MNP marker site, primer group, kit and identification method for identifying traditional Chinese medicinal materials of cynanchum atratum, cynanchum glaucescens, cynanchum komarovii and cynanchum paniculatum

Abstract

The invention belongs to the technical field of molecular identification, and particularly relates to an MNP marker site, a primer group, a kit and an identification method for identifying cynanchum atratum, cynanchum glaucescens, cynanchum komarovii and cynanchum paniculatum. The MNP marker sites are in genome regions with a plurality of nucleotide polymorphisms in cynanchum atratum, cynanchum glaucescens, cynanchum komarovii and cynanchum paniculatum, and comprise MNP-1 to MNP-20. The MNP marker provided by the invention has the advantages of large number of sites, high polymorphism and strong variety identification capability, and meets the requirements of distinguishing Chinese medicinal materials (cynanchum atratum, cynanchum glaucescens, cynanchum komarovii and cynanchum paniculatum) with similar shapes but actually different shapes. The DNA fingerprint data of the samples to be detected can be obtained by utilizing the primer group provided by the invention, and then the variety identification conclusion is obtained by comparing the DNA fingerprint data of the samples to be detected. According to the detection method, hundreds of to-be-detected samples can be compared at one time, a variety identification conclusion can be quickly obtained, and the accuracy and efficiency of identifying the traditional Chinese medicinal materials such as cynanchum atratum, cynanchum glaucescens, cynanchum komarovii and cynanchum paniculatum can be remarkably improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular identification, and particularly relates to MNP marker sites, primer sets, kits and identification methods for identifying Cynanchum atratum, Cynanchum glaucescens, Cynanchum komarovii and Cynanchum paniculatum. Background Art

[0002] Cynanchum atratum, Cynanchum glaucescens, Cynanchum komarovii and Cynanchum paniculatum are plants from the same family and genus, all belonging to the genus Cynanchum of the Asclepiadaceae family. Their appearance traits are very similar, and they are extremely prone to confusion during acquisition, distribution and application. Cynanchum glaucescens has the medicinal value of warming and resolving cold-phlegm, and is derived from the roots and rhizomes of the perennial herb Cynanchum stauntonii (Decne.) Schltr. ex H. Lév. and Cynanchum glaucescens (Decne.) Hand.-Mazz. of the Asclepiadaceae family. Cynanchum atratum has the medicinal value of clearing and removing deficiency-heat, and is the roots and rhizomes of the perennial herb Cynanchum atratum Bunge or Cynanchum versicolor Bge. of the Asclepiadaceae family. Cynanchum komarovii has the medicinal values of relieving cough and asthma, and anti-inflammatory and antibacterial effects, and is derived from the dried aerial parts of the perennial herb Cynanchum komarovii Al. Iljinski of the Asclepiadaceae family. Cynanchum paniculatum has the medicinal values of relieving pain, antibacterial and dampness-dispelling effects, and is derived from the dried roots or rhizomes of the plant Cynanchum paniculatum (Bunge) Kitagawa of the Asclepiadaceae family.

[0003] Cynanchum atratum, Cynanchum glaucescens, Cynanchum komarovii and Cynanchum paniculatum have similar morphological traits, but contain different medicinal components and their medicinal effects are not completely the same. Cynanchum glaucescens contains components such as triterpenoid saponins and flavonoid glycosides, and is a drug for lowering qi and resolving phlegm. Clinically, it is mostly used for the treatment of cold cough, wheezing phlegm, bronchitis and asthma. Cynanchum atratum contains components such as volatile oils and cardiac glycosides, and is a drug for clearing heat and cooling blood. It is mostly used for the treatment of fever caused by pathogenic warm factors injuring nutrient qi, yin deficiency fever, consumptive fever, heat strangury, blood strangury and carbuncles and sores. Cynanchum komarovii contains alkaloids and volatile oils, and has effects such as analgesia, anti-inflammatory and antibacterial, relieving cough and asthma, etc., and is used for the treatment of diseases such as fullness of qi in the lungs, profuse phlegm in cough, chest stuffiness 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 paniculatum contains components such as volatile oils, acetophenones and alkaloids, and has effects such as antiviral, protecting cardiovascular, immune regulation, anti-tumor analgesia, anti-inflammatory and antibacterial, lowering blood pressure, slowing heart rate, etc., and can be used for the treatment of symptoms such as stomachache and distension, rubella and eczema. Since Cynanchum atratum, Cynanchum glaucescens, Cynanchum komarovii and Cynanchum paniculatum are very similar in appearance, there are often phenomena such as miscollection, misapplication, being easily confused and difficult to distinguish, and substitution in the market, and they are also easily confused in clinical application. This not only affects the curative effect of medication, but because Cynanchum komarovii has neurotoxicity, mixing them is likely to cause medication safety problems.

