SNP marker-based DNA fingerprinting of rhizoma phrymarium, gene chip and application

Abstract

The application discloses a SNP marker-based genome DNA fingerprint of Herba Phrymatis, a gene chip and application thereof, and belongs to the field of molecular biology. The application obtains SNP of a genome of Herba Phrymatis based on GBS, identifies 248 core SNP sites, and develops a SNP liquid-phase gene chip through a targeted capture sequencing technology, so that the sites uniformly cover a chromosome set, are representative, have high polymorphism, high specificity, and strong universality, are suitable for being applied to identification of Herba Phrymatis varieties, provide scientific and technological support for Herba Phrymatis variety management and quality detection, and lay a technical foundation for Herba Phrymatis germplasm resource analysis.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology, specifically relating to a DNA fingerprinting of Nardostachys jatamansi based on SNP markers, a gene chip, and its applications. Background Technology

[0002] Nardostachys jatamansi is a plant belonging to the Valerianaceae family. Nardostachys jatamansi The dried root and rhizome of DC. are a medicinal material, which has been included in the Chinese Pharmacopoeia for many years. It has a pungent and sweet taste and is warm in nature. It has the effects of regulating qi and relieving pain, relieving depression and invigorating the spleen, and removing dampness and reducing swelling when used externally. It has significant effects on treating abdominal distension, vomiting, loss of appetite and other symptoms. At the same time, Nardostachys jatamansi is also one of the raw materials for Tibetan incense.

[0003] Nardostachys jatamansi, a perennial herb with significant ecological, medicinal, industrial, agricultural, and potential landscape value, possesses considerable market potential. However, current reliance on harvesting wild Nardostachys jatamansi is insufficient to meet sustainable needs, leading to the continuous depletion of natural resources. Nardostachys jatamansi has been listed as a critically endangered species by the International Union for Conservation of Nature (IUCN) and is included in Appendix II of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). Therefore, developing high-yielding and high-quality varieties and compatible cultivation techniques has become a crucial pillar for the sustainable development of this industry. Simultaneously, molecular identification of varieties is an essential requirement for the healthy development of the industry, making the establishment of technical standards for Nardostachys jatamansi variety identification absolutely necessary.

[0004] Early studies suggested that the genus *Nardostachys* consisted of three species, including *Nardostachys macrocarpa* (…). Nardostachys grandiflora ), spikenard ( Nardostachys chinensis ) and Nardostachys chinensis ( Nardostachys jatamansi In my country, only two species of *Nardostachys chinensis* are found: *Nardostachys japonica* and *Nardostachys spicata*. Most current research considers them to be the same plant. Morphological studies have revealed that *Nardostachys chinensis* has smooth seeds, oblanceolate leaves, and sheet-like leaf sheath remnants that are dark brown or darker; while *Nardostachys spicata* has pubescent seeds, long spoon-shaped leaves, and fibrous leaf sheath remnants that are yellowish-brown or lighter. Therefore, classifying and evaluating *Nardostachys chinensis* resources at the molecular level is of great significance for clarifying the germplasm classification and resource evaluation of *Nardostachys chinensis* in China.

[0005] In the field of variety identification, classification based on morphology, cytology, or molecular markers is often insufficient to resolve classification disputes concerning species boundaries and closely related groups. SNPs, as the most widely distributed genetic markers in the genome, possess high density, high genetic stability, and ease of automated analysis, making efficient and low-cost SNP genotyping technology the preferred choice for shared technology and platform development. Currently, gene chip methods are the most commonly used for SNP marker genotyping, including SNP solid-phase chips and SNP liquid-phase chips. High-throughput SNP liquid-phase chips are important tools for conducting molecular breeding research such as high-throughput genotyping of crops, germplasm resource identification, genuine and counterfeit seed identification, and fingerprinting. SNP molecular marker identification of crop varieties has become one of the important bases for new variety protection.

