Primer combination and kit of SSR molecular markers based on Dalbergia odorifera genome and their application

Abstract

The present invention belongs to the technical field of molecular biology, and specifically discloses a SSR molecular marker primer combination based on the Dalbergia odorifera genome, a kit and an application thereof, wherein the primer combination is shown in SEQ ID No.1-56. 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 Dalbergia odorifera.

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 Dalbergia odorifera genome. Background Art

[0002] Dalbergia odorifera T. Chen, commonly known as Hainan Huanghuali, is a semi-deciduous tree of the genus Dalbergia in the Leguminosae family. It is one of the rare species unique to Hainan. The heartwood of Dalbergia odorifera is brightly colored, densely structured, hard, with clear texture, resistant to wet decay, and has a unique fragrance. It is a top-quality material for making precious furniture, musical instruments, decorations, and high-end handicrafts. At the same time, the dried heartwood of the trunk and roots of Dalbergia odorifera is also a precious Chinese medicinal material - Dalbergia odorifera, which has the effects of removing blood stasis and stopping bleeding, regulating qi and relieving pain. Dalbergia odorifera was once overexploited, and its wild resources were severely damaged. In order to restore its population size, artificial breeding of Dalbergia odorifera has been widely carried out, but this has also led to the mixed genetic background of the existing Dalbergia odorifera, and the sources of some germplasm resources are unclear, which to a certain extent hindered the protection of its germplasm resources and the development of breeding work. At present, most of the research focuses on the seedling breeding, cultivation management and chemical composition analysis of Dalbergia odorifera. There is a lack of research on the genetic diversity analysis, germplasm resource identification and DNA fingerprint construction of Dalbergia odorifera, and there is an even greater lack of effective molecular markers to carry out these studies.

[0003] Simple sequence repeat (SSR) markers are tandemly repeated DNA fragments composed of nucleotides as repeating units, generally with 1-6 nucleotides repeated multiple times in tandem, and different numbers of repetitions at the same site cause the polymorphism of SSR markers. Among many molecular markers, SSR markers are widely used in plant genetic map construction, germplasm identification, and population genetic diversity due to their co-dominance, rich alleles, and easy detection. The development of SSR markers for Dalbergia odorifera can provide an effective molecular tool for analyzing the current status of its germplasm resources, individual identification, and fingerprint map construction, which is of great significance to the breeding of fine varieties and genetic improvement of Dalbergia odorifera. 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 Dalbergia odorifera genome. The primer combination has the advantages of good polymorphism, stable amplification, and clear and easy-to-interpret electrophoresis results, and can be applied to the research of genetic diversity analysis, fingerprint map construction and individual identification of Dalbergia odorifera.

[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 Dalbergia odorifera genome, wherein the SSR molecular markers include Do002, Do004, Do005, Do006, Do010, Do013, Do022, Do025, Do034, Do051, Do062, Do066, Do081, Do094, Do097, Do098, Do100, Do104, Do105, Do113, Do121, Do128, Do129, Do130, Do132, Do133, Do136 and Do137;

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

[0008] 1) The primers for amplifying the SSR molecular marker Do002 are Do002-F and Do002-R, and the nucleotide sequences of Do002-F and Do002-R are shown in SEQ ID No. 1 and SEQ ID No. 2, respectively;

[0009] 2) The primers for amplifying the SSR molecular marker Do004 are Do004-F and Do004-R, and the nucleotide sequences of Do004-F and Do004-R are shown in SEQ ID No. 3 and SEQ ID No. 4, respectively;

[0010] 3) The primers for amplifying the SSR molecular marker Do005 are Do005-F and Do005-R, and the nucleotide sequences of Do005-F and Do005-R are shown in SEQ ID No. 5 and SEQ ID No. 6, respectively;

[0011] 4) The primers for amplifying the SSR molecular marker Do006 are Do006-F and Do006-R, and the nucleotide sequences of Do006-F and Do006-R are shown in SEQ ID No.7 and SEQ ID No.8, respectively;

