Molecular identity card construction method based on aquilaria sinensis resequencing database development and application

Abstract

The invention provides a molecular identity card construction method based on aquilaria sinensis resequencing database development and application, and belongs to the technical field of molecular biology. The SSR loci are identified on the basis of a VCF file obtained through population genome re-sequencing analysis of 60 aquilaria sinensis individuals, 20 pairs of SSR molecular marker primer combinations are screened out after single nucleotide repetition is eliminated, and existing improved varieties, farmyard varieties and field germplasm of aquilaria sinensis can be effectively distinguished; through screening, six loci with high genetic variation degree and high PIC and many Na are obtained as core loci to construct an aquilaria sinensis molecular identity card, and corresponding two-dimensional code information is provided, so that existing improved varieties, farmyard varieties and individual germplasm of aquilaria sinensis can be accurately, efficiently and stably identified; and the phenomena of homonymous foreign matters, different names, same objects and counterfeit varieties on the market can be effectively solved, and effective technical guarantee is provided for effective protection and efficient utilization of high-quality aquilaria sinensis varieties. Compared with an existing aquilaria sinensis molecular marker, the aquilaria sinensis molecular marker is stable in result, good in repeatability and high in accuracy, and the identification rate reaches 95% or above.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and particularly relates to a set of SSR molecular marker primers for Aquilaria sinensis, a method for constructing a fingerprint map, a method for constructing a molecular identity card, and their applications in the identification of existing improved varieties, local varieties and wild germplasm resources of Aquilaria sinensis. In particular, it relates to a combination of SSR molecular marker primers, a kit and their applications developed based on the population genome re-sequencing database of Aquilaria sinensis. Background Art

[0002] Aquilariasinensis (Lour.) Gilg, also known as native agarwood, is a plant of the genus Aquilaria in the family Thymelaeaceae. It is a national second-class protected wild plant and the legal plant source of the precious aromatic medicine agarwood. It is mainly produced in Hainan, Guangdong, Guangxi and other places. Agarwood is a natural spice and precious medicine, with important economic utilization value. Aquilaria sinensis is widely planted in Hainan with a variety of cultivars. The identification of Aquilaria sinensis cultivars, the analysis of genetic diversity and the construction of a DNA fingerprint database can objectively and comprehensively understand the current status of Aquilaria sinensis cultivars, which is of great significance for cultivar management, cultivar breeding, and the collection and protection of germplasm resources.

[0003] Currently, a series of excellent germplasms with fast resin production, high yield, disease and insect resistance, colorful leaf ornamentation, and tea use have emerged on the market. Aquilaria sinensis is mainly distinguished and identified based on plant biological characteristics such as leaf morphology, leaf size, plant morphology, and fruit morphology characteristics. However, the differences between cultivars within Aquilaria sinensis are not obvious, and morphological traits are easily affected by external environmental conditions and vary, making it difficult to distinguish them visually. If these cultivars are confused, it will cause market chaos and the varietal advantages cannot be realized. Therefore, distinguishing these similar cultivars has practical significance for both the market value and scientific research value of Aquilaria sinensis.

[0004] Simple Sequence Repeats (SSR) marker technology has been widely regarded due to its characteristics such as a large number, high polymorphism, co-dominance in inheritance, stable amplification, and easy exchange of primer sequences. Simple sequence repeats, also known as microsatellites, are tandem repeat sequences with 1-6 nucleotides as repeat units, widely distributed in the coding and non-coding regions of eukaryotic genomes. In plant breeding, SSR has become an important molecular marker. Developing an SSR molecular marker that can distinguish existing improved varieties, local varieties and individual germplasms of Aquilaria sinensis, as well as different Aquilaria sinensis varieties with fast resin production, high yield, disease and insect resistance, colorful leaf ornamentation, and tea use widely planted in the market, is of great significance. Summary of the Invention

[0005] Based on the deficiencies and shortcomings of the existing rapid identification of Aquilaria sinensis varieties, the purpose of the present invention is to provide a set of SSR molecular marker primer combinations, kits and applications, and a method for constructing molecular identity cards based on the Aquilaria sinensis population genome resequencing database.

[0006] The first object of the present invention is to provide a set of SSR molecular marker primers developed based on the Aquilaria sinensis population genome resequencing database;

[0007] The second object of the present invention is to provide a method for constructing a fingerprint map based on the Aquilaria sinensis SSR molecular marker primer set;

[0008] The third object of the present invention is to provide the application of the Aquilaria sinensis SSR molecular marker primer set in the identification of improved varieties of Aquilaria sinensis forest trees, farmer varieties, and germplasm resources.

