SSR molecular marker combination for identifying gynura related species plants

Abstract

The present application relates to the technical field of plant molecular biology, and particularly relates to a SSR molecular marker combination for identifying Gynura related species plants, and provides a SSR molecular marker combination for identifying Gynura related species plants and a primer group, which can accurately distinguish Gynura related species plants, and on the basis of this, a method for identifying Gynura related species plants is constructed by using a fluorescent SSR-PCR technology, has the characteristics of high stability, good repeatability, high resolution, strong specificity and high sensitivity, and can realize efficient, scientific and accurate identification of Gynura related species plant seedlings, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of plant biotechnology, specifically to the field of plant molecular biology, and particularly to SSR molecular marker combinations for identifying closely related plants of the genus Gynostemma. Background Technology

[0002] Microsatellite markers, also known as simple tandem repeats (STRs) or simple sequence repeats (SSRs), are simple repetitive sequences uniformly distributed throughout the genomes of eukaryotes. They consist of tandem repeat fragments of 2–6 nucleotides. The number of repeat units in SSRs varies greatly, manifesting as euploidy or non-uniformity within the repeat unit sequence, thus causing site polymorphism. Specific primers are designed based on conserved SSR sequences, and fluorescent groups are added for fluorescent PCR amplification. The amplified products with fluorescent signals are then subjected to 3730 fluorescently labeled capillary electrophoresis. Fragments with different numbers of repeat units show peaks at different positions, and different alleles are identified based on the peak readings. Due to their co-dominance, stable amplification, high information content, low cost, and high efficiency, SSR molecular markers have been widely used as an ideal marker selection for plant genetic diversity analysis.

[0003] The chloroplast genome, a type of organelle genome, is independent of the nuclear genome and exhibits high conservation in sequence and structure, making it feasible for developing chloroplast microsatellite markers. The plant chloroplast genome generally has a four-segment structure: a large single copy (LSC), a small single copy (SSC), and two inverted repeats (IRs). The chloroplast genome is independent of the nuclear genome and is a type of organelle genome, exhibiting high conservation in sequence and structure. The chloroplast genome has a simple structure, smaller molecular weight and copy number, and is unisexually inherited. Its gene sequence and gene content are highly conserved, with almost no gene recombination, making it more conserved than the nuclear genome. Chloroplast microsatellite marker (cpSSR) loci are typically located in non-coding regions, exhibiting higher variability compared to coding regions, and possessing the advantages of a low evolutionary rate and almost zero recombination rate. Furthermore, the regions flanking the cpSSR loci are highly conserved, showing high universality among closely related species. Therefore, cpSSR markers developed based on chloroplast genome information can be used for phylogenetic studies at higher taxonomic levels, interspecific and intraspecific identification, and the construction of genetic maps and DNA fingerprints.

[0004] White cabbage ( G. divaricata ) and white phoenix vegetable ( G. formosana Both *Gynura* and *Gynura fusiforme* are perennial herbaceous plants belonging to the genus *Gynura* in the family Asteraceae, possessing both medicinal (heat-clearing and detoxifying, blood sugar-lowering) and edible value. However, *Gynura fusiforme* and *Gynura fusiforme* are highly similar in growth habits, plant morphology, stems and leaves, flowers and fruits, and can only be vaguely distinguished by minor differences in the fine veins on the underside of the leaves. This leads to confusion or misuse during resource surveys, production, distribution, and scientific research. Existing research indicates differences in their chemical composition and potential food safety, making accurate differentiation crucial. More importantly, some species in this genus (such as *Gynura fusiforme*) contain highly toxic pyrrolizidine alkaloids (PAs), which, if mistakenly ingested, can cause severe liver damage (HVOD). Furthermore, the genus *Gynura fusiforme* also contains several closely related species, such as *Gynura fusiforme*, *Gynura fusiforme* var. *fusiforme*, and *Gynura fusiforme* var. *fusiforme*. Particularly prominent is the serious problem of misuse, substitution, and counterfeiting of *Gynura fusiforme* var. *fusiforme* in the market, further exacerbating the confusion in identification. Lying-down Chrysanthemum Panax notoginseng ( G. procumbensIn medicinal herb markets, wild vegetable distribution, e-commerce sales, and seedling transactions, it is often colloquially known as "white-backed ginseng," "wild ginseng," "immortal herb," ​​"wealthy vegetable," and "bright moon herb," ​​leading to widespread confusion of the same thing with different names and the same thing being different. Research conducted by the project team in multiple locations revealed that over half of the "white-backed ginseng seedlings" and "edible ginseng vegetables" circulating in the market are actually *Gynostemma pentaphyllum*. Merchants often arbitrarily name plants based on marketing claims of "stronger efficacy," "higher yield," and "easier cultivation," lacking scientific basis. Different species are given the same name, resulting in widespread misuse of research materials, cultivation raw materials, and edible products.

