Molecular marker group, primer and kit related to identification of pseudomaize germplasm variety and detection method of pseudomaize germplasm variety

Abstract

The invention discloses a molecular marker group related to identification of germplasm and variety of false maize, and is characterized in that the molecular marker group is composed of the following 20 molecular markers: phi053k2, bnlg2305k4, umc1545y2, phi080k15, phi065k9, umc1492y13, umc1147y4, phi96100y1, bnlg490y3, umc2115k3, umc1429y7, phi299852y2, bnlg2235y5, phi2333376y1, umc2084w2, JAAS3637, JAAS3706, JAAS3715 and JAAS3790, and the molecular marker group is used for identifying the The corn variety identification and detection kit disclosed by the invention is simple to operate, and the name of an unknown variety can be identified by only carrying out one-time PCR (Polymerase Chain Reaction) operation. A large number of gene loci are arranged in the kit, tens of thousands of different varieties can be distinguished theoretically, the specificity of the primer pair is high, and stutter peaks, A-added incomplete peaks and non-specific amplification peaks are avoided. The invention also prepares an allele typing standard substance as a test standard for sample typing and quality control, and solves the problem that results obtained on different genetic analyzers are inconsistent.

Description

Technical Field

[0001] The invention relates to a primer set for plant variety identification, in particular to a primer set, a kit and a detection method thereof for false corn germplasm variety identification. Background Art

[0002] At present, how to identify the fake corn germplasm varieties is the focus of research and development that technicians in this field have been committed to. The existing corn germplasm fake evidence detection kit adopts a five-color fluorescent composite amplification detection system, and the number of loci arranged in the kit is limited. In the process of identifying the variety to be tested, the variety to be tested can only be compared with the known varieties to draw a conclusion on whether it is authentic. If the conclusion of unauthenticity is drawn, the true strain of the variety to be tested cannot be further determined. In addition, the locus primer pairs selected by the existing corn germplasm fake evidence detection kit are generally specific, and there are many impurity peaks after capillary electrophoresis detection, which brings difficulties to the result analysis, and there are differences in the results obtained on different genetic analyzers. In addition, the existing corn germplasm fake evidence detection kit requires two PCR operations, which is complicated.

[0003] Based on the shortcomings of the existing corn germplasm counterfeit evidence detection kit, it is imperative to invent a corn variety identification detection kit and its detection method.

[0004] This patent mainly solves the problems in the public security, food, drug and environmental protection fields, such as the complicated operation of the existing corn germplasm false evidence detection kit, the limited number of gene loci arranged in the kit, the general specificity of the primer pairs, the inability to determine the true strain of the tested variety after its authenticity is determined, and the inconsistent results obtained on different genetic analyzers. Summary of the invention

[0005] The primary technical problem to be solved by the present invention is to provide a molecular marker group for identifying fake corn germplasm varieties that is easy to operate, has good stability, has scientific and rigorous locus arrangement, has good primer specificity, and can further determine the true strain of the variety to be tested based on the judgment of whether it is authentic.

[0006] Another technical problem to be solved by the present invention is to provide a primer set for amplifying the above molecular marker set.

[0007] The third technical problem to be solved by the present invention is to provide an identification kit containing the above primer set.

[0008] The fourth technical problem to be solved by the present invention is to provide a method for identifying fake corn germplasm varieties using the above-mentioned identification kit.

[0009] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0010] A molecular marker group for identifying pseudo-corn germplasm varieties, the molecular marker group consisting of the following 20 molecular markers: phi 053k2, bnlg2305k4, umc 1545y2, phi 080k 15, phi 065k9, umc 1492y13, umc1147y4, phi96100y1, bnlg490y4, umc 1999y3, umc2115k3, umc 1429y7, phi 299852y2, bnlg2235y5, phi 233376y1, umc2084w2, JAAS3637, JAAS3706, JAAS3715 and JAAS3790.