[0006] GBS (Genome Bismuth Sequencing) is a restriction endonuclease-based genome sequencing method that efficiently and economically discovers genome-wide SNPs without the need for a reference genome. This makes GBS particularly suitable for non-type endangered plants lacking genomic resources, such as *Nardostachys japonica*. DNA fingerprinting has proven effective in accurately distinguishing species and varieties, supporting conservation efforts, and promoting breeding programs. Currently, no related technologies have been found for *Nardostachys japonica* variety identification, both domestically and internationally. Therefore, this invention provides a method for obtaining *Nardostachys japonica* genome SNPs through GBS and constructing a *Nardostachys japonica* DNA fingerprint map based on SNP markers. This provides scientific and technological support for *Nardostachys japonica* variety management and quality testing, and lays a technical foundation for *Nardostachys japonica* germplasm resource analysis. Summary of the Invention

[0007] This invention obtains SNPs from the *Pinus chinensis* genome based on GBS, identifying 248 core SNP loci. A liquid-phase SNP gene chip was developed using targeted capture sequencing technology. The loci uniformly cover the chromosome set, exhibiting high representativeness, high polymorphism, high specificity, and strong versatility, making it suitable for *Pinus chinensis* variety identification. The research results of this invention are of great significance for the protection and utilization of *Pinus chinensis* germplasm resources, laying the foundation for genetic research on *Pinus chinensis* and potentially guiding breeding programs to improve the resilience and adaptability of *Pinus chinensis* species. Simultaneously, this invention constructs a *Pinus chinensis* DNA fingerprint based on SNP markers, providing scientific and technological support for *Pinus chinensis* variety management and quality testing, and laying the technical foundation for *Pinus chinensis* germplasm resource analysis.

[0008] In a first aspect, the present invention provides the use of a reagent for detecting SNP sites in at least one of the following:

[0009] 1) Application in the identification of Nardostachys jatamansi varieties;

[0010] 2) Application in the preparation of gene chips for variety identification of Nardostachys jatamansi;

[0011] 3) Application in the genetic selection and breeding of Nardostachys jatamansi;

[0012] 4) Application in the construction of Nardostachys jatamansi fingerprint maps;

[0013] 5) Application in the genetic diversity analysis of Nardostachys jatamansi;

[0014] The characteristic feature is that the SNP site is selected from the combination of SNP sites located at CNP0006696 and CNA0504869 in the reference genome as shown in the table below:

[0015] Table 1. Combinations of SNP sites in Nardostachys jatamansi

[0016]

[0017]

[0018]

[0019] The reference genome described in this invention comes from the China National GeneBank Sequence Archive (CNSA, https: / / db.cngb.org / cnsa), with accession numbers CNP0006696 and CNA0504869.

[0020] The reagents include, but are not limited to, primers and / or probes, SNP solid-phase chips, or SNP liquid-phase chips.

[0021] In a specific embodiment of the present invention, the above-mentioned SNP sites are obtained by screening using the following method:

[0022] (1) Extract genomic DNA from the spikenard sample, test the quality of the DNA sample, and digest the DNA with restriction endonuclease;

[0023] (2) The enzyme fragments obtained in step (1) are ligated to the sequencing adapter, PCR amplification is performed, the samples are mixed, the fragments are recovered, and a GBS library is constructed. The library is quality checked, and sequencing is performed after the quality check is passed.

[0024] (3) The high-quality clean data obtained from GBS genome sequencing in step (2) are clustered using Stacks software to detect and screen SNPs, and high-quality SNP sites are selected for subsequent analysis.

[0025] (4) The SNP sites selected in step (3) are further screened to obtain 248 core SNP sites.

[0026] In step (3), SNP detection is performed using the cstacks, sstacks, tsv2bam, and gstacks programs to complete genome assembly and mutation detection.

[0027] The screening in step (3) uses vcftools software to screen the SNP typing results. The screening conditions are as follows: (1) missing rate <0.2; (2) MAF≥0.01; DP>4.

[0028] In step (4), the criteria for further screening of high-quality SNP sites are MAF > 0.1 and PIC > 0.3.

[0029] In a second aspect of the present invention, the present invention provides a Nardostachys jatamansi SNP liquid phase chip, the SNP liquid phase chip being used for variety identification of Nardostachys jatamansi, characterized in that the gene chip includes an SNP site probe array, the SNP site probe array being used to identify the genotype of the SNP site described in the first aspect of the present invention.

[0030] In a specific embodiment of the present invention, the SNP site probe assembly is single-stranded DNA synthesized from the SNP sites described in the first aspect of the present invention. Further, the 5' end of the probe has a biotinylate group, and the probe is coupled to fluorescent microspheres via a C12 molecular arm and amino modification, with each fluorescent microsphere coupled to one probe.

[0031] The spikenard SNP liquid-phase chip of the present invention is prepared by the following method:

[0032] (1) Using the SNP site described in the first aspect of the present invention as the center, select the 120bp nucleotide sequence with the GC content closest to 45% in the 120bp to its left and right as the probe;

[0033] (2) Based on the 120bp nucleotide sequence obtained in step (1), synthesize a single-stranded DNA with a biotinylated group at the 5' end to obtain an SNP site probe;

[0034] (3) The SNP site probe prepared in step (2) is coupled to the fluorescent microspheres, and a targeting capture reagent is added for targeting capture to obtain the Gan Song SNP liquid phase chip.

