Pinus sylvestris SSR (Simple Sequence Repeat) markers, method for constructing pinus sylvestris core germplasm by combining SSR markers and stable phenotypes and application

Abstract

The invention discloses a group of pinus sylvestris SSR markers, a method for constructing pinus sylvestris core germplasm in combination with the SSR markers and stable phenotypes and application. The SSR marker comprises 11 newly developed SSR loci, namely, Ps8, Ps10, Ps23, Ps36, Ps61, Ps63, Ps70, Ps82, Ps89, Ps163 and Ps164, and the loci have high polymorphism and good repeatability. Based on the markers, the pinus sylvestris core germplasm can be constructed, and the markers are further used for constructing SSR fingerprint spectrums. In addition, the invention also discloses a method for constructing the pinus sylvestris core germplasm by combining the SSR marker and the stable phenotype. The SSR marker and the method for constructing the pinus sylvestris core germplasm in combination with the SSR marker and the stable phenotype can scientifically guide afforestation and breeding work, and have important significance on germplasm research of timber species for long felling.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic breeding, and specifically relates to a group of mongolica pine SSR markers and a method and application thereof for constructing mongolica pine core germplasm by combining SSR markers and stable phenotypes. Background Art

[0002] In forest tree breeding, core germplasm plays a particularly important role. Core germplasm preserves genetic diversity to the maximum extent with the smallest germplasm quantity and is the core subset for germplasm resource management and utilization. When constructing core germplasm, it is usually necessary to type the individuals and try to select individuals with different typing results to form the core germplasm, so as to preserve genetic diversity to the maximum extent with the smallest germplasm quantity.

[0003] The data used to construct the core collection are mainly phenotypic traits and molecular markers, each of which has its own advantages and disadvantages. Molecular markers such as SSR and SNP are numerous and highly polymorphic, but they reflect genotypic differences and cannot directly reflect trait differences, that is, the phenotypic traits of two individuals with different typing results may be similar. Phenotypic traits directly reflect the growth, material characteristics and other direct utilization values ​​of forest trees, and are easy to measure, but are easily disturbed by plant growth stages and environmental effects. It is difficult to exclude the influence of age and environment and measure stable phenotypic traits. The phenotypic variation law of germplasm cannot be accurately evaluated based on the phenotypic traits of natural populations of different locations and ages or the phenotypic traits of young forests in the same environment. In addition, phenotypic differentiation and molecular variation usually respond to different evolutionary pressures and show relatively independent characteristics. Therefore, using phenotypic traits or molecular marker data alone to select core collections cannot fully reflect the diversity of the original collections. The core collections obtained may have different phenotypes with the same genotype or similar phenotypes with different genotypes, resulting in weak representativeness of the core collections.

[0004] The combination of phenotypic traits and molecular markers can more accurately assess the genetic diversity of germplasm resources, exclude germplasms with similar phenotypes or similar genetic backgrounds, and enhance the representativeness and effectiveness of core germplasm. However, currently only a few timber tree species use phenotypic traits and molecular markers together to construct core germplasm, and most of them adopt a segmented construction method, that is, first construct a primary core germplasm based on geographical distribution and phenotype, and then use molecular markers to screen to obtain the final core germplasm; or use phenotypic traits and molecular markers to screen separately, and then integrate to form a core germplasm.

[0005] Currently, only Pinus koraiensis and Pinus yunnanensis are used to construct core collections by directly combining phenotypic traits and molecular markers. The main characteristics are needles and seeds of natural populations of different ages. The key growth and morphological traits are only DBH or height below branches and crown width. The trees are not planted in the same environment and phenotypes are not measured. The effects of environment, age and growth stage on phenotypes are not excluded. Therefore, individuals with different phenotypes in the core collection may have similar genotypes, and individuals with the same phenotype may have completely different genotypes. That is, the phenotypic variation and genetic diversity of the core collection may be insufficiently representative and effective. At present, there are no reports on the methods and research cases of using SSR markers and stable phenotypes to construct core collections of long-rotation timber species.

