InDel marker primer for identifying authenticity or seed purity of zingun 81 and identification method thereof

Abstract

The application discloses InDel marker primers for identifying authenticity or seed purity of a cauliflower variety Jinpin 81 and a method for identifying the same. The application screens and designs specific InDel sites and selects five pairs of InDel marker primers from the specific InDel sites by analyzing parent resequencing data of the cauliflower variety 'Jinpin 81' in a whole genome range, and the nucleotide sequences of the five pairs of InDel marker primers are respectively shown in SEQ ID NO. 1-SEQ ID NO. 10. The identification method established by applying the five pairs of marker primers can quickly and accurately complete identification of authenticity of the cauliflower variety 'Jinpin 81' and purity of hybrid seeds thereof, solves problems of long cycle, high cost and low accuracy existing in traditional identification methods, has advantages of high identification accuracy, simple and fast operation, small sample requirement and the like, greatly improves identification efficiency and accuracy, and effectively guarantees seed production quality of the cauliflower variety 'Jinpin 81'.

Description

TECHNICAL FIELD

[0001] The present application relates to a molecular marker for identifying the authenticity of a cauliflower variety or the purity of a seed, in particular to an InDel marker primer for identifying the authenticity of a cauliflower variety 'Jinpin 81' or the purity of a seed and an identification method thereof, and belongs to the field of molecular markers for identifying the authenticity of a cauliflower variety or the purity of a seed. BACKGROUND

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

[0003] In recent years, the number of cauliflower varieties has been increasing, but some parents are frequently used, resulting in reduced genetic differences between varieties and increased identification difficulty. Therefore, it is particularly important to develop a rapid identification method based on the genome. Currently, the traditional identification method for cauliflower varieties relies on morphological markers, which is 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, and is not limited by growth stage, tissue type or environmental conditions, with high polymorphism and genetic stability. Insertion-deletion polymorphism (Insertion-Deletion Length Polymorphism, InDel) is one of the third-generation molecular marker types, which is generated by the insertion or deletion of nucleotides in alleles, and is developed based on whole-genome sequencing technology. InDel markers have higher density than SSR (Simple Sequence Repeat) markers, and have the characteristics of strong specificity, good stability, simple and economical detection method. InDel markers have been successfully applied in the identification of crop varieties such as rice, soybean and cotton, avoiding the uncertainty of identification based on morphological characteristics.

[0005] 'Jinpin 81' is a mid-late maturing cauliflower variety (Cauliflower variety 'Jinpin 81' is sold by Tianjin Kelun Vegetable Research Institute), which has good growth vigor and disease resistance, semi-erect plant type, white and compact flower balls, single ball weight about 1.9 kg, and needs to be harvested in time after the flower ball matures. However, so far there is still a lack of specific molecular markers and identification methods suitable for identifying the authenticity of cauliflower variety 'Jinpin 81' or the purity of its seeds. SUMMARY

[0006] One of the purposes of the present application is to provide an InDel molecular marker primer pair group for identifying the authenticity of the cauliflower variety 'Jinpin 81' or the seed purity thereof;

[0007] The second purpose of the present application is to provide a PCR detection kit for identifying the authenticity of the cauliflower variety 'Jinpin 81' or the seed purity of the cauliflower hybrid 'Jinpin 81'.

[0008] The third purpose of the present application is to apply the InDel marker primer pair group or the PCR detection kit containing the InDel marker primer pair group to identify the authenticity of the cauliflower variety 'Jinpin 81' or the seed purity thereof.

[0009] In order to achieve the above-mentioned purposes, the main technical solutions adopted by the present application include:

[0010] The present application provides an InDel molecular marker primer for identifying the authenticity of the cauliflower variety 'Jinpin 81' or the seed purity of the cauliflower variety 'Jinpin 81', which is selected from any one of the following (1)-(5) or a primer group consisting of more than one pair of primers:

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

[0012] (2) Primer pair 2: primer pair 2 consisting of the forward primer shown in SEQ ID NO. 3 and the 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 the forward primer shown in SEQ ID NO. 7 and the 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 application screens and designs specific InDel sites by analyzing the parent resequencing data of the cauliflower variety 'Jinpin 81' in the whole genome range, and selects five pairs of nucleotide sequence-specific InDel marker primer pairs. The application of the five pairs of InDel marker primer pairs can quickly and accurately identify the authenticity of the cauliflower variety 'Jinpin 81' or the purity of hybrid seeds.

