DNA bar code identification method for hypericum sampsonii hance and counterfeit product thereof

Abstract

The invention discloses a DNA bar code identification method for hypericum sampsonii hance and counterfeit products thereof, and belongs to the technical field of biology. The identification method comprises the following steps: taking DNA of a sample to be detected as a template, carrying out PCR amplification by using an ITS2 primer or a psbA-trnH primer, and carrying out sequencing and sequence splicing on an amplification product to obtain a DNA bar code of the sample to be detected. The amplification sequence is applied to construction of a hypericum phylogenetic tree or systematic analysis of the sequence, accurate identification of hypericum sampsonii hance is achieved, the identification method is simple, rapid, high in efficiency and good in repeatability, hypericum sampsonii hance, hypericum perforatum and other hypericum close-genetic plants can be accurately distinguished and identified, the identification effect is high, and the method is suitable for popularization and application. The identification success rate of hypericum sampsonii hance is greatly improved. The DNA barcode identification method provided by the invention lays a foundation for subsequent development of molecular biological detection technologies of hypericum sampsonii hance and counterfeit products thereof.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a DNA barcoding identification method for Hypericum sampsonii Hance and its counterfeits. Background Art

[0002] Hypericum sampsonii Hance, also known as Herba Hyperici Sampsonii, Herba Hyperici Oppositifolii, and Herba Hyperici Siphonostegii, etc., is a plant of the genus Hypericum in the family Hypericaceae. It is cold in nature and bitter in taste, and has the effects of regulating menstruation and dredging collaterals, stopping bleeding, and detoxifying. It is used for hematemesis, epistaxis, hematochezia, dysentery, enteritis, high fever in children, irregular menstruation, traumatic injury, external bleeding, burns and scalds, and snake bites.

[0003] After consulting relevant literature, there are currently about 50 species of Hypericum plants, and the main production areas are in the southwest. However, due to the large number of Hypericum plants and the influence of environmental factors and other factors, there are also differences in the morphological characteristics of the same species of plants. The research on Hypericum sampsonii mainly focuses on its chemical composition, pharmacological effects, and pharmacognostic characteristics. In terms of pharmacognostic identification, only traditional methods are currently used, which are difficult to fully meet the urgent needs of the authenticity identification of Hypericum sampsonii and market supervision. Therefore, it has become particularly urgent to explore a new, rapid, and accurate identification method.

[0004] With the progress of DNA molecular marker technology, in the exploration of the genetic relationship of medicinal plants, DNA barcoding technology has effectively solved the problem of the identity identification of multi-origin plant medicinal materials. Only one or a few genes can accurately identify the vast majority of species. The internationally widely recognized plant DNA barcodes mainly include ITS2, psbA-trnH, matK, and rbcL. These barcodes play a key role in the efficient identification of plant species and populations, can improve the accuracy of distinguishing closely related plant species, and are widely used in multiple links such as the planting, production, processing, circulation, and even clinical use of Chinese medicinal materials. However, there is currently no report on the DNA barcoding identification of Hypericum sampsonii and its counterfeits. Summary of the Invention

[0005] The purpose of the present invention is to provide a DNA barcoding identification method for Hypericum sampsonii and its counterfeits to solve the problems existing in the above-mentioned prior art. The present invention first provides a DNA barcoding identification method for Hypericum sampsonii based on ITS2 and psbA-trnH sequences. This identification method is simple, rapid, efficient, and has good repeatability. It can accurately distinguish and identify Hypericum sampsonii and closely related Hypericum plants such as Hypericum perforatum, and has a high identification effect, greatly improving the identification success rate of Hypericum sampsonii.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a method for DNA barcoding identification of Hypericum sampsonii Hance and its counterfeits. Using the DNA of the sample to be tested as a template, PCR amplification is carried out using ITS2 primers or psbA-trnH primers, and the amplification products are sequenced and sequence spliced to obtain the DNA barcode of the sample to be tested.

[0008] A phylogenetic tree is constructed using the DNA barcode sequence to determine whether the sample to be tested is Hypericum sampsonii Hance.

[0009] Alternatively, the GC content in the DNA barcode sequence is analyzed. When the GC content in the ITS2 sequence is 58%-62%, and / or the GC content in the psbA-trnH sequence is 24-27%, it is determined that the sample to be tested is Hypericum sampsonii Hance.

