Primer group, kit and identification method for identifying corn variety

Abstract

The invention provides a primer group and a kit for identifying corn varieties and application of the primer group and the kit. The primer group comprises a first primer pair to a 200th primer pair, each primer pair comprises a forward primer and a reverse primer, and the forward primer of the first primer pair, the reverse primer of the first primer pair to the forward primer of the 200th primer pair and the reverse primer of the 200th primer pair are sequentially shown as SEQ ID NO: 1 to SEQ ID NO: 400 in a sequence table. DNA fingerprint data of to-be-detected samples can be obtained through multiple PCR amplification and high-throughput sequencing by utilizing the primer group, then a variety identification conclusion is obtained by comparing the DNA fingerprint data of the to-be-detected samples, the accuracy and the digitization degree are high, hundreds of to-be-detected samples can be compared at a time through sequence analysis software, the variety identification conclusion is rapidly obtained, and the method is suitable for large-scale popularization and application. The accuracy and efficiency of corn variety identification can be obviously improved.

Description

Technical Field

[0001] The present disclosure relates to the field of molecular identification technology, and in particular to a primer set, a kit and an identification method for identifying corn varieties. Background Art

[0002] As a key global food crop, corn plays an important role in the food, feed, bioenergy and chemical industries, and has a significant impact on food security, sustainable agricultural development and energy transformation. The diversity of corn varieties and the richness of genetic resources provide the basis for breeding and improvement. Therefore, accurate and rapid identification of corn varieties is of great significance for exploring genetic potential, protecting germplasm resources and combating counterfeiting.

[0003] At present, DNA fingerprinting of corn varieties mainly relies on SSR (Simple Sequence Repeats) technology, but this faces a series of challenges. For example, SSR markers amplify one site through one PCR, resulting in limited detection throughput, making it difficult to complete the detection of a large number of marker sites in a short time. In addition, the slippage genotype that is easily produced during the DNA polymerase reaction makes it difficult to accurately distinguish between the true genotype and the slippage variation, increasing the risk of misjudgment. With the deepening of corn genetic research, the resolution of SSR markers has gradually been unable to meet the needs of fine identification of highly similar closely related varieties. At the same time, the cost of SSR technology is relatively high and the workload is large, which poses a challenge to the rapid screening and variety protection of large-scale germplasm resource banks. Therefore, it is necessary to develop more efficient, accurate and cost-effective DNA fingerprinting technology. 。

[0004] Public Content

[0005] In order to solve the problems of the prior art, the present disclosure provides a primer set, a kit and an identification method for identifying corn varieties. The technical solution is as follows:

[0006] On the one hand, an embodiment of the present invention provides a primer set for identifying corn varieties, the primer set comprising: the 1st primer pair to the 200th primer pair, each of the primer pairs comprising a forward primer and a reverse primer, the forward primer of the 1st primer pair, the reverse primer of the 1st primer pair to the forward primers of the 200th primer pair, and the reverse primer of the 200th primer pair are respectively as shown in SEQ ID NO: 1 to SEQ ID NO: 400 in the sequence listing.

[0007] On the other hand, an embodiment of the present invention provides a kit for identifying corn varieties, wherein the kit includes the above-mentioned primer set.

[0008] On the other hand, an embodiment of the present invention provides a method for identifying corn varieties, the method comprising: using the above primer set to identify corn varieties.

[0009] Specifically, the method comprises:

[0010] Performing multiple PCR amplification on the DNA of the sample to be tested using the primer set to obtain multiple PCR amplification products;

[0011] Purifying the multiplex PCR amplification products to obtain purified multiplex PCR amplification products;

[0012] Constructing a high-throughput sequencing library using the purified multiplex PCR amplification products to obtain a high-throughput sequencing library of the sample to be tested;

[0013] Purifying the high-throughput sequencing library of the sample to be tested to obtain a purified high-throughput sequencing library;

[0014] Sequencing the purified high-throughput sequencing library to obtain sequencing data;

[0015] Analyzing the sequencing data to obtain DNA fingerprint data;

[0016] Comparing the DNA fingerprint data with a control sample to obtain a genetic similarity coefficient;

[0017] According to the genetic similarity coefficient, the variety of the sample to be tested is identified.

[0018] Specifically, according to the genetic similarity coefficient, the variety identification conclusion of the sample to be tested is obtained: when the genetic similarity coefficient is greater than or equal to 96%, it is determined that the sample to be tested and the control sample are suspected to be the same variety.

