A method for identifying Trifolium, a kit and application thereof

Abstract

The present invention provides a method for identifying clover and a kit and application thereof, which belongs to the biological field and solves the problem of difficult accurate identification and differentiation of clover. 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, performs PCR amplification and sequencing on genomic DNA, and performs species identification and differentiation according to base sequences at specific sites. The clover genus includes red clover, white clover, ground clover, Caucasian clover, miscellaneous clover and wild fireball. The present invention realizes accurate identification and differentiation of clover genus, and has the advantages of simple, rapid, efficient and good repeatability in identification method.

Description

Technical Field

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

[0002] The genus Trifolium (Trifolium L.), an annual or perennial herb, is an important part of the leguminous plant family. Plants of the genus Trifolium have economic and medicinal value. In terms of economic value, plants of the genus Trifolium are an excellent forage grass, rich in protein and minerals, cold-resistant and heat-resistant, with strong adaptability. They can grow on acidic and alkaline soils, have high feeding value, and are suitable for breeding cattle, sheep, herbivorous fish, etc. In addition, plants of the genus Trifolium can also be used to build lawns, which have the effect of improving soil and water and soil moisture retention, and can be used to build green lawns in gardens, parks, golf courses, etc. The stems and leaves of clover contain aromatic oils, the main component of which is coumarin, which can be used as a raw material for blending flavors. In terms of medicinal value, plants of the genus Trifolium have the effects of clearing heat and relieving cough, relieving heat and dispersing blood stasis, cooling blood and stopping bleeding, and relaxing muscles and activating collaterals. They are mainly used to treat fever, blood stasis caused by falls, various bleeding, pain caused by meridian obstruction, and other symptoms. The whole plant of Trifolium affine can be used as medicine, which has the effects of clearing heat and cooling blood, calming the nerves and relieving pain, and expectorating and relieving cough. The tincture of its flowers can be used to treat colds. The whole plant tincture has astringent and hemostatic effects, can promote wound healing, and also contains isoflavones with anti-cancer effects. The leaves contain isoquercitrin, linseed glycosides, and radix scutellariae, and the whole plant contains coumestrol, tocopherol, saponins, genistein and other ingredients, among which genistein has a hormone-like effect. The genus Trifolium is rich in resources, but the varieties are mixed, so it is of great significance to accurately identify the species of the genus Trifolium. Summary of the invention

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

[0004] The technical scheme adopted by the present invention is as follows: a method for identifying different plant species in the genus Trifolium, wherein the genus Trifolium includes red clover, white clover, ground clover, Caucasian clover, miscellaneous clover, and wild fireball, and the identification method 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] PCR amplification of matk sequence fragments was performed using a DNA barcode primer set: primers shown in SEQ ID NO: 1 and SEQ ID NO: 2;

[0008] PCR amplification of rbcL sequence fragments was performed using a DNA barcode primer set: primers shown in SEQ ID NO: 3 and SEQ ID NO: 4;

[0009] The DNA barcode primer set: primers shown in SEQ ID NO: 5 and SEQ ID NO: 6 were used to perform PCR amplification of the ITS2 sequence fragment.

[0010] A DNA barcode primer set: primers as shown in SEQ ID NO: 7 and SEQ ID NO: 8 were used to perform PCR amplification of the psbA-trnH sequence fragment;

[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 DNA barcode of the ITS2 gene fragment has a nucleotide sequence as shown in SEQ ID NO: 9, and the base type at position 263 is G, then the species is determined to be red clover;

[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 position 160 is T, then the species is determined to be white clover;

[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 position 156 is A, then the species is determined to be Trifolium substantum;

[0016] If the DNA barcode of the ITS2 gene fragment has a nucleotide sequence as shown in SEQ ID NO: 12, with a T at base type 80, then the species is determined to be Caucasian clover;

[0017] If the DNA barcode of the ITS2 gene fragment has a nucleotide sequence as shown in SEQ ID NO: 13, and the base type at position 97 is A, then the species is determined to be Pyrophora pyrocaulifera;

