Application of SSR primer pair combination of slash pines in construction of core germplasm and fingerprint map of slash pines
By constructing the core germplasm and DNA fingerprint map of slash pine, and using SSR primer pair combinations and PCR technology, the problem of ambiguous genetic background of slash pine germplasm resources was solved, and efficient management and digitization of germplasm resources were achieved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- JIANGXI AGRICULTURAL UNIVERSITY
- Filing Date
- 2024-05-16
- Publication Date
- 2026-04-21
AI Technical Summary
In existing technologies, the genetic background of slash pine germplasm resources is ambiguous and mixed, leading to chaotic management of germplasm resources and making it difficult to achieve efficient preservation and utilization.
SSR primer pair combinations were used to construct core germplasm of Pinus slashii. SSR molecular marker data of Pinus slashii were obtained by SSR-PCR amplification. Core germplasm was selected using Power Core software, and DNA fingerprint and molecular identification of Pinus slashii were constructed.
It effectively removed genetic redundancy, improved genetic parameters, realized the digital and networked management of slash pine germplasm resources, and provided a basis for the preservation and identification of germplasm resources.
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Figure CN121896383A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of molecular biology, and in particular to the application of SSR primer pair combinations of slash pine in the construction of slash pine core germplasm and fingerprinting. Background Technology
[0002] *Pinus elliottii* Engelm., an evergreen tree belonging to the genus *Pinus* in the family Pinaceae, is native to the southeastern United States. Since the 1930s, it has been introduced and early provenance trials conducted in various southern provinces. Due to its rapid growth, good wood quality, strong adaptability, high resin yield, and excellent resin quality, it has been widely planted in southern provinces, becoming one of the important afforestation, resin-producing, and timber-producing tree species in the low hills and coastal areas of southern China. Jiangxi Province has established a slash pine gene bank through large-scale collection of slash pine germplasm resources, providing a large amount of high-quality material for the breeding of high-resin-producing and high-quality new varieties and the establishment of seed orchards. However, problems such as mixed existing slash pine germplasm and unclear genetic backgrounds limit the efficient preservation and utilization of slash pine germplasm resources. No mature solutions have yet been found to address these problems. This invention aims to solve these problems by constructing core germplasm to remove genetic redundancy and constructing fingerprint maps for germplasm identification.
[0003] Core collections refer to collections that represent the genetic diversity of an original population to the greatest extent possible with a minimal number of samples. Proposed by Frankel to address the contradiction between the vast and complex germplasm resources and the difficulties in their preservation, evaluation, identification, and utilization, core collections have been developed for numerous crops, fruit trees, and some forest trees. However, reports on the construction of core collections for slash pine are scarce.
[0004] DNA fingerprinting identifies differences in the DNA of individual organisms using molecular markers, presenting the results in the form of electrophoretic patterns. SSR (simple sequence repeats) markers are considered one of the standard marker methods for constructing DNA fingerprints due to their rich polymorphism, good stability, co-dominance, and strong specificity. DNA molecular identification cards are obtained by further encoding and assigning values based on fingerprint patterns and are widely used in food crops and fruit tree resources, enabling the digital and networked management of a large number of germplasm resources. As a major resin-producing tree species in Jiangxi Province, *Pinus slashii* has not yet had a DNA molecular identification card constructed; therefore, the digital and networked management of *Pinus slashii* germplasm resources has not yet been achieved. Summary of the Invention
[0005] The purpose of this invention is to provide the application of SSR primer pairs for *Pinus slashani* in constructing core germplasm and DNA fingerprinting, thereby addressing the problems existing in the prior art. The SSR primer pairs described in this invention can be used to construct core germplasm of *Pinus slashani*, DNA fingerprinting of *Pinus slashani*, and DNA molecular identification data for *Pinus slashani*.
[0006] To achieve the above objectives, the present invention provides the following solution:
[0007] This invention provides an SSR primer pair combination for pine slash, wherein the SSR primer pair combination includes 3 or more primer pairs as shown in the table below;
[0008]
[0009]
[0010] This invention provides the application of the above-mentioned SSR primer pair combination in constructing pine core germplasm, pine DNA fingerprinting, or pine DNA molecular identity card.
[0011] Preferably, the DNA molecular identity card includes one or more of the following: string DNA molecular identity card, barcode DNA molecular identity card, and QR code DNA molecular identity card.
