An MNP marker site, primer set, kit, and identification method for identifying *Cynanchum paniculatum* and *Trichosanthes kirilowii*.

Abstract

This invention belongs to the field of molecular identification technology, specifically relating to an MNP marker site, primer set, reagent kit, and identification method for identifying *Cynanchum paniculatum* and *Gnaphalium affine*. The primer set includes primer pairs 1 to 20, as shown in SEQ ID NO: 1 to SEQ ID NO: 40. The MNP marker site provided by this invention exhibits high polymorphism and strong variety identification ability, meeting the need to distinguish between similar but actually different Chinese medicinal materials (*Cynanchum paniculatum* and *Gnaphalium affine*). Using this primer set, DNA fingerprint data of the test samples can be obtained through multiplex PCR amplification and high-throughput sequencing. Variety identification conclusions are then obtained by comparing the DNA fingerprint data between the test samples. This detection method has high accuracy and a high degree of digitization. Multiple test samples can be compared simultaneously using sequence analysis software to quickly obtain variety identification conclusions, significantly improving the accuracy and efficiency of identifying the Chinese medicinal materials *Cynanchum paniculatum* and *Gnaphalium affine*.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular identification, and particularly relates to an MNP marker locus, a primer set, a kit and an identification method for identifying Cynanchum atratum and Cynanchum komarovii Background Art

[0002] Cynanchum komarovii and Cynanchum atratum are two traditional Chinese medicines with different functions and are easily confused in the market. Cynanchum komarovii has medicinal values of relieving cough and asthma, anti-inflammatory and antibacterial, and is derived from the dried aerial parts of the perennial herb Cynanchum komarovii of the Asclepiadaceae family. Cynanchum atratum has the medicinal value of clearing away deficiency-heat, and is the root and rhizome of the perennial herb Cynanchum atratum or Cynanchum versicolor of the Asclepiadaceae family. Cynanchum atratum and Cynanchum komarovii are similar in morphological characters, but contain different medicinal components and have completely different medicinal effects. Cynanchum komarovii contains alkaloids and volatile oils, and has effects such as analgesia, anti-inflammatory and antibacterial, relieving cough and asthma, and is used to treat diseases such as fullness of the lung qi, excessive phlegm in cough, chest fullness and dyspnea. The whole plant of Cynanchum komarovii contains a substance called 7-demethoxytylophorine, which has irreversible toxicity to the central nervous system of animals. Cynanchum atratum contains components such as volatile oils and cardiac glycosides, and is a heat-clearing and blood-cooling medicine, and is mostly used for the treatment of febrile diseases caused by pathogenic warm factors injuring nutrient qi, yin deficiency fever, bone-steaming fever, postpartum blood deficiency fever, heat strangury, blood strangury and carbuncles and sores. The rhizomes of Cynanchum komarovii and Cynanchum atratum are similar in shape, and the phenomenon of Cynanchum komarovii posing as Cynanchum atratum in the market is obvious. Due to the neurotoxicity of Cynanchum komarovii, accurate identification is required to ensure the safety of medication.

[0003] At present, the identification of Cynanchum atratum and Cynanchum komarovii can be carried out from the aspects of morphology and chemical analysis, but such identification methods are highly subjective and inaccurate. AFLP (Amplified Fragment Length Polymorphism) and RAPD (Random Amplified Polymorphic DNA) molecular marker techniques can also be used to identify the two, but the repeatability of RAPD is poor and it cannot distinguish homozygotes and heterozygotes; although AFLP has high repeatability and good stability, the operation steps are cumbersome and the consumable cost is high. Therefore, it is particularly important to develop a molecular marker technique with advantages such as high stability, strong pertinence and simple operation to identify the two more accurately and quickly, so as to ensure the safety of clinical medication and the stability of the market prices of the two. Summary of the Invention

[0004] To solve the problems in the background art, the present invention provides an MNP marker locus for identifying Cynanchum atratum and Cynanchum komarovii.