[0005] Current methods for identifying closely related species in the genus *Gynostemma* mainly rely on morphological characteristics or universal DNA barcode sequences, and there is no application of cpSSR markers for closely related species in the genus. However, morphological methods are easily affected by environmental conditions and growth stages, have poor stability, and depend on personal experience, resulting in high subjectivity and low accuracy. Conventional universal DNA barcodes (such as ITS and matK) have been shown in previous studies to have limited sequence differences among several closely related species within the same genus, making it difficult to provide sufficient resolution.

[0006] Therefore, there is an urgent need to establish a molecular marker and identification method that is highly stable, reproducible, high-resolution, highly specific, highly sensitive, and can effectively distinguish closely related species of the genus Gynostemma, in order to meet the practical needs for accurate differentiation of closely related species of the genus Gynostemma. Summary of the Invention

[0007] This invention addresses the current shortage of universally applicable polymorphic cpSSR primers among closely related species in the *Gynostemma* genus, the inability to distinguish *Gynostemma pentaphyllum* and *Gynostemma spp.* seedlings by phenotypic differentiation, and the lack of feasible molecular identification schemes for identifying closely related *Gynostemma* species. It provides SSR molecular markers, primers, and their applications for distinguishing closely related *Gynostemma* species. This invention utilizes Illumina novaseq X plus high-throughput sequencing technology to sequence the chloroplast genomes of *Gynostemma pentaphyllum* and *Gynostemma pentaphyllum*. Using comparative chloroplast genomics, variations at the same microsatellite loci in the chloroplast sequences of *Gynostemma pentaphyllum* and *Gynostemma spp.* were analyzed. Through screening and verification, effective cpSSR molecular markers for distinguishing *Gynostemma pentaphyllum* and *Gynostemma spp.* were obtained. Unexpectedly, these cpSSR molecular markers can also be used to identify other closely related species in the *Gynostemma* genus. Based on this, a method for identifying closely related *Gynostemma* species is proposed. The following technical solution is proposed based on this.

[0008] First, this invention provides a combination of SSR molecular markers (cpSSR molecular markers) for identifying closely related species of the genus *Gynostemma*. The SSR molecular markers are obtained by amplification using nine pairs of primers. These nine pairs of primers are shown in SEQ ID No. 1-2, SEQ ID No. 3-4, SEQ ID No. 5-6, SEQ ID No. 7-8, SEQ ID No. 9-10, SEQ ID No. 11-12, SEQ ID No. 13-14, SEQ ID No. 15-16, and SEQ ID No. 17-18, as detailed in Table 1. The closely related species of the genus *Gynostemma* described in this invention include *Gynostemma pentaphyllum*, *Gynostemma*, *Gynostemma sylvestris*, *Gynostemma floribunda*, and *Gynostemma rubrum*.

[0009] Table 1 Primer information for amplifying SSR molecular markers used to distinguish between *Gnaphalium affine* and *Gnaphalium affine*.

[0010] Furthermore, the present invention provides a primer set for amplifying SSR molecular markers for identifying closely related plants of the genus Gynostemma, the primer set comprising the above-mentioned 9 pairs of primers.

