Tilia amurensis primer combination, DNA fingerprint spectrum, variety identification method and application thereof
Through the construction of SSR molecular marker primer combination and DNA fingerprint map, the problem of titanium species identification was solved, efficient and accurate genetic diversity analysis and variety distinction were achieved, and it was suitable for titanium germplasm resource protection and endangered population monitoring.
Patent Information
- Application Number
- CN202510905020.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-02
- Publication Date
- 2025-08-01
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
The prior art is difficult to accurately distinguish linden and its relative species, and cannot effectively evaluate its genetic diversity, traditional morphological identification methods are insufficient, and molecular markers are time-consuming and costly.
The combination of SSR molecular marker primers was developed, including primers C110, C840, D150, and TC5. The linden DNA fingerprint was constructed by fluorescent PCR amplification and capillary electrophoresis. The primers were screened using core primer technology. Only 4 pairs of primers were needed to distinguish different linden individuals.
It has achieved efficient and accurate distinction between linden individuals and constructed a highly polymorphic DNA fingerprint map, which is suitable for linden variety identification, and supports germplasm resource protection, endangered population monitoring and molecular breeding.
Smart Images
Figure CN120400419A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the fields of molecular biology and plant genetics, and particularly relates to a primer combination for Tilia amurensis, a DNA fingerprint, a variety identification method and applications thereof. Background Art
[0002] Tilia amurensis is a national second-class protected endangered plant, an important nectar source plant, and provides habitats and food resources for insects and birds. Tilia amurensis is a well-known high-quality industrial timber tree species, and is an excellent mixed tree species for landscaping and complex communities, with important ecological and economic values. Due to habitat destruction and over-logging, its wild resources have decreased sharply. There are many variants in the morphological changes of Tilia amurensis caused by environmental and growth cycle effects, such as Tilia amurensis var. mongolica, Tilia amurensis var. dissecta, Tilia taishanensis, etc. Traditional morphological identification methods are difficult to distinguish Tilia amurensis and its related species, and cannot accurately evaluate the genetic diversity of populations.
[0003] In recent years, the commonly used genetic markers are divided into four types: morphological markers, cytological markers, biochemical markers, and molecular markers. The first three are indirect expressions of genes, with few marker loci, poor polymorphism, and are easily affected by environmental factors, so their development is relatively slow. Molecular markers, on the other hand, take less time, have low costs, are not affected by the environment, region, etc., and are widely used in related fields such as identification of plant germplasm resources, construction of molecular fingerprint maps, and research on genetic diversity.
[0004] Simple Sequence Repeats (SSR) markers have high polymorphism and good stability, have low requirements for DNA concentration, are conducive to DNA extraction of Tilia plants, and are widely used in aspects such as evaluation of germplasm resources, construction of fingerprint maps, and identification of true and false hybrids of hybrid offspring. However, there has been no report on the SSR marker combination for Tilia amurensis.
[0005] Therefore, it is an urgent problem to be solved at present to develop a primer combination of SSR molecular markers for Tilia amurensis, a DNA fingerprint of Tilia amurensis, a variety identification method for Tilia amurensis and applications thereof. Summary of the Invention
[0006] In view of the deficiencies of the prior art, a Tilia amurensis primer combination, a DNA fingerprint, a variety identification method and its application are provided. The primer combination is a primer combination of SSR molecular markers, including primer C110, primer C840, primer D150, and primer TC5. The primer sequences are shown in SEQ ID NO.1 to SEQ ID NO.8. It is used for the genetic diversity analysis of Tilia amurensis, has the characteristics of high polymorphism, can detect 70 polymorphic loci, can distinguish different Tilia amurensis individuals, and only 4 pairs of primers are needed to construct the DNA fingerprint of Tilia amurensis.