[0010] Furthermore, the sequences of the ITS2 primers are as shown in SEQ ID NO.1-2; the sequences of the psbA-trnH primers are as shown in SEQ ID NO.7-8.

[0011] Furthermore, when PCR amplification is carried out using ITS2 primers, the reaction system is: 15 μL of PCR Master Mix, 1.5 μL of each of the forward and reverse primers at 10 μmo / L, 6 μL of template DNA, and supplemented with ddH2O to 30 μL.

[0012] Furthermore, when PCR amplification is carried out using psbA-trnH primers, the reaction system is: 15 μL of PCR Master Mix, 1.5 μL of each of the forward and reverse primers at 10 μmo / L, 6 μL of template DNA, and supplemented with ddH2O to 30 μL.

[0013] Furthermore, when PCR amplification is carried out using ITS2 primers, the reaction program is: 94°C for 1 min; 94°C for 30 s, 55°C for 25 s, 72°C for 50 s, 33 cycles; 72°C for 5 min.

[0014] Furthermore, when PCR amplification is carried out using psbA-trnH primers, the reaction program is: 95°C for 4 min; 94°C for 30 s, 55°C for 1 min, 72°C for 1 min, 35 cycles; 72°C for 10 min.

[0015] The present invention also provides the application of the above DNA barcoding identification method in differentiating and identifying Hypericum sampsonii Hance and its counterfeits.

[0016] The present invention discloses the following technical effects:

[0017] In view of the problem that there is currently no DNA barcoding identification technology for Hypericum sampsonii Hance and its counterfeits, the present invention provides, for the first time, a DNA barcoding identification method. The ITS2 and psbA-trnH sequences of Hypericum sampsonii Hance are obtained by PCR amplification using ITS2 primers and psbA-trnH primers. Then, the amplified sequences are applied to construct a phylogenetic tree of Hypericum or to conduct a systematic analysis of the amplified sequences to achieve accurate identification of Hypericum sampsonii Hance. This identification method is simple, fast, highly efficient, and has good repeatability. It can accurately distinguish and identify Hypericum sampsonii Hance from closely related plants in the genus Hypericum such as Hypericum perforatum L., with high identification efficiency, greatly improving the success rate of identifying Hypericum sampsonii Hance. The DNA barcoding identification method of the present invention lays a foundation for the subsequent development of molecular biology detection technologies for Hypericum sampsonii Hance and its counterfeits. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0019] Figure 1 It is the electrophoresis result diagram of the amplification products in Example 1; among them, A is the amplification product of the ITS2 sequence; B is the rbcL amplification product; C is the psbA-trnH amplification product; D is the matK amplification product; in A-C, lanes 1-17 are samples s1-s17, and lanes 18-20 are samples GY1-GY3; in D, lanes 1-10 are samples s1-s10;

[0020] Figure 2 It is the peak diagram of partial fragments of the amplified product sequences in Example 1; among them, A is the ITS2 sequence; B is the rbcL sequence; C is the psbA-trnH sequence;

[0021] Figure 3 It is the intraspecific and interspecific genetic distance distribution diagram of the ITS2, rbcL, and psbA-trnH sequences of Hypericum sampsonii Hance;

[0022] Figure 4 It is the phylogenetic tree constructed based on the ITS2 sequence;

[0023] Figure 5 It is the phylogenetic tree constructed based on the rbcL sequence;

[0024] Figure 6 It is the phylogenetic tree constructed based on the psbA-trnH sequence. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0025] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation on the present invention, but rather as a more detailed description of certain aspects, features, and implementation schemes of the present invention.

[0026] It should be understood that the terms described in the present invention are only for describing specific embodiments and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0027] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0028] Without departing from the scope or spirit of the present invention, various improvements and variations can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0029] Regarding "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0030] Example 1

[0031] 1. Instruments and Materials

[0032] 1.1 Instruments

[0033] Full-wavelength microplate reader (Bio Tek, USA); Gel imaging system (Bio-Rad, USA); T100TM Thermal Cycler PCR instrument (Bio-Rad, USA); DYY-6C electrophoresis instrument (Beijing Liuyi Biology).

