A method for identifying the purity of cucumber hybrid and InDel primer combination used thereby

Abstract

The application discloses a method for identifying purity of cucumber hybrid and an InDel primer combination used in the method. The InDel primer combination provided by the application is composed of four primer groups; each primer group is composed of two primer sequences, and is used for amplifying an InDel site; and the nucleotide sequences of the primers are shown in SEQ ID NO:1 to SEQ ID NO:8 in sequence. The InDel primer combination can be used for early identification in the seed or seedling stage of the cucumber hybrid, so as to guarantee the purity of the hybrid, protect the rights and interests of producers and breeders, and provide technical support for seed quality management of the cucumber variety. The method provided by the application has the advantages of accuracy, low cost, simple operation, clear results, saving of manpower and material resources, and has a very wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and specifically relates to a method for identifying the purity of cucumber hybrid seeds and an InDel primer combination used therein. Hybrid seeds refer to hybrid first-generation seeds, which may be mixed with parent seeds. BACKGROUND

[0002] Cucumber, as an indispensable member of China's vegetable industry, is of paramount importance. It not only wins the favor of consumers with its unique taste and rich nutritional value, but also plays a crucial role in ensuring the stable supply of China's vegetable market due to its characteristics of being cultivated throughout the year and supplying all year round. According to statistics, the cultivation area of cucumber has reached 1.396 million hectares, accounting for 6.2% of the total area of vegetable cultivation in China. This figure fully demonstrates the important position of cucumber in China's agricultural economy. However, with the continuous progress of cucumber breeding technology and the continuous emergence of new varieties, we are also facing unprecedented challenges. Due to the relatively small scale and scattered distribution of cucumber breeding enterprises in China, seed quality management has become a problem that needs to be solved. Counterfeit and substandard cucumber seeds with unqualified purity are common on the market, which not only damages the interests of farmers, but also seriously affects the healthy development of the cucumber industry.

[0003] In order to regulate the cucumber seed market and ensure seed quality, China officially implemented the "Non-main crop variety registration method" in April 2017. The introduction of this policy has greatly promoted the standardized management of cucumber varieties. As of now, the number of cucumber varieties registered nationwide has reached 1607, of which more than 93% are hybrid first-generation seeds (referred to as hybrid seeds). Hybrid seeds, with their excellent genetic characteristics and production performance, have dominated the cucumber production. However, hybrid seeds must undergo strict purity identification before entering the market. According to the current crop seed inspection regulations (GB / T 3543.1-3543.7-1995), the purity of cucumber hybrid seeds needs to reach more than 98%, which is of great significance to ensure seed quality and maintain market order. However, traditional field purity identification methods have the disadvantages of high cost and long time-consuming, which cannot meet the needs of modern seed industry development. Under this background, DNA-based molecular detection technology has emerged as the times require. With its low cost and time-saving advantages, it has quickly become a new favorite for identifying the purity of cucumber hybrid seeds. By screening, we can obtain a molecular marker combination based on DNA detection technology for identifying the purity of cucumber hybrid seeds, which can more accurately and quickly complete the purity identification, effectively curb the circulation of counterfeit and substandard seeds, and protect the legitimate rights and interests of farmers and the healthy development of the cucumber industry. Therefore, it is particularly important to screen a molecular marker combination based on DNA detection technology for identifying the purity of cucumber hybrid seeds.

[0004] InDel (Insertion-Deletion) marker refers to the difference between the whole genome sequences of two parents, specifically, a certain number of nucleotide insertions or deletions in the genome of one parent compared to the other. This insertion-deletion marker is widely present in the genomes of different organisms, and specific InDel marker primers are usually designed according to the sequences on both sides of the insertion-deletion site. Compared with SNP, InDel is more efficient, can be detected quickly using ordinary electrophoresis technology platform, has simpler facility and method requirements and lower DNA sample quality requirements, and scholars have compared it with SSR amplification results and found that its stability and separation effect are better than SSR; has the characteristics of good stability, wide distribution, high polymorphism, strong universality, simple typing system and low cost. SUMMARY

[0005] The purpose of the present application is to identify the purity of cucumber hybrids.