[0007] The object of the present invention is achieved by the following technical solutions: In the first aspect, the present invention provides a primer combination of Tilia amurensis SSR molecular markers. The primer combination is a primer combination of SSR molecular markers, including primer C110, primer C840, primer D150, and primer TC5; The forward sequence of primer C110 is shown in SEQ ID NO.1, and the reverse sequence is shown in SEQ ID NO.2; The forward sequence of primer C840 is shown in SEQ ID NO.3, and the reverse sequence is shown in SEQ ID NO.4; The forward sequence of primer D150 is shown in SEQ ID NO.5, and the reverse sequence is shown in SEQ ID NO.6; The forward sequence of primer TC5 is shown in SEQ ID NO.7, and the reverse sequence is shown in SEQ ID NO.8.
[0008] In the second aspect, the present invention provides a DNA fingerprint of Tilia amurensis. The fingerprint is obtained by the following construction method. The construction method includes the following steps: S1. Extract the genomic DNA of Tilia amurensis; S2. Using the genomic DNA as a template, perform fluorescence PCR amplification with the primer combination of Tilia amurensis described in the first aspect to obtain an amplification product; S3. Perform capillary electrophoresis on the amplification product, and perform data analysis on the capillary electrophoresis results to obtain fluorescence data; S4. Generate a unique code according to the allele combination of the fluorescence data through the core primer technology to construct the DNA fingerprint of Tilia amurensis; The Tilia amurensis comes from Jilin, Heilongjiang, Liaoning, Hebei, and Shandong, and specifically comes from Wangqing, Hunchun, Helong, Dongning, Ning'an, Muling, Dandong, Fuxin, Lingyuan, Chengde, and Zibo.
[0009] In some specific embodiments of the present invention, the fluorescent PCR amplification system in S2 includes 10 μL of 2×PhantaMax Master Mix, 0.15 μL of each forward and reverse primers, 1 μL of DNA template, and ddH2O to 20 μL.
[0010] In some specific embodiments of the present invention, the procedure of the fluorescent PCR amplification in S2 includes: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 30 seconds, and 35 cycles; product end perfection at 72°C for 10 minutes; and finally storage at 4°C.
[0011] In some specific embodiments of the present invention, the capillary electrophoresis step in S3 includes taking 0.3 μL of PCR product, 0.5 μL of molecular weight internal standard and 9.5 μL of deionized formamide, mixing them and adding them to a PCR plate, denaturing at 95° C. for 5 minutes, cooling at 4° C. and then centrifuging.
[0012] In a third aspect, the present invention provides a method for identifying Tilia amurensis varieties, comprising: performing PCR amplification on the genomic DNA of a Tilia amurensis sample to be tested using the primer combination described in the first aspect of the present invention; subjecting the obtained PCR amplification product to capillary electrophoresis; performing data analysis on the electrophoresis results; obtaining a unique code based on the allele combination in the data; and comparing the code with the constructed Tilia amurensis DNA fingerprint to determine the Tilia amurensis variety.
[0013] In a fourth aspect, the present invention provides an application of the Tilia amurensis DNA fingerprint described in the second aspect of the present invention in the identification of Tilia amurensis varieties.
[0014] In some specific embodiments of the present invention, the Tilia amurensis variety comes from Jilin, Heilongjiang, Liaoning, Hebei, and Shandong, specifically from Wangqing, Hunchun, Helong, Dongning, Ning'an, Muling, Dandong, Fuxin, Lingyuan, Chengde, and Zibo.
[0015] In a fifth aspect, the present invention provides a kit comprising the Tilia amurensis primer combination described in the first aspect of the present invention.
[0016] In a sixth aspect, the present invention provides a DNA chip comprising the Tilia amurensis primer combination described in the first aspect of the present invention.
[0017] In a seventh aspect, the present invention provides an application of the Tilia amurensis primer combination described in the first aspect of the present invention in Tilia amurensis germplasm resource protection, genetic diversity assessment, endangered population monitoring, and molecular breeding.
[0018] The beneficial effects achieved by the present invention are: 1. The Tilia amurensis primer combination provided by the present invention is used for the genetic diversity analysis of Tilia amurensis, and has the characteristics of high polymorphism. It can detect 70 polymorphic loci and can distinguish different Tilia amurensis individuals.
