SRAP primer composition and application thereof in identification of Rabdosia medicinal plants

Abstract

The invention discloses an SRAP (sequence-related amplified polymorphism) primer composition and application of the SRAP primer composition in identification of Rabdosia medicinal plants. Through PCR amplification of 12 pairs of SRAP primer compositions, SRAP band analysis is carried out, so that the provenance of the tested Rabdosia medicinal plant can be distinguished, and especially, Rabdosia lanceolata and Rabdosia japonica can be completely distinguished from other Rabdosia plants. The method has the advantages that the identification result is not influenced by the age of the plant, the Rabdosia medicinal plants in the seedling stage can be accurately distinguished, the current cultivated species can be quickly and effectively distinguished and identified, planting enterprises and farmers are prevented from suffering from heavy loss caused by planting wrong varieties, and good technical guidance and theoretical support are provided for subsequent scientific research.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology and molecular marker technology, and particularly relates to a SRAP primer composition and its application in identifying medicinal plants of the genus Rabdosia. Background Art

[0002] Plants of the genus Rabdosia (Bl.) Hassk. are shrubs, semi-shrubs or perennial herbs of the Labiatae family. There are about 150 species existing in the world, and about 90 species in China, covering most parts of the country. Most plants of the genus Rabdosia are medicinal plants and are widely used clinically. For example, the Chinese medicinal material Rabdosia is the dried aerial part of Rabdosia amethystoides (Benth.) Hara, which has the effects of clearing heat and promoting diuresis, activating blood circulation to dissipate stasis, and relieving fever and swelling; the Chinese medicinal material Rabdosia rubescens is the dried aerial part of Isodon rubescens (Hemsl.) Hara of this genus, which is bitter, sweet and slightly cold. It belongs to the lung, stomach and liver meridians and has the effects of clearing heat and detoxifying, promoting blood circulation to relieve pain. It is mostly used for treating sore throat, mass in the abdomen, snake and insect bites, etc.; the Chinese medicinal material Rabdosia serra is the whole herb of Rabdosia serra (Maxim.) Hara and Rabdosia lophanthoides (Buch.-Ham. ex D. Don) Hara of the Labiatae family, which has the effects of clearing heat and detoxifying, promoting diuresis to reduce jaundice, and dispersing stasis and detumescence, and is mainly used for treating damp-heat jaundice, cholecystitis, diarrhea, dysentery, sores and boils, traumatic injuries, etc. It can be seen that Rabdosia and its congeners have good medicinal value, so they are widely used clinically and there is a huge market demand. However, Rabdosia has a wide ecological adaptation range and is distributed in most parts of China, which leads to the phenomenon of different plants with the same name and variety confusion between Rabdosia and its congeners, seriously affecting the safety of medication.

[0003] Molecular markers are a method for detecting genetic diversity based on the differences in biological DNA sequences. They have the characteristics of being unaffected by external environmental conditions, not affecting the traits of experimental materials, having a large number of markable sites, and high resolution. Currently, they are widely used in biological classification research. Sequence-related amplified polymorphism (SRAP) is a new PCR-based marker system, a dominant marker, developed by Dr. Li and Quiros from the Vegetable Department of the University of California in Brassica crops in 2001. This marker amplifies specific regions of the ORFs (open reading frames) of genes through unique dual primer design, and polymorphisms are generated due to the different lengths of introns, promoters, and spacer regions in different individuals and species. This marker has the characteristics of simplicity, high efficiency, high yield, and good repeatability, and is widely used in variety identification. However, currently, the identification of medicinal plants in the genus Rabdosia mainly relies on morphological identification, and there is no effective SRAP molecular identification method for medicinal plants in the genus Rabdosia. Therefore, it is necessary to establish a rapid and accurate SRAP molecular method for identifying medicinal plants in the genus Rabdosia. Summary of the Invention

[0004] The purpose of the present invention is to overcome the drawback that the identification of medicinal plants in the genus Rabdosia mainly relies on morphological identification, and the present invention provides an SRAP primer composition for identifying medicinal plants in the genus Rabdosia.

[0005] Another purpose of the present invention is to provide a method for identifying medicinal plants in the genus Rabdosia using SRAP primers.

