A method for simultaneously identifying three varieties of broccoli and primer pairs used therein

Abstract

The present invention discloses a method for simultaneously identifying three varieties of broccoli, namely, Jingsong No. 1, Jingsong No. 2 and Jingsong No. 3, and primer pairs used therein. The primer pair consists of a single-stranded DNA molecule shown in SEQ ID NO: 1 and a single-stranded DNA molecule shown in SEQ ID NO: 2. The authenticity of the three varieties can be identified by using the primer pair in the seedling stage. Compared with the traditional field identification method, the present invention can greatly shorten the time and save land, labor, water and fertilizer, etc. The present invention has important application value.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a method for simultaneously identifying three broccoli varieties and a primer pair used therein. Background Art

[0002] Variety identification is an important part of seed quality testing, and its main purpose is to determine the degree of conformity between the sample submitted for testing and the declared variety. SSR molecular marker technology is an emerging technology. It is a genetic marker technology based on nucleotide sequence variation within genetic material between individuals. It is a direct reflection of genetic polymorphism at the DNA level. Compared with other genetic marker technologies, SSR molecular marker technology has the characteristics of rich information, fast and accurate detection, and easy standardized operation. It is very suitable for plant variety identification and analysis. The new "Seed Law" clearly stipulates that the results of vegetable variety authenticity detection using the SSR molecular marker method can be used as the basis for determining counterfeit and inferior seeds.

[0003] Songhua cauliflower is a special variety of cauliflower of the Brassica oleracea species of the Cruciferae family. Because of its long pedicels and loose flower heads, it tastes crisper and better than traditional compact cauliflowers and is deeply loved by consumers. The cultivation area of ​​​​Songhua cauliflower in my country has been expanding in the past decade with the rapid growth of market demand. Correspondingly, more and more varieties have been bred and promoted, including homogeneous varieties with different names and the same species, which poses a severe challenge to the protection of intellectual property rights. At the same time, the seed purity of the variety is an important indicator affecting seed quality, which directly affects yield and economic benefits. Songhua cauliflower has obvious hybrid advantages, and the seeds used for production are basically hybrids. There are two main ways to produce seeds: self-incompatibility lines and male sterility lines. Seeds produced by the self-incompatibility line often produce a small amount of self-pollinated seeds due to the insufficient self-incompatibility of the parents. The seeds cannot be identified when harvested and are mixed in the hybrids to cause false hybrids. Although the production of hybrids through male sterile lines can overcome the false hybrids caused by the self-incompatibility line, if the seed production field is not isolated enough, the field management during the flowering period is improper, causing pollen contamination, or mechanical mixing during seed harvesting, drying, processing, and transportation, it will directly affect the purity of the seeds. Unqualified seed purity will cause huge economic losses to seed management companies and growers. Therefore, purity identification and seed quality assurance are necessary before seeds enter the market for sale.

[0004] The traditional method of purity identification is to use morphological differences such as seedlings or plant types to identify true and false hybrids. The growth cycle of broccoli is long, and the traditional morphological identification method takes a long time and is costly. It is also easily affected by environmental conditions and gene dominance, which reduces the accuracy of the identification results. The rapid development of modern molecular biotechnology has made it possible to select complex multi-traits at the DNA level in an early, accurate and efficient manner. The use of molecular marker technology to identify seed purity has the advantages of being fast, efficient, easy to repeat and not affected by the environment. The SSR (Simplesequence repeat) molecular marker, which is based on PCR technology, has site specificity and is widely, richly and evenly distributed in the genome. It has the characteristics of co-dominance, high polymorphism and good repeatability. It is used in plant genetic diversity analysis, genetic map construction, molecular marker-assisted breeding and variety identification. Although SSR molecular markers have been used in variety purity identification, basically one primer is used for one variety. To identify multiple varieties, multiple primers need to be developed, which affects cost and efficiency. Summary of the invention

[0005] The purpose of the present invention is to identify three varieties of Jingsong No. 1, Jingsong No. 2 and Jingsong No. 3 to ensure the purity of the Songhuacai variety.

[0006] The present invention firstly protects a method for identifying at least one of the three varieties of Jingsong No. 1, Jingsong No. 2 and Jingsong No. 3, which may include the following steps: using the genomic DNA of the tested Songhuacai as a template, using primer pair CH19-20 to perform PCR amplification, detection, and then performing the following judgment:

[0007] If only three amplified fragments are obtained and their sizes are 264 bp, 286 bp and 310 bp respectively, the Songhuacai to be tested is Jingsong No. 1;

[0008] If only three amplified fragments are obtained and their sizes are 269 bp, 288 bp and 312 bp respectively, the Songhuacai to be tested is Jingsong No. 2;

[0009] If only two amplified fragments are obtained and the sizes are 298 bp and 267 bp respectively, the Songhuacai to be tested is Jingsong No. 3;

[0010] The primer pair CH19-20 consists of an upstream primer and a downstream primer;

[0011] The nucleotide sequence of the upstream primer is shown in SEQ ID NO: 1;

[0012] The nucleotide sequence of the downstream primer is shown in SEQ ID NO:2.