[0017] Another aspect of the present application is to provide a PCR detection kit for identifying the authenticity of the broccoli variety 'Jinpin 81' or identifying the seed purity of the broccoli variety 'Jinpin 81', which comprises: dNTPs, Taq enzyme, MgCl2, 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 selected from any one of the following 5 pairs of InDel marker primer pairs or a primer group consisting of one or more 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] Still another aspect of the present application is to apply the InDel molecular marker primer or the PCR detection kit containing the InDel molecular marker primer to identify the authenticity of the broccoli variety 'Jinpin 81' or its seed purity, which comprises: using the extracted DNA of 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 identification purpose by detecting the polymorphism of the InDel site between different individuals.

[0020] Specifically, the present application provides a method for identifying the authenticity of the broccoli variety 'Jinpin 81', which comprises:

[0021] (1) extracting the genomic DNA of the broccoli plant sample to be detected;

[0022] (2) using the extracted sample genomic DNA as a template, using the 5 pairs of InDel molecular marker primer pairs as forward and reverse primers to establish a PCR amplification system and perform PCR amplification respectively;

[0023] (3) if the band types of the five amplified products simultaneously have the band types of the parent of the cauliflower variety ‘Jinpin 81’, the cauliflower variety to be detected is the cauliflower variety ‘Jinpin 81’; if the band types of only one of the five amplified products do not simultaneously have the band types of the parent of the cauliflower variety ‘Jinpin 81’, the cauliflower variety to be detected is not the cauliflower variety ‘Jinpin 81’.

[0024] In a preferred embodiment of the present application, the PCR amplification system is established by using the extracted sample genomic DNA as a template and using the primer pair 1 as the forward and reverse primers to perform PCR amplification, the length of the father band type of the amplified product is 247 bp, and the length of the mother band type of the amplified product is 399 bp; the PCR amplification system is established by using the extracted sample genomic DNA as a template and using the primer pair 2 as the forward and reverse primers to perform PCR amplification, the length of the father band type of the amplified product is 370 bp, and the length of the mother band type of the amplified product is 462 bp; the PCR amplification system is established by using the extracted sample genomic DNA as a template and using the primer pair 3 as the forward and reverse primers to perform PCR amplification, the length of the father band type of the amplified product is 314 bp, and the length of the mother band type of the amplified product is 354 bp; the PCR amplification system is established by using the extracted sample genomic DNA as a template and using the primer pair 4 as the forward and reverse primers to perform PCR amplification, the length of the father band type of the amplified product is 169 bp, and the length of the mother band type of the amplified product is 211 bp; and the PCR amplification system is established by using the extracted sample genomic DNA as a template and using the primer pair 5 as the forward and reverse primers to perform PCR amplification, the length of the father band type of the amplified product is 379 bp, and the length of the mother band type of the amplified product is 440 bp.

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

[0026] The PCR amplification program is preferably as follows: 95 ℃ for 3 min; 95 ℃ for 15 s, 58 ℃ for 15 s, 72 ℃ for 20 s, for 35 cycles; and 72 ℃ for 5 min.

[0027] The present application further provides a method for identifying the purity of the seeds of the cauliflower variety ‘Jinpin 81’, which comprises the following steps:

[0028] (1) extracting the genomic DNA of the cauliflower seed sample to be detected;

[0029] (2) using the extracted seed sample genomic DNA as a template, using 5 pairs of InDel molecular marker primer pairs as forward and reverse primers to establish a PCR amplification system respectively to perform PCR amplification;

[0030] (3) if the band types of the 5 kinds of amplification products simultaneously have the band types of the cauliflower variety 'Jinpin 81' parent, then the tested cauliflower seed is the cauliflower variety 'Jinpin 81' seed; if only one of the 5 kinds of amplification products has a band type different from the band type of the cauliflower variety 'Jinpin 81' parent, then the tested cauliflower seed is not the cauliflower variety 'Jinpin 81';

[0031] (4) calculating the ratio of the number of real 'Jinpin 81' cauliflower variety to the total number of detected seeds to obtain the seed purity of the tested 'Jinpin 81' cauliflower variety sample.