[0012] 5) The primers for amplifying the SSR molecular marker Do010 are Do010-F and Do010-R, and the nucleotide sequences of Do010-F and Do010-R are shown in SEQ ID No.9 and SEQ ID No.10, respectively;

[0013] 6) The primers for amplifying the SSR molecular marker Do013 are Do013-F and Do013-R, and the nucleotide sequences of Do013-F and Do013-R are shown in SEQ ID No.11 and SEQ ID No.12, respectively;

[0014] 7) The primers for amplifying the SSR molecular marker Do022 are Do022-F and Do022-R, and the nucleotide sequences of Do022-F and Do022-R are shown in SEQ ID No. 13 and SEQ ID No. 14, respectively;

[0015] 8) The primers for amplifying the SSR molecular marker Do025 are Do025-F and Do025-R, and the nucleotide sequences of Do025-F and Do025-R are shown in SEQ ID No. 15 and SEQ ID No. 16, respectively;

[0016] 9) The primers for amplifying the SSR molecular marker Do034 are Do034-F and Do034-R, and the nucleotide sequences of Do034-F and Do034-R are shown in SEQ ID No. 17 and SEQ ID No. 18, respectively;

[0017] 10) The primers for amplifying the SSR molecular marker Do051 are Do051-F and Do051-R, and the nucleotide sequences of Do051-F and Do051-R are shown in SEQ ID No. 19 and SEQ ID No. 20, respectively;

[0018] 11) The primers for amplifying the SSR molecular marker Do062 are Do062-F and Do062-R, and the nucleotide sequences of Do062-F and Do062-R are shown in SEQ ID No. 21 and SEQ ID No. 22, respectively;

[0019] 12) The primers for amplifying the SSR molecular marker Do066 are Do066-F and Do066-R, and the nucleotide sequences of Do066-F and Do066-R are shown in SEQ ID No. 23 and SEQ ID No. 24, respectively;

[0020] 13) The primers for amplifying the SSR molecular marker Do081 are Do081-F and Do081-R, and the nucleotide sequences of Do081-F and Do081-R are shown in SEQ ID No. 25 and SEQ ID No. 26, respectively;

[0021] 14) The primers for amplifying the SSR molecular marker Do094 are Do094-F and Do094-R, and the nucleotide sequences of Do094-F and Do094-R are shown in SEQ ID No. 27 and SEQ ID No. 28, respectively;

[0022] 15) The primers for amplifying the SSR molecular marker Do097 are Do097-F and Do097-R, and the nucleotide sequences of Do097-F and Do097-R are shown in SEQ ID No. 29 and SEQ ID No. 30, respectively;

[0023] 16) The primers for amplifying the SSR molecular marker Do098 are Do098-F and Do098-R, and the nucleotide sequences of Do098-F and Do098-R are shown in SEQ ID No.31 and SEQ ID No.32, respectively;

[0024] 17) The primers for amplifying the SSR molecular marker Do100 are Do100-F and Do100-R, and the nucleotide sequences of Do100-F and Do100-R are shown in SEQ ID No.33 and SEQ ID No.34, respectively;

[0025] 18) The primers for amplifying the SSR molecular marker Do104 are Do104-F and Do104-R, and the nucleotide sequences of Do104-F and Do104-R are shown in SEQ ID No.35 and SEQ ID No.36, respectively;

[0026] 19) The primers for amplifying the SSR molecular marker Do105 are Do105-F and Do105-R, and the nucleotide sequences of Do105-F and Do105-R are shown in SEQ ID No.37 and SEQ ID No.38, respectively;

[0027] 20) The primers for amplifying the SSR molecular marker Do113 are Do113-F and Do113-R, and the nucleotide sequences of Do113-F and Do113-R are shown in SEQ ID No.39 and SEQ ID No.40, respectively;

