A method for identifying Astragalus and Astragalus, and a kit and application thereof

Abstract

The present invention provides a method for identifying Astragalus and Astragalus genus, a kit and an application thereof, which belong to the biological field, solve the problem of the difficulty in accurately identifying and distinguishing Astragalus and Astragalus genus, and the present invention uses DNA barcodes: ITS2, psbA-trnH, rbcL and matk to design four primer pairs as shown in SEQ ID NO: 1-8, PCR amplification and sequencing of genomic DNA, species identification and differentiation according to the base sequence of specific sites, the Astragalus genus is Astragalus melaleuca, Astragalus davuri, Astragalus mongolicus, Astragalus oblique stem, and the Astragalus genus is Astragalus mountainus and Astragalus north Chinaus. The present invention realizes the accurate identification and differentiation of Astragalus and Astragalus genus, and has the advantages of simple, rapid, efficient and good repeatability of the identification method.

Description

Technical Field

[0001] The invention belongs to the biological field, and particularly relates to a method for identifying Astragalus and Astragalus, and a kit and application thereof. Background Art

[0002] Astragalus L. and Hedysarum L. are important components of the Leguminosae family, accounting for a large proportion. Both Astragalus and Hedysarum are annual or perennial herbs, and the roots of some species can be used as medicine, and the types of medicine are gradually expanding. In addition, Astragalus and Hedysarum species have important economic value, and most of the plants are favored by livestock and are important components of leguminous plants in natural pastures. Astragalus resources are abundant, but the varieties are mixed, so it is of great significance to accurately identify Astragalus and Hedysarum species. Summary of the invention

[0003] Based on the above shortcomings, the present invention provides a method for identifying different plant species within the genera Astragalus and Astragalus, which solves the problem of difficulty in accurately identifying and distinguishing the genera Astragalus and Astragalus.

[0004] The technical scheme adopted by the present invention is as follows: a method for identifying different plant species in the genera Astragalus and Astragalus, wherein the Astragalus genus includes Astragalus melaleuca, Astragalus davuri, Astragalus mongolicus, and Astragalus obliquus, and the Astragalus genus includes Astragalus montana and Astragalus huabei, the steps are as follows:

[0005] S1. Extract genomic DNA from the sample to be tested;

[0006] S2, using the genomic DNA of the sample to be tested as a template,

[0007] The DNA barcode primer set: primers shown in SEQ ID NO: 1 and SEQ ID NO: 2 were used to perform PCR amplification of the ITS2 sequence fragment.

[0008] A DNA barcode primer set: primers as shown in SEQ ID NO: 3 and SEQ ID NO: 4 were used to perform PCR amplification of the psbA-trnH sequence fragment;

[0009] PCR amplification of the rbcL sequence fragment was performed using a DNA barcode primer set: primers shown in SEQ ID NO: 5 and SEQ ID NO: 6;

[0010] PCR amplification of matk sequence fragment using DNA barcode primer set: primers shown in SEQ ID NO: 7 and SEQ ID NO: 8;

[0011] S3, obtaining the amplified product, and constructing an evolutionary tree for the amplified target DNA barcode sequence through sequencing, proofreading, and feature comparison;

[0012] S4. Determine the species of the sample to be tested:

[0013] If the nucleotide sequence of the DNA barcode of the ITS2 gene fragment is as shown in SEQ ID NO: 9, and the base type at position 27 is T, then the species is determined to be Astragalus melilotus.

[0014] If the DNA barcode of the ITS2 gene fragment has a nucleotide sequence as shown in SEQ ID NO: 10, and the base type at the second position is C and the base type at the 379th position is A, then the species is determined to be Astragalus davurii.

[0015] If the DNA barcode of the ITS2 gene fragment has a nucleotide sequence as shown in SEQ ID NO: 11, and the base type at the second position is C and the base type at the 379th position is C, then the species is determined to be Astragalus obliquus.