[0012] This invention provides a method for constructing core germplasm of slash pine based on the above-mentioned SSR primer pair combination, comprising the following steps:
[0013] Using the genomic DNA of the original population of slash pine as template DNA, SSR-PCR amplification was performed using the above-mentioned SSR primer pair combination to obtain slash pine SSR molecular marker data.
[0014] Based on the SSR molecular marker data of slash pine, the top 10.31% of slash pine germplasm resources were selected as core slash pine germplasm using the Power Core software.
[0015] Preferably, the SSR molecular marker data of slash pine is the band size of the original population of slash pine samples.
[0016] Preferably, the SSR-PCR amplification system, in 20 μL, includes 0.1 μL of Taq enzyme, 0.8 μL each of upstream and downstream SSR primers, 1.6 μL of dNTPs, 1 μL of template DNA, 2 μL of 10×PCR Buffer, and the remainder ddH2O.
[0017] The SSR-PCR reaction procedure is as follows: pre-denaturation at 94℃ for 5 min; denaturation at 94℃ for 30 s, annealing at 55-60℃ for 30 s, extension at 72℃ for 60 s, for 30 cycles; extension at 72℃ for 7 min, and storage at 4℃.
[0018] This invention provides a method for constructing a DNA fingerprint of Pine slashis based on the above-mentioned SSR primer pair combination, comprising the following steps:
[0019] Using the genomic DNA of the original population of slash pine as template DNA, SSR-PCR amplification was performed using the above-mentioned SSR primer pair combination. The electrophoresis results were observed, and statistical data were collected to obtain the DNA fingerprint of slash pine.
[0020] Preferably, the SSR-PCR amplification system, in 20 μL, includes 0.1 μL of Taq enzyme, 0.8 μL each of upstream and downstream SSR primers, 1.6 μL of dNTPs, 1 μL of template DNA, 2 μL of 10×PCR Buffer, and the remainder is made up with ddH2O.
[0021] The SSR-PCR reaction procedure is as follows: pre-denaturation at 94℃ for 5 min; denaturation at 94℃ for 30 s, annealing at 55-60℃ for 30 s, extension at 72℃ for 60 s, for 30 cycles; extension at 72℃ for 7 min, and storage at 4℃.
[0022] This invention provides a method for constructing a DNA molecular identity card for pine trees based on the above-mentioned SSR primer pair combination, comprising the following steps:
[0023] Using the genomic DNA of the original population of slash pine as template DNA, SSR-PCR amplification was performed using the above-mentioned SSR primer pair combination to obtain genotype data. The genotype data was then encoded and assigned values to generate the DNA molecular identity card of slash pine.
[0024] Preferably, the SSR-PCR amplification system, in 20 μL, includes 0.1 μL of Taq enzyme, 0.8 μL each of upstream and downstream SSR primers, 1.6 μL of dNTPs, 1 μL of template DNA, 2 μL of 10×PCR Buffer, and the remainder ddH2O.
[0025] The SSR-PCR reaction procedure was as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 55-60℃ annealing for 30 s, 72℃ extension for 60 s, repeated 30 times; 72℃ extension for 7 min, and storage at 4℃.
[0026] The present invention discloses the following technical effects:
[0027] This invention provides an SSR primer pair combination for *Pinus slashii*, the nucleotide sequence of which is shown in SEQ ID NO. 1-40. This invention utilizes this SSR primer pair combination to amplify template DNA, and then uses Power Core software to obtain core germplasm material with a sampling ratio of 10.31%. The effectiveness of the selected core set is then verified using a genetic parameter t-test and principal coordinate analysis. The verification results show that the core germplasm of *Pinus slashii* can eliminate genetic redundancy, resulting in improved genetic parameters. Finally, a germplasm fingerprint map and a DNA molecular identification map of *Pinus slashii* were successfully constructed using the SSR primer pair combination. Therefore, the SSR primer pair combination provided by this invention can be used to construct core germplasm of *Pinus slashii*, a DNA fingerprint map of *Pinus slashii*, and a DNA molecular identification map of *Pinus slashii*.