[0006] In order to achieve the above purposes, the specific technical solutions of the present invention are as follows:

[0007] An SRAP primer composition for identifying medicinal plants in the genus Rabdosia, comprising 12 pairs of primers, namely:

[0008] Name of primer composition Forward primer Reverse primer ME1-EM3: SEQ ID NO.1 SEQ ID NO.9 ME1-EM13: SEQ ID NO.1 SEQ ID NO.15 ME1-EM17: SEQ ID NO.1 SEQ ID NO.19 ME2-EM6: SEQ ID NO.2 SEQ ID NO.11 ME2-EM15: SEQ ID NO.2 SEQ ID NO.17 ME2-EM16: SEQ ID NO.2 SEQ ID NO.18 ME3-EM5: SEQ ID NO.3 SEQ ID NO.10 ME3-EM12: SEQ ID NO.3 SEQ ID NO.14 ME4-EM14: SEQ ID NO.4 SEQ ID NO.16 ME5-EM2: SEQ ID NO.5 SEQ ID NO.8 ME6-EM7: SEQ ID NO.6 SEQ ID NO.12 ME9-EM11 SEQ ID NO.7 SEQ ID NO.13 。

[0009] The present invention also provides the application of the SRAP primer composition in identifying medicinal plants in the genus Rabdosia or analyzing the genetic diversity of medicinal plants in the genus Rabdosia; the medicinal plants in the genus Rabdosia are selected from one or more of Isodon eriophyllus, Isodon japonicus var. glaucocalyx, Isodon tubulosus, Isodon lophanthoides, Isodon inflexus, Isodon macrocalyx, Isodon amethystoides, and Rabdosia japonica.

[0010] The present invention also provides a kit for identifying medicinal plants in the genus Rabdosia, comprising the SRAP primer composition.

[0011] The present invention also provides a method for identifying medicinal plants in the genus Rabdosia using SRAP primers, comprising the following steps:

[0012] (1) Extract the DNA of the sample to be identified;

[0013] (2) Using the DNA obtained in step (1) as a template, perform PCR amplification with the SRAP primer composition for identifying medicinal plants of the genus Rabdosia to obtain an amplification product;

[0014] (3) Electrophoretically detect the amplification product obtained in step (2), detect the band pattern of the amplification product, and perform genetic diversity analysis based on SRAP data to determine the variety of the sample to be identified.

[0015] Preferably, during the detection in step (2), each pair of primer compositions is separately subjected to PCR amplification.

[0016] Specifically, when each pair of primer compositions is separately subjected to PCR amplification, the PCR amplification system is: 1 μL of template, 6 μL of 2×Hieff TM PCR Master, 1 μL of upstream primer F, 1 μL of downstream primer R, 1 μL of ddH2O.

[0017] When each pair of primer compositions is separately subjected to PCR amplification, the PCR amplification program is: pre-denaturation at 94 °C for 5 min; 94 °C for 1 min, 35 °C for 1 min, 72 °C for 1 min, 5 cycles; 35 cycles of 94 °C for 1 min, 50 °C for 1 min, 72 °C for 1 min, 35 cycles; extension at 72 °C for 10 min; preservation at 4 °C.

[0018] Further, the method for performing genetic diversity analysis based on SRAP data in step (3) is specifically as follows: Read the band information obtained in step (3), record the clear and reproducible bands on the electrophoresis pattern as 1, and record no bands at the same position as 0, thereby generating an original matrix of 0 and 1; Count the total number of bands and polymorphic bands amplified by each pair of primers; Calculate the similarity coefficient matrix using the SimQual program in NTSYS-pc software, and perform UPGMA clustering with SHAN in the Clustering program; Generate a clustering map using the Tree plot module to construct a molecular phylogenetic tree. According to the 01 binary data matrix, count the total number of bands and polymorphic bands (NPB) of the SRAP amplification product, and calculate the ratio of polymorphic bands (PPB). Based on the "0 / 1" assignment matrix of the molecular marker SRAP, the number of alleles, effective number of alleles, Nei's (1973) gene diversity, Shannon's polymorphism information index, total gene diversity of the population, within-population gene diversity, Hei's gene differentiation coefficient, and gene flow of the samples were calculated.

