Molecular marker for distinguishing clematis plants and application thereof

Abstract

The invention discloses a molecular marker for distinguishing clematis plants and application of the molecular marker. The molecular marker is composed of a single-stranded DNA as shown in a sequence 1 and a single-stranded DNA as shown in a sequence 2, and PCR (Polymerase Chain Reaction) amplification is carried out on 15 clematis plants by using the molecular marker, so that the molecular marker has two banding patterns of 96 bp and 93 bp in the 15 clematis plants, but only has the banding pattern of 93 bp in Wuxing clematis and Clematis brevicaulis. And the banding patterns in the other 13 clematis plants are 96 bp banding patterns. In addition, by sequencing the PCR products of the clematis brevicaulis and the clematis brevicaulis, it is found that the sequences of the PCR products of the clematis brevicaulis and the clematis brevicaulis are different, and the clematis brevicaulis and the clematis brevicaulis can be further distinguished based on the sequence difference, so that identification of the clematis brevicaulis and the clematis brevicaulis is achieved. The invention has important significance for distinguishing, identifying, researching, utilizing and protecting clematis plants.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a molecular marker for distinguishing Clematis species and its application, particularly to an InDel molecular marker for distinguishing Clematis wuxingensis, Clematis breviculatum and other Clematis species and its application. Background Technology

[0002] Clematis genus ( Clematis Clematis is an important group within the Ranunculaceae family. Renowned for its beautiful flowers and rich colors, it is known as the "Queen of Vines" and possesses extremely high economic value in the global horticultural market. Furthermore, various Clematis species are also important medicinal resources in traditional Chinese medicine. Accurate identification is a prerequisite for their rational utilization.

[0003] Common plant identification methods include morphological identification, cellular identification, and molecular identification. Morphological identification is highly dependent on the experience of the identifyer and is greatly affected by the plant's growth stage and environmental factors. It is almost impossible to accurately identify specimens without flowers, plants in the vegetative stage, or specimen fragments. Furthermore, the genus *Clematis* is diverse, with complex morphologies and frequent interspecific hybridization, resulting in many closely related species exhibiting extremely similar morphological characteristics during the vegetative and flowering stages, making accurate differentiation difficult. Distinguishing based solely on morphology involves continuous variations and overlaps in leaf division, floral structures such as sepal color, and stamen characteristics. Traditional morphological identification methods are prone to misjudgment. Therefore, morphological identification cannot meet the rapid and accurate needs of modern agriculture, traceability of traditional Chinese medicinal materials, and plant quarantine. In addition, cellular identification is cumbersome, requires high-quality materials during cell division, and suffers from limited resolution due to the small differences in karyotypes among many closely related species, making it unsuitable for large-scale practical applications. As an important supplement to morphological and cellular identification, molecular identification offers advantages such as stability and high accuracy, and can be completed during the seedling stage, significantly reducing the identification cycle and cost. Therefore, developing molecular markers to distinguish different species of the genus Clematis is of great significance for the research and conservation of Clematis species.

[0004] Short-columnar Clematis ( Clematis cadmia Clematis chinensis is a plant belonging to the genus Clematis in the family Ranunculaceae. Its unique pale purple flowers can enrich the early spring garden landscape. (Clematis wuxingense) Clematis huchouensisThe flowers are white and delicate in shape; the whole plant is used medicinally for its wind-dispelling and swelling-reducing properties. Developing characteristic molecular markers for *Clematis short-columnae* and *Clematis wuxingensis* within the *Clematis* genus is currently a technical bottleneck in horticultural production, quality control of traditional Chinese medicinal materials, and plant taxonomy research. The market urgently needs a rapid, accurate, stable, low-cost, and easily scalable identification method. This method involves screening species-specific DNA fragments to construct a rapid and objective identification system. Developing InDel markers (insertion / deletion markers) offers significant advantages; this technology requires only conventional PCR and electrophoresis techniques to achieve low-cost, high-throughput detection, playing a crucial role in resource surveys, efficient assessment of population genetic diversity, and rapid identification in the seedling stage. Summary of the Invention

[0005] The technical problem to be solved by this invention is how to distinguish *Clematis wuxingensis*, *Clematis breviculata*, and other plants of the *Clematis* genus, as well as the identification of *Clematis wuxingensis* and *Clematis breviculata*.

