Primer combination and identification method for identifying varieties and producing areas of medicinal plants, namely, gynura and mukdenia, and identification method of variety and producing areas of medicinal plants, namely gynura and mukdenia

Abstract

The invention discloses a primer combination and an identification method for identifying the types and producing areas of medicinal plants, namely gynura and mukdenia, and belongs to the technical field of biology. Comprising a primer group 1 and a primer group 2, the primer group 1 is composed of a forward primer as shown in SEQ ID NO. 1 and a reverse primer as shown in SEQ ID NO. 2; and the primer group 2 is composed of a forward primer as shown in SEQ ID NO.3 and a reverse primer as shown in SEQ ID NO.4. The primer group 1 comprises a forward primer as shown in SEQ ID NO.3 and a reverse primer as shown in SEQ ID NO.4. The invention develops a primer combination and an identification method for identifying the types and the producing areas of the single-root grass and the mukdenia. The chloroplast molecular marker technology is applied to accurately identify species with a more sensitive and efficient identification means, and compared with traditional phenotype identification, the identification result is more accurate, reliable, rapid and simple, and the method is suitable for large-scale popularization and application. The method can be applied to the aspects of rapid identification, pedigree geography research, variety intellectual property protection, molecular marker-assisted breeding and the like of gynura and mukdenia species.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a primer combination and an identification method for identifying the species and origin of medicinal plants of the genus Unicot and the genus Acerola. Background Art

[0002] Both Oresitrophe and Mukdenia are perennial herbs of the Saxifragaceae family, usually growing in the cracks of rocks on cliffs in temperate deciduous forests. They are both ornamental plants and raw materials for traditional Chinese medicine. The genus Oresitrophe contains only one species, Oresitrophe rupifraga, which is distributed in central and northern China, in low to medium altitude areas (590-2050m). Oresitrophe has important medicinal value. Its roots, stems and leaves contain crude ash, crude fiber, crude fat, reducing sugar, some mineral elements and active ingredients, which can be used to treat kidney deficiency, cold pain in the waist and knees, impotence and spermatorrhea, neurosis, etc. Oresitrophe can effectively absorb carbon dioxide in rooms with insufficient light, reduce the concentration of carbon dioxide in indoor air, and ensure good health. The genus Mukdenia contains two species, Mukdenia rossii and Mukdenia acanthifolia. Mapleleaf is distributed from Northeast China to the Korean Peninsula, with an altitude of 300-900m. It has the effect of nourishing the heart and calming the mind, and can replenish heart yang and benefit heart blood. Rock mapleleaf is currently only found in Zhuanghe and Benxi, Liaoning, and is distributed at an altitude of about 110m. According to literature records, it is also distributed in North Hamgyong Province, North Korea. Due to over-exploitation and fragmentation of original habitats, the wild population of these plants has decreased sharply. Mapleleaf is listed as a rare and endangered protected plant in China, and rock mapleleaf is listed as a third-level protected plant in Liaoning.

[0003] In order to rationally utilize the resources of the genera of Unilobium and Acerola and meet the relevant medicinal and ornamental needs, it is necessary to scientifically and accurately identify the classification and origin of these species. At present, plants of the genera of Unilobium and Acerola can only be identified by observing the appearance of the plants. Due to the high morphological similarity of Unilobium, Acerola, and Rock Acer, especially in the non-flowering season, identification is very difficult, and misuse and deliberate adulteration often occur. In addition, the use of phenotypic identification technology cannot obtain the origin information of the plant. Therefore, when using these plants as raw materials for processing pharmaceutical products, their authenticity cannot be determined. Therefore, it is necessary to use molecular markers to compare them at the genetic level to quickly identify different species and origins. Summary of the invention

[0004] The purpose of the present invention is to provide a primer combination and identification method for identifying the species and origin of medicinal plants of the genus Unilobus and the genus Aglaonema. The present invention provides a primer combination and identification method for quickly identifying the species and origin of plants of the genus Unilobus and the genus Aglaonema, and identifying and screening the corresponding plants as raw materials for medicine.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] One of the technical solutions of the present invention is a primer combination for identifying the taxonomic status and origin of species of the genus Echinops and the genus Acerola, comprising a primer set 1 and a primer set 2;

[0007] The primer set 1 consists of a forward primer as shown in SEQ ID NO.1 and a reverse primer as shown in SEQ ID NO.2;

[0008] The primer set 2 consists of a forward primer as shown in SEQ ID NO.3 and a reverse primer as shown in SEQ ID NO.4.

