Molecular markers and methods for distinguishing codonopsis tangshen, codonopsis lactea and codonopsis subanceps
By using specific DNA barcoding markers and primer design, the identification challenges of Sichuan Codonopsis, Goat Milk Codonopsis, and Qinling Codonopsis species have been solved, achieving efficient and accurate species differentiation and ensuring the safe and effective use of medicinal plants.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- INST OF MEDICINAL PLANT DEV CHINESE ACADEMY OF MEDICAL SCI
- Filing Date
- 2022-07-05
- Publication Date
- 2026-04-14
AI Technical Summary
Existing technologies make it difficult to accurately distinguish between morphologically similar species such as Codonopsis pilosula, Codonopsis lanceolata, and Codonopsis pilosula from the Qinling Mountains, leading to confusion during medicinal use and affecting the safety and efficacy of medicinal plants.
Specific DNA barcoding markers, including molecular marker 1 and molecular marker 4, were used. PCR amplification and sequencing were performed using designed primers. SNP sites and INDEL sites were used to distinguish between Sichuan Codonopsis, Goat Milk Codonopsis and Qinling Codonopsis species.
It has enabled accurate identification of Sichuan Codonopsis, Goat Milk Codonopsis and Qinling Codonopsis species, improving the accuracy of medicinal plant identification and classification efficiency, and reducing costs and time requirements.
Smart Images
Figure CN115838817B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of plant molecular identification technology, specifically involving molecular markers and methods for distinguishing between Sichuan Codonopsis, Goat's Milk Codonopsis and Qinling Codonopsis species. Background Technology
[0002] Species of the genus Codonopsis (Codonopsis pilosula subsp. tangshen, Codonopsis lanceolata, and Codonopsis tsinlingensis) share highly similar morphological characteristics and are often used interchangeably in medicinal applications. To ensure the safe and effective use of Codonopsis medicinal materials, it is necessary to accurately identify easily confused species of the genus Codonopsis.
[0003] DNA-based markers are suitable for the identification of medicinal plants; among them, there are three main types of DNA barcode markers: universal, super, and specific DNA barcodes.
[0004] Universal DNA barcoding is applicable to all medicinal plants. These markers include ITS2, psbA-trnH, rbcL, and matK, and can serve as a first-line tool for identifying samples. However, they are mainly used for identifying distantly related species and lack the ability to identify closely related species.
[0005] Complete chloroplasts have been recognized as a super barcode for distinguishing related species, demonstrating high discriminative power and sufficient reliability. However, the use of super DNA barcodes is limited due to factors such as insufficient available DNA, the high cost of sequencing to assemble a complete genome and generate enough raw data to do so, and the complexity of data analysis.
[0006] Finding specific barcodes from highly variable regions allows for a trade-off between universal DNA barcodes and super DNA barcodes, ensuring accuracy in identifying closely related species while reducing time and costs. Therefore, there is an urgent need to provide a specific DNA barcode to distinguish between Sichuan Codonopsis, Goat's Milk Codonopsis, and Qinling Codonopsis species. Summary of the Invention
[0007] This invention discloses molecular markers and methods for distinguishing between Sichuan Codonopsis, Goat's Milk Codonopsis and Qinling Codonopsis species. When used for the identification of Sichuan Codonopsis, Goat's Milk Codonopsis and Qinling Codonopsis, the results are accurate and reliable.
[0008] To achieve the above objectives, the present invention adopts the following technical solution:
[0009] Molecular markers for distinguishing between Sichuan Codonopsis, Goat's Milk Codonopsis, and Qinling Codonopsis species.
[0010] Molecular marker 1 includes two SNP sites in the nucleotide sequence shown in SEQ ID NO: 1:
[0011] The 34th SNP site is C / A and the 38th SNP site is G / T;
[0012] SEQ ID NO: 1 is TCGTACAAGTATTTTAGCAATACTTTCACTGATMGTAKTAAATGCTATTCGAAC;
[0013] Molecular marker 4 includes the nucleotide sequence shown in SEQ ID NO: 2:
[0014] The INDEL sites at positions 4-6 are TCG / GAT, and the SNP site at position 127 is A / G.
[0015] SEQ ID NO: 2 is ATCKMKCCTCTATTTTCTACTTTTCTTGAAAAAGTGTTTTTCTCTCTTTGATTTCTTGTCTATCTTCTTTCATCTTCTCTAGTAGGGAATTCAATTAGTATTCTAATTGAATTGTTAATAGCAGTAARGATTCTG.
[0016] Furthermore, in molecular marker 1,
[0017] The two SNP sites of Codonopsis pilosula are A and T;
[0018] The two SNP sites in goat milk are C and G;
[0019] The two SNP sites of Codonopsis pilosula from the Qinling Mountains are A and G, respectively;
[0020] In molecular marker 4,
[0021] The INDEL site of Codonopsis pilosula is TCG, and the SNP site is A.