[0034] 1.2 Reagents

[0035] Polysaccharide and Polyphenol Plant Genomic DNA Extraction Kit (Lot No. A0103A, TIANGEN BIOTECH (BEIJING) CO., LTD.); HotStart PCR Master Mix (Lot No. H4402070, YEASEN BIOTECH (SHANGHAI) CO., LTD.); Goldview Nucleic Acid Dye (Lot No. 24088831, Beijing LanJieKe Biotechnology Co., Ltd.); 5×TBE Buffer (Lot No. 2310002, Solarbio); Gold Band 15000 DNA Marker (Lot No. G0315741, YEASEN BIOTECH (SHANGHAI) CO., LTD.); Sterile and Enzyme-Free Water (Lot No. 24054563, Beijing LanJieKe Biotechnology Co., Ltd.); Agarose (Lot No. A1406090, YEASEN BIOTECH (SHANGHAI) CO., LTD.). The primers were synthesized by Sangon Biotech (Shanghai).

[0036] 1.3 Samples

[0037] A total of 20 samples of the original plants of Hypericum sampsonii Hance and its adulterants were collected from 8 provinces (autonomous regions), namely Guangxi, Hunan, Guangdong, Sichuan, Jiangxi, Chongqing, Yunnan and Guizhou. The specific information is shown in Table 1. After selecting fresh leaves without pests and diseases, cleaning and sterilizing them, and drying them, they were stored at -20 °C for later use.

[0038] Table 1 Sample Information

[0039]

[0040] 2. DNA Extraction, PCR Amplification and Sequencing

[0041] Take 30 mg of dry sample leaves, place them in a centrifuge tube, add grinding magnetic beads, quickly freeze them in liquid nitrogen, and grind them at high speed in a centrifuge. Total DNA was extracted according to the kit instructions. The A 260 / A 280 concentration and purity of the 20 sample DNAs obtained by extraction were analyzed by a microplate reader to determine the content and concentration of genomic DNA. Samples with better total DNA extraction quality and suitable for primer fragment amplification were selected. Universal DNA barcode primers ITS2, matK, psbA-trnH and rbcL were selected, and each sample was amplified according to the standard procedure. The primer sequences are shown in Table 2. The PCR reaction system was 30 μL: 15 μL of PCR Master Mix, 1.5 μL each of forward and reverse primers (10 μmol / L), 6 μL of template DNA, and 6 μL of ddH2O. The PCR reaction program is shown in Table 3. The PCR amplification products were detected by 1% agarose gel electrophoresis, and the electrophoresis buffer was 1×TBE solution. It was found by staining with the gel staining method that the PCR products amplified by the primer matK had many heterozygous bands and serious trailing. For the other sequences, bright and single bands could be observed by gel imaging. The PCR products were sent to Sangon Biotech (Shanghai) for bidirectional sequencing.

[0042] Table 2 Primer Sequences

[0043]

[0044] Table 3 PCR reaction procedure

[0045] Gene fragment Reaction program ITS2 94°C for 1 min; 94°C for 30 s, 55°C for 25 s, 72°C for 50 s, 33 cycles; 72°C for 5 min Matk 94°C for 1 min; 94°C for 30 s, 52°C for 20 s, 72°C for 50 s, 35 cycles; 72°C for 5 min rbcL 95°C for 4 min; 94°C for 30 s, 55°C for 1 min, 72°C for 1 min, 35 cycles; 72°C for 10 min PsbA-trnH 95°C for 4 min; 94°C for 30 s, 55°C for 1 min, 72°C for 1 min, 35 cycles; 72°C for 10 min

[0046] 3. Data analysis

[0047] The sequencing results were spliced forward and backward to remove the low-quality parts, and the complete gene sequences were obtained. Four ITS2 sequences, five rbcL sequences, and four psbA-trnH sequences of plants with similar morphological characteristics of the genus Hypericum were downloaded from GenBank. The detailed information is shown in Table 4. All sequences were imported into MEGA 7.0 for alignment analysis, the genetic distances were calculated based on the K2P model, and the phylogenetic tree was constructed using the neighbor-joining (NJ) method.