[0011] Furthermore, the present invention provides a kit for identifying closely related plants of the genus Gynostemma, which contains the above-mentioned primer set.

[0012] Preferably, the kit is a fluorescent SSR-PCR amplification kit.

[0013] Furthermore, the present invention provides the application of the SSR molecular marker combination, the primer set, and the kit in identifying closely related species of the genus Gynostemma.

[0014] Furthermore, the present invention provides a method for identifying closely related plants of the genus Gynostemma, comprising: identifying the test sample by detecting the SSR molecular marker combination.

[0015] Preferably, the identification method includes: performing fluorescent SSR-PCR amplification on the DNA of the sample to be tested and a standard of closely related species of the genus Gynostemma using the primer set described above; calculating the genetic distance and performing cluster analysis after detecting the amplification products by capillary electrophoresis; and then identifying the sample to be tested; if the sample to be tested clusters together with the standard of closely related species of the genus Gynostemma, the sample to be tested is identified as a closely related species of the genus Gynostemma corresponding to the standard of closely related species of the genus Gynostemma.

[0016] For example, if the sample to be tested clusters together with the *Gnaphalium affine* standard, then the sample to be tested is *Gnaphalium affine*; if the sample to be tested clusters together with the *Gnaphalium affine* standard, then the sample to be tested is *Gnaphalium affine*.

[0017] Preferably, the identification method includes: performing fluorescent SSR-PCR amplification on the DNA of the sample to be tested using the primer set, analyzing the length of the amplification product, and then identifying the sample to be tested; If the lengths of the amplified products of the primers shown in SEQ ID No.1~SEQ ID No.2, SEQ ID No.3~SEQ ID No.4, SEQ ID No.5~SEQ ID No.6, SEQ ID No.7~SEQ ID No.8, SEQ ID No.9~SEQ ID No.10, SEQ ID No.11~SEQ ID No.12, SEQ ID No.13~SEQ ID No.14, SEQ ID No.15~SEQ ID No.16, and SEQ ID No.17~SEQ ID No.18 are 177 bp, 244 bp, 300 bp, 270 bp, 302 bp, 299 bp, 301 bp, 278 bp, and 270 bp respectively, then the sample to be tested is *Gynostemma pentaphyllum*. If the lengths of the amplified products are 173 bp, 243 bp, 294 bp, 268 bp, 293 bp, 294 bp, 299 bp, 281 bp and 272 bp respectively, then the sample to be tested is *Gnaphalium affine*. If the lengths of the amplified products are 153 bp, 244 bp, 301 bp, 268 bp, 296 bp, 294 bp, 301 bp, 278 bp and 272 bp respectively, then the sample to be tested is *Gnaphalium affine*. If the lengths of the amplified products are 173 bp, 243 bp, 299 bp, 272 bp, 296 bp, 292 bp, 301 bp, 278 bp and 271 bp respectively, then the sample to be tested is Gynostemma pentaphyllum. If the lengths of the amplified products are 177 bp, 243 bp, 301 bp, 268 bp, 296 bp, 297 bp, 300 bp, 279 bp and 272 bp respectively, then the sample to be tested is *Gynostemma pentaphyllum*.

[0018] Preferably, the procedure for fluorescent SSR-PCR amplification is as follows: pre-denaturation at 95℃ for 5 min; denaturation at 95℃ for 30 s, annealing at 62℃-52℃ for 30 s, extension at 72℃ for 30 s, for a total of 10 cycles, with a temperature decrease of 1℃ per cycle; denaturation at 95℃ for 30 s, annealing at 52℃ for 30 s, extension at 72℃ for 30 s, for a total of 25 cycles; and a final extension at 72℃ for 20 min.