[0022] The INDEL site for goat milk is GAT, and the SNP site is A.
[0023] The INDEL site of Codonopsis pilosula from the Qinling Mountains is TCG, and the SNP site is G.
[0024] Primers based on the molecular markers used to distinguish between Sichuan Codonopsis, Goat's Milk Codonopsis, and Qinling Codonopsis species:
[0025] The primers for molecular marker 1 are:
[0026] Upstream primer: 5'-GTCATTATCCCTCGAGAAGTAG-3', SEQ ID NO: 3;
[0027] Downstream primer: 5'-GCTGACCTGCTAACCTCTATAC-3', SEQ ID NO: 4;
[0028] The primers for molecular marker 4 are:
[0029] Upstream primer: 5'-TAGAAGGTGGGTTGAAAGGAGT-3', SEQ ID NO: 5;
[0030] Downstream primer: 5'-AGCCTACTCTCAAAATCGAACC-3', SEQ ID NO: 6.
[0031] The aforementioned molecular markers or primers can be used to distinguish between Sichuan Codonopsis, Goat Milk Codonopsis and Qinling Codonopsis species.
[0032] A kit for differentiating between Sichuan Codonopsis, Goat's Milk Codonopsis and Qinling Codonopsis species includes the primers mentioned above.
[0033] The method for distinguishing between Sichuan Codonopsis, Goat's Milk Codonopsis, and Qinling Codonopsis species includes the following steps:
[0034] (1) Take the sample to be tested and extract DNA;
[0035] (2) Using the DNA extracted in step (1) as a template, perform PCR amplification using the primers described above;
[0036] (3) Sequencing the amplification product from step (2).
[0037] Preferably, the amplification system in step (2) is as follows:
[0038] 25 μL of 2×Taq PCRMaster Mix, 1 μM of each primer, 1 μL of template DNA, and ddH2O to a final volume of 50 μL.
[0039] Preferably, the amplification procedure in step (2) is as follows:
[0040] Denaturation at 94℃ for 2 minutes;
[0041] 94℃ for 30s, annealing temperature for 30s, 72℃ for 60s, 35 cycles;
[0042] 72℃ for 2 minutes.
[0043] In summary, the molecular markers and methods of this invention can effectively distinguish between Codonopsis pilosula, Codonopsis lanceolata, and Codonopsis tangutica species, which is beneficial for the classification of Codonopsis pilosula species and the identification of medicinal products derived from Codonopsis pilosula species. Attached Figure Description
[0044] Figure 1The image shows five highly variable regions that were selected.
[0045] Figure 2 The results of PCR amplification are shown below;
[0046] Figure 3 The image shows the Com1 sequencing results;
[0047] Figure 4 The image shows the Com4 sequencing results. Detailed Implementation
[0048] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0049] Example
[0050] 1. Analysis of the highly variable region
[0051] Highly variable intergenic spacer regions (IGS) were extracted from the GenBank files of chloroplasts of three Codonopsis species (Codonopsis pilosula subsp. tangshen, Codonopsis lanceolata, and Codonopsis tsinlingensis), and IGS sequences were extracted using extractseq.
[0052] Use clustalw2 to align the extracted sequences with the options "-type=DNA-gapopen=10-gapext=2".
[0053] The K2p evolutionary model, implemented using the distmat program in the EMBOSS software package, was used to calculate the genetic distance between gene regions with the parameter "-nucmethod 2". A mapping threshold of 5 was set, visually displaying the top five K2p values in the graph. These five highly variable regions can serve as potential molecular markers to distinguish between three Codonopsis species. Figure 1 ).
[0054] 2. Extraction of template DNA
[0055] Five plants of each of the three varieties of Codonopsis pilosula (Sichuan Codonopsis), Codonopsis pilosula (Yangru Codonopsis), and Codonopsis pilosula (Qinling Codonopsis) were collected, for a total of 15 plants. DNA was extracted from each of the 15 plants using a DNA extraction kit as templates for PCR amplification.
[0056] 3. PCR amplification:
[0057] Primers were designed based on the conserved sequences at both ends of each highly variable region, as shown in Table 1.
[0058] Table 1 shows the 5 primer pairs used for marker development.
[0059]
[0060] The final volume of PCR amplification was 50 μL, including 25 μL of 2×Taq PCR Master Mix, 1 μM of each primer, 1 μL of template DNA, and 22 μL of ddH2O.
[0061] All amplifications were performed on a Pro-Flex PCR system (Applied Biosystems, Waltham, MA, USA) under the following conditions: denaturation at 94°C for 2 minutes, followed by 35 cycles of 94°C for 30 seconds, a specific annealing temperature (Tm) for 30 seconds, 72°C for 60 seconds, and 72°C for 2 minutes as a final extension.
