Primer combination and kit of SSR molecular markers based on the genome of Aquilaria sinensis and its application

Abstract

The present invention belongs to the technical field of molecular biology, and specifically discloses a primer combination, a kit and an application of SSR molecular markers based on the genome of Aquilaria sinensis, wherein the primer combination is shown in SEQ ID No. 1-32. The primer combination has the advantages of good polymorphism, stable amplification, clear and easy-to-read electrophoresis results, etc., and can be applied to the research of genetic diversity analysis, fingerprint map construction and individual identification of Aquilaria sinensis.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and particularly relates to an SSR molecular marker primer combination, a kit and an application, and more particularly to an SSR molecular marker primer combination, a kit and an application based on a genome of Aquilaria sinensis. Background Art

[0002] Aquilaria sinensis (Lour.) Spreng., commonly known as earth agarwood, is an evergreen tree of the genus Aquilaria in the family Thymelaeaceae. It is a rare tree species unique to my country. Aquilaria sinensis (Lour.) Spreng., a natural fragrance and precious medicinal material, is produced by plants of the genus Aquilaria in the family Thymelaeaceae after being subjected to biological or abiotic stress. It can be used to treat stomach cold, vomiting, chest and abdominal distension and pain, kidney deficiency, qi reversal and shortness of breath. Due to its extremely high economic and medicinal value, Aquilaria sinensis was once overexploited and its wild resources were gradually depleted. In order to restore its population size, artificial breeding of Aquilaria sinensis has been widely carried out. However, the source of the existing Aquilaria sinensis germplasm resources is unclear and the genetic background is mixed, which seriously hinders the protection of its germplasm resources and the development of breeding work. At present, most of the research on Aquilaria sinensis is focused on seedling breeding, cultivation management and chemical composition analysis of agarwood. There is a lack of research on genetic diversity analysis of Aquilaria sinensis, identification of germplasm resources and fingerprint map construction, and there is an even greater lack of effective molecular markers to promote these studies.

[0003] Simple sequence repeat (SSR) markers are tandemly repeated DNA fragments composed of 1-6 nucleotides as repeating units. SSR markers are polymorphic due to the different number of repetitions at the same site. Among many molecular markers, SSR markers are widely used in plant genetic map construction, germplasm identification and population genetic diversity analysis due to their high polymorphism, co-dominance, good repeatability and easy detection. The development of SSR molecular markers for Aquilaria sinensis can provide an effective tool for analyzing the current status of its germplasm resources, fingerprint map construction and individual identification, which plays an important role in promoting the breeding of fine varieties and genetic improvement of Aquilaria sinensis. Summary of the invention

[0004] Based on the deficiencies of the prior art, the purpose of the present invention is to provide an SSR molecular marker primer combination, a kit and an application based on the genome of Aquilaria sinensis. The primer combination has the advantages of good polymorphism, stable amplification, and clear and easy-to-interpret electrophoresis results, and can be applied to studies such as genetic diversity analysis, fingerprint map construction and individual identification of Aquilaria sinensis.

[0005] To this end, the technical solution of the present invention is as follows:

[0006] In a first aspect, the present invention provides an SSR molecular marker primer combination based on the genome of Aquilaria sinensis, wherein the SSR molecular markers include As002, As005, As015, As016, As024, As047, As048, As049, As062, As065, As066, As069, As079, As081, As089 and As096;

[0007] The primer combination is (as shown in Table 1):

[0008] 1) The primers for amplifying the SSR molecular marker As002 are As002-F and As002-R, and the nucleotide sequences of As002-F and As002-R are shown in SEQ ID No. 1 and SEQ ID No. 2, respectively;

[0009] 2) The primers for amplifying the SSR molecular marker As005 are As005-F and As005-R, and the nucleotide sequences of As005-F and As005-R are shown in SEQ ID No. 3 and SEQ ID No. 4, respectively;

[0010] 3) The primers for amplifying the SSR molecular marker As015 are As015-F and As015-R, and the nucleotide sequences of As015-F and As015-R are shown in SEQ ID No. 5 and SEQ ID No. 6, respectively;

