A primer set, a kit for identifying pinellia ternata varieties and application thereof

Abstract

This disclosure provides a primer set, a kit, and their applications for Pinellia ternata varieties. The primer set includes primer pairs 1 to 583, each primer pair including a forward primer and a reverse primer. The forward primer of primer pair 1, the reverse primer of primer pair 1, the forward primer of primer pair 583, and the reverse primer of primer pair 583 are shown in sequence as SEQ ID NO: 1 to SEQ ID NO: 1166 in the sequence listing. Using this primer set, DNA fingerprint data of the sample to be tested can be obtained through multiplex PCR amplification and high-throughput sequencing. The genetic similarity coefficient can then be obtained by comparing the sample with a control sample, leading to a variety identification conclusion between the sample to be tested and the control sample. The accuracy and digitization are both high. Sequence analysis software can be used to compare hundreds or thousands of samples at once to quickly obtain variety identification conclusions, which significantly improves the accuracy and efficiency of Pinellia ternata variety identification.

Description

Technical Field

[0001] This disclosure relates to the field of molecular identification technology, and in particular to a primer set, reagent kit, and application for identifying Pinellia ternata varieties. Background Technology

[0002] Pinellia ternata ( Pinellia ternata *Pinellia ternata* (Thunb.) Breit. is a perennial herb belonging to the genus *Pinellia* in the family Araceae. Its dried tubers are used medicinally and are a commonly used ingredient in Traditional Chinese Medicine. In recent years, with the increasing demand for *Pinellia ternata*, the market has seen the frequent sale of closely related species or counterfeit products such as *Pinellia ternata* and *Pinellia scabra*, seriously harming consumer rights and medication safety. Since plants in the *Pinellia* genus are highly similar in morphology, it is often difficult to distinguish different varieties with the naked eye. Therefore, research on the genetic background of *Pinellia ternata* and strengthening the management of new varieties have become particularly important.

[0003] Due to the lack of a standard method for identifying Pinellia ternata varieties, traditional morphological methods are often used. In recent years, research on the identification of Pinellia ternata species and analysis of genetic diversity based on molecular marker technology has largely employed SSR (Inter-simple sequence repeat) molecular marker technology. SSRs primarily utilize the highly conserved flanking sequences of SSRs to design specific primers for PCR (Polymerase Chain Reaction) amplification and agarose gel electrophoresis, followed by polymorphism analysis based on the electrophoretic bands. However, this method suffers from limited throughput and insufficient polymorphism, making it difficult to distinguish all varieties and meet the needs of large-scale sample testing. Furthermore, during the PCR reaction, DNA polymerase slippage upon binding to the template can lead to non-specific amplification, making it difficult to distinguish slipped genotype bands from the true genotype bands of the sample being tested, thus affecting the accuracy of genotyping. Therefore, developing a detection technology that can accurately identify Pinellia ternata varieties has become an urgent technical problem to be solved. Public content

[0004] To address the problems of existing technologies, this disclosure provides a primer set, a reagent kit, and their applications for identifying Pinellia ternata varieties. The technical solution is as follows: On one hand, embodiments of the present invention provide a primer set for identifying Pinellia ternata varieties, the primer set comprising: primer pair 1 to primer pair 583, each primer pair comprising a forward primer and a reverse primer, the forward primer of primer pair 1, the reverse primer of primer pair 1 to the forward primer of primer pair 583 and the reverse primer of primer pair 583 are as shown in SEQ ID NO: 1 to SEQ ID NO: 1166 in the sequence listing.

[0005] On the other hand, embodiments of the present invention provide a kit for identifying Pinellia ternata varieties, the kit comprising the aforementioned primer set.

[0006] In another aspect, embodiments of the present invention provide an application of the above-mentioned primer set in the identification of Pinellia ternata varieties, the application including: The DNA of the test sample was amplified by multiplex PCR using the above primer set to obtain multiplex PCR amplification products; Purify the multiplex PCR amplification product; A high-throughput sequencing library was constructed using the purified multiplex PCR amplification products to obtain the high-throughput library of the sample to be tested. Purify the high-throughput library of the test sample; The high-throughput library of the sample to be tested was sequenced to obtain the sequencing data; Analyze the sequencing data to obtain DNA fingerprint data; The DNA fingerprint data is compared with control samples to obtain the genetic similarity coefficient; Based on the genetic similarity coefficient, a variety identification conclusion is obtained between the tested sample and the control sample.