[0019] The present invention also provides a method for analyzing the genetic relationship of medicinal plants of the genus Rabdosia using SRAP primers, comprising the following steps:

[0020] (1) Extract the DNA of the sample to be analyzed;

[0021] (2) Using the DNA obtained in step (1) as a template, perform PCR amplification using the SRAP primer composition for identifying medicinal plants of the genus Rabdosia to obtain an amplification product;

[0022] (3) Electrophoretically detect the amplification product obtained in step (2), detect the band pattern of the amplification product, and compare the band patterns of the samples to be analyzed. The fewer the number of different band patterns at 12 positions, the closer the genetic relationship between the medicinal plants of the genus Rabdosia to be analyzed; the more the number of different band patterns at 12 positions, the farther the genetic relationship between the medicinal plants of the genus Rabdosia to be analyzed.

[0023] Preferably, when detecting in step (2), each pair of primer compositions is separately subjected to PCR amplification.

[0024] Specifically, when each pair of primer compositions is separately subjected to PCR amplification, the PCR amplification system is: 1 μL of template, 6 μL of 2×Hieff TM PCR Master, 1 μL of upstream primer F, 1 μL of downstream primer R, 1 μL of ddH2O;

[0025] When each pair of primer compositions is separately subjected to PCR amplification, the PCR amplification program is: pre-denaturation at 94 °C for 5 min; 94 °C for 1 min, 35 °C for 1 min, 72 °C for 1 min, for 5 cycles; 94 °C for 1 min, 50 °C for 1 min, 72 °C for 1 min, for 35 cycles; extension at 72 °C for 10 min; storage at 4 °C.

[0026] Advantages of the present invention:

[0027] The present invention provides an SRAP primer for identifying medicinal plants of the genus Rabdosia. Using this primer, the medicinal plants of the genus Rabdosia can be quickly and effectively identified. In particular, Isodon eriophyllus and Isodon japonicus var. glaucocalyx can be completely distinguished from other plants of the genus Rabdosia. And the identification result is not easily affected by the environment, and can be effectively distinguished through tissue samples at the seedling stage of the medicinal plants of the genus Rabdosia, which can prevent planting enterprises and farmers from suffering significant losses caused by planting the wrong varieties. Description of the Drawings

[0028] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention.

[0029] Figure 1It is the PCR amplification electrophoresis detection result diagram of 20 medicinal plant samples of Isodon with SRAP primer ME1-EM3;

[0030] Figure 2 It is the PCR amplification electrophoresis detection result diagram of 20 medicinal plant samples of Isodon with SRAP primer ME1-EM13;

[0031] Figure 3 It is the PCR amplification electrophoresis detection result diagram of 20 medicinal plant samples of Isodon with SRAP primer ME1-EM17;

[0032] Figure 4 It is the PCR amplification electrophoresis detection result diagram of 20 medicinal plant samples of Isodon with SRAP primer ME2-EM6;

[0033] Figure 5 It is the PCR amplification electrophoresis detection result diagram of 20 medicinal plant samples of Isodon with SRAP primer ME2-EM15;

[0034] Figure 6 It is the PCR amplification electrophoresis detection result diagram of 20 medicinal plant samples of Isodon with SRAP primer ME2-EM16;

[0035] Figure 7 It is the PCR amplification electrophoresis detection result diagram of 20 medicinal plant samples of Isodon with SRAP primer ME3-EM5;

[0036] Figure 8 It is the PCR amplification electrophoresis detection result diagram of 20 medicinal plant samples of Isodon with SRAP primer ME3-EM12;

[0037] Figure 9 It is the PCR amplification electrophoresis detection result diagram of 20 medicinal plant samples of Isodon with SRAP primer ME4-EM14;

[0038] Figure 10 It is the PCR amplification electrophoresis detection result diagram of 20 medicinal plant samples of Isodon with SRAP primer ME5-EM2;

[0039] Figure 11 It is the PCR amplification electrophoresis detection result diagram of 20 medicinal plant samples of Isodon with SRAP primer ME6-EM7;

[0040] Figure 12 It is the PCR amplification electrophoresis detection result diagram of 20 medicinal plant samples of Isodon with SRAP primer ME9-EM11;

[0041] Figure 13It is a phylogenetic tree diagram among 20 samples of medicinal plants of the genus Rabdosia based on SRAP markers. Detailed implementation mode

[0042] Example 1

[0043] First, 20 samples of medicinal plants of the genus Rabdosia from 8 species were selected. The sampling materials of the medicinal plants of the genus Rabdosia are specifically shown in Table 1. There is 1 sample for each variety. The sampling method and the sample preservation method both adopt the existing technologies and will not be elaborated here.