[0018] If the DNA barcode of the ITS2 gene fragment has a nucleotide sequence as shown in SEQ ID NO: 14, the base type at position 218 is A, and the base type at position 188 is C, then the species is determined to be Trifolium heterophyllum;

[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 22 is G, then the species is determined to be Trifolium repens;

[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 27 is C, then the species is determined to be white clover;

[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 214 is C, then the species is determined to be Trifolium substantum;

[0022] If the DNA barcode of the psbA-trnH gene fragment has a nucleotide sequence as shown in SEQ ID NO: 18, and the base type at position 121 is A, then the species is determined to be Trifolium heterophyllum;

[0023] If the DNA barcode of the psbA-trnH gene fragment has a nucleotide sequence as shown in SEQ ID NO: 19, and the base type at position 100 is T, then the species is determined to be Pyrophora pyrocaulifera;

[0024] If the DNA barcode of the matk gene fragment has a nucleotide sequence as shown in SEQ ID NO: 20, and the base type at position 39 is A, then the species is determined to be red clover;

[0025] If the DNA barcode of the matk gene fragment has a nucleotide sequence as shown in SEQ ID NO: 21, and the base type at position 47 is C, then the species is determined to be white clover;

[0026] 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 375 is C, then the species is determined to be Trifolium substantum;

[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 the 6th position is C, then the species is determined to be Caucasian clover;

[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 255 is G, then the species is determined to be heterophyllous;

[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 position 530 is C, then the species is determined to be Pyrophora pyrophylla;

[0030] If the DNA barcode of the rbcL gene fragment has a nucleotide sequence as shown in SEQ ID NO: 26, and the base type at the 4th position is T, then the species is determined to be Trifolium repens;

[0031] If the DNA barcode of the rbcL gene fragment has a nucleotide sequence as shown in SEQ ID NO: 27, and the base type at position 54 is G, then the species is determined to be white clover;

[0032] If the nucleotide sequence of the DNA barcode of the rbcL gene fragment is as shown in SEQ ID NO: 28, and the base type at position 348 is A, then the species is determined to be Trifolium pratense;

[0033] If the DNA barcode of the rbcL gene fragment has a nucleotide sequence as shown in SEQ ID NO: 29, and the base type at position 283 is C, then the species is determined to be Caucasian clover;

[0034] If the DNA barcode of the rbcL gene fragment has a nucleotide sequence as shown in SEQ ID NO: 30, and the base type at position 72 is C, then the species is determined to be heterophyllous;

[0035] If the nucleotide sequence of the DNA barcode of the rbcL gene fragment is as shown in SEQ ID NO: 31, and the base type at the 108th position is T, then the species is determined to be Pyrophora pyrocaulifera.

[0036] Further, the method described above, PCR amplification system: Taq DNA polymerase 16 μL, DNA template 2 μL, 10 umo1 / L upstream and downstream primers 1.2 μL each, add ddH2O to 40 μL.

[0037] Furthermore, as described above, the PCR reaction condition parameters are: 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; and storage at 4°C.

[0038] Another object of the present invention is to provide a primer set for identifying different plant species within the genus Trifolium, wherein the genus Trifolium includes red clover, white clover, subterranean clover, Caucasian clover, miscellaneous clover, and wild fireball, and the primer set includes: a primer pair as shown in SEQ ID NO: 1 and SEQ ID NO: 2, PCR amplification of matk sequence fragment.

[0039] Another object of the present invention is to provide a primer set for identifying different plant species within the genus Trifolium, wherein the genus Trifolium includes red clover, white clover, subterranean clover, Caucasian clover, miscellaneous clover, and wild fireball, and the primer set comprises: a primer pair as shown in SEQ ID NO: 3 and SEQ ID NO: 4, PCR amplification of rbcL sequence fragment.

[0040] Another object of the present invention is to provide a primer set for identifying different plant species within the genus Trifolium, wherein the genus Trifolium includes red clover, white clover, subterranean clover, Caucasian clover, miscellaneous clover, and wild fireball, and the primer set includes: a primer pair as shown in SEQ ID NO: 5 and SEQ ID NO: 6, PCR amplification of an ITS2 sequence fragment.