[0013] The present invention also protects a method for identifying which of the nine broccoli varieties or strains a broccoli variety or strain to be tested is, which may include the following steps: using the genomic DNA of the broccoli to be tested as a template, using primer pair CH19-20 to perform PCR amplification, testing, and then performing the following judgment:

[0014] If only three amplified fragments are obtained and the sizes are 264 bp, 286 bp and 310 bp respectively, the Songhuacai to be tested is identified as Jingsong No. 1;

[0015] If only one amplified fragment is obtained and the size is 264 bp, the tested Songhuacai is identified as the cytoplasmic male sterile line CMS060;

[0016] If only one amplified fragment is obtained and the size is 286 bp, the tested broccoli is identified as the inbred line 1028-1;

[0017] If only three amplified fragments are obtained and the sizes are 269 bp, 288 bp and 312 bp respectively, the Songhuacai to be tested is identified as Jingsong No. 2;

[0018] If only one amplified fragment is obtained and the size is 269 bp, the tested Songhuacai is identified as the cytoplasmic male sterile line CMS105;

[0019] If only one amplified fragment is obtained and the size is 288 bp, the tested broccoli is identified as inbred line 36B;

[0020] If only two amplified fragments are obtained and the sizes are 298 bp and 267 bp respectively, the Songhuacai to be tested is identified as Jingsong No. 3;

[0021] If only one amplified fragment is obtained and the size is 298 bp, the tested Songhuacai is identified as the cytoplasmic male sterile line CMS070;

[0022] If only one amplified fragment is obtained and the size is 267 bp, the tested broccoli is identified as the inbred line SH65-8;

[0023] The primer pair CH19-20 consists of an upstream primer and a downstream primer;

[0024] The nucleotide sequence of the upstream primer is shown in SEQ ID NO: 1;

[0025] The nucleotide sequence of the downstream primer is shown in SEQ ID NO: 2;

[0026] The nine broccoli varieties or lines are Jingsong 1, cytoplasmic male sterile line CMS060, inbred line 1028-1, Jingsong 2, cytoplasmic male sterile line CMS105, inbred line 36B, Jingsong 3, cytoplasmic male sterile line CMS070 and inbred line SH65-8.

[0027] The present invention also protects a method for identifying which of the nine broccoli varieties or strains a broccoli variety or strain to be tested is, comprising the following steps:

[0028] (1) Using the genomic DNA of the tested broccoli as a template, PCR amplification was performed using primer pair CH19-20; detection;

[0029] (2) using genomic DNA of nine Songhuacai varieties or strains as templates, respectively, and using the primer pair CH19-20 for PCR amplification; the nine Songhuacai varieties or strains are Jingsong No. 1, cytoplasmic male sterile line CMS060, inbred line 1028-1, Jingsong No. 2, cytoplasmic male sterile line CMS105, inbred line 36B, Jingsong No. 3, cytoplasmic male sterile line CMS070 and inbred line SH65-8; detection;

[0030] (3) After completing steps (1) and (2), make a judgment based on the following criteria:

[0031] If the PCR amplification product of the tested Songhuacai is consistent with the PCR amplification product of Jingsong No. 1, the tested Songhuacai is Jingsong No. 1;

[0032] If the PCR amplification product of the tested Songhuacai is consistent with the PCR amplification product of the cytoplasmic male sterile line CMS060, the tested Songhuacai is the cytoplasmic male sterile line CMS060;

[0033] If the PCR amplification product of the tested broccoli is consistent with the PCR amplification product of the inbred line 1028-1, the tested broccoli is the inbred line 1028-1;

[0034] If the PCR amplification product of the Songhuacai to be tested is consistent with the PCR amplification product of Jingsong No. 2, the Songhuacai to be tested is Jingsong No. 2;

[0035] If the PCR amplification product of the tested Songhuacai is consistent with the PCR amplification product of the cytoplasmic male sterile line CMS105, the tested Songhuacai is the cytoplasmic male sterile line CMS105;

[0036] If the PCR amplification product of the tested broccoli is consistent with the PCR amplification product of the inbred line 36B, the tested broccoli is the inbred line 36B;

[0037] If the PCR amplification product of the tested Songhuacai is consistent with the PCR amplification product of Jingsong No. 3, the tested Songhuacai is Jingsong No. 3;

[0038] If the PCR amplification product of the tested Songhuacai is consistent with the PCR amplification product of the cytoplasmic male sterile line CMS070, the tested Songhuacai is the cytoplasmic male sterile line CMS070;

[0039] If the PCR amplification product of the tested broccoli is consistent with the PCR amplification product of the inbred line SH65-8, the tested broccoli is the inbred line SH65-8;

[0040] The primer pair CH19-20 consists of an upstream primer and a downstream primer;

[0041] The nucleotide sequence of the upstream primer is shown in SEQ ID NO: 1;

[0042] The nucleotide sequence of the downstream primer is shown in SEQ ID NO:2.