[0044] Table 1 Information of samples of medicinal plants of the genus Rabdosia

[0045]

[0046]

[0047]

[0048] Extract the DNA of the test samples of medicinal plants of the genus Rabdosia.

[0049] In this example, the improved CTAB method was used to extract the total genomic DNA from the fresh leaves of the medicinal plants of the genus Rabdosia. The specific operation steps are as follows:

[0050] 1) Take 2 - 3 fresh and intact leaves of the medicinal plants of the genus Rabdosia (about 0.5 - 1.5 g);

[0051] 2) Cut off the main veins of the leaves and cut them into pieces. Then place the materials in a clean mortar, add an appropriate amount of liquid nitrogen, and quickly grind them into a fine powder for many times. Then quickly scrape the powder together with a spatula and transfer it to a 1.5 mL round - bottom centrifuge tube containing 800 μL of pre - heated CTAB∶β - mercaptoethanol (20∶1, V / V) at 65 °C. Cover the centrifuge tube cap and seal it with a sealing film;

[0052] 3) Immediately place it in a 65 °C water bath for water - bath heating. Take it out and shake it every 5 minutes to ensure that the sample is in full contact with the extraction solution. Take it out after 20 minutes to obtain a mixed solution;

[0053] 4) Cool to room temperature, add an equal volume (800 μL) of chloroform∶isoamyl alcohol (24∶1, V / V), mix evenly (slowly invert) for 15 - 30 minutes, and centrifuge at 10000 rpm at room temperature for 8 minutes;

[0054] 5) Pipette 600 μL of the supernatant into a new 1.5 mL pointed - tip centrifuge tube, add an equal amount of chloroform∶isoamyl alcohol (24∶1, V / V), and gently mix.

[0055] 6) Take out after centrifuging at 12,000 rpm for 8 min at room temperature in a centrifuge. Take 400 μL of the supernatant and transfer it to a new 1.5 mL pointed centrifuge tube. Add an equal volume of isopropanol pre-cooled at -20 °C, gently shake well, and let it stand for 3 h or overnight; centrifuge at 12,000 rpm for 3 min to make the DNA adhere to the bottom of the centrifuge tube, and discard the aqueous phase.

[0056] 7) Add 75% ethanol to wash 2 - 3 times, wash once with absolute ethanol, remove the ethanol. Then open the lid of the 1.5 mL pointed centrifuge tube, invert it on an inclined cardboard covered with absorbent paper, and air dry at room temperature for about 1 - 2 h.

[0057] 8) Dissolve the DNA precipitate with 100 μL of 1×TE buffer. After regularly tapping with forceps and mixing well, the required DNA extraction solution is obtained. The concentration and purity of the DNA solution are measured by a UV spectrophotometer in a microplate reader. Prepare the DNA solutions of each sample with labels into working solutions, with a concentration of about 150 ng / μL. Store at 4 °C for later use.

[0058] 9) The purity of the DNA solution is measured by a microplate reader (M200 pro Nanoquant); and detected by 1.5% agarose gel electrophoresis (Bio - Rad), with DL2000bp Marker as the standard molecular weight.

[0059] Perform a PCR amplification reaction using the SRAP primer composition.

[0060] The SRAP primer composition is 12 pairs, and its sequences are shown in Table 2.

[0061] Table 2 Information of 12 pairs of SRAP primers

[0062]

[0063] All PCR amplification reaction procedures are executed by a PCR amplifier (96 - well Thermal Cycler, Veriti), and an appropriate SRAP marker reaction system and procedure are optimized and screened out.