[0028] 21) The primers for amplifying the SSR molecular marker Do121 are Do121-F and Do121-R, and the nucleotide sequences of Do121-F and Do121-R are shown in SEQ ID No.41 and SEQ ID No.42, respectively;

[0029] 22) The primers for amplifying the SSR molecular marker Do128 are Do128-F and Do128-R, and the nucleotide sequences of Do128-F and Do128-R are shown in SEQ ID No.43 and SEQ ID No.44, respectively;

[0030] 23) The primers for amplifying the SSR molecular marker Do129 are Do129-F and Do129-R, and the nucleotide sequences of Do129-F and Do129-R are shown in SEQ ID No.45 and SEQ ID No.46, respectively;

[0031] 24) The primers for amplifying the SSR molecular marker Do130 are Do130-F and Do130-R, and the nucleotide sequences of Do130-F and Do130-R are shown in SEQ ID No.47 and SEQ ID No.48, respectively;

[0032] 25) The primers for amplifying the SSR molecular marker Do132 are Do132-F and Do132-R, and the nucleotide sequences of Do132-F and Do132-R are shown in SEQ ID No.49 and SEQ ID No.50, respectively;

[0033] 26) The primers for amplifying the SSR molecular marker Do133 are Do133-F and Do133-R, and the nucleotide sequences of Do133-F and Do133-R are shown in SEQ ID No.51 and SEQ ID No.52, respectively;

[0034] 27) The primers for amplifying the SSR molecular marker Do136 are Do136-F and Do136-R, and the nucleotide sequences of Do136-F and Do136-R are shown in SEQ ID No.53 and SEQ ID No.54, respectively;

[0035] 28) The primers for amplifying the SSR molecular marker Do137 are Do137-F and Do137-R. The nucleotide sequences of Do137-F and Do137-R are shown in SEQ ID No.55 and SEQ ID No.56, respectively.

[0036] Table 1 SSR primer combination sequences

[0037]

[0038]

[0039] In a second aspect, the present invention provides a kit for analyzing the genetic diversity of Dalbergia odorifera populations and constructing a fingerprint map of Dalbergia odorifera, characterized in that it comprises at least the primer combination SEQ ID No.1-SEQID No.56 for amplifying the first aspect.

[0040] Preferably, the PCR amplification system of the primer combination is a 15 μL system, including 7.5 μL of 2×TaqPCR MasterMix, 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. For example, SEQ ID No.1 and SEQ ID No.2 are a pair of forward and reverse primers labeled Do002, and so on, which will not be repeated.

[0041] 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.

[0042] 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 construction or individual identification of Dalbergia odorifera.

[0043] Preferably, the application comprises at least the following steps:

[0044] 1) Extracting genomic DNA from the tested Dalbergia odorifera;

[0045] 2) using the SSR molecular marker primer combination to perform PCR amplification on the genomic DNA of the tested Dalbergia odorifera;

[0046] 3) Perform electrophoresis on the PCR amplification product to obtain the test results;

[0047] 4) Using the results of step 3), performing population genetic diversity analysis, fingerprint construction or individual identification analysis of Dalbergia odorifera.

[0048] Preferably, the PCR amplification system of the primer combination is a 15 μL system, including 7.5 μL of 2×TaqPCR MasterMix, 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;

[0049] 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.

[0050] Preferably, the method for constructing the fingerprint of Dalbergia odorifera comprises at least the following steps:

[0051] 1) Use the software GeneMarkerv2.2.0 to perform band typing on the test results and determine the size of the amplified bands of each pair of primers in each sample;

[0052] 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 marked as "1", and the bands without bands were marked 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;

[0053] 3) Replace the positions marked as "1" in the matrix with blocks of different colors to draw the specific fingerprint of each sample.

