Primer combination of molecular marker development for juniperus and its application

Abstract

The application discloses a juniper SSR molecular marker development primer combination and application thereof. The application belongs to the field of plant molecular biology and particularly relates to a juniper SSR molecular marker development primer combination and application thereof. The juniper SSR molecular marker primer combination provided by the application comprises at least one of 18 pairs of primers, i.e., 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 are shown in SEQ ID No. 1-SEQ ID No. 36.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of plant molecular biology, and particularly relates to a Juniperus SSR molecular marker primer combination and application thereof. BACKGROUND

[0002] Juniperus and Sabina belong to Cupressaceae in gymnosperms, and were established by Linnaeus and Miller in 1753 and 1754 respectively, and belong to Juniperoideae Pilger. For a long time, the classification of Juniperus and Sabina has been controversial, and the morphology and growth of the leaves of the two genera are somewhat confused, which also brings trouble to breeders. At the same time, the protection of new plant variety rights is paid more and more attention by the state and breeders, and it is difficult to quickly and effectively distinguish variety resources only by morphological identification. Therefore, DNA molecular detection technologies represented by simple sequence repeats (SSR) and single nucleotide polymorphism (SNP) are gradually recognized, and are recommended by the UPOV molecular test guide as the preferred marker for variety authenticity identification and database construction. However, the DNA molecular marker technology of Juniperus plants is relatively backward, and there is no efficient and stable SSR molecular marker for application in the genus and intra-species of the genus for germplasm identification, genetic relationship analysis, fingerprint construction and the like. In order to better carry out the breeding of Cupressaceae plants and the protection of variety rights, and in order to better serve the breeders of Cupressaceae plants and protect the rights and interests of breeders, it is urgent to develop a universal SSR marker primer suitable for Juniperus and establish a DNA molecular detection technology. SUMMARY

[0003] The main problem to be solved by the application is how to identify the varieties of Juniperus and analyze the genetic relationship.

[0004] In order to solve the above problems, the application provides a Juniperus SSR molecular marker primer combination.

[0005] The Juniperus SSR molecular marker primer combination provided by the application comprises at least one 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 application provides a kit containing the SSR molecular marker primer group.

[0008] The application provides a DNA chip containing the SSR molecular marker primer group.

[0009] The application further provides the following applications of the SSR molecular marker primer group, the kit and / or the DNA chip:

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

[0011] A2) application in variety diversity analysis of Juniperus;

[0012] A3) application in genetic relationship analysis of Juniperus;

[0013] A4) application in variety identification of Juniperus;

[0014] A5) application in genetic map or DNA fingerprint map construction of Juniperus;

[0015] A6) application in germplasm improvement of Juniperus;

[0016] A7) application in gene location or functional gene mining of Juniperus;

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

[0018] The application further provides a screening method of the SSR molecular marker primer group, and the method comprises the following steps:

[0019] B1) detecting and designing primers for SSR sites of gene sequences (Unigene de) by taking the transcriptome sequences of P. ponderosa 'Blue Sky' and P. virginiana 'Grey Jay' as reference sequences to obtain SSR markers;

[0020] B2) screening the diversity of the SSR markers among germplasm resources materials according to 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 in B3) are: single nucleotide, di-nucleotide, tri-nucleotide, tetra-nucleotide, penta-nucleotide and hexa-nucleotide, and the minimum number of repetitions of each nucleotide is 12, 6, 5, 5, 4 and 4, respectively.

[0023] The application also provides a method for analyzing genetic diversity or genetic relationship of Juniperus, which comprises: using the genomic DNA of Juniperus to be tested as a template, performing PCR amplification by using the SSR molecular marker primer set described above, obtaining PCR amplification products, performing electrophoresis detection on the PCR amplification products, and analyzing genetic diversity or genetic relationship of Juniperus according to the results of electrophoresis detection.

[0024] Further, the analyzing genetic diversity or genetic relationship of Juniperus according to the results of electrophoresis detection comprises: counting the results of electrophoresis detection, recording "1" if there is a band at the same electrophoretic mobility position, and recording "0" if there is no band; further performing cluster analysis to construct a genetic relationship tree diagram, and analyzing genetic diversity or genetic relationship of Juniperus according to the counting results.

[0025] Further, Popgene 32 is used to analyze genetic diversity, and MEGA 11 is used to construct a genetic relationship cluster diagram.