[0004] At present, the identification of Cynanchum atratum, Cynanchum glaucescens, Cynanchum komarovii and Cynanchum paniculatum can be carried out through morphological identification, physicochemical identification and DNA barcoding identification. Morphological identification is limited by factors such as personal subjective consciousness and experience, and has strong limitations. Physicochemical identification methods such as ultraviolet spectral group method and chemical component analysis are easily restricted by the growth status of the sample itself and experimental conditions. The sensitivity of such identification methods is not high, and the reproducibility of the results is also difficult to guarantee. DNA barcoding technology cannot directly distinguish such closely related species and needs to be combined with other methods for joint discrimination. Therefore, it is particularly important to develop an accurate and efficient identification method to provide effective technical support for guiding the correct use of drugs in clinical practice. Summary of the Invention

[0005] To solve the problems existing in the prior art, the present invention provides MNP marker loci, primer compositions and kits for identifying Cynanchum atratum, Cynanchum glaucescens, Cynanchum komarovii and Cynanchum paniculatum, which can distinguish between them. This method has the characteristics of strong discrimination power, high identification throughput and accurate results.

[0006] The present invention also provides a method for identifying Cynanchum atratum, Cynanchum glaucescens, Cynanchum komarovii and Cynanchum paniculatum.

[0007] The technical solution of the present invention to solve the above technical problems is as follows: The present invention provides MNP marker loci for identifying traditional Chinese medicinal materials Cynanchum atratum, Cynanchum glaucescens, Cynanchum komarovii and Cynanchum paniculatum. The MNP marker loci are within genomic regions with multiple nucleotide polymorphisms in Cynanchum atratum, Cynanchum glaucescens, Cynanchum komarovii and Cynanchum paniculatum, including MNP-1 to MNP-20.

[0008] The present invention further provides a primer set for identifying traditional Chinese medicinal materials Cynanchum atratum, Cynanchum glaucescens, Cynanchum komarovii and Cynanchum paniculatum based on the above MNP loci. The primer set contains 150 pairs of primers; the nucleotide sequences of the primers are shown in SEQ ID NO: 1 to SEQ ID NO: 300.

[0009] The present invention also provides a kit for identifying Cynanchum atratum, Cynanchum glaucescens, Cynanchum komarovii and Cynanchum paniculatum, which includes the primer set described in claim 2.

[0010] Another object of the present invention is to provide a method for identifying Cynanchum atratum, Cynanchum glaucescens, Cynanchum komarovii and Cynanchum paniculatum. The method includes: using the above primer set or kit to identify Cynanchum atratum, Cynanchum glaucescens, Cynanchum komarovii and Cynanchum paniculatum.

[0011] Furthermore, it includes the following steps: (1) Using the primer set to perform multiplex PCR amplification on the DNA of the sample to be tested to obtain a multiplex PCR amplification product, and purifying the product; (2) Construct a high-throughput sequencing library based on the purified multiplex PCR amplification products to obtain the high-throughput library of the sample to be tested, and purify the high-throughput library; (3) Sequence the high-throughput library of the sample to be tested to obtain sequencing data; (4) Analyze the sequencing data to obtain DNA fingerprint data; (5) Compare the DNA fingerprint data of the sample to be tested, judge the genetic similarity coefficient of the sample to be tested according to the MNP marker locus, and identify the variety of the sample to be tested based on the obtained genetic similarity coefficient.