[0011] A primer set for detecting the above molecular marker group, the primer set consists of the following primer pairs:

[0012] The primer pair of SEQ ID No. 1-2 was used to amplify phi 053k2;

[0013] The primer pair of SEQ ID No. 3-4 was used to amplify bnl g2305k4;

[0014] The primer pair of SEQ ID No. 5-6 was used to amplify umc 1545y2;

[0015] The primer pair of SEQ ID No. 7-8 was used to amplify phi 080k15;

[0016] The primer pair of SEQ ID No. 9-10 was used to amplify phi 065k9;

[0017] The primer pair of SEQ ID No. 11-12 was used to amplify umc 1492y13;

[0018] The primer pair of SEQ ID No. 13-14 was used to amplify umc 1147y4;

[0019] The primer pair of SEQ ID No. 15-16 was used to amplify phi 96100y1;

[0020] The primer pair of SEQ ID No. 17-18 was used to amplify bnl g490y4;

[0021] The primer pair of SEQ ID No. 19-20 was used to amplify umc 1999y3;

[0022] The primer pair of SEQ ID No. 21-22 was used to amplify umc2115k3;

[0023] The primer pair of SEQ ID No. 23-24 was used to amplify umc 1429y7;

[0024] The primer pair of SEQ ID No. 25-26 was used to amplify phi 299852y2;

[0025] The primer pair of SEQ ID No. 27-28 was used to amplify bnl g2235y5;

[0026] The primer pair of SEQ ID No. 29-30 was used to amplify phi 233376y1;

[0027] The primer pair of SEQ ID No. 31-32 was used to amplify umc2084w2;

[0028] The primer pair of SEQ ID No. 33-34 was used to amplify JAAS3637;

[0029] The primer pair of SEQ ID No. 35-36 was used to amplify JAAS3706;

[0030] The primer pair of SEQ ID No. 37-38 was used to amplify JAAS3715;

[0031] The primer pair of SEQ ID No. 39-40 was used to amplify JAAS3790.

[0032] Preferably, the primers are connected to five fluorescent markers, which are FAM, HEX, L552, LSID and L592.

[0033] Preferably, the molecular markers are divided into five groups, and the primer pairs of each group of molecular markers are respectively combined with different fluorescent markers. The specific combinations are as follows:

[0034] The first group consists of the following molecular markers, linked to a FAM fluorescent marker:

[0035] JAAS3790, umc 1147y4, umc2084w2, phi96100y 1;

[0036] The second group consists of the following molecular markers, linked to a HEX fluorescent marker:

[0037] JAAS3715, JAAS3637, phi080k15, umc 1492y13;

[0038] The third group consists of the following molecular markers, linked to the L552 fluorescent marker:

[0039] JAAS3706, bnlg2235y5, phi 233376y1, bnlg2305k4;

[0040] The fourth group consists of the following molecular markers, linked to the LSID fluorescent marker:

[0041] umc 1999y3, phi299852y2, umc2115k3, phi 053k2;

[0042] The fifth group consists of the following molecular markers, linked to the L592 fluorescent marker:

[0043] umc 1429y7, umc 1545y2, bnl g490y4, phi065k9.

[0044] A kit for identifying pseudo-corn germplasm varieties, comprising the above-mentioned primer set.

[0045] A method for identifying fake corn germplasm varieties, the steps are as follows:

[0046] (1) Extracting DNA from corn samples to be tested;

[0047] (2) using the DNA extracted in step (1) as a template, and performing PCR amplification using the primer set described in any one of claims 2 to 4 of the present invention;

[0048] (3) detecting the amplified product by capillary electrophoresis;

[0049] (4) Obtain DNA fingerprint band data of the corn sample to be tested at 20 primer sites, and compare and analyze them with the standard DNA fingerprint band data.

[0050] Preferably, the PCR reaction system is:

[0051] DNA original concentration 1ng / μL, final concentration 0.1ng / μL, reaction volume 1μL; MIX reaction volume 4μL; primer set original concentration 10μM, final concentration 1μM, reaction volume 1μL; ddH 2 0 to make up to 10 μL.

[0052] Preferably, the PCR amplification procedure is:

[0053] Pre-denaturation at 95°C for 10 min for 1 cycle;

[0054] Denaturation at 95°C for 10 s, annealing at 60°C for 35 s, and extension at 72°C for 45 s, for 30 cycles;

[0055] The final extension was performed at 60°C for 20 min for one cycle and then stored at 4°C.