[0032] In a preferred embodiment of the present application, the extracted sample genomic DNA is used as a template, primer pair 1 is used as forward and reverse primers to establish a PCR amplification system to perform PCR amplification, the length of the father band type of the amplified product is 247 bp, and the length of the mother band type of the amplified product is 399 bp; the extracted sample genomic DNA is used as a template, primer pair 2 is used as forward and reverse primers to establish a PCR amplification system to perform PCR amplification, the length of the father band type of the amplified product is 370 bp, and the length of the mother band type of the amplified product is 462 bp; the extracted sample genomic DNA is used as a template, primer pair 3 is used as forward and reverse primers to establish a PCR amplification system to perform PCR amplification, the length of the father band type of the amplified product is 314 bp, and the length of the mother band type of the amplified product is 354 bp; the extracted sample genomic DNA is used as a template, primer pair 4 is used as forward and reverse primers to establish a PCR amplification system to perform PCR amplification, the length of the father band type of the amplified product is 169 bp, and the length of the mother band type of the amplified product is 211 bp; the extracted sample genomic DNA is used as a template, primer pair 5 is used as forward and reverse primers to establish a PCR amplification system to perform PCR amplification, the length of the father band type of the amplified product is 379 bp, and the length of the mother band type of the amplified product is 440 bp.

[0033] In step (2), the PCR amplification system is preferably: 10×buffer 1.0 μL, 25 mM MgCL2 1.0 μL, 2 mM dNTPs 1.0 μL, 10 μM forward and reverse primers each 1.0 μL, 0.5 units of Taq DNA polymerase 0.2 μL, 50 ng of template, and ddH2O to 10 μL;

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

[0035] Overall technical solution of the present application is described in detail

[0036] The genomic DNA of the female parent and the male parent of the broccoli variety 'Jinpin 81' is extracted, the genomic DNA of each sterile line and selfing line is subjected to Illumina double-end sequencing, after the raw data is removed from the adapter sequence and data quality control, the effective sequencing data is subjected to alignment with the broccoli reference genome by using BWA software, and the alignment result is removed from the repetition by using SAMTOOLS. The InDel variation in the sample is detected and analyzed by using the GATK HaplotypeCaller module; on the basis of the above-mentioned Illumina high-throughput sequencing, the difference InDel site screening and specific primer design are further adopted: the InDel sites in the genomic DNA of the male parent and the female parent of the broccoli variety 'Jinpin 81' are screened by using a Perl language script, 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 are located on the genome, and the nucleotide sequences of the upstream and downstream 200 bp of the InDel marker sites are extracted. Primers are designed in batches by using Primer3.0 and a self-programming Perl language script. The designed and synthesized primers are used for PCR amplification of the broccoli 'Jinpin 81' variety and its male parent and female parent, and 5 pairs of InDel marker primers are screened; the 5 pairs of InDel marker primers screened can be used for quickly, efficiently, stably and accurately identifying the authenticity of the broccoli variety 'Jinpin 81' or the purity of its seeds, and the problems of long identification period and large labor input of the existing broccoli 'Jinpin 81' variety identification are solved.

[0037] Main beneficial effects of the present application

[0038] 1. The parent and the male parent of the broccoli variety 'Jinpin 81' are resequenced, the difference InDel sites are screened in the whole genome, and specific primers are designed, so that the accuracy is high, and the result is stable and reliable.

[0039] 2. The agarose gel electrophoresis is used for detection, compared with polyacrylamide gel electrophoresis, the agarose gel electrophoresis has the advantages of simple operation, low cost, fast speed and wide application range.

[0040] 3. Based on the PCR amplification reaction, the InDel molecular markers at the 5 sites are used, the purity identification of the 'Jinpin 81' broccoli variety and hybrid seeds can be quickly and accurately completed in a short time, the method is simple and fast, the detection period is short, the labor cost is saved, and the method has a good application and promotion prospect.