[0035] The design principles of the SNP site probes described in this invention are as follows:

[0036] The probe length is 120bp, the probe GC content is between 25-49%, the average GC content is about 45%, the number of homology regions is ≤3, and the selected regions do not include SSR and GAP regions to the greatest extent possible; and the priority is: gene region > gene promoter region (2Kb) > gene downstream adjacent region (2Kb) > gene intergenic region, combined with the principle of uniform chromosome distribution.

[0037] In a third aspect of the present invention, the present invention provides a DNA fingerprint of Nardostachys jatamansi, the DNA fingerprint including the 248 SNP sites described in the first aspect of the present invention.

[0038] In a fourth aspect, the present invention provides a method for constructing a DNA fingerprint of Nardostachys jatamansi, comprising the following steps:

[0039] (1) Extract genomic DNA from the spikenard sample, test the quality of the DNA sample, and digest the DNA with restriction endonuclease;

[0040] (2) The enzyme fragments obtained in step (1) are ligated to the sequencing adapter, PCR amplification is performed, the samples are mixed, the fragments are recovered, and a GBS library is constructed. The library is quality checked, and sequencing is performed after the quality check is passed.

[0041] (3) The high-quality clean data obtained from GBS genome sequencing in step (2) were clustered using Stacks software to obtain clean reads. SNPs were constructed and mutations were detected using cstacks, sstacks, tsv2bam, and gstacks programs. Then, the SNP genotyping results were screened using vcftools software. The screening conditions were: deletion rate <0.2; MAF ≥0.01; DP >4. High-quality SNP sites were selected for subsequent analysis.

[0042] (4) The SNP sites selected in step (3) are further screened. The screening criteria are MAF > 0.1 and PIC > 0.3, resulting in 248 core SNP sites. The DNA fingerprint of *Gnaphalium affine* is successfully constructed.

[0043] In a fifth aspect, the present invention provides a method for identifying Nardostachys jatamansi varieties, comprising the following steps:

[0044] (1) Extract genomic DNA from the sample of Nardostachys jatamansi to be tested, test the quality of the DNA sample, and digest the DNA with restriction endonuclease;

[0045] (2) The enzyme fragments obtained in step (1) are ligated to the sequencing adapter, and PCR amplification, sample pooling, and fragment recovery are performed to construct a GBS library. The library is then subjected to quality control.

[0046] (3) Perform probe hybridization reaction between the GBS library that passed the quality inspection in step (2) and the Gan Song SNP liquid phase chip described in the second aspect of the present invention;

[0047] (4) Extract the genotyping information from the captured sequence after sequencing to form a genotyping file;

[0048] (5) Compare the genotyping file of the sample to be tested with the DNA fingerprint of *Nardostachys jatamansi* to determine that the sample to be tested is *Nardostachys jatamansi*. N. chinensis ) or Nardostachys chinensis ( N. jatamansi ).

[0049] Specifically, the DNA fingerprint of *Gnaphalium affine* provided by this invention has two characteristics: (1) SNP locus characteristics presented as a whole of the ecological populations of GJF, GJL1, GJL2, QHN1, QHN2, QHN3, QZC1, QZC2, SAB1, SAB2, SAR1, and SAR2, which are named *Gnaphalium affine* (Gnaphalium affine). N. chinensis (2) The SNP locus characteristics presented as a whole by the ecological populations of SMP, SMT1, SMT2, SMT3, XDK, XDY, YDX, YKD and YNG are named Spoonleaf Nardostachys ( N. jatamansi )-shaped SNP site characteristics.

[0050] Judgment criteria: If the genotype pattern of the sample to be tested is similar to that of spikenard (Nardostachys jatamansi) N. chinensis When the similarity of the SNP loci in the () shape is >88.00%, then the sample belongs to the genus *Nardostachys jatamansi*. N. chinensis If the genotype pattern of the sample to be tested matches that of Nardostachys chinensis in the fingerprint pattern (…), then… N. jatamansi When the similarity of the SNP loci in the shaped pattern is >88.00%, then the sample belongs to *Nardostachys chinensis* (spoonleaf pine). N. jatamansi ).