[0005] Another object of the present invention is to provide a primer set based on the above MNP marker locus and a kit containing the primer set, which can accurately distinguish Cynanchum atratum and Cynanchum komarovii.

[0006] This invention also provides a method for identifying *Cynanchum paniculatum* and *Trichosanthes kirilowii* using the above-mentioned primer set or kit. This method has strong distinguishing power, high identification throughput, and accurate results; and it greatly simplifies the operation steps and reduces the identification cost.

[0007] The technical solution of the present invention to solve the above-mentioned technical problems is as follows:

[0008] This invention provides an MNP marker site for identifying the Chinese medicinal herbs Baiwei and Laoguatou. The MNP marker site is located in a genomic region with multiple nucleotide polymorphisms in Baiwei and Laoguatou, including MNP-1 to MNP-20.

[0009] The present invention also provides a primer set for identifying the Chinese medicinal materials Baiwei and Laoguatou. The primer set is a multiplex PCR primer composition for detecting the above-mentioned MNP marker sites. The multiplex PCR primer composition includes 20 pairs of primers. The primer set includes primers as shown in SEQ ID NO: 1 to SEQ ID NO: 40 in the sequence listing.

[0010] Furthermore, the primers for each MNP marker site include an upstream primer and a downstream primer, as shown in Table 1 of the specification. Specifically, the upstream primer for sequence number 1 is SEQ ID NO.1, the downstream primer for sequence number 1 is SEQ ID NO.2, the upstream primer for sequence number 2 is SEQ ID NO.3, the downstream primer for sequence number 2 is SEQ ID NO.4, the upstream primer for sequence number 3 is SEQ ID NO.5, the downstream primer for sequence number 3 is SEQ ID NO.6, and so on.

[0011] The present invention further provides a kit containing the above-mentioned primer set for identifying Baiwei and Laoguatou.

[0012] This invention also provides a method for identifying *Cynanchum paniculatum* and *Trichosanthes kirilowii* using the above primer set, comprising the following steps:

[0013] (1) The DNA of the sample to be tested was amplified by multiplex PCR using the primer set to obtain the multiplex PCR amplification product, and the product was purified.

[0014] (2) Construct a high-throughput sequencing library based on the purified multiplex PCR amplification product, obtain a high-throughput library of the sample to be tested, and purify the high-throughput library;

[0015] (3) Sequencing the high-throughput library of the sample to be tested to obtain sequencing data;

[0016] (4) Analyze sequencing data to obtain DNA fingerprint data;

[0017] (5) Compare the DNA fingerprint data of the samples to be tested, determine the genetic similarity coefficient of the samples to be tested based on the MNP marker sites, and identify the variety of the samples to be tested based on the obtained genetic similarity coefficient;

[0018] This includes determining that the tested sample and the control sample are suspected to be of the same variety when the genetic similarity coefficient is greater than or equal to 96%. The formula for calculating the genetic similarity coefficient is:

[0019]

[0020] Where GS is the genetic similarity coefficient between the test sample and the control sample, n ij N represents the number of marker sites detected in both the test sample and the control sample, but with no difference in genotype. ij This represents the number of marker sites detected in both the test sample and the control sample.

[0021] The beneficial effects of this invention are:

[0022] (1) The primer set provided by the present invention is used to identify Baiwei and Laoguatou. Multiplex PCR amplification is used, and the amplification products are sequenced by integrating a second-generation sequencing platform to achieve the advantages of multi-target, high-throughput, high-efficiency and high-accuracy detection of Baiwei and Laoguatou. Moreover, the primer set has a high individual discrimination, and also has a high reproducibility and accuracy. Multiple targets can be detected at one time by multiplex PCR, thereby effectively avoiding the problems of high false negatives and low sensitivity caused by the failure of a single target amplification.