[0054] Compared with the prior art, the SSR molecular marker primer combination, kit and application based on the Dalbergia odorifera genome provided by the present invention have at least the following beneficial effects:

[0055] 1) The SSR molecular markers and primer combinations provided by the present invention are based on a large amount of Dalbergia odorifera genome data and are reliable. At the same time, the application of the present invention uses Dalbergia odorifera total DNA as material and is not affected or limited by plant tissues or materials at different growth stages.

[0056] 2) The Dalbergia odorifera-specific SSR primer combination provided by the present invention has been successfully applied to the analysis of the genetic diversity of different natural populations of Dalbergia odorifera in Hainan Island, and has achieved the specific differentiation and identification of 102 individual materials in different natural populations and the construction of fingerprints, and has strong applicability.

[0057] 3) The SSR primers provided by the present invention have the advantages of stable amplification, high polymorphism, and easy-to-distinguish detection results, and are simple to operate, low cost, and easy to promote. They can not only provide an effective molecular tool for analyzing the genetic diversity and individual identification of Dalbergia odorifera germplasm resources, but also create conditions for achieving early selection of traits and improving breeding efficiency, which is of great significance to the breeding of fine varieties and genetic improvement of Dalbergia odorifera. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 This is the capillary electrophoresis diagram of partial amplification results of SSR primer combination Do004 (SEQ ID No. 3 and SEQ ID No. 4) in Dalbergia odorifera sample.

[0059] Figure 2 This is a cluster analysis diagram among 102 Dalbergia odorifera individuals based on Nei's genetic distance.

[0060] Figure 3 Fingerprints of SSR markers Do002, Do004, Do005, Do006, Do010, Do013 and Do022 in 102 samples of Dalbergia odorifera.

[0061] Figure 4Fingerprints of SSR markers Do025, Do034, Do051, Do062, Do066, Do081, Do094, Do097 and Do098 in 102 samples of Dalbergia odorifera.

[0062] Figure 5 Fingerprints of SSR markers Do100, Do104, Do105, Do113 and Do121 in 102 samples of Dalbergia odorifera.

[0063] Figure 6 Fingerprints of SSR markers Do128, Do129, Do130 and Do132 in 102 samples of Dalbergia odorifera.

[0064] Figure 7 Fingerprints of SSR markers Do133, Do136 and Do137 in 102 samples of Dalbergia odorifera. DETAILED DESCRIPTION

[0065] 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.

[0066] 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.

[0067] Example 1: Development of highly polymorphic primers for Dalbergia odorifera SSR primers

[0068] (1) Polymorphic SSR primer development

[0069] First, the whole genome sequence of Dalbergia odorifera was downloaded (http: / / gigadb.org / dataset / 100760), and then the MISA software (https: / / webblast.ipk-gatersleben.de / misa / ) was used to search for SSR sites in the Dalbergia odorifera genome sequence. Primers were then designed based on the conserved sequences of 150 bp upstream and downstream of each SSR site, and 144 SSR primers were finally designed and synthesized. Ten Dalbergia odorifera leaves from Haikou (HN002 and HN007), Danzhou (HN032), Dongfang (HN048), Ledong (HN055 and HN056), Sanya (HN059 and HN060), Tunchang (HN095) and Ding'an (HN097) with distant geographical distribution were used as materials (Table 3), and the DNA of each sample was extracted using the Plant Genomic DNA Extraction Kit (Cat. No.: DP305) produced by Tiangen Biotechnology (Beijing) Co., Ltd. Then, the 10 DNA samples were subjected to PCR amplification and capillary electrophoresis detection with each pair of synthesized primers to detect the effectiveness and polymorphism of the primers.

[0070] (2) PCR amplification

[0071] The PCR amplification system was: 15 μL system, including 2×TaqPCRMasterMix 7.5 μL, mixed primer (with a fluorescent group that can bind to the M13 universal adapter sequence) 2.0 μL, DNA template 1 μL (50-200 ng), and ddH2O 4.5 μL.

[0072] 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.