[0051] The technical solution provided by this invention has the following beneficial contributions:

[0052] As an endangered plant with significant medicinal, industrial, ecological, and horticultural applications, the investigation, classification, and assessment of its species diversity are crucial. Selecting suitable core SNP markers for identifying *Nardostachys chinensis* populations is key to constructing a *Nardostachys chinensis* DNA fingerprint. The marker selection criteria vary depending on population size and genotype, resulting in different numbers of core SNP markers. This invention sets selection criteria based on SNP loci in 21 *Nardostachys chinensis* populations, ultimately obtaining a marker set of 248 core SNP loci to establish a species-specific fingerprint. This core SNP marker set exhibits strong representativeness and discriminative power, providing a scientific basis for promoting the application of SNP molecular markers in the precise and efficient identification of *Nardostachys chinensis* germplasm resources. Attached Figure Description

[0053] Figure 1 The number of SNPs for each mutation type.

[0054] Figure 2Population genetic analysis of Nardostachys jatamansi based on SNP loci, MAF(A) and PIC(B).

[0055] Figure 3 Phylogenetic tree of 126 Nardostachys jatamansi samples.

[0056] Figure 4 Fingerprint patterns of 126 Nardostachys jatamansi samples. Detailed Implementation

[0057] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0058] Example 1: Construction of Nardostachys jatamansi DNA fingerprint

[0059] (1) Plant materials

[0060] This study collected 126 *Nardostachys japonica* samples from five provinces in China (Gansu, Qinghai, Sichuan, Tibet, and Yunnan), with six replicates collected from each sample. Each collection site can be considered an ecological population, and the collected samples were named according to their collection sites as follows: GJL1, GJL2, GJF, QHN1, QHN2, QHN3, QZC1, QZC2, SAR1, SAR2, SAB1, SAB2, SMT1, SMT2, SMT3, SMP, XDY, XDK, YDX, YKD, and YNG. Collection information for the sample materials is shown in Table 2. Furthermore, the samples were classified into *Nardostachys japonica* (Gansu, Qinghai, Sichuan, Tibet, and Yunnan) according to current plant ecological classification methods. N. chinensis ) and Nardostachys chinensis ( N. jatamansi ).

[0061] Table 2. Sample collection information for Nardostachys jatamansi.

[0062]

[0063] (2) GBS library construction and sequencing

[0064] DNA was extracted from the *Nardostachys jatamansi* samples using the DNAsecure Plant Kit (Tiangen Biotech, Beijing, CHN). After DNA extraction and quality testing, sequencing libraries were constructed using SuperGBS technology. The library construction process is as follows:

[0065] DNA was digested using PstI-HF / MspI enzyme; the digested fragments were ligated at both ends using T4 ligase and barcodes; a modified magnetic bead recovery system was used to recover fragments of 300-700 bp by adjusting the volume ratio of magnetic bead solution to product; the recovered fragments were amplified by PCR using a high-fidelity enzyme, and the PCR product concentration was determined using the quantum bit method, with a concentration greater than 5 ng / μl to ensure library quality; after GBS library quality control, different libraries were mixed according to the effective concentration and target data volume requirements, and the mixed libraries were sequenced (Illumina Nova, PE150). Sequencing was performed by Shanghai Ouyi Biomedical Technology Co., Ltd.

[0066] (3) SNP detection and labeling

[0067] The high-quality Clean Data obtained from filtering the genome sequencing data in step (2) was used to cluster the sequencing reads (filtered high-quality Clean Reads obtained by splicing Clean Data) of each sample using the ustacks program in Stacks software. Then, the genome assembly and mutation detection were completed using programs such as cstacks, sstacks, tsv2bam, and gstacks. The obtained SNP genotyping results were screened using vcftools software. The screening criteria were as follows: excluding amorphous sites; read support number (DP) not less than 4; removing sites with MAF less than 0.01; and removing sites with SNP genotyping deletion rate higher than 20%.

[0068] SNPs developed from populations composed of non-genetic population materials are characterized by high marker quality, strong representativeness, high marker (combination) discriminability, uniform distribution across the genome, and high specificity. Loci with a depth of less than 8 are considered deletions, and only the second allele SNP is retained. The obtained SNPs are defined as the total SNPs of the sample. Genotypes of multiple samples from each variety are compared. If the deletion rate of a certain SNP locus in a variety is ≤0.0% and the genotype consistency rate is ≥100.0%, then that genotype is used as the genotype of that variety at that SNP locus. Otherwise, the missing data is used to represent the genotype of that variety at that SNP locus. Loci without polymorphism are removed; loci with a deletion rate higher than 100.0% are removed. Loci with a second allele proportion lower than 1.0% are excluded. Loci with a p-value lower than 0.01 in the Hardy-Weinberg test are excluded, as are loci with a polymorphism information content (PIC) lower than 0.30. Linkage filtering: The loci were delinked using Plink software (v 1.9) with parameters of indep-pairwise 50 10 0.95. The SNPs identified after filtering according to the above conditions were taken as the core SNPs of the variety.