[0006] To address the aforementioned technical problems, this invention first provides a primer pair.

[0007] The primer pair provided by this invention consists of a forward primer and a reverse primer; The forward primer is either a1) or a2) as follows. a1) The single-stranded DNA molecule shown in sequence 1; a2) A single-stranded DNA molecule that has undergone substitution and / or deletion and / or addition of one or more nucleotides of sequence 1 and has the same function as sequence 1; The reverse primer is either a3) or a4) as follows: a3) The single-stranded DNA molecule shown in sequence 2; a4) A single-stranded DNA molecule that has one or more nucleotides of sequence 2 replaced and / or deleted and / or added, and has the same function as sequence 2.

[0008] In the primer pair described above, the molar ratio of the forward primer to the reverse primer is 1:1.

[0009] To address the aforementioned technical problems, this invention also provides the uses of the primer pairs.

[0010] This invention provides the application of the above primer pairs in any of the following b1)-b8): b1) Distinguish between *Clematis wuxingensis* and other plants in the *Clematis* genus; b2) Prepare products that distinguish *Clematis wuxingensis* from other *Clematis* species; b3) Distinguish Clematis septemlobus from other Clematis species; b4) Prepare products that distinguish Clematis stylarii from other Clematis species; b5) Identification of Clematis wuxingensis; b6) Preparation and identification of products containing Clematis wuxingensis; b7) Identification of Clematis stubularis; b8) Prepare and identify products for the identification of short-columnar clematis.

[0011] Kits containing the above primer pairs are also within the scope of protection of this invention; the function of the kit is any one of c1)-c4) below: c1) Distinguish between *Clematis wuxingensis* and other plants in the *Clematis* genus; c2) Distinguish between Clematis short-columned and other Clematis species.

[0012] c3) Identification of Clematis wuxingensis; c4) Identification of short-columned clematis.

[0013] In some implementations, the kit also includes other reagents for PCR amplification, such as MIX (Nanjing Winozan Biotechnology Co., Ltd., catalog number p115-02).

[0014] In some implementations, the kit also includes a negative control (such as sterile ultrapure water) and a positive control (such as genomic DNA from Clematis wuxingensis or Clematis breviscapus).

[0015] To achieve the above objectives, the present invention also provides the use of an InDel site or a substance for detecting an InDel site; the InDel site is located at positions 174-176 of sequence 3.

[0016] This invention provides the use of InDel sites or substances for detecting InDel sites in any of the following b1)-b8): b1) Distinguish between *Clematis wuxingensis* and other plants in the *Clematis* genus; b2) Prepare products that distinguish *Clematis wuxingensis* from other *Clematis* species; b3) Distinguish Clematis septemlobus from other Clematis species; b4) Prepare products that distinguish Clematis stylarii from other Clematis species; b5) Identification of Clematis wuxingensis; b6) Preparation and identification of products containing Clematis wuxingensis; b7) Identification of Clematis stubularis; b8) Prepare and identify products for the identification of short-columnar clematis.

[0017] In the above applications, the substance used to detect InDel sites is a substance used to detect whether InDel sites are missing.

[0018] In some embodiments, the substance for detecting the InDel site includes a primer pair for amplifying a DNA fragment containing the InDel site. The primer pair consists of a forward primer and a reverse primer, wherein the forward primer is a single-stranded DNA that specifically binds upstream of the InDel site, and the reverse primer is a single-stranded DNA that specifically binds downstream of the InDel site.

