Pinus armandii EST-SSR molecular marker primer group and application thereof

Abstract

The invention discloses a pinus armandii EST-SSR (expressed sequence tag-simple sequence repeat) molecular marker primer group and application thereof, and belongs to the technical field of molecular markers. According to the invention, 15 pairs of EST-SSR molecular marker primers with high polymorphism are screened through transcriptome data, a pinus armandii resource fingerprint spectrum in Sichuan province is constructed, and genetic diversity of different populations is analyzed, so that a more efficient and accurate molecular marker tool is provided for protection of pinus armandii genetic resources, germplasm resource management and genetic improvement. The invention provides a high-polymorphism EST-SSR molecular marker primer group aiming at the current situations that pinus armandii transcriptome data is not widely applied to development of EST-SSR markers in the prior art, the screening effect of existing primers is not ideal, the number of available molecular markers is small, and particularly primer research aiming at pinus armandii germplasm resources in Sichuan province is not perfect. Theoretical support and technical guarantee are provided for germplasm innovation and genetic improvement of the pinus armandii.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular markers, and particularly relates to a primer set of Pinus armandii EST-SSR molecular markers and its application. Background Art

[0002] Pinus armandii Franch. is an evergreen tree of the genus Pinus in the family Pinaceae, named after its main distribution in the Huashan area. Pinus armandii is a unique timber and fruit economic tree species in China and an important afforestation tree species in the Yangtze River shelter forest system.

[0003] At present, domestic research on Pinus armandii mainly focuses on growth laws, seedling cultivation, tending management, and pest control, etc., while the research on its germplasm resources and population genetic diversity is still in its infancy. Sichuan Province is the main distribution area of the southern provenance of Pinus armandii, providing rich germplasm materials. Among them, the total area and pine nut yield of Pinus armandii in Huidong County rank first in the country. However, most of the Pinus armandii forests originated from aerial seeding afforestation in the 1970s, facing problems such as low seed production, weak growth, few high-yield stands, and serious pests and diseases, and it is difficult to meet the needs of industrial development. Therefore, there is an urgent need to genetically improve and innovate the germplasm. However, there is a lack of systematic genetic analysis of the germplasm resources of Pinus armandii in different distribution areas, and the research on the genetic diversity and genetic relationship of Pinus armandii is still in its infancy. Therefore, it is of great significance to deeply reveal the population genetic diversity and genetic differentiation of Pinus armandii for germplasm resource protection and genetic improvement.

[0004] Simple sequence repeats (SSRs), also known as short tandem repeats or microsatellite DNA, are DNA fragments composed of tandem repeat sequences with 1-6 nucleotides as the basic unit. Since the sequences on both sides of SSRs are highly conserved, primers based on these conserved sequences are often designed to amplify the repetitive core region with high polymorphism. SSR markers have become an important tool in molecular marker-assisted breeding due to their good stability, high polymorphism, and simple operation, and are widely used in fields such as tree variety identification, genetic diversity analysis, and fingerprint map construction.

[0005] Expression Sequence Tag - Simple Sequence Repeat (EST-SSR) is a molecular marker technology based on transcriptome data. It uses known EST sequences to screen regions containing SSRs and designs primers for PCR amplification. EST-SSR molecular markers have high polymorphism and stability, but the research on Pinus armandii germplasm resources is still relatively limited.

[0006] At present, the transcriptome data of Pinus armandii has not been widely applied to the development of EST-SSR molecular markers. Existing research shows that the banding pattern effect of current primer screening is not ideal, and the available molecular markers are still relatively limited. There is no reported research on primers applicable to the germplasm resources of Pinus armandii in Sichuan. Summary of the Invention

[0007] The object of the present invention is to provide a primer set of Pinus armandii EST-SSR molecular markers and its application to solve the problems existing in the above-mentioned prior art. By screening highly polymorphic EST-SSR primers from transcriptome data, constructing a fingerprint map of Pinus armandii resources in Sichuan Province, and analyzing the genetic diversity of different populations, a more efficient and accurate molecular marker tool can be provided for the protection of Pinus armandii genetic resources, germplasm resource management, and genetic improvement.