[0041] 4. The application can identify the cauliflower variety 'Jinpin 81' according to the band type amplified by the primer set, and can also distinguish different varieties including 'Zhenong Songhua 85', 'Zhenong Songhua 90', 'Shangmei 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', 'PK12', 'ID24', '20CP1', '20CP3', '20CP9', '21NY19', '19QK42', '19QK67', '19QK68', 'ID46', '16SY55', '16SY64', '16SY69', or '16SY96' according to the different band types of the primer set, so as to effectively regulate the variety registration, strengthen the market supervision, and enhance the protection of intellectual property rights. BRIEF DESCRIPTION OF DRAWINGS

[0042] Figure 1 The electrophoresis map of the PCR amplification products of the 5 pairs of InDel marker primer pairs of the parents of the cauliflower variety 'Jinpin 81' and 'Jinpin 81'; lanes 1, 2, and 3 in the figure are the amplification results of the parents of 'Jinpin 81', the female parent of 'Jinpin 81', and the mixed sample of the 5 single plants of 'Jinpin 81', respectively.

[0043] Figure 2Figure 1 is an electrophoretogram of PCR amplification products of 5 pairs of InDel marker primer pairs for detecting the purity of seeds of the cauliflower variety 'Jinpin 81' and the varieties Zhenongsonghua 85, 'Zhenongsonghua 90', 'Shangmei 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', 'PK12', 'ID24', '20CP1', '20CP3', '20CP9', '21NY19', '19QK42', '19QK67', '19QK68', 'ID46', '16SY55', '16SY64', '16SY69', or '16SY96'; in the figure, M is a Marker, and lanes 01-05 are the amplification products of the 5 pairs of InDel marker primers InD-JP81-1, InD-JP81-5, InD-JP81-6, InD-JP81-9, and InD-JP81-13, respectively.

[0044] Figure 3 Figure 2 is an electrophoretogram for detecting the purity of seeds of the cauliflower variety 'Jinpin 81' using the primer InD-JP81-1; 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 single plants of the sample to be tested.

[0045] Figure 4 Figure 3 is an electrophoretogram for detecting the purity of seeds of the cauliflower variety 'Jinpin 81' using the 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 single plants of the sample to be tested.

[0046] Figure 5 Figure 4 is an electrophoretogram for detecting the purity of seeds of the cauliflower variety 'Jinpin 81' using the 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 single plants of the sample to be tested.

[0047] Figure 6An electropherogram for detecting the seed purity of the cauliflower variety 'Jinpin 81'using primer InD-JP81-9; M is 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 single plants of the sample to be tested.

[0048] Figure 7 An electropherogram for detecting the seed purity of the cauliflower variety 'Jinpin 81'using primer InD-JP81-13; M is 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 single plants of the sample to be tested. DETAILED DESCRIPTION

[0049] The present application will be further described with reference to the following specific examples. The advantages and features of the present application will become apparent with the description. However, these examples are only exemplary and do not constitute any limitation on the scope of the present application. Those skilled in the art should understand that modifications or substitutions can be made to the details and forms of the present application without departing from the spirit and scope of the present application, and such modifications and substitutions fall within the scope of the present application.

[0050] The 5 pairs of InDel marker primer pairs screened in Test Example 1 below for identifying the 'Jinpin 81'cauliflower variety or the seed purity of the 'Jinpin 81'cauliflower hybrid and the 5 pairs of InDel marker primer pairs for identifying the authenticity of the 'Jinpin 81'cauliflower variety or the seed purity of the 'Jinpin 81'cauliflower hybrid in Test Example 2 and Test Example 3 are shown in Table 1.