[0009] In order to achieve the above invention objects, the present invention provides the following technical solutions:

[0010] The present invention provides a set of Aquilaria sinensis SSR molecular marker primer combinations developed based on the population genome resequencing database, characterized in that the molecular markers include: AquSSR07, AquSSR10, AquSSR14, AquSSR17, AquSSR18, AquSSR22, AquSSR27, AquSSR28, AquSSR29, AquSSR30, AquSSR34, AquSSR40, AquSSR42, AquSSR54, AquSSR58, AquSSR59, AquSSR62, AquSSR71, AquSSR89, AquSSR94, and their sequences are as SEQ ID NO.1 to SEQ ID NO.40 in sequence;

[0011] The present invention also provides a detection reagent or kit containing the Aquilaria sinensis SSR molecular marker primer set for analyzing the genetic structure of the Aquilaria sinensis population. It is characterized in that it at least includes the primer combination SEQ ID NO.1 to SEQ ID NO.40 for amplifying the first aspect;

[0012] The present invention also provides a set of Aquilaria sinensis SSR molecular marker primer sets and kits for constructing two-dimensional code molecular identity cards of Aquilaria sinensis. The molecular marker primer set includes: AquSSR14, AquSSR29, AquSSR30, AquSSR54, AquSSR62, and AquSSR94; the molecular markers are amplified by the following primers in sequence:

[0013] The primer sequence for amplifying the AquSSR14 molecular marker is SEQ ID NO.5 to SEQ ID NO.6;

[0014] The primer sequences for amplifying the AquSSR29 molecular marker are SEQ ID NO.17 to SEQ ID NO.18;

[0015] The primer sequences for amplifying the AquSSR30 molecular marker are SEQ ID NO.19 to SEQ ID NO.20;

[0016] The primer sequences for amplifying the AquSSR54 molecular marker are SEQ ID NO.27 to SEQ ID NO.28;

[0017] The primer sequences for amplifying the AquSSR62 molecular marker are SEQ ID NO.33 to SEQ ID NO.34;

[0018] The primer sequences for amplifying the AquSSR94 molecular marker are SEQ ID NO.39 to SEQ ID NO.40.

[0019] The present invention also provides a primer set for amplifying the Aquilaria sinensis SSR molecular marker described above, including the primers described above.

[0020] The present invention also provides a kit, including the primer set of the Aquilaria sinensis SSR molecular marker described above.

[0021] The present invention also provides the application of the primer set of the Aquilaria sinensis SSR molecular marker described above or the kit according to claim 4 in at least one of the following:

[0022] (1) Application in the identification of Aquilaria sinensis varieties and / or the analysis of genetic relationships of Aquilaria sinensis;

[0023] (2) Application in the cluster analysis among Aquilaria sinensis germplasms;

[0024] (3) Application in the analysis of the population genetic structure of Aquilaria sinensis germplasms;

[0025] (4) Application in the construction of Aquilaria sinensis fingerprint maps;

[0026] (5) Application in the construction of molecular identity cards for Aquilaria sinensis germplasms.

[0027] Preferably, the application includes the following steps:

[0028] (1) Extract the genomic DNA of the test Aquilaria sinensis;

[0029] (2) Perform PCR amplification on the genomic DNA of the test Aquilaria sinensis using the primer set of the Aquilaria sinensis SSR molecular marker described above;

[0030] (3) Perform electrophoresis detection on the PCR amplification product to obtain a detection result;

[0031] (4) Use the results of step (3) for the analysis of Aquilaria sinensis population genetic diversity, fingerprint construction, or individual identification analysis.

[0032] Preferably, the reaction system for the PCR amplification is as follows: 30 ng of DNA template, 5 μL of 2×Taq PCRMix, 0.5 μL of upstream primer, 0.5 μL of downstream primer, and supplemented with ddH2O to 10 μL; the amplification program is: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, for 35 cycles; extension at 72°C for 20 min.

[0033] Preferably, the electrophoresis detection includes polyacrylamide gel electrophoresis or capillary electrophoresis.

[0034] Preferably, the method for constructing the fingerprint of Aquilaria sinensis is as follows:

[0035] (1) According to the amplification detection results of each SSR locus, mark the positions corresponding to the amplified bands in the electrophoresis result as "1", and mark those without amplified bands as "0";

[0036] (2) Establish a 0, 1 matrix of SSR markers for each sample in the order of the allelic fragments amplified by each pair of SSR primers from small to large;

[0037] (3) Replace the positions marked as "1" in the matrix with color blocks of different colors to draw the specific fingerprint of each sample.