[0043] In any of the above methods, the detection can be performed by agarose gel electrophoresis. In an embodiment of the present invention, agarose gel electrophoresis can be performed in a 0.5 mg / mL TM The samples were electrophoresed on an agarose gel with a specific gel concentration of 3%.

[0044] The present invention also protects a primer pair CH19-20, which is composed of an upstream primer and a downstream primer;

[0045] The nucleotide sequence of the upstream primer is shown in SEQ ID NO: 1;

[0046] The nucleotide sequence of the downstream primer is shown in SEQ ID NO:2.

[0047] The present invention also protects the use of the primer pair CH19-20 in identifying at least one of the three varieties of Jingsong No. 1, Jingsong No. 2 and Jingsong No. 3.

[0048] The present invention also protects the use of the primer pair CH19-20 in identifying 9 varieties or strains of Songhuacai; the 9 varieties or strains of Songhuacai are Jingsong No. 1, cytoplasmic male sterile line CMS060, inbred line 1028-1, Jingsong No. 2, cytoplasmic male sterile line CMS105, inbred line 36B, Jingsong No. 3, cytoplasmic male sterile line CMS070 and inbred line SH65-8.

[0049] The present invention also protects a kit containing the primer pair CH19-20.

[0050] The present invention also protects a method for preparing the kit, including the step of individually packaging each primer in the primer pair CH19-20.

[0051] The present invention also protects the use of the kit, which may be a1) or a2):

[0052] a1) Identify at least one of the three varieties: Jingsong No. 1, Jingsong No. 2 and Jingsong No. 3;

[0053] a2) Application in identifying 9 varieties or strains of Songhuacai; the 9 varieties or strains of Songhuacai are Jingsong No. 1, cytoplasmic male sterile line CMS060, inbred line 1028-1, Jingsong No. 2, cytoplasmic male sterile line CMS105, inbred line 36B, Jingsong No. 3, cytoplasmic male sterile line CMS070 and inbred line SH65-8.

[0054] The present invention has developed an SSR primer pair that can simultaneously identify three pine cauliflower varieties, "Jingsong No. 1", "Jingsong No. 2" and "Jingsong No. 3", which are promoted and applied in production. The authenticity of the three varieties can be identified in the seedling stage. Compared with the traditional field identification method, the present invention can greatly shorten the time (from 4 months to 1-2 weeks), and can also save land, labor, water and fertilizer. The present invention has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 This is a partial agarose gel electrophoresis result of PCR amplification of Jingsong No. 1 and its male and female parents using primer pair CH19-20 in Example 2.

[0056] Figure 2 This is a partial agarose gel electrophoresis result of PCR amplification of Jingsong 2 and its male and female parents by primer pair CH19-20 in Example 2.

[0057] Figure 3 This is a partial agarose gel electrophoresis result of PCR amplification of Jingsong No. 3 and its male and female parents by primer pair CH19-20 in Example 2.

[0058] Figure 4 This is a partial agarose gel electrophoresis result of PCR amplification of Jingsong No. 1 and its male and female parents using primer pair CH19-20 in Example 3.

[0059] Figure 5 This is a partial agarose gel electrophoresis result of PCR amplification of Jingsong 2 and its male and female parents using primer pair CH19-20 in Example 3. DETAILED DESCRIPTION

[0060] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0061] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0062] Jingsong No. 1 is a loose type first-generation cauliflower hybrid with cytoplasmic male sterile line CMS060 as the female parent and inbred line 1028-1 as the male parent. Jingsong No. 2 is a loose type first-generation cauliflower hybrid with cytoplasmic male sterile line CMS105 as the female parent and inbred line 36B as the male parent. Jingsong No. 3 is a loose type first-generation cauliflower hybrid with cytoplasmic male sterile line CMS070 as the female parent and inbred line SH65-8 as the male parent.

[0063] In the following examples, Jingsong 1, cytoplasmic male sterile line CMS060, inbred line 1028-1, Jingsong 2, cytoplasmic male sterile line CMS105, inbred line 36B, Jingsong 3, cytoplasmic male sterile line CMS070 and inbred line SH65-8 were all provided by Jingyan Yinong (Beijing) Seed Technology Co., Ltd.