[0008] To achieve the above object, the present invention provides the following solutions:

[0009] The present invention provides a primer set of Pinus armandii EST-SSR molecular markers, which includes 15 pairs of primers. The nucleotide sequences of the 15 pairs of primers are:

[0010] 。

[0011] The present invention also provides a kit, including the primer set.

[0012] The present invention also provides the application of the primer set or the kit in the analysis of genetic diversity or genetic differentiation characteristics of Pinus armandii germplasm resources.

[0013] The present invention also provides the application of the primer set or the kit in constructing a fingerprint map of Pinus armandii.

[0014] The present invention also provides a method for analyzing the genetic diversity or genetic differentiation characteristics of Pinus armandii germplasm resources, including: extracting genomic DNA of the Pinus armandii sample to be detected; performing PCR amplification using the primer set, and detecting the PCR amplification product by capillary electrophoresis, and analyzing the genetic diversity or genetic differentiation characteristics of Pinus armandii germplasm resources according to the detection results.

[0015] Preferably, the reaction system of the PCR amplification includes the following components: ddH 2 O 14.8 μL, dNTP 0.4 μL, PCR amplification buffer 2 μL, upstream primer F 0.3 μL (10 μM), downstream primer R 0.3 μL, DNA template 2 μL, and Taq enzyme 0.2 μL.

[0016] Preferably, the reaction procedure for PCR amplification is as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 54°C for 35 s, extension at 72°C for 40 s, for a total of 35 cycles; and finally extension at 72°C for 3 min.

[0017] The present invention discloses the following technical effects:

[0018] The present invention develops EST-SSR molecular markers with strong specificity, high polymorphism, and stable amplification through the transcriptome data of Pinus armandii. Compared with traditional genomic SSR markers, they have higher universality and conservativeness, expanding the application scope of transcriptome data in the development of SSR markers for Pinus armandii.

[0019] Based on the EST-SSR molecular marker technology, the present invention screens out 15 pairs of EST-SSR molecular marker primer sets, and conducts a genetic diversity analysis on 128 germplasm resources of Pinus armandii, revealing the genetic relationship and population genetic differentiation characteristics of Pinus armandii germplasm. It further expands the application of EST-SSR primers in the genetic diversity analysis of Pinus armandii and their potential in molecular assisted breeding, providing strong technical support for the scientific management of germplasm resources and the cultivation of new varieties.

[0020] Through capillary electrophoresis detection, the present invention constructs DNA fingerprint maps of 128 germplasm resources of Pinus armandii using 15 pairs of primers. This method has the characteristics of being fast, accurate, highly precise, and having good reproducibility and stability, and can efficiently complete the identification and classification of Pinus armandii germplasm resources, providing theoretical support and technical guarantee for the germplasm innovation and genetic improvement of Pinus armandii. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the following described drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0022] Figure 1 It is the agarose gel electrophoresis band pattern diagram of PCR amplification of some tested primers;

[0023] Figure 2 It is the partial amplification capillary fluorescence electrophoresis detection peak diagram of the ERT-SSR molecular marker primer P32 of the present invention. a - e respectively represent the detection peak diagrams of primer P32 for samples HY-2, HD-T-3, KD-3, ML-7, and TJ-25;

[0024] Figure 3 It is the cluster analysis diagram of 128 germplasm resources of Pinus armandii of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0025] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be construed as a limitation on the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terms used in the present invention are only for describing particular embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0027] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0028] Without departing from the scope or spirit of the present invention, various modifications and variations can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and embodiments of the present invention are merely exemplary.

[0029] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0030] Example 1 Development and Screening of Polymorphic Primers for Pinus armandii

[0031] 1. Transcriptome Sequencing

[0032] Samples were collected from Pinus armandii in the natural forest of Huidong County. Young and healthy needles of Pinus armandii without pests and diseases were selected for RNA extraction. The library was constructed using the Illumina TruseqTM RNA sample prep Kit. After passing the inspection, it was sequenced on the Illumina NovaSeq X Plus platform. The obtained sequences were assembled using Trinity software (https: / / github.com / trinityrnaseq / trinityrnaseq / wiki) to generate an independent gene set, that is, the transcriptome assembled from EST sequences.