[0048] Table 4 Outgroup information downloaded from NCBI

[0049]

[0050] 4. Results and analysis

[0051] 4.1 Analysis of PCR amplification and sequencing results

[0052] The gel electrophoresis results of the PCR products are as Figure 1 shown. For the PCR products of the matK sequence samples, there were multiple miscellaneous bands after gel electrophoresis, which did not meet the sequencing standards, so this primer was discarded. The sequence characteristics of the remaining 3 barcodes showed that the amplification success rates were 100% (ITS2), 100% (rbcL), and 100% (psbA-trnH), respectively. The sequencing success rates of ITS2, rbcL, and psbA-trnH were all 100%. Their chromatogram peak shapes were relatively complete, basically single peak shapes, the peaks and troughs were clear, the distances between the peaks were uniform, and there was no interference of miscellaneous peaks at the bottom (see Figure 2 ). This result indicates that the amplification and sequencing effects of ITS2, rbcL, and psbA-trnH sequences are good, and they can be preliminarily used as candidate DNA barcodes.

[0053] 4.2 Sequence characteristic analysis

[0054] From the sequence characteristics of primers within and between different species of *Hypericum sampsonii* (Table 5), it can be seen that the sequence lengths are all less than 1000 bp, and the length relationship is RBCL > psbA-trnH > its2. The ideal genetic distance between species for DNA barcoding should be significantly higher than that within species, with a clear "barcoding gap" boundary. The rbcL sequences of *Hypericum sampsonii* and its adulterants have fewer mutations, and there is an overlap between the within-species K2P distance and the between-species K2P distance, without an obvious barcoding gap, so it is not suitable for differentiating *Hypericum sampsonii* from its adulterants. In the ITS2 sequence and the psbA sequence, the barcoding gap is obvious, which can distinguish them from the adulterant varieties of *Hypericum sampsonii*. It conforms to the characteristics of ideal DNA barcodes and can be used as the DNA barcode for the identification of *Hypericum sampsonii* and its adulterants.

[0055] Table 5 Sequence characteristics of primers within and between different species of *Hypericum sampsonii*

[0056] Gene fragment ITS2 Rbcl psbA-trnH Aligned sequence length (bp) 471 716 590 Number of conserved sites 363 697 539 Number of variable sites 108 19 51 Number of parsimony informative sites 58 18 41 Single mutation site 50 1 10 K2P distance within species 0.0000～0.0484 0.0000～1.9013 0.0000～0.0151 Average value 0.02073 0.74889 0.00597 K2P distance between species 0.1015～0.2459 0～1.8074 0.0680～0.2246 Average value 0.18253 1.8074 0.115

[0057] 4.3 Genetic distances of ITS2, rbcl, and psbA-trnH sequences

[0058] According to the distribution map of genetic distances within and between species of *Hypericum sampsonii* ( Figure 3 ), among the candidate DNA barcode sequences of *Hypericum sampsonii* and its easily confused species, the ITS2 and psbA-trnH sequences show smaller within-species variation and larger between-species variation, have high sequence conservation, and exhibit a significant barcoding gap, and these characteristics all meet the criteria of ideal DNA barcodes.

[0059] 4.4 Phylogenetic trees of ITS2, rbcl, and psbA-trnH sequences

[0060] Using MEGA7.0, with the K2P model, the phylogenetic trees were constructed 1000 times by the Bootstrap test for the ITS2, rbcL, and psbA-trnH sequences respectively by the neighbor-joining method (NJ). The results show ( Figures 4 - 6 ), in the phylogenetic trees, the nuclear gene ITS2 sequence and the chloroplast gene psbA-trnH sequence show more significant clustering effects. The tested *Hypericum sampsonii* is clustered into a single branch alone, with a clear boundary from other easily confused varieties. This indicates that the nuclear gene ITS2 sequence and the chloroplast gene psbA-trnH sequence have clear grouping within the genus and can successfully identify *Hypericum sampsonii* from the materials of the genus *Hypericum*.

[0061] 4.5 GC content of ITS2 and psbA-trnH sequences

[0062] The BioEdit software was used to count the base composition of each sequence, calculate the average GC content, and count the GC content of the amplification products. The results are shown in Table 6.