[0064] The DNA Marker in the following embodiments is a 50bp Ladder DNA Marker, which is a product of Beijing Saibaisheng Gene Technology Co., Ltd. with a product number of MD111-01. The 50bp Ladder DNA Marker consists of 10 DNA bands, and the DNA bands are: 50bp, 100bp, 150bp, 200bp, 250bp, 300bp, 400bp, 500bp, 600bp, and 700bp.

[0065] Example 1. Development of SSR primer pairs for simultaneous identification of three pine cauliflower varieties, Jingsong 1, Jingsong 2 and Jingsong 3

[0066] 1. Evenly sow the seeds of Jingsong No. 1 in the germination box, then place the germination box in an incubator, culture at 15-25°C with alternating light and dark (10 hours of light / 14 hours of darkness), collect young leaves of 5 seedlings after 14 days, mix samples of equal mass, and obtain sample 1.

[0067] According to the above method, Jingsong No. 1 was replaced by cytoplasmic male sterile line CMS060, inbred line 1028-1, Jingsong No. 2, cytoplasmic male sterile line CMS105, inbred line 36B, Jingsong No. 3, cytoplasmic male sterile line CMS070 and inbred line SH65-8, respectively, and the other steps were the same, and samples 2 to 9 were obtained in sequence.

[0068] 2. The 9 samples obtained in step 1 were subjected to DNA extraction, enzyme digestion, adapter ligation, adapter library amplification, sequencing, etc., to obtain sequencing results with DNA fragment lengths between 200-300bp. Further, primers were designed and screened based on the sequencing results, and finally a polymorphic, stable amplification, and easy-to-count SSR primer pair, primer pair CH19-20, was obtained for the detection of three varieties of broccoli.

[0069] Primer pair CH19-20 consists of an upstream primer and a downstream primer.

[0070] The nucleotide sequence of the upstream primer is 5'-AAACGAGGCTTCCACAGAGA-3' (SEQ ID NO: 1).

[0071] The nucleotide sequence of the downstream primer is 5'-GGGTACCCGTTCGGTTCT-3' (SEQ ID NO: 2).

[0072] Artificially synthesize upstream primers and downstream primers.

[0073] Example 2: Using the primer pair CH19-20 developed in Example 1 to identify three varieties of broccoli, namely Jingsong 1, Jingsong 2 and Jingsong 3

[0074] 1. Extraction of genomic DNA from samples

[0075] (1) Take 40 mg of the sample obtained in step 1 of Example 1 (sample 1, sample 2, sample 3, sample 4, sample 5, sample 6, sample 7, sample 8 or sample 9), add 400 μL of SDS-DNA extraction solution, grind with a high-throughput tissue grinder, and mix well.

[0076] The solutes and concentrations of the SDS-DNA extraction solution are NaCl 250mmol / L, EDTA 2.5mmol / L, SDS 0.5% (m / v), and the solvent is pH 7.5, 200mmol / L Tris-HCl buffer.

[0077] (2) After completing step (1), place the mixture in a 65°C water bath for 15 min (shake and mix three times during this period), then add 200 μL of a 5 mol / L potassium acetate aqueous solution and mix.

[0078] (3) After completing step (2), place the mixture in an ice bath for 10 min, then centrifuge at 13,000 rpm for 30 min and collect the supernatant.

[0079] (4) After completing step (3), add an equal volume of -20°C precooled isopropanol to the supernatant and place on ice for 20 min (to precipitate DNA) to obtain a precipitate. Wash the precipitate 2-3 times with 70% (v / v) ethanol aqueous solution, then dissolve in 60-80 μL of sterilized double distilled water overnight to obtain the genomic DNA of the sample.

[0080] After testing, the OD260 / OD230 ratio of the genomic DNA of the sample was between 1.9-2.0, and the OD260 / OD280 ratio was between 1.8-2.0.

[0081] The genomic DNA of the sample was detected by 1.0% Agrose gel electrophoresis. The results showed that the genomic DNA of the sample was a clear band, indicating that the genomic DNA of the sample was not broken or degraded, and was a complete double-stranded molecule, which met the requirements of PCR amplification.

[0082] 2. PCR amplification

[0083] Using the genomic DNA of the sample as a template, PCR amplification was performed on CH19-20 using the primers synthesized in Example 1 to obtain a PCR amplification product.

[0084] The reaction system was 20 μL, consisting of 0.4 μL dNTP solution (concentration of 10 mmol / L), 2 μL 10×PCR buffer, 0.3 μL Taq DNA polymerase (concentration of 2.5 U / μL), 2 μL sample genomic DNA (concentration of 20-200 ng / μL), 2 μL upstream primer (concentration of 10 μM), 2 μL downstream primer (concentration of 10 μM) and water.