[0016] If the DNA barcode of the ITS2 gene fragment has a nucleotide sequence as shown in SEQ ID NO: 12, and the base type at the 81st position is A and the base type at the 238th position is T, then the species is determined to be Astragalus mongolica;

[0017] If the DNA barcode of the psbA-trnH gene fragment has a nucleotide sequence as shown in SEQ ID NO: 13, and the base type at the 5th position is A, then the species is determined to be Astragalus melilotus;

[0018] If the DNA barcode of the psbA-trnH gene fragment has a nucleotide sequence as shown in SEQ ID NO: 14, and the base type at position 160 is C, then the species is determined to be Astragalus membranaceus;

[0019] If the DNA barcode of the psbA-trnH gene fragment has a nucleotide sequence as shown in SEQ ID NO: 15, and the base type at position 169 is C, then the species is determined to be Astragalus membranaceus;

[0020] If the DNA barcode of the psbA-trnH gene fragment has a nucleotide sequence as shown in SEQ ID NO: 16, and the base type at position 176 is G, then the species is determined to be Astragalus davuriensis;

[0021] If the DNA barcode of the psbA-trnH gene fragment has a nucleotide sequence as shown in SEQ ID NO: 17, and the base type at position 83 is G, then the species is determined to be Astragalus obliquus;

[0022] If the nucleotide sequence of the DNA barcode of the rbcL gene fragment is as shown in SEQ ID NO: 18, and the base type at position 37 is G, then the species is determined to be Astragalus melilotus;

[0023] If the DNA barcode of the rbcL gene fragment has a nucleotide sequence as shown in SEQ ID NO: 19, and the base type at position 37 is A, then the species is determined to be Astragalus mongolica;

[0024] If the DNA barcode of the rbcL gene fragment has a nucleotide sequence as shown in SEQ ID NO: 20, and the base type at position 37 is C, then the species is determined to be Astragalus obliquus;

[0025] If the DNA barcode of the rbcL gene fragment has a nucleotide sequence as shown in SEQ ID NO: 21, and the base type at position 38 is G, then the species is determined to be Astragalus davurii;

[0026] If the DNA barcode of the matk gene fragment has a nucleotide sequence as shown in SEQ ID NO: 232, and the base type at position 16 is G, then the species is determined to be Astragalus davuriensis;

[0027] If the DNA barcode of the matk gene fragment has a nucleotide sequence as shown in SEQ ID NO: 23, and the base type at position 204 is C, then the species is determined to be Astragalus obliquus;

[0028] If the DNA barcode of the matk gene fragment has a nucleotide sequence as shown in SEQ ID NO: 24, and the base type at position 189 is A, then the species is determined to be Astragalus melilotus;

[0029] If the DNA barcode of the matk gene fragment has a nucleotide sequence as shown in SEQ ID NO: 25, and the base type at the 94th base is C and the base type at the 670th base is T, then the species is determined to be Astragalus mongolica.

[0030] Furthermore, in the method described above, the PCR amplification system includes the following: 18 μL of Taq DNA polymerase, 2 μL of DNA template, 1.2 μL of 10 umo1 / L upstream and downstream primers, and ddH2O to 40 μL.

[0031] Further, in the method described above, the PCR reaction condition parameters include the following: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 54°C-62°C for 15 s, extension at 72°C for 30 s, a total of 35 cycles, extension at 72°C for 10 min; storage at 4°C.

[0032] Another object of the present invention is to provide a primer set for identifying different plant species within the genera Astragalus and Astragalus, the primer set comprising: a primer pair as shown in SEQ ID NO: 1 and SEQ ID NO: 2, for amplifying an ITS2 sequence fragment, and a primer pair as shown in SEQ ID NO: 3 and SEQ ID NO: 4, for amplifying a psbA-trnH sequence fragment.

[0033] Another object of the present invention is to provide a primer set for identifying different plant species within the genera Astragalus and Astragalus, the primer set comprising: a primer pair as shown in SEQ ID NO: 5 and SEQ ID NO: 6, for amplifying a rbcL sequence fragment, and a primer pair as shown in SEQ ID NO: 3 and SEQ ID NO: 4, for amplifying a psbA-trnH sequence fragment.