[0033] 2. Identification of SSR loci and primer design and synthesis

[0034] Based on the transcriptome sequencing data, the MISA software (http: / / pgrc.ipk-gatersleben.de / misa / misa.html, default parameters) was used to identify EST-SSR loci, and primers were designed through Primer 3 (version 2.3.4). A total of 1498 primers were designed in batches. Primers with the following characteristics were screened: the primer length was 18 - 25 bases, the annealing temperature (Tm) was between 55 - 65 °C, and the length of the amplification product was between 100 - 300 bp. Further screening was carried out for primers with the following characteristics: more than 9 repeats of dinucleotide repeats, more than 6 repeats of trinucleotide repeats, and more than 5 repeats of tetranucleotide repeats. After removing duplicates of forward and reverse primers, primers with a target amplification fragment larger than 110 bp were randomly selected, and finally 100 pairs of primers were selected for synthesis.

[0035] 3. DNA extraction

[0036] One Pinus armandii clone was selected from each of the 4 provenances distributed from northeastern Sichuan to southwestern Sichuan to extract genomic DNA. Approximately 0.5 g of Pinus armandii samples were taken from each sample for DNA extraction, and the concentration and purity of DNA were detected by 1.5% agarose gel electrophoresis and ultra-micro spectrophotometer. 260 / A 280 All were between 1.8 and 2.0, indicating high DNA purity. Finally, the DNA concentration was adjusted to 50 - 200 ng / L and stored in a -20 °C refrigerator for later use.

[0037] 4. PCR amplification reaction

[0038] 100 pairs of primers were used to perform PCR amplification on the DNA of one Pinus armandii clone from each of the 4 provenances. The PCR reaction system was 20 μL: ddH 214.8 μL of O, 0.4 μL of dNTP, 2 μL of PCR amplification buffer (Buffer), 0.3 μL of upstream primer F (10 μM), 0.3 μL of downstream primer R (10 μM), 2 μL of DNA template, and 0.2 μL of Taq enzyme. The PCR amplification program was as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 54°C (annealing temperature) for 35 s, extension at 72°C for 40 s, for a total of 35 cycles; finally, extension at 72°C for 3 min. The amplified products were stored at 4°C.

[0039] 5. Screening of polymorphic primers

[0040] The PCR amplification products were detected by 1.5% agarose electrophoresis, and a total of 60 pairs amplified obvious and single bands. Subsequently, 6% polyacrylamide gel electrophoresis (PAGE) was used for further screening. Finally, 36 pairs of primers with high polymorphism were selected, and 15 pairs of primers with clear bands and good polymorphism were selected from them (see Figure 1 ).

[0041] Among the EST-SSR molecular marker primers developed based on the transcriptome sequence of Pinus armandii, 36 pairs were marked as highly polymorphic sites, and 15 pairs of primers with clear bands were selected for further study.

[0042] The 15 pairs of primers were named P32, P33, P34, P35, P44, P50, P55, P80, P86, P92, P93, P96, P113, P117, P125 respectively, and the sequences included:

[0043] Table 1 Basic characteristics of 15 pairs of primers

[0044]

[0045] Example 2 Application of EST-SSR molecular marker primers in genetic diversity analysis and genetic differentiation characteristics analysis of Pinus armandii germplasm resources

[0046] 1. DNA extraction

[0047] Genomic DNA was extracted from 128 leaf samples of Pinus armandii clones from 12 provenances in Sichuan Province in Table 2. Approximately 0.5 g of Pinus armandii samples were taken from each sample for DNA extraction. The concentration and purity of the extracted DNA were detected by 1.5% agarose gel electrophoresis and a ultra-micro spectrophotometer. The A260 / A280 was all between 1.8 and 2.0, indicating a high purity of DNA extraction. Finally, the DNA concentration was adjusted to 50 - 200 ng / L and stored in a -20°C refrigerator for later use.