[0085] Taq DNA polymerase is a product of Shanghai Sangon Biotechnology Co., Ltd., and 10×PCR buffer is a component of Taq DNA polymerase.

[0086] Reaction conditions: pre-denaturation at 94°C for 5 min, 1 cycle; denaturation at 94°C for 15 s, annealing at 55°C for 15 s, extension at 72°C for 30 s, 35 cycles; extension at 72°C for 4 min; storage at 4°C.

[0087] 3. Agarose gel electrophoresis

[0088] Take the PCR amplification product obtained in step 2 and add it to a 0.5 mg / mL TM The samples were electrophoresed on agarose gel (gel concentration was 3% (m / v), agarose SFRTM purchased from AMRESCO, USA), and the electrophoresis results were scanned and imaged using a G:BOX-Chemi XR2 gel imager (Gene Co., Ltd.).

[0089] The above experiment was repeated 3 times. The results of agarose gel electrophoresis are shown in Figure 1 (The lanes from left to right are 4 female copies of Kyungsong 1, 4 male copies of Kyungsong 1, Kyungsong 1, blank control, Kyungsong 1, Kyungsong 1, 3 female copies of Kyungsong 1, 3 male copies of Kyungsong 1, DNA Marker), Figure 2 (The lanes from left to right are 3 female copies of Jingsong 2, blank control, 4 male copies of Jingsong 2, 4 Jingsong 2, 6 female copies of Jingsong 2, and DNA Marker) and Figure 3 (The lanes from left to right are 4 female parents of Jingsong No. 3, 2 male parents of Jingsong No. 3, DNA Marker, 2 male parents of Jingsong No. 3, and 4 Jingsong No. 3).

[0090] The target specific bands of the PCR amplification products were sequenced, and the statistical bands of the sequencing results are shown in Table 1. The results showed that Jingsong 1, Jingsong 2 and Jingsong 3 all had complementary band patterns of their parents. It can be seen that the primer pair CH19-20 can identify the three Songhuacai varieties Jingsong 1, Jingsong 2 and Jingsong 3. Since the band patterns of Jingsong 1, cytoplasmic male sterile line CMS060, inbred line 1028-1, Jingsong 2, cytoplasmic male sterile line CMS105, inbred line 36B, Jingsong 3, cytoplasmic male sterile line CMS070 and inbred line SH65-8 are all different, these 9 Songhuacai varieties or lines can also be identified.

[0091] Table 1

[0092]

[0093]

[0094] Example 3: Accuracy Experiment

[0095] The seeds to be tested are seeds of the identified cytoplasmic male sterile line CMS060 (the female parent of Jingsong No. 1), seeds of the inbred line 1028-1 (the male parent of Jingsong No. 1), seeds of Jingsong No. 1, seeds of the cytoplasmic male sterile line CMS105 (the female parent of Jingsong No. 2), seeds of the inbred line 36B (the male parent of Jingsong No. 2), seeds of Jingsong No. 2, seeds of the cytoplasmic male sterile line CMS070 (the female parent of Jingsong No. 3), seeds of the inbred line SH65-8 (the male parent of Jingsong No. 3) and seeds of Jingsong No. 3.

[0096] 1. Evenly sow 20 seeds of cytoplasmic male sterile line CMS060 in a germination box, then place the germination box in an incubator at 15-25°C with alternating light and dark (10 hours of light / 14 hours of darkness). After 14 days, collect young leaves from 5 seedlings, mix them with equal mass, and obtain 4 samples to be tested, which are named sample 1 to sample 4 to be tested.

[0097] According to the above method, the cytoplasmic male sterile line CMS060 was replaced by the inbred line 1028-1, the cytoplasmic male sterile line CMS105 and the inbred line 36B respectively, and the other steps were the same, and the test samples 5 to 16 were obtained in sequence.

[0098] According to the above method, the cytoplasmic male sterile line CMS060 was replaced by the cytoplasmic male sterile line CMS070 and the inbred line SH65-8, respectively, and the other steps were the same, and the test samples 97 to 104 were obtained in sequence.

[0099] 2. Plant 40 seeds of Jingsong No. 1 in a seed box, then place the seed box in an incubator at 15-25°C with alternating light and dark (10 hours of light / 14 hours of darkness). After 14 days, collect young leaves of individual seedlings to obtain test samples 17 to 56.

[0100] According to the above method, Jingsong No. 1 was replaced by Jingsong No. 2, and the other steps were the same, to obtain test samples 57 to test samples 96.

[0101] According to the above method, Jingsong No. 1 is replaced by Jingsong No. 3, and the other steps are the same, and the samples to be tested 105-144 are obtained.

[0102] 3. After completing step 2, according to the method of step 1 in Example 2, the sample is replaced with the sample to be tested (sample 1 to sample 144) to obtain the genomic DNA of the sample to be tested.