[0041] Another object of the present invention is to provide a kit for identifying different plant species within the genus Trifolium, comprising the primer set described above.

[0042] Another object of the present invention is to provide the use of the primer set as described above for different plant species within the genus Trifolium, wherein the genus Trifolium includes red clover, white clover, ground clover, Caucasian clover, miscellaneous clover, and wild ball of fire.

[0043] Advantages and beneficial effects of the present invention: The present invention realizes accurate identification and differentiation of different plant species within the genus Trifolium, and has the advantages of simple, rapid, efficient and good repeatability of the identification method. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 This is the result of ITS2 multiple sequence alignment;

[0045] Figure 2 This is the result of multiple sequence alignment of rbcL;

[0046] Figure 3 This is the result of multiple sequence alignment of psbA-trnH;

[0047] Figure 4 This is the result of matk multiple sequence alignment;

[0048] Figure 5 This is the gel electrophoresis of ITS2 sequence samples, from left to right: M: Marker; 1: Red clover; 2: White clover; 3: Trifolium repens; 4: Caucasian clover; 5: Trifolium variegatum; 6: Pyrocalyx pyrophyllum;

[0049] Figure 6 This is the gel electrophoresis diagram of the rbcL sequence sample. From left to right, the order is: M: Marker; 1: Red clover; 2: White clover; 3: Trifolium repens; 4: Caucasian clover; 5: Trifolium variegatum; 6: Pyrocalyx pyrophyllum;

[0050] Figure 7 This is the gel electrophoresis diagram of the matk sequence sample. From left to right, the order is: M: Marker; 1: Red clover; 2: White clover; 3: Ground clover; 4: Caucasian clover; 5: Miscellaneous clover; 6: Pyrocalyx pyrophylla;

[0051] Figure 8This is the gel electrophoresis diagram of the psbA-trnH sequence sample. The order from left to right is: M: Marker; 1: Red clover; 2: White clover; 3: Trifolium repens; 4: Caucasian clover; 5: Trifolium variegatum; 6: Pyrocalyx pyrophyllum.

[0052] Fig. 9 NJ tree of Trifolium species constructed based on ITS2 sequences;

[0053] Fig.10 is the NJ tree of Trifolium species constructed based on rbcL sequences;

[0054] Fig.11 is the NJ tree of Trifolium species constructed based on matk sequences;

[0055] Fig.12 NJ tree of Trifolium species constructed based on psbA-trnH sequences. DETAILED DESCRIPTION

[0056] DNA barcoding technology is also known as DNA barcoding. Its principle is to use one or several standard DNA fragments to construct a biological identification database based on the intraspecificity and interspecific diversity of different species, so as 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 ability. Therefore, it is a feasible method to identify plant species based on chloroplasts. Among the species of Poaceae, matk, rbcL, ITS2 and psbA-trnH are widely studied as universal barcodes, while the research on leguminous species is superficial. Therefore, four primers, matk, rbcL, ITS2 and psbA-trnH, were used to perform PCR amplification on the genomic DNA of Trifolium species, and then sequence alignment and manual correction were performed. The interspecific distance was calculated using the Kimura-2-parameter distance (K2P) model, and the intraspecific and interspecific K2P distances were calculated using MEGA11. Then, molecular systematics methods were used to analyze the barcodes by constructing phylogenetic trees (such as NJ, UPGMA, ML and MP, etc.) and to verify the monophyly of each species. The present invention is further described below in conjunction with the embodiments.

[0057] Example 1

[0058] 1.1 Sample:

[0059] A total of 6 samples of 6 species of Trifolium were collected. The sample information is shown in Table 1.

