A primer set, kit and identification method for identifying corn varieties

Abstract

The present disclosure provides a primer set, a kit, and applications thereof for identifying corn varieties. The primer set includes: primer pairs 1 to 200, each primer pair including 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 primers of the 200th primer pair, and the reverse primer of the 200th primer pair are shown in sequence as SEQ ID NO: 1 to SEQ ID NO: 400 in the sequence listing. Using this primer set, DNA fingerprint data of a sample to be tested can be obtained through multiplex PCR amplification and high-throughput sequencing. Variety identification conclusions can then be obtained by comparing the DNA fingerprint data between the samples to be tested, with high accuracy and a high degree of digitization. Hundreds or thousands of samples to be tested can be compared at one time using sequence analysis software to quickly obtain variety identification conclusions, significantly improving the accuracy and efficiency of corn variety identification.

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 a vital role in the food, feed, bioenergy, and chemical industries, significantly impacting food security, sustainable agricultural development, and energy transition. The diversity of corn varieties and the richness of its genetic resources provide the foundation for breeding and improvement. Therefore, accurate and rapid identification of corn varieties is crucial for exploring genetic potential, preserving germplasm resources, and combating counterfeiting.

[0003] At present, DNA fingerprinting of maize varieties mainly relies on SSR (Simple Sequence Repeats) technology, but this faces a series of challenges. For example, SSR markers amplify one site through a single PCR, resulting in limited detection throughput and difficulty in completing the detection of a large number of marker sites in a short period of 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 maize genetic research, the resolution of SSR markers has gradually become 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] To address 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 includes:

[0010] Performing multiplex PCR amplification on the DNA of the sample to be tested using the primer set to obtain multiplex 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] The variety of the sample to be tested is identified according to the genetic similarity coefficient.

[0018] Specifically, based on the genetic similarity coefficient, a 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 of the same variety.

[0019] The technical solutions provided by the embodiments of the present disclosure have the following beneficial effects: The embodiments of the present disclosure provide a primer set, a kit, and their applications for identifying corn varieties. This primer set, used for corn variety identification, has excellent application value in corn variety identification and DNA fingerprint database construction, providing technical support for intellectual property protection of corn varieties in my country and promoting the healthy development of the industry. This identification method is based on MNP marker typing technology using high-throughput sequencing. A single PCR run can detect 200 marker sites, and a single sequencing run can detect hundreds or thousands of test samples, making it relatively low-cost. Bioinformatics analysis software then rapidly analyzes the marker difference information between hundreds or thousands of test samples, allowing for rapid identification of corn varieties. Furthermore, this method achieves a typing accuracy of 99.95% for each marker site, enabling precise 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 following briefly introduces the drawings required for use in the description of the embodiments. 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 any creative work.

[0021] Figure 1 : is a distribution diagram of the MNP marker detection rate of the test sample provided by the embodiment of the present disclosure, wherein the horizontal axis is the test sample and the vertical axis is the MNP marker site detection rate;

[0022] Figure 2 3 is a distribution diagram of the difference ratios of MNP marker 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] Example 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 primers of the 200th primer pair, and the reverse primer of the 200th primer pair being respectively as shown in SEQ ID NO: 1 to SEQ ID NO: 400 in the sequence listing, 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] Example 2

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

[0037] Example 3

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

[0039] Performing multiplex PCR amplification on the DNA of the sample to be tested using the primer set to obtain multiplex 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] DNA fingerprint data were compared with control samples to obtain genetic similarity coefficients;

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

[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] DNA extraction from the test samples: DNA from the 20 corn leaf samples was extracted using the Plant Genomic DNA Extraction Kit (Cat. No. DP320) manufactured by Tiangen Biochemical Technology (Beijing) Co., Ltd., yielding 20 test samples. For detailed procedures, refer to the kit's instructions. 1 μL of DNA from each test sample was then taken and its concentration was measured using a Qubit fluorescence quantifier. In this example, the measured DNA concentrations of the test samples were between 20 ng / μL and 50 ng / μL.

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

[0051] Specifically, the amplification reaction for each sample to be tested included: 4 μL of the primer set provided in the examples of the present invention (each forward primer and each reverse primer had a concentration of 0.2 μM), 4 μL of the DNA of the sample to be tested, 10 μL of GenoPlexs 3×T MasterMix (manufacturer: Shijiazhuang Boruidi Biotechnology Co., Ltd.), and 12 μL of water. The mixture was shaken and mixed to obtain a mixture, which was 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 Novozymes Biotechnology Co., Ltd.) to purify the obtained multiplex PCR amplification products. The operation steps are detailed in the product manual.

