Microsatellite molecular marker, specific primer of chrysanthemum, preparation method and application thereof

Abstract

The application belongs to the technical field of biology and provides a molecular marker of microsatellite of Flourensia, specific primers, a preparation method and application. The application develops the molecular marker of microsatellite of Flourensia by using a restriction enzyme site related DNA genome scanning technology and obtains corresponding primers, establishes a method for preparing the molecular marker of microsatellite of Flourensia, and provides the molecular marker for researches on Flourensia germplasm resource identification, genetic diversity analysis, plant genetic map construction, population genetic structure analysis, gene positioning, quantitative trait gene analysis, evolution and genetic relationship, and lays a foundation for genetic researches on Flourensia germplasm resource genetic diversity, population structure and population historical dynamics, and is favorable for reasonable development of Flourensia resources.

Description

Technical Field

[0001] This application belongs to the field of biological technology, and in particular relates to microsatellite molecular markers, specific primers, preparation methods and applications of Chrysanthemum indicum. Background Technology

[0002] Yellow chrysanthemum (Xanthopappus subacaulis), also known as yellow crown chrysanthemum, is a perennial herb belonging to the genus Xanthopappus in the family Asteraceae. It typically grows in alpine meadows, grasslands, and dry mountain slopes at altitudes of 2230–4150 m, exhibiting strong cold, drought, and salt tolerance. It is mainly distributed in northwestern Yunnan, northern and western Sichuan, western Qinghai, and southeastern Gansu in my country, and is a monotypic species endemic to the central and eastern Qinghai-Tibet Plateau. The whole plant of Xanthopappus is used medicinally, possessing cooling and hemostatic effects. It is effective in treating hematemesis, uterine bleeding, food poisoning, and allergic purpura, demonstrating high medicinal value. Developing appropriate SSR molecular markers can provide effective technical means for genetic research on the genetic diversity, population structure, and population history dynamics of Xanthopappus, laying the foundation for the rational development of Xanthopappus resources in the future.

[0003] SSRs (simple sequence repeats) are widely distributed in eukaryotic genomes. Molecular markers based on specific primer PCR, also known as microsatellite DNA, are typically tandem repeat sequences composed of 1-6 nucleotide repeat units, such as (AC)n, (GA)n, (AT)n, (AAG)n, and (AAT)n, where n represents the number of repeats, ranging from a few to dozens. Their length is generally between 100 and 200 bp. The number of repeats among different alleles exhibits high variability, manifesting as integer fold variations in the number of microsatellites or sequence differences within repeat units, thus demonstrating SSR polymorphism. SSR markers possess advantages such as wide distribution, specificity, co-dominance, and ease of operation, making them widely used in plant molecular marker research.

[0004] However, current technologies do not cover the use of SSR molecular markers for genetic research on *Chrysanthemum indicum*. Therefore, developing SSR microsatellite molecular markers could provide an effective technical means for conducting genetic research on the genetic diversity, population structure, and population history dynamics of *Chrysanthemum indicum* germplasm resources. Summary of the Invention

[0005] The purpose of this application is to provide microsatellite molecular markers for Chrysanthemum indicum, aiming to fill the gap in current technology that does not involve the use of SSR molecular markers for genetic research on Chrysanthemum indicum.

[0006] The embodiments of this application are implemented as follows: a microsatellite molecular marker for *Chloranthus chinensis*, wherein the nucleotide sequence of the microsatellite molecular marker for *Chloranthus chinensis* is shown in one or more of SEQ.ID.No.1-SEQ.ID.No.15.

[0007] Another objective of this application is to provide specific primers for microsatellite molecular markers of Chrysanthemum indicum, wherein the specific primers are one or more of the following: T22-4-1, T31-4-1, T48-4-1, T60-4-1, T2-5-1, T25-5-1, T29-5-1, T42-5-1, T51-5-1, T5-6-1, T9-6-1, T34-6-1, T36-6-1, T56-6-1, and T59-6-1.