[0034] Another object of the present invention is to provide a primer set for identifying different plant species within the genera Astragalus and Astragalus, the primer set comprising: a primer pair as shown in SEQ ID NO: 7 and SEQ ID NO: 8, amplifying a matk sequence fragment, and a primer pair as shown in SEQ ID NO: 3 and SEQ ID NO: 4, amplifying a psbA-trnH sequence fragment.

[0035] The Astragalus species that can be identified by the above four primer sets are Astragalus melilotus, Astragalus davuri, Astragalus mongolicus, and Astragalus obliquus, and the Astragalus genus are Astragalus montana and Astragalus huabei.

[0036] Another object of the present invention is to provide a kit for identifying different plant species within the genera Astragalus and Astragalus, comprising the primer set as described in any one of the above items.

[0037] Another object of the present invention is to provide an application of a primer set as described in any of the above items for identifying different plant species within the genera Astragalus and Astragalus, wherein the Astragalus genus includes Astragalus melilotiformis, Astragalus davuri, Astragalus mongolicus, and Astragalus obliquus, and the Astragalus genus includes Astragalus montana and Astragalus huabei.

[0038] Advantages and beneficial effects of the present invention: The present invention realizes accurate identification and differentiation of different plant species within the genera Astragalus and Astragalus, and has the advantages of simple, rapid, efficient and good repeatability of the identification method. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is the gel electrophoresis diagram of ITS2 sequence primer amplification, and the order from left to right is: M: Marker; 1: Astragalus melaleuca; 2: Astragalus davuri; 3: Astragalus huabei; 4: Astragalus mongolicus; 5: Astragalus shanica; 6: Astragalus tatarica; 7: Astragalus truncatus; 8: Astragalus obliquus;

[0040] Figure 2 The gel electrophoresis diagram of rbcL sequence primer amplification; from left to right: M: Marker; 1: Astragalus melaleuca; 2: Astragalus davuri; 3: Astragalus huabei; 4: Astragalus mongolicus; 5: Astragalus shanica; 6: Astragalus tatarica; 7: Astragalus truncatus; 8: Astragalus obliquus;

[0041] Figure 3 The gel electrophoresis diagram of matk sequence primer amplification; from left to right: M: Marker; 1: Astragalus melaleuca; 2: Astragalus davuri; 3: Astragalus huabei; 4: Astragalus mongolicus; 5: Astragalus shanica; 6: Astragalus tatarica; 7: Astragalus truncatus; 8: Astragalus obliquus;

[0042] Figure 4 The gel electrophoresis diagram of psbA-trnH sequence primer amplification; from left to right: M: Marker; 1: Astragalus melaleuca; 2: Astragalus davurica; 3: Astragalus huabei; 4: Astragalus mongolica; 5: Astragalus shanica; 6: Astragalus tatarica; 7: Astragalus truncatus; 8: Astragalus obliquus;

[0043] Figure 5 is the phylogenetic tree based on ITS2 sequences;

[0044] Figure 6 Phylogenetic tree based on rbcL sequences

[0045] Figure 7 Phylogenetic tree based on matk sequences

[0046] Figure 8 is the phylogenetic tree based on psbA-trnH sequences;

[0047] Fig. 9 This is the result of ITS2 multiple sequence alignment;

[0048] Fig.10 This is the result of multiple sequence alignment of rbcL;

[0049] Fig.11 This is the result of multiple sequence alignment of psbA-trnH;

[0050] Fig.12 This is the result of matk multiple sequence alignment; DETAILED DESCRIPTION

[0051] DNA barcoding technology is also called DNA barcoding. Its principle is to construct a biological identification database based on the intraspecificity and interspecific diversity of different species using one or several standard DNA fragments to quickly, easily and accurately identify different species. Studies have found that although the chloroplast genes in plants are relatively conservative, their uniparental inheritance avoids gene recombination, and they are rich in content and have strong amplification capabilities. Therefore, identifying plant species on the chloroplast genome has become a feasible method. Among the Gramineae species, matk, rbcL, ITS2 and psbA-trnH are studied in depth as universal barcodes, while the research on Leguminosae species is superficial. Therefore, matk, rbcL, ITS2 and psbA-trnH four primers are selected to identify Astragalus and Astragalus species. The present invention is further described below in conjunction with the accompanying drawings and examples.