[0048] Table 2 Information table of Pinus armandii samples

[0049]

[0050] 2. PCR Amplification Reaction

[0051] Using the above DNA as a template, 15 pairs of polymorphic primers screened in Example 1 were used for PCR amplification. The PCR reaction system was a total of 20 μL: ddH 2 O 14.8 μL, dNTP 0.4 μL, PCR amplification buffer (Buffer) 2 μL, upstream primer F 0.3 μL (10 μM), downstream primer R 0.3 μL (10 μM), DNA template 2 μL, and Taq enzyme 0.2 μL. The forward primer needs to be labeled with FAM. The PCR amplification program was: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 54°C for 35 s, extension at 72°C for 40 s, for a total of 35 cycles; finally, extension at 72°C for 3 min.

[0052] 3. Genetic Diversity Analysis and Genetic Differentiation Characteristic Analysis

[0053] (1) Polymorphism of SSR Primers: Capillary electrophoresis was used to detect and analyze the PCR amplification products. The specific operation was as follows: Formamide and the molecular weight internal standard ROX500 were mixed at a volume ratio of 100:1. 15 μL of the mixed solution was added to the sample loading plate, and then 1 μL of the 10-fold diluted PCR product was added. After mixing, capillary electrophoresis detection was performed using a 3730XL sequencer. Taking P32 as an example, part of the electrophoresis diagram is shown as Figure 2 shown.

[0054] The capillary electrophoresis detection results were read and analyzed. The genetic diversity of Pinus armandii was analyzed by comparing the amplified polymorphic bands. Specifically: The Popgen32 software was used to calculate the genetic diversity parameters of each primer and population, including the number of alleles (Na), effective number of alleles (Ne), Shannon's information index (I), observed heterozygosity (Ho), expected heterozygosity (He), Nei's gene diversity (H), and PIC value at each SSR locus; Nei's (1972) genetic identity (I) and genetic distance (D) were used to evaluate the degree of genetic differentiation among populations. Based on the genetic distance coefficient, the UPGMA cluster diagram was drawn using MEGA 11 software and further modified to generate the cluster diagram.

[0055] As shown in Table 3, the results showed that 15 pairs of SSR primers amplified 239 Ns in 128 tested materials in total a, on average, each pair of primers amplified 15.933 loci, with a variation range of 4 - 39. The Ne variation range was 1.782 - 14.899, with an average of 3.322. The proportion of effective alleles was 44.98%. The I value varied between 0.804 - 3.104, with an average of 0.569. The Ho variation range was 0.067 - 0.685, with an average of 0.178. The He variation range was 0.441 - 0.937, with an average of 0.820 (He > 0.5), indicating a relatively high genetic diversity of the tested Pinus armandii germplasms. Except for the He of only 2 primers being less than Ho, the He of the remaining primers was greater than Ho, indicating a low phenomenon of heterozygote excess within the tested germplasms. The average PIC was 0.799, showing a high polymorphism (PIC > 0.5). Primers with high PIC values accounted for 93.3%, indicating a relatively high polymorphism of the selected primers. These SSR loci can explain genotype differences at the molecular level and have rich genetic differences.

[0056] Table 3 Primers and Polymorphism Information of 15 SSR Loci in the Genome of Pinus armandii

[0057]

[0058] (2) Population genetic diversity: As can be seen from Table 4, the number of alleles Na of 12 provenances ranged from 5.200 to 6.933, with an average of 6.372. The range of effective allele numbers Ne was 3.413 - 5.022, and the average effective allele number was 4.405. The range of Shannon's information index was 1.297 - 1.673, and all populations were greater than 1, indicating that all 12 provenances had relatively high polymorphism. The range of Nei's gene diversity was 0.633 - 0.771, with an average of 0.714, indicating that although there were certain differences in various genetic parameters among the 12 provenances, each provenance had relatively high genetic diversity.

