Primer pair for identifying coptis chinensis species, identification method and application thereof

Abstract

The invention provides a primer pair for identifying coptis chinensis species, an identification method and application thereof, and belongs to the technical field of molecular identification. The primer pair for identifying the coptis chinensis species comprises an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer is as shown in SEQ ID NO.1, and the nucleotide sequence of the downstream primer is as shown in SEQ ID NO.2. Specific primers are designed aiming at coptis chloroplast ycf1 genes, target fragments can be accurately amplified, sequences obtained based on amplification of the primers can be used for constructing phylogenetic trees, and then the seven species of coptis chinensis, coptis triangularis, coptis omeiensis, coptis wuzhi, coptis yunnanensis, coptis short calyx and coptis japonica can be clearly distinguished. The identification method provided by the invention is simple and convenient in steps, is not limited by plant growth stages and sample forms, does not need complex equipment, and is convenient to popularize in laboratories and grassroots institutions. And molecular level evidence can be provided for rhizoma coptidis classification and resource protection.

Description

Technical Field

[0001] This invention belongs to the field of molecular identification technology, and in particular relates to a primer pair, identification method and application for identifying Coptis chinensis species. Background Technology

[0002] Plants of the genus *Coptis* are an important group of traditional medicinal plants. Their rhizomes are rich in alkaloids and possess significant medicinal effects such as clearing heat and drying dampness, purging fire and detoxifying. They have a long history of application in traditional Chinese medicine and have outstanding clinical value. This genus includes many species and varieties, such as *Coptis chinensis* (also known as *Coptis yunnanensis*), *Coptis triangularis* (also known as *Coptis yalian*), *Coptis embryophylla*, *Coptis chinensis* var. *yunnanensis*, *Coptis bryophylla* var. *yunnanensis*, and *Coptis rubrum*. The content of medicinal components, pharmacological effects, and clinical applications vary significantly among different species. The *Chinese Pharmacopoeia* specifies *Coptis chinensis*, *Coptis yalian*, and *Coptis rubrum* as the original sources of *Coptis* medicinal materials. Therefore, accurate species identification is of great significance for ensuring the quality of medicinal materials, guaranteeing the safety of clinical medication, and promoting the rational development and utilization of *Coptis* plant resources.

[0003] Currently, the identification of species in the genus *Coptis* mainly relies on traditional morphological methods, distinguishing them by observing phenotypic differences such as leaf shape, petal characteristics, and rhizome morphology. However, the morphological characteristics of plants in this genus are easily affected by factors such as growth environment and developmental stage, resulting in complex phenotypic variations and difficulty in distinguishing similar species. Especially after processing into medicinal materials, the morphological characteristics of the rhizome are destroyed, significantly reducing the accuracy of traditional identification methods. Furthermore, some closely related species have highly similar morphological characteristics, making accurate identification difficult even for experienced taxonomists. This leads to frequent species confusion and the substitution of inferior products for superior ones in the market, seriously affecting the quality control and industrial development of *Coptis* medicinal materials.

[0004] With the development of molecular biology techniques, DNA molecular marker-based species identification methods have gradually become important tools for plant classification and identification due to their high stability and accuracy. Chloroplast genes, in particular, are widely used in plant phylogenetic and species identification research due to their moderate evolutionary rate, stable genetic information, and ease of amplification. However, existing technologies lack specific molecular markers for *Coptis* species, and many reported markers suffer from low amplification efficiency, insufficient polymorphism, or limited identification range, failing to meet the need for rapid and accurate identification of multiple closely related species within this genus. Therefore, developing a species-specific identification method for *Coptis* based on highly polymorphic chloroplast genes has significant theoretical and practical value. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a primer pair for identifying Coptis chinensis species, which can accurately identify whether the plant to be tested belongs to the Coptis genus and has high sensitivity.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] A primer pair for identifying Coptis chinensis species, the primer pair comprising an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer being shown in SEQ ID NO.1 and the nucleotide sequence of the downstream primer being shown in SEQ ID NO.2.

[0008] Another object of the present invention is to provide a reagent for identifying species of Coptis chinensis, the reagent comprising the above-mentioned primer pair.

[0009] Another object of the present invention is to provide a kit for identifying species of Coptis chinensis, the kit comprising the above-described primer pairs or the above-described reagents.

[0010] Another objective of this invention is to provide a method for identifying Coptis chinensis species, comprising the following steps: extracting DNA from the leaves of the plant to be tested; performing PCR amplification using the primer pair, the reagent, or the kit described herein; after amplification, performing agarose gel electrophoresis on the amplified product to determine whether the plant to be tested belongs to the genus Coptis; constructing a phylogenetic tree from the amplified sequence to determine the species of the plant to be tested.