[0012] Further, judging 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 99%, determining that the sample to be tested and the control variety are extremely similar varieties or the same variety; 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 the same variety.

[0013] In the technical solution provided by the present invention, the primers for each MNP marker locus include an upstream primer and a downstream primer, as specifically shown in Table 1 of the specification. Among them, the upstream primer of No. 1 is SEQ ID NO.1, the downstream primer of No. 1 is SEQ ID NO.2, the upstream primer of No. 2 is SEQ ID NO.3, the downstream primer of No. 2 is SEQ ID NO.4, the upstream primer of No. 3 is SEQ ID NO.5, and the downstream primer of No. 3 is SEQ ID NO.6, and so on.

[0014] When judging the variety of the sample to be tested by the genetic similarity coefficient provided by the present invention: including: when the genetic similarity coefficient is greater than or equal to 99%, determining that the sample to be tested and the control variety are extremely similar varieties or the same variety; 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 the same variety. The calculation formula of the genetic similarity is: Among them, GS is the genetic similarity coefficient between the sample to be tested and the control variety, n ij is the number of marker loci that are both detected in the sample to be tested and the control variety but have no genotype difference, and N ij is the number of marker loci that are both detected in the sample to be tested and the control variety.

[0015] The beneficial effects of the present invention are: (1)The primer set provided by the present invention is used for identifying Cynanchum atratum, Cynanchum stauntonii, Cynanchum komarovii, and Cynanchum paniculatum. By using multiplex PCR amplification and integrating the next-generation sequencing platform for sequencing the amplification products, the advantages of multi-target, high-throughput, high-efficiency, and high-accuracy detection of Cynanchum atratum, Cynanchum stauntonii, Cynanchum komarovii, and Cynanchum paniculatum are realized. Moreover, this primer set has a high individual discrimination degree, as well as a high reproducibility rate and accuracy rate. Using multiplex PCR, multiple targets can be detected at one time, thus effectively avoiding the problems of high false negatives and low sensitivity caused by the failure of a single target amplification. (2)When using the primer set provided by the present invention for detection, the accuracy is high. In combination with a next-generation high-throughput sequencer, the amplification products are sequenced hundreds of times, and the output result is a base sequence. Therefore, parallel experiments are not required, and the data can be compared arbitrarily, with strong data sharing. The kit using this primer set can simultaneously have the above advantages, effectively ensuring the reproducibility rate and accuracy rate of detection, thus ensuring the safety of clinical medication and further protecting the interests of consumers. Description of the Drawings

[0016] Figure 1 It is the distribution diagram of the detected number of MNP marker sites of Cynanchum atratum, Cynanchum stauntonii, Cynanchum komarovii, and Cynanchum paniculatum in Example 2 of the present invention; Figure 2 It is the distribution diagram of the difference ratio of MNP markers of Cynanchum atratum, Cynanchum stauntonii, Cynanchum komarovii, and Cynanchum paniculatum in Example 2 of the present invention; Figure 3 It is the genetic clustering diagram of Cynanchum atratum, Cynanchum stauntonii, Cynanchum komarovii, and Cynanchum paniculatum based on MNP markers in Example 2 of the present invention. Detailed Embodiments

[0017] 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.

[0018] Example 1 Screening of MNP Marker Sites for Identifying Cynanchum atratum, Cynanchum stauntonii, Cynanchum komarovii, and Cynanchum paniculatum and Design of Multiplex PCR Amplification Primers First of all, the present invention uses Cynanchum atratum as the reference genome, combines the sequencing data of Cynanchum atratum, Cynanchum stauntonii, Cynanchum komarovii, and Cynanchum paniculatum, and uses Samtools (Version 1.2) and BCFtools (Version: 1.2) for site polymorphism comparative analysis. The MNP markers are screened according to the following principles: (1) The marker sequence is common in Cynanchum atratum, Cynanchum stauntonii, Cynanchum komarovii, and Cynanchum paniculatum, but does not appear in other species; (2) There are multiple discontinuous SNP differences in the sequence; (3) The length of the marker sequence is between 200 and 300 bp. Through the above screening principles, a total of 150 MNP marker sites with high polymorphism are finally screened out.