[0026] The application also provides a method for analyzing Juniperus variety fingerprint to identify Juniperus varieties, which comprises: using the genomic DNA of a Juniperus variety to be tested and a standard Juniperus variety as templates, respectively, performing PCR amplification by using the SSR molecular marker primer set described above, performing electrophoresis detection on the PCR amplification products, obtaining a fingerprint of the Juniperus germplasm resource to be tested and a fingerprint of the standard variety, and identifying the Juniperus variety according to the comparison and analysis results of the fingerprints.

[0027] The application also provides a method for analyzing Sabina variety fingerprint to identify Sabina varieties, which comprises: using the genomic DNA of a Sabina variety to be tested and a standard Sabina variety as templates, respectively, performing PCR amplification by using the SSR molecular marker primer set described above, performing electrophoresis detection on the PCR amplification products, obtaining a fingerprint of the Sabina variety to be tested and a fingerprint of the standard variety, and identifying the Sabina variety according to the comparison and analysis results of the fingerprints.

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

[0029] The PCR amplification procedure is: 94 DEG C pre-denaturation for 5 min, 1 cycle; 94 DEG C denaturation for 30 s, 60 DEG C annealing for 40 s, 72 DEG C extension for 50 s, 10 cycles; 94 DEG C denaturation for 30 s, 53 DEG C annealing for 40 s, 72 DEG C extension for 50 s, 27 cycles; final 72 DEG C extension for 10 min.

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

[0031] In the present application, the Sabina chinensis (L.) Ant is mainly Sabina chinensis (L.) Ant, and the Thuja includes Thuja plicata, Thuja plicata, Thuja plicata, Thuja plicata, Thuja plicata, and the like.

[0032] In order to solve the technical problem of lack of DNA molecular marker primers of Cupressaceae plants, a set of SSR primers suitable for germplasm identification of Thuja and Sabina plants is developed based on the transcriptome sequence of Thuja plicata and Thuja plicata. Compared with the DUS test technology mainly based on phenotypic characteristics, the developed complete set of SSR primers has the advantages of stable amplification, high efficiency polymorphism, and the like, and is not limited by tree age, environment and the like, so that the germplasm identification between different species and intraspecific levels of Thuja can be effectively realized. BRIEF DESCRIPTION OF DRAWINGS

[0033] Fig. 1 Figure 4 is a capillary electrophoresis result of part of the SSR primers in the present application. Wherein A is the amplification result of primer SSR-J283 (BHE); B is the amplification result of primer SSR-J231 (YM); C is the amplification result of primer SSR-J28 (BLA).

[0034] Fig. 2 Figure 5 is a cluster diagram of 19 Thuja and Sabina variety resources constructed by using the complete set of SSR primers in the present application. DETAILED DESCRIPTION

[0035] The present application will be further described in detail below in conjunction with specific embodiments. The examples provided below are only for illustrating the present application, and are not intended to limit the scope of the present application. The examples provided below can serve as a guide for further improvement by those skilled in the art, and do not constitute any limitation on the present application in any way.

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

[0037] Quantitative experiments in the following examples were set up with three replicates, and the results were averaged.

[0038] The materials of ‘Lantian’, ‘Jin Hua’, ‘Jingyi No.3’, ‘Jingyi No.6’, ‘Y18’, ‘Y23’ in the following examples were from the Important Tree Species Germplasm Repository of Beijing Academy of Agriculture and Forestry Sciences, and were also collected in the National Forestry and Grassland Germplasm Repository (https: / / www.nfgrp.cn / ). The public can obtain the biological materials from the National Forestry and Grassland Germplasm Repository or the applicant. The biological materials are only for repeating the experiments of the present application 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 method

[0041] According to the transcriptome sequencing data of the leaves, female flowers and cones of Juniperus scopulorum Blue Heaven' and J. virginiana ‘Grey Owl’, the Unigene was detected by MISA software, and the detection standard was: single nucleotide, di-nucleotide, tri-nucleotide, tetra-nucleotide, penta-nucleotide and hexa-nucleotide, and the minimum number of repeats of each nucleotide was 12, 6, 5, 5, 4 and 4, respectively. Then, the detected SSR primers were designed by Primer 3.0.