[0060] Table 1 Trifolium sample information

[0061]

[0062] In this embodiment, according to the nucleotide sequences of matk, rbcL, ITS2 and psbA-trnH genes of Trifolium species in GenBank, 4 pairs of universal primers were designed, and PCR amplification conditions for the target fragments of 6 samples of 6 species of Trifolium were established for amplification, and then the amplified products were sequenced and analyzed. After comparison, the 5' and 3' conservative sequences of the four marker sites of Trifolium were screened out, and the haplotype analysis of single nucleotide polymorphisms (SNPs) was performed on the nucleotides in the conservative region of each marker site. The four marker sites screened according to matk, rbcL, ITS2 and psbA-trnH genes were used to establish corresponding specific DNA identification codes for 6 species of Trifolium, so that the 6 species of Trifolium can be accurately identified. The method steps are as follows:

[0063] 1.2 Genomic DNA extraction, PCR amplification and sequencing

[0064] About 10 mg of plant leaves were weighed, ground into powder with liquid nitrogen, and genomic DNA was extracted using a kit method.

[0065] 1.3 PCR amplification

[0066] Primers were designed using Primer5.0 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 2.

[0067] Table 2 Four pairs of primers for identifying Trifolium species

[0068]

[0069] PCR amplification system: 16 μL of Taq DNA polymerase, 2 μL of DNA template, 1.2 μL of 10 μm / L upstream and downstream primers, and ddH2O to 40 μL.

[0070] PCR reaction conditions: 95℃ pre-denaturation for 3min; 95℃ denaturation for 15s, 54℃-62℃ annealing for 15s, 72℃ extension for 30s, 35 cycles in total, 72℃ extension for 10min; 4℃ storage. PCR products were spotted into agarose gel electrophoresis wells, electrophoresed at 145V for 5min, and detected by UV gel analysis. Figure 5-8 Shown are sample gel electrophoresis images of ITS2, rbcL, matk and psbA-trnH sequences, respectively.

[0071] 1.4 PCR amplification and sequencing

[0072] The PCR amplification products were sent to Ruiboxingke Biotechnology Co., Ltd. for sequencing.

[0073] 1.5 Data processing

[0074] The sequencing results were proofread and edited using chromas2.33, and sequence alignment was performed using MEGA11, and analysis was performed using Dnasp5.10.

[0075] Example 2

[0076] Results and Analysis

[0077] 2.1 Primer screening

[0078] Based on the designed 4 pairs of universal primers, PCR amplification conditions for the target fragments were established and optimized for 6 species of 6 Trifolium species. The obtained optimal annealing temperatures are shown in Table 3.

[0079] Table 3 Optimal annealing temperature of primers for PCR reaction

[0080]

[0081] Using the genomic DNA of Trifolium plants as a template, PCR amplification and sequencing were performed using the above primers. The intra-species and inter-species K2P distances of the high-quality sequences obtained by sequencing were calculated using MEGA11, and the NJ phylogenetic tree was constructed.

[0082] 2.2K2P distance

[0083] As shown in Table 4-7, the K2P distance of Trifolium species in the psbA-trnH region is the largest, indicating that the difference between Trifolium species in the psbA-trnH region is the largest. The K2P distance of Trifolium species in the rbcL region is the smallest, indicating that the difference between Trifolium species in the rbcL region is the smallest.

[0084] Table 4 K2P distances in the psbA-trnH region of Trifolium

[0085] Species Red Clover White Clover Clover Caucasian clover Trifolium variegatum Wild Fire Ball Red Clover White Clover 0.094 Clover 0.986 0.878 Caucasian clover 0.984 0.883 0.006 Trifolium variegatum 0.971 0.872 0.011 0.004 Wild Fire Ball 0.973 0.883 0.013 0.006 0.011

[0086] Table 5 K2P distances of Trifolium in matk region

[0087] Species Red Clover White Clover Clover Caucasian clover Trifolium variegatum Wild Fire Ball Red Clover White Clover 0.032 Clover 0.017 0.027 Caucasian clover 0.029 0.035 0.029 Trifolium variegatum 0.029 0.025 0.026 0.029 Wild Fire Ball 0.029 0.030 0.023 0.025 0.027