[0011] 4) The primers for amplifying the SSR molecular marker As016 are As016-F and As016-R, and the nucleotide sequences of As016-F and As016-R are shown in SEQ ID No.7 and SEQ ID No.8, respectively;

[0012] 5) The primers for amplifying the SSR molecular marker As024 are As024-F and As024-R, and the nucleotide sequences of As024-F and As024-R are shown in SEQ ID No.9 and SEQ ID No.10, respectively;

[0013] 6) The primers for amplifying the SSR molecular marker As047 are As047-F and As047-R, and the nucleotide sequences of As047-F and As047-R are shown in SEQ ID No.11 and SEQ ID No.12, respectively;

[0014] 7) The primers for amplifying the SSR molecular marker As048 are As048-F and As048-R, and the nucleotide sequences of As048-F and As048-R are shown in SEQ ID No. 13 and SEQ ID No. 14, respectively;

[0015] 8) The primers for amplifying the SSR molecular marker As049 are As049-F and As049-R, and the nucleotide sequences of As049-F and As049-R are shown in SEQ ID No.15 and SEQ ID No.16, respectively;

[0016] 9) The primers for amplifying the SSR molecular marker As062 are As062-F and As062-R, and the nucleotide sequences of As062-F and As062-R are shown in SEQ ID No. 17 and SEQ ID No. 18, respectively;

[0017] 10) The primers for amplifying the SSR molecular marker As065 are As065-F and As065-R, and the nucleotide sequences of As065-F and As065-R are shown in SEQ ID No. 19 and SEQ ID No. 20, respectively;

[0018] 11) The primers for amplifying the SSR molecular marker As066 are As066-F and As066-R, and the nucleotide sequences of As066-F and As066-R are shown in SEQ ID No.21 and SEQ ID No.22, respectively;

[0019] 12) The primers for amplifying the SSR molecular marker As069 are As069-F and As069-R, and the nucleotide sequences of As069-F and As069-R are shown in SEQ ID No. 23 and SEQ ID No. 24, respectively;

[0020] 13) The primers for amplifying the SSR molecular marker As079 are As079-F and As079-R, and the nucleotide sequences of As079-F and As079-R are shown in SEQ ID No. 25 and SEQ ID No. 26, respectively;

[0021] 14) The primers for amplifying the SSR molecular marker As081 are As081-F and As081-R, and the nucleotide sequences of As081-F and As081-R are shown in SEQ ID No. 27 and SEQ ID No. 28, respectively;

[0022] 15) The primers for amplifying the SSR molecular marker As089 are As089-F and As089-R, and the nucleotide sequences of As089-F and As089-R are shown in SEQ ID No. 29 and SEQ ID No. 30, respectively;

[0023] 16) The primers for amplifying the SSR molecular marker As096 are As096-F and As096-R. The nucleotide sequences of As096-F and As096-R are shown in SEQ ID No.31 and SEQ ID No.32, respectively.

[0024] Table 1 SSR primer combination sequences

[0025]

[0026]

[0027] In a second aspect, the present invention provides a kit for analyzing the genetic diversity of Aquilaria sinensis populations and constructing a fingerprint map of Aquilaria sinensis, characterized in that it comprises at least the primer combination SEQ ID No.1-SEQ ID No.32 for amplifying the first aspect.

[0028] Preferably, the PCR amplification system of the primer combination is a 15 μL system, including 7.5 μL of 2×Taq PCR Master Mix, 1.0 μL of each forward and reverse primer of each primer pair, 1 μL of DNA template and 4.5 μL of ddH2O. For example, SEQ ID No.1 and SEQ ID No.2 are a pair of forward and reverse primers labeled As002, and so on.

[0029] Preferably, the PCR amplification program of the primer combination is: preheating at 96°C for 3 min; performing 30 cycles of 96°C, 30s, 60°C, annealing for 30s, and extending at 72°C for 1 min; extending at 72°C for 10 min; and storing at 12°C.

[0030] In a third aspect, the present invention provides the use of the SSR molecular marker primer combination as described in the first aspect in genetic diversity analysis, fingerprint map construction or individual identification of Aquilaria sinensis.