[0028] The SSR primer pair combinations provided by this invention construct core germplasm of slash pine, DNA fingerprint map of slash pine, and DNA molecular identity card of slash pine, laying a research foundation for the preservation, germplasm identification and innovation of slash pine germplasm resources, and also providing research basis and technical support for the construction of core germplasm and fingerprint maps of other forest trees. Attached Figure Description
[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.
[0030] Figure 1 Principal coordinate distribution map of core and primitive germplasm of slash pine;
[0031] Figure 2 DNA molecular identity cards for 43 core germplasm of slash pine. Detailed Implementation
[0032] Unless otherwise specified, the experimental methods used in this invention are all conventional methods; the materials and reagents used are all commercially available unless otherwise specified.
[0033] The samples collected in this invention use 417 slash pine germplasm resources currently collected and preserved in Jiangxi Province as experimental materials, mainly divided into three groups:
[0034] 1. Introduced family population (IP): The slash pine germplasm / family experimental forest established in 1990 by Baiyunshan Forest Farm in Qingyuan District, Ji'an City, contained 113 families introduced from seed orchards in Georgia, Mississippi, and Florida, USA, ultimately resulting in the effective amplification of 112 samples; the slash pine family experimental forests established in 2011 and 2018 by Fengshushan Forest Farm in Jingdezhen contained 30 and 10 families respectively, introduced from Florida, USA, ultimately resulting in the effective amplification of 18 samples; the slash pine superior family testing forest established by Ganzhou Forestry Research Institute in 2018 contained 40 families introduced from Arkansas, USA, ultimately resulting in the effective amplification of 40 samples.
[0035] 2. First-generation superior trees breeding population (FP): mainly from early introduced slash pine plantation stands in Jiangxi Province, including 44 samples from the slash pine first-generation seed orchard established in May 1992 by Baiyunshan Forest Farm in Qingyuan District, Ji'an City; and 77 samples from the slash pine first-generation seed orchard established in 1980 by Xiajiang County Forest Seed Farm.
[0036] 3. Improved generation superior trees breeding population (IGP): mainly from 126 samples selected and established experimental forests throughout the province, including 79 samples from Baiyunshan Forest Farm in Qingyuan District, Ji'an City, and 47 samples from Xiajiang County Forest Seed Farm.
[0037] Example 1: Construction of core germplasm of slash pine
[0038] 1. Materials and Methods
[0039] 1.1 Materials
[0040] It includes 417 slash pine germplasm resources.
[0041] 1.2 Methods
[0042] 1.2.1 Construction of SSR core germplasm of slash pine
[0043] The core germplasm constructed based on SSR molecular marker data of pine slashis is called the Core Collection. The specific steps are as follows:
[0044] (1) DNA extraction and detection from slash pine
[0045] Genomic DNA was extracted from 417 samples of pine shoots or leaves using the DNAsecure Plant Kit (DP320) from Tiangen Biotech.
[0046] (2) Detection of SSR-PCR amplification products of slash pine
[0047] SSR-PCR amplification was performed using the primer pairs listed in Table 1. The SSR-PCR reaction system for *Pinus slashii* consisted of a 20 μL system containing 0.1 μL Taq enzyme, 0.8 μL each of forward and reverse SSR primers, 1.6 μL of 10 mol / L dNTPs, and 1 μL of 50 ng / μL DNA template and 10×PCR Buffer (containing Mg2+) per tube. 2 +2μL, the remainder is made up with ddH2O;
[0048] SSR-PCR reaction procedure for pine: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 60 s, 30 cycles; 72℃ extension for 7 min, storage at 4℃.
[0049] The PCR products were then subjected to capillary electrophoresis on an ABI 3730XL DNA sequencer.
[0050] Table 1. Detailed information about the primers.
[0051]
[0052] (3) Data collection
[0053] The raw peak diagram results for each pine sample were analyzed using GeneMarker V1.91 software.
[0054] (4) Construction Strategies: The M strategy (Power Core and Core Finder) for maximizing allele count automatically generates reasonable sampling ratios; the Core Hunter method sets EN (Average Entry to Nearest Entry) and SH (Shannons Diversity Index) to each have a weight of 50%, selecting a sampling ratio of 5%-40%; based on the simulated annealing algorithm, the software PowerMarker Version 3.25 is used with the SANA (maximizing allele richness) method and the SAGD (maximizing genetic diversity) method to select a sampling ratio of 5%-40%. A total of 5 different methods with 3 sampling strategies and different sampling ratios are used, resulting in 26 combinations, as detailed in Table 3. Core subsets are constructed using these 26 methods, and their genetic parameters are calculated. A t-test is performed on the genetic parameters using SPSS 26.0 to screen the most suitable method for constructing the core germplasm of Pinus slashii.