[0103] 4. After completing step 3, the genomic DNA of the sample to be tested is used as a template, and PCR amplification is performed on CH19-20 using the primers synthesized in Example 1 to obtain a PCR amplification product.

[0104] The reaction system is 20 μL, consisting of 0.4 μL dNTP solution (concentration of 10 mmol / L), 2 μL 10×PCR buffer, 0.3 μL Taq DNA polymerase (concentration of 2.5 U / μL), 2 μL genomic DNA of the sample to be tested (concentration of 20-200 ng / μL), 2 μL upstream primer (concentration of 10 μM), 2 μL downstream primer (concentration of 10 μM) and water.

[0105] Taq DNA polymerase is a product of Shanghai Sangon Biotechnology Co., Ltd., and 10×PCR buffer is a component of Taq DNA polymerase.

[0106] Reaction conditions: pre-denaturation at 94°C for 5 min, 1 cycle; denaturation at 94°C for 15 s, annealing at 55°C for 15 s, extension at 72°C for 30 s, 35 cycles; extension at 72°C for 4 min; storage at 4°C.

[0107] 5. Take the PCR amplification product obtained in step 4 and add it to a 0.5 mg / mL solution of Good View TM The samples were electrophoresed on agarose gel (gel concentration was 3% (m / v), agarose SFRTM was purchased from AMRESCO, USA), and the electrophoresis results were scanned and imaged using a G:BOX-Chemi XR2 gel imager (Gene Co., Ltd.).

[0108] Agarose gel electrophoresis results are shown in Figure 4 (The lanes from left to right are 2 female parents of Jingsong No. 1, 2 male parents of Jingsong No. 1, 14 Jingsong No. 1, DNA Marker, and 6 Jingsong No. 1) and Figure 5 (The lanes from left to right are 2 female parents of Jingsong No. 2, 2 male parents of Jingsong No. 2, 14 Jingsong No. 2, DNA Marker, and 6 Jingsong No. 2).

[0109] The results are as follows:

[0110] All the seeds of the female parent of Jingsong No. 1 tested were PCR amplified using primer pair CH19-20, and the electrophoretic bands in the obtained electrophoretic patterns all had bands with the same composition as the specific amplified bands in the electrophoretic pattern of the cytoplasmic male sterile line CMS060 shown in Table 1 of Example 2, indicating that the seeds of the female parent of Jingsong No. 1 tested were indeed seeds of the cytoplasmic male sterile line CMS060;

[0111] The seeds of the male parent of all tested Jingsong No. 1 were PCR amplified using primer pair CH19-20, and the electrophoretic bands in the obtained electrophoretic patterns all had bands with the same composition as the specific amplified bands in the electrophoretic pattern of the inbred line 1028-1 shown in Table 1 of Example 2, indicating that the seeds of the male parent of Jingsong No. 1 tested were indeed seeds of the inbred line 1028-1;

[0112] All tested seeds of Jingsong No. 1 were amplified by PCR using primer pair CH19-20, and the electrophoretic bands in the obtained electrophoretic patterns all had bands with the same composition as the specific amplified bands in the electrophoretic pattern of Jingsong No. 1 shown in Table 1 of the corresponding Example 2, indicating that the tested seeds of Jingsong No. 1 were indeed seeds of Jingsong No. 1, and the purity was 100%;

[0113] All the seeds of the female parent of Jingsong No. 2 tested were PCR amplified using primer pair CH19-20, and the electrophoretic bands in the obtained electrophoretic pattern all had bands with the same composition as the specific amplified bands in the electrophoretic pattern of the cytoplasmic male sterile line CMS105 shown in Table 1 of Example 2, indicating that the seeds of the female parent of Jingsong No. 2 tested were indeed seeds of the cytoplasmic male sterile line CMS105;

[0114] All seeds of the male parent of Jingsong No. 2 tested were PCR amplified using primer pair CH19-20, and the electrophoretic bands in the obtained electrophoretic patterns all had bands with the same composition as the specific amplified bands in the electrophoretic pattern of the inbred line 36B shown in Table 1 of Example 2, indicating that the seeds of the male parent of Jingsong No. 2 tested were indeed seeds of the inbred line 36B;

[0115] All the seeds of Jingsong No. 2 tested were PCR amplified using primer pair CH19-20, and the electrophoretic bands in the obtained electrophoretic patterns all had bands with the same composition as the specific amplified bands in the electrophoretic pattern of Jingsong No. 2 shown in Table 1 of the corresponding Example 2, indicating that the seeds of Jingsong No. 2 tested were indeed seeds of Jingsong No. 2, and the purity was 100%;