[0019] Secondly, according to the above MNP marker loci, a multiplex PCR primer set is designed. The primer design follows the principle that primers do not interfere with each other. All primers can be combined into a primer pool for multiplex PCR amplification, and finally a primer composition for the 150 MNP loci described in Table 1 is screened out. The primer set includes the 1st primer pair to the 150th primer pair. Each primer pair contains a forward primer and a reverse primer. The nucleotide sequences of the forward primer and the reverse primer of the 1st primer pair are shown in SEQ ID NO:1 and SEQ ID NO:2 respectively, and so on. The nucleotide sequences of the forward primer and the reverse primer of the 150th primer pair are shown in SEQ ID NO:299 and SEQ ID NO:300 respectively. The primer set has high amplification efficiency and high identification accuracy, meeting the requirements for identifying Cynanchum atratum, Cynanchum stauntonii, Cynanchum komarovii, and Pyrola decorata.

[0020] Table 1 Primer sequences corresponding to 20 MNP marker loci regions Example 2 Evaluation of MNP markers, primer compositions, and kits for identifying Cynanchum atratum, Cynanchum stauntonii, Cynanchum komarovii, and Pyrola decorata After synthesizing 150 pairs of primers, 5 μL of each primer was taken for equal - volume mixing to form a 1:1 equal - volume mixture of forward and reverse primers. The developed MNP markers, primers, and kits were evaluated using 2 samples of Cynanchum atratum, 2 samples of Cynanchum stauntonii, 2 samples of Cynanchum komarovii, and 2 samples of Pyrola decorata provided by the unit to test the detection rate, accuracy, and discrimination of MNP marker loci.

[0021] To evaluate the above parameters, first, the DNA sequence information of the MNP markers of the above samples needs to be obtained. The specific experimental procedure is as follows: DNA extraction to obtain the DNA of the samples to be tested. Specifically, a plant genomic DNA extraction kit (manufacturer: Tiangen Biochemical Technology (Beijing) Co., Ltd., product number: DP320) was used to extract the DNA of the above Cynanchum atratum, Cynanchum stauntonii, Cynanchum komarovii, and Pyrola decorata samples. The detailed operation steps can be found in the instruction manual of this kit. After obtaining the DNA of the above 8 samples to be tested, 1 μL of each was taken to measure the concentration (using a Qubit fluorescence quantifier), and the DNA concentrations were all within the range of 20 ng / μL - 50 ng / μL.

[0022] Multiple PCR amplification of MNP marker loci to obtain multiple PCR amplification products. Specifically, in the amplification reaction of each sample to be tested, add 4 μL of the primer set provided in the examples of the present invention, 4 μL of the DNA of the sample to be tested (the DNA amount needs to reach 200 ng), and 10 μL of GenoPlexs 3×T Master Mix (manufacturer: Shijiazhuang Borui Biotechnology Co., Ltd.). The total reaction system is 30 μL, and the insufficient part is made up with water. After mixing well by oscillation, perform multiple PCR amplification. The multiple PCR amplification program is: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 60°C for 4 min, and this step is repeated for 17 cycles; extension at 72°C for 4 min. After the reaction is completed, store the PCR amplification product at 4°C.

[0023] Purify the PCR product. Purify the amplified DNA using the magnetic bead method (manufacturer: Nanjing Novoprotein Scientific Inc., product number: N411), and the specific operation refers to the product instruction manual.

[0024] Construct a high-throughput sequencing library. Specifically, add the following reaction reagents to the purified multiple PCR amplification product: 10 μL of GenoPlexs 3×T Master Mix, 2 μL of Illumina sequencing adapter primer with a concentration of 5 μM, and 16 μL of water. Perform PCR reaction according to the following program: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 60°C for 15 s, extension at 70°C for 30 s, and this step is repeated for 8 cycles; final extension at 72°C for 5 min. After the reaction is completed, obtain the high-throughput sequencing library of the sample to be tested.