[0055] 1.2.2 Evaluation of core germplasm of slash pine
[0056] Genetic parameters such as average allele count (Na), average effective allele count (Ne), average Shannon information index (I), average observed heterozygosity (Ho), and average expected heterozygosity (He) were used to evaluate the genetic diversity of the core collection. The ratio of the genetic diversity parameters between the core germplasm and the original germplasm reflects the amount of genetic diversity retained by the core germplasm compared to the original germplasm. This ratio is called the retaining ratio (RR), which is calculated as: Retention ratio = Parameter of core germplasm / Genetic parameters of the original germplasm population. The retention ratios of the core collection across these five parameters were calculated to determine whether it has well preserved the molecular genetic diversity of the original pine germplasm. The evaluation parameters were calculated using Popgene32 software.
[0057] This invention performs principal coordinate analysis on the SSR molecular marker data of the original germplasm and the core germplasm, respectively, and draws two-dimensional scatter plots based on the scores of various germplasm materials on the first and second principal coordinates to intuitively show the retention of the original germplasm by the core germplasm.
[0058] 2. Experimental Results
[0059] 2.1 Different Construction Strategies for Core Germplasm of Pine slashii
[0060] The core set of 417 slash pine materials was selected according to 26 construction strategies, as shown in Table 2.
[0061] Table 2. Sample sources for constructing core sets using different strategies
[0062]
[0063] Note: IP: Introduced family lineage; FP: First generation superior tree breeding group; IGP: Improved generation superior tree breeding group.
[0064] Five different methods (M-strategy, Core Hunter, and simulated annealing algorithm-based strategy) were employed with varying sampling ratios (5%-40%), resulting in 26 combinations. Core subsets were constructed using each of these 26 methods, and their genetic parameters were calculated and parametric t-tests were performed. The results are shown in Tables 3 and 4.
[0065] The retention ratio of the core set relative to the original germplasm in five parameters—number of alleles (Na), effective number of alleles (Ne), Shannon information index (I), observed heterozygosity (Ho), and expected heterozygosity (He)—is used as a standard to measure the retention rate of the genetic diversity of the original population by the core set. The higher the retention ratio, the better the core set preserves the genetic diversity of the original germplasm.
[0066] Table 3 Comparison of genetic diversity indicators for constructing core sets using different strategies
[0067] Construction method Na Ne I Ho He Original germplasm 5.250 2.009 0.835 0.390 0.442 PowerCore 5.250 2.343 1.013 0.409 0.527 CoreFinder 5.250 2.220 0.961 0.379 0.498 CoreHunter (5%) 3.750* 2.066 0.845 0.377 0.456 CoreHunter (10%) 4.100 2.126 0.880 0.407 0.471 CoreHunter (15%) 4.300 2.066 0.857 0.405 0.456 CoreHunter (20%) 4.450 2.087 0.868 0.399 0.460 CoreHunter (25%) 4.600 2.082 0.866 0.397 0.460 CoreHunter (30%) 4.650 2.043 0.848 0.397 0.450 CoreHunter (35%) 4.850 2.083 0.869 0.402 0.460 CoreHunter (40%) 4.800 2.059 0.858 0.399 0.454 SANA (5%) 3.300* 1.894 0.735 0.385 0.404 SANA (10%) 4.000 2.057 0.831 0.408 0.450 SANA (15%) 4.150 1.979 0.803 0.405 0.433 SANA (20%) 4.450 2.092 0.866 0.380 0.461 SANA (25%) 4.550 1.960 0.800 0.377 0.424 SANA (30%) 4.600 2.010 0.832 0.388 0.444 SANA (35%) 4.650 2.065 0.847 0.392 0.452 SANA (40%) 4.750 2.005 0.829 0.384 0.441 SAGD (5%) 3.650* 1.954 0.785 0.373 0.428 SAGD (10%) 4.100 1.993 0.830 0.356 0.447 SAGD (15%) 4.400 1.986 0.830 0.363 0.442 SAGD (20%) 4.500 1.982 0.824 0.374 0.439 SAGD (25%) 4.800 2.016 0.849 0.377 0.449 SAGD (30%) 4.600 1.978 0.820 0.369 0.435 SAGD (35%) 4.850 2.081 0.869 0.397 0.460 SAGD (40%) 4.850 2.038 0.848 0.383 0.448
[0068] Table 4. Retention rate of genetic parameters for different construction strategies
[0069]
[0070]
[0071] As shown in Table 4, except for the Core Hunter method and the SANA method where the Na retention rate was less than 70% at a sampling ratio of 40%, the genetic parameter retention rates of the other construction strategies were all greater than 70%, indicating that the core sets constructed by different construction strategies preserved the original germplasm genetic diversity well.