[0007] Specifically, obtaining the variety identification conclusion between the test sample and the control sample based on the genetic similarity coefficient includes: when the genetic similarity coefficient is greater than or equal to 99%, determining that the test sample and the control sample are extremely similar varieties or the same variety.

[0008] The beneficial effects of the technical solution provided in this disclosure are as follows: This invention provides a primer set, kit, and application for identifying Pinellia ternata varieties. Using this primer set, DNA fingerprint data of the sample to be tested can be obtained through multiplex PCR amplification and high-throughput sequencing. The genetic similarity coefficient is then obtained by comparing the DNA fingerprint data with a control sample, and the variety identification conclusion is derived based on the genetic similarity coefficient. The DNA fingerprint data consists of multiple MNP-labeled base sequences obtained after sequencing, achieving single-base resolution with high accuracy and digitization. Hundreds or thousands of samples can be compared simultaneously using sequence analysis software to quickly obtain variety identification conclusions, significantly improving the accuracy and efficiency of Pinellia ternata variety identification. The primer set provided by this invention has good application value in Pinellia ternata variety identification and DNA fingerprint database construction, providing technical support for the intellectual property protection of Pinellia ternata varieties in my country and promoting the healthy development of the industry. Attached Figure Description

[0009] To more clearly illustrate the technical solutions in the embodiments of this disclosure, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this disclosure. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0010] Figure 1 This is a distribution map of the number of MNP marker detection sites in the sample to be tested provided in Embodiment 3 of this disclosure, where the horizontal axis represents the sample to be tested and the vertical axis represents the number of MNP sites. Figure 2 This is a differential proportion distribution map of MNP marker sites provided in Embodiment 3 of this disclosure. Detailed Implementation

[0011] To make the objectives, technical solutions, and advantages of this disclosure clearer, the embodiments of this disclosure will be described in further detail below with reference to the accompanying drawings. Example 1

[0012] This invention provides a primer set for identifying Pinellia ternata varieties. The primer set includes primer pairs 1 to 583, each primer pair including a forward primer and a reverse primer. The forward primer of primer pair 1, the reverse primer of primer pair 1 to the forward primer of primer pair 583 and the reverse primer of primer pair 583 are shown in sequence as SEQ ID NO: 1 to SEQ ID NO: 1166 in the sequence listing, as shown in Table 1.

[0013] Table 1 shows the sequences of 583 primer pairs.

[0014] 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. Example 2

[0015] This invention provides a kit for identifying Pinellia ternata varieties, which uses the primer set provided in Example 1. Example 3

[0016] This invention provides an application for identifying Pinellia ternata varieties using the primer set provided in Example 1. This application includes: Multiplex PCR amplification was performed on the DNA of the sample to be tested using a primer set to obtain multiplex PCR amplification products; Purify multiplex PCR amplification products; High-throughput sequencing libraries were constructed using purified multiplex PCR amplification products to obtain high-throughput libraries of the samples to be tested. Purifying high-throughput libraries of test samples; Sequencing data was obtained by sequencing the high-throughput library of the sample to be tested. Analyze sequencing data to obtain DNA fingerprint data; The genetic similarity coefficient is obtained by comparing DNA fingerprint data with control samples; Based on the genetic similarity coefficient, the variety identification conclusion between the test sample and the control variety is obtained.

[0017] Specifically, based on the genetic similarity coefficient, the variety identification conclusion between the test sample and the control sample is obtained, including: when the genetic similarity coefficient is greater than or equal to 99%, the test sample and the control sample are determined to be extremely similar varieties or the same varieties.

[0018] In this embodiment, the samples to be tested are 30 Pinellia ternata varieties collected by the project unit. The samples mainly come from Qianjiang, Tianmen, Xiantao, Jingshan, Zhongxiang and Songzi in Hubei Province. The medicinal materials are mainly wild resources. Samples from different places of origin are distinguished as varieties. The information of the 30 Pinellia ternata samples to be tested provided in this embodiment is shown in Table 2.

[0019] Table 2 contains information on 30 samples of Pinellia ternata to be tested.