[0056] The present invention has the following technical effects:

[0057] The molecular marker group for identifying and detecting corn varieties and the primer group and kit for amplifying the marker group of the present invention are simple to operate, and the name of an unknown variety can be identified by performing a PCR operation once. The kit has a large number of gene loci arranged in a scientific and reasonable manner, and can distinguish tens of thousands of different varieties. The primer pairs have high specificity, and no stutter peaks, incomplete A addition peaks, or non-specific amplification peaks will appear. The present invention also prepares allele typing standards as test standards for sample typing and quality control, solving the problem of inconsistent results obtained on different genetic analyzers.

[0058] The present invention is further described below in conjunction with specific examples. These examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually performed under conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise defined, all professional and scientific terms used in the text have the same meanings as those familiar to those skilled in the art. In addition, any method and material similar or equivalent to the described content can be applied to the method of the present invention. The preferred implementation methods and materials described in the text are for demonstration purposes only. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 The DNA fingerprint band data of Zhengdan 958 in Example 3;

[0060] Figure 2 This is the DNA fingerprint band data of Jingke 968 in Example 4. DETAILED DESCRIPTION

[0061] The technical content of the present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0062] In order to solve the problems of existing corn germplasm false evidence detection kits in the field of public security food, medicine and environment, such as complicated operation, limited number of loci arrangement, general primer pair specificity, inability to determine the true strain of the tested variety after its authenticity is determined, and inconsistent results obtained on different genetic analyzers, the present invention provides a kit comprising 20 primers and a test method thereof after a large amount of primer screening, redesign, system exploration and experimental verification.

[0063] Example 1 Screening of molecular markers for identification and detection of pseudo-corn germplasm varieties of the present invention

[0064] The SSR primer combination kit for corn variety identification and detection of the present invention is determined by screening and optimizing a large number of primers, taking into account both the uniform distribution of primers on corn chromosomes and the annealing temperatures of different primers in multiplex PCR and the length of amplified products.

[0065] Example 2 Molecular marker set and primer set for identification and detection of pseudo-corn germplasm varieties of the present invention

[0066] The corn variety identification and detection kit provided by the present invention comprises the following 20 primer sets, as shown in Table 1, and also comprises an amplification MIX.

[0067] Table 1

[0068]

[0069]

[0070] The present invention has no special provisions for the synthesis method of the primer, and the primer can be prepared using existing standard techniques. According to the primer sequence provided by the present invention, the synthesis can be achieved by a DNA synthesizer. If you have the corresponding equipment, you can synthesize it yourself; if you do not have it, you can entrust a professional primer synthesis service company to complete it. In short, you only need to master the sequence information of the primer to obtain the corresponding primer.

[0071] The present invention uses capillary electrophoresis with a six-color fluorescence detection system, and each set of primer sequences needs to be labeled with five different fluorescent labels, namely FAM, HEX, L552, LS ID, and L592. The amplification MIX is NUHIR Pro NGS PCR MIX produced by Suzhou Xinhai Company, which contains DNA polymerase, dNTP, buffer, etc.

[0072] The corn variety identification and detection kit provided by the present invention also contains 109 allele typing fragments of the following 20 loci, as shown in Table 2.

[0073] Table 2

[0074]

[0075]

[0076] The present invention has no special provisions for the synthesis method of different types of the above-mentioned loci, and can be prepared using existing standard technologies. The present invention first selects a suitable primer pair to amplify and purify the allele DNA fragments appearing in each SSR locus of each crop variety to be tested. The TA cloning technology is used to construct a recombinant plasmid of the corresponding fragment, and the target fragment is confirmed by DNA sequencing. PCR amplification obtains the fluorescently labeled DNA fragment products of each allele of SSR, mixes each allele DNA fragment in a certain proportion, and performs electrophoresis detection to ensure that the size of each fragment is consistent with the expectation and the peak height on gene analyzers such as GA118 and ABI3130 is basically consistent.

[0077] The corn variety identification and detection kit provided by the present invention contains corn germplasm strains in the promotion catalog of excellent crop varieties in my country / leading varieties in grain and oil production, and has determined the molecular identity cards of more than 100 corn varieties including the current main cultivated varieties.

[0078] The identification and detection method using the corn variety identification and detection kit of the present invention adopts capillary electrophoresis combined with a six-color fluorescence detection system. Twenty pairs of primers are labeled with five different types of fluorescence according to the following quadruple combination, as shown in Table 3.

