Juniperus SSR molecular marker development primer combination and application thereof

Abstract

The invention discloses a Juniperus SSR molecular marker development primer combination and application thereof. The invention belongs to the field of plant molecular biology, and particularly relates to a Juniperus SSR molecular marker development primer combination and application thereof. The SSR molecular marker primer group of the juniperus, disclosed by the invention, comprises at least one pair of 18 pairs of primers which are numbered as SSR-J16, SSR-J28, SSR-J31, SSR-J57, SSR-J69, SSR-J80, SSR-J160, SSR-J187, SSR-J198, SSR-J210, SSR-J211, SSR-J222, SSR-J231, SSR-J234, SSR-J237, SSR-J246, SSR-J255 and SSR-J283, and the nucleotide sequences of the 18 pairs of primers are respectively shown as SEQ ID No. 1 to SEQ ID No. 36.

Description

Technical Field

[0001] The invention belongs to the field of plant molecular biology, and in particular relates to a primer combination for developing SSR molecular markers of Juniperus and application thereof. Background Art

[0002] Juniperus and Sabina belong to the gymnosperm Cupressaceae in classification. They were established by Linnaeus and Miller in 1753 and 1754 respectively, and belong to the subfamily Juniperoideae Pilger. For a long time, there has been controversy over the classification of Juniperus and Sabina. The morphology and growth pattern of the leaves of the two genera are somewhat confusing, which has also caused trouble for breeders. At the same time, the protection of new varieties of forest and grass plants has received more and more attention from the state and breeders. It is difficult to quickly and effectively distinguish variety resources only by morphological identification. Therefore, DNA molecular detection technologies represented by microsatellites (Simple Sequence Repeats, SSR) and SNPs (Single Nucleotide Polymorphism) have gradually been recognized and recommended by the UPOV molecular testing guidelines as preferred markers for variety authenticity identification and database construction. However, DNA molecular marker technology for Juniperus plants is relatively backward, and there has been no application of efficient and stable SSR molecular markers in germplasm identification, kinship analysis, fingerprint map construction, etc. between and within species of this genus. In order to better carry out the breeding of new varieties of Cupressaceae plants and protect variety rights, and to better serve Cupressaceae plant breeders and protect the rights of breeders, it is urgent to develop universal SSR marker primers that are also applicable to the genus Juniperus and establish DNA molecular detection technology. Summary of the invention

[0003] The main problem to be solved by the invention is how to identify varieties of Juniperus species and perform kinship analysis.

[0004] In order to solve the above problems, the present invention provides a SSR molecular marker primer set for Juniperus.

[0005] The present invention provides a Juniperus SSR molecular marker primer set, which includes at least one pair of 18 pairs of primers, and the nucleotide sequences of the 18 pairs of primers are shown in SEQ ID No.1-SEQ ID No.36.

[0006] The 18 pairs of primers are specifically SSR-J16, SSR-J28, SSR-J31, SSR-J57, SSR-J69, SSR-J80, SSR-J160, SSR-J187, SSR-J198, SSR-J210, SSR-J211, SSR-J222, SSR-J231, SSR-J234, SSR-J237, SSR-J246, SSR-J255, and SSR-J283.

[0007] The present invention provides a kit, comprising the SSR molecular marker primer set described above.

[0008] The present invention provides a DNA chip, wherein the DNA chip comprises the SSR molecular marker primer set described above.

[0009] The present invention also provides any of the following applications of the aforementioned SSR molecular marker primer set, and / or the aforementioned kit, and / or the aforementioned DNA chip:

[0010] A1) Application in genetic diversity analysis of Juniperus;

[0011] A2) Application in the analysis of species diversity of Juniperus;

[0012] A3) Application in the analysis of phylogenetic relationships in the genus Juniperus;

[0013] A4) Application in identification of Juniperus species;

[0014] A5) Application in constructing a genetic map or DNA fingerprint map of Juniperus;

[0015] A6) Application in the improvement of Juniperus germplasm resources;

[0016] A7) Application in gene localization or functional gene mining in Juniperus;

[0017] A8) in molecular marker-assisted breeding of Juniperus .