[0019] In some preferred embodiments, the forward primer is a single-stranded DNA molecule as shown in Sequence 1; and the reverse primer is a single-stranded DNA molecule as shown in Sequence 2.

[0020] To address the aforementioned technical problems, this invention also provides a method for distinguishing *Clematis wuxingensis* from other *Clematis* species.

[0021] The method for distinguishing *Clematis wuxingensis* from other *Clematis* species provided by the present invention includes the following steps: detecting whether the plant to be tested lacks the InDel site, and distinguishing *Clematis wuxingensis* from other *Clematis* species based on whether the plant to be tested lacks the InDel site; the InDel site is located at positions 174-176 of sequence 3.

[0022] The above method for distinguishing *Clematis wuxingensis* from other *Clematis* species is as follows: if the plant to be tested lacks the InDel locus, then the plant to be tested is *Clematis wuxingensis*; if the plant to be tested does not lack the InDel locus, then the plant to be tested is another *Clematis* species.

[0023] To address the aforementioned technical problems, the present invention also provides a method for distinguishing Clematis short-columnarii from other Clematis species.

[0024] The method for distinguishing Clematis brevichorata from other Clematis species provided by the present invention includes the following steps: detecting whether the plant to be tested lacks the InDel site, and distinguishing Clematis brevichorata from other Clematis species based on whether the plant to be tested lacks the InDel site; the InDel site is located at positions 174-176 of sequence 3.

[0025] The above method for distinguishing Clematis brevichorata from other Clematis species is as follows: if the plant to be tested lacks the InDel site, the plant to be tested is Clematis brevichorata; if the plant to be tested does not lack the InDel site, the plant to be tested is another Clematis species.

[0026] In any of the methods described above, the DNA fragment shown in sequence 3 is used as a reference sequence when detecting whether the plant to be tested lacks the InDel site.

[0027] Any of the methods described above may include the following steps: using the genomic DNA of the plant to be tested as a template, performing PCR amplification with the above primer pairs to obtain PCR products; detecting the PCR products by capillary electrophoresis, and distinguishing *Clematis wuxingensis*, *Clematis breviculata* and other *Clematis* species based on the size of the PCR products.

[0028] Furthermore, the method for distinguishing *Clematis wuxingensis* from other *Clematis* species based on the size of the PCR product is as follows: if the PCR product shows a band size of 93 bp after capillary electrophoresis, the plant to be tested is *Clematis wuxingensis*; otherwise, the plant to be tested is another *Clematis* species.

[0029] The method for distinguishing Clematis brevicula from other Clematis species based on the size of the PCR product is as follows: if the PCR product shows a band size of 93 bp after capillary electrophoresis, the plant to be tested is Clematis brevicula; otherwise, the plant to be tested is another Clematis species.

[0030] Furthermore, in Clematis septemlobus, the nucleotide sequence of the PCR product corresponding to the 93 bp band pattern is shown in Sequence 5.

[0031] In Clematis wuxingensis, the nucleotide sequence of the PCR product corresponding to the band pattern of 93 bp is shown in Sequence 6.

[0032] Furthermore, the PCR amplification reaction system is as follows: 2 μL genomic DNA, 5 μL MIX, 2.6 μL water, 0.2 μL forward primer, and 0.2 μL reverse primer. The final concentration of both the forward and reverse primers in the PCR amplification reaction system is 0.2 µmol / L.

[0033] The PCR amplification procedure is as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 35 cycles; 72℃ extension for 5 min; storage at 25℃.

[0034] The capillary electrophoresis was performed using an ABI 3730xl DNA Analyzer.

[0035] The other Clematis species mentioned above are at least one of the following Clematis species: Clematis stalkae, Clematis zeylanica, Clematis russula, Clematis coarse-toothed, Clematis halostae, Clematis breviscapus, Clematis short-tailed, Clematis macrophylla, Clematis celery-leaved, Clematis shrubby, Clematis chrysantha, Clematis pubescens, Clematis pinnata, Clematis acerifolia, and Clematis pubescens.