[0079] Table 3

[0080]

[0081]

[0082] Then, DNA was extracted using conventional methods, and 20-plex PCR reactions were performed in PCR tubes. The reaction products were detected using capillary electrophoresis combined with a six-color fluorescence detection system, in which the primers occupied a total of 5 different fluorescences and the molecular weight internal standard occupied 1 color fluorescence.

[0083] From the above detection method, we can see that this method only requires one PCR reaction to complete the detection of all 20 primers, obtain a specific molecular ID card for each variety, and complete the task of variety identification and detection.

[0084] The DNA extraction in the detection method of the present invention can be carried out according to various methods provided in the prior art to obtain DNA of quality that meets the requirements of PCR amplification. The test materials can be seeds, seedlings, leaves, etc.

[0085] The PCR amplification method in the detection method of the present invention is specifically as follows:

[0086] Reaction system: The reaction liquid volume is 10 ml, and the composition should comply with the provisions of the following table.

[0087] Table 4 PCR amplification reaction system

[0088]

[0089] Reaction procedure:

[0090] Table 5 PCR amplification program

[0091]

[0092]

[0093] The electrophoresis detection in the detection method of the present invention needs to use capillary electrophoresis combined with fluorescence detection.

[0094] The specific method is as follows:

[0095] 1. Sex change

[0096] 9 μL of formamide-internal standard mixture (996 μL formamide + 4 μL Liz500) and 1 μL of PCR product were added to each well of a 96-well plate, and the mixture was centrifuged briefly at 3000 rpm.

[0097] 2. Electrophoresis

[0098] After the sample is taken out, capillary electrophoresis is performed on a genetic analyzer (such as GA118-16A, GA118-16B, GA118-24A, GA118-24B, GA118-24C).

[0099] 3. Data Collection and Analysis

[0100] The raw data were collected using data acquisition software and analyzed using fragment analysis software.

[0101] The standards for detecting and determining corn varieties using the corn variety identification test kit of the present invention are as follows:

[0102] The primers of the present invention are used for detection, and the DNA fingerprint band data of the tested variety at 20 primer sites are obtained, and the DNA molecular identity card specific to each variety is obtained, so as to determine which corn variety the tested variety is. Currently, the only available test kit for detecting fake corn germplasm evidence on the market is the variety authenticity identification test kit, and there is no kit for variety identification detection.

[0103] The efficacy verification experimental data of the present invention are as follows:

[0104] Example 3 Using the kit of the present invention to identify whether the corn seeds sold by seed dealers are Zhengdan 958 There is a phenomenon of OEM sales

[0105] The operation process is as follows:

[0106] (1) DNA extraction

[0107] Five individual DNAs were extracted from the unknown sample A. The extraction method was as follows: the embryo of a single dry seed was peeled off, placed in a 1.5 ml centrifuge tube, 100 μL of chloroform was added and then ground, 300 μL of DNA extraction solution (1 mol / L Tris-HCl 50 ml, 0.5 mol / lEDTA 50 ml, 5 mol / l NaCl 50 ml, SDS 7.5 g, fixed to 500 ml) was added, mixed and centrifuged at 10000 rpm for 2 min, the supernatant was aspirated and added to a 1.5 ml centrifuge tube pre-filled with 300 μL of isopropanol and 300 μL of NaCl solution, the DNA was picked out after being pelleted, washed with 70% ethanol, and 200 μL of TE buffer (1 mol / l Tris-HCl 5 ml, 0.5 mol / l EDTA 1 ml, HCl was added to adjust the pH to 8.0, and the volume was fixed to 500 ml) was added, and it was used after being fully dissolved.

[0108] (2) SSR primer synthesis

[0109] Five different colors of fluorescent FAM, HEX, L552, LS ID, and L592 were labeled at the 5' end of the upstream primer, and the fluorescent primer sequences were synthesized by China Huada Liuhe Co., Ltd. The fluorescent primer sequences are shown in Table 1 above.

[0110] (3) PCR amplification

[0111] The DNA samples were amplified by 20-plex PCR using the SSR primer pairs provided above to form a 20-plex PCR reaction system, and the 20-plex PCR combination is shown in Table 3. The reaction system is shown in Table 4, and the amplification program is shown in Table 5.