[0023] (2) Furthermore, the primer set provided by this invention provides high accuracy for detection. Combined with a second-generation high-throughput sequencer, the amplified products can be sequenced hundreds of times, outputting a base sequence. Therefore, parallel experiments are unnecessary, and data can be compared arbitrarily, resulting in strong data sharing. The kit using this primer set possesses all the above advantages, effectively ensuring the reproducibility and accuracy of the detection, thereby ensuring clinical drug safety and protecting consumer interests.

[0024] (3) The detection kit provided by the present invention has high repeatability and good stability, is easy to operate, and has low consumable cost. Attached Figure Description

[0025] Figure 1 This is a distribution map of the number of MNP marker sites detected in Baiwei and Laoguatou in Example 2 of the present invention;

[0026] Figure 2 This is a genetic clustering diagram of *Cynanchum paniculatum* and *Gnaphalium affine* based on MNP markers in Embodiment 2 of the present invention. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of the present invention clearer, the embodiments of the present invention will be described in further detail below.

[0028] Example 1: Screening of MNP marker sites for identification of Baiwei and Laoguatou and design of primers for multiplex PCR amplification.

[0029] First, this invention uses *Cynanchum paniculatum* as a reference genome. Combining the sequencing data of *Cynanchum paniculatum* and *Gnaphalium affine*, it employs Samtools (Version 1.2) and BCFtools (Version 1.2) to perform site polymorphism comparison analysis, and screens MNP markers according to the following principles:

[0030] (1) The marker sequence is shared by Baiwei and Laoguatou, but does not appear in other species;

[0031] (2) There are multiple discontinuous SNPs in the sequence;

[0032] (3) The marker sequence length is between 200 and 300 bp. Based on the above screening principles, 20 MNP marker sites with high polymorphism were finally selected.

[0033] Secondly, multiplex PCR primer sets were designed based on the aforementioned MNP marker sites. Primer design followed the principle of non-interference between primers, and all primers could be combined into a primer pool for multiplex PCR amplification. Finally, primer combinations for the 20 MNP sites described in Table 1 were screened. This primer set includes primer pairs 1 to 20, each containing a forward primer and a reverse primer. The nucleotide sequences of the forward and reverse primers of primer pair 1 are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively, and so on. The nucleotide sequences of the forward and reverse primers of primer pair 20 are shown in SEQ ID:39 and SEQ ID NO:40, respectively. This primer set exhibits high amplification efficiency and high identification accuracy, meeting the requirements for distinguishing between *Baiwei* and *Laoguatou*.

[0034] Example 2: Evaluation of MNP markers, primer compositions, and kits for identifying Baiwei and Laoguatou.

[0035] After synthesizing 20 primer pairs, 5 μL of each primer was mixed in equal volumes to form a 1:1 mixture of forward and reverse primers. The developed MNP markers, primers, and kit were evaluated using 5 samples of *Baiwei* and 4 samples of *Laoguatou* provided by the institution. The detection rate, accuracy, and discrimination of the MNP marker sites were tested. Specific primers are shown in Table 1.

[0036] Table 1. 20 MNP marker sites and their corresponding primer sequences

[0037]

[0038] To evaluate the above parameters, it is first necessary to obtain the DNA sequence information of the MNP markers in the above samples. The specific experimental procedure is as follows:

[0039] DNA extraction was performed to obtain DNA from the samples to be tested. Specifically, DNA was extracted from the above-mentioned *Cynanchum paniculatum* and *Cucumis melo* var. *mongolicum* using a plant genomic DNA extraction kit (manufacturer: Tiangen Biotech (Beijing) Co., Ltd., catalog number: DP320). Detailed operating procedures are described in the kit's instruction manual. After obtaining DNA from the nine samples, 1 μL of each sample was taken to determine the concentration (using a Qubit fluorescence quantitative quantitation system). The DNA concentrations were all within the range of 20 ng / μL–50 ng / μL.