[0073] Table 228 Polymorphic characteristics of SSR primers of Dalbergia odorifera

[0074]

[0075]

[0076] 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.

[0077] The obtained fluorescent PCR product was detected by fluorescence electrophoresis using ABI3730xl, 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. After the electrophoresis, the detection results were analyzed by band typing using the software GeneMarker v2.2.0, and primers with stable amplification results, no impurity peaks, good repeatability and polymorphism were screened.

[0078] (3) Experimental results

[0079] The amplification results showed that among the 144 pairs of SSR primers, one pair of primers could not amplify products in some DNA templates, 37 pairs of primers had interference from mixed peaks, and 79 pairs of primers could only amplify products of 1-2 lengths. The remaining 28 pairs of primers could amplify more than 2 specific bands, and the polymorphic information content of the loci was PIC>0.25. These results show that the 28 pairs of SSR primers screened have good polymorphism and are of application value. The 28 pairs of primers were named Do002, Do004, Do005, Do006, Do010, Do013, Do022, Do025, Do034, Do051, Do062, Do066, Do081, Do094, Do097, Do098, Do100, Do104, Do105, Do113, Do121, Do128, Do129, Do130, Do132, Do133, Do136 and Do137. 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 Figure 1 shown.

[0080] Example 2: Genetic clustering and population diversity analysis of natural populations of Dalbergia odorifera

[0081]

[0082] (1) Experimental Materials

[0083] Table 3102 Dalbergia odorifera sampling location information

[0084] The experimental materials of this embodiment are 102 samples of Dalbergia odorifera collected from 17 regions including Haikou, Danzhou, Dongfang, Ledong, Sanya, Wenchang, Wanning, Chengmai, Baoting, Qionghai, Lingao, Tunchang, Ding'an, Lingshui, Wuzhishan, Changjiang and Baisha (Table 3). Healthy young leaves of each tree were collected and placed in a numbered plastic bag filled with absorbent paper. After the samples were brought back to the laboratory, they were classified according to the collection location and stored in a -80°C refrigerator until DNA was extracted.

[0085] The collected materials are mainly: ① the ancient and famous trees of Dalbergia odorifera recorded in the existing public data; ② the old Dalbergia odorifera trees transplanted from the original mountain forest about 40 years ago; ③ the old Dalbergia odorifera trees over 50 years old preserved in parks, government agencies and relatively primitive villages; ④ the Dalbergia odorifera trees with an age of over 30 years scattered in the mountain forest; ⑤ due to the small distribution of Dalbergia odorifera in some areas, it is difficult to collect germplasm resources, the present invention also collects a small number of Dalbergia odorifera samples with an age of more than 30 years and a breast diameter of more than 10 cm that were artificially planted in the early years but grown under natural conditions. And in order to ensure that the individual materials of Dalbergia odorifera collected have no genetic relationship, the distance between the collection sites of different individuals during collection is greater than 10km.

[0086] (2) DNA extraction from Dalbergia odorifera

[0087] The DNA of each Dalbergia odorifera natural population sample was extracted separately, using the same DNA extraction method as in Example 1.

[0088] (3) PCR amplification and capillary electrophoresis detection

[0089] The DNA of 102 natural population samples of Dalbergia odorifera obtained in step (2) was used as a template material, and PCR amplification was performed with 28 pairs of SSR primers developed by the present invention, and the obtained amplified products were detected by capillary electrophoresis. After the electrophoresis, the detection results were subjected to band typing analysis using the software GeneMarker v2.2.0. The specific method steps were the same as those of the PCR and capillary electrophoresis detection methods in Example 1.

[0090] (4) Data analysis

[0091] ① The GenAIex software was used to calculate and count genetic diversity parameters such as 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) of each population.

[0092] 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.