[0006] The present application first protects the InDel site combination, including four InDel sites of cucumber genome; the four sites are as follows: the InD_Cu1 site is located at the 400412th nucleotide on chromosome 4; the InD_Cu2 site is located at the 18170370th nucleotide on chromosome 6; the InD_Cu3 site is located at the 183336th nucleotide on chromosome 7; and the InD_Cu4 site is located at the 3748553th nucleotide on chromosome 5.

[0007] The InDel site combination specifically consists of the above-mentioned four InDel sites.

[0008] The present application also protects four pairs of InDel primer combinations, including primer group 1 for amplifying InD_Cu1; primer group 2 for amplifying InD_Cu2; primer group 3 for amplifying InD_Cu3; and primer group 4 for amplifying InD_Cu4.

[0009] In the four pairs of InDel primer combinations, the primer group 1 consists of the forward primer F1 shown in SEQ ID NO: 1 and the reverse primer R1 shown in SEQ ID NO: 2. The primer group 2 consists of the forward primer F2 shown in SEQ ID NO: 3 and the reverse primer R2 shown in SEQ ID NO: 4. The primer group 3 consists of the forward primer F3 shown in SEQ ID NO: 5 and the reverse primer R3 shown in SEQ ID NO: 6. The primer group 4 consists of the forward primer F4 shown in SEQ ID NO: 7 and the reverse primer R4 shown in SEQ ID NO: 8.

[0010] The molar ratio of the primer containing "F" in the name to the primer containing "R" in the name in the above primer set can be 1:1.

[0011] The InDel primer combination can be specifically composed of the primer set 1, the primer set 2, the primer set 3 and the primer set 4.

[0012] The method for identifying the purity of the to-be-tested cucumber hybrid can specifically include the following steps:

[0013] (1) Obtain the genomic DNA of N to-be-tested cucumber hybrids;

[0014] (2) Take the genomic DNA of 8-12 (such as 8-10, 10-12, 8, 10 or 12) to-be-tested cucumber hybrids obtained in step (1) as the template, and respectively use four primer sets in the InDel primer combination for PCR amplification to obtain the corresponding PCR amplification products;

[0015] (3) After step (2) is completed, perform agarose gel electrophoresis, and use the instrument to view the results, and count the number of strains of hybrid bands of the four primer sets; the primer set with more and clearer hybrid bands is the target primer set;

[0016] (4) Take the genomic DNA of N to-be-tested cucumber hybrids obtained in step (1) as the template, and respectively use the target primer set for PCR amplification to obtain the corresponding PCR amplification products;

[0017] (5) After step (4) is completed, use the instrument to view the bands of each PCR amplification product, and obtain the purity of the to-be-tested cucumber hybrid according to the number of hybrid bands.

[0018] In the above method, the method for obtaining the purity of the to-be-tested cucumber hybrid according to the number of hybrid bands can be: counting the number of strains of hybrid bands and the number of strains of no bands displayed by each target primer set, respectively calculating the purity, and then averaging.

[0019] Purity = Number of strains of hybrid bands displayed by the target primer set / (N - Number of strains of no bands displayed by the target primer set) x 100%.

[0020] In the above method, the reaction system of the PCR amplification can be specifically 5 µl of template DNA (50 ng / µl), 10 µl of 2x Mix, 1 µl of forward primer and 1 µl of reverse primer (20 µmol / L), and 3 µl of ultrapure water. The reaction program of the PCR amplification can be specifically 95℃ pre-denaturation for 5 min, 95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 1 min, 35 cycles, and 72℃ extension for 10 min.