[0006] Scots pine (Pinus sylvestris Linn. var. mongolica Litv.) is a geographical variant of Scots pine (Pinus sylvestris Linn.), which is naturally distributed in the northern part of Greater Khingan Range, Hulunbuir Desert, the transition zone between Greater and Lesser Khingan Range, along the Heilongjiang River, and parts of Mongolia and northeastern Russia. Scots pine is fast-growing, cold-resistant, drought-resistant, and barren-resistant, and is an excellent ecological and economic tree species in the arid and semi-arid areas of the north. It plays an irreplaceable role in windbreak and sand fixation, soil and water conservation, and other important functions in the "Three Norths" shelterbelt project. Its wood and turpentine are also widely used in furniture, house construction, shipbuilding, medicine, health care, environmental protection and other fields.

[0007] my country has vigorously developed Larix gmelinii plantations. So far, the planting area has exceeded 800,000 hectares. In order to promote the genetic improvement of Larix gmelinii, predecessors have carried out large-scale germplasm resource collection, studied the genetic diversity of Larix gmelinii natural populations, selected a batch of excellent germplasms through provenance experiments, and established a primary seed garden. However, due to the large scale of its breeding resources and unclear genetic background, the management and utilization costs of germplasm resources and the risk of inbreeding depression in seed gardens have increased. Therefore, it is urgent to carry out a comprehensive evaluation of Larix gmelinii germplasm resources and construct core germplasm to effectively promote the genetic improvement of Larix gmelinii. Summary of the invention

[0008] To this end, the technical problem to be solved by the present invention is to provide a group of SSR markers with high polymorphism and good repeatability for Larix gmelinii and a method and application of constructing Larix gmelinii core germplasm by combining SSR markers and stable phenotypes. These SSR markers can be used to construct the SSR fingerprint map of Larix gmelinii, and can be combined with stable phenotypes to construct Larix gmelinii core germplasm, providing technical support for the evaluation, breeding and variety right protection of Larix gmelinii germplasm resources.

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

[0010] A group of Pinus sylvestris SSR markers, the SSR markers comprising Ps8, Ps10, Ps23, Ps36, Ps61, Ps63, Ps70, Ps82, Ps89, Ps163 and Ps164;

[0011] The nucleotide sequence of Ps8 is shown in SEQ ID NO.1;

[0012] The nucleotide sequence of Ps10 is shown in SEQ ID NO.2;

[0013] The nucleotide sequence of Ps23 is shown in SEQ ID NO.3;

[0014] The nucleotide sequence of Ps36 is shown in SEQ ID NO.4;

[0015] The nucleotide sequence of Ps61 is shown in SEQ ID NO.5;

[0016] The nucleotide sequence of Ps63 is shown in SEQ ID NO.6;

[0017] The nucleotide sequence of Ps70 is shown in SEQ ID NO.7;

[0018] The nucleotide sequence of Ps82 is shown in SEQ ID NO.8;

[0019] The nucleotide sequence of Ps89 is shown in SEQ ID NO.9;

[0020] The nucleotide sequence of Ps163 is shown in SEQ ID NO.10;

[0021] The nucleotide sequence of Ps164 is shown in SEQ ID NO.11.

[0022] A primer set for specific amplification of the above-mentioned Pinus sylvestris SSR marker, the primer set comprising 11 pairs of SSR marker core primer pairs and M13 primers; wherein:

[0023] The SSR marker core primer pair for specific amplification of Ps8 consists of primers whose sequences are shown in SEQ ID NO.12 and SEQ ID NO.13, respectively;

[0024] The SSR marker core primer pair for specific amplification of Ps10 consists of primers whose sequences are shown in SEQ ID NO.14 and SEQ ID NO.15, respectively;

[0025] The SSR marker core primer pair for specific amplification of Ps23 consists of primers whose sequences are shown in SEQ ID NO.16 and SEQ ID NO.17, respectively;

[0026] The SSR marker core primer pair for specific amplification of Ps36 consists of primers whose sequences are shown in SEQ ID NO.18 and SEQ ID NO.19, respectively;

[0027] The SSR marker core primer pair for specific amplification of Ps61 consists of primers whose sequences are shown in SEQ ID NO.20 and SEQ ID NO.21, respectively;

[0028] The SSR marker core primer pair for specific amplification of Ps63 consists of primers whose sequences are shown in SEQ ID NO.22 and SEQ ID NO.23, respectively;

[0029] The SSR marker core primer pair for specific amplification of Ps70 consists of primers whose sequences are shown in SEQ ID NO.24 and SEQ ID NO.25, respectively;

