Sabina chinensis SSR molecular marker primer combination and application thereof

Abstract

The invention discloses a sabina chinensis SSR molecular marker primer combination and application thereof. The invention belongs to the technical field of biology, and particularly relates to a sabina chinensis SSR molecular marker primer combination and application thereof. The SSR molecular marker primer group developed by the invention comprises at least one pair of 17 pairs of primers, namely SSR-G34, SSR-G46, SSR-G102, SSR-G104, SSR-G108, SSR-G109, SSR-G111, SSR-G124, SSR-G149, SSR-G161, SSR-G164, SSR-G165, SSR-G173, SSR-G193, SSR-G197, SSR-G223 and SSR-G245, and the nucleotide sequences of the 17 pairs of primers are as shown in SEQ ID No. 1 to SEQ ID No. 34. The SSR molecular marker primer group disclosed by the invention has the advantages that the SSR molecular marker primer group disclosed by the invention can be used for identifying the Sabina;

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a juniper SSR molecular marker primer combination and an application thereof. Background Art

[0002] Sabina chinensis (Linn.) Ant., formerly known as "juniper", is called juniper in Beijing and other places. It belongs to the genus Sabina of the Cupressaceae family. It is widely distributed in my country and is an important garden greening tree species. At the same time, Sabina is drought-resistant and barren-resistant, and is an important ecological stress-resistant tree species in my country. my country has a long history of Sabina cultivation and rich germplasm resources. In Beijing alone, there are more than 6,000 first- and second-level ancient Sabina trees. The collection and preservation of Sabina germplasm resources in my country began in the 1980s. The germplasm resource evaluation technology is still mainly based on morphological evaluation, and the morphological differences between different varieties are identified through the phenotypic traits in the DUS test guide for new plant varieties. With the continuous development of Sabina germplasm innovation research, new varieties and new germplasms are increasing, and the application and protection of new Sabina varieties face severe challenges of similar phenotypic traits.

[0003] As the core technology for rapid DUS testing of new plant varieties, DNA molecular detection technology has gradually received attention. Microsatellites (Simple Sequence Repeats, SSR) and SNPs (single nucleotide polymorphism) are recommended by the UPOV molecular testing guidelines as preferred markers for variety authenticity identification and database construction. However, there is a lack of microsatellite molecular markers for Sabina plants, and there is no application of efficient and stable SSR molecular markers in the identification of this tree germplasm and the construction of fingerprint maps. In order to better carry out the breeding of new varieties of Sabina plants, and to better serve the protection of new varieties of Sabina plants and the protection of breeders' rights, it is urgent to develop more efficient, stable, and polymorphic specific microsatellite molecular marker primers. Summary of the invention

[0004] The main problem to be solved by the invention is how to identify and evaluate the variety resources of the genus Sabina.

[0005] In order to solve the above problems, the present invention provides a SSR molecular marker primer set for the genus Sabina.

[0006] The SSR molecular marker primer set for the genus Sabina provided by the present invention includes at least one pair of the 17 pairs of primers numbered SSR-G34, SSR-G46, SSR-G102, SSR-G104, SSR-G108, SSR-G109, SSR-G111, SSR-G124, SSR-G149, SSR-G161, SSR-G164, SSR-G165, SSR-G173, SSR-G193, SSR-G197, SSR-G223, and SSR-G245, and the nucleotide sequences of the 17 pairs of primers are shown in SEQ ID No.1 to SEQ ID No.34.

[0007] Furthermore, in the 17 pairs of primers, the 5' end of the forward primer sequence can be modified with a 6-FAM fluorescent group to synthesize 17 pairs of fluorescent SSR primers for easy identification.

[0008] The present invention also provides a kit, which comprises the SSR molecular marker primer set described above.

[0009] The present invention also provides a DNA chip, which comprises the SSR molecular marker primer set mentioned above.

[0010] The present invention also provides a method for screening the SSR molecular marker primer set described above, the method comprising the following steps:

[0011] B1) Using the transcriptome sequence of the juniper variety 'Fenghui' as a reference sequence, the SSR loci of the gene sequence (Unigene) were detected and primers were designed to obtain SSR markers;

[0012] B2) screening the diversity among germplasm resources materials using the SSR markers described in B1);

[0013] B3) Designing primers for the SSR markers screened in B2) to obtain the SSR molecular marker primer set.

[0014] 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.

[0015] The present invention also provides a method for genetic diversity analysis or phylogenetic analysis of different germplasm resources of the genus Sabina, the method comprising: using the genomic DNA of the juniper germplasm resource to be tested as a template, performing PCR amplification using the SSR molecular marker primer set described above to obtain PCR amplification products, performing electrophoresis detection on the PCR amplification products, and performing genetic diversity analysis or phylogenetic analysis on the genus Sabina based on the electrophoresis detection results.