[0018] The present invention also provides a method for screening the SSR molecular marker primer set described above, the method comprising the following steps:

[0019] B1) Using the transcriptome sequences of Sabina 'Blue Sky' and Sabina 'Gray Owl' as reference sequences, the Unigenede SSR loci were detected and primers were designed to obtain SSR markers;

[0020] B2) screening the diversity among germplasm resources materials using the SSR markers described in B1);

[0021] B3) Designing primers for the SSR markers screened in B2) to obtain the SSR molecular marker primer set.

[0022] In the above method, the screening criteria described in B3) are: mononucleotide, dinucleotide, trinucleotide, tetranucleotide, pentanucleotide and hexanucleotide, and the minimum number of repetitions of each nucleotide is 12, 6, 5, 5, 4, and 4 respectively.

[0023] The present invention also provides a method for genetic diversity analysis or phylogenetic analysis of Juniper, the method comprising: using the genomic DNA of Juniper to be tested as a template, performing PCR amplification using the SSR molecular marker primer set described above to obtain a PCR amplification product, performing electrophoresis detection on the PCR amplification product, and performing genetic diversity analysis or phylogenetic analysis of Juniper based on the electrophoresis detection result.

[0024] Furthermore, the genetic diversity analysis or phylogenetic analysis of Juniperus genus according to the electrophoresis detection results includes statistically analyzing the electrophoresis detection results, and recording "1" if there is a band at the same electrophoretic mobility position, and "0" if there is no band. Cluster analysis is further performed to construct a phylogenetic tree diagram, and the genetic diversity analysis or phylogenetic analysis of Juniperus genus is performed according to the statistical results.

[0025] Furthermore, Popgene 32 was used to statistically analyze genetic diversity, and MEGA 11 was used to construct a phylogenetic cluster diagram.

[0026] The present invention also provides a method for analyzing the fingerprint of Juniper varieties to identify Juniper varieties, the method comprising: using the genomic DNA of the Juniper variety to be tested and the standard Juniper variety as templates, respectively, performing PCR amplification using the SSR molecular marker primer set described above, detecting the PCR amplification product by electrophoresis to obtain the fingerprint of the Juniper germplasm resources to be tested and the fingerprint of the standard variety, and identifying the Juniper variety based on the fingerprint comparison analysis results.

[0027] The present invention also provides a method for analyzing the fingerprint of Sabina varieties to identify Sabina varieties, the method comprising: using the genomic DNA of the Sabina variety to be tested and the standard Sabina variety as templates, respectively, performing PCR amplification using the SSR molecular marker primer set described above, detecting the PCR amplification products by electrophoresis to obtain the fingerprint of the Sabina variety to be tested and the fingerprint of the standard variety, and identifying the Sabina variety based on the fingerprint comparison analysis results.

[0028] In the above method, the PCR amplification reaction system (20 μL): 10 μL of PCR mix, 0.3 μL of forward primer with fluorescent label (20 μM), 0.3 μL of reverse primer (20 μM), 2 μL of template DNA, and 7.4 μL of ddH2O;

[0029] The PCR amplification program was as follows: pre-denaturation at 94°C for 5 min, 1 cycle; denaturation at 94°C for 30 s, annealing at 60°C for 40 s, extension at 72°C for 50 s, 10 cycles; denaturation at 94°C for 30 s, annealing at 53°C for 40 s, extension at 72°C for 50 s, 27 cycles; and final extension at 72°C for 10 min.

[0030] In the above method, the electrophoresis detection is agarose gel electrophoresis detection, polyacrylamide gel electrophoresis detection, or / and capillary electrophoresis detection.