[0038] Preferably, the construction of the molecular identity card of Aquilaria sinensis germplasm is as follows: According to the amplified fragment length, assign values to each locus using "1-9" and "A-Z", and perform fingerprint coding on each Aquilaria sinensis germplasm resource. And use the barcode generation website (https: / / www.ecjson.com / barcode / ) to generate the corresponding molecular identity card barcode, and use the QR code generation website (https: / / cli.im / ) to generate the QR code.

[0039] Compared with the prior art, by adopting the above technical solutions, the present invention has the following beneficial effects:

[0040] (1) Based on the VCF file obtained from the population genome re-sequencing analysis of 60 Aquilaria sinensis individuals, the present invention identified SSR loci. Based on a large amount of Aquilaria sinensis genome data, it has stronger reliability. 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.

[0041] (2) The specific SSR primer combination provided by the present invention has been successfully applied to the genetic diversity analysis of Aquilaria sinensis varieties in Hainan and Guangdong, widely planted local varieties, and natural populations, achieving the specific differentiation of 149 individual materials of improved varieties, local varieties, and natural populations and the construction of fingerprint maps, with strong applicability.

[0042] (3) The present invention screened 6 groups of Aquilaria sinensis SSR molecular marker primer sets, with stable results, good repeatability, high accuracy, and a discrimination rate of over 95%. It can accurately, efficiently, and stably identify existing improved varieties, local varieties, and individual germplasms of Aquilaria sinensis, effectively solve the phenomena of different plants with the same name, the same plant with different names, and counterfeit varieties in the market, and provide effective technical guarantees for the effective protection and efficient utilization of high-quality Aquilaria sinensis varieties. Description of the Drawings

[0043] Figure 1 It is a capillary electrophoresis diagram of partial amplification results of the SSR primer combination AquSSR07 (SEQ ID No.1 and SEQ ID No.2) in Aquilaria sinensis samples.

[0044] Figure 2 It is a cluster analysis diagram among 149 Aquilaria sinensis germplasms based on Nei's genetic distance.

[0045] Figure 3 It is a fingerprint map of 149 Aquilaria sinensis germplasms drawn by 20 SSR loci.

[0046] Figure 4 It is the molecular identity two-dimensional code of Aquilaria sinensis germplasms CX001 - CX090.

[0047] Figure 5 It is the molecular identity two-dimensional code of Aquilaria sinensis germplasms CX091 - CX149. Detailed Embodiments

[0048] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0049] In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiments or examples. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, those skilled in the art can combine and combine the different embodiments or examples described in this specification.

[0050] Example 1. Development and Screening of SSR Primers Based on Aquilaria sinensis Resequencing

[0051] 1. Experimental Materials

[0052] The materials of this embodiment are 149 portions collected from Dianbai area of Maoming, Guangdong, Haikou, Hainan, Tunchang, Hainan, Ding'an, Hainan, Chengmai, Hainan, Wuzhishan, Hainan, Qiongzhong, Hainan, and Lingao, Hainan, covering 10 Hainan provincial improved tree varieties, 124 common local varieties on the market, and 15 individual plants. The variety characteristics cover excellent varieties or germplasms such as easy to form agarwood, tea use, colorful leaf ornamental, insect resistance, etc., and also cover common Aquilaria sinensis.

[0053] Table 1 Information of Aquilaria sinensis Samples

[0054]

[0055]

[0056]

[0057]

[0058]

[0059] 2. DNA Extraction

[0060] Take fresh leaf samples of different excellent germplasm Aquilaria sinensis individual plants that have been collected, with a wet weight of about 0.6 g. First, place the cryotube in liquid nitrogen for precooling, put the taken leaves into liquid nitrogen for quick freezing, then pack them in the cryotube and store them in a -80°C refrigerator. Subsequently, use the Plant Genomic DNA Extraction Kit (product number: DP305) of Tiangen Biotech (Beijing) Co., Ltd. for DNA extraction and detection.