[0069] (4) GBS sequencing to identify SNPs and screen core SNP sites

[0070] GBS sequencing of 126 *Nardostachys jatamansi* samples from 21 populations yielded 689.4 million raw reads. After quality filtering, 672 million clean reads were retained, with an average validity of 97.08%, indicating high sequencing quality (average Q20: 96.45%, average Q30: 90.47%) and an average GC content of 41.67%. The average sequencing depth was 42.83×, and the GBS library contained 1,152,811 tags. SNP sites in each population were screened according to the following criteria: (1) deletion rate <0.2; (2) MAF ≥0.01; DP >4. After rigorous screening, 24,356 high-quality SNPs were identified for subsequent analysis.

[0071] Analysis of predicted mutation types for the 24,356 SNPs showed that among the six possible single-base mutations, CT / TC and AG / GA mutations were the most frequent, accounting for 25.93% and 25.48% of the total, respectively. CG / GC mutations were the least frequent, accounting for only 8.21%. Of the 24,356 SNPs, 12,520 were transition mutations and 11,836 were transversion mutations. The ratio of transition to transversion was 1.06 (…). Figure 1 ).

[0072] Based on the core site screening method, 17,975 SNPs with MAF values ​​less than 0.1 were removed. Figure 2 A), removing 23,162 SNPs with a PIC value less than 0.3 ( Figure 2 B). Combining other screening indicators, 248 core SNP sites were finally identified (Table 1) for further analysis.

[0073] Develop fingerprinting to distinguish Nardostachys jatamansi ( N. chinensis ) and Nardostachys chinensis ( N. jatamansi )

[0074] DNA fingerprinting is based on nucleotide polymorphisms at multiple molecular marker sites distributed throughout the genome. DNA fingerprinting is a powerful tool for identifying varieties and strains, and is also highly suitable for identifying plant germplasm resources. To meet the needs of large-scale detection and fingerprint database construction, it is necessary to develop and screen a large number of SNP markers to achieve ideal variety identification capabilities. The selection of core fingerprint markers depends on the complexity of the species' genome, the marker type, the marker detection technology, and the number of varieties.

[0075] Based on the 248 core SNP loci selected above, a phylogenetic tree of 126 *Nardostachys japonica* samples was constructed using the nearest neighbor method. Using appropriate cluster analysis, the 21 populations corresponding to the 126 *Nardostachys japonica* samples were clearly divided into two categories. Figure 3 The first category includes GJF, GJL1, GJL2, QHN1, QHN2, QHN3, QZC1, QZC2, SAB1, SAB2, SAR1, and SAR2. These 12 ecological populations of *Nardostachys chinensis* belong to the *Nardostachys chinensis* ( N. chinensis The second category includes nine species of Nardostachys japonica: SMP, SMT1, SMT2, SMT3, XDK, XDY, YDX, YKD, and YNG. These nine ecological populations belong to the Nardostachys chinensis species. N. jatamansi )variety.

[0076] Simultaneously, using Microsoft Excel to plot data, each row represents a SNP locus, and each column represents a sample. Based on the obtained 248 core SNP loci, DNA fingerprinting patterns were constructed for 126 *Nardostachys japonica* samples. Figure 4 In the figure, each small square represents the type of variation at that SNP site. Red, light blue, green, and yellow represent A, T, C, and G, respectively, while dark blue is marked N, indicating no SNP variation at that location. This DNA fingerprint is compared with the classification results of the phylogenetic tree. Figure 3 The high consistency further verifies the effectiveness of the core SNP sites provided by this invention in distinguishing Nardostachys jatamansi varieties molecularly, enabling the differentiation of Nardostachys jatamansi (…). N. chinensis ) and Nardostachys chinensis ( N. jatamansi Precise molecular identification of two varieties. For Nardostachys genus test materials, the overall predictive accuracy of the DNA fingerprint provided by this invention is 100%.