[0051] Table 1 5 pairs of InDel marker primer pairs for identifying the cauliflower 'Jinpin 81'variety

[0052]

[0053]

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

[0055] 1 Test method

[0056] 1.1 Illumina high-throughput sequencing

[0057] Genomic DNA of the parents of the broccoli variety 'Jinpin 81' (the seeds of the broccoli variety 'Jinpin 81' are sold by Tianjin Keyun Vegetable Research Institute to the outside world; in addition, Tianjin Keyun Vegetable Research Institute sells the seeds of the parents of the broccoli variety 'Jinpin 81' to the outside world) was extracted, and Illumina double-end sequencing was performed on the genomic DNA. After removing the adapter sequences and data quality control from the obtained raw data, the effective sequencing data was aligned with the broccoli reference genome by using the BWA software, and the alignment results were removed by SAMTOOLS. The InDel variation in the sample was detected and analyzed by using the GATK HaplotypeCaller module;

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

[0059] Perl language scripts were used to screen the InDel sites in the genomes of the parents of the broccoli variety 'Jinpin 81', and the InDel size was set to 20-500 bp, 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 200 bp upstream and downstream of the InDel marker sites were extracted. Primers were designed in batches by using Primer3.0 and self-programming Perl language scripts. The designed and synthesized primers were used for PCR amplification of the broccoli 'Jinpin 81' variety and its parents.

[0060] 2 Test results

[0061] Figure 1 PCR amplification products of 5 pairs of InDel marker primer pairs of the parents of the broccoli variety 'Jinpin 81' and the broccoli variety 'Jinpin 81' were electrophoresed; according to Figure 1 It can be seen that the 5 pairs of InDel marker primer pairs screened can simultaneously amplify the characteristic bands of the parents in the broccoli variety 'Jinpin 81', and can be used to identify the broccoli variety 'Jinpin 81' and the purity of its seeds Figure 1 ). Test example 2 Application of the 5 pairs of InDel marker primer pairs screened to identify the authenticity of the broccoli variety 'Jinpin 81'

[0062] 1 Test method

[0063] 1.1 Extraction of leaf genomic DNA

[0064] Each 5 single plants of broccoli hybrid 'Jinpin 81' and its parents, 'Zhenong Songhua 85', 'Zhenong Songhua 90', 'Shangmei 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', 'PK12', 'ID24', '20CP1', '20CP3', '20CP9', '21NY19', '19QK42', '19QK67', '19QK68', 'ID46', '16SY55', '16SY64', '16SY69' or '16SY96' were mixed to extract DNA.

[0065] 1.2 PCR amplification

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

[0067] The PCR amplification system was as follows: 10x 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, and ddH2O to 10 μL;

[0068] The PCR amplification program was as follows: 95℃ 3 min, (95℃ 15 s, 58℃ 15 s, 72℃ 20 s) for 35 cycles, and 72℃ 5 min.

[0069] 1.3 Detection and analysis of PCR amplification products

[0070] 1 μL of 10x Loding Buffer was added to the amplified products described in 1.2, and electrophoresis was performed on a 2% agarose gel at a constant voltage of 120 V / cm for 90 min, followed by Gelred staining and imaging with a gel imaging system. According to the electrophoretic band pattern, if the band patterns of the 5 InDel sites simultaneously have those of the parents of broccoli variety 'Jinpin 81', the tested broccoli variety is determined to be broccoli variety 'Jinpin 81'.

[0071] 2Identification results

[0072] The above method was used to identify the hybrid broccoli cultivars 'Jinpin 81', 'Zhenongsonghua 85', 'Zhenongsonghua 90', 'Shangmei 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', 'PK12', 'ID24', '20CP1', '20CP3', '20CP9', '21NY19', '19QK42', '19QK67', '19QK68', 'ID46', '16SY55', '16SY64', '16SY69', or '16SY96'; 5 InDel marker primers were verified to be polymorphic in the broccoli cultivars, all with 3 bands, for intuitive representation, the same primer pair amplification map was generally classified by the smallest amplification product molecular weight, when the smallest product molecular weight was the same, the second smallest molecular weight was classified, and so on. The type of the low molecular weight band was defined as 1, the type of the high molecular weight band was defined as 2, and the type of the hybrid co-band was defined as 3. The corresponding map type was converted into a unique code like an identity card number, and the code was used to distinguish the differences between the cultivars. The hybrid broccoli cultivars 'Jinpin 81', 'Zhenongsonghua 85', 'Zhenongsonghua 90', 'Shangmei 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', 'PK12', 'ID24', '20CP1', '20CP3', '20CP9', '21NY19', '19QK42', '19QK67', '19QK68', 'ID46', '16SY55', '16SY64', '16SY69', or '16SY96' were identified as follows:

[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] The agarose gel electrophoresis image of the amplification using five pairs of InDel-labeled primers ('ID46', '16SY55', '16SY64', '16SY69', or '16SY96') is shown below. Figure 2 As shown. Figure 2 In the middle, M is the 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. Their 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 shown in Table 2, the banding patterns at five InDel loci of the cauliflower hybrid 'Jinpin 81' simultaneously exhibit the banding patterns of both parents of the cauliflower hybrid 'Jinpin 81'. The following varieties also show similar patterns: 'Zhenong Songhua 85', 'Zhenong Songhua 90', 'Shangmei 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 five InDel loci (D51', 'ID53', 'ID62', 'PK4', 'PK5', 'PK10', 'PK12', 'ID24', '20CP1', '20CP3', '20CP9', '21NY19', '19QK42', '19QK67', '19QK68', 'ID46', '16SY55', '16SY64', '16SY69', and '16SY96') do not simultaneously possess the banding patterns of the parent cauliflower variety 'Jinpin 81'. Therefore, the five InDel marker pairs designed in this invention can accurately identify the cauliflower variety 'Jinpin 81' from other cauliflower varieties. Example 3: Experiment using the selected five InDel marker primer pairs to identify the purity of 'Jinpin 81' cauliflower hybrid seeds.

[0083] 1 Test method

[0084] 1.1 Leaf genomic DNA extraction

[0085] A batch of 94 hybrid seed samples of 'Jinpin 81' hybrid cauliflower seeds were randomly selected.

[0086] The seeds of the parents of the hybrid cauliflower '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 single plants of each parent were mixed and sampled, and the 94 hybrid sample seeds were sampled individually. Genomic DNA was extracted according to the conventional CTAB method, and stored at -20°C for later use.

[0087] 1.2 PCR amplification

[0088] The PCR amplification was the same as that of Test Example 2.

[0089] 1.3 Detection and analysis of PCR amplification products

[0090] The detection of PCR amplification products and the identification method of hybrid varieties were the same as those of Test Example 2.

[0091] 2 Identification results

[0092] The agarose gel electrophoresis part of the results of the seed purity identification of the hybrid cauliflower 'Jinpin 81' using the above method is shown in Figure 3 、 Figure 4 、 Figure 5 、 Figure 6 and Figure 7 . In the figure, M is Marker, lane 01 is the father of the hybrid cauliflower 'Jinpin 81', lane 02 is the mother of the hybrid cauliflower 'Jinpin 81', and lanes 03-96 are the 94 single plants of the samples to be tested.

[0093] According to the identification results of Figures 3-7 , it can be seen that there are 94 true hybrid seeds in the 94 sample seeds, so the purity of the batch of 'Jinpin 81' hybrid seeds is 100%, which meets the national standard and is consistent with the field investigation results; the identification test results prove that the 5 pairs of InDel marker primers selected by the present application can accurately identify the purity of the hybrid seed of 'Jinpin 81' hybrid cauliflower.

Claims

1. An InDel molecular marker primer for identifying the authenticity of cauliflower variety 'Jinpin 81' or the purity of cauliflower variety 'Jinpin 81' seeds, characterized in that, The InDel molecular marker primers consist of a primer set composed of the primer pairs described in (1)-(5) below: (1) Primer pair 1: Primer pair 1 consists of the forward primer shown in SEQ ID NO.1 and the reverse primer shown in SEQ ID NO.2; (2) Primer pair 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; (3) Primer pair 3: Primer pair 3 consists 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 consists 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 consists 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 cauliflower variety 'Jinpin 81' seeds, comprising: The PCR primers consist of dNTPs, Taq polymerase, MgCl2, a PCR primer set consisting of forward and reverse primers, amplification buffer, and sterile water; characterized in that the PCR primer set consists of the five pairs of InDel-labeled primers as described in claim 1.