[0116] All the seeds of the female parent of Jingsong No. 3 tested were PCR amplified using primer pair CH19-20, and the electrophoretic bands in the obtained electrophoretic pattern all had bands with the same composition as the specific amplified bands in the electrophoretic pattern of the cytoplasmic male sterile line CMS070 shown in Table 1 of Example 2, indicating that the seeds of the female parent of Jingsong No. 3 tested were indeed seeds of the cytoplasmic male sterile line CMS070;

[0117] All the seeds of the male parent of Jingsong No. 3 tested were PCR amplified using primer pair CH19-20, and the electrophoretic bands in the obtained electrophoretic patterns all had bands with the same composition as the specific amplified bands in the electrophoretic pattern of the inbred line SH65-8 shown in Table 1 of the corresponding Example 2, indicating that the seeds of the male parent inbred line SH65-8 of Jingsong No. 3 tested were indeed seeds of the inbred line SH65-8;

[0118] All tested seeds of Jingsong No. 3 were amplified by PCR using primer pair CH19-20, and the electrophoretic bands in the obtained electrophoretic patterns all had bands with the same composition as the specific amplified bands in the electrophoretic pattern of Jingsong No. 3 shown in Table 1 of the corresponding Example 2, indicating that the tested seeds of Jingsong No. 3 were indeed seeds of Jingsong No. 3 with a purity of 100%.

[0119] The above results show that the method provided by the present invention can identify whether the tested broccoli is Jingsong No. 1, cytoplasmic male sterile line CMS060, inbred line 1028-1, Jingsong No. 2, cytoplasmic male sterile line CMS105, inbred line 36B, Jingsong No. 3, cytoplasmic male sterile line CMS070 or inbred line SH65-8, thereby verifying the authenticity of 9 broccoli varieties or strains and the purity of the three broccoli varieties of Jingsong No. 1, Jingsong No. 2 and Jingsong No. 3.

[0120] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art. <110> Beijing Academy of Agricultural and Forestry Sciences <120> A method for simultaneously identifying three varieties of broccoli and primer pairs used therein <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 20 <212> DNA <213> Artificial sequence <400> 1 aaacgaggct tccacagaga 20 <210> 2 <211> 18 <212> DNA <213> Artificial sequence <400> 2 gggtacccgt tcggttct 18

Claims

1. A method for identifying at least one of the three varieties of Jingsong No. 1, Jingsong No. 2 and Jingsong No. 3, comprising the following steps: using the genomic DNA of the tested Songhuacai as a template, using primer pair CH19-20 for PCR amplification, detection, and then making the following judgment: If only three amplified fragments are obtained and their sizes are 264 bp, 286 bp and 310 bp respectively, the Songhuacai to be tested is Jingsong No. 1; If only three amplified fragments are obtained and their sizes are 269 bp, 288 bp and 312 bp respectively, the Songhuacai to be tested is Jingsong No. 2; If only two amplified fragments are obtained and the sizes are 298 bp and 267 bp respectively, the Songhuacai to be tested is Jingsong No. 3; The primer pair CH19-20 consists of an upstream primer and a downstream primer; The nucleotide sequence of the upstream primer is shown in SEQ ID NO: 1; The nucleotide sequence of the downstream primer is shown in SEQ ID NO:

2.

2. A method for identifying which of nine broccoli varieties or strains a broccoli variety or strain to be tested is, comprising the following steps: using the genomic DNA of the broccoli to be tested as a template, using a primer pair to perform PCR amplification, detecting, and then performing the following judgment: If only three amplified fragments are obtained and the sizes are 264 bp, 286 bp and 310 bp respectively, the Songhuacai to be tested is identified as Jingsong No. 1; If only one amplified fragment is obtained and the size is 264 bp, the tested Songhuacai is identified as the cytoplasmic male sterile line CMS060; If only one amplified fragment is obtained and the size is 286 bp, the tested broccoli is identified as the inbred line 1028-1; If only three amplified fragments are obtained and the sizes are 269 bp, 288 bp and 312 bp respectively, the Songhuacai to be tested is identified as Jingsong No. 2; If only one amplified fragment is obtained and the size is 269 bp, the tested Songhuacai is identified as the cytoplasmic male sterile line CMS105; If only one amplified fragment is obtained and the size is 288 bp, the tested broccoli is identified as inbred line 36B; If only two amplified fragments are obtained and the sizes are 298 bp and 267 bp respectively, the Songhuacai to be tested is identified as Jingsong No. 3; If only one amplified fragment is obtained and the size is 298 bp, the tested Songhuacai is identified as the cytoplasmic male sterile line CMS070; If only one amplified fragment is obtained and the size is 267 bp, the tested broccoli is identified as the inbred line SH65-8; The primer pair CH19-20 consists of an upstream primer and a downstream primer; The nucleotide sequence of the upstream primer is shown in SEQ ID NO: 1; The nucleotide sequence of the downstream primer is shown in SEQ ID NO: 2; The nine broccoli varieties or lines are Jingsong 1, cytoplasmic male sterile line CMS060, inbred line 1028-1, Jingsong 2, cytoplasmic male sterile line CMS105, inbred line 36B, Jingsong 3, cytoplasmic male sterile line CMS070 and inbred line SH65-8.