[0016] Furthermore, the genetic diversity analysis or phylogenetic analysis of the genus Sabina based on 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 the genus Sabina is performed based on the statistical results.

[0017] Furthermore, Popgene 32 was used to statistically analyze genetic diversity, and MEGA 11 was used to construct a phylogenetic cluster diagram.

[0018] The present invention also provides a method for performing fingerprint analysis on genus Sabina varieties to identify genus Sabina varieties, the method comprising: using the genomic DNA of the genus Sabina variety to be tested and the standard genus Sabina variety as templates, respectively, performing PCR amplification using the SSR molecular marker primer set described above, detecting the PCR amplification products by electrophoresis, obtaining the fingerprint of the genus Sabina germplasm resources to be tested and the fingerprint of the standard genus Sabina variety, and identifying the genus Sabina variety based on the fingerprint comparison analysis results.

[0019] 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;

[0020] 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.

[0021] In the above method, the electrophoresis detection is agarose gel electrophoresis detection, polyacrylamide gel electrophoresis detection, or / and capillary electrophoresis detection.

[0022] Furthermore, the SSR molecular marker primer set may be 9 pairs of SSR primers: SSR-G34, SSR-G46, SSR-G104, SSR-G124, SSR-G149, SSR-G164, SSR-G197, SSR-G223, and SSR-G245.

[0023] 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:

[0024] A1) Application in the analysis of genetic diversity of Sabina;

[0025] A2) Application in the analysis of species diversity of the genus Sabina;

[0026] A3) Application in the analysis of phylogenetic relationships in the genus Sabina;

[0027] A4) Application in identification of Sabina species;

[0028] A5) Application in constructing a genetic map or DNA fingerprint map of the genus Sabina;

[0029] A6) Application in improving the germplasm resources of Sabina genus;

[0030] A7) Application in gene localization or functional gene mining of Sabina;

[0031] A8) Application of molecular marker-assisted breeding in Sabina.

[0032] Herein, the juniper varieties may be: 'Feng Juniper', 'Rong Tuan', 'Lanta', 'Menglin Lanbai' and 'Menglin Zhongbai', as well as 'Jinhua', 'Yunmi' and 'Diecui'.

[0033] The present invention develops a set of SSR primers using the transcriptome sequence of the juniper variety 'Fenghui'. The set of SSR primers developed by the present invention has the advantages of stable amplification, high efficiency and polymorphism, and can effectively realize the germplasm identification and differentiation of juniper and its closely related species and the construction of variety fingerprints. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The figure is the result of polyacrylamide gel electrophoresis using some of the SSR primers described in the present invention.

[0035] Figure 2 The present invention is a phylogenetic cluster diagram of the genome amplification results of 77 juniper variety resources using the set of SSR primers described in the present invention. DETAILED DESCRIPTION

[0036] 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.

[0037] 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.

[0038] Unless otherwise specified, the quantitative tests in the following examples were performed three times and the results were averaged.

[0039] The test materials in the following embodiments are 8 varieties of juniper, including 5 female plants: 'Fenghui', 'Rongtuan', 'Lanta', 'Menglinlanbai' and 'Menglinzhongbai', and 3 male plants: 'Jinhua', 'Yunmi' and 'Diecui'; the materials come from the important tree germplasm resource bank of Beijing Academy of Agriculture and Forestry Sciences (platform number see Table 1 for details), 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, and the biological material is only used for repeating the experiment of the present invention and cannot be used for other purposes.

[0040] Table 1. Information on germplasm resources for testing

[0041]

[0042]

[0043] Table 2. Information on the 9 closely related germplasm resources tested

[0044]

[0045]

[0046] Example 1: Development and screening method of Sabina SSR primer set

[0047] 1. Primer development and design methods

[0048] Based on the transcriptome sequencing data of needles, female flowers and cones of the juniper variety 'Fenghui', MISA (Microsatellite) software was used to detect Unigene, and the detection standards were set as: 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.

[0049] 2. Polymorphic primer screening method

[0050] The test materials were 8 varieties of Juniperus chinensis, including 5 female plants: 'Fenghui', 'Rongtuan', 'Lanta', 'Menglinlanbai' and 'Menglinzhongbai', and 3 male plants: 'Jinhua', 'Yunmi' and 'Diecui' (Table 1).

[0051] 1) DNA extraction and detection

[0052] The DNA of juniper needles was extracted using a plant genomic 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.

[0053] 2) PCR amplification

[0054] According to the primer design results of step 1, 250 pairs of primers were synthesized mainly with dinucleotides and trinucleotides for polymorphic primer screening.

[0055] The reaction system used for primer screening was 20 μL, including 10 μL of PCR mix, 0.3 μL of forward primer (20 μM), 0.3 μL of reverse primer (20 μM), 2 μL of template DNA, and 7.4 μL of ddH2O.