[0019] The plant standards of closely related species of the genus Gynostemma mentioned in this invention refer to materials whose species have been determined by other methods.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides an SSR molecular marker combination and primer set for identifying closely related species of the genus Gynostemma. It can accurately distinguish closely related species of the genus Gynostemma. Based on this, a method for identifying closely related species of the genus Gynostemma is constructed using fluorescent SSR-PCR technology. It has the characteristics of high stability, good repeatability, high resolution, strong specificity and high sensitivity. It can realize efficient, scientific and accurate identification of seedlings of closely related species of the genus Gynostemma and has broad application prospects. Attached Figure Description

[0021] Figure 1 This is a gel image of partial PCR product detection from *Gnaphalium affine* and *Gnaphalium affine* germplasm.

[0022] Figure 2 This is an electrophoresis peak diagram of the germplasm of *Gnaphalium affine* and *Gnaphalium affine* after amplification using primers CPSSR024.

[0023] Figure 3 It is a clustering graph based on UPGMA. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0025] In the embodiments provided in this specification, unless specific techniques or conditions are specified, the techniques or conditions described in the literature in this field, or the product instructions, shall be followed. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0026] This invention relates to molecular biology experiments. Unless otherwise specified, reference can be made to the book *Molecular Cloning* (J. Sambrook, E.F. Fritsch, and T. Maniatis, Science Press, 1994). This book and its subsequent editions are the most commonly used and guiding reference books for those skilled in the art when performing experiments related to molecular biology. In addition, depending on the experimental purpose, those skilled in the art complete the corresponding experiments under the guidance of the operating manuals accompanying various commercial reagent kits or entrust them to specialized companies, such as primer synthesis and gene sequencing.

[0027] The *Gynostemma pentaphyllum* material used in the following embodiments was sourced from Xinan Village, Zhuqi Township, Fuzhou City, Fujian Province, whose taxonomic status is well-established. The *Gynostemma pentaphyllum* material was sourced from the Lizhihai Cultivation Base in Zhangzhou City, Fujian Province, whose taxonomic status is well-established. This invention can also be verified using *Gynostemma pentaphyllum* plant materials from other sources (e.g., commercially available materials).

[0028] Example 1: Screening of cpSSR molecular markers for differentiating between *Gnaphalium affine* and *Gnaphalium affine* I. DNA Extraction Total DNA was extracted from two plant tissue samples using a modified CTAB method. The specific steps are as follows: 1. Take appropriate amounts of tender leaves of white cabbage and white phoenix cabbage respectively and grind them into powder in a mortar that has been pre-cooled and contains an appropriate amount of liquid nitrogen (the liquid nitrogen in the mortar must be kept sufficient during this process to ensure the quality of DNA extraction). Take an appropriate amount of powder and quickly transfer it to a 2ml centrifuge tube that has been pre-cooled by liquid nitrogen. 2. Add 1500 μl of preheated CTAB (at 65°C) and 100 μl of β-mercaptoethanol to a centrifuge tube. Seal the tube tightly and shake vigorously to mix thoroughly, ensuring the powder at the bottom of the tube is evenly dispersed in the CTAB buffer. Place the centrifuge tube in a 65°C water bath for 50 minutes. During this time, remove the tube every 5 minutes and shake vigorously for 30 seconds. 3. After the water bath, remove the centrifuge tubes and let them cool to room temperature before placing them in a high-speed refrigerated centrifuge and centrifuging at 4 ℃ and 12000 rpm for 12 min. 4. Transfer the supernatant to another 2ml centrifuge tube, add an equal volume of 24:1 chloroform / isoamyl alcohol, and shake vigorously upside down for 10 minutes. 5. After centrifuging at 12000 rpm for 15 minutes at 4°C in a high-speed refrigerated centrifuge, repeat step 4; 6. Repeat steps 4 and 5 twice to thoroughly remove proteins and polyphenols; 7. Transfer the supernatant to another 2ml centrifuge tube, add an equal volume of pre-cooled anhydrous ethanol at -20℃, gently invert to mix, and then place in a -20℃ freezer for 30 minutes to allow the DNA to precipitate fully. 8. Use a 200μl pick to remove the flocculent DNA precipitate from the centrifuge tube into another 1.5ml centrifuge tube. Add 1000μl of 70% anhydrous ethanol to rinse the precipitate. Gently flick the precipitate with your finger to remove it from the bottom of the centrifuge tube. Gently invert the tube several times and repeat the washing process 3 times. 9. Place the rinsed DNA centrifuge tubes in a clean bench and air dry until they are translucent. Then add 200-300 μl of sterile double-distilled water to dissolve them, add 2 μl of RNase to remove RNA from the total DNA, incubate at 37°C for 1 hour, and then store at -20°C for later use.