[0031] Herein, the juniper species are mainly juniper species Sabina chinensis (L.) Ant; the genus Juniper includes North American juniper, Rocky Mountain juniper, Alpine juniper, Staghorn cypress, and flat juniper.

[0032] In order to solve the technical problem of lack of DNA molecular marker primers for Cupressaceae plants, the present invention has developed a set of SSR primers suitable for germplasm identification of Juniperus and Juniperus plants based on the transcriptome sequences of Rocky Mountain Juniperus and North American Juniperus. Compared with the DUS test technology based on phenotypic characteristics, the set of SSR primer combinations developed in the present invention has the advantages of stable amplification, efficient polymorphism, etc., and is not limited by factors such as tree age and environment, and can effectively realize the germplasm identification of different inter-species and intra-species levels of Juniperus. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The capillary electrophoresis results of some SSR primers described in the present invention are shown in Figure 1. A is the amplification result of primer SSR-J283 (BHE); B is the amplification result of primer SSR-J231 (YM); and C is the amplification result of primer SSR-J28 (BLA).

[0034] Figure 2 The present invention provides a cluster diagram of 19 Juniperus and Sabina variety resources constructed using the complete set of SSR primers described in the present invention. DETAILED DESCRIPTION

[0035] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0036] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0037] Unless otherwise specified, the quantitative tests in the following examples were performed three times and the results were averaged.

[0038] The materials of 'Lanta', 'Jinhua', 'Jinghui No. 3', 'Jinghui No. 6', 'Y18', and 'Y23' in the following embodiments are from the important tree germplasm resource bank of Beijing Academy of Agriculture and Forestry Sciences, and are also included in the National Forestry and Grassland Germplasm Resource Bank (https: / / www.nfgrp.cn / ). The public can obtain the biological material from the National Forestry and Grassland Germplasm Resource Bank or the applicant. The biological material is only used to repeat the experiment of the present invention and cannot be used for other purposes.

[0039] Example 1. Development and screening method of Juniperus SSR primer set

[0040] 1. Primer development and design methods

[0041] Based on the transcriptome sequencing data of leaves, female flowers and cones of Juniperus scopulorum Blue Heaven' and J. virginiana 'Grey Owl', MISA software was used to detect Unigene, and the detection standards were: mononucleotide, dinucleotide, trinucleotide, tetranucleotide, pentanucleotide and hexanucleotide, and the minimum number of repetitions of each nucleotide was 12, 6, 5, 5, 4, and 4, respectively. Then Primer 3.0 was used to design the detected SSR primers.

[0042] 2. Polymorphic primer screening method

[0043] The test materials are 6 varieties of juniper: 'Lan Ta', 'Jin Hua', 'Jing Hui No. 3', 'Jing Hui No. 6', Y18, and Y23; the materials come from the important tree germplasm resource bank of Beijing Academy of Agriculture and Forestry Sciences and are also included in the National Forestry and Grassland Germplasm Resource Bank ( https: / / www.nfgrp.cn / ), see Table 1 for specific platform resource numbers.

[0044] Table 1. Materials used for primer screening

[0045]

[0046] 1) DNA extraction and detection

[0047] The DNA was extracted from the leaves using a DNA extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., DP305) according to the instructions, and the DNA purity was detected by 2.0% agarose gel electrophoresis.

[0048] 2) PCR amplification

[0049] According to the primer design results of step 1, 300 pairs of primers were synthesized mainly with dinucleotides and trinucleotides for polymorphic primer screening.

[0050] The reaction system used for primer screening (20 μL): PCR mix 10 μL, forward primer 0.3 μL (20 μM), reverse primer 0.3 μL (20 μM), template DNA 2 μL, ddH2O 7.4 μL.

[0051] The amplification program used for primer screening was: pre-denaturation at 94°C for 5 min, 1 cycle; denaturation at 94°C for 30 s, annealing at 60°C for 40 s, and extension at 72°C for 50 s, 10 cycles; denaturation at 94°C for 30 s, annealing at 53°C for 40 s, and extension at 72°C for 50 s, 27 cycles; and final extension at 72°C for 10 min.