[0072] As shown in Tables 3 and 4, the Na allele values ranged from 3.300 to 5.250, with retention rates ranging from 63% to 100%. The Power Core and Core Finder methods achieved the highest retention rate of 100% for constructing core sets, while the SANA method, with a 5% sampling ratio, had the lowest Na retention rate of 63%. The number of effective alleles (Ne) ranged from 1.894 to 2.343, with retention rates ranging from 94% to 117%. The Power Core method achieved the highest retention rate of 117% for constructing core sets, while the SANA method, with a 5% sampling ratio, had the lowest Ne retention rate of 94%. The Shannon information index (I) values ranged from 0.735 to 1.013, with retention rates ranging from 88% to 121%. The Power Core method achieved the highest retention rate of 121% for constructing core sets, while the SANA method, with a 5% sampling ratio, had the lowest I retention rate of 88%. The observed heterozygosity (Ho) values ranged from 0.356 to 0.409, with retention rates between 91% and 105%. The highest retention rate (105%) was achieved with the Power Core method for constructing the core set, while the lowest (91%) was achieved with the SANA method at a 10% sampling rate. The expected heterozygosity (He) values ranged from 0.404 to 0.527, with retention rates between 91% and 119%. The highest retention rate (119%) was achieved with the Power Core method for constructing the core set, while the lowest (91%) was achieved with the SANA method at a 5% sampling rate.
[0073] Comprehensive analysis revealed that the Power Core method was the optimal one, with the highest retention rate of all genetic parameters and no significant differences from the original germplasm parameters, making it the most suitable method for constructing core germplasm of slash pine.
[0074] 2.2 Evaluation of core germplasm of slash pine
[0075] 2.2.1 Evaluation of genetic diversity parameters
[0076] The results of the t-test for the diversity parameters of core germplasm, preserved germplasm, and original germplasm are shown in Table 5.
[0077] Table 5. t-test of diversity parameters of core germplasm, preserved germplasm, and original germplasm.
[0078]
[0079] As shown in Table 5, the genetic diversity parameters of the core germplasm and the preserved germplasm were not significantly different from those of the original germplasm at the 0.05 level (P>0.05).
[0080] 2.2.2 Principal Coordinate Analysis
[0081] To examine whether the core germplasm retains the genetic structure of the original germplasm population, principal coordinate analysis was performed on the core germplasm and the original germplasm. Figure 1 The results are from the principal coordinate analysis. Figure 1 It can be seen that the cumulative contribution rate of the first principal coordinate (principal coordinate 1) and the second principal coordinate (principal coordinate 2) is 29.55%. The core germplasm set of slash pine constructed in the figure is evenly distributed in the original germplasm, indicating that the constructed core germplasm of slash pine effectively removed redundant materials. This core germplasm of slash pine is a qualified core germplasm and is representative of the original germplasm.
[0082] 3. Conclusion:
[0083] Based on the principle of maximizing alleles, 43 core germplasms were selected from 417 pine germplasm materials using Power Core software, with a sampling ratio of 10.31%. After genetic parameter t-test and principal coordinate analysis, the constructed core germplasms can well represent the genetic diversity of the original germplasms.
[0084] Example 2: Construction of Molecular Identity Cards for Pine Germplasm Resources
[0085] 1. Materials and Methods
[0086] 1.1 Materials
[0087] The test materials were the same as in Example 1, consisting of 417 pine germplasm resources.