[0036] To address the aforementioned technical problems, this invention also provides a method for identifying Clematis wuxingensis.

[0037] The method for identifying Clematis wuxingensis provided by the present invention includes the following steps: using the genomic DNA of the plant to be tested as a template, performing PCR amplification using the above primer pair to obtain PCR products; and identifying whether the plant to be tested is Clematis wuxingensis based on the size and sequence of the PCR products.

[0038] In the above method for identifying Clematis wuxingensis, the method for identifying whether the plant to be tested is Clematis wuxingensis based on the size and sequence of the PCR product is as follows: if the PCR product obtains a band of 93 bp by capillary electrophoresis and the nucleotide sequence of the PCR product corresponding to the 93 bp band is as shown in Sequence 6, then the plant to be tested is Clematis wuxingensis; otherwise, the plant to be tested is not Clematis wuxingensis.

[0039] To address the aforementioned technical problems, this invention ultimately provides a method for identifying short-column clematis.

[0040] The method for identifying Clematis short-columnus provided by the present invention includes the following steps: using the genomic DNA of the plant to be tested as a template, performing PCR amplification using the above primer pair to obtain PCR products; and identifying whether the plant to be tested is Clematis short-columnus based on the size and sequence of the PCR products.

[0041] In the above method for identifying Clematis septemlobus, the method for identifying whether the plant to be tested is Clematis septemlobus based on the size and sequence of the PCR product is as follows: if the PCR product obtains a band of 93 bp by capillary electrophoresis and the nucleotide sequence of the PCR product corresponding to the 93 bp band is as shown in Sequence 5, then the plant to be tested is Clematis septemlobus; otherwise, the plant to be tested is not Clematis septemlobus.

[0042] This invention provides an InDel site for distinguishing *Clematis wuxingensis*, *Clematis breviscapus*, and other *Clematis* species. This InDel site is located at positions 174-176 of sequence 3. Based on this InDel site, this invention also developed the InDel molecular marker CA25292, which consists of single-stranded DNA molecules shown in sequence 1 and sequence 2. PCR amplification of 15 *Clematis* species using the InDel molecular marker CA25292 and capillary electrophoresis of the PCR products revealed that the InDel molecular marker CA25292 exhibited two band patterns (96 bp and 93 bp) across the 15 *Clematis* species. However, the 93 bp band pattern was only present in *Clematis wuxingensis* and *Clematis breviscapus*, while the band pattern in the other 13 *Clematis* species was 96 bp. Therefore, in practical applications, the InDel molecular marker CA25292 can be used to distinguish *Clematis wuxingensis*, *Clematis breviscapus*, and other *Clematis* species by detecting whether the InDel site of the test plant is deleted. Furthermore, this invention, through sequencing of PCR products corresponding to 93 bp band patterns in *Clematis wuxingensis* and *Clematis breviscapus*, found that the base after the InDel site in *Clematis breviscapus* is G, while that in *Clematis wuxingensis* is T. This sequence difference in PCR products can further distinguish *Clematis breviscapus* and *Clematis wuxingensis*, enabling identification of these two species, especially at the seedling stage. This invention is of great significance for the differentiation, identification, research, utilization, and protection of *Clematis* species. Attached Figure Description

[0043] Figure 1 Capillary electrophoresis images of two band types found in 15 species of Clematis.

[0044] Figure 2 This is a comparison of the sequencing results of PCR products from 15 species of Clematis. Note: The number before the first "-" in each sequence name corresponds to the number in Table 1. Detailed Implementation

[0045] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0046] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0047] The Clematis stalk described in the following examples is described in the literature “Zheng Weilie, Xing Zhen, Bianba Dorje, et al. Germplasm resources and habitat types of Clematis stalk in Sejila Mountain, Tibet [J]. Journal of Horticulture, 1999, (04): 47-50.”