[0025] Purify the PCR product. Purify the high-throughput sequencing library using the magnetic bead method (manufacturer: Nanjing Novoprotein Scientific Inc., product number: N411), and the purification method refers to the instruction manual of this product.

[0026] Library sequencing. Use the Illumina NextSeq1000 sequencer to sequence the high-throughput sequencing library to obtain the sequencing data of the sample to be tested. The detailed sequencing steps refer to the instruction manual of this sequencer. After the sequencing is completed, copy the sequencing data to a portable hard drive.

[0027] Sequencing data analysis. Use the data alignment software Bowtie2 (version number 2.1.0) to align the sequencing data of the sample to be tested to the Cynanchum atratum reference genome. The alignment result is saved in the SAM (The Sequence Alignment / Map format), and finally obtain the DNA sequences of the MNP markers of each sample to be tested. By comparing these DNA base sequences, the detection rate, accuracy, and discrimination of the MNP marker loci can be analyzed.

[0028] Analysis of the detection rate of MNP markers Using the primer set provided in Example 1 of the present invention, multiplex PCR amplification and construction of a sequencing library were carried out, and multiplex PCR amplification, second-generation high-throughput sequencing and data analysis were performed on the DNA of these 8 test samples. On average, 143.7 MNP markers could be detected in each test sample, and the average detection rate reached 95.8%. The distribution of the number of detected MNP marker sites in the test samples is as Figure 1 shown. In the national standard GB / T 38551-2020, it is required that the site detection ratio is not less than 95% during variety identification, indicating that the MNP markers developed by the present invention meet the requirements for the detection rate of markers in variety identification applications.

[0029] (2)Analysis of the accuracy of the MNP marker method The accuracy of variety identification ultimately depends on the accuracy of genotype typing of marker sites. The reproducibility experiment results were used to calculate the precision, and then the accuracy of genotyping was calculated. Among them, the reproducibility experiment refers to two independent repeated experiments carried out by different personnel, reagents of different batches, and different instruments. The precision refers to the proportion of marker sites with consistent genotyping results in the two experiments, and the accuracy = 1 - (1 - precision) / 2.

[0030] To test the accuracy of the MNP marker method for identifying Cynanchum atratum, Cynanchum glaucescens, Cynanchum komarovii and Cynanchum paniculatum, the present invention conducted a reproducibility experiment on these 8 samples. As can be seen from Table 2, a total of 1150 MNP marker sites were compared in the reproducibility experiment, and the accuracy of genotyping of the MNP marker method for marker sites was 99.57%. The high marker accuracy indicates that DNA fingerprint data collected by different laboratories or at different times can be accurately compared with each other, providing a technical guarantee for the sharing of DNA fingerprint data.

[0031] Table 2 Reproducibility of the genotyping results of MNP marker sites (3)Analysis of the variety discrimination ability of the MNP marker method All the MNP marker genotypes detected in 2 samples of Cynanchum atratum, 2 samples of Cynanchum glaucescens, 2 samples of Cynanchum komarovii and 2 samples of Cynanchum paniculatum were compared pairwise, and a total of 28 pairs of comparison results were obtained. The proportion of MNP markers that are different between each pair of samples is called the distance between samples, and the distance between samples directly shows the ability of MNP markers to distinguish varieties. The number of different MNP markers in the pairwise comparison of these two types of samples was counted. The results showed that on average, there were 117.9 different marker sites in each pair of samples, and the average difference ratio was 88%. The distribution of the difference ratio is as Figure 2 shown, indicating that the MNP markers screened by the present invention have high polymorphism and can significantly distinguish Cynanchum atratum, Cynanchum glaucescens, Cynanchum komarovii and Cynanchum paniculatum.

