A set of Pinus armandii EST-SSR molecular marker primers and its application

Abstract

The present invention discloses a primer set of EST-SSR molecular markers for Pinus armandii and its application, belonging to the technical field of molecular markers. Through transcriptome data screening, the present invention selects 15 pairs of EST-SSR molecular marker primers with high polymorphism, constructs a fingerprint map of Pinus armandii resources in Sichuan Province, analyzes the genetic diversity of different populations, and further provides a more efficient and accurate molecular marker tool for the protection of Pinus armandii genetic resources, germplasm resource management and genetic improvement. In view of the current situation that the transcriptome data of Pinus armandii in the prior art has not been widely applied to the development of EST-SSR markers, the existing primer screening effect is not ideal, and the number of available molecular markers is small, especially the primer research on the germplasm resources of Pinus armandii in Sichuan Province is not perfect, the present invention provides a primer set of EST-SSR molecular markers with high polymorphism, providing theoretical support and technical guarantee for the germplasm innovation and genetic improvement of Pinus armandii.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular markers, and particularly to a primer set of EST-SSR molecular markers for Pinus armandii 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 of Pinus armandii and the yield of pine nuts 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 setting, 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 (SSR), 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. Due to the highly conserved sequences on both sides of SSR, 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. No reports have been found on the primer-related research applicable to the germplasm resources of Pinus armandii in Sichuan. Summary of the Invention

[0007] The purpose 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 purpose, 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: 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.

[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; 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 Pinus armandii SSR markers.

[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 genetic diversity analysis on 128 germplasm resources of Pinus armandii, revealing the genetic relationship and population genetic differentiation characteristics of Pinus armandii germplasm. Further expanding 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, high-precision, 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. 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 drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also 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 the P32 primer pair 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 Embodiments

[0025] A variety of exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of 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 specific 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 those 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 examples 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 double-base repeats, more than 6 triple-base repeats, and more than 5 quadruple-base 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 clone of Pinus armandii 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 a 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] PCR amplification was performed on the DNA of one Chinese pine clone from each of the 4 provenances using 100 pairs of primers. The PCR reaction system was 20 μL: 14.8 μL of ddH2O, 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 gel electrophoresis, and 60 pairs of them 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 Chinese pine transcriptome sequence above, 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, and the sequences are as follows:

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

[0044]

[0045] Example 2 Application of EST-SSR molecular marker primers to genetic diversity analysis and genetic differentiation characteristics analysis of Chinese pine germplasm resources

[0046] 1. DNA extraction

[0047] Genomic DNA was extracted from the leaf samples of 128 Chinese pine clones from 12 provenances in Sichuan Province in Table 2. Approximately 0.5 g of Chinese pine 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 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: 14.8 μL of ddH2O, 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 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, renaturation 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, a partial electrophoresis diagram was 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 clustering map was drawn using the MEGA 11 software and was secondarily modified to generate the clustering map.

[0055] As shown in Table 3, the results showed that: 15 pairs of SSR primers amplified 239 Ns in a total of 128 tested materials a, on average, each pair of primers amplified 15.933 loci, with a range of 4 - 39. The range of Ne was 1.782 - 14.899, with an average of 3.322, and the proportion of effective alleles was 44.98%. The I value ranged from 0.804 to 3.104, with an average of 0.569. The range of Ho was 0.067 - 0.685, with an average of 0.178. The range of He was 0.441 - 0.937, with an average of 0.820 (He > 0.5), indicating a relatively high genetic diversity of the tested Chinese white pine germplasms. For all primers except two, He was greater than Ho, indicating a low phenomenon of heterozygote excess in the tested germplasms. The average PIC was 0.799, showing 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 Chinese White Pine Genome

[0057]

[0058] (2) Population genetic diversity: As shown in Table 4, the number of alleles Na in 12 provenances ranged from 5.200 to 6.933, with an average of 6.372. The number of effective alleles Ne ranged from 3.413 to 5.022, with an average number of effective alleles of 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 Chinese White Pine

[0060]

[0061] (3) Cluster analysis: Using the UPGMA method, the genetic distances of 128 Chinese white pine samples from 12 provenances were analyzed to generate a cluster diagram ( Figure 3). The results showed that the samples can be clearly divided into two groups according to the difference in genetic distance: 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 (YGGY), including samples distributed in Yuexi County, Butuo County, Muli Tibetan Autonomous County, and Huidong County. In addition, samples from Kangding City and Hanyuan County in the Sichuan-Tibet Plateau (CZGY) showed more complex genetic characteristics. These samples have genetic characteristics of both northeastern Sichuan (Qinba Mountain Area) and southwestern Sichuan (Yunnan-Guizhou Plateau), reflecting the important geographical role of the Sichuan-Tibet Plateau as a transition zone for genetic exchange.

[0062] Example 3 Application of EST-SSR molecular marker primers in the construction of Pinus armandii fingerprint

[0063] Construct 128 fingerprints of Armand pine clones with 15 pairs of SSR primers; perform capillary electrophoresis detection on the obtained PCR amplification products as in Example 2, and record the allele marker configuration of each sample at each SSR site according to the detection results. Encode the 15 pairs of SSR primers in sequence, combine the encoding information, and form the SSR fingerprint of the Armand pine samples. The specific operations are:

[0064] Primers P32, P33, P34, P 35, P 44, P 50, P 55, P 80, P 86, P 92, P 93, P 96, P 113, P117, and P 125 were coded as letters A to O, respectively, and the allele base length (unit: bp) of each Armand pine sample in the amplification product of these 15 pairs of SSR primers was recorded to generate the fingerprint codes of 128 Armand pine samples. The code format was “locus letter-allele 1 size, allele 2 size”, and different loci were separated by “ / ”, as shown in Table 5. For example, the fingerprint code of the BT-1 sample 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 means that the base length of the allele amplified by primer P32 is 165bp, 165 bp; L-0, 0 means that the P96 primer did not amplify a valid site on the BT-1 sample. Combining the capillary electrophoresis results, band positions and information of each sample, the fingerprint codes of 128 Armand pine samples were converted into ID card QR codes for rapid identification using online QR code generation software.

[0065] Table 5 Fingerprint codes of Pinus armandii samples (part)

[0066]

[0067] The embodiments described above are only descriptions of the preferred embodiments 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 Pinus armandii EST-SSR molecular markers, characterized in that, It includes 15 pairs of primers, and the nucleotide sequences of the 15 pairs of primers are as follows:

2. A kit, characterized in that, It includes the primer set described in claim 1.

3. Use of the primer set described in claim 1 or the kit described in claim 2 in the analysis of genetic diversity or genetic differentiation characteristics of Pinus armandii Franch. germplasm resources in Sichuan Province.

4. Use of the primer set described in claim 1 or the kit described in claim 2 in constructing the fingerprint map of Pinus armandii Franch. in Sichuan Province.

5. A method for analyzing the genetic diversity or genetic differentiation characteristics of Pinus armandii Franch. germplasm resources in Sichuan Province, characterized in that, It includes: Extract the genomic DNA of the Pinus armandii Franch. sample to be detected; Perform PCR amplification using the primer set described in claim 1, and perform capillary electrophoresis detection on the PCR amplification product, and analyze the genetic diversity or genetic differentiation characteristics of Pinus armandii Franch. germplasm resources according to the detection results.

6. The method according to claim 5, wherein The reaction system of the PCR amplification includes the following components: 14.8 μL of ddH2O, 0.4 μL of dNTP, 2 μL of PCR amplification buffer, 0.3 μL of 10 μM upstream primer F, 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: 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.

Images