[0021] In the above method, the to-be-tested cucumber hybrid can be Zhongnong 15, Zhongnong 9, Jinxu 12, Jinxu 4, Jinxu 2, Jinxu 35, Zhongnong 26, Bomixi 11, Jinxu 48, Jingyan 107, Jinxu 108, Huannai H1104, Jingren 3, Bomixi 5032, Bomixi 517, Jinxu 10, Dedete D19, Bomixi 10, Bomixi 74, Zhongnong 50, Jinxu 308, Dedete 4, Bomixi 6913, Dedete 79, Jinxu 335, Jinxu 316, Jinxu 358, Jinxu 3, Zhongnong 37, Qiande 2, Qiande 117, Jingyan 403, Jingyan 207, Jingyan 402, Jingyan 1568, Jingyan 15163, Jingyan 108, Jingyan 118, Jingyan Green Arrow, Jingyan Green Arrow 2, Jingyan 407, Jingyan 109, Huza 6, Tianjia 7, Tianjia 8, Jingyan Hanbao 5, Ningjia 3, Zhongnong 19, Shenglu 72, Shenglu 64, Biju, Ningyun 3, Dongnong 806, Jingyan Mini 1, Jingyan Cuiyu Mini 2, Jingyan Mini 2, Qiande 1217, Jingyan Mini White, Jingyan Yutan 156, Jingyan Mini White 1, Jingyan Mini 5, Jingyan Jingbao, Jingyan Mini 8, Jingyan Mini 9, Jingyan Mini 10, or any one of the Shengyan Mini.

[0022] In the above method, the greater the value of N, the higher the accuracy of identifying the purity of the to-be-tested cucumber hybrid.

[0023] It should be noted that the present application focuses on the purity guarantee of cucumber hybrids, and specifically refers to hybrid first-generation seeds, and the core challenge is to avoid parent hybridization rather than mechanical hybridization, that is, not to be simply mixed by multiple cucumber hybrids.

[0024] The InDel primer combination provided by the present application aims to realize early identification of cucumber hybrids at the seed or seedling stage, thereby ensuring the purity of the hybrid, effectively maintaining the legal rights and interests of producers and breeders, and providing technical support for seed quality management of cucumber varieties. The method provided by the present application has the advantages of high throughput, accuracy, low cost, simple operation, saving of manpower and material resources, and has a very broad application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 FIG. 4 is the InDel typing effect of the four primer groups in part of the test cucumber hybrids.

[0026] Figure 2 FIG. 5 is the distribution of 66 cucumber hybrid loci for the four primer groups.

[0027] Figure 3 FIG. 6 is the band distribution effect of primer group 2 in 96 Jingyan 118 hybrid seeds. DETAILED DESCRIPTION

[0028] The following examples facilitate a better understanding of the present application, but do not limit the present application.

[0029] The experimental methods in the following examples are all conventional methods, unless otherwise specified.

[0030] The experimental materials used in the following examples are all purchased from conventional biochemical reagent stores, unless otherwise specified.

[0031] In the quantitative experiments in the following examples, three repeated experiments are set up, and the average value is taken.

[0032] Example 1, obtaining of InDel primer combination for identifying purity of cucumber hybrid.

[0033] I. Discovery of four InDel sites

[0034] Based on the resequencing data of 182 representative cucumber resources, the present application develops InDel markers of the whole genome of cucumber and uses them for variety purity identification, and four InDel sites are obtained. The 182 cucumber resources are rich in types, covering North China type, South China type, European fruit type and other types, basically including the main ecological types and agronomic traits of cucumber, as much as possible to reflect the representativeness of germplasm, and have high genetic diversity.

[0035] Specifically, the screening criteria of the InDel sites are as follows: first, InDel sites belonging to the biallelic type in the whole genome chromosome are screened, InDel sites with 200 conservative wings and > 30 bp are selected, then InDel sites with Miss < 0.2, MAF > 0.2, He < 0.05 are screened, subsequently, InDel sites with MAF > 0.2 in two populations are screened, finally, blast specificity analysis is performed, and primers are designed.

[0036] The basic information of the four InDel sites is shown in Table 1, columns 1 to 4. The position of the InDel site on the chromosome is determined based on the alignment of the cucumber 9930 reference genome sequence, the version number of which is V2 (download address: http: / / cucurbitgenomics.org / organism / 2 ).

[0037] Table 1. Basic information of four InDel sites

[0038]

[0039] II. Obtaining of InDel primer combination for identifying purity of cucumber hybrid

[0040] Based on the four InDel sites discovered in step one, the inventors of the present application developed an InDel primer combination with high polymorphism for identifying the purity of cucumber hybrid.

[0041] The InDel primer combination consists of 4 primer groups, and the name of each primer group is shown in column 2 of Table 2. Each primer group consists of 2 primer sequences for amplifying one InDel site. The nucleotide sequences of the primers in the 4 primer groups are shown in column 4 of Table 2.