[0052] 3) Polymorphic SSR primer screening

[0053] Screening was performed by agarose gel electrophoresis, polyacrylamide gel electrophoresis and capillary electrophoresis respectively.

[0054] Agarose gel electrophoresis detection is to use 2.0% agarose gel electrophoresis to detect the PCR amplification products of six samples, namely, 'Lanta', 'Jinhua', 'Jinghui No. 3', 'Jinghui No. 6', Y18, and Y23, and preliminarily select primers with target genes.

[0055] Polyacrylamide gel electrophoresis detection is to detect the PCR amplification products of 6 samples using 6.0% polyacrylamide gel electrophoresis on the basis of the preliminary selection, and to re-select primers containing clear target bands and having polymorphism.

[0056] Capillary electrophoresis detection is based on the re-selection, and further capillary electrophoresis detection is performed on the PCR amplification products of varieties such as 'Lanta', 'Jinhua', 'Jinghui No. 3', 'Jinghui No. 6', Y18, and Y23 to screen out efficient primers with rich sites, clear peak shapes, and stable amplification.

[0057] 3. Polymorphic primers

[0058] According to the primer development and screening methods of steps 1 and 2, 20 pairs of polymorphic primers were obtained (Table 2), and then the 20 pairs of primers were used to perform genetic diversity analysis on 19 Juniperus and Sabina variety resources (Table 3).

[0059] As shown in Table 4, the total number of bands amplified by the 20 pairs of primers ranged from 3 to 23, and the number of polymorphic bands ranged from 2 to 23. Primer SSR-J116 amplified the least number of bands and had a lower polymorphism; although primer SSR-J150 had a better polymorphism, the amplification was weaker. The remaining 18 pairs of primers amplified bands that were all polymorphic, and the percentage of polymorphism of the primers reached 100%. Finally, a set of efficient microsatellite molecular marker primers based on the transcriptome sequences of Juniperus were obtained, totaling 18 pairs, namely primer sets SSR-J16, SSR-J28, SSR-J31, SSR-J57, SSR-J69, SSR-J80, SSR-J160, SSR-J187, SSR-J198, SSR-J210, SSR-J211, SSR-J222, SSR-J231, SSR-J234, SSR-J237, SSR-J246, SSR-J255 and SSR-J283.

[0060] Table 2. Information of 20 pairs of Juniperus SSR marker primer sets

[0061]

[0062]

[0063]

[0064] Table 3. Information on 19 varieties

[0065]

[0066]

[0067] Table 4. Polymorphism results of SSR primers

[0068]

[0069]

[0070] Example 2: Method for identifying Juniperus germplasm using SSR marker primer sets

[0071] The 19 Juniperus and Juniperus species resources collected and preserved in the National Forestry and Grassland Germplasm Resource Bank were amplified using the fluorescent primer capillary electrophoresis method using the Juniperus SSR primers numbered SSR-J16, SSR-J28, SSR-J31, SSR-J57, SSR-J69, SSR-J80, SSR-J160, SSR-J187, SSR-J198, SSR-J210, SSR-J211, SSR-J222, SSR-J231, SSR-J234, SSR-J237, SSR-J246, SSR-J255 and SSR-J283 screened in Example 1. The size of the fluorescent PCR product was detected by capillary electrophoresis, the genetic diversity was statistically analyzed by Popgene 32, and the kinship cluster diagram was constructed by MEGA 11.

[0072] The specific steps are as follows:

[0073] 1. DNA extraction and detection

[0074] The genomic DNA of needles was extracted using a DNA extraction kit, and the purity and integrity of the DNA were tested using 2.0% agarose gel electrophoresis.

[0075] 2. Synthesis of fluorescent primers

[0076] The 5' end of the forward primer sequence of the 18 pairs of Juniperus SSR marker primers (Table 2) screened above was modified with a 6-FAM fluorescent group to synthesize 18 pairs of fluorescent SSR primers.