[0009] The second technical solution of the present invention is a product for identifying the species and origin of medicinal plants of the genus Ulmus pumila and the genus Acerola, comprising the primer combination.

[0010] The third technical solution of the present invention is the application of the primer combination or the product in identifying the taxonomic status and origin of species of the genera Ulmus pumila and Smilax glabra.

[0011] A fourth technical solution of the present invention is a method for identifying the taxonomic status and origin of species of the genus Sophora and the genus Acerola, comprising the following steps:

[0012] (1) extracting the DNA of the sample to be tested, using the DNA of the sample to be tested as a template, and performing PCR amplification and sequencing using the primer combination or the product described in 1;

[0013] (2) The .ab1 files obtained by sequencing were assembled de novo in Geneious software, and then the MAFFT program in Geneious R9 software was used for sequence alignment. The aligned matrix was imported into DNASP 5 to calculate the haplotype number, nucleotide diversity, and haplotype diversity of different species, different genetic lineages, and different populations. Then, a simplified network map was constructed using TCS software to illustrate the genetic structure of haplotypes.

[0014] (3) Determine the species and origin of the sample to be tested based on the genetic structure of the haplotype.

[0015] Based on the above technical solution, the present invention has the following technical effects:

[0016] (1) The present invention collected 222 samples of single-rooted grass, 139 samples of maple leaf grass, and 50 samples of rock maple leaf grass (Table 1). The test materials basically covered their natural distribution range ( Figure 1 ).

[0017] (2) One sample each from Sphaerocephalum indica, Sphaerocephalum truncatum, and Sphaerocephalum truncatum was selected for shallow genome resequencing.

[0018] (3) Perform chloroplast genome assembly, annotation, and genetic diversity analysis on the sequencing data to identify highly variable regions.

[0019] (4) Primers were designed for the highly variable regions, and DNA was extracted and PCR amplified for all samples.

[0020] (5) After the amplified sequences are aligned, the number, type, and diversity of haplotypes are counted, a haplotype network diagram and a neighbor-joining tree are constructed, and the primer combination is judged based on the results to be reliable, accurate, and effective in identifying species and origins. In the process of identifying the species and origins of the genus Selenicereus and Selenicereus, the two pairs of DNA primer combinations of the present invention do not need to consider the growth and development stage and plant morphology of the plants, and the identification success rate is higher than that of phenotypic identification technology, and the cost is lower. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0022] Figure 1 This is the distribution range of the single-root grass, maple leaf grass and rock maple leaf grass groups.

[0023] Figure 2 This is a haplotype network diagram constructed based on sequences amplified by two pairs of primers. DETAILED DESCRIPTION

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0026] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0027] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0028] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0029] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0030] The embodiment of the present invention provides a primer combination for identifying the taxonomic status and origin of species of the genus Echinops and the genus Acerola, including primer set 1 and primer set 2;

[0031] The primer set 1 consists of a forward primer as shown in SEQ ID NO.1 and a reverse primer as shown in SEQ ID NO.2;

[0032] The primer set 2 consists of a forward primer as shown in SEQ ID NO.3 and a reverse primer as shown in SEQ ID NO.4.

[0033] The embodiment of the present invention also provides a product for identifying the species and origin of medicinal plants of the genus Ulmus pumila and the genus Acerola, including the primer combination.

[0034] In some specific embodiments, reagents, kits and chips are included.

[0035] The embodiment of the present invention also provides the use of the primer combination or the product in identifying the taxonomic status and origin of species of the genus Sesamum and Sesamum.