[0031] Preferably, the application comprises at least the following steps:

[0032] 1) Extracting genomic DNA from the tested Aquilaria sinensis;

[0033] 2) using the SSR molecular marker primer combination to perform PCR amplification on the genomic DNA of the tested Aquilaria sinensis;

[0034] 3) Perform electrophoresis on the PCR amplification product to obtain the test results;

[0035] 4) Using the results of step 3), performing genetic diversity analysis, fingerprint construction or individual identification analysis of Aquilaria sinensis population.

[0036] Preferably, the PCR amplification system of the primer combination is a 15 μL system, including 7.5 μL of 2×Taq PCR Master Mix, 1.0 μL of each forward and reverse primer of each pair of primers, 1 μL of DNA template and 4.5 μL of ddH2O;

[0037] The PCR amplification program of the primer combination is: preheating at 96°C for 3 minutes; performing 30 cycles with the program of 96°C, 30 seconds, 60°C, annealing for 30 seconds, and extending at 72°C for 1 minute; extending at 72°C for 10 minutes; and storing at 12°C.

[0038] Preferably, the method for constructing the fingerprint of Aquilaria sinensis comprises at least the following steps:

[0039] 1) Use GeneMarker v2.2.0 software to perform band typing on the test results and determine the size of the amplified band of each primer pair in each sample;

[0040] 2) The "0, 1" system was used to record the positions of the amplified bands. The bands with obvious peaks at the same position were recorded as "1", and the bands without bands were recorded as "0". The 0, 1 matrix of the SSR markers of each sample was established according to the order of the allelic fragments amplified by each pair of SSR primers from small to large;

[0041] 3) Replace the positions marked as "1" in the matrix with blocks of different colors to draw the specific fingerprint of each sample.

[0042] Compared with the prior art, the SSR molecular marker primer combination, kit and application based on the genome of Aquilaria sinensis provided by the present invention have at least the following beneficial effects:

[0043] 1) The amount of Aquilaria sinensis genome data based on the SSR molecular markers and primer combinations provided by the present invention is large and reliable. At the same time, the application of the present invention uses the total DNA of Aquilaria sinensis as the material, which is not affected by plant tissues or materials at different growth stages.

[0044] 2) The Aquilaria sinensis-specific SSR primer combination provided by the present invention has been successfully applied to the genetic diversity analysis of the natural population of Aquilaria sinensis in Hainan Island, achieving the specific differentiation of 188 individual materials in the natural population and the construction of fingerprint maps, and has strong applicability.

[0045] 3) The SSR primers provided by the present invention have the advantages of stable amplification, high polymorphism, easy to distinguish detection results, etc., and are simple to operate, low cost, and easy to promote. The present invention provides an effective molecular tool for analyzing the genetic diversity and individual identification of white agarwood germplasm resources, and also creates conditions for early selection of traits and improving breeding efficiency, which is of great significance to the breeding and genetic improvement of white agarwood. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is the capillary electrophoresis diagram of partial amplification results of SSR primer combination As049 (SEQ ID No.15 and SEQ ID No.16) in white aquilaria samples.

[0047] Figure 2 This is a cluster analysis diagram of 188 Aquilaria sinensis individuals based on Nei's genetic distance.

[0048] Figure 3 Fingerprints of SSR markers As002, As005, As015, As016, As024, As047, As048 and As049 in 188 samples of Aquilaria sinensis.

[0049] Figure 4 Fingerprints of SSR markers As062, As065, As066, As069, As079, As081, As089 and As096 in 188 samples of Aquilaria sinensis. DETAILED DESCRIPTION

[0050] Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and should not be construed as limiting the present invention.

[0051] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example" or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine different embodiments or examples described in this specification.