[0048] The Clematis wuxingensis in the following examples is described in the literature “Liu Zhigao, Shao Weili, Wang Yu, et al. Study on photosynthetic characteristics of Clematis species [J]. Northwest Agriculture Journal, 2016, 25(11):1704-1709.”

[0049] The *Clematis pubescens* in the following examples is described in the literature “Cao Kun, Liu Yaoqi, Yang Longfei, et al. Study on chemical composition of *Clematis pubescens* [J]. Chinese Medicinal Herbs, 2021, 44(04):858-862.”

[0050] The *Clematis chinensis* species used in the following examples are described in the literature “Yu Bing, Yao Zhensheng. Medicinal plant resources of the genus *Clematis* in Zhejiang Province [J]. Jiangxi Science, 2006, (01): 89-92.”

[0051] The Clematis chamaejas in the following examples is described in the literature “Wang Ruolan, Zhang Dequan. Chloroplast genome analysis and phylogenetic analysis of Clematis chamaejas [J]. Journal of Jishou University (Natural Science Edition), 2024, 45(01):66-76.”

[0052] The *Clematis dentata* in the following examples is described in the literature “Yang Bijia, Wu Yulan, Bao Zhijuan, et al. Preliminary study on the propagation conditions of *Clematis dentata* by cuttings [J]. Hubei Agricultural Sciences, 2014, 53(07):1579-1582.”

[0053] The Clematis spp. in the following examples is described in the literature “Liu Jingjing, Gao Yike. Survey and study of germplasm resources of wild Clematis species in Beijing area [J]. Heilongjiang Agricultural Sciences, 2013, (04): 65-69.”

[0054] The Clematis septemlobus in the following examples is described in the literature “Sheng Lu, Yang Yingjie, Ji Kongshu. Callus culture and browning inhibition of Clematis septemlobus [J]. Molecular Plant Breeding, 2015, 13(10):2380-2387.”

[0055] The Clematis breviculae in the following examples are described in the literature “Lin Dong, Feng Chaoyang, Lü Shihai, et al. Study on chlorophyll fluorescence characteristics of Clematis breviculae [J]. Northwest Botanical Journal, 2008, (11): 2299-2305.”

[0056] The Clematis macrocarpa described in the following examples is described in the literature “Yang Yi, Wang Na, Wang Kuiling, et al. Microsporogenesis and gametophyte development of Clematis macrocarpa [J]. Acta Botanica Sinica, 2019, 54(05): 596-605.”

[0057] The Clematis celeryensis in the following examples is described in the literature “Tian Xiaolin. Study on insecticidal active ingredients of Clematis celeryensis [D]. Northwest A&F University, 2019.”.

[0058] The shrub Clematis described in the following examples is described in the literature “Liu Zhijie, Zheng Yan, Li Mingyang, et al. Polyploid induction and identification of shrub Clematis [J]. Forestry and Ecological Science, 2023, 38(03):338-344.”

[0059] The Clematis floridae used in the following examples is described in the literature “Zhao Ke, Xie Hongxia, Zhang Na. Optimization of ultrasonic extraction process of total saponins from Clematis floridae using response surface methodology [J]. Chinese Journal of Traditional Chinese Medicine, 2015, 30(04):1239-1241.”

[0060] The *Clematis pedunculata* in the following examples is described in the literature “Lü Rudan, He Jian, Liu Huijie, et al. Analysis of distribution area and ecological niche model of *Clematis pedunculata* [J]. Journal of Beijing Forestry University, 2019, 41(02):70-79.”

[0061] The *Clematis acerifolia* in the following examples is described in the literature “Huang Chunxiao, Liu Quanru, Du Yuxuan, et al. Population age structure and population dynamics of *Clematis acerifolia*, a rare and endangered plant endemic to the Taihang Mountains [J]. Journal of Ecology, 2023, 42(12):2911-2917.”