[0036] The embodiment of the present invention also provides a method for identifying the taxonomic status and origin of species of the genus Sophora and the genus Aglaonema, comprising the following steps:

[0037] (1) extracting the DNA of the sample to be tested, using the DNA of the sample to be tested as a template, and performing PCR amplification and sequencing using the primer combination or the product;

[0038] (2) The .ab1 files obtained by sequencing were assembled de novo in Geneious software, and then the MAFFT program in Geneious R9 software was used for sequence alignment. The aligned matrix was imported into DNASP 5 to calculate the haplotype number, nucleotide diversity, and haplotype diversity of different species, different genetic lineages, and different populations. Then, a simplified network map was constructed using TCS software to illustrate the genetic structure of haplotypes.

[0039] (3) Determine the species and origin of the sample to be tested based on the genetic structure of the haplotype.

[0040] In some specific embodiments, the reaction system for PCR amplification is: 35 μL: 1 μL of 50 ng / μL template DNA, 13.7 μL ddH2O, 1.4 μL forward primer, 1.4 μL reverse primer and 17.5 μL PCR Mix;

[0041] Reaction procedure: denaturation at 94°C for 2 min; 94°C for 30 s, annealing at 55°C for 30 s, and 72°C for 90 s for 28 cycles; extension at 72°C for 2 min, and storage at 10°C.

[0042] In some specific embodiments, the method for determining the species and origin of the sample to be tested is: if the genetic structure of the haplotype is OR1-OR13, then the sample to be tested is of the genus Solitaria;

[0043] If the genetic structure of the haplotype is MA1-MA2, then the sample to be tested is rock maple leaf grass;

[0044] If the genetic structure of the haplotype is MR1-MR7, the sample to be tested is maple grass.

[0045] In some specific embodiments, the method for determining the species and origin of the sample to be tested is: if the genetic structure of the haplotype is OR1-OR4, then the origin of the sample to be tested is Wuling Mountain area;

[0046] If the genetic structure of the haplotype is OR5-OR7, the origin of the sample to be tested is the southern area of ​​Taihang Mountain;

[0047] If the genetic structure of the haplotype is OR8-OR11, the origin of the sample to be tested is Yanshan area;

[0048] If the genetic structure of the haplotype is OR12-OR13, the origin of the sample to be tested is the middle area of ​​Taihang Mountain;

[0049] If the genetic structure of the haplotype is MR1-MR3, the origin of the sample to be tested is Changbai Mountain area;

[0050] If the genetic structure of the haplotype is MR4-MR7, the origin of the sample to be tested is the Korean Peninsula.

[0051] The present invention develops a primer combination and an identification method for identifying species and origins of the genus Eupatorium and the genus Aglaonema. The present invention uses chloroplast molecular marker technology to accurately identify species with a more sensitive and efficient identification method. Compared with traditional phenotypic identification, the identification result is more accurate, reliable, rapid and simple, and can be applied to rapid identification of species of the genus Eupatorium and the genus Aglaonema, phylogenetic geography research, variety intellectual property protection, and molecular marker-assisted breeding.

[0052] Example 1

[0053] (1) Experimental samples and genome resequencing

[0054] One sample was selected from each of the species of Herba Solitariae, Herba Acerolae and Herba Acerolae, and the genome was resequenced on the Illumina HiSeq 2500 platform with a sequencing fragment length of 150 bp. The original read length data was obtained for subsequent analysis.

[0055] (2) Chloroplast genome assembly

[0056] The CLC v4.06 software was used to assemble the chloroplast genome from scratch on the raw sequencing data. The CLC parameters were set as follows: deletion and insertion costs of 3, mismatch costs of 2, minimum continuous length of 200, bubble size of 98, length score and similarity score of 0.9. The assembled contigs were compared with the reference chloroplast genome in NCBI BLAST v2.2.31, and representative chloroplast sequences were selected for sorting and positioning. The sequenced data were then remapped to the chloroplast genome draft to obtain the complete chloroplast genome sequence.

[0057] (3) Chloroplast genome annotation

[0058] The three assembled chloroplast genomes were preliminarily annotated using the online software Dual Organellar Genome Annotator (Wyman et al., 2004), and the start, end and intron positions were checked using Geneious R9 (Kearse et al., 2012), and the positions of tRNA genes were checked using tRNAscan-SE v1.21.