[0052] Example 1: Development of highly polymorphic primers for SSR primers of Aquilaria sinensis

[0053] (1) Polymorphic SSR primer development

[0054] First, the whole genome sequence of Aquilaria sinensis was downloaded (http: / / gigadb.org / dataset / view / id / 100702), and then the SSR sites in the genome sequence of Aquilaria sinensis were searched using MISA software (https: / / webblast.ipk-gatersleben.de / misa / ). Then, primers were designed for the conserved sequences of 150 bp upstream and downstream of each SSR site, and finally 96 pairs of SSR primers were designed and synthesized. Leaves of Aquilaria sinensis from 10 geographically distant places, including Haikou (M003), Sanya (M026), Danzhou (M035), Dongfang (M083), Ledong (M093), Wenchang (M113 and M131), Ding'an (M138), Wanning (M145) and Baoting (M167), were used as materials (Table 3), and the plant genomic DNA extraction kit (Cat. No.: DP305) of Tiangen Biotechnology (Beijing) Co., Ltd. was used to extract sample DNA. Then, the 10 DNA samples were subjected to PCR amplification and capillary electrophoresis with 96 pairs of synthesized primers to detect the effectiveness and polymorphism of the primers.

[0055] (2) PCR amplification

[0056] The PCR amplification system is 15 μL, including 7.5 μL of 2×Taq PCR Master Mix, 2.0 μL of mixed primers (with fluorescent groups that can bind to the M13 universal adapter sequence added), 1 μL of DNA template (50-200 ng), and 4.5 μL of ddH2O.

[0057] The PCR amplification program was as follows: ① preheating at 96°C for 3 min, ② 30 cycles of 96°C for 30 s, 60°C for annealing for 30 s, and 72°C for extension for 1 min, ③ extension at 72°C for 10 min, and ④ storage at 12°C.

[0058] Table 2 Polymorphic characteristics of 16 pairs of SSR primers of Aquilaria sinensis

[0059]

[0060]

[0061] Note: N: effective number of individuals; Na: number of alleles; Ne: effective number of alleles; I: Shannon's information index; Ho: observed heterozygosity; He: expected heterozygosity; F: fixation index; PIC: polymorphism information content index.

[0062] The obtained fluorescent PCR product was detected by fluorescence electrophoresis using ABI 3730xl, and the detection system was: Hi-DiFormamide 10μL, internal standard 0.5μL (a mixture of formamide and internal standard prepared in advance), and PCR product 1.0μL. The detection results were then band typed using GeneMarker v2.2.0 software, and primers with stable amplification results, no impurity peaks, and good polymorphism were screened.

[0063] (3) Experimental results

[0064] The amplification results showed that among the 96 pairs of SSR primers, 13 pairs of primers failed to amplify, 1 pair of primers had poor peak shape, 1 pair of primers had continuous peaks, 9 pairs of primers had interference from miscellaneous peaks, and 48 pairs of primers could only amplify products of 1-2 lengths. The remaining 16 pairs of primers can amplify more than 2 specific bands, and the site polymorphism information content PIC>0.30. This shows that the 16 pairs of SSR primers screened have good polymorphism and have application value. These 16 pairs of primers are As002, As005, As015, As016, As024, As047, As048, As049, As062, As065, As066, As069, As079, As081, As089 and As096. The primer sequence information is shown in Table 1, the primer polymorphism information is shown in Table 2, and the capillary electrophoresis diagrams of some primers are shown in Table 1. Figure 1 .

[0065] Example 2: Genetic clustering and population diversity analysis of natural populations of Aquilaria sinensis

[0066] (1) Experimental Materials

[0067] Table 3 Information on the sampling locations of 188 samples of Aquilaria sinensis

[0068]

[0069]

[0070] The experimental materials of this embodiment are 188 samples of Aucklandia odorata collected from 16 regions including Haikou, Sanya, Danzhou, Dongfang, Ledong, Wenchang, Ding'an, Tunchang, Chengmai, Wanning, Lingao, Qionghai, Baoting, Lingshui, Changjiang and Baisha (Table 3). Healthy young leaves of each tree were collected, put into numbered collection bags, and then classified by collection location and put into -80°C refrigerator for standby use.

[0071] The materials collected mainly include: ① the ancient and famous trees of Aquilaria sinensis recorded in the existing public data; ② the materials with an age of more than 30 years and a breast diameter of more than 10 cm according to the geographical division of Hainan; ③ the scattered Aquilaria sinensis trees with an age of more than 30 years in the mountains and forests. In order to ensure that the individual materials of Aquilaria sinensis collected have no kinship, the geographical distance between different individuals during collection is greater than 10 km.