[0077] 3. PCR amplification of microsatellite fluorescent primers

[0078] PCR amplification reaction system (20 μL): PCR mix 10 μL, forward primer with fluorescent label 0.3 μL (20 μM), reverse primer 0.3 μL (20 μM), template DNA 2 μL, ddH2O 7.4 μL.

[0079] The PCR amplification program was as follows: pre-denaturation at 94°C for 5 min, 1 cycle; denaturation at 94°C for 30 s, annealing at 60°C for 40 s, and extension at 72°C for 50 s, 10 cycles; denaturation at 94°C for 30 s, annealing at 53°C for 40 s, and extension at 72°C for 50 s, 27 cycles; and final extension at 72°C for 10 min.

[0080] 4. Capillary fluorescence electrophoresis detection

[0081] After formamide and molecular weight internal standard were mixed at a volume ratio of 100:1, 9 μL was added to the sample plate, and then 1 μL of the PCR product obtained in step 3, which was diluted 10 times, was added. Then, capillary electrophoresis was performed using an ABI 3730XL fully automatic DNA sequencer, and the raw data obtained by the sequencer was analyzed using the Fragment (Plant) fragment analysis software in Genemarker. The position of the molecular weight internal standard in each lane was compared with the position of the peak of each sample to obtain the fragment size.

[0082] 5. Germplasm resource identification

[0083] Convert 1.31 software was used for format conversion, Popgene32 software was used for statistical analysis of genetic consistency and genetic distance, UPGMA method was used for germplasm resource cluster analysis, and phylogenetic tree was constructed.

[0084] According to the above 18 pairs of SSR primers with fluorescent labels, the 19 varieties were clustered and analyzed for their kinship, and the 19 varieties were able to be completely distinguished. At the same time, it can be seen from the cluster diagram that the six varieties of the genus Juniperus, RT, FG, LT, DC, JH, and YM, are clustered together as a branch, the two varieties of the genus Juniperus, BHE and BAR, are clustered together, the two varieties of the genus Juniperus, SVI-x and SVI-c, are clustered together, the varieties of the genus Juniperus, TIS and BDA, are clustered together, and the remaining varieties belong to a separate branch, which further illustrates that the SSR primer combination developed in this application is highly efficient in the identification and kinship analysis of varieties within the species of Juniperus, Juniperus, Juniperus, and Juniperus ( Figure 2 ).

[0085] Example 3: Method for constructing DNA molecular ID card of new species of Sabina plants using SSR marker primer set

[0086] Using the Juniperus SSR primers numbered SSR-J16, SSR-J28, SSR-J31, SSR-J57, SSR-J69, SSR-J80, SSR-J160, SSR-J187, SSR-J198, SSR-J210, SSR-J211, SSR-J222, SSR-J231, SSR-J234, SSR-J237, SSR-J246, SSR-J255 and SSR-J283 screened in Example 2, the four new Juniperus varieties that have obtained the national plant variety rights: 'Yunmi', 'Diecui', 'Menglin Lanbai' and 'Menglin Zhongbai' were amplified by fluorescent primer capillary electrophoresis. The size of the fluorescent PCR product was detected by capillary electrophoresis.

[0087] 1. DNA extraction and detection

[0088] The genomic DNA of juniper needles was extracted using a DNA extraction kit, and the purity and integrity of the DNA were detected by 2.0% agarose gel electrophoresis.

[0089] 2. Synthesis of fluorescent primers

[0090] The 5' end of the forward primer sequence of the above 18 pairs of juniper SSR marker primers (specific primer information is shown in Table 2) was modified with a 6-FAM fluorescent group to synthesize 18 pairs of fluorescent SSR primers.