[0019] The technical solution provided by the embodiment of the present disclosure has the following beneficial effects: the embodiment of the present disclosure provides a primer set, a kit and its application for identifying corn varieties. The primer set is used for corn variety identification and has good application value in corn variety identification and DNA fingerprint database construction, providing technical support for the intellectual property protection of corn varieties in my country and promoting the healthy development of the industry. The identification method is based on the MNP marker typing technology of high-throughput sequencing. One PCR can detect 200 marker sites, and one sequencing can detect hundreds of samples to be tested, making its cost low. Then, the marker difference information between hundreds of samples to be tested is obtained by a quick analysis of bioinformatics analysis software, so as to quickly obtain the corn variety identification conclusion, and the typing accuracy of each marker site obtained by the identification method reaches 99.95%, thereby realizing accurate and efficient corn variety identification. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present disclosure, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present disclosure. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0021] Figure 1 is a distribution diagram of the MNP marker detection rate of the sample to be tested provided by the embodiment of the present disclosure, wherein the abscissa is the sample to be tested and the ordinate is the MNP marker site detection rate;

[0022] Figure 2 It is a distribution diagram of the difference ratio of MNP labeling sites between the samples to be tested provided by the embodiments of the present disclosure. DETAILED DESCRIPTION

[0023] In order to make the objectives, technical solutions and advantages of the present disclosure more clear, the embodiments of the present disclosure will be further described in detail below with reference to the accompanying drawings.

[0024] Embodiment 1

[0025] An embodiment of the present invention provides a primer set for identifying corn varieties, the primer set comprising: a first primer pair to a 200th primer pair, each primer pair comprising a forward primer and a reverse primer, the forward primer of the first primer pair, the reverse primer of the first primer pair to the forward primer of the 200th primer pair, and the reverse primer of the 200th primer pair are respectively as shown in SEQ ID NO: 1 to SEQ ID NO: 400 in the sequence table, as specifically shown in Table 1.

[0026] Table 1 shows the sequences of 200 primer pairs

[0027]

[0028]

[0029]

[0030]

[0031]

[0032]

[0033]

[0034] The primer pairs in the above primer set do not interfere with each other, ensuring that each primer pair can react normally in the same amplification reaction and specifically amplify the target gene sequence.

[0035] Embodiment 2

[0036] An embodiment of the present invention provides a kit for identifying corn varieties, wherein the kit adopts and implements a provided primer set.

[0037] Embodiment 3

[0038] The embodiment of the present invention provides a method for identifying corn varieties using the primer set provided in embodiment 1, the method comprising:

[0039] Performing multiple PCR amplification on the DNA of the sample to be tested using the primer set to obtain multiple PCR amplification products;

[0040] purifying the multiplex PCR amplification products to obtain purified multiplex PCR amplification products;

[0041] A high-throughput sequencing library is constructed using the purified multiplex PCR amplification products to obtain a high-throughput sequencing library of the sample to be tested;

[0042] Purifying the high-throughput library of the sample to be tested to obtain a purified high-throughput sequencing library;

[0043] Sequencing the purified high-throughput sequencing library of the sample to be tested to obtain sequencing data;

[0044] Analyze sequencing data to obtain DNA fingerprint data;

[0045] The DNA fingerprint data were compared with the control samples to obtain the genetic similarity coefficient;

[0046] According to the genetic similarity coefficient, the variety of the sample to be tested is identified, specifically, a variety identification conclusion between the sample to be tested and the control variety is obtained.

[0047] Specifically, based on the genetic similarity coefficient, the variety identification conclusion of the sample to be tested includes: when the genetic similarity coefficient is greater than or equal to 96%, it is determined that the sample to be tested and the control sample are suspected to be the same variety.

[0048] In this embodiment, the samples to be tested are 20 corn samples collected by Jianghan University.

[0049] Extracting DNA from the samples to be tested: The DNA of the 20 corn sample leaves was extracted using a plant genomic DNA extraction kit (Cat. No.: DP320) produced by Tiangen Biochemical Technology (Beijing) Co., Ltd. to obtain the DNA of 20 samples to be tested. The operation steps are detailed in the instructions of the kit. Then, 1 μL of the DNA of the samples to be tested was taken and the DNA concentration of the samples to be tested was measured using a Qubit fluorescence quantifier. In this embodiment, the DNA concentration of the samples to be tested was measured to be between 20 ng / μL and 50 ng / μL.

[0050] The primer set provided in the embodiment of the present invention is used to perform multiple PCR amplification on the DNA of the sample to be tested to obtain multiple PCR amplification products.