[0019] 2. The Tilia amurensis primer combination provided by the present invention only requires 4 pairs of primers to construct a DNA fingerprint map of Tilia amurensis for the rapid identification of Tilia amurensis, and has a high accuracy rate, and can effectively distinguish 170 Tilia amurensis individuals.
[0020] 3. The SSR molecular marker primer combination provided by the present invention has a wide range of applications and is suitable for the protection of Tilia amurensis germplasm resources, the monitoring of endangered populations and molecular breeding. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] In order to more clearly illustrate the technical solutions of the present application, the drawings required for use in the embodiments will be briefly introduced below. Obviously, for those of ordinary skill in the art, other drawings can also be obtained based on these drawings without creative efforts.
[0022] Figure 1 It is a capillary electrophoresis detection result diagram of the Tilia amurensis SSR molecular marker primer combination in Tilia amurensis individuals in Example 3 of the present invention.
[0023] Figure 2 It is a cluster analysis diagram of the Tilia amurensis SSR molecular marker primer combination for 16 provenance Tilia amurensis individuals in Example 3 of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS
[0024] The present invention will be further described in detail below in conjunction with specific embodiments. The following embodiments are not used to limit the present invention, but only to illustrate the present invention. The experimental methods used in the following embodiments are not specifically described, and the experimental methods without specific conditions in the embodiments are usually in accordance with conventional conditions. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels without specific description.
[0025] Example 1 Collection of Tilia amurensis Plant Samples Resources were collected from the main regions where Tilia amurensis is distributed across the country, and a total of 16 provenances and 173 samples were collected. The Tilia amurensis samples of different provenances are shown in Table 1.
[0026] Table 1 Tilia amurensis samples of different provenances
[0027] Example 2 Screening of Tilia amurensis SSR Primers Based on the research on the genetic diversity of Tilia plants, 12 pairs of SSR primers were obtained. These primers were subjected to PCR amplification screening, and primer pairs were preferably selected according to the peak shape quality and polymorphism level of the amplification products. Among them, the primer pairs B2410, B2550, B2915, B485, C840, C110, D150, and TC5 had good peak shapes and high polymorphism, and a high amplification success rate, while the primer pairs Tc915, Tc943, B2405, and B75 had poor peak shapes and a low amplification success rate. Therefore, the primer pairs B2410, B2550, B2915, B485, C840, C110, D150, and TC5 were preferably selected.
[0028] Using the core primer technology, the primer pairs were further screened. Key parameters such as Nei's genetic diversity, the number of alleles, and the polymorphism information content were comprehensively considered and statistically analyzed using the Powermarker V3.25 software. Among them, the primer pairs C840, C110, D150, and TC5 had a high polymorphism information content (>0.7) and a large number of alleles (≥10 for each primer), while the primer pairs B2410, B2550, B2915, and B485 had a lower polymorphism information content and a smaller number of alleles. Therefore, the primer pairs C840, C110, D150, and TC5 were preferably selected.
[0029] The basic information of the Tilia amurensis SSR marker primers is shown in Table 2. The forward sequence of primer B2410 is as shown in SEQ ID NO.9, and the reverse sequence is as shown in SEQ ID NO.10. The forward sequence of primer B2550 is as shown in SEQ ID NO.11, and the reverse sequence is as shown in SEQ ID NO.12. The forward sequence of primer B2915 is as shown in SEQ ID NO.13, and the reverse sequence is as shown in SEQ ID NO.14. The forward sequence of primer B485 is as shown in SEQ ID NO.15, and the reverse sequence is as shown in SEQ ID NO.16. The forward sequence of primer Tc915 is as shown in SEQ ID NO.17, and the reverse sequence is as shown in SEQ ID NO.18. The forward sequence of primer Tc943 is as shown in SEQ ID NO.19, and the reverse sequence is as shown in SEQ ID NO.20. The forward sequence of primer B2405 is as shown in SEQ ID NO.21, and the reverse sequence is as shown in SEQ ID NO.22. The forward sequence of primer B75 is as shown in SEQ ID NO.23, and the reverse sequence is as shown in SEQ ID NO.24.