[0072] (2) DNA extraction from Aquilaria sinensis

[0073] The DNA of each natural population sample of Aquilaria sinensis was extracted separately, using the same DNA extraction method as in Example 1.

[0074] (3) PCR amplification and capillary electrophoresis detection

[0075] The DNA of 188 samples of natural population of Aquilaria sinensis obtained in step (2) was used as template material, and PCR amplification was performed with 16 pairs of SSR primers developed by the present invention, and the obtained amplified products were detected by capillary electrophoresis. Then, the detection results were subjected to band typing analysis using GeneMarker v2.2.0 software. PCR amplification and capillary electrophoresis detection were the same as those in Example 1.

[0076] (4) Data analysis

[0077] ① The GenAIex software was used to calculate and statistically analyze the genetic diversity parameters of each population, including the number of alleles (Na), allele frequency, effective number of alleles (Ne), expected heterozygosity (He), observed heterozygosity (Ho), Shannon's information index (I) and fixation index (F).

[0078] ②The aboot method of the R package poppr was used to perform cluster analysis among individuals based on the Nei's genetic distance between samples.

[0079] (5) Experimental results

[0080] Nei's genetic distance cluster analysis results of 188 natural population germplasm resources of Aquilaria sinensis and the number of samples in each category are shown in Figure 2 Figure 2 M128, M129 and M130 (Wenchang) were first clustered into the first category, followed by M108 (Wenchang) into the second category, and M122 and M123 (Wenchang) into the third category. These three categories of Aucklandia odora were the farthest genetically from all other samples.

[0081] The 188 samples of Aquilaria sinensis were divided into 13 groups according to the collection sites. The detailed information of the groups is shown in Table 3. The results of the genetic diversity analysis of Aquilaria sinensis populations are shown in Table 4. The Ho and He of the 13 populations were 0.475-0.719 and 0.510-0.655, with average values ​​of 0.590 and 0.586, respectively. Among them, the Ho value of population VIII (Tunchang, Chengmai) was the largest, and the He value of population VI (Wenchang) was the largest. The F value was -0.322-0.169, among which the F values ​​of populations V (Ledong), VII (Ding'an), VIII (Tunchang, Chengmai), IX (Wanning), X (Lingao) and XIII (Changjiang, Baisha) were less than 0, indicating that these 6 populations were more heterozygous. The I values ​​of the 13 populations were 0.869-1.301, with an average value of 1.066, among which the I value of population VI was the largest, and the population differentiation degree was the highest (Table 4). The above examples show that the primers provided by the present invention have good polymorphism and high amplification efficiency, and can be applied to the population genetic diversity analysis of Aquilaria sinensis. The analysis results can more accurately evaluate the current status of germplasm resources of the natural population of Aquilaria sinensis in Hainan.

[0082] Table 4 Analysis of genetic diversity of Aquilaria sinensis population

[0083]

[0084] Note: N: effective number of individuals; Na: number of alleles; Ne: effective number of alleles; I: Shannon's information index; Ho: observed heterozygosity; He: expected heterozygosity; F: fixation index. The information of each population corresponding to population numbers Ⅰ to XIII is shown in Table 3.

[0085] Example 3: Construction of fingerprints of natural populations of Aquilaria sinensis and identification of individuals

[0086] (1) Experimental materials and construction methods

[0087] According to the PCR and capillary electrophoresis test results of 16 pairs of SSR primers provided by the present invention in 188 samples of Aucklandia lappa, the "0, 1" system is used to record the position of the amplified bands. The bands with obvious peaks at the same position are recorded as "1", and no bands are recorded as "0". The 0, 1 matrix of 188 samples is constructed in the order of small to large allele fragments amplified by SSR primers. Then, the position of "1" in the matrix is ​​replaced by a colored block, and the specific fingerprint of each sample is drawn. The differences in fingerprints between different individual materials are compared to identify different individuals.