[0091] 3. PCR amplification of microsatellite fluorescent primers

[0092] PCR amplification reaction system (20 μL): PCR mix 10 μL, forward primer with fluorescent label 0.3 μL (20 μM), reverse primer 0.3 μL (20 μM), template DNA 2 μL, ddH2O 7.4 μL.

[0093] The PCR amplification program was as follows: pre-denaturation at 94°C for 5 min, 1 cycle; denaturation at 94°C for 30 s, annealing at 60°C for 40 s, and extension at 72°C for 50 s, 10 cycles; denaturation at 94°C for 30 s, annealing at 53°C for 40 s, and extension at 72°C for 50 s, 27 cycles; and final extension at 72°C for 10 min.

[0094] 4. Capillary fluorescence electrophoresis detection

[0095] After formamide and molecular weight internal standard were mixed at a volume ratio of 100:1, 9 μL was added to the sample plate, and then 1 μL of the PCR product obtained in step 3, which was diluted 10 times, was added. Then, capillary electrophoresis was performed using an ABI 3730XL fully automatic DNA sequencer, and the raw data obtained by the sequencer was analyzed using the Fragment (Plant) fragment analysis software in Genemarker. The position of the molecular weight internal standard in each lane was compared with the position of the peak of each sample to obtain the fragment size.

[0096] 5. Construction of DNA molecular ID card

[0097] The sizes of fragments amplified by 18 pairs of SSR primers SSR-J16, SSR-J28, SSR-J31, SSR-J57, SSR-J69, SSR-J80, SSR-J160, SSR-J187, SSR-J198, SSR-J210, SSR-J211, SSR-J222, SSR-J231, SSR-J234, SSR-J237, SSR-J246, SSR-J255 and SSR-J283 on four improved varieties of Sabina were counted. The sizes of the fragments amplified at the SSR loci of each material were arranged in order from small to large, and the amplification of different varieties at the loci was counted. According to the principle of small to large, "1" was marked if there was a band and "0" was marked if there was no band (Table 5).

[0098] The number of fragments of the 18 pairs of SSR primers in the four juniper varieties ranged from 2 to 11, and the amplification band type at the SSR locus of each variety was 1 to 4, which can be inferred that the tested samples were polyploid.

[0099] In addition, it can be seen from the amplification results (Table 5) that among the 18 pairs of SSR primers, any one pair of primers including SSR-J16, SSR-J31, SSR-J57, SSR-J69, SSR-J160, SSR-J210, SSR-J222, SSR-J231, SSR-J237, SSR-J246, SSR-J255, and SSR-J283 can distinguish the four new varieties, indicating that these 12 pairs of primers have a high identification efficiency.

[0100] Table 5. Amplified bands and corresponding codes of 12 pairs of efficient SSR primers in four new varieties of Sabina

[0101]

[0102]

[0103]

[0104] Furthermore, using the 12 pairs of SSR primers with high identification efficiency in Table 5, the fragments that did not exist in the four varieties were eliminated, and then the fragments were arranged from small to large. According to the 0,1 method, a DNA molecular identity card consisting of 60 bits of code was constructed for the four new varieties of juniper plants (Table 6).

[0105] Table 6. DNA molecular ID of new juniper varieties based on 60 codes from 12 pairs of SSR primers

[0106] Juniper variety DNA molecule ID card encoding 'Green' 100000101011100101001011101000001010000111110010110000111000 'Dense clouds' 001001010011001000011000011000000100001011010101011000000110 'Montrin Cypress' 011100100100011111000000100001110011011001001000000110111001 'Mongolian cypress' 001110100101011011110100000111010110110101010000010011001000

[0107] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A set of SSR molecular marker primers for Juniperus, characterized in that: The SSR molecular marker primer set includes at least one pair of the 18 pairs of primers numbered SSR-J16, SSR-J28, SSR-J31, SSR-J57, SSR-J69, SSR-J80, SSR-J160, SSR-J187, SSR-J198, SSR-J210, SSR-J211, SSR-J222, SSR-J231, SSR-J234, SSR-J237, SSR-J246, SSR-J255, and SSR-J283, and the nucleotide sequences of the 18 pairs of primers are shown in SEQ ID No.1 to SEQ ID No.