[0042] Table 2

[0043]

[0044] Example 2, verification of the effectiveness of the InDel primer combination developed in Example 1

[0045] The basic information of the 66 test cucumber hybrids in this example is shown in Table 3. The 66 test cucumber hybrids are all common excellent hybrids or foreign introduced hybrids.

[0046] Table 3. Basic information of 66 test cucumber hybrids

[0047]

[0048] 1. Obtaining of genomic DNA of the test cucumber hybrids (DNA extraction by magnetic beads method)

[0049] Each cucumber material takes an appropriate amount of leaf sample collected in a centrifuge tube, add steel ball, and placed in liquid nitrogen for freezing, then use the ultra-high throughput grinder for grinding; after grinding, add 800 μl of SDS extraction solution to each sample; 60°C oven for 30-40 min, during which time mix well for 3 times; add 240 μl (i.e. 0.3 times the volume of SDS extraction solution) of 3M potassium acetate solution to each sample, mix well for 5 times, then place in the 4°C refrigerator for 20 min; centrifuge at 4000 rpm for 10 min; prepare new centrifuge tubes or deep well plates in advance, add 600 μl of magnetic bead isopropanol to them (ensure the volume is equal to the supernatant), note the operation sequence is to add magnetic bead isopropanol first, then add the supernatant, and the volume of the supernatant should not exceed 2 / 3 of the total volume; mix well, stand at -20°C until the magnetic beads precipitate for 30 min, mix well again, use a magnetic stand to absorb, discard the supernatant, and stick to the residual liquid on the inverted absorption paper; add 200 μl of 75% ethanol solution to the sample, mix well, and stand until the magnetic beads completely precipitate; mix well again, use a magnetic stand to absorb, discard the supernatant, and stick to the residual liquid on the inverted absorption paper; after the magnetic beads are completely dried, add 100 μl of pure water to them, mix well to ensure that the DNA is completely dissolved; use a Nanodrop (microspectrophotometer) to take 2 μl of DNA extraction solution for detection, measure the OD260 / 280 and OD260 / 230 ratios (ideal range is 1.8-2.0), and record the concentration of the DNA. Then, adjust the DNA concentration to 50 ng / μl for subsequent use.

[0050] 2. Using the genomic DNA of 66 test cucumber hybrids as templates, respectively, and using four primer groups for PCR amplification, respectively, to obtain the corresponding PCR amplification products. In each PCR reaction system, the concentration ratio of the primer with "F" in the name to the primer with "R" in the name is 1:1.

[0051] The reaction program is: 95°C pre-denaturation for 5 min; 95°C denaturation for 30 s; 58°C annealing for 30 s; 72°C extension for 1 min, 35 cycles; 72°C extension for 10 min.

[0052] 3. After completing step 2, perform agarose gel electrophoresis on the PCR amplification products, and take pictures and read the results through a gel imaging system. Determine the genotype of each InDel site based on the 66 test cucumber hybrids through the banding. The specific judgment principle is as follows: if a test cucumber hybrid shows heterozygous banding results based on a certain InDel site, then the genotype of the test cucumber hybrid based on the InDel site is heterozygous; if a test cucumber hybrid shows single banding results based on a certain InDel site, then the genotype of the test cucumber hybrid based on the InDel site is homozygous.

[0053] Some results are shown in Table 1 Figure 1 The results show that each primer group can obtain good typing effect in the tested cucumber hybrids.

[0054] 4. Distribution of hybrid sites and efficiency evaluation

[0055] (1) According to the genotypes of 66 tested cucumber hybrids based on 4 InDel sites, the number of hybrid sites of each tested cucumber hybrid was counted.

[0056] The distribution of the number of hybrid sites of 66 tested cucumber hybrids based on 4 primer groups is shown in Table 2 Figure 2 The results show that 4 primer groups can make each tested cucumber hybrid have at least one hybrid site.

[0057] (2) Hybrid purity identification can use multiplex PCR to reduce workload.

[0058] The results show that the hybrid site coverage rate of multiplex PCR (4 primer groups) in 66 tested cucumber hybrids reaches 100%.