[0032] (4)MNP marker method for variety identification of Cynanchum atratum, Cynanchum glaucescens, Cynanchum komarovii and Cynanchum paniculatum Compare the DNA fingerprint data with the control samples to obtain the genetic similarity coefficient; according to the genetic similarity coefficient, identify the variety of the sample to be tested. Identifying the variety of the sample to be tested according to the genetic similarity coefficient specifically 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. Use the 150 MNP marker loci and the kit provided by the embodiments of the present invention to perform MNP marker locus difference analysis on 3 samples of Cynanchum atratum, 2 samples of Cynanchum glaucescens, 2 samples of Cynanchum komarovii and 2 samples of Cynanchum paniculatum provided by the Shandong Academy of Agricultural Sciences. The identification results of Cynanchum atratum, Cynanchum glaucescens, Cynanchum komarovii and Cynanchum paniculatum are shown in Table 3.

[0033] Table 3 Variety identification of Cynanchum atratum, Cynanchum glaucescens, Cynanchum komarovii and Cynanchum paniculatum As can be seen from Table 3, among the pairwise comparison results of 3 samples of Cynanchum atratum, 2 samples of Cynanchum glaucescens, 2 samples of Cynanchum komarovii and 2 samples of Cynanchum paniculatum, the highest value of the genetic similarity coefficient is 7.81%, and they are determined to be different varieties. In addition, through cluster analysis, it is found that ( Figure 3 ), Cynanchum atratum, Cynanchum glaucescens, Cynanchum komarovii and Cynanchum paniculatum can be divided into 4 major categories respectively, and each sample under the major categories of Cynanchum atratum, Cynanchum glaucescens, Cynanchum komarovii and Cynanchum paniculatum can also be clustered into one category respectively. The above results show that the primer group and the identification method provided by the present invention can significantly distinguish varieties between species and within species, and can accurately identify Cynanchum atratum, Cynanchum glaucescens, Cynanchum komarovii and Cynanchum paniculatum, which is of great significance for guiding the correct clinical medication and standardizing the order of the Chinese medicinal materials market.

[0034] The above are only the preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A MNP marker site for identifying the Chinese medicinal materials Bletilla striata, Bletilla striata, Laogenia officinalis and Cynanchum paniculatum, characterized in that: The MNP marker site is within a genome region having multiple nucleotide polymorphisms in Bletilla striata, Bletilla striata, Laoguatou and Cynanchum paniculatum, including MNP-1 to MNP-20.

2. A primer set for identifying the Chinese medicinal materials Rhizoma Cynanchum, Rhizoma Cynanchum, Rhizoma Cynanchum and Rhizoma Cynanchum, characterized in that: The primer set comprises 150 pairs of primers; the nucleotide sequences of the primers are shown in SEQ ID NO: 1 to SEQ ID NO:

300.

3. A kit for identifying Rhizoma Cynanchum, Rhizoma Cynanchum, Rhizoma Cynanchum and Rhizoma Cynanchum, characterized in that: The kit comprises the primer set according to claim 2.

4. A method for identifying Bletilla striata, Bletilla striata, Laogenia officinalis and Cynanchum paniculatum, characterized in that: The method comprises: using the primer set described in claim 2 or the kit described in claim 3 to identify Bletilla striata, Bletilla striata, Laogenia officinalis and Cynanchum cyrtonema.

5. The method according to claim 4, characterized in that The following steps are involved: (1) performing multiple PCR amplification on the DNA of the sample to be tested using the primer set to obtain multiple PCR amplification products, and purifying the products; (2) constructing a high-throughput sequencing library based on the purified multiplex PCR amplification products, obtaining a high-throughput library of the sample to be tested, and purifying the high-throughput library; (3) Sequencing the high-throughput library of the sample to be tested to obtain sequencing data; (4) Analyze sequencing data and 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.

6. The method according to claim 5, characterized in that The variety of the sample to be tested is determined according to the genetic similarity coefficient, including: when the genetic similarity coefficient is greater than or equal to 99%, the sample to be tested and the control sample are determined to be very similar varieties or the same variety; 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.

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