36.

2. A kit, characterized in that: The kit comprises the SSR molecular marker primer set according to claim 1.

3. A DNA chip, characterized in that The DNA chip comprises the SSR molecular marker primer set according to claim 1.

4. Any of the following uses of the SSR molecular marker primer set of claim 1, and / or the kit of claim 2, and / or the DNA chip of claim 3: A1) Application in genetic diversity analysis of Juniperus; A2) Application in the analysis of species diversity of Juniperus; A3) Application in the analysis of phylogenetic relationships in the genus Juniperus; A4) Application in identification of Juniperus species; A5) Application in constructing a genetic map or DNA fingerprint map of Juniperus; A6) Application in the improvement of Juniperus germplasm resources; A7) Application in gene localization or functional gene mining in Juniperus; A8) in molecular marker-assisted breeding of Juniperus .

5. The screening method of the SSR molecular marker primer set according to claim 1, characterized in that: The method comprises the following steps: B1) Using the transcriptome sequences of the Rocky Mountain juniper species Juniperus scopulorum Blue Heaven' and the North American juniper species J. virginiana 'Grey Owl' as reference sequences, the SSR loci of Unigene were detected and primers were designed to obtain SSR markers; B2) screening the diversity among germplasm resources materials using the SSR markers described in B1); B3) Designing primers for the SSR markers screened in B2) to obtain the SSR molecular marker primer set.

6. The method according to claim 5, characterized in that The screening criteria described in B3) are: mononucleotide, dinucleotide, trinucleotide, tetranucleotide, pentanucleotide and hexanucleotide, and the minimum number of repetitions of each nucleotide is 12, 6, 5, 5, 4, and 4, respectively.

7. A method for genetic diversity analysis or phylogenetic relationship analysis of Juniperus using the SSR molecular marker primer set of claim 1, characterized in that: The method comprises: using the genomic DNA of the genus Juniperus to be tested as a template, performing PCR amplification using the SSR molecular marker primer set of claim 1 to obtain a PCR amplification product, performing electrophoresis detection on the PCR amplification product, and performing genetic diversity analysis or kinship analysis on the genus Juniperus according to the electrophoresis detection result.

8. A method for identifying Juniperus species by fingerprint analysis, the method comprising: The genomic DNA of the Juniperus species to be tested and the standard Juniperus species are used as templates respectively, and PCR amplification is performed using the SSR molecular marker primer set described in claim 1. The PCR amplification products are detected by electrophoresis to obtain the fingerprint map of the Juniperus species to be tested and the fingerprint map of the standard species. The Juniperus species is identified based on the fingerprint map comparison and analysis results.

9. A method for identifying Sabina species by analyzing their fingerprints, the method comprising: The genomic DNA of the tested juniper variety and the standard juniper variety are used as templates respectively, and PCR amplification is performed using the SSR molecular marker primer set described in claim 1. The PCR amplification products are detected by electrophoresis to obtain the fingerprint map of the tested juniper variety and the fingerprint map of the standard variety. The juniper variety is identified based on the fingerprint map comparison and analysis results.

10. The method according to any one of claims 7 to 9, characterized in that: The PCR amplification reaction system (20 μL): 10 μL of PCRmix, 0.3 μL of forward primer with fluorescent label (20 μM), 0.3 μL of reverse primer (20 μM), 2 μL of template DNA, and 7.4 μL of ddH2O; The PCR amplification program was as follows: pre-denaturation at 94°C for 5 min, 1 cycle; denaturation at 94°C for 30 s, annealing at 60°C for 40 s, extension at 72°C for 50 s, 10 cycles; denaturation at 94°C for 30 s, annealing at 53°C for 40 s, extension at 72°C for 50 s, 27 cycles; and final extension at 72°C for 10 min.

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