[0055] A high-throughput sequencing library was 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 of GenoPlexs 3×T Master Mix, 2 μL of 5 μM BGI sequencer adapter primers (Boridi Biotechnology Co., Ltd.), and 16 μL of water were added to the purified multiplex PCR amplification product. PCR amplification was performed according to the following protocol: 95°C for 3 minutes, followed by 8 cycles of (95°C for 15 seconds, 58°C for 15 seconds, and 70°C for 30 seconds), followed by a final extension at 72°C for 5 minutes, and termination at 16°C. After completion of the reaction, a high-throughput sequencing library for the sample 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. For specific purification methods, refer 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, please refer to the instruction manual of the sequencer.

[0059] The sequencing data were analyzed to obtain DNA fingerprint data. Specifically, the sequencing data were aligned to the maize reference genome using the data alignment software Bowtie2 (version 2.1.0) 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 using the primer sets provided in the embodiments of the present invention. DNA from these 20 test samples was subjected to multiplex PCR amplification, second-generation high-throughput sequencing, and data analysis. 194 to 200 MNP markers were detected in each test sample. The statistical results are shown in Table 3.

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

[0063] serial number Variety name Number of detected sites M000898 Agricultural University GY798 197 M007308 Long Ping 2008 200 M009706 Haiyu 13 200 M011129 Sukenuo 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 Hongnuo 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 is 99.4%. The distribution of the MNP marker detection rates of the samples to be tested is as follows: Figure 1 shown.

[0065] Accuracy analysis

[0066] To verify the accuracy of the primer pairs, reproducibility experiments were conducted on 10 maize samples (including two independent experiments performed by different personnel, using different batches of reagents, and using different instruments). The data from the two experiments for each sample were compared and analyzed, and typing accuracy was calculated using the formula: accuracy = 1 - (1 - reproducibility) / 2. Reproducibility refers to the proportion of MNP-labeled sites with consistent typing results between the two experiments, out of all MNP-labeled 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 2,000 MNP markers were compared, with only two unique sites, resulting in a typing accuracy of 99.95%. This high marker accuracy indicates that the results provided by this example are unaffected by differences in personnel, reagent batches, and instrumentation, providing technical support for the sharing of DNA fingerprint data.

[0070] MNP marker genotype discrimination: All detected MNP marker genotypes of the 20 test samples were compared pairwise. Based on the principle that the allele genotype of the same MNP marker locus in different varieties differed by at least one SNP, the number of MNP markers that differed in the 20 test samples was counted. A total of 190 pairs of comparison results were obtained. On average, each test sample differed by 133.2 marker loci, and the average difference ratio was 67.36%. The difference ratio distribution is shown below: Figure 2 As shown. Figure 2 It can be seen that the screened MNP markers are highly polymorphic and can significantly distinguish any corn variety.

[0071] Corn variety identification

[0072] DNA fingerprint data is compared with a control sample to obtain a genetic similarity coefficient. Based on this coefficient, the test sample is identified as the same variety as the control. The identification conclusions based on the genetic similarity coefficient include: if the genetic similarity coefficient is greater than or equal to 96%, the test sample and the control sample are considered to be the same variety; if the genetic similarity coefficient is less than 96%, the test sample and the control sample are considered to be different varieties.

[0073] DNA was re-extracted from the collected maize variety M012235, designated M012235-21, and tested according to the experimental procedure provided in Example 1. The DNA fingerprint data were then compared with the DNA fingerprint data from 10 of the 20 test samples provided in Example 1 to obtain genetic similarity coefficients. The results are shown in Table 5. The genetic similarity coefficients were used as the basis for variety identification. When the genetic similarity coefficient was greater than or equal to 96%, the test sample and the control sample were considered "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 differential 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 to be the same variety"; while the number of differential sites between them and other corn samples is significant, and the GS value is less than 96%, which is determined to be "different varieties". These results indicate that the primer set and identification method provided by the present invention can compare and analyze the DNA fingerprints of the collected test samples with existing data to accurately identify corn varieties.

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

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

Claims

1. A primer set for identifying corn varieties, characterized in that: The primer set consists of 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 shown in sequence 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 according to claim 1.

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

4. The method according to claim 3, characterized in that The method comprises: Performing multiplex PCR amplification on the DNA of the sample to be tested using the primer set to obtain multiplex 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; The variety of the sample to be tested is identified according to the genetic similarity coefficient.

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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