[0064] The reaction system is as follows: in 10 μL, it contains 1 μL of template DNA with a concentration of 150 ng / μL, 6 μL of 2×Hieff TM PCRMaster, 1 μL of F - primer with a concentration of 10 μM, 1 μL of R - primer with a concentration of 10 μM, and 1 μL of ddH2O.

[0065] The reaction procedure was as follows: pre-denaturation at 94°C for 5 min; 5 cycles of 94°C for 1 min, 35°C for 1 min, and 72°C for 1 min; 35 cycles of 94°C for 1 min, 50°C for 1 min, and 72°C for 1 min; extension at 72°C for 10 min; storage at 4°C. After the PCR amplification reaction was completed, 2 μL of 1× Loading buffer was added, and the mixture was thoroughly vortexed and then stored in a 4°C refrigerator for later use.

[0066] Agarose gel electrophoresis analysis:

[0067] Electrophoresis separation was carried out using a 1.5% agarose gel in a horizontal electrophoresis tank (Bio-Rad) with 1× TAE as the medium. DL 2000bp Marker (Yeasen) was used as the standard molecular weight marker for fragment size. The electrophoresis products were recorded in a gel imager (gel Doc-XR, Bio-Rad). Among them, the method of electrophoresis separation was as follows: 5.0 μL of the PCR product with added buffer was loaded onto the sample well using a pipette tip, and electrophoresis was carried out at a constant voltage of 120V for 1 h. The glass plate was placed in the gel imager for observation, and finally, photos were taken and recorded. The PCR amplification electrophoresis detection results of 11 primer pairs for 20 medicinal plants of the genus Rabdosia are shown in Figures 1 to 12 .

[0068] SRAP band analysis:

[0069] For the obtained Figures 1~12 The gel image data was interpreted by multiple people for comparison. All electrophoresis bands were accurately read. Bands with clear or weak bands were recorded as "1", and bands without were recorded as "0", thus generating a raw matrix of 0 and 1. After the band analysis was completed, all the results were input into the corresponding software according to the software format requirements for experimental data processing and statistics. The similarity coefficient matrix was calculated using the SimQual program in the NTSYS-pc software, and UPGMA clustering was performed using SHAN in the Clustering program; a clustering diagram was generated using the Tree plot module to construct a molecular phylogenetic tree. According to the 01 binary data matrix, the total number of bands and the number of polymorphic bands (NPB) of the SRAP amplification products were statistically analyzed, and the ratio of polymorphic bands (PPB) was calculated. Based on the "0 / 1" assignment matrix of the molecular marker SRAP, the number of alleles, effective number of alleles, Nei's (1973) gene diversity, Shannon's polymorphism information index, total gene diversity of the population, gene diversity within the population, Hei's gene differentiation coefficient, and gene flow of the samples were calculated.

[0070] In this example, all 12 pairs of SRAP primer combinations achieved good polymorphic amplification in the test samples. A total of 155 bands were amplified from 20 samples of Isodon medicinal plants from different origins, among which 145 were polymorphic bands. On average, each primer amplified 12.91 bands, and the proportion of polymorphic bands for each primer ranged from 83.33% to 100%, with an average of 93.55%. The primer combinations ME2-EM15, ME2-EM16, and ME3-EM5 amplified the most bands, all with 18 bands, while the primer combination ME1-EM17 amplified the fewest bands, only 6 bands. See Table 3 for details.

[0071] The overall species-level Na of Isodon and its related species was 1.9871, Ne was 1.4127, H was 0.2595, and I was 0.4111. For Isodon, Rabdosia japonica var. glaucocalyx, and Rabdosia excisa with multiple populations, the range of Na was 1.3484 - 1.6516, with an average of 1.5011; the range of Ne was 1.2338 - 1.3402, with an average of 1.2840; the range of H was 0.1348 - 0.2048, with an average of 0.1688; the range of I was 0.1990 - 0.3137, with an average of 0.2554. The overall levels of Na and Ne in the samples were higher than the average level, indicating rich genetic diversity among Isodon and its related species. Moreover, the relatively higher Na, Ne, H, and I of Isodon suggest higher genetic diversity. The Ht among the 8 species was 0.2761, Hs was 0.0627, Gst was 0.7728 (>0.5000), indicating a high degree of genetic differentiation among species, and Nm was 0.1470, suggesting a certain degree of gene flow among Isodon plants. (Table 4)