[0088] 1.2 Methods
[0089] (1) DNA extraction and detection from slash pine
[0090] Genomic DNA was extracted from 417 samples of pine shoots or leaves using the DNAsecure Plant Kit (DP320) from Tiangen Biotech.
[0091] (2) Detection of SSR-PCR amplification products of pine trees using different combinations of SSR primer pairs.
[0092] Primer pairs Pe145169, Pe103802, Pe130187, Pe132622, Pe144426, Pe106732, Pe138370, Pe131259, Pe145380, Pe113019, Pe119033, Pe140688, Pe110222, Pe134815, Pe146453, Pe114582, Pe135178, Pe144142, Pe139538, and Pe123077 were numbered with the letters AT and combined in different ways. The combinations are detailed in Table 8.
[0093] SSR-PCR reaction system for slash pine: A 20 μL system contains 0.1 μL Taq enzyme, 0.8 μL each of forward and reverse SSR primers, 1.6 μL of 10 mol / L dNTPs, and 1 μL of 50 ng / μL DNA template and 10× PCR buffer (containing Mg2+) per tube. 2+ 2 μL, the remainder was made up with ddH2O; the nucleotide sequences of the upstream and downstream primers are shown in Table 1. The SSR-PCR reaction program for pine slashis was as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 60 s, 30 cycles; 72℃ extension for 7 min, and storage at 4℃.
[0094] The PCR products were subjected to capillary electrophoresis on an ABI 3730XL DNA sequencer.
[0095] (3) Statistical analysis and numbering of genotype data of slash pine
[0096] The number of genotypes amplified at SSR loci (band size and quantity) was counted and numbered in ascending order, generating a string of numbers and letters. This string was then encoded with germplasm information such as sampling location, preservation method, and collection or preservation time to generate a molecular identification number for the slash pine. This number was then output as a QR code or barcode using a generator (https: / / qr-batch.com / index.php). The germplasm information consists of three parts: the first three digits indicate the sampling location of the germplasm material (e.g., 001 for Baiyunshan Forest Farm, Ji'an City, Jiangxi Province; 002 for Xiajiang Forest Seed Farm, Ji'an City, Jiangxi Province; 003 for Ganzhou Forestry Research Institute, Jiangxi Province; 004 for Fengshushan Forest Farm, Jingdezhen City, Jiangxi Province); the fourth digit indicates the preservation method (e.g., 1 for an introduced family experimental forest; 2 for a first-generation seed orchard; 3 for an improved seed orchard); and the last two digits indicate the collection and preservation time of the germplasm material (e.g., 1980 is recorded as 80). If the above information is unknown or unclear, it is replaced with X.
[0097] 2. Experimental Results
[0098] 2.1 Construction of Molecular Identity Cards for Pine Germplasm Resources
[0099] The discrimination rate of primer pair combinations is shown in Table 6.
[0100] Table 6 shows the identification rate of different primer combinations for pine germplasm resources.
[0101]
[0102]
[0103]
[0104] Table 8350 ID numbers of slash pine molecules
[0105]
[0106]
[0107]
[0108] 2.2 Construction of Molecular Identity Card for Core Germplasm of Pine slashii
[0109] Primer pairs Pe145169, Pe103802, and Pe144426 were numbered sequentially with the letters AC and combined as shown in Table 9. The genotypes of the three primer pairs were sorted and assigned values (Table 10). Using these three primer pairs (Pe145169, Pe103802, and Pe144426), 43 core germplasm materials could be distinguished with a 100% identification rate. These were then combined with 6-character germplasm information to generate a 9-digit alphanumeric molecular ID number for the core germplasm (Table 11) and a QR code barcode molecular ID number. Figure 2 ).
[0110] Table 9 shows the identification rate of different primer combinations for core germplasm resources of Pinus slashii.
[0111]
[0112] Table 103 lists primer banding codes.