[0052] Example 1

[0053] 1.1 Sample:

[0054] A total of 8 samples from 8 species of 2 genera, Astragalus and Astragalus were collected. The sample information is shown in Table 1.

[0055] Table 1 - Astragalus and Astragalus sample information

[0056]

[0057] 1.2 Design primer sequences:

[0058] Primers were designed using the chloroplast genome sequence as a template, providing four pairs of primers for identifying Astragalus and Astragalus, denoted as mF / mR, rF / rR, IF / IR, and tF / tR. The nucleotide sequences of the primers are shown in Table 2 below:

[0059] Table 2 Four pairs of primers used to identify Astragalus and Astragalus species

[0060]

[0061] 1.3 Genomic DNA extraction, PCR amplification and sequencing

[0062] About 10 mg of plant leaves were weighed, ground into powder with liquid nitrogen, and genomic DNA was extracted using a kit method.

[0063] 1.4 PCR amplification

[0064] Primers were designed using Primer 5.0 software with reference to the nucleotide sequences of chloroplast matk, rbcL and trnH-psbA genes and ITS2 nuclear gene in GenBank. Primers were synthesized by Ruibo Xingke Biotechnology Co., Ltd. The primer sequences are shown in Table 1.

[0065] PCR amplification uses a 40u1 system: 18μL Taq DNA polymerase, 2μL DNA template, 1.2μL each of 10umo1 / L upstream and downstream primers, and add ddH2O to 40μL.

[0066] PCR reaction conditions: 95°C pre-denaturation for 3 min; 95°C denaturation for 15 s, 54°C-62°C annealing for 15 s, 72°C extension for 30 s, 35 cycles in total, 72°C extension for 10 min; storage at 4°C. PCR products were spotted into agarose gel electrophoresis wells, electrophoresed at 145 V for 5 min, and detected by UV gel analysis.

[0067] 1.5 PCR amplification and sequencing

[0068] The PCR amplification products were sent to Ruiboxingke Biotechnology Co., Ltd. for sequencing.

[0069] 1.6 Data Processing

[0070] The sequencing results were proofread and edited using chromas 2.33 software, and sequence alignment was performed using MEGA11 software, and analysis was performed using Dnasp 5.10 software.

[0071] Example 2

[0072] Results and Analysis

[0073] 2.1 Primer screening

[0074] Based on the designed 4 pairs of universal primers, PCR amplification conditions for the target fragments were established and optimized for 8 species in 8 genera of Astragalus and Astragalus. The optimal annealing temperatures obtained are shown in Table 3.

[0075] Table 3- Optimal annealing temperature of primers for PCR reaction

[0076]

[0077] 2.2K2P distance

[0078] As shown in Table 4-7, the K2P distance between Astragalus and Astragalus in the psbA-trnH region is the largest, indicating that the difference between Astragalus and Astragalus in the psbA-trnH region is the largest. The K2P distance between Astragalus and Astragalus in the matk region is the smallest, indicating that the difference between Astragalus and Astragalus in the matk region is the smallest.

[0079] Table 4: K2P distances between Astragalus and Lithospermum in the psbA-trnH region

[0080]

[0081] Table 5: K2P distances between Astragalus and Astragalus in the matk region

[0082]

[0083] Table 6: K2P distances between Astragalus and Astragalus in ITS2 region

[0084]

[0085] Table 7: K2P distances between Astragalus and Astragalus in the rbcL region

[0086]

[0087] 2.3 Multiple sequence alignment

[0088] 2.3.1 ITS2 gene fragment

[0089] like Fig. 9 As shown, if the ITS2 gene fragment is T at the 27th base type, the species is determined to be Astragalus melilotus; if the ITS2 gene fragment is C at the 2nd base type and A at the 379th base type, the species is determined to be Astragalus davuri; if the ITS2 gene fragment is C at the 2nd base type and C at the 379th base type, the species is determined to be Astragalus obliquus; if the ITS2 gene fragment is A at the 81st base type and T at the 238th base type, the species is determined to be Astragalus mongolicus; if the ITS2 gene fragment is G at the 242nd base type and A at the 162nd base type, the species is determined to be Astragalus huabei or Astragalus mountainus; if the ITS2 gene fragment is A at the 324th base type, the species is determined to be Astragalus taluo or Astragalus fine-branched.