[0088] Table 6 K2P distance of Trifolium in ITS2 region

[0089] Species Red Clover White Clover Clover Caucasian clover Trifolium variegatum Wild Fire Ball Red Clover White Clover 0.049 Clover 0.062 0.065 Caucasian clover 0.101 0.108 0.101 Trifolium variegatum 0.027 0.033 0.046 0.101 Wild Fire Ball 0.024 0.055 0.071 0.108 0.036

[0090] Table 7 K2P distances of Trifolium in rbcL region

[0091] Species Red Clover White Clover Clover Caucasian clover Trifolium variegatum Wild Fire Ball Red Clover White Clover 0.020 Clover 0.006 0.022 Caucasian clover 0.010 0.022 0.010 Trifolium variegatum 0.018 0.008 0.020 0.020 Wild Fire Ball 0.018 0.016 0.020 0.020 0.018

[0092] 2.3 Multiple sequence alignment

[0093] 2.3.1ITS2

[0094] like Figure 1 As shown, if the base type of the ITS2 gene fragment at the 160th position is T, the species is determined to be white clover; if the base type of the ITS2 gene fragment at the 156th position is A, the species is determined to be ground clover; if the base type of the ITS2 gene fragment at the 80th position is T, the species is determined to be Caucasian clover; if the base type of the ITS2 gene fragment at the 97th position is A, the species is determined to be wild fireball; if the base type of the ITS2 gene fragment at the 263rd position is G, the species is determined to be red clover; if the base type of the ITS2 gene fragment at the 218th position is A and the base type at the 188th position is C, the species is determined to be hybrid clover.

[0095] 2.3.2rbcL

[0096] like Figure 2 As shown, if the 4th base type of the rbcL gene fragment is T, the species is determined to be red clover; if the 54th base type of the rbcL gene fragment is G, the species is determined to be white clover; if the 72nd base type of the rbcL gene fragment is C, the species is determined to be hybrid clover; if the 108th base type of the rbcL gene fragment is T, the species is determined to be wild fireball; if the 348th base type of the rbcL gene fragment is A, the species is determined to be ground clover; if the 283rd base type of the rbcL gene fragment is C, the species is determined to be Caucasian clover.

[0097] 2.3.3psbA-trnH

[0098] like Figure 3 As shown, if the base type of the psbA-trnH gene fragment at the 22nd position is G, the species is determined to be red clover; if the base type of the psbA-trnH gene fragment at the 27th position is C, the species is determined to be white clover; if the base type of the psbA-trnH gene fragment at the 214th position is C, the species is determined to be ground clover; if the base type of the psbA-trnH gene fragment at the 100th position is T, the species is determined to be wild fireball; if the base type of the psbA-trnH gene fragment at the 121st position is A, the species is determined to be hybrid clover.

[0099] 2.3.4matk

[0100] like Figure 4As shown, if the matk gene fragment is C at the 47th base type, the species is determined to be white clover; if the matk gene fragment is C at the 530th base type, the species is determined to be wild fireball; if the matk gene fragment is G at the 255th base type, the species is determined to be hybrid clover; if the matk gene fragment is C at the 6th base type, the species is determined to be Caucasian clover; if the matk gene fragment is A at the 39th base type, the species is determined to be red clover; if the matk gene fragment is C at the 375th base type, the species is determined to be subterranean clover.

[0101] The present invention analyzes the characteristics of the relevant sequences of ITS2, psbA-trnH, rbcL and matK of six Trifolium samples, calculates the genetic distance, and constructs a phylogenetic tree (such as Figure 9-12 ) and other aspects of analysis, the results showed that ITS2, rbcL and matK sequences can be used as the preferred species for differentiation and identification of Trifolium species, providing a certain theoretical reference for the molecular identification of Trifolium.