[0113]
[0114]
[0115] Table 1.143 molecular identification numbers of core germplasm of Pine slashis
[0116] coding Molecular ID number coding Molecular ID number coding molecular ID number 21 001190JAA 216 002280DBA 346 001311JB4 42 001190M4A 245 002280GD6 347 001311KAA 79 001190AH7 285 002280KA4 353 001311AGA 85 001190JI4 290 002280CAE 361 001311FA5 93 001190BA4 305 002315OF6 372 001311JA4 96 001190DB3 309 002315OA2 374 00131114B 97 001190F2A 311 0023157A6 383 0013116AA 100 001190MBA 322 0023152CB 384 001311CJA 108 001190C4D 325 002315OA6 387 001311J1A 141 003118DEE 335 002315GA4 391 001311HBA 147 003118MFA 336 002315O46 398 001311CA4 156 003118K37 337 002315MAA 407 001311NK4 163 003118HA7 338 002315IB5 411 001311IAB 181 0012920A1 339 00131175C 215 002280A42 340 001311NAA
[0117] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.
Claims
1. An SSR primer pair combination for slash pine, characterized in that, The SSR primer pair combination includes 3 or more of the primer pairs shown in the table below; 2. The application of the SSR primer pair combination as described in claim 1 in constructing pine core germplasm, pine DNA fingerprinting, or pine DNA molecular identity card.
3. The application according to claim 2, characterized in that, The DNA molecular identity card includes one or more of the following: string DNA molecular identity card, barcode DNA molecular identity card, and QR code DNA molecular identity card.
4. A method for constructing core germplasm of *Pinus slashii* based on the SSR primer pair combination described in claim 1, characterized in that, Includes the following steps: Using the genomic DNA of the original population of slash pine as template DNA, SSR-PCR amplification was performed using the SSR primer pair combination described in claim 1 to obtain slash pine SSR molecular marker data. Based on the SSR molecular marker data of pine, the top 10.31% of pine germplasm resources were selected as core pine germplasm using the Power Core software.
5. The method according to claim 4, characterized in that, The SSR molecular marker data for slash pine refers to the band size of the original population of slash pine samples.
6. The method according to claim 4, characterized in that, The SSR-PCR amplification system, in 20 μL increments, includes 0.1 μL of Taq enzyme, 0.8 μL each of upstream and downstream SSR primers, 1.6 μL of dNTPs, 1 μL of template DNA, 2 μL of 10×PCR Buffer, and the remainder ddH2O. The SSR-PCR reaction procedure is as follows: pre-denaturation at 94℃ for 5 min; denaturation at 94℃ for 30 s, annealing at 55-60℃ for 30 s, extension at 72℃ for 60 s, for 30 cycles; extension at 72℃ for 7 min, and storage at 4℃.
7. A method for constructing a DNA fingerprint of *Pinus slashii* based on the SSR primer pair combination described in claim 1, characterized in that, Includes the following steps: Using the genomic DNA of the original population of slash pine as template DNA, SSR-PCR amplification was performed using the SSR primer pair combination described in claim 1. The electrophoresis results were observed, and statistical data were collected to obtain the DNA fingerprint of slash pine.
8. The method according to claim 7, characterized in that, The SSR-PCR amplification system, in 20 μL increments, includes 0.1 μL of Taq enzyme, 0.8 μL each of upstream and downstream SSR primers, 1.6 μL of dNTPs, 1 μL of template DNA, 2 μL of 10×PCR Buffer, and the remainder ddH2O. The SSR-PCR reaction procedure is as follows: pre-denaturation at 94℃ for 5 min; denaturation at 94℃ for 30 s, annealing at 55-60℃ for 30 s, extension at 72℃ for 60 s, for 30 cycles; extension at 72℃ for 7 min, and storage at 4℃.
9. A method for constructing a DNA molecular identity card for pine trees based on the SSR primer pair combination described in claim 1, characterized in that, Includes the following steps: Using the genomic DNA of the original population of slash pine as template DNA, SSR-PCR amplification was performed using the SSR primer pair combination described in claim 1 to obtain genotype data. The genotype data was then encoded and assigned values to generate a DNA molecular identity card for slash pine.
10. The method according to claim 9, characterized in that, The SSR-PCR amplification system, in 20 μL increments, includes 0.1 μL of Taq enzyme, 0.8 μL each of upstream and downstream SSR primers, 1.6 μL of dNTPs, 1 μL of template DNA, 2 μL of 10×PCR Buffer, and the remainder ddH2O. The SSR-PCR reaction procedure is as follows: pre-denaturation at 94℃ for 5 min; denaturation at 94℃ for 30 s, annealing at 55-60℃ for 30 s, extension at 72℃ for 60 s, for 30 cycles; extension at 72℃ for 7 min, and storage at 4℃.