[0093] (5) Experimental results

[0094] The Nei's genetic distance cluster analysis results of the natural population germplasm resources of Dalbergia odorifera showed that the 102 Dalbergia odorifera samples were clustered into 15 small categories, and the number of samples in each category was as follows: Figure 2HN086 (Qionghai) was the farthest genetically distant from all other samples and was first clustered into cluster 1. Subsequently, HN102 (Wuzhishan), HN049 (Dongfang, Gushu), HN003 (Haikou, Gushu), HN002 (Haikou, Gushu), HN005 (Haikou, Gushu) and HN004 (Haikou, Gushu) were clustered into cluster 2.

[0095] The 102 Dalbergia odorifera samples were divided into 11 groups according to the collection sites, and the detailed information of the groups is shown in Table 3. The results of the genetic diversity analysis of Dalbergia odorifera populations are shown in Table 4. The Ho and He values ​​of the 11 populations were 0.321-0.582 and 0.381-0.591, with average values ​​of 0.489 and 0.527, respectively. Among them, the Ho value of population XI (others) was the largest, and the He value of population I (Haikou, Ding'an) was the largest. The F value was -0.065-0.165, among which the F values ​​of populations II (Danzhou) and VIII (Chengmai) were less than 0, indicating that these two populations had higher heterozygosity. The I values ​​of the 11 populations were 0.602-1.173, with an average value of 0.963, among which the I value of population I was the largest, and the degree of population differentiation was the highest (Table 4). In summary, the genetic diversity of the 11 Dalbergia odorifera populations was moderate overall. The above examples illustrate 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 Dalbergia odorifera. The analysis results can more accurately evaluate the current status of germplasm resources of natural populations of Dalbergia odorifera in Hainan.

[0096] Table 4 Analysis of genetic diversity of Dalbergia odorifera population

[0097]

[0098] 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 Ⅺ is shown in Table 3.

[0099] Example 3: Construction of fingerprints of natural populations of Dalbergia odorifera and identification of individuals

[0100] (1) Experimental materials and construction methods

[0101] According to the PCR and capillary electrophoresis test results of 28 pairs of SSR primers provided by the present invention in 102 samples of Dalbergia odorifera, the "0, 1" system is used to record the position of the amplified bands, and the bands with obvious peaks at the same position are marked as "1", and the bands without bands are marked as "0". The 0, 1 matrix of the SSR markers of each sample is established in the order from small to large of the allelic fragments amplified by each pair of SSR primers. Then, the positions marked as "1" in the matrix are replaced with colored blocks to draw the specific fingerprints of each sample. Further compare whether there are differences in the fingerprints between different individual materials, and identify different individuals.

[0102] (2) Experimental results

[0103] 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, such as Figures 3 to 7 As shown. By comparing these fingerprints, the results show that the 28 pairs of SSR primers provided by the present invention can completely distinguish the 102 tested Dalbergia odorifera materials (discrimination is 100%). Further comparison shows that the combination of 3 pairs of SSR primers, Do132, Do136 and Do137, can achieve specific differentiation of the 102 tested Dalbergia odorifera materials.

[0104] The above results also show that the 28 pairs of SSR molecular marker 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 analysis of Dalbergia odorifera. At the same time, the invented primers can accurately distinguish individuals in the natural population of Dalbergia odorifera, which also shows that the developed primers are highly efficient and have application value in the identification of Dalbergia odorifera individuals.

[0105] In summary, the SSR molecular markers and primer combinations provided by the present invention are based on a large amount of Dalbergia odorifera genome data and are reliable. At the same time, the application of the present invention uses Dalbergia odorifera total DNA as material, and is not affected or limited by plant tissues or materials in different growth periods. The Dalbergia odorifera-specific SSR primer combination provided by the present invention has been successfully applied to the analysis of genetic diversity of different natural populations of Dalbergia odorifera in Hainan Island, and has achieved the specific differentiation and identification of 102 individual materials in different natural populations and the construction of fingerprint maps, and has strong applicability. The SSR primers provided by the present invention have the advantages of stable amplification, high polymorphism, and easy to distinguish test results, and are simple to operate, low cost, and easy to promote; it can not only provide an effective molecular tool for analyzing the genetic diversity of Dalbergia odorifera germplasm resources and individual identification, but also create conditions for realizing early selection of traits and improving breeding efficiency, which is of great significance to the breeding and genetic improvement of Dalbergia odorifera.