[0056] 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.

[0057] 3) Triple detection method to screen polymorphic SSR primers

[0058] Screening was carried out by agarose gel electrophoresis, polyacrylamide gel electrophoresis and capillary electrophoresis respectively.

[0059] Agarose gel electrophoresis detection is to use 2.0% agarose gel electrophoresis to detect the PCR amplification products of 8 juniper samples, and preliminarily select primers with target genes.

[0060] Polyacrylamide gel electrophoresis detection is to detect the PCR amplification products of 8 juniper samples using 6.0% polyacrylamide gel electrophoresis on the basis of the preliminary selection, and to re-select primers containing clear target bands and polymorphism.

[0061] Capillary electrophoresis detection was performed on the PCR amplification products of 8 juniper samples on the basis of re-selection to screen out efficient primers with rich sites, clear peak shapes and stable amplification.

[0062] 3. Obtaining polymorphic primers

[0063] According to the primer development and screening methods of steps 1 and 2, polymorphism analysis was performed on a total of 86 resources of Sabina and its closely related species (Y1-Y77 in Table 1; B02-B11 in Table 2), and finally a set of efficient microsatellite molecular marker primers based on the transcriptome sequence of Sabina was obtained, totaling 17 pairs (Table 3). As can be seen from Table 4, the total number of amplified bands of the 17 pairs of SSR primers obtained according to steps 1 and 2 ranged from 3 to 22, the number of polymorphic bands ranged from 3 to 22, and the polymorphism percentage of each pair of primers reached 100%.

[0064] Table 3. Information of 17 pairs of SSR marker primer sets for Sabina

[0065]

[0066] Table 4. Polymorphism results of 86 juniper and its related species amplified by SSR primers

[0067]

[0068]

[0069] Example 2: A method for identifying Sabina germplasm using SSR marker primer sets

[0070] The 77 juniper germplasm resources collected and preserved in the National Forestry and Grassland Germplasm Resource Bank (https: / / www.nfgrp.cn / , see Table 1 for specific information) were amplified using the juniper SSR primers numbered SSR-G34, SSR-G46, SSR-G102, SSR-G104, SSR-G108, SSR-G109, SSR-G111, SSR-G124, SSR-G149, SSR-G161, SSR-G164, SSR-G165, SSR-G173, SSR-G193, SSR-G197, SSR-G223, and SSR-G245 screened in Example 1. The fluorescent primer capillary electrophoresis method was used to amplify the 77 juniper germplasm resources collected and preserved in the National Forestry and Grassland Germplasm Resource Bank (https: / / www.nfgrp.cn / , see Table 1 for specific information). 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.

[0071] The specific steps are as follows:

[0072] 1. DNA extraction and detection

[0073] The genomic DNA of the needles of the juniper sample to be tested was extracted using a DNA extraction kit, and the purity and integrity of the DNA were detected using 2.0% agarose gel electrophoresis.

[0074] 2. Synthesis of fluorescent primers

[0075] The 5' end of the forward primer sequence of the 17 pairs of Sabina SSR marker primers developed in Example 1 was modified with a 6-FAM fluorescent group to synthesize 17 pairs of fluorescent SSR primers.

[0076] 3. PCR amplification of microsatellite fluorescent primers

[0077] The PCR amplification reaction system was 20 μL, including 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.

[0078] 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.

[0079] 4. Capillary fluorescence electrophoresis detection

[0080] 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.

[0081] 5. Germplasm resource identification

[0082] 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.

[0083] According to the clustering results of 17 pairs of SSR primers with fluorescent tags for 77 Sabina germplasm resources (see Table 1 for details), ( Figure 2 ): Except for Y32, Y46, Y32, Y48, Y49 and Y53, Y17 and Y33, Y16 and Y25, the remaining 67 germplasms can be completely distinguished and identified; and among the 10 germplasms that could not be completely distinguished, Y32, Y46, Y32, Y48, Y49 and Y53 belong to the same asexual line, Y17 and Y33 belong to the same asexual line, and Y16 and Y25 belong to the same asexual line. This further illustrates the reliability of the SSR primers developed by the present invention in germplasm identification.

[0084] Example 3: Application of SSR marker primer set in the construction of DNA molecular identity card of Sabina cultivars

[0085] The 17 pairs of juniper SSR primer sets numbered SSR-G34, SSR-G46, SSR-G102, SSR-G104, SSR-G108, SSR-G109, SSR-G111, SSR-G124, SSR-G149, SSR-G161, SSR-G164, SSR-G165, SSR-G173, SSR-G193, SSR-G197, SSR-G223, and SSR-G245 selected in Example 1 were used to amplify 4 juniper local varieties recorded and preserved in the National Forestry and Grassland Germplasm Resource Bank: 'Fenghui', 'Jinhua', 'Rongtuan', and 'Lanta'. The size of the fluorescent PCR product was detected by capillary electrophoresis.

