', '21NY19', '19QK42', '19QK67', '19QK68', 'ID46', '16SY55', '16SY64', '16SY69' and '16SY96' do not have the banding patterns of the parents of the cauliflower variety 'Jinpin 81' at the same time; therefore, the five pairs of InDel marker pairs designed by the present invention can accurately identify the cauliflower variety 'Jinpin 81' from other cauliflower varieties. Experimental Example 3 Experimental identification of the purity of 'Jinpin 81' cauliflower hybrid seeds using the five pairs of InDel marker primer pairs selected

[0083] 1 Test method

[0084] 1.1 Extraction of leaf genomic DNA

[0085] 94 random hybrid sample seeds from a batch of 'Jinpin 81' cauliflower hybrid seeds.

[0086] The seeds of the parental plants of the cauliflower hybrid 'Jinpin 81' and the sample seeds were sown in a 72-hole tray at the same time, and watered regularly until two true leaves grew; 5 individual plants of each parental plant were mixed and sampled, and leaves of 94 hybrid sample seeds were sampled individually. Genomic DNA was extracted according to the conventional CTAB method and stored at -20℃ for later use.

[0087] 1.2 PCR amplification

[0088] The PCR amplification was the same as that in Experimental Example 2.

[0089] 1.3 Detection and analysis of PCR amplification products

[0090] The PCR amplification product detection and hybrid variety identification methods are the same as those in Experimental Example 2.

[0091] 2 Identification results

[0092] The results of agarose gel electrophoresis of seed purity identification of cauliflower hybrid 'Jinpin 81' using the above method are shown in part. Figure 3 , Figure 4 , Figure 5 , Figure 6 and Figure 7 In the figure, M is a marker, lane 01 is the male parent of the cauliflower hybrid 'Jinpin 81', lane 02 is the female parent of the cauliflower hybrid 'Jinpin 81', and lanes 03-96 are 94 individual plants of the sample to be tested.

[0093] according to Figure 3-Figure 7 The identification results show that there are 94 true hybrid seeds among the 94 sample seeds, so the purity of this batch of 'Jinpin 81' hybrid seeds is 100%, which meets the national standards and is consistent with the field investigation results; the identification test results prove that the 5 pairs of InDel marker primers screened by the present invention can accurately identify the purity of 'Jinpin 81' cauliflower hybrid seeds.

Claims

1. An InDel molecular marker primer for identifying the authenticity of cauliflower variety 'Jinpin 81' or identifying the seed purity of cauliflower variety 'Jinpin 81', characterized in that: The InDel molecular marker primers are selected from any one of the primer pairs or a primer set consisting of more than one primer pair described in (1) to (5) below: (1) Primer pair 1: Primer pair 1 consisting of a forward primer shown in SEQ ID NO. 1 and a reverse primer shown in SEQ ID NO. 2; (2) Primer pair 2: Primer pair 2 consisting of a forward primer shown in SEQ ID NO. 3 and a reverse primer shown in SEQ ID NO. 4; (3) Primer pair 3: Primer pair 3 consisting of the forward primer shown in SEQ ID NO. 5 and the reverse primer shown in SEQ ID NO. 6; (4) Primer pair 4: Primer pair 4 consisting of the forward primer shown in SEQ ID NO. 7 and the reverse primer shown in SEQ ID NO. 8; (5) Primer pair 5: Primer pair 5 consisting of the forward primer shown in SEQ ID NO. 9 and the reverse primer shown in SEQ ID NO.

10.

2. A PCR detection kit for identifying the authenticity of cauliflower variety 'Jinpin 81' or the purity of seeds of cauliflower variety 'Jinpin 81', comprising: dNTPs, Taq enzyme, MgCl2, a PCR primer pair consisting of a forward primer and a reverse primer, an amplification buffer and sterile water; characterized in that the PCR primer pair is selected from a primer set consisting of any one or more primer pairs among the five pairs of InDel labeled primer pairs described in claim 1.

3. Use of the InDel molecular marker primers according to claim 1 in identifying the authenticity of the cauliflower variety 'Jinpin 81' or the seed purity of the cauliflower variety 'Jinpin 81'.

4. Use of the PCR detection kit according to claim 2 in identifying the authenticity of the cauliflower variety 'Jinpin 81' or the seed purity of the cauliflower variety 'Jinpin 81'.

5. A method for authenticating the authenticity of cauliflower variety 'Jinpin 81', characterized in that: include: (1) extracting genomic DNA from a cauliflower plant sample to be tested; (2) using the extracted sample genomic DNA as a template and using the five pairs of InDel molecular marker primers described in claim 1 as forward and reverse primers to establish a PCR amplification system and perform PCR amplification respectively; (3) If the banding patterns of the five amplified products all have the banding patterns of the parent strain of the cauliflower variety 'Jinpin 81', the cauliflower variety to be tested is the cauliflower variety 'Jinpin 81'; if any of the five amplified products does not have the banding patterns of the parent strain of the cauliflower variety 'Jinpin 81', the cauliflower variety to be tested is not the cauliflower variety 'Jinpin 81'.