[0077] according to Figure 4 From the fingerprint pattern shown, we can clearly see that the SNP locus characteristics of 21 ecological populations (126 samples) present two situations: (1) The SNP locus characteristics presented as a whole for the ecological populations GJF, GJL1, GJL2, QHN1, QHN2, QHN3, QZC1, QZC2, SAB1, SAB2, SAR1 and SAR2 are named Gan Song (Gan Song) N. chinensis (2) The SNP locus characteristics presented as a whole by the ecological populations of SMP, SMT1, SMT2, SMT3, XDK, XDY, YDX, YKD and YNG are named Spoonleaf Nardostachys (SMP). N. jatamansi )-shaped SNP site characteristics.

[0078] The detection and species determination of unknown test samples can be performed as follows: Detect the 248 core SNP loci of the test sample to obtain the genotyping file, and then compare it with... Figure 4 Comparing the DNA fingerprints of the sample shown, if the genotype pattern of the sample to be tested matches that of Nardostachys japonica (…), the genotype pattern of the sample to be tested will match that of Nardostachys japonica (…). N. chinensis When the similarity of the SNP loci in the () shape is >88.00%, then the sample belongs to the genus *Nardostachys jatamansi*. N. chinensis If the genotype pattern of the sample to be tested matches that of Nardostachys chinensis in the fingerprint pattern (…), then… N. jatamansi When the similarity of the SNP loci in the shaped pattern is >88.00%, then the sample belongs to *Nardostachys chinensis* (spoonleaf pine). N. jatamansi ).

[0079] The above specific embodiments are merely illustrative of the invention and do not represent a limitation thereof. Those skilled in the art will recognize that other variations of the specific structure of this invention are possible.

Claims

1. The use of a reagent for detecting SNP sites in at least one of the following: 1) Application in the identification of Nardostachys jatamansi varieties; 2) Application in the preparation of gene chips for the identification of Nardostachys jatamansi varieties; 3) Application in the genetic selection and breeding of Nardostachys jatamansi; 4) Application in the construction of Nardostachys jatamansi fingerprint maps; 5) Application in the genetic diversity analysis of Nardostachys jatamansi; Its features are, The SNP sites consist of the SNP sites located at reference genome CNP0006696 and CNA0504869, as shown in the table below: The reagents include primers and probes; the gene chip includes an SNP solid-phase chip or an SNP liquid-phase chip; and the *Nardostachys japonica* variety is *Nardostachys japonica* (…). N. chinensis ) or Nardostachys chinensis ( N. jatamansi ).

2. A SNP liquid phase chip for identifying the variety of *Nardostachys chinensis*, wherein the *Nardostachys chinensis* is *Nardostachys chinensis* (…). N. chinensis ) or Nardostachys chinensis ( N. jatamansi ), characterized in that, The gene chip includes an SNP site probe array, which is used to identify the genotype of the SNP site described in claim 1.

3. The Nardostachys jatamansi SNP liquid-phase chip according to claim 2, characterized in that, The SNP site probe assembly is a single-stranded DNA synthesized from the SNP sites as described in claim 1.

4. The Nardostachys jatamansi SNP liquid-phase chip according to claim 3, characterized in that, The spikenard SNP liquid phase chip was prepared by the following method: (1) Using the SNP site described in claim 1 as the center, select the 120bp nucleotide sequence with the GC content closest to 45% in the 120bp to its left and right as the probe; (2) Based on the 120bp nucleotide sequence obtained in step (1), synthesize a single-stranded DNA with a biotinylated group at the 5' end to obtain an SNP site probe; (3) The SNP site probes prepared in step (2) are coupled to fluorescent microspheres for targeted capture to obtain the Gan Song SNP liquid phase chip.

5. A method for identifying varieties of Nardostachys jatamansi, comprising the following steps: (1) Extract genomic DNA from the sample of Nardostachys jatamansi to be tested, test the quality of the DNA sample, and digest the DNA with restriction endonuclease; (2) The enzyme fragments obtained in step (1) are ligated to the sequencing adapter, and PCR amplification, sample pooling, and fragment recovery are performed to construct a GBS library. The library is then subjected to quality control. (3) Perform probe hybridization reaction between the GBS library that passed the quality inspection in step (2) and the Gan Song SNP liquid phase chip as described in claim 2; (4) Extract the genotyping information from the captured sequence after sequencing to form a genotyping file; (5) Compare the genotyping file of the sample to be tested with the DNA fingerprint of *Nardostachys jatamansi* to determine that the sample to be tested is *Nardostachys jatamansi*. N. chinensis ) or Nardostachys chinensis ( N. jatamansi The spikenard DNA fingerprint contains the 248 SNP sites described in claim 1.

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