[0029] II. Detection of Total DNA Integrity, Purity and Concentration DNA integrity was assessed using 1% agarose gel electrophoresis, and purity and concentration were determined using a Nanodrop 2000 micro spectrophotometer.

[0030] III. Obtaining the Chloroplast Genome Sequence 1. The total plant DNA obtained in step 2 was fragmented using the VAHTS™ Universal DNA Library Prep Kit for Illumina® V2 to construct an Illumina sequencing library with a size of 300 fragments; 2. Paired-end sequencing of sample DNA was performed using Illumina novaseq X plus sequencing technology, with a sequencing length of 150 bp; 3. Use FastP v0.23.4 (https: / / github.com / OpenGene / fastp) software to perform quality filtering on the raw sequencing data; 4. The chloroplast genome was assembled from scratch using GetOrganelle[1]v1.7.7.1 (https: / / github.com / Kinggerm / GetOrganelle); finally, after de novo splicing, sequence alignment and assembly, a complete chloroplast genome map was drawn.

[0031] IV. Site identification of cpSSR and design of cpSSR primers: 1. Using MISA v1.0 (MIcroSAtellite identification tool, https: / / webblast.ipk-gatersleben.de / misa / ) software, search for SSR sites distributed in the chloroplast genomes of *Gnaphalium affine* and *Gnaphalium affine*. The parameters were set as follows: mononucleotide repeats, ≥8 repeat units; dinucleotide repeats, ≥5 repeat units; trinucleotide, tetranucleotide, pentanucleotide, and hexanucleotide repeats, ≥3 repeat units.

[0032] 2. Primers for cpSSR were designed using Primer Premier 5 software, with manual adjustments made as well. Primer design parameters followed these principles: primer length 18-25 bp, GC content 40-55%, primer annealing temperature 50-60℃, and amplified product size ideally concentrated between 250-1000 bp. To ensure the accuracy of Sanger sequencing results, the primer design site should be at least 40 bp away from both upstream and downstream primers. The selected cpSSR primers were then filtered, and duplicate primer sequences were removed.

[0033] 3. Using comparative chloroplast genomics, homology analysis was performed on the chloroplast genome sequences of the two plants. Polymorphic cpSSR sites were screened and cpSSR marker primers were designed. Candidate polymorphic chloroplast genome microsatellite molecular marker primers were obtained, as shown in Table 2.

[0034] Table 2. Characteristics of the preliminary SSR primers

[0035] Example 2: Screening of cpSSR marker primers 1. Collect tender leaves from multiple plant samples with certain differences in plant phenotype. The collected tender leaves should be intact and free of disease. Use the modified CTAB method to extract total DNA from the plant leaves.

[0036] 2. Use the method described in Example 1 to detect the integrity, purity, and concentration of DNA to meet experimental requirements.

[0037] 3. Dilute the total DNA with elution buffer TB (Tiangen) in equal portions (20 ng / μl) and store immediately at -20°C for later use.

[0038] 4. After mixing equal amounts of DNA from 8-10 randomly selected plants, each mixture is divided into 3 portions. The cpSSR primers obtained by PCR are used to screen the DNA of the samples to be tested.

[0039] 5. The capillary electrophoresis PCR amplification system involved in this invention is as follows: 10 μl, including 1.0 μl of genomic DNA (~20 ng), 5.0 μl of 2×Taq PCR Master Mix, 0.5 μl each of upstream and downstream primers (concentration 10 pmol / μl), and 3 μl of ddH2O.