[0030] The SSR marker core primer pair for specific amplification of Ps82 consists of primers whose sequences are shown in SEQ ID NO.26 and SEQ ID NO.27, respectively;

[0031] The SSR marker core primer pair for specific amplification of Ps89 consists of primers whose sequences are shown in SEQ ID NO.28 and SEQ ID NO.29, respectively;

[0032] The SSR marker core primer pair for specific amplification of Ps163 consists of primers whose sequences are shown in SEQ ID NO.30 and SEQ ID NO.31, respectively;

[0033] The SSR marker core primer pair for specific amplification of Ps164 consists of primers whose sequences are shown in SEQ ID NO.32 and SEQ ID NO.33, respectively;

[0034] The primers shown in SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16, SEQ ID NO.18, SEQ ID NO.20, SEQ ID NO.22, SEQ ID NO.24, SEQ ID NO.26, SEQ ID NO.28, SEQ ID NO.30 and SEQ ID NO.32 are forward primers with M13 sequence; SEQ ID NO.13, SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO.19, SEQ ID NO.21, SEQ ID NO.23, SEQ ID NO.25, SEQ ID NO.27, SEQ ID NO.29, SEQ ID NO.31 and SEQ ID NO.33 are reverse primers;

[0035] The sequence of the M13 primer is shown in SEQ ID NO.34, and a fluorescent marker is connected to the M13 primer.

[0036] In the above primer set, the fluorescent label connected to the M13 primer is any one of FAM, NED, VI C and ROX.

[0037] A method for constructing a core germplasm of Pinus sylvestris var. mongolica by combining SSR markers and stable phenotypes, the method comprising: using the above-mentioned SSR markers to perform typing on original germplasms of Pinus sylvestris var. mongolica that are located in the same site and are 27 to 41 years old and of the same age, to obtain SSR typing results; measuring the phenotypic traits of the original germplasm of Pinus sylvestris var. mongolica to obtain phenotypic trait measurement results; and selecting a core germplasm from the original germplasm of Pinus sylvestris var. mongolica based on the phenotypic trait measurement results and the SSR typing results;

[0038] Among them, the original germplasm of Pinus sylvestris var. mongolica constitutes the Pinus sylvestris var. mongolica provenance experimental forest, and the Pinus sylvestris var. mongolica provenance experimental forest is an experimental forest adopting a completely randomized block design.

[0039] Preferably, when selecting core germplasm from the original germplasm of Pinus sylvestris var. mongolica, the screening is carried out according to the strategy of maximizing the phenotypic variation and the number of alleles, the principle of phenotypic extremes and the principle of prioritizing rare alleles.

[0040] The so-called stable phenotype refers to the measurement results of phenotypic traits after the original germplasm of Pinus sylvestris is planted under the same site conditions and reaches 27 years old or more. When the measured age is insufficient, the phenotypic traits are not stable enough and the genetic characteristics are not fully manifested. The rotation period of Pinus sylvestris is generally 41 years. According to the standard of not less than two-thirds of the rotation period of the successfully introduced tree species, preferably, the inventor measures the calibration traits after the original germplasm of Pinus sylvestris under the same site conditions reaches 31 years old. At this time, the growth traits and morphological traits of various qualities basically no longer change greatly (i.e., they tend to be stable), and the environmental effect and age effect are eliminated to the greatest extent, and the genetic variation law of each phenotypic trait can be reflected truthfully.

[0041] It takes a long time to obtain stable phenotypic data of long-rotation tree species, and the phenotypic evaluation of germplasm requires experimental design to ensure environmental consistency. The provenance test forest of Pinus sylvestris var. mongolica adopts a completely randomized block design, which eliminates the environmental influence of phenotype to the greatest extent and is conducive to obtaining more accurate phenotypic trait measurement results.

[0042] The so-called strategy of maximizing phenotypic variation and the number of alleles means that when selecting core germplasm, the core germplasm should preserve all alleles and contain all types of variation of phenotypic traits, so that the core germplasm population can represent the phenotypic characteristics and genetic diversity of the entire germplasm pool as comprehensively as possible, while keeping the number of individuals in the core germplasm as small as possible. The principle of phenotypic extreme value means that on the premise that the core germplasm contains all types of alleles and phenotypic shape variation, those individuals that show the maximum and minimum values ​​in a certain trait are selected into the core germplasm to ensure that the core germplasm can represent the genetic diversity of the entire germplasm pool and contain individuals with particularly important traits in actual breeding or research. The principle of rare allele priority means that when the types of alleles contained in two individuals overlap, individuals containing rare alleles (rare alleles refer to alleles with lower gene frequencies in the original germplasm population) are preferentially selected as individuals in the core germplasm.