[0059] Therefore, the InDel primer combination developed in Example 1 can be applied to the purity identification of cucumber hybrids.

[0060] Example 3: Detection of the purity of Jingyan 118 hybrid using the InDel primer combination developed in Example 1

[0061] I. Detection of the purity of Jingyan 118 hybrid using the InDel primer combination developed in Example 1

[0062] 1. Obtaining of genomic DNA of Jingyan 118 hybrid

[0063] (1) Plant 200 seeds of Jingyan 118 hybrid to obtain Jingyan 118 hybrid seedlings.

[0064] (2) Randomly take 96 leaf or root samples of Jingyan 118 hybrid seedlings, and extract genomic DNA using magnetic beads method to obtain 96 genomic DNA samples of Jingyan 118 hybrid, respectively.

[0065] 2. Screening of primer groups

[0066] (1) Take 4 genomic DNA samples of Jingyan 118 hybrid as templates, respectively, and use 4 primer groups in the InDel primer combination developed in Example 1 to perform PCR amplification, respectively, to obtain corresponding PCR amplification products. In each PCR reaction system, the concentration ratio of the primer containing "F" in the name to the primer containing "R" in the name is 1:1.

[0067] The reaction system for PCR amplification can be specifically: 5 μl of template DNA (50 ng / μl), 10 μl of 2x Mix, 1 μl of forward primer and 1 μl of reverse primer (20 μmol / L), and 3 μl of ultrapure water.

[0068] The reaction procedure for PCR amplification can be specifically: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s; 58℃ annealing for 30 s; 72℃ extension for 1 min, 35 cycles; and 72℃ extension for 10 min.

[0069] (2) After step (1) is completed, agarose gel electrophoresis is performed, and the electrophoresis results are photographed and recorded using a gel imaging system to obtain the results. The number of strains of hybrid sites of the four primer groups is compared, and the primer group with the largest number of hybrid sites is the selected primer group.

[0070] The results show that the primer group 2 has the largest number of hybrid sites. Therefore, the primer group 2 is the selected primer group for subsequent experiments.

[0071] 3. Obtain the purity of Jingyan 118 hybrid

[0072] (1) Four genomic DNAs of Jingyan 118 hybrid are used as templates, respectively, and four primer groups in the InDel primer combination developed in Example 1 are used for PCR amplification, respectively, to obtain corresponding PCR amplification products. In each PCR reaction system, the concentration ratio of the primer containing “F” in the name to the primer containing “R” in the name is 1:1.

[0073] The reaction system for PCR amplification can be specifically: 5 μl of template DNA (50 ng / μl), 10 μl of 2x Mix, 1 μl of forward primer and 1 μl of reverse primer (20 μmol / L), and 3 μl of ultrapure water.

[0074] The reaction procedure for PCR amplification can be specifically: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s; 58℃ annealing for 30 s; 72℃ extension for 1 min, 35 cycles; and 72℃ extension for 10 min.

[0075] (2) After step (1) is completed, agarose gel electrophoresis is performed, and the electrophoresis results are photographed and recorded using a gel imaging system to obtain the results.

[0076] The InDel typing results are shown in Figure 3 .

[0077] (3) After step (2) is completed, the number of strains of hybrid sites and the number of strains of non-hybrid sites displayed by the primer group 2 are counted; the purity of Jingyan 118 hybrid is calculated according to the following formula; and the average value is further calculated to obtain the average purity.

[0078] Purity = number of strains showing heterozygous sites by primer set / (96 - number of strains showing no band by primer set) x 100%.

[0079] The results show that the number of strains showing heterozygous bands by primer set 2 is 96, the number of strains showing no band is 0, and the purity is 96 / (96-0) x 100% = 100%.