[0020] DNA extraction from the test samples: The rhizome DNA of the above 30 Pinellia ternata varieties was extracted using the Plant Genomic DNA Extraction Kit (catalog number: DP320) produced by Tiangen Biotech (Beijing) Co., Ltd. For detailed operation steps, please refer to the instruction manual of the kit. DNA from 30 test samples was obtained. Then, 1 μL of DNA from each test sample was taken and the DNA concentration of the test sample was determined using a Qubit fluorescence quantitative quantitation instrument. In this example, the DNA concentration of the test samples was measured to be between 20 ng / μL and 50 ng / μL.

[0021] The DNA of the test sample was amplified by multiplex PCR using the primer set provided in the embodiments of the present invention to obtain multiplex PCR amplification products.

[0022] Specifically, each amplification reaction for a test sample included: 4 μL of the primer set provided in this embodiment of the invention, 4 μL of DNA from the test sample, 10 μL of GenoPlexs 3×T Master Mix (manufacturer: Shijiazhuang Borui Biotechnology Co., Ltd.), and 12 μL of water. The mixture was vortexed and mixed to obtain a homogenate. This homogenate was used as the multiplex PCR amplification system for multiplex PCR amplification. The multiplex PCR amplification program was: 95℃ for 3 min; (95℃ for 20 sec, 60℃ for 4 min) × 17 cycles; 72℃ for 4 min to obtain the multiplex PCR amplification product.

[0023] Purify the multiplex PCR amplification products.

[0024] Specifically, the amplification products after the reaction were then purified using DNA purification magnetic beads (manufacturer: Nanjing Novizan Biotechnology Co., Ltd.). For detailed operating procedures, please refer to the product's instruction manual.

[0025] High-throughput sequencing libraries were constructed using purified multiplex PCR amplification products to obtain high-throughput libraries of the samples to be tested.

[0026] Specifically, 10 μL of GenoPlexs 3×T Master Mix, 2 μL of 5 μM Illumina sequencing adapter primers (manufactured by Borui Biotechnology Co., Ltd.), and 16 μL of water were added to the purified multiplex PCR amplification product, and the PCR amplification reaction was performed according to the following program: 95℃ for 3 min; (95℃ for 15 s, 58℃ for 15 s, 70℃ for 30 s) × 8 cycles; a final extension at 72℃ for 5 min, and the reaction was terminated at 16℃. After the reaction, a high-throughput sequencing library of the sample to be tested was obtained.

[0027] Purify the high-throughput library of the sample to be tested. Specifically, use DNA purification magnetic beads to purify the high-throughput sequencing library to obtain the purified high-throughput sequencing library. Refer to the instructions for the DNA purification magnetic bead product for specific purification methods.

[0028] The high-throughput library of the sample to be tested was sequenced to obtain sequencing data. Specifically, the high-throughput sequencing library was sequenced using an Illumina NextSeq1000 sequencer to obtain sequencing data of the sample to be tested. For detailed sequencing steps, please refer to the instruction manual of the sequencer.

[0029] Sequencing data was analyzed to obtain DNA fingerprint data. Specifically, sequence analysis was performed using Bowtie2 (version 2.1.0) software to obtain the MNP-tagged DNA sequence for each sample.

[0030] Detection rate of MNP markers Multiplex PCR amplification and sequencing library construction were performed using the primer set provided in the embodiments of the present invention. Multiplex amplification, second-generation high-throughput sequencing and data analysis were performed on these 30 Pinellia ternata DNA samples. The 583 markers were detected in all 30 samples to be tested. For specific detection information, please refer to Table 3.

[0031] Table 3 shows the detection information for 30 Pinellia ternata varieties. Table 3 shows that the highest number of detected markers was 576 for the Pinellia ternata variety, with an average of 542.33 MNP markers detected per variety, resulting in an average detection rate of 93.02%. The distribution of MNP marker detection sites in the tested samples is shown below. Figure 1 As shown.

[0032] Accuracy Analysis To verify the accuracy of the primer pairs, reproducibility experiments were performed on eight test samples (including two independent experiments conducted by different personnel, using different batches of reagents, and using different instruments). The data from the two experiments for each test sample were compared and analyzed, and the accuracy of the genotyping was calculated using the formula: accuracy = 1 - (1 - precision) / 2. Precision refers to the proportion of MNP marker sites whose genotyping results are consistent between the two experiments. The statistical results are shown in Table 4.