3. The application of the InDel molecular marker primers as described in claim 1 in identifying the authenticity of the cauliflower variety 'Jinpin 81' or the seed purity of the cauliflower variety 'Jinpin 81'.

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

5. A method for identifying the authenticity of the cauliflower variety 'Jinpin 81', characterized in that, include: (1) Extract genomic DNA from the cauliflower plant samples to be tested; (2) Using the extracted genomic DNA of the sample as a template, PCR amplification systems were established using the 5 pairs of InDel molecular marker primers described in claim 1 as forward and reverse primers, respectively, and PCR amplification was performed. (3) If all five amplification products have the same band pattern as the parent cauliflower variety 'Jinpin 81', then the cauliflower variety to be tested is cauliflower variety 'Jinpin 81'; if any one of the five amplification products does not have the same band pattern as the parent cauliflower variety 'Jinpin 81', then the cauliflower variety to be tested is not cauliflower variety 'Jinpin 81'.

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

7. The identification method according to claim 5, characterized in that, The PCR amplification system described in step (2) is as follows: 10×buffer 1.0μL, 25 mM MgCl2 1.0μL, 2 mM dNTPs 1.0μL, 10μM forward primer and reverse primer 1.0μL each, 0.5 units of Taq DNA polymerase 0.2μL, 50ng of sample template to be tested, and ddH2O added to 10μL; The PCR amplification program described in step (2) is as follows: 95℃ for 3 min; 95℃ for 15 s, 58℃ for 15 s, 72℃ for 20 s, for 35 cycles; 72℃ for 5 min.

8. A method for identifying the seed purity of a cauliflower variety 'Jinpin 81', characterized in that, include: (1) Extract genomic DNA from the cauliflower seed samples to be tested; (2) Using the extracted seed sample genomic DNA as a template, PCR amplification systems were established using the 5 primer pairs described in claim 1 as forward and reverse primers, respectively, and PCR amplification was performed. (3) If the band patterns of all five amplification products are simultaneously the band patterns of the parents of the cauliflower variety 'Jinpin 81', then the cauliflower seed to be tested is the seed of the cauliflower hybrid 'Jinpin 81'; if the band pattern of any one of the five amplification products is not simultaneously the band pattern of the parents of the cauliflower variety 'Jinpin 81', then the cauliflower seed to be tested is not the cauliflower variety 'Jinpin 81'. (4) Calculate the ratio of the number of real 'Jinpin 81' cauliflower hybrids to the total number of seeds tested, and 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 genomic DNA as a template, PCR amplification was performed using primer pair 1 as the forward and reverse primers of claim 1. The paternal band length of the amplified product was 247 bp, and the maternal band length was 399 bp. Using the extracted genomic DNA as a template, PCR amplification was performed using primer pair 2 as the forward and reverse primers of claim 1. The paternal band length of the amplified product was 370 bp, and the maternal band length was 462 bp. Using the extracted genomic DNA as a template, PCR amplification was performed using primer pair 3 as the forward and reverse primers of claim 1. PCR amplification was performed, and the length of the paternal band of the amplified product was 314 bp, while the length of the maternal band of the amplified product was 354 bp. Using the extracted sample genomic DNA as a template, PCR amplification was performed using primer pair 4 as the forward and reverse primers as described in claim 1. The length of the paternal band of the amplified product was 169 bp, while the length of the maternal band of the amplified product was 211 bp. Using the extracted sample genomic DNA as a template, PCR amplification was performed using primer pair 5 as the forward and reverse primers as described in claim 1. The length of the paternal band of the amplified product was 379 bp, while the length of the maternal band of the amplified product was 440 bp.

10. The identification method according to claim 8, characterized in that, The PCR amplification system described in step (2) is as follows: 10×buffer 1.0μL, 25 mM MgCl2 1.0μL, 2 mM dNTPs 1.0μL, 10μM forward primer and reverse primer 1.0μL each, 0.5 units of Taq DNA polymerase 0.2μL, template 50ng, and ddH2O added to 10μL; The PCR amplification program is as follows: 95℃ for 3 min; 95℃ for 15 s, 58℃ for 15 s, 72℃ for 20 s, for 35 cycles; 72℃ for 5 min.

Images