[0090] 2.3.2rbcL gene fragment

[0091] like Fig.10 As shown, if the 37th base type of the rbcL gene fragment is G, the species is determined to be Astragalus melilotus; if the 37th base type of the rbcL gene fragment is A, the species is determined to be Astragalus mongolicus; if the 37th base type of the rbcL gene fragment is C, the species is determined to be Astragalus oblique stem; if the 38th base type of the rbcL gene fragment is G, the species is determined to be Astragalus davuricus; if the 323rd base type of the rbcL gene fragment is A, the species is determined to be Astragalus shanyanensis or Astragalus huabeiyanensis; if the 16th base type of the rbcL gene fragment is C, the species is determined to be Astragalus taluoyanensis or Astragalus fine-branched.

[0092] 2.3.3psbA-trnH gene fragment

[0093] like Fig.11As shown, if the base type of the psbA-trnH gene fragment at the 5th position is A, the species is determined to be Astragalus melilotus; if the base type of the psbA-trnH gene fragment at the 160th position is C, the species is determined to be Astragalus montana; if the base type of the psbA-rnH gene fragment at the 169th position is C, the species is determined to be Astragalus dawuliensis; if the base type of the psbA-trnH gene fragment at the 83rd position is G, the species is determined to be Astragalus oblique stem; if the base type of the psbA-trnH gene fragment at the 323rd position is A, the species is determined to be Astragalus montana or Astragalus montana; if the base type of the psbA-trnH gene fragment at the 13th position is T, the species is determined to be Astragalus talouensis or Astragalus fine-branched.

[0094] 2.3.4 matk gene fragment

[0095] like Fig.12 As shown, if the matk gene fragment is G at the 16th base type, the species is determined to be Astragalus dauricus; if the matk gene fragment is C at the 204th base type, the species is determined to be Astragalus obliquus; if the matk gene fragment is A at the 189th base type, the species is determined to be Astragalus melilotus; if the matk gene fragment is C at the 94th base type and T at the 670th base type, the species is determined to be Astragalus mongolicus; if the matk gene fragment is C at the 94th base type and T at the 100th base type, the species is determined to be Astragalus shanyanensis or Astragalus huabeiyanensis; if the matk gene fragment is A at the 100th base type, the species is determined to be Astragalus taluoyanensis or Astragalus fine-branched.

[0096] Example 3

[0097] 3. Establishment of DNA Barcoding Database

[0098] As shown in Table 8, the ITS2 fragment was analyzed based on DNAsp 6.0 software. The results showed that the eight samples of the eight Astragalus and Astragalus species had eight variant sites, thus defining six haplotypes. A 、H1 B 、H1 D 、H1 F The unique haplotypes of Astragalus melaleuca, Astragalus davuri, Astragalus mongolicus and Astragalus obliquus are H1 C It is the shared haplotype of Astragalus membranaceus and Astragalus membranaceus in North China, H1 E It is a shared haplotype of Astragalus membranaceus and Astragalus membranaceus.

[0099] The rbcL fragment was analyzed based on DNAsp 6.0 software. The results showed that there were 8 variant sites among 8 samples of 8 species of Astragalus and Astragalus, thus defining 6 haplotypes. A 、H2 B 、H2 D 、H2 F The unique haplotypes of Astragalus melaleuca, Astragalus davuri, Astragalus mongolicus and Astragalus obliquus are H2 C It is the shared haplotype of Astragalus membranaceus and Astragalus membranaceus in North China. E It is a shared haplotype of Astragalus membranaceus and Astragalus membranaceus.