[0030] Description: Core Primer Technology is a primer design strategy in molecular biology for efficiently amplifying target DNA sequences. Its core idea is to design universal or semi-universal primers (i.e., "core primers") to amplify multiple target regions, thereby improving amplification efficiency and reducing the number of primers.
[0031] Table 2 Basic information of Tilia amurensis SSR marker primers
[0032] Example 3 Construction of Tilia amurensis DNA fingerprint For each Tilia amurensis provenance collected in Example 1, the following operations were performed on each Tilia amurensis sample.
[0033] 3.1 Extraction of Tilia amurensis genomic DNA (1) Weigh about 20 mg of dry Tilia amurensis leaf tissue and thoroughly crush it at low temperature in a pre-cooled mortar; quantitatively transfer the plant tissue powder to a centrifuge tube containing 1 mL of pre-cooled SET nuclear separation solution (SET nuclear separation solution: Tris•Hcl, 0.1 mol / L; EDTA, 0.05 mol / L; Nacl, 0.25 mol / L; β-mercaptoethanol, 1% (V / V)), mix well, and incubate on ice for 15 min; After the ice bath, centrifuge at 12000 rpm for 5 min to remove the supernatant; add 700 μL of preheated CTAB nuclear lysis solution and incubate in a water bath at 65 °C for 30 min, shaking the centrifuge tube to fully mix the materials.
[0034] (3) After taking it out and allowing it to reach room temperature, add 700 μL of nucleic acid extraction solution, gently invert and mix, and centrifuge at 12000 rpm for 15 min. Add an equal volume of isopropanol to the supernatant, gently invert 25 - 30 times, and a flocculent substance without wrapping will appear. Place it in a -20 °C refrigerator for 2 - 3 hours.
[0035] (4) Primary precipitation: Centrifuge at 12000 rpm for 2 min, discard the supernatant, inject 1 mL of absolute ethanol and let it stand for 1 min. Secondary precipitation: Centrifuge at the same parameters for 1 min, remove the supernatant, add 1 mL of 75% alcohol and let it stand for 1 min, centrifuge again for 1 min, and pour out the supernatant. Drying: After pouring out the supernatant, centrifuge at 12000 rpm for 2 min, precisely aspirate the excess liquid with a pipette gun, and place it in a fume hood for about 5 min.
[0036] (5) Preheat the eluent at 65 °C, add 60 μL of eluent to dissolve the precipitate, place it in a 4 °C refrigerator overnight for dissolution, and store it in a -20 °C refrigerator after complete dissolution.
[0037] (6)The concentration and purity of DNA were detected using a Nanodrop 2000 ultraviolet spectrophotometry system, and the sharpness of the bands and the quality of DNA were observed by 1% agarose gel electrophoresis. The qualified DNA samples were stored at -20 °C in a constant temperature refrigerator for later use.
[0038] 3.2 PCR Amplification Experimental method: Four pairs of Tilia amurensis SSR primer combinations screened were used for amplification to obtain PCR amplification products.
[0039] The fluorescence primer PCR amplification system was: 2×Phanta Max Master Mix 10 μL, 0.15 μL each of the forward and reverse primers, 1 μL of DNA template, and supplemented with ddH2O to 20 μL.
[0040] The PCR reaction program was: pre-denaturation at 95 °C for 5 min to fully unwind the strands; denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 30 s, for 35 cycles; final extension at 72 °C for 10 min to perfect the product ends; and finally stored at 4 °C to prevent product degradation.
[0041] 3.3 Capillary Electrophoresis Detection Experimental method: Within 24 h after the PCR amplification reaction was completed, 0.3 μL of the PCR product, 0.5 μL of the molecular weight internal standard, and 9.5 μL of deionized formamide were mixed and added to the PCR plate, denatured at 95 °C for 5 min, centrifuged after cooling at 4 °C, and detected on the machine using 1×Buffer buffer to record the allele size.