6. The identification method according to claim 5, characterized in that: Using the extracted sample genomic DNA as a template, the primer pair 1 described in claim 1 as the forward and reverse primers to establish a PCR amplification system for PCR amplification, and the length of the paternal band type of the amplified product is 247bp, and the length of the maternal band type of the amplified product is 399bp; using the extracted sample genomic DNA as a template, the primer pair 2 described in claim 1 as the forward and reverse primers to establish a PCR amplification system for PCR amplification, and the length of the paternal band type of the amplified product is 370bp, and the length of the maternal band type of the amplified product is 462bp; using the extracted sample genomic DNA as a template, the primer pair 3 described in claim 1 as the forward and reverse primers to establish a PCR amplification system for PCR amplification, and the length of the paternal band type of the amplified product is 370bp, and the length of the maternal band type of the amplified product is 462bp. PCR amplification was performed, and the length of the paternal band type of the amplified product was 314bp, and the length of the maternal band type of the amplified product was 354bp; using the extracted sample genomic DNA as a template, and using the primer pair 4 described in claim 1 as the forward and reverse primers to establish a PCR amplification system, and PCR amplification was performed respectively, and the length of the paternal band type of the amplified product was 169bp, and the length of the maternal band type of the amplified product was 211bp; using the extracted sample genomic DNA as a template, and using the primer pair 5 described in claim 1 as the forward and reverse primers to establish a PCR amplification system, and PCR amplification was performed respectively, and the length of the paternal band type of the amplified product was 379bp, and the length of the maternal band type of the amplified product was 440bp.

7. The identification method according to claim 5, characterized in that: The PCR amplification system described in step (2) is: 10× buffer 1.0 μL, 25 mM MgCL2 1.0 μL, 2 mM dNTPs 1.0 μL, 10 μM forward primer and reverse primer 1.0 μL each, 0.5 unit Taq DNA polymerase 0.2 μL, 50 ng of sample template to be detected, and ddH2O added to 10 μL; The PCR amplification procedure described in step (2) is: 95°C for 3 min; 95°C for 15 s, 58°C for 15 s, 72°C for 20 s, 35 cycles; 72°C for 5 min.

8. A method for identifying the purity of seeds of cauliflower variety 'Jinpin 81', characterized in that: include: (1) extracting genomic DNA from a cauliflower seed sample to be tested; (2) using the extracted genomic DNA of the seed sample as a template and using the five pairs of primers described in claim 1 as forward and reverse primers to establish a PCR amplification system and perform PCR amplification respectively; (3) If the band patterns of the five amplified products all have the band patterns of the parents of the cauliflower variety 'Jinpin 81', the cauliflower seeds to be tested are the seeds of the cauliflower hybrid 'Jinpin 81'; if the band patterns of only one of the five amplified products do not have the band patterns of the parents of the cauliflower variety 'Jinpin 81', the cauliflower seeds to be tested are not the cauliflower variety 'Jinpin 81'; (4) Calculate the ratio of the number of true 'Jinpin 81' cauliflower hybrid seeds to the total number of tested seeds to obtain the seed purity of the 'Jinpin 81' cauliflower variety sample to be tested.

9. The identification method according to claim 8, characterized in that: Using the extracted sample genomic DNA as a template, the primer pair 1 described in claim 1 as the forward and reverse primers to establish a PCR amplification system for PCR amplification, and the length of the paternal band type of the amplified product is 247bp, and the length of the maternal band type of the amplified product is 399bp; using the extracted sample genomic DNA as a template, the primer pair 2 described in claim 1 as the forward and reverse primers to establish a PCR amplification system for PCR amplification, and the length of the paternal band type of the amplified product is 370bp, and the length of the maternal band type of the amplified product is 462bp; using the extracted sample genomic DNA as a template, the primer pair 3 described in claim 1 as the forward and reverse primers to establish a PCR amplification system for PCR amplification, and the length of the paternal band type of the amplified product is 370bp, and the length of the maternal band type of the amplified product is 462bp. PCR amplification was performed, and the length of the paternal band type of the amplified product was 314bp, and the length of the maternal band type of the amplified product was 354bp; using the extracted sample genomic DNA as a template, and using the primer pair 4 described in claim 1 as the forward and reverse primers to establish a PCR amplification system, and PCR amplification was performed respectively, and the length of the paternal band type of the amplified product was 169bp, and the length of the maternal band type of the amplified product was 211bp; using the extracted sample genomic DNA as a template, and using the primer pair 5 described in claim 1 as the forward and reverse primers to establish a PCR amplification system, and PCR amplification was performed respectively, and the length of the paternal band type of the amplified product was 379bp, and the length of the maternal band type of the amplified product was 440bp.

10. The identification method according to claim 8, characterized in that: The PCR amplification system described in step (2) is: 10× buffer 1.0 μL, 25 mM MgCL2 1.0 μL, 2 mM dNTPs 1.0 μL, 10 μM forward primer and reverse primer 1.0 μL each, 0.5 unit Taq DNA polymerase 0.2 μL, template 50 ng, add ddH2O to 10 μL; The PCR amplification procedure is: 95°C for 3 min; 95°C for 15 s, 58°C for 15 s, 72°C for 20 s, 35 cycles; 72°C for 5 min.

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