[0008] Further, the forward primer sequence of T22-4-1 is shown in SEQ.ID.No.16; the reverse primer sequence of T22-4-1 is shown in SEQ.ID.No.17; the forward primer sequence of T31-4-1 is shown in SEQ.ID.No.18; the reverse primer sequence of T31-4-1 is shown in SEQ.ID.No.19; the forward primer sequence of T48-4-1 is shown in SEQ.ID.No.20; the reverse primer sequence of T48-4-1 is shown in SEQ.ID.No.21; and the forward primer sequence of T60-4-1 is shown in SE. The reverse primer sequence of T60-4-1 is shown in SEQ ID No. 22; the forward primer sequence of T2-5-1 is shown in SEQ ID No. 24; the reverse primer sequence of T2-5-1 is shown in SEQ ID No. 25; the forward primer sequence of T25-5-1 is shown in SEQ ID No. 26; the reverse primer sequence of T25-5-1 is shown in SEQ ID No. 27; the forward primer sequence of T29-5-1 is shown in SEQ ID No. 28; the reverse primer sequence of T29-5-1 is shown in SEQ ID No. 29. The forward primer sequence of T42-5-1 is shown in Q.ID.No.29; the reverse primer sequence of T42-5-1 is shown in SEQ.ID.No.30; the forward primer sequence of T42-5-1 is shown in SEQ.ID.No.31; the forward primer sequence of T51-5-1 is shown in SEQ.ID.No.32; the reverse primer sequence of T51-5-1 is shown in SEQ.ID.No.33; the forward primer sequence of T5-6-1 is shown in SEQ.ID.No.34; the reverse primer sequence of T5-6-1 is shown in SEQ.ID.No.35; the forward primer sequence of T9-6-1 is shown in SE. The reverse primer sequence of T9-6-1 is shown in SEQ ID No. 37; the forward primer sequence of T34-6-1 is shown in SEQ ID No. 38; the reverse primer sequence of T34-6-1 is shown in SEQ ID No. 39; the forward primer sequence of T36-6-1 is shown in SEQ ID No. 40; the reverse primer sequence of T36-6-1 is shown in SEQ ID No. 41; the forward primer sequence of T56-6-1 is shown in SEQ ID No. 42; the reverse primer sequence of T56-6-1 is shown in SEQ ID No. 43; the forward primer sequence of T59-6-1 is shown in SEQ ID No. 44; and the reverse primer sequence of T59-6-1 is shown in SEQ ID No. 45.

[0009] Another objective of this application is a method for preparing microsatellite molecular markers for Chrysanthemum indicum, comprising:

[0010] RNA extraction and quality testing: RNA was extracted from the samples, and the total RNA quality was tested using 1% agarose gel electrophoresis, Nanodrop 2000, and Agilent 2100.

[0011] Construction of cDNA library: Total RNA was reverse transcribed into cDNA, a portion of the cDNA was used for fragment screening with BluePippin to enrich fragments larger than 4kb, and the screened fragments were amplified by PCR. Then the full-length cDNA was purified, end-repaired and adapters were ligated to obtain cDNA library.

[0012] Quality checks of cDNA libraries: Quantification was performed using a Qubit 2.0 fluorometer, and then the library size was checked using an Agilent 2100 to ensure the quality of the sequencing library;

[0013] Transcriptome sequencing: Full-length transcriptome sequencing analysis was performed using SMRT single-molecule real-time sequencing technology on the PacBio Sequel sequencing platform.

[0014] SSR site detection and search: SSR detection and search were performed using MISA software, and sequences containing SSR sites were selected as candidate sequences for SSR primers.

[0015] SSR primer design: SSR primers were designed using Primer Premier 5 software, and their polymorphism in different individuals of Chrysanthemum indicum was identified, resulting in polymorphic primers for Chrysanthemum indicum microsatellite molecular markers.