[0072] Taking the genetic matrix as the analysis object, cluster analysis was performed using the unweighted pair-group method with arithmetic means (UPGMA) in NTSYS-pc 2.1 software. Please refer to Figure 13 to establish a phylogenetic dendrogram among 20 samples of Isodon medicinal plants. From the clustering results, at a genetic similarity coefficient of 0.768, Rabdosia excisa and Rabdosia japonica var. glaucocalyx can be distinguished from other Isodon plants. Further, at a genetic similarity coefficient of 0.794, Rabdosia excisa can be distinguished from Rabdosia japonica var. glaucocalyx, and it is possible to distinguish the sources of Isodon medicinal plants tested using 12 pairs of SRAP primers.

[0073] Table 3 Amplification results of 12 pairs of SRAP primers

[0074]

[0075]

[0076] Table 4 Genetic diversity analysis of Isodon medicinal plants

[0077] Species Number of individuals Na Ne H I <![CDATA[H t > <![CDATA[H s > <![CDATA[G st > <![CDATA[N m > Rabdosia amethystoides 8 1.6516 1.3402 0.2048 0.3137 / / / / Rabdosia incisa 4 1.4516 1.2757 0.1624 0.2433 / / / / Rabdosia eriophylla 3 1.3484 1.2338 0.1348 0.1990 / / / / Rabdosia japonica var. glaucocalyx 1 / / / / / / / / Rabdosia lophanthoides 1 / / / / / / / / Rabdosia longituba 1 / / / / / / / / Rabdosia inflexa 1 / / / / / / / / Rabdosia macrocalyx 1 / / / / / / / / Average level 1.5011 1.2840 0.1688 0.2554 / / / / Overall level 20 1.9871 1.4127 0.2595 0.4111 0.2761 0.0627 0.7728 0.1470

[0078] Note: Number of alleles (N a ), effective number of alleles (N e ), Nei's (1973) gene diversity (gene diversity, H), Shannon's information index (Shannon's information index, I), total gene diversity of populations (total gene diversity, H t ), gene diversity with provenances within populations (H s ), Hei's coefficient of gene differentiation (G st ) and estimate of gene flow from G st , N m )

[0079] The embodiment of the present invention provides an application of the above method, which distinguishes the provenance of the tested medicinal plants of the genus Camellia by SRAP band analysis after PCR amplification with 12 pairs of SRAP primers, and can especially completely distinguish the hairy-leaved Camellia and the blue-calyx Camellia from other Camellia plants, and the identification result is not easily affected by the age of the plant, and can accurately distinguish the medicinal plants of the genus Camellia in the seedling stage, and can quickly and effectively distinguish and identify the current cultivated species. Planting companies and farmers can avoid heavy losses from planting the wrong varieties, and the SRAP fingerprint of medicinal plants of the genus Camellia established based on the above method is of great significance.

[0080] The above description shows and describes several preferred embodiments of the invention, but as mentioned above, it should be understood that the invention is not limited to the form disclosed herein, and should not be regarded as excluding other embodiments, but can be used in various other combinations, modifications and environments, and can be modified within the scope of the invention concept described herein through the above teachings or the technology or knowledge of the relevant field. Changes and modifications made by those skilled in the art should be within the scope of protection of the claims attached to the invention without departing from the spirit and scope of the invention.

Claims

1. A SRAP primer composition for identifying medicinal plants of the genus Rabdosia, characterized in that, Any one or more pairs selected from the following 12 pairs of primer compositions, and the primer compositions are respectively: 。 2. The application of the SRAP primer composition according to claim 1 in identifying medicinal plants of the genus Rabdosia or analyzing the genetic diversity of medicinal plants of the genus Rabdosia; the medicinal plants of the genus Rabdosia are selected from one or more of Rabdosia eriophylla, Rabdosia japonica var. glaucocalyx, Rabdosia longituba, Rabdosia lophanthoides, Rabdosia inflexa, Rabdosia macrocalyx, Rabdosia amethystoides, and Rabdosia japonica.

3. A kit for identifying medicinal plants of the genus Isodon, characterized in that, Comprising the SRAP primer composition according to claim 1.