[0061] 3. SSR Marker Analysis

[0062] Based on the VCF file obtained from the population genomic resequencing analysis of 60 individuals of Aquilaria sinensis (Table 2) in the early stage of the project, variant sequences with a length greater than 10 nt were screened out from the population genomic variant sites. Then, the MISA software was used to identify SSR sequences in the variant sequences - SSR definition (unit size, min repeats): 1-10, 2-6, 3-5, 4-5, 5-5, 6-5. Among the total 168,459 sequences identified, a total of 56,657 SSR sequences were obtained, including 4,747 mononucleotide repeats, 41,695 dinucleotide repeats, 9,550 trinucleotide repeats, 644 tetranucleotide repeats, 13 pentanucleotide repeats, and 8 hexanucleotide repeats. After folding and counting the SSR sequences at the same locus, it was found that these 51,910 SSR sequences were generated by 24,430 SSR loci, that is, 24,430 SSR loci of Aquilaria sinensis were identified using the VCF file obtained from the population genomic resequencing analysis of Aquilaria sinensis. After excluding mononucleotide repeats, a total of 22,486 SSR loci were obtained. Subsequently, SSR primers will be designed based on these loci and related work will be carried out.

[0063] Table 2 List of population genomic resequencing

[0064]

[0065] 4. Genotype frequency statistics

[0066] Using these SSR loci, the individuals in the population were genotyped, and the frequencies of different genotypes were calculated, and finally the SSR locus information was obtained. The SSR loci are mainly trinucleotide repeats, followed by dinucleotide repeats, and the tetranucleotide repeat type is less. The genotype information of SSR loci in each individual of Aquilaria sinensis was statistically analyzed, and the frequency of SSR genotypes in the population was calculated. The numerical change range of the most common SSR gene frequency is 0.15 - 0.87, the numerical change range of the second most common SSR gene frequency is 0.08 - 0.47, and the numerical change range of the third most common SSR gene frequency is 0.00 - 0.28. Among them, the base repeat unit of SSR number Scaffold_5532_109254228 is CTT, and its most common gene frequency value is equal to the second most common gene frequency (frequency value is 0.15), and the difference from the third most common gene frequency is not large (frequency value is 0.13). The results of Scaffold_5532_8308141, Scaffold_8152_16679434, and Scaffold_3585_64368533 are similar. The remaining SSR loci all have the main SSR genotypes in the population. These SSRs information can be used to guide the development of molecular marker-assisted selection and breeding of Aquilaria sinensis.

[0067] Table 3 Statistical results of SSR gene frequencies

[0068]

[0069]

[0070]

[0071]

[0072]

[0073] 5. Development and screening of SSR primers

[0074] SSR primers were designed based on the second most common genotype. First, the sites without SSR in the second most common genotype were removed from the SSR loci, and 3 pairs of primers were designed for each remaining SSR locus. Primers with short amplified target fragments, more than 4 consecutive base repeats, and too high GC content were filtered out. The SSR primers obtained by screening were used for subsequent genetic diversity analysis. To obtain polymorphic SSR loci, 93 pairs of synthesized primers were subjected to PCR amplification and capillary electrophoresis detection using 3 samples of Aquilaria sinensis DNA, namely CX10, CX15, and CX66, for the first round of primer screening (Table 4). The PCR amplification system was as follows: 30 ng of DNA template, 5 μL of 2×Taq PCRMix, 0.5 μL of upstream primer, 0.5 μL of downstream primer, and ddH2O was added to make up to 10 μL; the PCR amplification program was: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, for 35 cycles; extension at 72°C for 20 min.

[0075] Primers with less than 3 product lengths in the amplification results, amplification failures, and interference from continuous peaks and miscellaneous peaks were excluded. SSR primers with good amplification results and capable of stably amplifying 3 or more specific bands were selected, and 8 randomly selected samples of Aquilaria sinensis DNA were used for the second round of primer screening (Table 5). Similarly, primers that could amplify 3 or more specific bands and with a polymorphic information content (PIC) > 0.30 of the locus were selected. Finally, 20 pairs of SSR primers were obtained for the genetic diversity analysis of the Aquilaria sinensis population. The repeat types of these 20 pairs of polymorphic primers were mostly tri-nucleotide repeats, followed by di-nucleotide repeats, and only AquSSR34 was a tetra-nucleotide repeat type, as shown in Table 6.

[0076] Table 4 Results of the first round of screening of 93 pairs of SSR primers

[0077]

[0078]

[0079]

[0080] Table 5 Results of the Preliminary Screening II of SSR Primers

[0081]

[0082]

[0083] Table 6 Information of 20 Aquilaria sinensis SSR Primers

[0084]

[0085]

[0086] 6. Polymorphism Evaluation of 20 SSR Loci in the Aquilaria sinensis Population

[0087] A total of 149 Aquilaria sinensis germplasms were amplified and detected at 20 SSR loci, and the polymorphism of these loci in this population was analyzed. The following parameters were calculated using GenAlex 6.5 software: the number of observed alleles (Na), the number of effective alleles (Ne), Shannon's information index (I), expected heterozygosity (He), observed heterozygosity (Ho), and fixation index (F). The polymorphic information content (PIC) of the loci was calculated using Cervus 3.0 software, and the results are shown in Table 7.