[0040] Multiplex PCR was used to amplify the MNP marker sites, yielding multiplex PCR amplification products. Specifically, 4 μL of the primer set provided in this embodiment, 4 μL of DNA (the amount of DNA needs to reach 200 ng), and 10 μL of GenoPlexs 3×T Master Mix (manufacturer: Shijiazhuang Borui Biotechnology Co., Ltd.) were added to the amplification reaction of each sample, for a total reaction volume of 30 μL. Any remaining volume less than 30 μL was made up with water. After vortexing and mixing, multiplex PCR amplification was performed. The multiplex PCR amplification program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 60℃ annealing for 4 min, for a total of 17 cycles; and 72℃ extension for 4 min. After the reaction, the PCR amplification products were stored at 4℃.

[0041] Purify the PCR product. Purify the amplified DNA (manufacturer: Nanjing Novizan Biotechnology Co., Ltd., product number: N411) using the magnetic bead method. Refer to the product instructions for specific procedures.

[0042] Construct a high-throughput sequencing library. Specifically, the following reagents were added to the purified multiplex PCR amplification product: 10 μL GenoPlexs 3×T Master Mix, 2 μL 5 μM Illumina sequencing adapter primers, and 16 μL water. The PCR reaction was performed according to the following program: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 60℃ annealing for 15 s, 70℃ extension for 30 s, for a total of 8 cycles; final extension at 72℃ for 5 min. After the reaction, a high-throughput sequencing library of the sample was obtained.

[0043] Purify the PCR products. Purify the high-throughput sequencing library (manufacturer: Nanjing Novizan Biotechnology Co., Ltd., catalog number: N411) using magnetic beads, following the product's instructions.

[0044] Library sequencing. The high-throughput sequencing library was sequenced using an Illumina NextSeq1000 sequencer to obtain sequencing data for the sample. Detailed sequencing procedures can be found in the sequencer's manual. After sequencing, the sequencing data was copied to an external hard drive.

[0045] Sequencing data analysis. Using Bowtie2 (version 2.1.0) software, the sequencing data of the samples to be tested were aligned to the *Cynanchum paniculatum* reference genome. The alignment results were saved in SAM (Sequence Alignment / Map format), ultimately obtaining the MNP-tagged DNA sequence for each sample. By comparing these DNA base sequences, the detection rate, accuracy, and discrimination of the MNP marker sites could be analyzed.

[0046] (1) Analysis of MNP marker detection rate

[0047] Multiplex PCR amplification and sequencing library construction were performed using the primer set provided in this embodiment of the invention. Multiplex PCR amplification, next-generation high-throughput sequencing, and data analysis were conducted on the DNA of these 9 test samples. An average of 19.6 MNP markers were detected per test sample, with an average detection rate of 98.3%. The distribution of MNP marker detection sites in the test samples is shown below. Figure 1 As shown. The national standard GB / T 38551-2020 requires that the detection rate of markers during variety identification should not be less than 95%, indicating that the marker developed by this patent meets the requirements for marker detection rate in variety identification applications.

[0048] (2) Accuracy analysis of MNP labeling method

[0049] The accuracy of variety identification ultimately depends on the accuracy of marker locus genotyping. We use the results of reproducibility experiments to calculate precision, and then calculate the accuracy of genotyping. A reproducibility experiment refers to two independent replicate experiments performed by different personnel, using different batches of reagents, and different instruments. Precision refers to the proportion of marker loci whose genotyping results are consistent between the two experiments. Accuracy = 1 - (1 - precision) / 2.

[0050] To verify the accuracy of the MNP labeling method for identifying Baiwei and Laoguatou, this invention conducted a reproducibility experiment on these 9 samples. As shown in Table 2, a total of 177 MNP labeling sites were compared in the reproducibility experiment, and the accuracy rate of the MNP labeling method for labeling site genotyping was 99.72%. The high labeling accuracy indicates that DNA fingerprint data collected from different laboratories or at different times can be accurately compared, providing technical support for the sharing of DNA fingerprint data.