[0051] Specifically, the amplification reaction of each sample to be tested includes: 4 μL of the primer set provided in the embodiment of the present invention (the concentration of each forward primer and each reverse primer is 0.2 μM), 4 μL of the DNA of the sample to be tested, 10 μL of GenoPlexs 3×T MasterMix (manufacturer: Shijiazhuang Boridi Biotechnology Co., Ltd.) and 12 μL of water, oscillated and mixed to obtain a mixture, and the mixture is used as a multiplex PCR amplification system for multiplex PCR amplification. The multiplex PCR amplification program is shown in Table 2.

[0052] Table 2 shows the multiplex PCR amplification program.

[0053]

[0054] Purify the multiplex PCR amplification products to obtain purified multiplex PCR amplification products. Specifically, use DNA purification magnetic beads (manufacturer: Nanjing Novozyme Biotechnology Co., Ltd.) to purify the obtained multiplex PCR amplification products. The operation steps are detailed in the instructions of the product.

[0055] A high-throughput sequencing library is constructed using the purified multiplex PCR amplification products to obtain a high-throughput sequencing library of the sample to be tested.

[0056] Specifically, 10 μL GenoPlexs 3×T Master Mix, 2 μL 5 μM BGI sequencer adapter primer (produced by Boredy Biotechnology Co., Ltd.) and 16 μL water were added to the purified multiple PCR amplification product, and the PCR amplification reaction was performed according to the following procedure: 95°C for 3 min; (95°C for 15 s, 58°C for 15 s, 70°C for 30 s) × 8 cycles; 72°C for the final extension for 5 min, and the reaction was terminated at 16°C. After the reaction was completed, a high-throughput sequencing library of the sample to be tested was obtained.

[0057] Purify the high-throughput sequencing library of the sample to be tested to obtain a purified high-throughput sequencing library. Specifically, use DNA purification magnetic beads to purify the high-throughput sequencing library of the sample to be tested. The specific purification method refers to the instructions of the DNA purification magnetic beads product.

[0058] The high-throughput sequencing library of the sample to be tested is sequenced to obtain sequencing data. Specifically, the high-throughput sequencing library is sequenced using a BGI sequencer to obtain sequencing data. For detailed sequencing steps, refer to the instruction manual of the sequencer.

[0059] Analyze the sequencing data to obtain DNA fingerprint data. Specifically, use the data alignment software Bowtie2 (version 2.1.0) to align the sequencing data to the maize reference genome to obtain the MNP-labeled DNA sequence of each sample to be tested.

[0060] Detection rate of MNP labeling

[0061] Multiplex PCR amplification and sequencing library construction were performed according to the primer set provided in the embodiment of the present invention. Multiplex PCR amplification, second-generation high-throughput sequencing and data analysis were performed on the DNA of the 20 samples to be tested. 194 to 200 MNP markers could be detected in each sample to be tested. The statistical results are shown in Table 3.

[0062] Table 3 shows the statistical results of MNP marker detection rate of 20 corn samples

[0063] serial number Variety name Number of detected sites M000898 Agricultural University GY798 197 M007308 Long Ping 2008 200 M009706 Haiyu13 200 M011129 Sukeno No.2 197 M011289 Wei Ke 7 195 M011291 Wei Ke 606 200 M011928 LP04 199 M012235 Longdan 53 200 M012384 Fuyuan No. 2 200 M012839 Shannong 201 198 M013655 Beijing 464 194 M016757 DH34 198 M018759 Smooth Jade 168 200 M018805 Longyu No. 11 200 M018851 Xixing Red Glutinous Rice No. 4 199 M018919 Huawan 611 200 M019034 Gold 1510 200 M019044 JH13106 200 M019046 ZF045 199 M019323 Liaodan 588 200

[0064] As shown in Table 3, the average detection rate of the markers in the above corn samples reached 99.4%, and the MNP marker detection rate distribution of the samples to be tested was as follows: Figure 1 shown.

[0065] Accuracy Analysis

[0066] In order to test the accuracy of the primer pair, a reproducibility experiment was conducted on 10 corn samples (including two independent experiments conducted by different personnel, different batches of reagents and different instruments), and the two experimental data of each sample to be tested were compared and analyzed, and the typing accuracy was calculated according to the formula of accuracy = 1-(1-reproducibility) / 2. Among them, reproducibility refers to the proportion of MNP marker sites with consistent typing results in two experiments to all MNP marker sites. The statistical results are shown in Table 4.