[0059] (4) Genetic diversity analysis

[0060] The three sequenced chloroplast genomes were aligned using the MAFFT function in Geneious R9 software. The aligned matrix was sheared, and each CDS region, tRNA region, rRNA region, and intergenic spacer (IGS) region was used to calculate genetic diversity. The sliding window analysis in DNASP 5 software was used to count the nucleotide diversity of fragments containing >1 mutation site and length >100bp (Librado and Rozas, 2009). The results were plotted in Excel. Regions with genetic diversity higher than 0.2 were screened as high-variability regions: trnH-psbA, psbC-trnS, trnM-atpE, petA-psbJ, ccsA-ndhD, rbcL, and finally accurate and reliable chloroplast genome sequences ccsA-ndhD and rbcL were obtained.

[0061] (5) Primer design

[0062] Primers were designed using Primer6 for the screened high-variability regions to obtain oligonucleotide primer sequences with scores higher than 90 and sequence lengths less than 30 bp. The primer sequences were then sent to a biotechnology company for primer synthesis and purification. The primer sequences are as follows:

[0063] Primer set 1 (ccsA-ndhD):

[0064] Forward primer ccsA5F (SEQ ID NO. 1: AGTATGGGCTAATGAGGC);

[0065] The reverse primer is ndhD3R (SEQ ID NO. 2: GCAGGAACCAGTTATGAT), and the annealing temperature is 55°C.

[0066] Primer set 2 (rbcL):

[0067] Forward primer rbcL5F (SEQ ID NO. 3: CAACTGTGTGGACCGATG);

[0068] The reverse primer was rbcL3R (SEQ ID NO. 4: CAACTGTGTGGACCGATG), and the annealing temperature was 55°C.

[0069] (6) DNA extraction

[0070] In order to verify the universality of the primers, 222 samples of Glechoma longituba, 139 samples of Glechoma sutchuenensis, and 50 samples of Glechoma sutchuenensis were collected for DNA extraction.

[0071] The steps are as follows: preheat the CTAB extract and the corresponding amount of β-mercaptoethanol in a 65°C water bath; grind about 0.1g of the dried leaf material into powder, quickly transfer it into a 2ml centrifuge tube, and quickly mix it; place it in a 65°C water bath for 45 minutes, and shake it three times to mix it; after cooling to room temperature, add an equal volume of chloroform: isoamyl alcohol (24:1), gently invert and mix to emulsify for 10 minutes; centrifuge at 10000 rpm for 10 minutes; aspirate the supernatant into another clean 2mL centrifuge tube; aspirate the supernatant and add an equal volume of chloroform: isoamyl alcohol (24:1) to the supernatant. Pre-cooled isopropanol at -20 degrees, mix by inversion, and place at room temperature for 15 minutes until white flocculent precipitate appears; pick up DNA with a glass hook and place it in a 2mL tube containing 1mL of 75% ethanol by volume; rinse the DNA precipitate with 75% ethanol by volume, and remove the ethanol with a Tip; rinse again with 1ml of 75% ethanol by volume; rinse again with anhydrous ethanol; dry the precipitate in a constant temperature box at room temperature or below 60℃ for a while until it just becomes translucent; dissolve the precipitate with 10-30μL of TE. Use 1% agarose gel electrophoresis at 95V for 30min to detect the quality and integrity of the extracted DNA, and store it in a refrigerator at -20℃.

[0072] (7) PCR and sequencing

[0073] The sample DNA was extracted and PCR amplification was performed using primer sets 1 and 2 with the DNA as a template.

[0074] The reaction system was 35 μL: 1 μL genomic DNA (concentration was 50 ng / μL), 13.7 μL pure water (ddH2O), 1.4 μL forward primer, 1.4 μL reverse primer and 17.5 μL PCR Mix.

[0075] PCR reaction program: denaturation at 94°C for 2 min; 94°C for 30 s, annealing at 55°C for 30 s, 72°C for 90 s, for 28 cycles; extension at 72°C for 2 min, and storage at 10°C. The amplified product was sent to a sequencing company for Sanger sequencing.