[0051] Table 2 Reproducibility of MNP marker site genotyping results

[0052] (3) Variety differentiation analysis using MNP marker method

[0053] We performed pairwise comparisons of all MNP marker genotypes detected in the five *Baiwei* and four *Laoguatou* samples, obtaining a total of 20 comparison pairs. The proportion of differing MNP markers between each pair of samples was called the inter-sample distance, which directly reflects the discriminatory ability of MNP markers for varieties. The number of differing MNP markers in each pair of these two types of samples was counted. The results showed that the average difference proportion between each pair of samples was 100%, indicating that the MNP markers screened in this invention have high polymorphism and can significantly distinguish between *Baiwei* and *Laoguatou*.

[0054] (4) MNP marker method is used for variety identification of Baiwei and Laoguatou.

[0055] DNA fingerprint data was compared with control samples to obtain genetic similarity coefficients. Based on these coefficients, the variety of the test sample was identified. Specifically, identifying the variety of the test sample based on the genetic similarity coefficient includes: when the genetic similarity coefficient is greater than or equal to 96%, the test sample and control sample are considered to be of similar varieties. Using the 20 MNP marker loci and kit provided in this embodiment of the invention, differential MNP marker loci analysis was performed on 4 samples of *Cynanchum paniculatum* and 3 samples of *Gnaphalium affine* provided by the Shandong Academy of Agricultural Sciences. The identification results of *Cynanchum paniculatum* and *Gnaphalium affine* are shown in Table 3.

[0056] Table 3 Variety identification of Baiwei and Laoguatou

[0057]

[0058] Table 3 shows that, in pairwise comparisons between the four *Cynanchum paniculatum* samples and the three *Gnaphalium affine* samples, the genetic similarity coefficient between *Cynanchum paniculatum* and *Gnaphalium affine* was 0%, indicating they are different varieties. Furthermore, cluster analysis revealed (…). Figure 2 The four samples of *Cynanchum paniculatum* and three samples of *Trichosanthes kirilowii* can be divided into two main categories, with each sample within each of these categories further clustered into its own subcategory. These results demonstrate that the primer set and identification method provided by this invention can significantly distinguish between interspecific and intraspecific varieties, and accurately identify *Cynanchum paniculatum* and *Trichosanthes kirilowii*. This is of great significance for guiding correct clinical medication and standardizing the market order of traditional Chinese medicinal materials.

[0059] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A primer set for identifying the traditional Chinese medicinal materials *Baiwei* and *Laoguatou*, characterized in that, The primer set includes primers as shown in the sequence listing SEQ ID NO: 1 to SEQ ID NO:

40.

2. A reagent kit for identifying *Cynanchum paniculatum* and *Trichosanthes kirilowii*, characterized in that, The kit includes the primer set as described in claim 1.

3. A method for identifying *Cynanchum paniculatum* and *Gnaphalium affine* using the primer set described in claim 1, characterized in that, Includes the following steps: (1) The DNA of the sample to be tested was amplified by multiplex PCR using the primer set to obtain the multiplex PCR amplification product, and the product was purified. (2) Construct a high-throughput sequencing library based on the purified multiplex PCR amplification product, obtain a high-throughput library of the sample to be tested, and purify the high-throughput library; (3) Sequencing the high-throughput library of the sample to be tested to obtain sequencing data; (4) Analyze sequencing data to obtain DNA fingerprint data; (5) Compare the DNA fingerprint data of the samples to be tested, determine the genetic similarity coefficient of the samples to be tested based on the MNP marker sites, and identify the variety of the samples to be tested based on the obtained genetic similarity coefficient.

4. The method according to claim 3, characterized in that, In step (5), when the genetic similarity coefficient is greater than or equal to 96%, the test sample and the control sample are determined to be of the same variety.

5. The method according to claim 3 or 4, characterized in that, The formula for calculating the genetic similarity coefficient is: Where GS is the genetic similarity coefficient between the test sample and the control sample, n ij N represents the number of marker sites detected in both the test sample and the control sample, but with no difference in genotype. ij This represents the number of marker sites detected in both the test sample and the control sample.

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