[0043] The core germplasm constructed using this method has rich phenotypic variation and genetic diversity, and can be directly used in breeding work such as breeding population construction, hybrid parent configuration, and trait molecular association analysis, which greatly promotes the genetic improvement process of tree species and can scientifically guide germplasm configuration in afforestation.

[0044] When typing the original germplasm of Larix gmelinii using the above method, the genomic DNA of the original germplasm of Larix gmelinii is extracted from individual plants and used as a PCR template. For each SSR site in the genomic DNA, PCR is performed using the SSR marker core primer pair and M13 primer in the above primer set that specifically amplify the SSR site.

[0045] In the above method, when performing PCR on the SSR site, the volume of the PCR reaction system is 10 μL, and the PCR reaction system contains 1×PCR buffer and 0.25U Taq DNA polymerase; in the PCR reaction system, the concentration of the forward primer with the M13 sequence, the concentration of the reverse primer and the concentration of the M13 primer are all 0.5 μM, the concentration of dNTPs is 150 μM, and the concentration of MgCl2 is 2.0 μM.

[0046] In the above method, the PCR reaction procedure is: pre-denaturation at 94°C for 4 minutes; denaturation at 94°C for 30 seconds, annealing at 50-55°C for 30 seconds, extension at 72°C for 30 seconds, for a total of 31 cycles; and finally extension at 72°C for 10 minutes.

[0047] In the above method, the phenotypic traits are divided into growth traits and morphological traits, wherein the growth traits include breast diameter, tree height, height under branches, crown width and single plant volume, and the morphological traits include crown shape, trunk shape and branch characteristics. The above growth traits and morphological traits are key criteria for measuring the utilization value of Pinus sylvestris var. mongolica, and the above growth traits and morphological traits basically remain stable after Pinus sylvestris var. mongolica reaches 27 years old, is less affected by the environment, and can more accurately reflect the genetic variation of germplasm.

[0048] Application of Pinus sylvestris SSR markers: using the above-mentioned Pinus sylvestris SSR markers and / or the above-mentioned primer sets for genetic diversity analysis, variety identification, fingerprint map construction, molecular-assisted breeding and / or construction of Pinus sylvestris core germplasm resources.

[0049] When the above application is used to construct a fingerprint map, the genomic DNA of the tested Larix gmelinii individual is extracted, and the SSR sites in the genomic DNA are PCR-treated using the above primer set to obtain SSR typing results, and a fingerprint map is constructed based on the SSR typing results.

[0050] The technical solution of the present invention achieves the following beneficial technical effects:

[0051] 1. Among the currently available SSR markers, including 4 SSR markers and 10 EST-SSR markers developed by Belletti et al. and Sebastiani et al. from the original species Pinus sylvestris, and 10 EST-SSR markers developed by Fang et al. from the closely related species Pinus tabuliformis, only one site, lwisotig07383, can be stably amplified in a large number of Pinus sylvestris samples. The 11 highly polymorphic SSR markers developed by the present invention can all be stably amplified in Pinus sylvestris samples, significantly improving the genetic diversity analysis capability of Pinus sylvestris germplasm resources. Through these markers, the genetic diversity of Pinus sylvestris can be accurately evaluated, thereby effectively protecting and scientifically managing the germplasm resources of Pinus sylvestris, and ensuring that precious germplasm resources are rationally utilized in the breeding process.

[0052] 2. Based on a 31-year-old provenance test forest of Pinus sylvestris, the present invention investigated and analyzed the growth and morphological trait variations of 517 germplasms; used SSR markers to evaluate the genetic diversity of germplasm resources, and then drew the SSR fingerprint map of germplasm resources. The SSR markers provided in the present invention can provide a scientific basis for the effective protection, management and genetic improvement of Pinus sylvestris germplasm resources, and also provide a reference for the construction of core germplasm of other timber species.