[0086] 1. DNA extraction and detection

[0087] The genomic DNA of the four tested juniper needles was extracted using a DNA extraction kit, and the purity and integrity of the DNA were detected using 2.0% agarose gel electrophoresis.

[0088] 2. Synthesis of fluorescent primers

[0089] The 5' end of the forward primer sequence of the above 17 pairs of juniper SSR marker primers was modified with 6-FAM fluorescent group to synthesize 17 pairs of fluorescent SSR primers.

[0090] 3. PCR amplification of microsatellite fluorescent primers

[0091] The PCR amplification reaction system was 20 μL, including 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.

[0092] 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.

[0093] 4. Capillary fluorescence electrophoresis detection

[0094] 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.

[0095] 5. Construction of DNA molecular ID card

[0096] The sizes of fragments amplified by 17 pairs of SSR primers on four improved varieties of Sabina were counted, and the sizes of the amplified fragments of each material at the SSR site were arranged in order from small to large. The amplification of different variety resources at this site was counted, and the presence of a band was marked with "1" and the absence of a band was marked with "0".

[0097] The number of amplified bands of the 17 pairs of SSR primers in the four certified varieties of Sabina varied from 1 to 4, which indicates that the tested samples were polyploid. From the amplification results (Table 5), it can be seen that among the 17 pairs of SSR primers selected, any pair of primers from SSR-G34, SSR-G46, SSR-G104, SSR-G124, SSR-G149, SSR-G164, SSR-G197, SSR-G223, and SSR-G245 can identify and distinguish the four varieties.

[0098] Table 5. Locus information of 17 pairs of SSR primers in four improved varieties of Sabina

[0099]

[0100]

[0101] Furthermore, using nine pairs of SSR primers, namely SSR-G34, SSR-G46, SSR-G104, SSR-G124, SSR-G149, SSR-G164, SSR-G197, SSR-G223, and SSR-G245, the 0,1 method was used to construct DNA molecular identity cards consisting of 42 bits of code for four certified varieties of Juniperus chinensis (Tables 6 and 7).

[0102] Table 6. Amplified bands and corresponding codes of 9 pairs of SSR primers in four improved varieties of Sabina

[0103]

[0104]

[0105] Table 7. DNA molecular identity cards of four approved varieties of Juniperus chinensis

[0106] Juniperus chinensis DNA molecular ID card encoding 'Juniperus chinensis' 110011001110111000100011101110110100110011 'Rongtuan juniper' 001010010101000100011100110011010111111000 'Blue Tower Juniper' 010010111000010011010001010111111100110100 'Peak cypress' 011110000110111010110010110110000111100010

[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 primer set for SSR molecular markers of the genus Sabina, characterized in that: The SSR molecular marker primer set includes at least one pair of the 17 pairs of primers numbered SSR-G34, SSR-G46, SSR-G102, SSR-G104, SSR-G108, SSR-G109, SSR-G111, SSR-G124, SSR-G149, SSR-G161, SSR-G164, SSR-G165, SSR-G173, SSR-G193, SSR-G197, SSR-G223, and SSR-G245, and the nucleotide sequences of the 17 pairs of primers are shown in SEQ ID No.1 to SEQ ID No.

34.

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. 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 sequence of the juniper variety 'Fenghui' as a reference sequence, the Unigene SSR loci were detected 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.

5. The method according to claim 4, 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.

6. A method for analyzing genetic diversity or kinship of Sabina germplasm resources, the method comprising: Using the genomic DNA of the juniper germplasm resource to be tested as a template, PCR amplification is performed using the SSR molecular marker primer set described in claim 1 to obtain a PCR amplification product, the PCR amplification product is subjected to electrophoresis detection, and genetic diversity analysis or kinship analysis of the juniper germplasm resource is performed based on the electrophoresis detection result.

7. A method for identifying a genus Sabina species by fingerprint analysis, 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 germplasm resources and the fingerprint map of the standard juniper variety, and the variety is identified based on the fingerprint map comparison and analysis results.

8. The method according to claim 6 or 7, characterized in that: The 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; 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.

9. The method according to any one of claims 6 to 8, characterized in that: The electrophoresis detection is agarose gel electrophoresis detection, polyacrylamide gel electrophoresis detection, or / and capillary electrophoresis detection.

10. 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 the analysis of genetic diversity of Sabina; A2) Application in the analysis of the diversity of genus Sabina species resources; A3) Application in the analysis of phylogenetic relationships in the genus Sabina; A4) Application in identification of Sabina species; A5) Application in constructing a genetic map or DNA fingerprint map of the genus Sabina; A6) Application in improving the germplasm resources of Sabina genus; A7) Application in gene localization or functional gene mining of Sabina; A8) Application of molecular marker-assisted breeding in Sabina.

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