4. A method for identifying medicinal plants of the genus Rabdosia using SRAP primers, characterized in that, Comprising the following steps: (1) Extract the DNA of the sample to be identified by the CTAB method; (2) Using the DNA obtained in step (1) as a template, perform PCR amplification using the SRAP primer composition for identifying medicinal plants of the genus Rabdosia according to claim 1 to obtain an amplification product; (3) Perform electrophoresis detection on the amplification product obtained in step (2), detect the band pattern of the amplification product, and perform genetic diversity analysis based on SRAP data to determine the variety of the sample to be identified.

5. The method according to claim 4, wherein The genetic diversity analysis based on SRAP data in step (3) is specifically as follows: Read the band information obtained in step (3), record the clear and reproducible bands on the electrophoresis map as 1, and record 0 for no bands at the same position, thereby generating an original matrix of 0 and 1; Count the total number of bands and polymorphic bands amplified by each pair of primers; Calculate the similarity coefficient matrix using the SimQual program in the NTSYS-pc software, and perform UPGMA clustering using SHAN in the Clustering program; Generate a clustering map using the Tree plot module to construct a molecular phylogenetic tree; According to the 01 binary data matrix, count the total number of bands and the number of polymorphic bands NPB of the SRAP amplification product, and calculate the ratio PPB of the polymorphic bands; Calculate the number of alleles, effective number of alleles, Nei's (1973) gene diversity, Shannon's polymorphism information index, total gene diversity of the population, within-population gene diversity, Hei's gene differentiation coefficient, and gene flow of the samples based on the "0 / 1" assignment matrix of the molecular marker SRAP.

6. The method for identifying medicinal plants of the genus Isodon using SRAP primers according to claim 4, characterized in that, During the detection in step (2), each pair of primer compositions is separately subjected to PCR amplification.

7. The method for identifying medicinal plants of Rabdosia using SRAP primers according to claim 4, characterized in that When each pair of primer compositions is subjected to PCR amplification individually, the PCR amplification system is as follows: 1 μL of template, 6 μL of 2×Hieff TM PCR Master, 1 μL of upstream primer F, 1 μL of downstream primer R, 1 μL of ddH2O; the PCR amplification program is: pre-denaturation at 94°C for 5 min; 94°C for 1 min, 35°C for 1 min, 72°C for 1 min, for 5 cycles; 35 cycles of 94°C for 1 min, 50°C for 1 min, 72°C for 1 min; extension at 72°C for 10 min; preservation at 4°C.

8. A method for analyzing the genetic distance of medicinal plants of the genus Rabdosia using SRAP primers, characterized in that, Comprising the following steps: (1) Extract the DNA of the sample to be analyzed; (2) Using the DNA obtained in step (1) as a template, perform PCR amplification using the SRAP primer for identifying medicinal plants of the genus Rabdosia according to claim 1 to obtain an amplification product; (3) Perform electrophoresis detection on the amplification product obtained in step (2), detect the band pattern of the amplification product, and compare the band patterns of the samples to be analyzed. The fewer the number of different band patterns at 12 positions, the closer the genetic relationship between the medicinal plants of the genus Rabdosia to be analyzed, and the more the number of different band patterns at 12 positions, the farther the genetic relationship between the medicinal plants of the genus Rabdosia to be analyzed.

9. The method for analyzing the genetic relationship of medicinal plants of the genus Rabdosia using SRAP primers according to claim 8, characterized in that During the detection in step (2), each pair of primers is detected once separately.

10. The method for analyzing the genetic relationship of medicinal plants of Rabdosia using SRAP primers according to claim 9, wherein When each pair of primers was detected separately, the PCR amplification system was as follows: 1 μL of template, 6 μL of 2×Hieff TM PCR Master, 1 μL of upstream primer F, 1 μL of downstream primer R, 1 μL of ddH2O; when each pair of primers was detected separately, the PCR amplification procedure was as follows: pre-denaturation at 94°C for 5 min; 94°C for 1 min, 35°C for 1 min, 72°C for 1 min, for 5 cycles; 35 cycles of 94°C for 1 min, 50°C for 1 min, 72°C for 1 min; extension at 72°C for 10 min; preservation at 4°C.

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