[0062] The harvesting license number for the Clematis maple samples in the following examples is 11BJ20230421000063.

[0063] Example 1: Obtaining the InDel site and developing the InDel molecular marker CA25292 I. Assembly of a very long segment of Clematis macrantha Acer palmatum leaves were collected, and DNA was extracted and subjected to quality testing. After passing quality testing, the DNA sequence was fragmented into random fragments using ultrasound. The fragmented DNA underwent end repair, 3′ A addition, and sequencing adapter ligation. Fragments of approximately 300 bp in length were then enriched using magnetic beads, amplified by PCR to form a sequencing library, and subjected to quality testing. After passing quality testing, sequencing was performed using the Illumina NovaSeq™ platform with Illumina PE150 sequencing mode. The total read length was 300 bp, yielding 398G of next-generation sequencing data. The sequences were assembled using SPAdes genome assembler v3.15.2 software. The assembled sequences were filtered according to length, retaining scaffolds longer than 1000 bp as the reference genome ultra-long fragment for this experiment. The accession number of this ultra-long fragment sequence in NCBI is PRJNA1179677.

[0064] II. Simplified Genome Sequencing GBS (Genotyping-By-Sequencing) was performed on the following 15 species of *Clematis*: *Clematis stalkata*, *Clematis wuxingensis*, *Clematis pubescens*, *Clematis zeylanica*, *Clematis davidii*, *Clematis stylanica*, *Clematis short-columnarii*, *Clematis short-tailedii*, *Clematis macrophylla*, *Clematis semi-bellifera*, *Clematis celery-leavedii*, *Clematis shrubbyii*, *Clematis chrysotricha*, *Clematis pinnata*, and *Clematis acerifolia*. The assembled *Clematis acerifolia* reference genome ultra-long fragment was used as a reference for silicon electronic enzyme digestion evaluation and enzyme digestion effect verification to obtain information including enzyme digestion combination, screening fragment size, tag number, and reference genome coverage. The specific steps of GBS sequencing are as follows: Genomic DNA was extracted from the 15 species of *Clematis*, with 3 plants from each species sampled at the *Clematis* Resource Nursery of the Beijing Academy of Landscape Architecture and Forestry Sciences. After passing quality control, 0.1-1 μg of DNA was subjected to double enzyme digestion (MseI + TaqAI) to obtain the GBS library. Following quality control, the GBS library was sequenced using the X-plus platform with PE150 sequencing mode to obtain simplified genome sequencing data. The accession numbers for this simplified genome sequencing data in NCBI are PRJNA1184821 and PRJNA1298512.

[0065] III. Obtaining the InDel site After the sequencing data (Raw Data) is processed, Fastp is used to perform quality control on the data, filtering out low-quality data to obtain high-quality data (Clean Data). The Clean Data is then aligned to the reference genome sequence using BWA-MEME software. The alignment results are evaluated, key parameters are statistically analyzed, and the sequence location is determined (i.e., the BAM file). The alignment results (BAM file) are processed using GATK's Best Practices workflow. InDel detection and filtering are performed using GATK's Haplotyper method, with filtering conditions following GATK's recommended parameters. The obtained InDel data are statistically analyzed. Loci with a depth greater than 4x, a deletion rate less than 30%, and a maf greater than 0.05 are retained. Then, InDel loci are selected using the following method: 3 samples from each of 15 Clematis species are used, resulting in 45 samples divided into two groups (Group A and Group B). Group A consists of 3 data sets from the same species, while Group B consists of 42 data sets from 14 other species. The selection criteria are that the genotypes in Group A are consistent, and the genotypes in Group A and Group B are inconsistent. The present invention obtained an InDel site through screening, which is located at positions 174-176 of sequence 3.