3. A method for identifying which of nine broccoli varieties or strains a broccoli variety or strain is to be tested, comprising the following steps: (1) Using the genomic DNA of the tested broccoli as a template, PCR amplification was performed using primer pair CH19-20; detection; (2) Using genomic DNA of nine Songhuacai varieties or strains as templates, the primer pair CH19-20 was used for PCR amplification; the nine Songhuacai varieties or strains were Jingsong No. 1, cytoplasmic male sterile line CMS060, inbred line 1028-1, Jingsong No. 2, cytoplasmic male sterile line CMS105, inbred line 36B, Jingsong No. 3, cytoplasmic male sterile line CMS070 and inbred line SH65-8; detection; (3) After completing steps (1) and (2), make a judgment based on the following criteria: If the PCR amplification product of the tested Songhuacai is consistent with the PCR amplification product of Jingsong No. 1, the tested Songhuacai is Jingsong No. 1; If the PCR amplification product of the tested Songhuacai is consistent with the PCR amplification product of the cytoplasmic male sterile line CMS060, the tested Songhuacai is the cytoplasmic male sterile line CMS060; If the PCR amplification product of the tested broccoli is consistent with the PCR amplification product of the inbred line 1028-1, the tested broccoli is the inbred line 1028-1; If the PCR amplification product of the Songhuacai to be tested is consistent with the PCR amplification product of Jingsong No. 2, the Songhuacai to be tested is Jingsong No. 2; If the PCR amplification product of the tested Songhuacai is consistent with the PCR amplification product of the cytoplasmic male sterile line CMS105, the tested Songhuacai is the cytoplasmic male sterile line CMS105; If the PCR amplification product of the tested broccoli is consistent with the PCR amplification product of the inbred line 36B, the tested broccoli is the inbred line 36B; If the PCR amplification product of the tested Songhuacai is consistent with the PCR amplification product of Jingsong No. 3, the tested Songhuacai is Jingsong No. 3; If the PCR amplification product of the tested Songhuacai is consistent with the PCR amplification product of the cytoplasmic male sterile line CMS070, the tested Songhuacai is the cytoplasmic male sterile line CMS070; If the PCR amplification product of the tested broccoli is consistent with the PCR amplification product of the inbred line SH65-8, the tested broccoli is the inbred line SH65-8; The primer pair CH19-20 consists of an upstream primer and a downstream primer; The nucleotide sequence of the upstream primer is shown in SEQ ID NO: 1; The nucleotide sequence of the downstream primer is shown in SEQ ID NO:

2.

4. The method according to any one of claims 1 to 3, characterized in that: The detection is agarose gel electrophoresis detection.

5. Application of primer pair CH19-20 in identifying at least one of the three varieties Jingsong 1, Jingsong 2 and Jingsong 3; The primer pair CH19-20 consists of an upstream primer and a downstream primer; The nucleotide sequence of the upstream primer is shown in SEQ ID NO: 1; The nucleotide sequence of the downstream primer is shown in SEQ ID NO:

2.

6. Application of primer pair CH19-20 in identifying 9 varieties or strains of Songhuacai; the 9 varieties or strains of Songhuacai are Jingsong No. 1, cytoplasmic male sterile line CMS060, inbred line 1028-1, Jingsong No. 2, cytoplasmic male sterile line CMS105, inbred line 36B, Jingsong No. 3, cytoplasmic male sterile line CMS070 and inbred line SH65-8; The primer pair CH19-20 consists of an upstream primer and a downstream primer; The nucleotide sequence of the upstream primer is shown in SEQ ID NO: 1; The nucleotide sequence of the downstream primer is shown in SEQ ID NO:

2.

7. Application of the kit containing primer pair CH19-20 is a1) or a2): a1) Identify at least one of the three varieties: Jingsong No. 1, Jingsong No. 2 and Jingsong No. 3; a2) Application in identifying 9 varieties or strains of Songhuacai; the 9 varieties or strains of Songhuacai are Jingsong No. 1, cytoplasmic male sterile line CMS060, inbred line 1028-1, Jingsong No. 2, cytoplasmic male sterile line CMS105, inbred line 36B, Jingsong No. 3, cytoplasmic male sterile line CMS070 and inbred line SH65-8; The primer pair CH19-20 consists of an upstream primer and a downstream primer; The nucleotide sequence of the upstream primer is shown in SEQ ID NO: 1; The nucleotide sequence of the downstream primer is shown in SEQ ID NO:2.

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