[0100] The matk fragment was analyzed based on DNAsp6.0 software. The results showed that there were 8 mutation sites among 8 samples of 8 species of Astragalus and Astragalus, thus defining 6 haplotypes. A 、H3 B 、H3 D 、H3 F The unique haplotypes of Astragalus melaleuca, Astragalus davuri, Astragalus mongolicus and Astragalus obliquus are H3 C It is the shared haplotype of Astragalus membranaceus and Astragalus membranaceus in North China, H3 E It is a shared haplotype of Astragalus membranaceus and Astragalus membranaceus.

[0101] The psbA-trnH fragment was analyzed based on DNAsp6.0 software. The results showed that there were 8 variant sites among 8 samples of 8 species of Astragalus and Astragalus, thus defining 6 haplotypes. A 、H4 B 、H4 D 、H4 F The unique haplotypes of Astragalus melaleuca, Astragalus davuri, Astragalus mongolicus and Astragalus obliquus are H4 C It is the shared haplotype of Astragalus membranaceus and Astragalus membranaceus in North China, H4 E It is a shared haplotype of Astragalus membranaceus and Astragalus membranaceus.

[0102] In addition, based on the expression specificity of matk, rbcL, ITS2 and psbA-trnH genes among Astragalus and Astragalus species, a DNA barcode database of 8 Astragalus and Astragalus species was established. The database consists of specific primers and DNA identification codes of matk, rbcL, ITS2 and psbA-trnH genes.

[0103] Table 8: DNA Barcoding database of 8 species of Astragalus and Astragalus

[0104]

[0105] (Note: "I" stands for "ITS2 gene", "R" stands for "rbcl gene", "M" stands for "matk gene", "P" stands for "trnH-psbA gene", "H" stands for "haplotype", and the following "1, 2, 3 and 4" stand for the four marker sites "ITS2, rbcl, matk and trnH-psbA" respectively, and the superscript letters represent the nucleotide haplotype categories of the corresponding marker sites.)

[0106] in conclusion:

[0107] The present invention performs feature analysis, calculates genetic distance, constructs phylogenetic trees and other multi-faceted analyses on the relevant sequences of ITS2, psbA-trnH, rbcL and matK of 8 samples of Astragalus and Astragalus. The results show that ITS2 and psbA-trnH sequences can effectively distinguish the two major categories of Astragalus and Astragalus, and can distinguish species of the same genus but different species. Based on the results of this experimental study, ITS2 and psbA-trnH sequences can be used as species differentiation and identification of Astragalus and Astragalus, and the present invention provides a certain theoretical reference for the molecular identification of Astragalus and Astragalus.