[0016] Another objective of this application is a method for applying microsatellite molecular markers of *Chrysanthemum indicum* in detecting the genetic diversity of *Chrysanthemum indicum* populations, including:

[0017] DNA was extracted from individual specimens of the Chrysanthemum 'Yellow' species.

[0018] The DNA was subjected to PCR amplification using the specific primers for the microsatellite molecular markers of Chrysanthemum indicum described above.

[0019] The selected primers with high polymorphism were fluorescently labeled, and the 5' end was fluorescently modified with 6-FAM. The fluorescently labeled forward primer and the ordinary reverse primer were used for capillary electrophoresis amplification on an ABI3730XL DNA analyzer.

[0020] Genotyping was performed on the amplified products.

[0021] Another objective of this application is to apply the above-mentioned microsatellite molecular markers of Chrysanthemum in the analysis of genetic diversity of Chrysanthemum in Chrysanthemum, construction of plant genetic maps, gene mapping, variety identification, germplasm preservation, analysis of quantitative trait genes and / or research on evolution and phylogenetic relationships.

[0022] This application provides a method for preparing microsatellite molecular markers for *Chloranthus chinensis* by developing microsatellite molecular markers using restriction enzyme site-related DNA genome scanning technology and obtaining corresponding primers. This provides molecular markers for research on *Chloranthus chinensis* germplasm resource identification, genetic diversity analysis, plant genetic map construction, population genetic structure analysis, gene mapping, quantitative trait gene analysis, evolution and phylogenetic relationships. It lays the foundation for conducting genetic research on the genetic diversity, population structure, and population history dynamics of *Chloranthus chinensis* germplasm resources and is conducive to the rational development of *Chloranthus chinensis* resources. Attached Figure Description

[0023] Figure 1 This is a schematic diagram illustrating the experimental principle of sequencing the full-length transcriptome of Chrysanthemum indicum provided in this embodiment of the invention.

[0024] Figure 2 A CSS sequence length distribution diagram provided for embodiments of the present invention.

[0025] Figure 3 The length distribution diagram of the full-length non-chimeric sequence provided in the embodiments of the present invention.

[0026] Figure 4 This is a quality distribution diagram of the corrected sequence provided in an embodiment of the present invention.

[0027] Figure 5 The length distribution diagram of *Chrysanthemum indicum* transcripts after redundancy removal is provided in an embodiment of the present invention.

[0028] Figure 6 The figure shows the statistical results of different types of SSR classification provided in the embodiments of the present invention.

[0029] Figure 7 Electrophoresis images of T60-4-1 and T59-6-1 primers provided for embodiments of the present invention.

[0030] Figure 8 Capillary electrophoresis images of primers T29-5-1 and T60-4-1 provided for embodiments of the present invention. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of this application clearer, the present invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0032] This application addresses the current lack of microsatellite markers for *Chlorophytum comosum*. It utilizes single-molecule real-time sequencing technology to perform full-length transcriptome sequencing on *Chlorophytum comosum*, develops SSR molecular markers for *Chlorophytum comosum*, obtains corresponding primers, and establishes a method for preparing microsatellite molecular markers for *Chlorophytum comosum*. This provides molecular markers for research on *Chlorophytum comosum* germplasm resource identification, genetic diversity analysis, plant genetic map construction, population genetic structure analysis, gene mapping, quantitative trait gene analysis, evolution, and phylogenetic relationships.

[0033] Example 1: Preparation method of microsatellite molecular markers for Chrysanthemum indicum, the specific steps are as follows:

[0034] S1: RNA extraction and quality testing: RNA was extracted from the samples using the Tiangen Biotech (Beijing) Polysaccharide and Polyphenol Total RNA Extraction Kit (DP441); the quality of total RNA was tested using 1% agarose gel electrophoresis, Nanodrop 2000 micro spectrophotometer, and Agilent 2100 bioanalyzer.