[0053] 3. The present invention provides a method for constructing a core germplasm of Pinus sylvestris by combining phenotypic traits and SSR markers. While effectively maintaining the phenotypic variation and genetic diversity of the core germplasm, it provides a scientific basis for the selection of improved varieties of Pinus sylvestris and the parent configuration of high-generation breeding and hybrid breeding, significantly improves the breeding efficiency, and promotes the cultivation and promotion of excellent forest stands. The method provided by the present invention combines the phenotypic traits and SSR markers of the same individual, and also lays a solid foundation for molecular association analysis and molecular marker-assisted selection of key traits. The use of the method provided in the present invention to construct a core germplasm significantly shortens the breeding cycle and accelerates the genetic improvement and new variety creation process of Pinus sylvestris. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 The SSR fingerprints of 46 core germplasms of Pinus sylvestris var. mongolica constructed in Example 3 of the present invention. DETAILED DESCRIPTION

[0055] Example 1 Development of polymorphic SSR loci (SSR markers) in Pinus sylvestris

[0056] First, the total DNA (genomic DNA) of Pinus sylvestris was broken into 350bp fragments using ultrasound. A double-end library was constructed using the NEBNext UltraTM DNA library construction kit (purchased from New England BioLabs, item number E7370S), and then sequenced in PE150 mode on the Illumina HiSeq X Ten platform. A total of 10Gb of data was obtained. Data quality control was performed using NGS QC Toolkit 2.3.2 software. Sequence assembly was performed using the SPAdes 3.6.1 program, and a total of 6429 SSR loci (SSR markers) were identified using the whole genome microsatellite analysis (GMATA) software, from which SSR loci with high polymorphism and good stability were screened and primers were designed.

[0057] Through screening, the inventors finally obtained 11 SSR loci and designed primers for these 11 SSR loci.

[0058] Table 1 shows the detailed information of the 11 SSR loci of Pinus sylvestris var. mongolica, i.e. the primers corresponding to each locus.

[0059] Table 1 Detailed information of 11 SSR loci in Pinus sylvestris

[0060]

[0061] Example 2 Construction of core collection of Pinus sylvestris var. mongolica based on phenotypic traits and SSR markers

[0062] 1. Phenotypic trait investigation

[0063] The phenotypic traits of Scots pine are divided into growth traits and quality traits. In this step, the diameter at breast height, tree height, height under branches and crown width of Scots pine germplasm are measured, and the volume of individual plants is calculated according to the binary volume formula of Scots pine. The diameter at breast height, tree height, height under branches, crown width and individual plant volume all belong to growth traits, and the measurement results of these traits are all results in numerical form. For the three morphological and qualitative traits of the trunk shape, crown shape and branch characteristics of Scots pine, they are rated respectively, wherein grade 1 is the best and grade 4 is the worst, and the rating results are standardized to obtain the measurement results of morphological and qualitative traits in numerical form, which are used for statistical analysis and as the basis for the selection of core germplasm.

[0064] The above 8 kinds of phenotypic traits of various qualities are all measured at the same location (Zhanggutai Experimental Forest Farm of Liaoning Province Sandy Land Management and Utilization Research Institute), which is a pine provenance test forest composed of the original germplasm of pine, and the pine in the forest farm is located under the same site condition. The distribution of trees in the forest farm adopts a completely randomized block design (i.e., the test forest is divided into 3 blocks for repetition, and the original germplasm of pine in each block is randomly arranged), and a fixed spacing of 1.5m × 3.0m is arranged to eliminate the environmental impact of the phenotype to the greatest extent. And the age of the test forest has reached 31 years (rotation period is 41 years), and various phenotypic traits have been stable, so the phenotypic traits used in the present embodiment are stable phenotypes that have eliminated environmental effects and age effects to the greatest extent.

[0065] 2. Leaf DNA Extraction and SSR Amplification

[0066] 517 fresh young leaves of the original germplasm of Pinus sylvestris were collected from individual plants, dried quickly with silica gel and preserved. Total DNA (genomic DNA) was extracted using the CTAB method, and the concentration and quality of DNA were tested by 1.0% agarose gel electrophoresis and NanoDrop-2000 ultra-micro spectrophotometer. Finally, the DNA was diluted to 50 ng·μL -1 , stored at -20℃ for future use. Primers were designed for the SSR sites (SSR markers) Ps8, Ps10, Ps23, Ps36, Ps61, Ps63, Ps70, Ps82, Ps89, Ps163 and Ps164 of Pinus sylvestris var. mongolica, and PCR amplification was performed on the corresponding SSR sites in the genomic DNA of the original germplasm of Pinus sylvestris var. mongolica. After PCR amplification, the length of the PCR product of each SSR site was checked. For a certain SSR site, the number of lengths of its PCR product was considered to be the number of alleles at the SSR site. For an individual, the composition of its alleles at each SSR site is the SSR typing result of the individual. This is the typing of the original germplasm of Pinus sylvestris var. mongolica using SSR markers.