[0088] Table 7 Polymorphism Characteristics of 20 SSR Loci in 149 Aquilaria sinensis Germplasms

[0089]

[0090]

[0091] Note: N: number of effective individuals; Na: number of alleles; Ne: number of effective alleles; I: Shannon's information index; Ho: observed heterozygosity; He: expected heterozygosity; F: fixation index; PIC: polymorphic information content index.

[0092] Example 2. Cluster Analysis among 149 Aquilaria sinensis Germplasms

[0093] Based on the Nei's genetic distance between individuals, the UPGMA method was used to perform a cluster analysis on 149 Aquilaria sinensis germplasm resources. The clustering results showed that 149 Aquilaria sinensis germplasms were successively clustered into 4 major categories, as Figure 1As shown, different colors represent different classifications. Analysis of the clustering results revealed that the four major categories accounted for 18.79%, 0.67%, 12.08%, and 68.46% of the total number of species, respectively. According to the clustering results, 28 germplasms, namely CX052, CX038, CX012, CX123, CX032, CX079, CX013, CX010, CX005, CX058, CX011, CX087, CX149, CX053, CX108, CX037, CX095, CX009, CX041, CX014, CX025, CX046, CX034, CX018, CX008, CX125, CX020, CX019, were clustered into the first category, CX114 was separately clustered into the second category, 18 germplasms, namely CX112, CX106, CX026, CX120, CX042, CX078, CX069, CX081, CX147, CX044, CX129, CX135, CX130, CX132, CX093, CX143, CX119, CX145, were clustered into the third category, and the remaining 102 germplasms were clustered into the fourth category. Among them, CX114 was separately clustered into one category, indicating that it has higher genetic differences than other germplasms and can be preferentially considered in the construction of the core germplasm bank in the later stage, such as Figure 2 as shown

[0094] Example 3. Population genetic structure analysis of 149 Aquilaria sinensis germplasms

[0095] The software Structure2.3.4 was used to analyze the population genetic structure of 149 Aquilaria sinensis germplasms, and the Evanno ΔK method was used to statistically analyze the data. It was found that when K = 3, the ΔK value was the largest, 116.890, indicating that theoretically, the 149 Aquilaria sinensis could be divided into three genetic structure groups( Figure 2 A). The three different genetic structure groups are represented by yellow (Ⅰ), orange (Ⅱ), and blue (Ⅲ), respectively. Among them, 71 germplasms are mainly yellow, 30 germplasms are mainly orange, and 48 germplasms are mainly blue( Figure 2B). According to the genetic structure, 149 Aquilaria germplasms can be divided into three groups, namely Group Ⅰ (including CX001, CX003, CX004, CX006, CX007, CX021, CX022, CX023, CX024, CX026, CX027, CX031, CX032, CX039, CX042, CX043, CX044, CX048, CX050, CX054, CX055, CX057, CX059, CX060, CX064, CX066, CX068, CX069, CX070, CX074, CX077, CX078, CX080, CX081, CX083, CX085, CX088, CX089, CX091, CX092, CX093, CX094, CX097, CX099, CX100, CX101, CX103, CX106, CX107, CX109, CX111, CX112, CX113, CX115, CX116, CX119, CX120, CX121, CX123, CX127, CX128, CX129, CX130, CX132, CX134, CX135, CX136, CX138, CX139, CX140, CX141, a total of 71 germplasms), Group Ⅱ (including CX005, CX008, CX009, CX010, CX011, CX012, CX013, CX014, CX017, CX018, CX019, CX020, CX025, CX034, CX037, CX038, CX041, CX046, CX052, CX053, CX058, CX079, CX087, CX090, CX095, CX108, CX125, CX144, CX147, CX149, a total of 30 germplasms), and Group Ⅲ (including CX002, CX015, CX016, CX028, CX029, CX030, CX033, CX035, CX036, CX040, CX045, CX047, CX049, CX051, CX056, CX061, CX062, CX063, CX065, CX067, CX071, CX072, CX073, CX075, CX076, CX082, CX084, CX086, CX096, CX098, CX102, CX104, CX105, CX110, CX114, CX117, CX118, CX122, CX124, CX126, CX131, CX133, CX137, CX142, CX143, CX145, CX146, CX148, a total of 48 germplasms).