[0042] 2. Polymorphic primer screening method

[0043] The test materials were 6 Juniperus varieties: ‘Lantian’, ‘Jin Hua’, ‘Jingyi No.3’, ‘Jingyi No.6’, Y18 and Y23. The materials were from the Important Tree Species Germplasm Repository of Beijing Academy of Agriculture and Forestry Sciences, and were also collected in the National Forestry and Grassland Germplasm Repository (https: / / www.nfgrp.cn / ). The specific platform resource number is shown in Table 1. https: / / www.nfgrp.cn /

[0044] Table 1, materials used for primer screening

[0045]

[0046] 1) DNA extraction and detection

[0047] The DNA of the leaves was extracted by using the DNA extraction kit (Tiangen Biotech (Beijing) Co., Ltd., DP305) according to the instruction steps, and the DNA purity was detected by 2.0% agarose gel electrophoresis.

[0048] 2) PCR amplification

[0049] ​According to the primer design result of step 1, 300 pairs of primers were synthesized for polymorphic primer screening, mainly dinucleotide and trinucleotide.

[0050] The reaction system (20 μL) used for primer screening: 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: 94℃ pre-denaturation for 5 min, 1 cycle; 94℃ denaturation for 30 s, 60℃ annealing for 40 s, 72℃ extension for 50 s, 10 cycles; 94℃ denaturation for 30 s, 53℃ annealing for 40 s, 72℃ extension for 50 s, 27 cycles; final 72℃ extension for 10 min.

[0052] 3) Polymorphic SSR primer screening

[0053] The screening was carried out by agarose gel electrophoresis, polyacrylamide gel electrophoresis and capillary electrophoresis, respectively.

[0054] Agarose gel electrophoresis detection: the PCR amplification products of 6 samples, i.e. 'Lantian', 'Jin Hua', 'Jingyi No. 3', 'Jingyi No. 6', Y18 and Y23, were detected by 2.0% agarose gel electrophoresis to initially select primers with target genes.

[0055] Polyacrylamide gel electrophoresis detection: on the basis of initial selection, the PCR amplification products of 6 samples were detected by 6.0% polyacrylamide gel electrophoresis to reselect primers containing clear target bands and polymorphisms.

[0056] Capillary electrophoresis detection: on the basis of reselection, the PCR amplification products of 'Lantian', 'Jin Hua', 'Jingyi No. 3', 'Jingyi No. 6', Y18 and Y23 were further detected by capillary electrophoresis to screen out high-efficiency primers with rich loci, clear peak type and stable amplification.

[0057] 3, Polymorphic primers

[0058] According to the primer development and screening method of steps 1 and 2, 20 pairs of polymorphic primers were obtained (Table 2), and then 19 varieties of Juniperus and Sabina (Table 3) were subjected to genetic diversity analysis using the 20 pairs of primers.

[0059] From Table 4, the total number of bands amplified by 20 pairs of primers was between 3 and 23, and the number of polymorphic bands was between 2 and 23. Among them, primer SSR-J116 amplified the least number of bands and had lower polymorphism; primer SSR-J150 had good polymorphism but weak amplification. The bands amplified by the remaining 18 pairs of primers were all polymorphic bands, and the polymorphism percentage of the primers all reached 100%. Finally, a set of high-efficiency microsatellite molecular marker primers based on the transcriptome sequences of Juniperus was obtained, a total of 18 pairs, which were primer groups 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, respectively.

[0060] Table 2, information of 20 pairs of SSR marker primer groups of Juniperus

[0061]

[0062]

[0063]

[0064] Table 3, information of 19 varieties of resources

[0065]

[0066]

[0067] Table 4, polymorphism results of SSR primers

[0068]

[0069]

[0070] Example 2, method for identifying Juniperus germplasm by using SSR marker primer groups

[0071] The juniper SSR primers screened in Example 1 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 were used to amplify 19 varieties of Sabina and Juniperus resources collected and preserved in the National Forestry and Grassland Germplasm Bank (Table 3) using fluorescent primer capillary electrophoresis. The size of the fluorescent PCR product was detected using capillary electrophoresis, the genetic diversity was statistically analyzed using Popgene 32, and the phylogenetic clustering diagram was constructed using MEGA 11.

[0072] The specific steps are as follows:

[0073] 1. DNA extraction and detection

[0074] Genomic DNA of needle leaves was extracted using a DNA extraction kit. The purity and integrity of the DNA were detected using 2.0% agarose gel electrophoresis.