[0088] (2) Experimental results

[0089] The specific fingerprint of each sample was constructed based on the "0, 1" matrix constructed according to the amplification test results of each pair of SSR primers. The results are shown in Figure 3 to Figure 4. By comparing these fingerprint maps, the results show that the 16 pairs of SSR primers provided by the present invention can distinguish 176 samples out of 188 tested white agarwood materials, with a discrimination rate of 93.62%. The above results also show that the 16 pairs of SSR molecular marker primers provided by the present invention have good polymorphism and stable amplification, and can be applied to white agarwood genetic clustering, population genetic diversity analysis, fingerprint map construction analysis, etc. At the same time, the primers invented also achieve accurate distinction between individuals in the natural population of white agarwood, indicating that the developed primers are highly efficient and have application value in the identification of white agarwood individuals.

[0090] The above results also show that the 16 pairs of SSR primers provided by the present invention have good polymorphism and stable amplification, and can be applied to genetic clustering, population genetic diversity analysis and fingerprint map construction of Aquilaria sinensis. At the same time, the primers of the present invention can accurately distinguish individuals in the natural population of Aquilaria sinensis, which also shows that the developed primers are highly efficient and have application value in the identification of Aquilaria sinensis individuals.

[0091] In summary, the amount of Aquilaria sinensis genome data based on the SSR molecular markers and primer combinations provided by the present invention is large and reliable. At the same time, the application of the present invention uses the total DNA of Aquilaria sinensis as the material, which is not affected by plant tissues or materials in different growth periods. The Aquilaria sinensis-specific SSR primer combination provided by the present invention has been successfully applied to the analysis of the genetic diversity of the natural population of Aquilaria sinensis in Hainan Island, and has achieved the specific distinction and fingerprint map construction of 188 individual materials in the natural population, and has strong applicability. The SSR primers provided by the present invention have the advantages of high polymorphism, stable amplification, and easy to distinguish test results, and are simple to operate, low in cost, and easy to promote. It can provide an effective molecular tool for analyzing the genetic diversity of Aquilaria sinensis germplasm resources and individual identification, and can also create conditions for realizing early selection of traits and improving breeding efficiency, which has a driving effect on the breeding and genetic improvement of Aquilaria sinensis.

[0092] It should be noted and understood that various modifications and improvements can be made to the present invention described in detail above without departing from the spirit and scope of the present invention required by the appended claims. Therefore, the scope of the technical solution claimed is not limited by any specific exemplary teaching given.

[0093] The applicant declares that the above contents are further detailed descriptions of the present invention in combination with specific preferred embodiments, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, which should be regarded as falling within the scope of protection of the present invention.