Claims

1. A method for identifying different plant species in the genera Astragalus and Astragalus, wherein the Astragalus genus includes Astragalus melaleuca, Astragalus davuri, Astragalus mongolicus, and Astragalus obliquus, and the Astragalus genus includes Astragalus montana and Astragalus huabei, characterized in that: Here are the steps: S1. Extract genomic DNA from the sample to be tested; S2, using the genomic DNA of the sample to be tested as a template, The DNA barcode primer set: primers shown in SEQ ID NO: 1 and SEQ ID NO: 2 were used to perform PCR amplification of the ITS2 sequence fragment. A DNA barcode primer set: primers as shown in SEQ ID NO: 3 and SEQ ID NO: 4 were used to perform PCR amplification of the psbA-trnH sequence fragment; PCR amplification of rbcL sequence fragments was performed using a DNA barcode primer set: primers shown in SEQ ID NO: 5 and SEQ ID NO: 6; Using a DNA barcode primer set: primers as shown in SEQ ID NO: 7 and SEQ ID NO: 8 to perform PCR amplification of a matk sequence fragment; S3, obtaining the amplified product, and constructing an evolutionary tree for the amplified target DNA barcode sequence through sequencing, proofreading, and feature comparison; S4. Determine the species of the sample to be tested: If the nucleotide sequence of the DNA barcode of the ITS2 gene fragment is as shown in SEQ ID NO: 9, and the base type at position 27 is T, then the species is determined to be Astragalus melilotus. If the DNA barcode of the ITS2 gene fragment has a nucleotide sequence as shown in SEQ ID NO: 10, and the base type at the second position is C and the base type at the 379th position is A, then the species is determined to be Astragalus davurii. If the DNA barcode of the ITS2 gene fragment has a nucleotide sequence as shown in SEQ ID NO: 11, and the base type at the second position is C and the base type at the 379th position is C, then the species is determined to be Astragalus obliquus. If the DNA barcode of the ITS2 gene fragment has a nucleotide sequence as shown in SEQ ID NO: 12, and the base type at the 81st position is A and the base type at the 238th position is T, then the species is determined to be Astragalus mongolica; If the DNA barcode of the psbA-trnH gene fragment has a nucleotide sequence as shown in SEQ ID NO: 13, and the base type at the 5th position is A, then the species is determined to be Astragalus melilotus; If the DNA barcode of the psbA-trnH gene fragment has a nucleotide sequence as shown in SEQ ID NO: 14, and the base type at position 160 is C, then the species is determined to be Astragalus membranaceus; If the DNA barcode of the psbA-trnH gene fragment has a nucleotide sequence as shown in SEQ ID NO: 15, and the base type at position 169 is C, then the species is determined to be Astragalus membranaceus; If the DNA barcode of the psbA-trnH gene fragment has a nucleotide sequence as shown in SEQ ID NO: 16, and the base type at position 176 is G, then the species is determined to be Astragalus davuriensis; If the DNA barcode of the psbA-trnH gene fragment has a nucleotide sequence as shown in SEQ ID NO: 17, and the base type at position 83 is G, then the species is determined to be Astragalus obliquus; If the nucleotide sequence of the DNA barcode of the rbcL gene fragment is as shown in SEQ ID NO: 18, and the base type at position 37 is G, then the species is determined to be Astragalus melilotus; If the DNA barcode of the rbcL gene fragment has a nucleotide sequence as shown in SEQ ID NO: 19, and the base type at position 37 is A, then the species is determined to be Astragalus mongolica; If the DNA barcode of the rbcL gene fragment has a nucleotide sequence as shown in SEQ ID NO: 20, and the base type at position 37 is C, then the species is determined to be Astragalus obliquus; If the DNA barcode of the rbcL gene fragment has a nucleotide sequence as shown in SEQ ID NO: 21, and the base type at position 38 is G, then the species is determined to be Astragalus davurii; If the DNA barcode of the matk gene fragment has a nucleotide sequence as shown in SEQ ID NO: 22, and the base type at position 16 is G, then the species is determined to be Astragalus davuriensis; If the DNA barcode of the matk gene fragment has a nucleotide sequence as shown in SEQ ID NO: 23, and the base type at position 204 is C, then the species is determined to be Astragalus obliquus; If the DNA barcode of the matk gene fragment has a nucleotide sequence as shown in SEQ ID NO: 24, and the base type at position 189 is A, then the species is determined to be Astragalus melilotus; If the DNA barcode of the matk gene fragment has a nucleotide sequence as shown in SEQ ID NO: 25, and the base type at the 94th base is C and the base type at the 670th base is T, then the species is determined to be Astragalus mongolica.

2. The method according to claim 1, characterized in that: PCR amplification system: Taq DNA polymerase 18 μL, DNA template 2 μL, 1.2 μL each of 10 umo1 / L upstream and downstream primers, add ddH2O to 40 μL.

3. The method according to claim 2, characterized in that: PCR reaction condition parameters: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 54°C-62°C for 15 s, extension at 72°C for 30 s, a total of 35 cycles, extension at 72°C for 10 min; storage at 4°C.

4. The use of the method according to any one of claims 1-3 in identifying or assisting in identifying different plant species within the genera Astragalus and Astragalus, wherein the Astragalus genus includes Astragalus melilotiformis, Astragalus davuri, Astragalus mongolicus, and Astragalus obliquus, and the Astragalus genus includes Astragalus montana and Astragalus huabei.

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