[0075] 2. Fluorescent primer synthesis

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

[0077] 3. Microsatellite fluorescent primer PCR amplification

[0078] The PCR amplification reaction system (20 μL) was as follows: 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, and ddH2O 7.4 μL.

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

[0080] 4. Capillary fluorescent electrophoresis detection

[0081] Mix formamide with molecular weight marker in a volume ratio of 100:1, then take 9 μL and add to the loading plate, and then add 1 μL of 10-fold diluted PCR product obtained in step 3. Then use ABI 3730XL automatic DNA sequencer for capillary electrophoresis, and use Fragment (Plant) fragment analysis software in Genemarker to analyze the raw data obtained by the sequencer, compare the positions of the molecular weight markers in each lane with the positions of the peak values of each sample, and obtain the fragment size.

[0082] 5. Germplasm identification

[0083] Use Convert 1.31 software for format conversion, use Popgene 32 software for statistical analysis of genetic identity and genetic distance, use UPGMA method for germplasm clustering analysis, and construct a phylogenetic tree.

[0084] According to the above-mentioned 18 pairs of SSR primers with fluorescent labels, the genetic relationship of 19 varieties of resources is clustered and analyzed, and the 19 varieties of resources can be completely distinguished. At the same time, from the cluster diagram, it can be seen that the 6 varieties of Sabina of RT, FG, LT, DC, JH and YM are clustered together, the 2 varieties of BHE and BAR of Juniperus of the Rocky Mountain Juniper species are clustered together, the 2 varieties of SVI-x and SVI-c of the North American Juniper species are clustered together, the varieties of TIS and BDA of the Forked Juniper species are clustered together, and the remaining varieties are independently clustered, which further illustrates that the SSR primer composition developed in the present application has high efficiency in the identification of intraspecific varieties of Sabina, Rocky Mountain Juniper, North American Juniper and Forked Juniper, and genetic relationship analysis. Fig. 2 ).

[0085] Example 3, method for constructing DNA molecular identity card of Sabina plant new variety by using SSR marker primer group

[0086] Using the Juniperus SSR primers screened in Example 2, 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, 4 new varieties of Sabina obtained the national plant new variety right: 'Yunmi', 'Diecui', 'Menglin Lanbai' and 'Menglin Zhongbai'. The size of the fluorescent PCR product is detected by capillary electrophoresis.

[0087] 1. DNA extraction and detection

[0088] Genomic DNA of Juniperus chinensis needles was extracted by using DNA extraction kit. DNA purity and integrity were detected by 2.0% agarose gel electrophoresis.

[0089] 2. Fluorescent primer synthesis

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

[0091] 3. Microsatellite fluorescent primer PCR amplification

[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: 94°C pre-denaturation for 5 min, 1 cycle; 94°C denaturation for 30 s, 60°C annealing for 40 s, 72°C extension for 50 s, 10 cycles; 94°C denaturation for 30 s, 53°C annealing for 40 s, 72°C extension for 50 s, 27 cycles; final 72°C extension for 10 min.

[0094] 4. Capillary fluorescence electrophoresis detection

[0095] Formamide and molecular weight marker were mixed at a volume ratio of 100:1, 9 μL of which was added to the loading plate, and 1 μL of 10-fold diluted PCR product obtained in step 3 was added. Then capillary electrophoresis was performed using ABI 3730XL automatic DNA sequencer, and the raw data obtained by the sequencer were analyzed using Fragment (Plant) fragment analysis software in Genemarker. The positions of the molecular weight markers in each lane were compared with the positions of the peak values of each sample to obtain the fragment size.

[0096] 5. DNA molecular identity card construction

[0097] The fragment sizes amplified by 18 pairs of SSR primers SSRI6, 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 Juniperus plants were counted, and the amplified fragments of each material were arranged in order from small to large. The amplification of different varieties of resources at the site was counted, and according to the principle of from small to large, 1 was marked with, and 0 was marked without (Table 5).

[0098] The number of fragments of 18 pairs of SSR primers in four Juniperus varieties ranged from 2 to 11, and the amplification band type of each variety at the SSR site was 1-4. It can be inferred that the detection sample is a polyploid.