[0035] S2: Constructing a cDNA library: After the RNA samples passed the testing, total RNA was reverse transcribed into cDNA using the SMARTer PCR cDNA Synthesis Kit (Clontech, 634925). A portion of the cDNA was used for fragment screening with BluePippin to enrich fragments larger than 4kb. The screened fragments were then subjected to large-scale PCR. The full-length cDNA was then purified, end-repaired, and adapters were ligated to obtain the final library. The experimental principle of *Chrysanthemum indicum* full-length transcriptome library construction and sequencing is as follows: Figure 1 As shown;

[0036] S3: After the library is constructed, the library is first initially quantified using Qubit 2.0 to dilute it. Then, the size of the inserted fragments in the library is detected using Agilent 2100. After the inserted fragments meet the expectations, the effective concentration of the library is accurately quantified using the Q-PCR method to ensure the quality of the library.

[0037] S4: Transcriptome Sequencing: After the library passed the test, full-length transcriptome sequencing analysis was performed using the SMRT (Singlemolecule real-time) molecular real-time sequencing technology (third-generation sequencing) on ​​the PacBio Sequel sequencing platform. Before sequencing, adapter sequences and primers were added to both ends of the transcript. During sequencing on the PacBio Sequel platform, the ultra-long read length was used to sequence the inserted fragment sequence multiple times in succession. Each successful sequence is called a Full pass and generates a Subread. The data format after sequencing is a Bam file, which contains the sequence information of all the Subreads obtained from sequencing. The raw data of Huang Yingju after sequencing is shown in Table 1.

[0038] Table 1: Statistics of raw sequence data

[0039]

[0040] S5: Sequencing data quality assessment: The analysis process of obtaining full-length transcriptomes using the SMRTLink software Iso-Seq pipeline mainly includes three stages: obtaining CCS sequences (CCS), full-length sequence identification (Classify), and Isoform-level clustering (Cluster).

[0041] S5.1: CCS: Extract CCS (Circular consensus sequences) sequences from the Bam file of the downloaded Subreads. See Table 2 for CCS sequence statistics, and see the CCS sequence length distribution. Figure 3 ;

[0042] Table 2: Statistics of raw sequence data

[0043]

[0044] S5.2: Classify: Based on the presence of 3' primer, 5' primer and PolyA in the sequence, the CCS sequence is divided into full-length sequence (FL) and non-full-length sequence (NFL). Primers and PolyA / T tail sequences at both ends of the CCS sequence are removed, and artificial concatenators in the full-length sequence are filtered out to obtain full-length non-chimeric sequence (FLNC) and non-full-length sequence (NFL).

[0045] The classification criteria include the following four parts:

[0046] (1) Includes 5' and 3' primers;

[0047] (2) The sequence length is greater than 300bp;

[0048] (3) Contains a PolyA tail;

[0049] (4) Does not contain chimeric sequences;

[0050] The transcriptome classification results of *Chrysanthemum indicum* are shown in Table 3. The statistical analysis of the obtained full-length non-chimeric sequences is also shown in Table 3.

[0051] Table 3: Statistical analysis of classification results of full-length non-chimeric sequences of *Chrysanthemum indicum*

[0052]

[0053] S5.3: Cluster: The ICE (Iterative isoform-clustering) algorithm is used to cluster similar FLNC sequences into a cluster by comparison. Each cluster yields a consistent sequence.

[0054] By combining non-full-length sequences, the Quiver (or Arrow) algorithm is used to correct (Polish) the Consensusisoforms obtained from the clustering pairs, and high-quality (HQ, Polished high quality) sequences are selected for subsequent analysis;

[0055] The screening criteria for high-quality transcripts are as follows:

[0056] (1) Full-length sequence (containing both cDNA primer and PolyA signal tail);

[0057] (2) High quality (Predicted accuracy >= 0.99);

[0058] (3) Support for more than two full-length sequences;

[0059] The statistical results of HQ transcripts obtained from library construction are shown in Table 4:

[0060] Table 4: Statistical analysis of HQ transcript sequence results

[0061]

[0062] S6: Redundancy Removal Analysis: Due to potential degradation at the 5' end of the RNA sequence during library construction, and the high sensitivity and specificity requirements of the Cluster algorithm (ICE), redundant transcripts may exist in the polished sequences obtained from the Iso-Seq process. The polished transcripts were screened using CD-HIT software to remove redundancy from sequences with >95% similarity, yielding 184,076 unigenes with an average length of 2620 bp, a total length of 120,791,357 bp, and an N50 length of 2976 bp. The length distribution of the transcripts after redundancy removal is shown in [Figure number missing]. Figure 5 The statistics for redundancy removal are shown in Table 5:

[0063] Table 5: Distribution of transcript lengths after redundancy removal

[0064]

[0065] S7: SSR Site Detection and Search: SSR detection and search were performed using MISA (MIcroSAtellite: http: / / pgrc.ipk-gatersleben.de / misa / ). Sequences containing SSR sites were used as candidate sequences for SSR primers. The minimum repeat counts for each repeat unit were 1-10, 2-6, 3-5, 4-5, 5-5, and 6-5 (e.g., 1-10 requires at least 10 repeats for single nucleotide repeats to be detectable; 2-6 requires at least 6 repeats for double nucleotide repeats). A total of 16,441 SSR sites were found, with 4,220, 4,991, 6,045, 493, 202, and 490 single, di, tri, tetra, penta, and hexanucleotide SSRs, respectively. Statistics on the number of SSRs for different nucleotide types are shown below. Figure 6 See Table 6.

[0066] Table 6: Distribution characteristics of SSR site repetitive motif types in the transcriptome of *Chrysanthemum indicum*

[0067]

[0068]

[0069] S8: SSR Primer Design: SSR primers were designed in batches using Primer Premier 5 software. A total of 145 SSR primer pairs were designed based on transcriptome data. Twenty individuals from geographically distant locations were randomly selected for PCR amplification. Fifteen primer pairs amplified products with clear bands and high polymorphism. The PCR amplification reaction program was as follows: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 51-65℃ annealing for 30 s, 72℃ extension for 30 s, repeated 36 times, followed by a final extension at 72℃ for 5 min, and storage at 4℃. Some primer agarose gel electrophoresis images are shown below. Figure 7 As shown;

[0070] The polymorphism of *Chlorophytum comosum* in different individuals was identified, and polymorphic primers for microsatellite molecular markers of *Chlorophytum* species were obtained. The characteristics of each primer and the correspondence of microsatellite molecular markers are shown in Table 7.

[0071] Table 7: Correspondence between microsatellite primer characteristics and microsatellite molecular markers of Chrysanthemum indicum

[0072]

[0073]

[0074] The primer numbers for the 15 pairs of polymorphic primers for microsatellite molecular markers of the genus *Chloranthus* shown in Table 7 above are: T22-4-1, T31-4-1, T48-4-1, T60-4-1, T2-5-1, T29-5-1, T25-5-1, T42-5-1, T51-5-1, T5-6-1, T9-6-1, T34-6-1, T36-6-1, T56-6-1, and T59-6-1, respectively. The nucleotide sequences are shown in SEQ.ID.No.1-SEQ.ID.No.15, respectively.

[0075] Example 2: A method for analyzing the genetic diversity of *Chrysanthemum indicum* using microsatellite molecular markers, comprising the following steps:

[0076] S1: Field sampling of *Chrysanthemum indicum* was conducted. Leaves of *Chrysanthemum indicum* growing well in the wild were collected and placed in small envelopes pre-filled with color-changing silica gel. These were then brought back to the laboratory for preservation. DNA was extracted using the Plant Genome Extraction Kit (DP350) from Tiangen Biotech (Beijing) Co., Ltd. This study used 200 individuals from 40 populations as experimental materials, as shown in Table 8.