[0106] 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.

[0107] 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 Dalbergia odorifera genome is characterized by: The SSR molecular markers are: Do002, Do004, Do005, Do006, Do010, Do013, Do022, Do025, Do034, Do051, Do062, Do066, Do081, Do094, Do097, Do098, Do100, Do104, Do105, Do113, Do121, Do128, Do129, Do130, Do1322, Do133, Do136 and Do137; The primer combination is: 1) The primers for amplifying the SSR molecular marker Do002 are Do002-F and Do002-R, and the nucleotide sequences of Do002-F and Do002-R are shown in SEQ ID No. 1 and SEQ ID No. 2, respectively; 2) The primers for amplifying the SSR molecular marker Do004 are Do004-F and Do004-R, and the nucleotide sequences of Do004-F and Do004-R are shown in SEQ ID No. 3 and SEQ ID No. 4, respectively; 3) The primers for amplifying the SSR molecular marker Do005 are Do005-F and Do005-R, and the nucleotide sequences of Do005-F and Do005-R are shown in SEQ ID No. 5 and SEQ ID No. 6, respectively; 4) The primers for amplifying the SSR molecular marker Do006 are Do006-F and Do006-R, and the nucleotide sequences of Do006-F and Do006-R are shown in SEQ ID No.7 and SEQ ID No.8, respectively; 5) The primers for amplifying the SSR molecular marker Do010 are Do010-F and Do010-R, and the nucleotide sequences of Do010-F and Do010-R are shown in SEQ ID No.9 and SEQ ID No.10, respectively; 6) The primers for amplifying the SSR molecular marker Do013 are Do013-F and Do013-R, and the nucleotide sequences of Do013-F and Do013-R are shown in SEQ ID No.11 and SEQ ID No.12, respectively; 7) The primers for amplifying the SSR molecular marker Do022 are Do022-F and Do022-R, and the nucleotide sequences of Do022-F and Do022-R are shown in SEQ ID No. 13 and SEQ ID No. 14, respectively; 8) The primers for amplifying the SSR molecular marker Do025 are Do025-F and Do025-R, and the nucleotide sequences of Do025-F and Do025-R are shown in SEQ ID No. 15 and SEQ ID No. 16, respectively; 9) The primers for amplifying the SSR molecular marker Do034 are Do034-F and Do034-R, and the nucleotide sequences of Do034-F and Do034-R are shown in SEQ ID No. 17 and SEQ ID No. 18, respectively; 10) The primers for amplifying the SSR molecular marker Do051 are Do051-F and Do051-R, and the nucleotide sequences of Do051-F and Do051-R are shown in SEQ ID No. 19 and SEQ ID No. 20, respectively; 11) The primers for amplifying the SSR molecular marker Do062 are Do062-F and Do062-R, and the nucleotide sequences of Do062-F and Do062-R are shown in SEQ ID No. 21 and SEQ ID No. 22, respectively; 12) The primers for amplifying the SSR molecular marker Do066 are Do066-F and Do066-R, and the nucleotide sequences of Do066-F and Do066-R are shown in SEQ ID No. 23 and SEQ ID No. 24, respectively; 13) The primers for amplifying the SSR molecular marker Do081 are Do81-F and Do081-R, and the nucleotide sequences of Do081-F and Do081-R are shown in SEQ ID No. 25 and SEQ ID No. 26, respectively; 14) The primers for amplifying the SSR molecular marker Do094 are Do094-F and Do094-R, and the nucleotide sequences of Do094-F and Do094-R are shown in SEQ ID No. 27 and SEQ ID No. 28, respectively; 15) The primers for amplifying the SSR molecular marker Do097 are Do097-F and Do097-R, and the nucleotide sequences of Do097-F and Do097-R are shown in SEQ ID No. 29 and SEQ ID No. 30, respectively; 16) The primers for amplifying the SSR molecular marker Do098 are Do098-F and Do098-R, and the nucleotide