[0042] Specific parameter settings for machine detection: Module file GeneMapper50_POP7_1, running voltage 15.0 kv, injection voltage 1.6 kv, injection time 15 s, temperature 63 °C, current stability 30.0 uA, and each experiment was repeated three times.
[0043] Among them, the capillary electrophoresis detection results of the PCR amplification of primer D150 for individuals JWQ1-10 in the Tilia amurensis population are as Figure 1 shown.
[0044] 3.4 SSR Fluorescence Capillary Electrophoresis Data Analysis Capillary tube analysis results from all Tilia provenances and samples were collated and statistically analyzed using Excel 2010 software. PopGene 1.32 software was used to calculate the following parameters: observed number of alleles (Na), effective number of alleles (Ne), Shannon information index (I), observed heterozygosity (Ho), expected heterozygosity (He), polymorphic information content (PIC), and Nei's diversity index (H). The amplification products and their polymorphisms using the four SSR primer combinations for Tilia are shown in Table 3. The results showed that this primer combination was able to detect 70 polymorphic loci, exhibiting high polymorphism and making it suitable for genetic diversity analysis in Tilia.
[0045] Genalex 6.501 and Powermarker V3.25 software were used to calculate molecular variance analysis (AMOVA), genetic differentiation coefficient (Fst), gene flow (Nm), genetic distance (D) and genetic identity (In) between different populations, and the Mantel test for the correlation between geographical distance and genetic distance was performed.
[0046] The NJ method of MEGA 11 software was used to perform cluster analysis on Tilia amurensis populations from 16 provenances, and a cluster analysis dendrogram was drawn, as shown in the figure. Figure 2 As shown in the figure, the cluster analysis dendrogram divided the 16 Tilia amurensis populations into 5 categories, which was consistent with the geographical distribution and verified the reliability of the markers.
[0047] Table 3 Amplification products and their polymorphisms of four SSR primer pairs in Tilia amurensis
[0048] 3.5 Construction of SSR fingerprint of Tilia amurensis method: Fingerprints were constructed using core primer technology. Key parameters such as Nei's genetic diversity, number of alleles, and polymorphism information content were comprehensively considered and statistically analyzed using Powermarker V3.25 software. Finally, core primers were selected.
[0049] Alleles were named according to their size (e.g., C110 249 bp / 249 bp). The uniqueness of the fingerprint was assessed using the formula P = 1 / 2 n, where n is the total number of polymorphic sites in the core primers and P is the probability of identical fingerprints between any two varieties. A unique code (e.g., A249 / 249-B182 / 182-C158 / 158-D150 / 150) was generated based on the allele combination to construct the Tilia amurensis SSR fingerprint. Table 4 shows the DNA fingerprint codes for some Tilia amurensis individuals.
[0050] Table 4 DNA fingerprint codes of some Tilia amurensis individuals
[0051] 3.6 Identification of Tilia amurensis Samples The 170 Tilia amurensis samples were identified using the Tilia amurensis SSR fingerprint map constructed above.
[0052] Identification method: The genomic DNA of the Tilia amurensis samples to be tested was subjected to PCR amplification using the 4 primer combinations screened in Example 2. The obtained PCR amplification products were subjected to capillary electrophoresis, and the electrophoresis results were subjected to data analysis. The unique code was obtained according to the allele combinations in the data and compared with the constructed Tilia amurensis DNA fingerprint map to determine the Tilia amurensis variety.
[0053] Results: The constructed Tilia amurensis SSR fingerprint map could successfully distinguish 170 Tilia amurensis samples, and the genetic diversity parameters were He = 0.528 and PIC = 0.739, indicating that the Tilia amurensis SSR marker combination had high efficiency, high specificity, and high polymorphism.