[0112] (4) Capillary electrophoresis detection

[0113] Denaturation: Add 9 μL of formamide-internal standard mixture (996 μL formamide + 4 μL Liz500) and 1 μL of PCR product to each well of a 96-well plate, and centrifuge briefly at 3000 rpm.

[0114] Capillary electrophoresis: After taking out the sample, perform capillary electrophoresis on a genetic analyzer (such as GA118-16A, GA118-16B, GA118-24A, GA118-24B, GA118-24C).

[0115] (1) Data collection and analysis

[0116] The raw data were collected using data acquisition software and analyzed using fragment analysis software.

[0117] (6) Test results

[0118] The primer set of the present invention was used for testing, and the DNA fingerprint band data of the tested variety at 20 primer sites were obtained, which were inconsistent with the DNA fingerprint band of Zhengdan 958. According to the identification standard, it was identified that this seed distributor had OEM sales. Figure 1 shown.

[0119] Example 4 Identification of a batch of seeds whose variety names were lost due to human error

[0120] The operation process is as follows:

[0121] (1) DNA extraction

[0122] Five individual DNAs were extracted from the unknown sample A. The extraction method was as follows: the embryo of a single dry seed was peeled off, placed in a 1.5 ml centrifuge tube, 100 μL of chloroform was added and then ground, 300 μL of DNA extraction solution (1 mol / L Tris-HCl 50 ml, 0.5 mol / lEDTA 50 ml, 5 mol / l NaCl 50 ml, SDS 7.5 g, fixed to 500 ml) was added, mixed and centrifuged at 10000 rpm for 2 min, the supernatant was aspirated and added to a 1.5 ml centrifuge tube pre-filled with 300 μL of isopropanol and 300 μL of NaCl solution, the DNA was picked out after being pelleted, washed with 70% ethanol, and 200 μL of TE buffer (1 mol / l Tris-HCl 5 ml, 0.5 mol / l EDTA 1 ml, HCl was added to adjust the pH to 8.0, and the volume was fixed to 500 ml) was added, and it was used after being fully dissolved.

[0123] (2) SSR primer synthesis

[0124] Five different colors of fluorescent FAM, HEX, L552, LS ID, and L592 were labeled at the 5' end of the upstream primer, and the fluorescent primer sequences were synthesized by China Huada Liuhe Co., Ltd. The fluorescent primer sequences are shown in Table 1 above.

[0125] (3) PCR amplification

[0126] The DNA samples were amplified by 20-plex PCR using the SSR primer pairs provided above to form a 20-plex PCR reaction system, and the 20-plex PCR combination is shown in Table 3. The reaction system is shown in Table 4, and the amplification program is shown in Table 5.

[0127] (4) Capillary electrophoresis detection

[0128] Denaturation: Add 9 μL of formamide-internal standard mixture (996 μL formamide + 4 μL Liz500) and 1 μL of PCR product to each well of a 96-well plate, and centrifuge briefly at 3000 rpm.

[0129] Capillary electrophoresis: After taking out the sample, perform capillary electrophoresis on a genetic analyzer (such as GA118-16A, GA118-16B, GA118-24A, GA118-24B, GA118-24C).

[0130] (5) Data collection and analysis

[0131] The raw data were collected using data acquisition software and analyzed using fragment analysis software. Figure 2 shown.

[0132] (6) Test results

[0133] The DNA fingerprint band data of the tested variety at 20 primer sites were obtained using this set of primers, which were consistent with the DNA fingerprint band data of Jingke 968. According to the identification criteria, this batch of seeds was identified as Jingke 968.

Claims

1. A molecular marker group for identifying pseudo-corn germplasm varieties, characterized in that: The molecular marker group consists of the following 20 molecular markers: phi 053k2, bnlg2305k4, umc 1545y2, phi 080k15, phi 065k9, umc1492y13, umc 1147y4, phi96100y1, bnlg490y4, umc 1999y3, umc2115k3, umc 1429y7, phi299852y2, bnlg2235y5, phi 233376y1, umc2084w2, JAAS3637, JAAS3706, JAAS3715 and JAAS3790.