[0076] (8) Genetic diversity and genetic structure analysis

[0077] The .ab1 files obtained by sequencing were assembled de novo in Geneious software, and then the MAFFT program in Geneious R9 software was used for sequence alignment. The aligned matrix was imported into DNASP 5 to calculate the number of haplotypes, nucleotide diversity (π), and haplotype diversity (Hd) of different species, different genetic lineages, and different populations. Then, a simple network map was constructed using TCS software to illustrate the genetic structure of haplotypes.

[0078] The results showed that the assembled and annotated chloroplast genome sequences of Glechoma longituba, Glechoma chinensis and Glechoma chinensis all showed tetrad structures, with lengths of 156775bp, 156420bp and 156935bp, respectively. Secondly, the two chloroplast genome sequences were compared and analyzed, revealing some variable regions that could become DNA markers and serve as potential DNA barcodes to identify ginger species. Among these regions, ccsA-ndhD and rbcL were selected, and two pairs of primers were designed for the two DNA regions, which had good amplification versatility in Glechoma longituba and Glechoma chinensis.

[0079] The PCR amplification results of 411 samples (including 222 samples of Solitaria, 139 samples of Acer truncatum, and 50 samples of Rock Acer truncatum) were sequenced, and the obtained sequencing data were assembled and aligned. The resulting matrix was 1,696 bp in length, resulting in 30 single nucleotide substitutions, 4 insertions and deletions, and 1 inversion.

[0080] Genetic diversity analysis revealed that the overall nucleotide diversity (π) of the plant was 0.00456, and the haplotype diversity (H d ) is 0.878, and the π of each group is 0-0.00056, H d is 0-0.533; the overall nucleotide diversity (π) of maple grass is 0.00256, and the haplotype diversity (H d ) is 0.665, and the π of each group is 0-0.004, H d is 0-0.511; the overall nucleotide diversity (π) of rock maple is 0.00193, and the haplotype diversity (H d ) is 0.327, and the π of each group is 0, H d is 0 (Table 1).

[0081] Table 1 Sample information and genetic diversity of the single-rooted grass, maple leaf grass and rock maple leaf grass used in the present invention

[0082]

[0083]

[0084] A total of 22 haplotypes were identified in the three species. The 13 haplotypes of the single-rooted grass were OR1-OR13, the 7 haplotypes of the maple leaf grass were MR1-MR7, and the 2 haplotypes of the rock maple leaf grass were MA1-MA2 ( Figure 2 ). The haplotypes of each species are unique, and the haplotypes of different species differ by 12 genetic mutation steps, indicating that the two cpDNAs can be used to correctly identify the three species.

[0085] According to the spatial distribution information of the collected populations, each haplotype was geographically divided, and it was found that there were 4 subgroups in the single-root grass. The unique haplotypes in Wuling Mountain were OR1-OR4, and the distribution groups were HNBD and HNYD; the unique haplotypes in the southern Taihang Mountain were OR5-OR7, and the distribution groups were HNXW, HNWX, SXFH and SXWM; the unique haplotypes in the central Taihang Mountain were OR12-OR13, and the distribution groups were HBYM and SXXZ. The unique haplotypes in Yanshan were OR8-OR11, and the distribution groups were BJAJ, BJBH, BJBL, BJCP, BJKP, BJQL, BJSF, HBDM, HBHX, HBJC, HBLL, HBQL, TJJL, and TJLZ. There are two subgroups in the maple grass. The unique haplotypes in Changbai Mountain are MR1-MR3, and the distribution groups are JLBS, JLFS, JLJH, LNFH, and LNTQ. The main haplotypes in the Korean Peninsula are MR4-MR7, and the distribution groups are SKBM, SKDJ, SKGM, SKHG, SKOG, SKOR, and SKYS. In addition, MR7 is also distributed in LNMD and LNTQ. Because of its narrow distribution range, there are no obvious subgroups in the rock maple grass.

[0086] In summary, the primer set of this implementation case has the advantage of better identifying the relationship between the species of the genus Solitaria and the genus Acerola and the origin of the samples:

[0087] (1) The present invention collected 222 samples of single-rooted grass, 139 samples of maple leaf grass, and 50 samples of rock maple leaf grass (Table 1). The test materials basically covered their natural distribution range ( Figure 1 ).