[0040] 6. The DNA of the sample to be tested was amplified using PCR primers. The amplification program is shown in Table 3 below.

[0041] Table 3 PCR amplification program

[0042] 7. After the fluorescent PCR amplification is complete, take 1.0 μL of the fluorescent PCR product for fluorescent capillary electrophoresis detection. The system is shown in Table 4. The amplification electrophoresis gel image is shown in Table 4. Figure 1 The specificity of each SSR primer amplification is determined by the band pattern of the PCR product, and the amplification efficiency of each SSR primer is determined by the brightness of the PCR product band. According to the concentration requirements for sample testing, each fluorescent PCR product is diluted to obtain fluorescent PCR products with uniform concentration.

[0043] Table 4. Fluorescent capillary electrophoresis detection system

[0044] 8. All 74 primer pairs were used for SSR-PCR amplification in 6 mixed materials (3 parts of *Gnaphalium affine* and 3 parts of *Gnaphalium affine*). After centrifugation, the test plates with the added samples and reagents were placed on a PCR instrument and the denaturation program was run (95℃, 3 min). After denaturation, the plates were cooled immediately. The amplification products were analyzed and detected using an ABI 3730xL DNA Analyzer.

[0045] 9. Remove primers with low polymorphism PIC values ​​and retain 9 pairs of primers with high polymorphism, good stability and effective differentiation. Finally, a primer set with high polymorphism that can be used to amplify and identify SSR molecular markers of *Gnaphalium affine* and *Gnaphalium affine* is obtained. Primer information is shown in Table 1.

[0046] Example 3: Application of primer sets in identifying closely related species of the genus Gynostemma. 1. In this embodiment, 26 samples of *Gnaphalium affine* and 21 samples of *Gnaphalium affine*, different from those in Example 2, were used to verify the accuracy of the primer set used in Example 2 in identifying *Gnaphalium affine* and *Gnaphalium affine*. The steps are as follows: 2. Extract DNA from the above-mentioned sample according to the method in Example 1; 3. Using the primer set from Example 2 (primers fluorescently labeled), fluorescent SSR-PCR amplification was performed on the DNA sample to be tested, *Gnaphalium affine* standard, and *Gnaphalium affine* standard (*Gnaphalium affine* and *Gnaphalium affine* standards refer to materials whose species have been identified by other methods). The PCR system and PCR procedure were the same as in Example 2. The amplification products were detected using an ABI 3730xL DNA Analyzer fully automated capillary electrophoresis analyzer. The electrophoretic typing peaks of *Gnaphalium affine* and *Gnaphalium affine* germplasm amplified using CPSSR024 primers are shown below. Figure 2 As shown.

[0047] 4. Genetic distance was calculated and cluster analysis was performed. All the above-mentioned samples of *Gnaphalium affine* were clustered together with the *Gnaphalium affine* standard, and all the samples of *Gnaphalium affine* were clustered together with the *Gnaphalium affine* standard. The results were consistent with those of *Gnaphalium affine* and *Gnaphalium affine* identified by other methods.

[0048] 5. While using 9 primer pairs to identify *Gnaphalium affine* and *Gnaphalium affine*, this invention also found that the above 9 primer pairs can also be used to distinguish closely related species in the same genus in the market. Therefore, in this embodiment, the same primers were also used to detect three other closely related species in the same genus. The raw data of the samples were analyzed to obtain the length of the amplified fragments of different samples. Further analysis of the data using GenAlEx 6.5 was used to obtain primer polymorphism: the total number of different alleles (Na), the effective number of alleles (Ne), the observed heterozygosity (Ho), the expected heterozygosity (He), the Shannon's info (I), the fixation index (F, an index used to assess the degree of deviation between the actual observed value and the theoretical value), the average expected heterozygosity (Hs, reflecting the level of genetic diversity in the population), and the PIC value (polymorphism information index) were calculated. The results are shown in Table 5 below (N is the number of samples).