Claims

1. A method for identifying different plant species within the genus Trifolium, wherein the genus Trifolium is red clover, white clover, ground clover, Caucasian clover, miscellaneous clover, and wild clover, characterized in that: The identification method steps are as follows: S1. Extract genomic DNA from the sample to be tested; S2, using the genomic DNA of the sample to be tested as a template, PCR amplification of matk sequence fragments was performed using a DNA barcode primer set: primers shown in SEQ ID NO: 1 and SEQ ID NO: 2; PCR amplification of rbcL sequence fragments was performed using a DNA barcode primer set: primers shown in SEQ ID NO: 3 and SEQ ID NO: 4; The DNA barcode primer set: primers shown in SEQ ID NO: 5 and SEQ ID NO: 6 were used to perform PCR amplification of the ITS2 sequence fragment. A DNA barcode primer set: primers as shown in SEQ ID NO: 7 and SEQ ID NO: 8 were used to perform PCR amplification of the psbA-trnH 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 DNA barcode of the ITS2 gene fragment has a nucleotide sequence as shown in SEQ ID NO: 9, and the base type at position 263 is G, then the species is determined to be red clover; 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 position 160 is T, then the species is determined to be white clover; 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 position 156 is A, then the species is determined to be Trifolium substantum; If the DNA barcode of the ITS2 gene fragment has a nucleotide sequence as shown in SEQ ID NO: 12, with a T at base type 80, then the species is determined to be Caucasian clover; If the DNA barcode of the ITS2 gene fragment has a nucleotide sequence as shown in SEQ ID NO: 13, and the base type at position 97 is A, then the species is determined to be Pyrophora pyrocaulifera; If the DNA barcode of the ITS2 gene fragment has a nucleotide sequence as shown in SEQ ID NO: 14, the base type at position 218 is A, and the base type at position 188 is C, then the species is determined to be Trifolium heterophyllum; 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 22 is G, then the species is determined to be Trifolium repens; 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 27 is C, then the species is determined to be white clover; 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 214 is C, then the species is determined to be Trifolium substantum; If the DNA barcode of the psbA-trnH gene fragment has a nucleotide sequence as shown in SEQ ID NO: 18, and the base type at position 121 is A, then the species is determined to be Trifolium heterophyllum; If the DNA barcode of the psbA-trnH gene fragment has a nucleotide sequence as shown in SEQ ID NO: 19, and the base type at position 100 is T, then the species is determined to be Pyrophora pyrocaulifera; If the DNA barcode of the matk gene fragment has a nucleotide sequence as shown in SEQ ID NO: 20, and the base type at position 39 is A, then the species is determined to be red clover; If the DNA barcode of the matk gene fragment has a nucleotide sequence as shown in SEQ ID NO: 21, and the base type at position 47 is C, then the species is determined to be white clover; 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 375 is C, then the species is determined to be Trifolium substantum; 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 the 6th position is C, then the species is determined to be Caucasian clover; 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 255 is G, then the species is determined to be heterophyllous; 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 position 530 is C, then the species is determined to be Pyrophora pyrophylla; If the DNA barcode of the rbcL gene fragment has a nucleotide sequence as shown in SEQ ID NO: 26, and the base type at the 4th position is T, then the species is determined to be Trifolium repens; If the DNA barcode of the rbcL gene fragment has a nucleotide sequence as shown in SEQ ID NO: 27, and the base type at position 54 is G, then the species is determined to be white clover; If the nucleotide sequence of the DNA barcode of the rbcL gene fragment is as shown in SEQ ID NO: 28, and the base type at position 348 is A, then the species is determined to be Trifolium pratense; If the DNA barcode of the rbcL gene fragment has a nucleotide sequence as shown in SEQ ID NO: 29, and the base type at position 283 is C, then the species is determined to be Caucasian clover; If the DNA barcode of the rbcL gene fragment has a nucleotide sequence as shown in SEQ ID NO: 30, and the base type at position 72 is C, then the species is determined to be heterophyllous; If the nucleotide sequence of the DNA barcode of the rbcL gene fragment is as shown in SEQ ID NO: 31, and the base type at the 108th position is T, then the species is determined to be Pyrophora pyrocaulifera.

2. The method according to claim 1, characterized in that: PCR amplification system: 16 μL of Taq DNA polymerase, 2 μL of DNA template, 1.2 μL of 10 μm of upstream and downstream primers, and 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.

Images