[0096] Example 4.149 Aquilaria sinensis germplasm core loci screening

[0097] The number of alleles (Na) and polymorphic information content (PIC) are important indicators for measuring population diversity. A higher PIC and more Na indicate a high degree of genetic variation at the site and a large selection margin. These polymorphic sites can be used for marker-assisted selection.

[0098] First, 9 loci (AquSSR14, AquSSR34, AquSSR07, AquSSR17, AquSSR18, AquSSR28, AquSSR58, AquSSR71, AquSSR89; PIC<0.5) showing low to moderate polymorphism were deleted. After deletion, germplasm CX065 could not be identified, and the identification rate of 149 germplasms dropped to 95.30%. Germplasm CX065 could be identified using locus AquSSR14, so locus AquSSR14 was retained. Then, 3 loci (AquSSR22, AquSSR42, AquSSR59) with lower values ​​were deleted based on the number of alleles and heterozygosity, and no more varieties that could not be identified were added. Similarly, when two loci with low allele numbers and heterozygosity (AquSSR10 and AquSSR62) were deleted, the newly added germplasms CX131 and CX133 could not be identified, and the identification rate dropped to 94.63%. Germplasms CX131 and CX133 could be identified using locus AquSSR62, so locus AquSSR62 was retained. Next, when loci AquSSR27 and AquSSR40 were deleted based on the number of alleles and heterozygosity, no more varieties could be identified, and the identification rate was 95.97%. Continuing to delete based on the number of alleles or polymorphic information content, it was found that the identification rate decreased significantly.

[0099] In summary, in order to ensure that the most germplasms are identified through the least number of loci, 6 loci (AquSSR14, AquSSR29, AquSSR30, AquSSR54, AquSSR62, and AquSSR94) with high genetic variation that show higher PIC and more Na were screened and used as core loci to establish a molecular identity card. The identification rate of the 6 core loci for 149 agarwood germplasms was 95.97%.

[0100] Table 8 Core site screening

[0101]

[0102] Example 5. Construction of fingerprints of 149 Aquilaria sinensis germplasms

[0103] Using the 20 pairs of SSR primers provided by the present invention for PCR amplification in 149 Aquilaria sinensis, the system is as follows: DNA template is greater than 30 ng, 2×TaqPCR Mix is 5 μL, upstream primer is 0.5 μL, downstream primer is 0.5 μL, and ddH₂O is added to make up to 10 μL; the procedure is: pre-denaturation at 95 °C for 5 min; denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 30 s, for 35 cycles; extension at 72 °C for 20 min.

[0104] Then, according to the amplification detection results of each SSR locus, those with amplified bands at the corresponding positions in the electrophoresis results are marked as "1", and those without amplified bands are marked as "0", and the amplification results of each locus are converted into a specific "0, 1" matrix fingerprint map that can represent each Aquilaria germplasm. Then, the positions of "1" in the matrix are replaced with colored blocks to draw the specific fingerprint maps of each sample. Compare the differences in fingerprint maps between different individual materials to identify different individuals.

[0105] The fingerprint maps of 149 Aquilaria germplasms are as Figure 3 shown, Figure 3 It shows that the amplification results composed of 126 alleles at 20 SSR loci show that all germplasms except CX002 and CX075, CX015 and CX084, CX103 and CX111 are clearly identified, and the identification rate reaches 95.97%. It shows that the 20 pairs of SSR molecular marker primers provided by the present invention have good polymorphism and stable amplification, and can be applied to genetic clustering of Aquilaria sinensis, population genetic diversity analysis, fingerprint map construction analysis, etc. At the same time, the primers of the invention also achieve precise differentiation between individuals within the natural population of Aquilaria sinensis, indicating that the developed primers are efficient and have the application value for individual identification of Aquilaria sinensis.

[0106] Example 6. Construction of a fingerprint map database for 149 Aquilaria sinensis germplasms

[0107] According to the assignment of amplified fragment lengths (Table 9), fingerprint coding is performed on 149 Aquilaria germplasm resources.