[0049] Table 5 Calculation Results

[0050] 6. Calculate the polymorphism information content (PIC) and construct the clustering tree file using Power Marker 3.25. Perform cluster analysis using the UPGMA method and draw the cluster diagram. The UPGMA-based cluster diagram shows that there are differences in the phylogenetic relationships between the tested species *Gnaphalium affine*, *Gnaphalium affine*, and *Gnaphalium affine* and *Gnaphalium affine*, with the phylogenetic relationship from most distant to most intimate being: *Gnaphalium affine* > *Gnaphalium affine* > *Gnaphalium affine* > *Gnaphalium affine* > *Gnaphalium affine*. Figure 3 ).

[0051] 7. Perform capillary electrophoresis on the PCR products, and compare the capillary electrophoresis results with the fingerprint pattern to identify the results; the fingerprint pattern is shown in Table 6 below.

[0052] Table 6 Fingerprint patterns

[0053] Where A represents the letter designation of locus CPSSR001, B represents the letter designation of locus CPSSR003, C represents the letter designation of locus CPSSR004, D represents the letter designation of locus CPSSR005, E represents the letter designation of locus CPSSR022, F represents the letter designation of locus CPSSR024, G represents the letter designation of locus CPSSR039, H represents the letter designation of locus CPSSR049, and I represents the letter designation of locus CPSSR058; the numbers represent the fragment length of the PCR amplification product, in bp; in this study, all species showed homozygous specific single bands at their corresponding loci, with no fragment length ranges.

[0054] Fingerprint mapping enables precise identification of target species: *Gnaphalium affine* identification characteristics: a specific band of 302 bp at the CPSSR022 marker and a 270 bp band at the CPSSR005 marker; this combination completely distinguishes it from *Gnaphalium affine* and other closely related species. *Gnaphalium affine* identification characteristics: a specific band of 281 bp at the CPSSR049 marker and a 293 bp band at the CPSSR022 marker, exhibiting significant species specificity. CPSSR001 shows extremely high polymorphism among *Gnaphalium affine* (153 bp), *Gnaphalium affine* (177 bp), and *Gnaphalium affine* (173 bp), and can be used as the preferred identification marker.

[0055] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An SSR molecular marker combination for identifying closely related plant species of the genus *Gynostemma*, characterized in that, The SSR molecular marker combination was amplified using 9 pairs of primers; the 9 pairs of primers are shown in SEQ ID No. 1~SEQ ID No. 2, SEQ ID No. 3~SEQ ID No. 4, SEQ ID No. 5~SEQ ID No. 6, SEQ ID No. 7~SEQ ID No. 8, SEQ ID No. 9~SEQ ID No. 10, SEQ ID No. 11~SEQ ID No. 12, SEQ ID No. 13~SEQ ID No. 14, SEQ ID No. 15~SEQ ID No. 16, and SEQ ID No. 17~SEQ ID No. 18; the closely related species of the genus Gynostemma include Gynostemma pentaphyllum, Gynostemma pentaphyllum, Gynostemma pentaphyllum, Gynostemma pentaphyllum var. praecox, and Gynostemma pentaphyllum var. praecox.

2. A primer set for amplifying SSR molecular markers for identifying closely related plants of the genus *Gynostemma*, characterized in that... The primer set includes 9 pairs of primers; the 9 pairs of primers are shown in SEQ ID No. 1~SEQ ID No. 2, SEQ ID No. 3~SEQ ID No. 4, SEQ ID No. 5~SEQ ID No. 6, SEQ ID No. 7~SEQ ID No. 8, SEQ ID No. 9~SEQ ID No. 10, SEQ ID No. 11~SEQ ID No. 12, SEQ ID No. 13~SEQ ID No. 14, SEQ ID No. 15~SEQ ID No. 16, and SEQ ID No. 17~SEQ ID No. 18; the closely related species of the genus Gynostemma include Gynostemma pentaphyllum, Gynostemma pentaphyllum, Gynostemma pentaphyllum var. ...