Claims

1. An InDel primer combination comprising: Primer group 1 for amplifying InDel site InD_Cu1 in cucumber genome; Primer group 2 for amplifying InDel site InD_Cu2 in cucumber genome; Primer group 3 for amplifying InDel site InD_Cu3 in cucumber genome; Primer group 4 for amplifying InDel site InD_Cu4 in cucumber genome; InD_Cu1 is located at 400412-400453 of chromosome 4 of cucumber genome, and the base type is shown in SEQ ID NO:1 or C; InD_Cu2 is located at 18170370 of chromosome 6 of cucumber genome, and the base type is shown in SEQ ID NO:2 or G; InD_Cu3 is located at 183336 of chromosome 7 of cucumber genome, and the base type is shown in SEQ ID NO:3 or T; InD_Cu4 is located at 3748553 of chromosome 5 of cucumber genome, and the base type is shown in SEQ ID NO:4 or T; The positions of InDel sites InD_Cu1, InD_Cu2, InD_Cu3 and InD_Cu4 in the genome are determined based on the cucumber 9930 reference genome V2 version; The primer group 1 consists of a forward primer F1 shown in SEQ ID NO:5 and a reverse primer R1 shown in SEQ ID NO:6; The primer group 2 consists of a forward primer F2 shown in SEQ ID NO:7 and a reverse primer R2 shown in SEQ ID NO:8; The primer group 3 consists of a forward primer F3 shown in SEQ ID NO:9 and a reverse primer R3 shown in SEQ ID NO:10; The primer group 4 consists of a forward primer F4 shown in SEQ ID NO:11 and a reverse primer R4 shown in SEQ ID NO:

12.

2. A method for identifying the purity of a to-be-tested cucumber hybrid, comprising the following steps: (1) obtaining genomic DNA of N to-be-tested cucumber hybrids; (2) respectively taking the genomic DNA of 8-12 to-be-tested cucumber hybrids obtained in step (1) as a template, and respectively using four primer groups in the InDel primer combination of claim 1 to perform PCR amplification to obtain corresponding PCR amplification products; (3) after step (2) is completed, performing agarose gel electrophoresis, and using an instrument to view the results, and counting the number of strains of hybrid bands of the four primer groups; the primer group with more and clearer hybrid bands is the target primer group; (4) respectively taking the genomic DNA of N to-be-tested cucumber hybrids obtained in step (1) as a template, and respectively using the target primer group to perform PCR amplification to obtain corresponding PCR amplification products; (5) after step (4) is completed, using an instrument to view the bands of each PCR amplification product, and obtaining the purity of the to-be-tested cucumber hybrid according to the number of hybrid bands. The to-be-tested cucumber hybrids are Zhongnong 15, Zhongnong 9, Jinyou 12, Jinchun 4, Jinyou 2, Jinyou 35, Zhongnong 26, Bomai 11, Jinyou 48, Jingyan 107, Jinyou 108, Huannai H1104, Jingren 3, Bomai 5032, Bomai 517, Jinyou 10, Deduite D19, Bomai 10, Bomai 74, Zhongnong 50, Jinyou 308, Deduite 4, Bomai 6913, Deduite 79, Jinyou 335, Jinyou 316, Jinyou 358, Jinchun 3, Zhongnong 37, Qiantai 2, Qiantai 117, Jingyan 403, Jingyan 207, Jingyan 402, Jingyan 1568, Jingyan 15163, Jingyan 108, Jingyan 118, Jingyan Green Arrow, Jingyan Green Arrow 2, Jingyan 407, Jingyan 109, Huza 6, Tianjia 7, Tianjia 8, Jingyan Hanbao 5, Ningjia 3, Zhongnong 19, Shenglu 72, Shenglu 64, Biju, Ningyun 3, Dongnong 806, Jingyan Mini 1, Jingyan Cuiyu Mini 2, Jingyan Mini 2, Qiantai 1217, Jingyan Mini White, Jingyan Yutan 156, Jingyan Mini White 1, Jingyan Mini 5, Jingyan Jingbao, Jingyan Mini 8, Jingyan Mini 9, Jingyan Mini 10, Shengyan Mini, or any one of the above.

3. The method of claim 2, wherein: The method for obtaining the purity of the to-be-tested cucumber hybrid according to the number of hybridization bands is as follows: counting the number of plants showing hybridization bands and the number of plants showing no bands for each target primer group, respectively calculating the purity, and then averaging the values; Purity = (number of plants showing hybridization bands for the target primer group) / (N - number of plants showing no bands for the target primer group) x 100%.

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