2. A primer set for detecting the molecular marker set according to claim 1, characterized in that: The primer set consists of the following primer pairs: The primer pair of SEQ ID No. 1-2 was used to amplify phi 053k2; The primer pair of SEQ ID No. 3-4 was used to amplify bnlg2305k4; The primer pair of SEQ ID No. 5-6 was used to amplify umc 1545y2; The primer pair of SEQ ID No. 7-8 was used to amplify phi 080k15; The primer pair of SEQ ID No. 9-10 was used to amplify phi 065k9; The primer pair of SEQ ID No. 11-12 was used to amplify umc 1492y13; The primer pair of SEQ ID No. 13-14 was used to amplify umc 1147y4; The primer pair of SEQ ID No. 15-16 was used to amplify phi 96100y1; The primer pair of SEQ ID No. 17-18 was used to amplify bnlg490y4; The primer pair of SEQ ID No. 19-20 was used to amplify umc 1999y3; The primer pair of SEQ ID No. 21-22 was used to amplify umc2115k3; The primer pair of SEQ ID No. 23-24 was used to amplify umc 1429y7; The primer pair of SEQ ID No. 25-26 was used to amplify phi 299852y2; The primer pair of SEQ ID No. 27-28 was used to amplify bnlg2235y5; The primer pair of SEQ ID No. 29-30 was used to amplify phi 233376y1; The primer pair of SEQ ID No. 31-32 was used to amplify umc2084w2; The primer pair of SEQ ID No. 33-34 was used to amplify JAAS3637; The primer pair of SEQ ID No. 35-36 was used to amplify JAAS3706; The primer pair of SEQ ID No. 37-38 was used to amplify JAAS3715; The primer pair of SEQ ID No. 39-40 was used to amplify JAAS3790.

3. The primer set according to claim 2, characterized in that The primers were linked to five fluorescent labels, namely FAM, HEX, L552, LSID and L592.

4. The primer set according to claim 3, characterized in that The molecular markers are divided into five groups, each group is combined with a different fluorescent marker, and the specific combinations are as follows: The first group consists of the following molecular markers, linked to a FAM fluorescent marker: JAAS3790, umc 1147y4, umc2084w2, phi96100y 1; The second group consists of the following molecular markers, linked to a HEX fluorescent marker: JAAS3715, JAAS3637, phi080k15, umc 1492y13; The third group consists of the following molecular markers, linked to the L552 fluorescent marker: JAAS3706, bnlg2235y5, phi 233376y1, bnlg2305k4; The fourth group consists of the following molecular markers, linked to the LSID fluorescent marker: umc 1999y3, phi299852y2, umc2115k3, phi 053k2; The fifth group consists of the following molecular markers, linked to the L592 fluorescent marker: umc 1429y7, umc 1545y2, bnlg490y4, phi065k9.

5. A kit for identifying fake corn germplasm varieties, characterized in that: The kit comprises the primer set according to any one of claims 2 to 4.

6. A method for identifying fake corn germplasm varieties, characterized in that: Here are the steps: (5) extracting DNA from corn samples to be tested; (6) using the DNA extracted in step (1) as a template, and performing PCR amplification using the primer set according to any one of claims 2 to 4 of the present invention; (7) detecting the amplified product by capillary electrophoresis; (8) Obtain DNA fingerprint band data of the corn sample to be tested at 20 primer sites, and compare and analyze them with the standard DNA fingerprint band data.

7. The method for identifying fake corn germplasm varieties according to claim 6, characterized in that: The PCR reaction system is: The original concentration of DNA was 1 ng / μL, the final concentration was 0.1 ng / μL, and the reaction volume was 1 μL; the MIX reaction volume was 4 μL; the original concentration of the primer set was 10 μM, and the final concentration was 1 μM, and the reaction volume was 1 μL; ddH20 was supplemented to 10 μL.

8. The method for identifying fake corn germplasm varieties according to claim 6, characterized in that: The PCR amplification procedure is: Pre-denaturation at 95°C for 10 min for 1 cycle; Denaturation at 95°C for 10 s, annealing at 60°C for 35 s, and extension at 72°C for 45 s, for 30 cycles; The final extension was performed at 60°C for 20 min for one cycle and then stored at 4°C.

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