[0054] The above specific implementation manners further detailed the purpose, technical solutions, and beneficial effects of the present application. It should be understood that the above are only specific implementation manners of the present application and are not used to limit the protection scope of the present application. Any modifications, equivalent replacements, improvements, etc. made on the basis of the technical solutions of the present application shall be included in the protection scope of the present application.
Claims
1. A primer combination for Tilia amurensis, characterized in that, The primer combination is a primer combination for SSR molecular markers, comprising primer C110, primer C840, primer D150 and primer TC5; The forward sequence of the primer C110 is shown as SEQ ID NO.1, and the reverse sequence is shown as SEQ ID NO.2; The forward sequence of the primer C840 is shown in SEQ ID NO.3, and the reverse sequence is shown in SEQ ID NO.4; The forward sequence of the primer D150 is shown in SEQ ID NO.5, and the reverse sequence is shown in SEQ ID NO.6; The forward sequence of the primer TC5 is shown in SEQ ID NO.7, and the reverse sequence is shown in SEQ ID NO.
8.
2. A DNA fingerprinting map of Tilia amurensis, characterized in that, The fingerprint spectrum is obtained by the following construction method, which includes the following steps: S1. Extract genomic DNA from Tilia amurensis; S2. Using the genomic DNA as a template, performing fluorescent PCR amplification using the Tilia amurensis primer combination described in claim 1 to obtain an amplified product; S3, performing capillary electrophoresis on the amplified product and performing data analysis on the capillary electrophoresis results to obtain fluorescence data; S4. Using core primer technology, the fluorescence data is used to generate a unique code according to the allele combination to construct a DNA fingerprint of Tilia amurensis; The described Tilia amurensis comes from Jilin, Heilongjiang, Liaoning, Hebei and Shandong, specifically from Wangqing, Hunchun, Helong, Dongning, Ning'an, Muling, Dandong, Fuxin, Lingyuan, Chengde and Zibo.
3. The Tilia amurensis Rupr. DNA fingerprinting map according to claim 2, characterized in that, The fluorescent PCR amplification system in S2 includes 10 μL of 2×Phanta Max Master Mix, 0.15 μL of each of forward and reverse primers, 1 μL of DNA template, and ddH 2 O to make up to 20 μL.
4. The Tilia amurensis Rupr. DNA fingerprint map according to claim 3, characterized in that, The procedure of the fluorescent PCR amplification in S2 includes: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 30 seconds, 35 cycles; product end completion at 72°C for 10 minutes; and finally storage at 4°C.
5. The Tilia amurensis Rupr. DNA fingerprint map according to claim 2, wherein The specific steps of the capillary electrophoresis in S3 include taking 0.3 μL of PCR product, 0.5 μL of molecular weight internal standard and 9.5 μL of deionized formamide, mixing them and adding them to a PCR plate, denaturing at 95° C. for 5 minutes, cooling at 4° C. and then centrifuging.
6. A method for identifying Tilia amurensis varieties, characterized in that, The primer combination according to claim 1 is used to perform PCR amplification on the genomic DNA of the Tilia amurensis sample to be tested, the obtained PCR amplification product is subjected to capillary electrophoresis, the electrophoresis result is subjected to data analysis, a unique code is obtained according to the allele combination in the data, and the unique code is compared with the constructed Tilia amurensis DNA fingerprint to determine the Tilia amurensis variety.
7. Use of the Tilia amurensis DNA fingerprinting map according to claim 2 in the identification of Tilia amurensis varieties, characterized in that, The described Tilia amurensis varieties come from Jilin, Heilongjiang, Liaoning, Hebei and Shandong, specifically from Wangqing, Hunchun, Helong, Dongning, Ning'an, Muling, Dandong, Fuxin, Lingyuan, Chengde and Zibo. A kit comprising the Tilia amurensis primer combination according to claim 1 . 9 . A DNA chip comprising the Tilia amurensis primer combination according to claim 1 .
10. Use of the Tilia amurensis primer combination according to claim 1 in the protection of Tilia amurensis germplasm resources, assessment of genetic diversity, monitoring of endangered populations, and molecular breeding.
Citation Information
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