[0108] Table 9 Assignment of amplified fragment lengths at 20 SSR loci

[0109]

[0110]

[0111] During the encoding process, according to the allele encoding standard, if the sizes of the amplified allele fragments are the same, both two-digit numbers in the fingerprint encoding are represented by the numbers corresponding to the fragments. Taking the germplasm CX001 as an example, its fingerprint data is 1234112222114446881322446648454424352338, indicating that there are "peaks" at 146bp(1) and 168bp(2) of AquSSR07, 144bp(3) and 147bp(4) of AquSSR10, 201bp(1) of AquSSR14, 221bp(2) of AquSSR17, 175bp(2) of AquSSR18, 220bp(1) of AquSSR22, 146bp(4) of AquSSR27, 239bp(4) and 248bp(6) of AquSSR28, 142bp(8) of AquSSR29, 213bp(1) and 232bp(3) of AquSSR30, 222bp(2) of AquSSR34, 159bp(4) of AquSSR40, 203bp(6) of AquSSR42, 252bp(4) and 266(8) of AquSSR54, 198bp(4) and 201bp(5) of AquSSR58, 198bp(4) of AquSSR59, 243bp(2) and 246bp(4) of AquSSR62, 275bp(3) and 291bp(5) of AquSSR71, 128bp(2) and 134bp(3) of AquSSR89, 132bp(3) and 139bp(8) of AquSSR94. The fingerprint database of 149 Aquilaria sinensis germplasms is shown in Table 10.

[0112] Table 10 Fingerprint database of 149 Aquilaria sinensis germplasms

[0113]

[0114]

[0115]

[0116] Example 7. Construction of molecular identity cards for 149 Aquilaria sinensis germplasms

[0117] Referring to the encoding method in Example 9, molecular identity cards were constructed for 149 Aquilaria sinensis germplasms using the 6 core loci screened in Example 4, as shown in Table 11. And QR codes were generated using the website (https: / / cli.im / ), as Figure 4 and Figure 5 shown.

[0118] Table 11 Molecular identity coding of 149 Aquilaria sinensis germplasms

[0119]

[0120]

[0121]

[0122] As can be seen from the above embodiments, the present invention provides a set of Aquilaria sinensis SSR molecular markers, primer sets and their applications, which can accurately, efficiently and stably identify existing improved varieties, farmer varieties and wild individual germplasms of Aquilaria sinensis, with a discrimination rate of over 95%. It can effectively solve the problems of different plants with the same name, the same plant with different names and counterfeit varieties in the market, and provide effective technical guarantee for the effective protection and efficient utilization of high-quality varieties of Aquilaria sinensis. The results of the molecular markers of Aquilaria sinensis of the present invention are more stable, have better repeatability and higher accuracy than the existing ones, and the discrimination rate is over 95%.

[0123] The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A set of Aquilaria sinensis SSR molecular marker primer combinations developed based on a population genomic resequencing database, characterized in that, The molecular markers include: AquSSR07, AquSSR10, AquSSR14, AquSSR17, AquSSR18, AquSSR22, AquSSR27, AquSSR28, AquSSR29, AquSSR30, AquSSR34, AquSSR40, AquSSR42, AquSSR54, AquSSR58, AquSSR59, AquSSR62, AquSSR71, AquSSR89, AquSSR94; the molecular markers are sequentially amplified by the following primers: The primer sequences for amplifying the AquSSR07 molecular marker are SEQ ID NO.1 to SEQ ID NO.2; The primer sequences for amplifying the AquSSR10 molecular marker are SEQ ID NO.3 to SEQ ID NO.4; The primer sequences for amplifying the AquSSR14 molecular marker are SEQ ID NO.5 to SEQ ID NO.6; The primer sequences for amplifying the AquSSR17 molecular marker are SEQ ID NO.7 to SEQ ID NO.8; The primer sequences for amplifying the AquSSR18 molecular marker are SEQ ID NO.9 to SEQ ID NO.10; The primer sequences for amplifying the AquSSR22 molecular marker are SEQ ID NO.11 to SEQ ID NO.12; The primer sequences for amplifying the AquSSR27 molecular marker are SEQ ID NO.13 to SEQ ID NO.14; The primer sequences for amplifying the AquSSR28 molecular marker are SEQ ID NO.15 to SEQ ID NO.16; The primer sequences for amplifying the AquSSR29 molecular marker are SEQ ID NO.17 to SEQ ID NO.18; The primer sequences for amplifying the AquSSR30 molecular marker are SEQ ID NO.19 to SEQ ID NO.20; The primer sequences for amplifying the AquSSR34 molecular marker are SEQ ID NO.21 to SEQ ID NO.22; The primer sequences for amplifying the AquSSR40 molecular marker are SEQ ID NO.23 to SEQ ID NO.24; The primer sequences for amplifying the AquSSR42 molecular marker are SEQ ID NO.25 to SEQ ID NO.26; The primer sequences for amplifying the AquSSR54 molecular marker are SEQ ID NO.27 to SEQ ID NO.28; The primer sequences for amplifying the AquSSR58 molecular marker are SEQ ID NO.29 to SEQ ID NO.30; The primer sequences for amplifying the AquSSR59 molecular marker are SEQ ID NO.31 to SEQ ID NO.32; The primer sequences for amplifying the AquSSR62 molecular marker are SEQ ID NO.33 to SEQ ID NO.34; The primer sequences for amplifying the AquSSR71 molecular marker are SEQ ID NO.35 to SEQ ID NO.36; The primer sequences for amplifying the AquSSR89 molecular marker are SEQ ID NO.37 to SEQ ID NO.38; The primer sequences for amplifying the AquSSR94 molecular marker are SEQ ID NO.39 to SEQ ID NO.