[0077] Table 8: Information on 40 population samples

[0078]

[0079]

[0080] S2: Using any one or more of the above primer pairs, perform PCR amplification on the extracted DNA from different individuals of Chrysanthemum indicum. The PCR amplification reaction program is as follows: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 51-65℃ annealing for 30 s, 72℃ extension for 30 s, run for 36 cycles, 72℃ extension for 5 min, and store at 4℃.

[0081] S3: Capillary electrophoresis detection: Selected primers with high polymorphism were fluorescently labeled, with the 5' end fluorescently modified with 6-FAM. Amplification was then performed by capillary electrophoresis using fluorescently labeled positive primers and ordinary reverse primers on an ABI 3730XL DNA analyzer. Figure 8 As shown;

[0082] S4: The amplified product was sent to Genewiz Biotechnology Co., Ltd. (Suzhou) for genotyping;

[0083] S5: Data Processing: The results of capillary electrophoresis were analyzed using GeneMapper V5.0. At the same time, each locus was converted into an intuitive map by comparing the size and number of fragments of the amplified product. DataFormater software was used to convert the obtained fragment length values ​​into a format that Popgene and GenAlEx software could recognize. Popgene V1.32 and GenAlEx V6.501 software were used to calculate the genetic diversity index.

[0084] SSR amplification was performed on 200 individuals from 40 populations of *Chrysanthemum indicum*. The results showed that the expected heterozygosity (He) and observed heterozygosity (Ho) of microsatellite loci at the population level ranged from 0.501 to 0.856 and 0.031 to 1.000, respectively; the number of alleles (Na) ranged from 3 to 21, with an average of 6.8; the mean number of effective alleles (Ne) was 3.654, with all loci ranging from 1.988 to 6.845; and Shannon's information index (I) was [missing information]. The values ​​ranged from 0.722 to 2.242, with a mean of 1.364; the genetic differentiation coefficient (Fst) ranged from 0.356 to 0.957, with a mean of 0.642; the percentage of polymorphic sites was 100%; and the polymorphism information index (PIC) ranged from 0.381 to 0.839, with a mean of 0.635 (see Table 9 for details). These results indicate that the screened SSR primers have high polymorphism and high genetic diversity, which can provide a new research method for the future development and utilization of Chrysanthemum indicum resources.

[0085] Table 9: Genetic diversity index information of 15 SSR loci from 40 *Chlorophytum comosum* populations

[0086]

[0087] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

[0088] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. Specific primers for microsatellite molecular markers of Chrysanthemum indicum, characterized in that, The specific primers are the following 15 pairs of primers: T22-4-1, T31-4-1, T48-4-1, T60-4-1, T2-5-1, T25-5-1, T29-5-1, T42-5-1, T51-5-1, T5-6-1, T9-6-1, T34-6-1, T36-6-1, T56-6-1, and T59-6-1; among which, The forward primer sequence of T22-4-1 is shown in SEQ.ID.No.16; the reverse primer sequence of T22-4-1 is shown in SEQ.ID.No.17; the forward primer sequence of T31-4-1 is shown in SEQ.ID.No.18; the reverse primer sequence of T31-4-1 is shown in SEQ.ID.No.19; the forward primer sequence of T48-4-1 is shown in SEQ.ID.No.20; the reverse primer sequence of T48-4-1 is shown in SEQ.ID.No.21; the forward primer sequence of T60-4-1 is shown in SEQ.ID.No.22; the reverse primer sequence of T60-4-1 is shown in SEQ.ID.No.22; the reverse primer sequence of T60-4-1 is shown in SEQ.ID.No.17; the forward primer sequence of T31-4-1 is shown in SEQ.ID.No.18; the reverse primer sequence of T31-4-1 is shown in SEQ.ID.No.19; the forward primer sequence of T48-4-1 is shown in SEQ.ID.No.20; the reverse primer sequence of T60-4-1 is shown in SEQ.ID.No.21; the reverse primer sequence of T60-4-1 is shown in SEQ.ID.No.22; the forward primer sequence of T31-4-1 is shown in SEQ.ID.No.19; the reverse primer sequence of T31-4-1 is shown in SEQ.ID.No.19; the forward primer sequence of T31-4-1 is shown in SEQ.ID.No.20; the reverse primer sequence of T31-4-1 is shown in SEQ.ID.No.21; the forward primer sequence of T31-4-1 is shown in SEQ.ID.No.22; the reverse primer sequence of T31-4-1 is shown in SEQ.ID.No.19; the forward primer sequence of T31-4- The sequences are shown in SEQ ID No. 23; the forward primer sequence of T2-5-1 is shown in SEQ ID No. 24; the reverse primer sequence of T2-5-1 is shown in SEQ ID No. 25; the forward primer sequence of T25-5-1 is shown in SEQ ID No. 26; the reverse primer sequence of T25-5-1 is shown in SEQ ID No. 27; the forward primer sequence of T29-5-1 is shown in SEQ ID No. 28; the reverse primer sequence of T29-5-1 is shown in SEQ ID No. 29; and the forward primer sequence of T42-5-1 is shown in SEQ ID No.