[0066] IV. Design of InDel molecular marker CA25292 For the InDel site obtained in step three, 200 bp sequences were extracted from the genome upstream and downstream. The primer annealing temperature was set to 57-63 ℃, and the InDel molecular marker CA25292 was designed. The primer sequences are as follows: Forward primer: 5'-ATCACCTACTGCAATCCGAAAC-3' (sequence 1); Reverse primer: 5'-CAAAGAAAGCAACAAAGCCAG-3' (sequence 2).

[0067] Example 2: Application of InDel molecular marker CA25292 I. Obtaining the test sample Fifteen species of Clematis were selected as test samples. The number, name, collection location and total number of each species are shown in Table 1.

[0068] Table 1

[0069] II. PCR Amplification and Capillary Electrophoresis Detection 1. Extraction of genomic DNA Genomic DNA was extracted from each test sample and quality testing was performed.

[0070] 2. PCR amplification Using the genomic DNA obtained in step 1 as a template, PCR amplification was performed using the InDel molecular marker CA25292 from Example 1 to obtain the PCR product.

[0071] The PCR amplification system was as follows: 2 μL genomic DNA, 5 μL MIX (Nanjing Winozan Biotechnology Co., Ltd., catalog number p115-02), 2.6 μL water, 0.2 μL forward primer, and 0.2 μL reverse primer. The final concentration of both the forward and reverse primers in the PCR amplification system was 0.2 μmol / L.

[0072] The PCR amplification program is as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 30 s; 35 cycles in total; 72℃ extension for 5 min; store at 25℃.

[0073] 3. Detection of PCR products Take 1 μL of DNA (PCR product) from each well and perform capillary electrophoresis using an ABI 3730xl DNA Analyzer.

[0074] 4. Results Analysis The SSR fingerprint analyzer software SSR analyzer was used to analyze the detection results in FSA format.

[0075] The results are shown in Table 2. As can be seen from Table 2, the InDel molecular marker CA25292 has two band patterns of 96 bp and 93 bp in 15 species of Clematis. However, the 93 bp band pattern is only present in Clematis breviculata and Clematis wuxingensis, while the band pattern in the other 13 species of Clematis is 96 bp.

[0076] Table 2

[0077] Note: The numbers before and after " / " in the table indicate the band size obtained by amplifying the Indel molecular marker CA25292 in the corresponding Clematis stalk. For example, the Indel molecular marker CA25292 amplifies a 96 bp band in Clematis stalk.

[0078] III. First-generation sequencing validation 1. Primer design The PCR products of 15 Clematis species obtained in step two were subjected to first-generation sequencing, which was carried out by Shanghai Sangon Biotech.

[0079] The results showed that the nucleotide sequence of the PCR product with a size of 96 bp is shown in Sequence 4, and the nucleotide sequence of the PCR product with a size of 93 bp is shown in Sequence 5 or Sequence 6.

[0080] 2. Sequence Analysis After first-generation sequencing was completed, the results were compared and analyzed using Mega11 software. Sequence analysis showed that the InDel site (positions 174-176 of sequence 3) was deleted only in Clematis brevicornu and Clematis wuxingensis, while the InDel site was not deleted in the other 13 species of Clematis. Figure 2 Therefore, in practical applications, the InDel molecular marker CA25292 can be used to distinguish Clematis brevicula and Clematis wuxingensis from other Clematis species by detecting whether the plant being tested lacks the InDel site.

[0081] Furthermore, sequencing revealed that the base following the InDel site in *Clematis short-column* is G, while the base following the InDel site in *Clematis wuxingensis* is T. Figure 2 In practical applications, the sequence differences of PCR products can be further used to distinguish between Clematis short-column and Clematis wuxingensis, thus enabling the identification of Clematis short-column and Clematis wuxingensis.