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2. A molecular identity card constructed based on Aquilaria sinensis SSR molecular markers, characterized in that, The molecular marker combination includes: AquSSR14, AquSSR29, AquSSR30, AquSSR54, AquSSR62 and AquSSR94; the molecular markers are amplified by the following primers in sequence: The primer sequences for amplifying the AquSSR14 molecular marker are SEQ ID NO.5 to SEQ ID NO.6; The primer sequences for amplifying the AquSSR29 molecular marker are SEQ ID NO.17 to SEQ ID NO.18; The primer sequences for amplifying the AquSSR30 molecular marker are SEQ ID NO.19 to SEQ ID NO.20; The primer sequences for amplifying the AquSSR54 molecular marker are SEQ ID NO.27 to SEQ ID NO.28; The primer sequences for amplifying the AquSSR62 molecular marker are SEQ ID NO.33 to SEQ ID NO.34; The primer sequences for amplifying the AquSSR94 molecular marker are SEQ ID NO.39 to SEQ ID NO.

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3. A primer set for amplifying the Aquilaria sinensis SSR molecular marker, characterized in that, Comprising the primer described in claim 1 or claim 2.

4. A kit, characterized in that, A primer set comprising the Aquilaria sinensis SSR molecular marker according to claim 3.

5. Use of the primer set for SSR molecular markers of Aquilaria sinensis according to claim 3 or the kit according to claim 4 in at least one of the following: (1) Application in identification of Aquilaria sinensis species and / or analysis of scent kinship; (2) Application in cluster analysis among Aquilaria sinensis germplasm; (3) Application in population genetic structure analysis of Aquilaria sinensis germplasm; (4) Application in the construction of fingerprint of Aquilaria sinensis; (5) Application in the construction of molecular identity card of Aquilaria sinensis germplasm.

6. The application according to claim 5, wherein The following steps are involved: (1) Extracting genomic DNA of the tested Aquilaria sinensis; (2) using the primer set of the SSR molecular marker of Aquilaria sinensis to perform PCR amplification on the genomic DNA of the tested Aquilaria sinensis; (3) Performing electrophoresis on the PCR amplification product to obtain the test results; (4) Using the results of step (3) to conduct genetic diversity analysis of the Aquilaria sinensis population, fingerprint construction or individual identification analysis.

7. The application according to claim 6, characterized in that, The reaction system of the PCR amplification is: 30 ng DNA template, 5 μL 2×Taq PCR Mix, 0.5 μL upstream primer, 0.5 μL downstream primer, and ddH2O to make up to 10 μL; the amplification program is: 95°C pre-denaturation for 5 min; 95°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 30 s, 35 cycles; 72°C extension for 20 min.

8. The application according to claim 5, wherein The electrophoresis detection includes polyacrylamide gel electrophoresis or capillary electrophoresis.

9. The application according to claim 5, wherein The method for constructing the fingerprint spectrum of Aquilaria sinensis is as follows: (1) According to the amplification detection results of each SSR locus, the corresponding position in the electrophoresis result with an amplified band is marked as "1", and the position without an amplified band is marked as "0"; (2) Establish the 0, 1 matrix of SSR markers of each sample 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.

10. The application according to claim 5, wherein The molecular ID card of the agarwood germplasm is constructed as follows: according to the length of the amplified fragment, each site is assigned a value using "1-9" and "A-Z", each agarwood germplasm resource is fingerprinted, the corresponding molecular ID barcode is generated using a barcode generation website, and a QR code is generated using a QR code generation website.

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