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

[0100] Table 5, 12 pairs of high-efficiency SSR primers in four new varieties of Juniperus amplification bands and corresponding codes

[0101]

[0102]

[0103]

[0104] Further, using the 12 pairs of SSR primers with high identification efficiency in Table 5, the fragments that do not exist in the four varieties are removed, and then arranged in order from small to large. According to the 0, 1 method, a DNA molecular identity card composed of 60 codes for four new varieties of Juniperus plants was constructed (Table 6).

[0105] Table 6, 60 code-based DNA molecular identity card of new varieties of Juniperus

[0106] Improved variety of Sabina chinensis DNA molecular identity certificate code ‘Diecui’ 100000101011100101001011101000001010000111110010110000111000 ‘Yunmi’ 001001010011001000011000011000000100001011010101011000000110 ‘Menglin Lanbai’ 011100100100011111000000100001110011011001001000000110111001 ‘Menglin Zhongbai’ 001110100101011011110100000111010110110101010000010011001000

[0107] The application has been described in detail. For those skilled in the art, the application can be implemented in a wider range under the same parameters, concentrations and conditions without departing from the spirit and scope of the application and without unnecessary experiments. Although the application gives a special example, it should be understood that the application can be further improved. In summary, according to the principle of the application, the application intends to include any change, use or improvement of the application, including the change made by the conventional technology known in the art, which is out of the range disclosed in the application.

Claims

1. A set of SSR molecular marker primers for Juniperus, characterized in that, The SSR molecular marker primer set comprises 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 as SEQ ID No. 1-SEQ ID No.

36.

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

3. A DNA chip characterized by The DNA chip comprises the SSR molecular marker primer set of claim 1.

4. The SSR molecular marker primer set of claim 1, and / or the kit of claim 2, and / or the DNA chip of claim 3, for any one of the following applications: A1) application in genetic diversity analysis of Juniperus; A2) application in variety diversity analysis of Juniperus; A3) application in genetic relationship analysis of Juniperus; A4) application in variety identification of Juniperus; A5) application in construction of genetic map or DNA fingerprint of Juniperus; A6) application in improvement of germplasm resources of Juniperus; A7) application in gene location or functional gene mining of Juniperus; A8) application in molecular marker assisted breeding of Juniperus.

5. A method for analyzing genetic diversity or relationship of the genus Juniperus using the primer set of the SSR molecular marker according to claim 1, wherein, The method comprises: using the genomic DNA of the to-be-tested Juniperus as a template, performing PCR amplification by using the SSR molecular marker primer set of claim 1 to obtain PCR amplification products, performing electrophoretic detection on the PCR amplification products, and performing genetic diversity analysis or genetic relationship analysis of Juniperus according to the electrophoretic detection results.

6. A method of analyzing a Juniperus cultivar fingerprint to identify a Juniperus cultivar, the method comprising: The genomic DNA of the to-be-tested Juniperus and the standard Juniperus variety are used as templates, respectively, PCR amplification is performed by using the SSR molecular marker primer set of claim 1, the PCR amplification products are subjected to electrophoretic detection, the fingerprint of the to-be-tested Juniperus variety and the fingerprint of the standard variety are obtained, and the identification of the Juniperus variety is performed according to the comparison and analysis results of the fingerprints.

7. A method of analyzing a Sabina variety fingerprint to identify a Sabina variety, the method comprising: The genomic DNA of the to-be-tested Sabina variety and the standard Sabina variety are used as templates, respectively, PCR amplification is performed by using the SSR molecular marker primer set of claim 1, the PCR amplification products are subjected to electrophoretic detection, the fingerprint of the to-be-tested Sabina variety and the fingerprint of the standard variety are obtained, and the identification of the Sabina variety is performed according to the comparison and analysis results of the fingerprints.

8. The method according to any of claims 5-7, characterized by, The PCR amplification reaction system is 20 μL: PCRmix 10 μL, 20 μM forward primer with a fluorescent label 0.3 μL, 20 μM reverse primer 0.3 μL, template DNA 2 μL, and ddH2O 7.4 μL. The PCR amplification procedure is: 94℃ pre-denaturation for 5 min, 1 cycle; 94℃ denaturation for 30 s, 60℃ annealing for 40 s, 72℃ extension for 50 s, 10 cycles; 94℃ denaturation for 30 s, 53℃ annealing for 40 s, 72℃ extension for 50 s, 27 cycles; final 72℃ extension for 10 min.

Images