Claims

1. The SSR molecular marker primer combination based on the genome of Aquilaria sinensis is characterized by: The SSR molecular markers are: As002, As005, As015, As016, As024, As047, As048, As049, As062, As065, As066, As069, As079, As081, As089 and As096; The primer combination is: 1) The primers for amplifying the SSR molecular marker As002 are As002-F and As002-R, and the nucleotide sequences of As002-F and As002-R are shown in SEQ ID No. 1 and SEQ ID No. 2, respectively; 2) The primers for amplifying the SSR molecular marker As005 are As005-F and As005-R, and the nucleotide sequences of As005-F and As005-R are shown in SEQ ID No. 3 and SEQ ID No. 4, respectively; 3) The primers for amplifying the SSR molecular marker As015 are As015-F and As015-R, and the nucleotide sequences of As015-F and As015-R are shown in SEQ ID No. 5 and SEQ ID No. 6, respectively; 4) The primers for amplifying the SSR molecular marker As016 are As016-F and As016-R, and the nucleotide sequences of As016-F and As016-R are shown in SEQ ID No.7 and SEQ ID No.8, respectively; 5) The primers for amplifying the SSR molecular marker As024 are As024-F and As024-R, and the nucleotide sequences of As024-F and As024-R are shown in SEQ ID No.9 and SEQ ID No.10, respectively; 6) The primers for amplifying the SSR molecular marker As047 are As047-F and As047-R, and the nucleotide sequences of As047-F and As047-R are shown in SEQ ID No.11 and SEQ ID No.12, respectively; 7) The primers for amplifying the SSR molecular marker As048 are As048-F and As048-R, and the nucleotide sequences of As048-F and As048-R are shown in SEQ ID No. 13 and SEQ ID No. 14, respectively; 8) The primers for amplifying the SSR molecular marker As049 are As049-F and As049-R, and the nucleotide sequences of As049-F and As049-R are shown in SEQ ID No.15 and SEQ ID No.16, respectively; 9) The primers for amplifying the SSR molecular marker As062 are As062-F and As062-R, and the nucleotide sequences of As062-F and As062-R are shown in SEQ ID No. 17 and SEQ ID No. 18, respectively; 10) The primers for amplifying the SSR molecular marker As065 are As065-F and As065-R, and the nucleotide sequences of As065-F and As065-R are shown in SEQ ID No. 19 and SEQ ID No. 20, respectively; 11) The primers for amplifying the SSR molecular marker As066 are As066-F and As066-R, and the nucleotide sequences of As066-F and As066-R are shown in SEQ ID No.21 and SEQ ID No.22, respectively; 12) The primers for amplifying the SSR molecular marker As069 are As069-F and As069-R, and the nucleotide sequences of As069-F and As069-R are shown in SEQ ID No. 23 and SEQ ID No. 24, respectively; 13) The primers for amplifying the SSR molecular marker As079 are As079-F and As079-R, and the nucleotide sequences of As079-F and As079-R are shown in SEQ ID No. 25 and SEQ ID No. 26, respectively; 14) The primers for amplifying the SSR molecular marker As081 are As081-F and As081-R, and the nucleotide sequences of As081-F and As081-R are shown in SEQ ID No. 27 and SEQ ID No. 28, respectively; 15) The primers for amplifying the SSR molecular marker As089 are As089-F and As089-R, and the nucleotide sequences of As089-F and As089-R are shown in SEQ ID No. 29 and SEQ ID No. 30, respectively; 16) The primers for amplifying the SSR molecular marker As096 are As096-F and As096-R. The nucleotide sequences of As096-F and As096-R are shown in SEQ ID No.31 and SEQ ID No.32, respectively.

2. A kit for analyzing the genetic diversity of Aquilaria sinensis populations and constructing a fingerprint of Aquilaria sinensis, characterized in that: At least comprises the primer combination SEQ ID No.1-SEQ ID No.32 described in claim 1.

3. Application of the SSR molecular marker primer combination described in claim 1 in genetic diversity analysis, fingerprint construction or individual identification of Aquilaria sinensis.

4. The use according to claim 3, characterized in that: At least the following steps are included: 1) Extracting genomic DNA from the tested Aquilaria sinensis; 2) using the SSR molecular marker primer combination to perform PCR amplification on the genomic DNA of the tested Aquilaria sinensis; 3) Perform electrophoresis on the PCR amplification product to obtain the test results; 4) Using the results of step 3), performing genetic diversity analysis, fingerprint construction or individual identification analysis of Aquilaria sinensis population.

5. The use according to claim 4, characterized in that: The PCR amplification system of the primer combination is 15 μL, including 7.5 μL of 2×Taq PCR Master Mix, 1.0 μL of each forward and reverse primer of each primer pair, 1 μL of DNA template and 4.5 μL of ddH2O; The PCR amplification program of the primer combination is: preheating at 96°C for 3 minutes; performing 30 cycles with the program of 96°C, 30 seconds, 60°C, annealing for 30 seconds, and extending at 72°C for 1 minute; extending at 72°C for 10 minutes; and storing at 12°C.

6. The use according to claim 4, characterized in that: The method for constructing the fingerprint spectrum of Aquilaria sinensis comprises at least the following steps: 1) Use GeneMarker v2.2.0 software to perform band typing on the test results and determine the size of the amplified band of each primer pair in each sample; 2) The "0, 1" system was used to record the positions of the amplified bands. The bands with obvious peaks at the same position were recorded as "1", and the bands without bands were recorded as "0". The 0, 1 matrix of the SSR markers of each sample was established according to the order of the allelic fragments amplified by each pair of SSR primers from small to large; 3) Replace the positions marked as "1" in the matrix with blocks of different colors to draw the specific fingerprint of each sample.

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