[0082] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. Primer pair, wherein the primer pair consists of a forward primer and a reverse primer; The forward primer is either a1) or a2) as follows. a1) The single-stranded DNA molecule shown in sequence 1; a2) A single-stranded DNA molecule that has undergone substitution and / or deletion and / or addition of one or more nucleotides of sequence 1 and has the same function as sequence 1; The reverse primer is either a3) or a4) as follows: a3) The single-stranded DNA molecule shown in sequence 2; a4) A single-stranded DNA molecule that has one or more nucleotides of sequence 2 replaced and / or deleted and / or added, and has the same function as sequence 2.

2. The primer pair according to claim 1, characterized in that: The molar ratio of the forward primer to the reverse primer is 1:

1.

3. The use of the primer pair according to claim 1 or 2 in any of the following b1)-b8): b1) Distinguish between *Clematis wuxingensis* and other plants in the *Clematis* genus; b2) Prepare products that distinguish *Clematis wuxingensis* from other *Clematis* species; b3) Distinguish Clematis septemlobus from other Clematis species; b4) Prepare products that distinguish Clematis stylarii from other Clematis species; b5) Identification of Clematis wuxingensis; b6) Preparation and identification of products containing Clematis wuxingensis; b7) Identification of Clematis stubularis; b8) Prepare and identify products for the identification of short-columnar clematis.

4. A kit containing the primer pair of claim 1 or 2; the kit having the function of any one of c1)-c4): c1) Distinguish between *Clematis wuxingensis* and other plants in the *Clematis* genus; c2) Distinguish *Clematis styracifolium* from other *Clematis* species; c3) Identification of Clematis wuxingensis; c4) Identification of short-columned clematis.

5. Application of InDel sites or substances that detect InDel sites in any of the following b1)-b8): b1) Distinguish between *Clematis wuxingensis* and other plants in the *Clematis* genus; b2) Prepare products that distinguish *Clematis wuxingensis* from other *Clematis* species; b3) Distinguish Clematis septemlobus from other Clematis species; b4) Prepare products that distinguish Clematis stylarii from other Clematis species; b5) Identification of Clematis wuxingensis; b6) Preparation and identification of products containing Clematis wuxingensis; b7) Identification of Clematis stubularis; b8) Prepare and identify products for the identification of short-columnar clematis.

6. A method for distinguishing *Clematis wuxingensis* from other *Clematis* species, the method comprising the following steps: detecting whether the plant to be tested lacks the InDel site, and distinguishing *Clematis wuxingensis* from other *Clematis* species based on whether the plant to be tested lacks the InDel site; wherein the InDel site is located at positions 174-176 of sequence 3.

7. A method for distinguishing Clematis brevichorata from other Clematis species, the method comprising the following steps: detecting whether the plant to be tested lacks the InDel site, and distinguishing Clematis brevichorata from other Clematis species based on whether the plant to be tested lacks the InDel site; wherein the InDel site is located at positions 174-176 of sequence 3.

8. The application according to claim 3, the kit according to claim 4, the application according to claim 5, or the method according to claim 6 or 7, characterized in that: The other Clematis species mentioned are at least one of the following Clematis species: Clematis stalkae, Clematis zeylanica, Clematis russula, Clematis coarse-toothed, Clematis halostae, Clematis short-tailed, Clematis macrophylla, Clematis celery-leaved, Clematis shrubby, Clematis chrysantha, Clematis pubescens, Clematis pinnata, Clematis acerifolia, and Clematis pubescens.

9. A method for identifying Clematis wuxingensis, the method comprising the following steps: using the genomic DNA of the plant to be tested as a template, performing PCR amplification using the primer pair described in claim 1 or 2 to obtain PCR products; and identifying whether the plant to be tested is Clematis wuxingensis based on the size and sequence of the PCR products.

10. A method for identifying Clematis brevichorata, the method comprising the following steps: using the genomic DNA of the plant to be tested as a template, performing PCR amplification using the primer pair described in claim 1 or 2 to obtain PCR products; and identifying whether the plant to be tested is Clematis brevichorata based on the size and sequence of the PCR products.

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