[0059] Table 4 Population Genetic Diversity Parameters of Pinus armandii

[0060]

[0061] (3) Cluster analysis: Using the UPGMA method, the genetic distances of 128 Pinus armandii samples from 12 provenances were analyzed to generate a cluster diagram ( Figure 3). The results showed that according to the differences in genetic distances, the samples could be clearly divided into two major groups: Group I: Qinba Mountain Area (QBSQ), including samples distributed in Xuanhan County, Chaotian District, Tongjiang County, Wangcang County, Pingwu County, and Mao County; Group II: Yunnan-Guizhou Plateau Area (YGGY), including samples distributed in Yuexi County, Butuo County, Muli Tibetan Autonomous County, and Huidong County. In addition, the samples from Kangding City and Hanyuan County in the Sichuan-Tibet Plateau Area (CZGY) showed relatively complex genetic characteristics. These samples simultaneously had the genetic characteristics of northeastern Sichuan (Qinba Mountain Area) and southwestern Sichuan (Yunnan-Guizhou Plateau Area), reflecting the important geographical role of the Sichuan-Tibet Plateau Area as a genetic exchange transition zone.

[0062] Example 3 Application of EST-SSR Molecular Marker Primers in the Construction of Pinus armandii Fingerprint Maps

[0063] Construct the fingerprint maps of 128 Pinus armandii clones with 15 pairs of SSR primers; perform capillary electrophoresis detection on the obtained PCR amplification products as in Example 2, and record the allelic marker configurations of each sample at each SSR locus according to the detection results. Code the 15 pairs of SSR primers in sequence and combine the coding information to form the SSR fingerprint maps of Pinus armandii samples. The specific operation is as follows:

[0064] Primers P32, P33, P34, P35, P44, P50, P55, P80, P86, P92, P93, P96, P113, P117, P125 are coded as letters A to O in sequence. Record the allelic gene base lengths (unit: bp) of each Pinus armandii sample in the amplification products of these 15 pairs of SSR primers to generate the fingerprint map codes of 128 Pinus armandii samples. The code format is "locus letter - allele 1 size, allele 2 size", separated by " / " between different loci, as shown in Table 5. For example, the fingerprint map code of sample BT-1 is: A-165, 165 / B-167, 167 / C-272, 272 / D-202, 202 / E-218, 231 / F-254, 254 / G-178, 184 / H-198, 198 / I-284, 284 / J-281, 281 / K-240, 240 / L-0, 0 / M-150, 152 / N-260, 260 / O-159, 165, where A-165, 165 indicates that the allelic gene base lengths amplified by primer P32 are 165 bp and 165 bp; L-0, 0 indicates that primer P96 did not amplify an effective locus on sample BT-1. Combining the capillary electrophoresis results, band positions, and the information of each sample, use online two-dimensional code generation software to convert the fingerprint map codes of 128 Pinus armandii samples into ID two-dimensional codes for rapid identification.

[0065] Table 5 Fingerprint Codes of Pinus armandii Samples (Partial)

[0066]

[0067] The embodiments described above are only descriptions of the preferred modes of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A primer set for EST-SSR molecular markers of Pinus armandii, characterized in that: It includes 15 pairs of primers, and the nucleotide sequences of the 15 pairs of primers are: 。 2. A kit, characterized in that: Comprising the primer set of claim 1.

3. Use of the primer set according to claim 1 or the kit according to claim 2 in genetic diversity analysis or genetic differentiation characteristics analysis of Pinus armandii germplasm resources.

4. Use of the primer set according to claim 1 or the kit according to claim 2 in constructing a fingerprint of Pinus armandii.

5. A method for analyzing the genetic diversity or genetic differentiation characteristics of Pinus armandii germplasm resources, characterized in that: include: Extract genomic DNA of the Pinus armandii samples to be tested; PCR amplification is performed using the primer set described in claim 1, and the PCR amplification products are detected by capillary electrophoresis, and the genetic diversity or genetic differentiation characteristics of Armand pine germplasm resources are analyzed based on the detection results.

6. The method according to claim 5, characterized in that The PCR amplification reaction system includes the following components: 14.8 μL of ddH2O, 0.4 μL of dNTP, 2 μL of PCR amplification buffer, 0.3 μL of upstream primer F (10 μM), 0.3 μL of downstream primer R, 2 μL of DNA template and 0.2 μL of Taq enzyme.

7. The method according to claim 5, characterized in that The reaction procedure of the PCR amplification was as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 54°C for 35 s, and extension at 72°C for 40 s, for a total of 35 cycles; and final extension at 72°C for 3 min.

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