[0011] Preferably, the PCR amplification reaction system is as follows: 1 μL DNA template, 3 μL 10× Buffer, 3 μL dNTPs, 3 μL MgSO4; 1 μL each of upstream and downstream primers, 1 μL KOD-Plus-Neo, and ddH2O added to 30 μL.

[0012] Preferably, the concentration of the primer is 0.1–1 μM.

[0013] Preferably, the concentration of DNA in the DNA template is 20–100 ng / μL.

[0014] Preferably, the PCR amplification reaction program is as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 58℃ annealing for 30 s, 68℃ extension for 80 s, 35 cycles; 68℃ extension for 8 min, and storage at 4℃.

[0015] Preferably, the method for determining whether the plant to be tested belongs to the Coptis genus includes: if a 1300bp specific identification band appears in the electrophoretic pattern, then the plant to be tested belongs to the Coptis genus.

[0016] Methods for determining the species of a plant to be tested include: determining the species of the plant to be tested based on its position in the phylogenetic tree; if the plant to be tested clusters together with a known species in the phylogenetic tree, then the plant to be tested and the known species are determined to be the same species.

[0017] Another object of the present invention is to provide the application of the primer pair, reagent, kit, or method described herein in the identification of Coptis chinensis species.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] This invention provides a primer pair for identifying Coptis species. The invention designs specific primers for the ycf1 gene of chloroplasts in the genus Coptis. Due to the high polymorphism of this gene in the genus Coptis, the target fragment can be accurately amplified. The sequence amplified based on the primers of this application can be used to construct a phylogenetic tree, thereby clearly distinguishing seven species: Coptis chinensis, Coptis triangularis, Coptis meiyanensis, Coptis chinensis pentadactylis, Coptis yunnanensis, Coptis brevicornu, and Coptis japonica. This solves the problem of misjudgment caused by high similarity in traditional morphological identification and greatly improves the accuracy of identification.

[0020] The identification method provided by this invention is based on mature PCR amplification and sequence analysis techniques. The steps are simple, requiring no complex equipment, making it easy to promote in laboratories and grassroots institutions. Furthermore, it is not limited by plant growth stage or sample morphology, making it applicable to a wide range of scenarios. Simultaneously, it provides molecular-level evidence for the classification and resource conservation of the *Coptis* genus, aiding in systematic classification and phylogenetic studies, laying the foundation for resource conservation, development, utilization, and variety improvement. It fills a technical gap in the molecular identification of species in this genus and also provides a reference for the development of identification methods for closely related genera. Attached Figure Description

[0021] Figure 1 The results of agarose gel electrophoresis of PCR amplification products are shown; lanes 1-7 are Coptis chinensis, Coptis triangularis, Coptis chinensis 'Emei', Coptis chinensis 'Five-lobed', Coptis yunnanensis, Coptis chinensis 'Short-calyx', and Coptis japonica, respectively; lanes 8-10 are DNA concentrations of 100, 50, and 20 ng / μL, respectively.

[0022] Figure 2 A phylogenetic tree was constructed for the amplified sequences. Detailed Implementation

[0023] This invention provides a primer pair for identifying Coptis chinensis species. The primer pair includes an upstream primer and a downstream primer. The nucleotide sequence of the upstream primer is shown in SEQ ID NO.1: 5'-CCTGAGCAACGAACTTATCAACCG-3', and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.2: 5'-TGTAGCTTAGATCTGTGCCACGGT-3'.

[0024] The primers used in this invention are amplification primers for the chloroplast gene ycf1. The ycf1 gene is a highly polymorphic gene in plants of the genus *Coptis*. This application is based on the following species: *Coptis chinensis* (ycf1 gene nucleotide sequence as shown in SEQ ID NO. 3), *C. chinensis var. brevisepala* (ycf1 gene nucleotide sequence as shown in SEQ ID NO. 4), *C. deltoidea* (ycf1 gene nucleotide sequence as shown in SEQ ID NO. 5), *C. japonica* (ycf1 gene nucleotide sequence as shown in SEQ ID NO. 6), *C. omeiensis* (ycf1 gene nucleotide sequence as shown in SEQ ID NO. 7), *C. quin quesecta* (ycf1 gene nucleotide sequence as shown in SEQ ID NO. 8), and *C. teeta* (ycf1 gene nucleotide sequence as shown in SEQ ID NO. 8). Primers designed using the ycf1 gene (shown in NO.9) can be used for specific amplification, which can accurately determine whether the plant to be tested belongs to the Coptis genus. The amplified sequence can be used to construct a phylogenetic tree to further determine the species to which the plant to be tested belongs.