3. A kit for identifying closely related plant species of the genus *Gynostemma*, characterized in that, It contains the primer set as described in claim 2.

4. The reagent kit according to claim 3, characterized in that, The kit is a fluorescent SSR-PCR amplification kit.

5. The application of the SSR molecular marker combination of claim 1, the primer set of claim 2, and the kit of claim 3 or 4 in the identification of closely related species of the genus Gynostemma; wherein the closely related species of the genus Gynostemma include Gynostemma pentaphyllum, Gynostemma pentaphyllum, Gynostemma pentaphyllum, Gynostemma pentaphyllum var. praecox, and Gynostemma pentaphyllum var. praecox.

6. The application of the SSR molecular marker combination of claim 1, the primer set of claim 2, and the kit of claim 3 or 4 in the genetic breeding of closely related species of the genus Gynostemma; wherein the closely related species of the genus Gynostemma include Gynostemma pentaphyllum, Gynostemma pentaphyllum, Gynostemma pentaphyllum, Gynostemma pentaphyllum var. praecox, and Gynostemma pentaphyllum var. praecox.

7. A method for identifying closely related plant species of the genus *Gynostemma*, characterized in that, include: The test sample is then identified by detecting the SSR molecular marker combination described in claim 1.

8. The identification method according to claim 7, characterized in that, include: The primer set described in claim 2 was used to amplify the DNA of the test sample and the standards of closely related species of the genus Gynostemma. After the amplification products were detected by capillary electrophoresis, the genetic distance was calculated and cluster analysis was performed. Then the test sample was identified. If the sample to be tested clusters together with the standard of a closely related species of the genus Gynostemma, then the sample to be tested is identified as a closely related species of the genus Gynostemma corresponding to the standard of a closely related species of the genus Gynostemma.

9. The identification method according to claim 7, characterized in that, include: The primer set described in claim 2 was used to amplify the DNA of the test sample by fluorescent SSR-PCR, the length of the amplification product was analyzed, and then the test sample was identified. If the lengths of the amplified products of the primers shown in SEQ ID No.1~SEQ ID No.2, SEQ ID No.3~SEQ ID No.4, SEQ ID No.5~SEQ ID No.6, SEQ ID No.7~SEQ ID No.8, SEQ ID No.9~SEQ ID No.10, SEQ ID No.11~SEQ ID No.12, SEQ ID No.13~SEQ ID No.14, SEQ ID No.15~SEQ ID No.16, and SEQ ID No.17~SEQ ID No.18 are 177 bp, 244 bp, 300 bp, 270 bp, 302 bp, 299 bp, 301 bp, 278 bp, and 270 bp respectively, then the sample to be tested is *Gnaphalium affine*. If the lengths of the amplified products are 173 bp, 243 bp, 294 bp, 268 bp, 293 bp, 294 bp, 299 bp, 281 bp and 272 bp respectively, then the sample to be tested is *Gnaphalium affine*. If the lengths of the amplified products are 153 bp, 244 bp, 301 bp, 268 bp, 296 bp, 294 bp, 301 bp, 278 bp and 272 bp respectively, then the sample to be tested is *Gnaphalium affine*. If the lengths of the amplified products are 173 bp, 243 bp, 299 bp, 272 bp, 296 bp, 292 bp, 301 bp, 278 bp and 271 bp respectively, then the sample to be tested is Gynostemma pentaphyllum. If the lengths of the amplified products are 177 bp, 243 bp, 301 bp, 268 bp, 296 bp, 297 bp, 300 bp, 279 bp and 272 bp respectively, then the sample to be tested is *Gynostemma pentaphyllum*.

10. The identification method according to claim 8 or 9, characterized in that, The procedure for fluorescent SSR-PCR amplification is as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 62℃-52℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 10 cycles, with a temperature decrease of 1℃ per cycle; 95℃ denaturation for 30 s, 52℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 25 cycles; and finally, 72℃ extension for 20 min.

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