sequences of Do098-F and Do098-R are shown in SEQ ID No.31 and SEQ ID No.32, respectively; 17) The primers for amplifying the SSR molecular marker Do100 are Do100-F and Do100-R, and the nucleotide sequences of Do100-F and Do100-R are shown in SEQ ID No.33 and SEQ ID No.34, respectively; 18) The primers for amplifying the SSR molecular marker Do104 are Do104-F and Do104-R, and the nucleotide sequences of Do104-F and Do104-R are shown in SEQ ID No.35 and SEQ ID No.36, respectively; 19) The primers for amplifying the SSR molecular marker Do105 are Do105-F and Do105-R, and the nucleotide sequences of Do105-F and Do105-R are shown in SEQ ID No.37 and SEQ ID No.38, respectively; 20) The primers for amplifying the SSR molecular marker Do113 are Do113-F and Do113-R, and the nucleotide sequences of Do113-F and Do113-R are shown in SEQ ID No.39 and SEQ ID No.40, respectively; 21) The primers for amplifying the SSR molecular marker Do121 are Do121-F and Do121-R, and the nucleotide sequences of Do121-F and Do121-R are shown in SEQ ID No.41 and SEQ ID No.42, respectively; 22) The primers for amplifying the SSR molecular marker Do128 are Do128-F and Do128-R, and the nucleotide sequences of Do128-F and Do128-R are shown in SEQ ID No.43 and SEQ ID No.44, respectively; 23) The primers for amplifying the SSR molecular marker Do129 are Do129-F and Do129-R, and the nucleotide sequences of Do129-F and Do129-R are shown in SEQ ID No.45 and SEQ ID No.46, respectively; 24) The primers for amplifying the SSR molecular marker Do130 are Do130-F and Do130-R, and the nucleotide sequences of Do130-F and Do130-R are shown in SEQ ID No.47 and SEQ ID No.48, respectively; 25) The primers for amplifying the SSR molecular marker Do132 are Do132-F and Do132-R, and the nucleotide sequences of Do132-F and Do132-R are shown in SEQ ID No.49 and SEQ ID No.50, respectively; 26) The primers for amplifying the SSR molecular marker Do133 are Do133-F and Do133-R, and the nucleotide sequences of Do133-F and Do133-R are shown in SEQ ID No.51 and SEQ ID No.52, respectively; 27) The primers for amplifying the SSR molecular marker Do136 are Do136-F and Do136-R, and the nucleotide sequences of Do136-F and Do136-R are shown in SEQ ID No.53 and SEQ ID No.54, respectively; 28) The primers for amplifying the SSR molecular marker Do137 are Do137-F and Do137-R. The nucleotide sequences of Do137-F and Do137-R are shown in SEQ ID No.55 and SEQ ID No.56, respectively.

2. A kit for analyzing the genetic diversity of Dalbergia odorifera populations and constructing a fingerprint of Dalbergia odorifera, characterized in that: At least comprises the primer combination SEQ ID No.1-SEQ ID No.56 described in claim 1.

3. Use of the SSR molecular marker primer combination of claim 1 in genetic diversity analysis, fingerprint construction or individual identification of Dalbergia odorifera.

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

5. The use according to claim 4, characterized in that: 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; 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 Dalbergia odorifera comprises at least the following steps: 1) Use the software GeneMarker v2.2.0 to perform band typing on the test results and determine the size of the amplified bands 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 marked as "1", and the bands without bands were marked 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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