30. The reverse primer sequence of T42-5-1 is shown in SEQ.ID.No.31; the forward primer sequence of T51-5-1 is shown in SEQ.ID.No.32; the reverse primer sequence of T51-5-1 is shown in SEQ.ID.No.33; the forward primer sequence of T5-6-1 is shown in SEQ.ID.No.34; the reverse primer sequence of T5-6-1 is shown in SEQ.ID.No.35; the forward primer sequence of T9-6-1 is shown in SEQ.ID.No.36; the reverse primer sequence of T9-6-1 is shown in SEQ.ID.No.37; the forward primer sequence of T34-6-1 is shown in SEQ.ID.No.

37. The reverse primer sequence of T34-6-1 is shown in SEQ.ID.No.38; the forward primer sequence of T36-6-1 is shown in SEQ.ID.No.39; the forward primer sequence of T36-6-1 is shown in SEQ.ID.No.40; the reverse primer sequence of T36-6-1 is shown in SEQ.ID.No.41; the forward primer sequence of T56-6-1 is shown in SEQ.ID.No.42; the reverse primer sequence of T56-6-1 is shown in SEQ.ID.No.43; the forward primer sequence of T59-6-1 is shown in SEQ.ID.No.44; and the reverse primer sequence of T59-6-1 is shown in SEQ.ID.No.

45.

2. The method for applying the specific primers for the microsatellite molecular markers of *Chloranthus chinensis* as described in claim 1 to the detection of genetic diversity in *Chloranthus chinensis* populations, characterized in that, include: DNA was extracted from individual specimens of the Chrysanthemum 'Yellow' species. The DNA was subjected to PCR amplification using the specific primers for the microsatellite molecular markers of Chrysanthemum indicum as described in claim 1. The forward primer of the specific primer was labeled with 5' end 6-FAM fluorescent label, and the fluorescently labeled forward primer and ordinary reverse primer were used for capillary electrophoresis amplification on an ABI3730XL DNA analyzer. Genotyping was performed on the amplified products.

3. The method for applying the specific primers for the microsatellite molecular markers of *Chloranthus chinensis* according to claim 2 in the detection of genetic diversity in *Chloranthus chinensis* populations, characterized in that, The PCR amplification reaction program is as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 51-65℃ annealing for 30 s, 72℃ extension for 30 s, 36 cycles; 72℃ extension for 5 min; storage at 4℃.

4. The application of the specific primers for the microsatellite molecular markers of Chrysanthemum in claim 1 in the genetic diversity analysis, construction of plant genetic maps, gene mapping, variety identification, germplasm preservation, analysis of quantitative trait genes and / or evolution and phylogenetic studies of Chrysanthemum in ...

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