[0063] Table 6 GC content of amplification products of each sample

[0064]

[0065]

[0066] As can be seen from Table 6, there are significant differences in the average GC content between the ITS2 amplification products and the psbA-trnH amplification products of Hypericum sampsonii and Hypericum perforatum, indicating that ITS2 sequences and psbA-trnH sequences can be used as DNA barcodes to accurately identify Hypericum sampsonii and Hypericum perforatum.

[0067] 5. Summary

[0068] Since there are differences in the active ingredients among Hypericum plants, but their external morphological characteristics are relatively similar. Therefore, four commonly used DNA barcodes (ITS2, psbA-trnH, matK, rbcL) were used in this invention for exploration. Among the 20 samples collected, PCR amplification was successful for the other 3 barcodes except matK, and they had a high sequencing success rate. In the DNA barcode evaluation criteria of this invention, an ideal candidate sequence needs to have a significant Barcoding gap (support rate > 90%) and a large number of interspecific variable sites. At the same time, the phylogenetic tree established by this barcode can separate Hypericum sampsonii and Hypericum perforatum and cluster them into a single branch separately. After analysis, in the genetic distance analysis of Hypericum sequences, the intraspecific genetic distance and the interspecific genetic distance between Hypericum sampsonii and Hypericum perforatum are concentrated in the region of 0 - 1.9013 and there is an overlap. And the constructed phylogenetic tree cannot distinguish Hypericum sampsonii and Hypericum perforatum, so the rbcL sequence cannot be used to distinguish Hypericum sampsonii and Hypericum perforatum. The minimum interspecific genetic distance of the psbA-trnH sequence and the ITS2 sequence of Hypericum sampsonii and Hypericum perforatum is greater than the maximum intraspecific genetic distance. The genetic distance analysis of these two sequences shows that there is an obvious boundary in the genetic relationship between Hypericum sampsonii and Hypericum perforatum, and the phylogenetic tree can distinguish Hypericum sampsonii and Hypericum perforatum. Therefore, they can be used as DNA barcode sequences for identifying Hypericum sampsonii and Hypericum perforatum.

[0069] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention should fall within the protection scope determined by the claims of the present invention.

Claims

1. A DNA barcoding identification method for Hypericum sampsonii Hance and its counterfeits, characterized in that, Using the DNA of the sample to be tested as a template, PCR amplification is carried out using ITS2 primers or psbA-trnH primers, and the amplified product is sequenced and sequence spliced to obtain the DNA barcode of the sample to be tested. A phylogenetic tree is constructed using the DNA barcode sequence to determine whether the sample to be tested is *Hypericum sampsonii* Hance. Alternatively, the GC content in the DNA barcode sequence is analyzed. When the GC content in the ITS2 sequence is 58%-62%, and / or the GC content in the psbA-trnH sequence is 24-27%, it is determined that the sample to be tested is *Hypericum sampsonii* Hance.

2. The DNA barcoding identification method according to claim 1, wherein The sequences of the ITS2 primers are shown in SEQ ID NO.1-2; the sequences of the psbA-trnH primers are shown in SEQ ID NO.7-8.

3. The DNA barcoding identification method according to claim 2, characterized in that, When PCR amplification is carried out using ITS2 primers, the reaction system is: 15 μL of PCR Master Mix, 1.5 μL of each of the forward and reverse primers at 10 μmol / L, 6 μL of template DNA, and supplemented with ddH2O to 30 μL.

4. The DNA barcode identification method according to claim 2, wherein When PCR amplification is carried out using psbA-trnH primers, the reaction system is: 15 μL of PCR Master Mix, 1.5 μL of each of the forward and reverse primers at 10 μmol / L, 6 μL of template DNA, and supplemented with ddH2O to 30 μL.

5. The DNA barcoding identification method according to claim 2, wherein When PCR amplification is carried out using ITS2 primers, the reaction program is: 94°C for 1 min; 94°C for 30 s, 55°C for 25 s, 72°C for 50 s, 33 cycles; 72°C for 5 min.

6. The DNA barcode identification method according to claim 2, wherein When PCR amplification is carried out using psbA-trnH primers, the reaction program is: 95°C for 4 min; 94°C for 30 s, 55°C for 1 min, 72°C for 1 min, 35 cycles; 72°C for 10 min.

7. Application of the DNA barcode identification method according to any one of claims 1-6 in differentiating and identifying *Hypericum sampsonii* Hance and its counterfeits.

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