[0088] (2) One sample each from Sphaerocephalum indica, Sphaerocephalum truncatum, and Sphaerocephalum truncatum was selected for shallow genome resequencing.

[0089] (3) Perform chloroplast genome assembly, annotation, and genetic diversity analysis on the sequencing data to identify highly variable regions.

[0090] (4) Primers were designed for the highly variable regions, and DNA was extracted and PCR amplified for all samples.

[0091] (5) After the amplified sequences are aligned, the number, type, and diversity of haplotypes are counted, a haplotype network diagram and a neighbor-joining tree are constructed, and the primer combination is judged based on the results to be reliable, accurate, and effective in identifying species and origins. In the process of identifying the species and origins of the genus Selenicereus and Selenicereus, the two pairs of DNA primer combinations of the present invention do not need to consider the growth and development stage and plant morphology of the plants, and the identification success rate is higher than that of phenotypic identification technology, and the cost is lower.

[0092] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A primer combination for identifying the taxonomic status and origin of species of the genus Echinops and Echinops, characterized in that: Comprising primer set 1 and primer set 2; The primer set 1 consists of a forward primer as shown in SEQ ID NO.1 and a reverse primer as shown in SEQ ID NO.2; The primer set 2 consists of a forward primer as shown in SEQ ID NO.3 and a reverse primer as shown in SEQ ID NO.

4.

2. A product for identifying the species and origin of medicinal plants of the genus Unicot and the genus Acerola, characterized in that: Comprising the primer combination according to claim 1.

3. The product according to claim 2, characterized in that Includes reagents, kits and chips.

4. Use of the primer combination as claimed in claim 1 or the product as claimed in claim 2 in identifying the taxonomic status and origin of species of the genus Ulmus and Ulmus.

5. A method for identifying the taxonomic status and origin of species of the genus Echeveria and Echeveria, characterized in that: The following steps are involved: (1) extracting the DNA of the sample to be tested, using the DNA of the sample to be tested as a template, and performing PCR amplification and sequencing using the primer combination described in claim 1 or the product described in claim 2; (2) The .ab1 files obtained by sequencing were assembled de novo in Geneious software, and then the MAFFT program in Geneious R9 software was used for sequence alignment. The aligned matrix was imported into DNASP 5 to calculate the haplotype number, nucleotide diversity, and haplotype diversity of different species, different genetic lineages, and different populations. Then, a simplified network map was constructed using TCS software to illustrate the genetic structure of haplotypes. (3) Determine the species and origin of the sample to be tested based on the genetic structure of the haplotype.

6. The method according to claim 5, characterized in that The reaction system of the PCR amplification is 35 μL: 1 μL of template DNA with a concentration of 50 ng / μL, 13.7 μL of ddH2O, 1.4 μL of forward primer, 1.4 μL of reverse primer and 17.5 μL of PCRMix; Reaction procedure: denaturation at 94°C for 2 min; 94°C for 30 s, annealing at 55°C for 30 s, and 72°C for 90 s for 28 cycles; extension at 72°C for 2 min, and storage at 10°C.

7. The method according to claim 5, characterized in that The method for determining the species and origin of the sample to be tested is as follows: if the genetic structure of the haplotype is OR1-OR13, the sample to be tested is of the genus Solitaria; If the genetic structure of the haplotype is MA1-MA2, then the sample to be tested is rock maple leaf grass; If the genetic structure of the haplotype is MR1-MR7, the sample to be tested is maple grass.

8. The method according to claim 7, characterized in that The method for determining the species and origin of the sample to be tested is: if the genetic structure of the haplotype is OR1-OR4, the origin of the sample to be tested is Wuling Mountain area; If the genetic structure of the haplotype is OR5-OR7, the origin of the sample to be tested is the southern area of ​​Taihang Mountain; If the genetic structure of the haplotype is OR8-OR11, the origin of the sample to be tested is Yanshan area; If the genetic structure of the haplotype is OR12-OR13, the origin of the sample to be tested is the middle area of ​​Taihang Mountain; If the genetic structure of the haplotype is MR1-MR3, the origin of the sample to be tested is Changbai Mountain area; If the genetic structure of the haplotype is MR4-MR7, the origin of the sample to be tested is the Korean Peninsula.

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