[0025] The present invention also provides a reagent for identifying species of Coptis chinensis, the reagent comprising the above-mentioned primer pair.

[0026] The present invention also provides a kit for identifying species of Coptis chinensis, the kit comprising the above-described primer pairs or the above-described reagents.

[0027] This invention also provides a method for identifying Coptis chinensis species, comprising the following steps: extracting DNA from the leaves of the plant to be tested; performing PCR amplification using the primer pair, the reagent, or the kit described herein; after amplification, performing agarose gel electrophoresis on the amplification product to determine whether the plant to be tested belongs to the genus Coptis; constructing a phylogenetic tree from the amplified sequence to determine the species of the plant to be tested.

[0028] In the method for identifying Coptis chinensis species according to the present invention, the preferred PCR amplification reaction system is as follows: 1 μL DNA template, 3 μL 10× Buffer, 3 μL dNTPs, 3 μL MgSO4; 1 μL each of upstream and downstream primers, 1 μL KOD-Plus-Neo, and ddH2O added to a final volume of 30 μL. The primer concentrations used in the present invention are preferably 0.1–1 μM, more preferably 0.5 μM, which effectively improves the detection sensitivity. The DNA concentration in the DNA template used in the present invention is preferably 20–100 ng / μL, more preferably 50 ng / μL; the detection method of the present invention can still amplify clear specific bands even at DNA concentrations as low as 20 ng / μL, and the amplification products are sufficient for subsequent phylogenetic tree construction, demonstrating high detection sensitivity and accuracy.

[0029] In the method for identifying Coptis chinensis species of the present invention, the preferred PCR amplification reaction program is as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 58℃ annealing for 30 s, 68℃ extension for 80 s, 35 cycles; 68℃ extension for 8 min, and storage at 4℃.

[0030] In the method for identifying species of Coptis chinensis in this invention, the method for determining whether a plant to be tested belongs to the Coptis chinensis species includes: if a 1300bp specific identification band appears in the electrophoretic pattern, then the plant to be tested belongs to the Coptis genus; the method for determining the species of the plant to be tested includes: determining the species of the plant to be tested based on its position in the phylogenetic tree; if the plant to be tested clusters together with a known species in the phylogenetic tree, then the plant to be tested and the known species are determined to be the same species.

[0031] This invention also provides the application of the described primer pairs, reagents, kits, or methods in the identification of Coptis chinensis species. This invention can be used to identify Coptis chinensis species, providing molecular-level evidence for the classification and resource conservation of the genus Coptis.

[0032] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0033] Example 1

[0034] A primer pair for identifying species of Coptis chinensis.

[0035] The nucleotide sequence of the upstream primer in the primer pair is shown in SEQ ID NO.1: 5'-CCTGAGCAACGAACTTATCAACCG-3'; the nucleotide sequence of the downstream primer is shown in SEQ ID NO.2: 5'-TGTAGCTTAGATCTGTGCCACGGT-3'.

[0036] Example 2

[0037] A method for identifying species of Coptis chinensis, the steps of which are as follows:

[0038] DNA was extracted from the leaves of the plant to be tested.

[0039] PCR amplification was performed using the primer pairs from Example 1, the reagents described herein, or the kit described herein:

[0040] The PCR amplification reaction system was as follows: 1 μL DNA template (50 ng / μL), 3 μL 10× Buffer, 3 μL dNTPs, 3 μL MgSO4; 1 μL each of upstream and downstream primers, 1 μL KOD-Plus-Neo, and ddH2O added to a final volume of 30 μL; the working concentration of both upstream and downstream primers was 0.5 μM.

[0041] The PCR amplification reaction program was as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 58℃ annealing for 30 s, 68℃ extension for 80 s, 35 cycles; 68℃ extension for 8 min, and storage at 4℃.

[0042] After amplification, the amplification products are subjected to agarose gel electrophoresis to determine whether the plant belongs to the genus Coptis: if a 1300bp specific identification band appears in the electrophoresis pattern, the plant belongs to the genus Coptis.

[0043] The amplified sequences are used to construct a phylogenetic tree to determine the species of the plant to be tested: the species of the plant to be tested is determined based on its position in the phylogenetic tree. If the plant to be tested is clustered with a known species in the phylogenetic tree, then the plant to be tested and the known species are determined to be the same species.