[0033] Table 4 shows the accuracy of 583 Pinellia ternata MNP marker sites. As shown in Table 4, a total of 4299 MNP markers were compared, with 13 non-repeating sites, resulting in a typing accuracy of 99.85%. High marker accuracy indicates that the identification results are not affected by different personnel, different batches of reagents, or different instruments, providing technical support for the sharing of DNA fingerprint data.

[0034] Distinguishing properties of MNP-marked varieties All detected MNP marker genotypes from the 30 test samples were compared pairwise. Based on the principle that a difference of at least one SNP at the same MNP marker locus across different varieties is considered significant, the number of differentially expressed MNP markers in each pairwise comparison of the 30 test samples was counted, resulting in 435 pairs of comparisons. The average number of differentially expressed marker loci per test sample was 316.31, with an average differential proportion of 60.78%. The differential proportion distribution is shown below. Figure 2 As shown. By Figure 2 It can be seen that the selected MNP markers have high polymorphism and can significantly distinguish between any half of the summer varieties.

[0035] Pinellia ternata variety identification DNA fingerprint data is compared with control samples to obtain a genetic similarity coefficient. Based on the genetic similarity coefficient, the variety of the test sample is identified. A variety identification conclusion is obtained between the test sample and the control sample based on the genetic similarity coefficient, specifically: when the genetic similarity coefficient is greater than or equal to 99%, the test sample and the control sample are determined to be very similar varieties or the same variety. The control sample can be amplified and sequenced together with the test sample to obtain DNA fingerprint data, or it can be amplified and sequenced beforehand and then compared with the test sample, depending on the actual situation.

[0036] Specifically, a new rhizome was collected from the test sample BX008, named BX-CS, and tested according to the above experimental procedure. The DNA fingerprint data was then compared with that of the 30 test samples to obtain the genetic similarity coefficient. Referring to the existing national standard "MNP Marker Method for Plant Variety Identification," the genetic similarity coefficient was used as the basis for determining the variety identification conclusion. When the genetic similarity coefficient (GS) was greater than or equal to 99%, the test sample and the control sample were determined to be "very similar varieties or the same variety." The results are shown in Table 5.

[0037] Table 5 shows the comparison results of DNA fingerprint data between BX-CS and 30 samples to be tested. As shown in Table 5, if the number of differing loci between BX-CS and BX-008 samples from the same source is 0 and the genetic similarity (GS value) reaches 100%, they are identified as potentially similar varieties. However, if the number of differing loci between these samples and other Pinellia ternata samples is significant (221-368 loci) and the GS value is extremely low (less than or equal to 59.82%), they are identified as different varieties. Therefore, the primer set provided in this embodiment of the invention can be used to compare and analyze the DNA fingerprint data of the collected samples with existing data for accurate identification of Pinellia ternata varieties.

[0038] The above description is merely an optional embodiment of this disclosure and is not intended to limit this disclosure. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this disclosure should be included within the protection scope of this disclosure.

Claims

1. A primer set for identifying Pinellia ternata varieties, characterized in that, The primer set includes primer pairs 1 to 583, each primer pair including a forward primer and a reverse primer. The forward primer of primer pair 1, the reverse primer of primer pair 1 to the forward primer of primer pair 583 and the reverse primer of primer pair 583 are shown in sequence as SEQ ID NO: 1 to SEQ ID NO: 1166 in the sequence listing.

2. A reagent kit for identifying Pinellia ternata varieties, characterized in that, The kit includes the primer set as described in claim 1.

3. The application of a primer set as described in claim 1 in the identification of Pinellia ternata varieties, characterized in that, The applications include: Multiplex PCR amplification of the DNA of the test sample was performed using the primer set described in claim 1 to obtain multiplex PCR amplification products; Purify the multiplex PCR amplification product; A high-throughput sequencing library was constructed using the purified multiplex PCR amplification products to obtain the high-throughput library of the sample to be tested. Purify the high-throughput library of the test sample; The high-throughput library of the sample to be tested was sequenced to obtain the sequencing data; Analyze the sequencing data to obtain DNA fingerprint data; The DNA fingerprint data is compared with control samples to obtain the genetic similarity coefficient; Based on the genetic similarity coefficient, a variety identification conclusion is obtained between the tested sample and the control sample.

4. The application according to claim 3, characterized in that, Based on the genetic similarity coefficient, a variety identification conclusion is obtained between the test sample and the control sample, including: when the genetic similarity coefficient is greater than or equal to 99%, the test sample and the control sample are determined to be extremely similar varieties or the same varieties.

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