[0067] Table 4 shows the statistical results of the reproducibility experiment of 10 corn samples

[0068]

[0069] As shown in Table 4, a total of 2000 MNP markers were compared, the number of non-reproducible sites was 2, and the typing accuracy was 99.95%. The high marker accuracy indicates that the results provided by this embodiment are not affected by different personnel, different batches of reagents and different instruments, which provides technical guarantee for the sharing of DNA fingerprint data.

[0070] Discrimination of MNP-marked varieties: All the MNP-marked genotypes detected in the above 20 samples were compared in pairs. According to the principle that the allele genotype of the same MNP marker locus in different varieties is different by at least 1 SNP, the number of different MNP markers in the 20 samples was counted, and a total of 190 pairs of comparison results were obtained. On average, each sample had 133.2 different marker loci, and the average difference ratio was 67.36%. The difference ratio distribution is as follows: Figure 2 As shown. Figure 2 It can be seen that the screened MNP markers have high polymorphism and can significantly distinguish any corn variety.

[0071] Corn Variety Identification

[0072] The DNA fingerprint data is compared with the control sample to obtain the genetic similarity coefficient; based on the genetic similarity coefficient, it is determined whether the sample to be tested is the same variety as the control sample. Based on the genetic similarity coefficient, the identification conclusion specifically includes: when the genetic similarity coefficient is greater than or equal to 96%, the sample to be tested and the control sample are determined to be suspected to be the same variety; when the genetic similarity coefficient is less than 96%, the sample to be tested and the control sample are determined to be different varieties.

[0073] The DNA of the collected corn variety M012235 was re-extracted and named M012235-21, and tested according to the experimental process provided in Example 1, and then compared with the DNA fingerprint data of 10 samples to be tested among the 20 samples to be tested provided in Example 1 to obtain the genetic similarity coefficient, and the results are shown in Table 5. The genetic similarity coefficient value is used as the basis for the conclusion judgment during variety identification. When the GS is greater than or equal to 96%, the sample to be tested and the control sample are judged to be "suspected to be the same variety".

[0074] Table 5 shows the conclusion of corn variety identification

[0075]

[0076] As shown in Table 5, the number of difference sites between corn variety M012235-21 and corn variety M012235 is 0, and the GS value reaches 100.00%, which is determined to be "suspected same variety"; while the number of difference sites between them and other corn samples is significant, and the GS value is less than 96%, which is determined to be "different varieties"; the results show that the DNA fingerprint of the collected sample to be tested can be compared and analyzed with the existing data through the primer set and identification method provided by the present invention, so as to accurately identify the corn variety.

[0077] According to the primer set and identification method provided in this embodiment, multiple PCR amplification and sequencing can be performed on other corn varieties to obtain DNA fingerprint data, which can then be compared with the sequencing data of the same batch or the existing sequencing data to analyze the marker difference ratio between varieties and obtain variety identification conclusions.

[0078] The above description is only an optional embodiment of the present disclosure and is not intended to limit the present disclosure. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present disclosure shall be included in the protection scope of the present disclosure.

Claims

1. A primer set for identifying corn varieties, characterized in that: The primer set includes: the 1st primer pair to the 200th primer pair, each of the primer pairs includes a forward primer and a reverse primer, and the forward primer of the 1st primer pair, the reverse primer of the 1st primer pair to the forward primers of the 200th primer pair, and the reverse primer of the 200th primer pair are respectively shown as SEQ ID NO: 1 to SEQ ID NO: 400 in the sequence listing.

2. A kit for identifying corn varieties, characterized in that: The kit comprises the primer set as claimed in claim 1.

3. A method for identifying corn varieties, characterized in that: The method comprises: using the primer set of claim 1 to identify corn varieties.

4. The method according to claim 3, characterized in that The method comprises: Performing multiple PCR amplification on the DNA of the sample to be tested using the primer set to obtain multiple PCR amplification products; Purifying the multiplex PCR amplification products to obtain purified multiplex PCR amplification products; Constructing a high-throughput sequencing library using the purified multiplex PCR amplification products to obtain a high-throughput sequencing library of the sample to be tested; Purifying the high-throughput sequencing library of the sample to be tested to obtain a purified high-throughput sequencing library; Sequencing the purified high-throughput sequencing library to obtain sequencing data; Analyzing the sequencing data to obtain DNA fingerprint data; Comparing the DNA fingerprint data with a control sample to obtain a genetic similarity coefficient; According to the genetic similarity coefficient, the variety of the sample to be tested is identified.

5. The method according to claim 4, characterized in that Identifying the variety of the sample to be tested according to the genetic similarity coefficient includes: when the genetic similarity coefficient is greater than or equal to 96%, determining that the sample to be tested and the control sample are suspected to be of the same variety.

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