[0044] Example 3

[0045] Plants of the genus Coptis were identified using the method described in Example 2.

[0046] DNA was extracted from the leaves of *Coptis chinensis*, *C. deltoidea*, *C. omeiensis*, *C. quinquesecta*, *C. teeta*, *C. chinensis var. brevisepala*, and *C. japonica*. PCR amplification was performed using the method described in Example 2. The amplified products were then subjected to agarose gel electrophoresis. The results are as follows: Figure 1 As shown, lanes 1-7 are Coptis chinensis, Coptis triangularis, Coptis chinensis 'Emei', Coptis chinensis 'Five-lobed', Coptis yunnanensis, Coptis brevicornu, and Coptis japonica, respectively. The primers of this invention can specifically amplify the bands of Coptis genus plants.

[0047] DNA extracted from Coptis chinensis leaves was diluted to concentrations of 100, 50, and 20 ng / μL. PCR amplification was performed using the method described in Example 2. The amplified products were then subjected to agarose gel electrophoresis, and the results are as follows: Figure 1 As shown, lanes 8-10 represent the results when the DNA concentration is 100, 50, and 20 ng / μL, respectively. It can be seen that when the DNA concentration is diluted to 20 ng / μL, a specific band can still be amplified, indicating that the present invention has high detection sensitivity.

[0048] The products amplified from *Coptis chinensis*, *Coptis triangularis*, *Coptis embryophylla*, *Coptis chinensis var. chinensis*, *Coptis yunnanensis*, *Coptis brevis*, and *Coptis jasminoides* were sequenced (by Sangon Biotech) to obtain amplified sequences. A phylogenetic tree was constructed using these sequences, and the results are as follows: Figure 2 As shown.

[0049] according to Figure 2 The results showed that Coptis chinensis and its variant, Coptis short-calyx, are sister groups; Coptis triangularis and Coptis meiensis are sister groups, and these two sister groups are also sister groups of each other. Coptis japonica and Coptis yunnanensis are sister groups of each other; and Coptis chinensis var. chinensis is located at the base of the phylogenetic tree, forming a separate branch.

[0050] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A primer pair for identifying the species of Coptis, characterized in that, The primer pair comprises an upstream primer and a downstream primer, wherein the nucleotide sequence of the upstream primer is shown as SEQ ID NO. 1, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO.

2.

2. A reagent for identifying the species of Coptis, characterized in that, The reagent comprises the primer pair of claim 1.

3. A kit for identifying species of Coptis comprising, The kit comprises the primer pair of claim 1 or the reagent of claim 2.

4. A method of identifying a species of Coptis, comprising, The method comprises the following steps: extracting DNA from leaves of the plant to be tested; performing PCR amplification by using the primer pair of claim 1 or the reagent of claim 2 or the kit of claim 3; after the amplification, performing agarose gel electrophoresis on the amplification product to determine whether the plant to be tested belongs to the genus Coptis; constructing a phylogenetic tree based on the obtained sequence to determine the species of the plant to be tested.

5. The method of claim 4, wherein, The reaction system of the PCR amplification is as follows: 1 μL of DNA template, 3 μL of 10×Buffer, 3 μL of dNTPs, 3 μL of MgSO4, 1 μL of upstream primer, 1 μL of downstream primer, 1 μL of KOD-Plus-Neo, and ddH2O to 30 μL.

6. The method of claim 5, wherein, The concentration of the primer is 0.1-1 μM.

7. The method of claim 5, wherein, The concentration of DNA in the DNA template is 20-100 ng / μL.

8. The method of claim 4, wherein, The reaction procedure of the PCR amplification is as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30 s, annealing at 58 ℃ for 30 s, extension at 68 ℃ for 80 s, 35 cycles; extension at 68 ℃ for 8 min, and preservation at 4 ℃.

9. The method of claim 4, wherein, The method for determining whether the plant to be tested belongs to the genus Coptis comprises: if a specific identification band of about 1300 bp appears in the electrophoretogram, then the plant to be tested belongs to the genus Coptis. The method for determining the species of the plant to be tested comprises: determining the species to be tested according to the position of the plant to be tested in the phylogenetic tree, for example, if the plant to be tested and the known species are clustered together in the phylogenetic tree, then the plant to be tested and the known species are determined to be the same species.

10. The primer pair of claim 1 or the reagent of claim 2 or the kit of claim 3 or the method of any one of claims 4-9 for use in the identification of the species of Coptis.

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