[0062] PCR amplification products were observed on a 1.2% agarose gel, and only two primer pairs (Com1 and Com4) yielded amplification products. Figure 2 ).
[0063] 4. Sanger sequencing:
[0064] The successfully amplified products were Sanger sequenced using the same primers as those used in PCR amplification on an ABI 3730XL instrument (Applied Biosystems, USA). Two molecular markers were ultimately obtained that can distinguish the three Codonopsis species. Sequencing results are shown below. Figure 3 and Figure 4 . Figure 3 The SNPs marked in the two boxes can be combined to distinguish three species of the genus Codonopsis. Figure 4 A boxed INDEL and a boxed SNP can be used to distinguish three species of Codonopsis pilosula.
[0065] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.
[0066] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to the above embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims
1. The application of molecular markers for distinguishing the species of Codonopsis pilosula, Codonopsis lanceolata, and Codonopsis qinlingense in the differentiation of these three species, characterized in that... The molecular markers include molecular marker 1 and / or molecular marker 4. The molecular marker 1 includes two SNP sites in the nucleotide sequence shown in SEQ ID NO: 1: The 34th SNP site is C / A and the 38th SNP site is G / T; SEQ ID NO: 1 is TCGTACAAGTATTTTAGCAATACTTTCACTGATMGTAKTAAATGCTATTCGAAC; The molecular marker 4 includes the nucleotide sequence shown in SEQ ID NO: 2: The SNPs at positions 4-6 are TCG / GAT and the SNP at position 127 is A / G; SEQ ID NO: 2 is ATCKMKCCTCTATTTTCTACTTTTCTTGAAAAAGTGTTTTCTCRTCTTTGATTTCTTGTCTATCTTCTTTCATCTTCTCTAGTAGGGAATTCAATTAGTATTCTAATTGAATTGTTAATAGCAGTAARGATTCTG; In the molecular marker 1, The two SNP sites of Codonopsis pilosula are A and T; The two SNP sites in goat milk are C and G; The two SNP sites of Codonopsis pilosula from the Qinling Mountains are A and G, respectively; In the molecular marker 4, The SNP sites of Codonopsis pilosula are TCG and A, respectively; The SNP sites in goat milk are GAT and A, respectively; The SNP sites of Codonopsis pilosula from the Qinling Mountains are TCG and G.
2. The application of primers used to amplify the molecular marker described in claim 1 in distinguishing between *Codonopsis pilosula*, *Codonopsis lanceolata*, and *Codonopsis qinlingense* species, characterized in that... The primers for molecular marker 1 are: Upstream primer: 5'-GTCATTATCCCTCGAGAAGTAG-3', SEQ ID NO: 3; Downstream primer: 5'-GCTGACCTGCTAACCTCTATAC-3', SEQ ID NO: 4; The primers for molecular marker 4 are: Upstream primer: 5'-TAGAAGGTGGGTTGAAAGGAGT-3', SEQ ID NO: 5; Downstream primer: 5'-AGCCTACTCTCAAAATCGAACC-3', SEQ ID NO: 6; In the molecular marker 1, The two SNP sites of Codonopsis pilosula are A and T; The two SNP sites in goat milk are C and G; The two SNP sites of Codonopsis pilosula from the Qinling Mountains are A and G, respectively; In the molecular marker 4, The SNP sites of Codonopsis pilosula are TCG and A, respectively; The SNP sites in goat milk are GAT and A, respectively; The SNP sites of Codonopsis pilosula from the Qinling Mountains are TCG and G.
3. A method for distinguishing between Sichuan Codonopsis, Goat's Milk Codonopsis, and Qinling Codonopsis species, characterized in that... Distinguishing using the molecular markers described in claim 1 includes the following steps: (1) Take the sample to be tested and extract DNA; (2) Using the DNA extracted in step (1) as a template, perform PCR amplification using the primers described in claim 2; (3) Sequencing the amplification product from step (2); (4) Based on the sequencing results, the species of Codonopsis pilosula, Codonopsis pilosula var. chuanxiong, Codonopsis pilosula var. yangnu, and Codonopsis pilosula var. qinling were distinguished; In the molecular marker 1, The two SNP sites of Codonopsis pilosula are A and T; The two SNP sites in goat milk are C and G; The two SNP sites of Codonopsis pilosula from the Qinling Mountains are A and G, respectively; In the molecular marker 4, The SNP sites of Codonopsis pilosula are TCG and A, respectively; The SNP sites in goat milk are GAT and A, respectively; The SNP sites of Codonopsis pilosula from the Qinling Mountains are TCG and G.
4. The method for distinguishing between Sichuan Codonopsis, Goat's Milk Codonopsis, and Qinling Codonopsis species according to claim 3, characterized in that, The PCR amplification system in step (2) is as follows: 25 µL of 2×Taq PCR Master Mix, 1 µM of each primer, 1 µL of template DNA, and ddH2O to a final volume of 50 µL.