[0067] The DNA sequences corresponding to each SSR locus are as follows:

[0068] SEQ ID NO.1[Pinus sylvestris var.mongolica]Ps8:

[0069] CACATTTCTTTTATTTTTCTAATATATCCTTCAACATTGCATGTTCATAATATAAGCATGGGCACAATTATGGTAATTATAATAACATGATATCAAAATCAGAAATATGCCAGGGCTACCTCTTTCCTAATTGATTCC TCACACAACCTATAATATTTTCATGCCATCTTTACCATCATATCCCACAATATATATATATATGAACATATCACATATTTACATCTGTAAATAGATTCAAGGTCAAGAGAACCTCTTGTGCACCATTTTCATGGCTTC

[0070] SEQ ID NO.2[Pinus sylvestris var.mongolica]Ps10:

[0071] GTATAGATGAATTTGGAAACTCTCTCTCTCATAAGGAAGAATTTTACCGTTTTCCCCTTGTGGGTTTTGCCCTGGTTTGGTGATTATTCTGCTATTGTTATTTATGATGTATAGATGAATCTGGAAACTCTCTCTCTCTCTCCGAAAGGAAAAATTTTACTGGGTTTTTCCCTGGCTGTGTGCCTAATTTTTGTGTCTTTTGTTTCTCCATTTCATGGTTACTTTACTTCTCTTTCATGTTTATTAGTTGACGTTTT

[0072] SEQ ID NO.3[Pinus sylvestris var.mongolica]Ps23:

[0073] CCCAGCAGGCCTTCCTTAGGATCACTTGTGAAGGAGGAAAGTTAGAAGGGTCAGGGTTTAGATATGTGTGTGTGTGTGTGTATACATGTGCATTAATGGTGAAGACCCATGCCATGAATGGGTCTACATTTAATACGCTAATGTTAAGTGGGCTAGGAGGTTGGCTAGGATAGTTACATGGGTGCCACATGGCAAGGTGATAGTGTGGCACCTTCTAATTCGTCAAAAAATCAAATGGTCATATCT

[0074] SEQ I D NO.4[Pinus sylvestris var.mongolica]Ps36:

[0075] CACCTTGTATCGGTTGGTAATTAAGTATAGTTGAAGGAAAATCACTTAACTTATCATTAATCAATCCTATTAGTTCAATCTCAACTTAATCATCAATATTATTATTATTATTATTATCCTTTATGATAGTATGAATTATGATATTATTATAAACCCCAAGGGTTAGCTCAACTGGTCAAATAAGTGCCTCGCTGTTCGGGAGGTCACAGGGCCAATTCTCCCCGAAGCCCTTGGGTTTAGGCTTAGCTATTAACACGGGGCTTGATACCCTTGATCA

[0076] SEQ I D NO.5[Pinus sylvestris var.mongolica]Ps61:

[0077] TAGTTAATTATTGATGAAACGTTTTTGAAAAGTGAATTTGTCACTACACTAATTCAACCCCCTCTGAGTGTGATCTTTCTTCTAGCACATTCACACACACACACACGCACACACACACTCACACATGAGAGAGAGAGAGAGAGATTTCACCGAATCCATTTGTTTGTTCAAGGCCAGACATGGAATTGAATTATTTTGAGAGAGGAATTAGTTTGTTATGAATTTTGGGGTTGGGTTATACCTCACATACATATATGCGTACAAAC

[0078] SEQ I D NO.6[Pinus sylvestris var.mongolica]Ps63:

[0079] TTATTCTACTCATTGTGCTTCTACAATTTTGTGAGTGTCTTTTATATTTCTTTGAAGCGATTCCCTATTTTT TTACCAATCTTTGATCATCCAACTTCATGAATTGTTATGTGTGTGTGTGTGTGTAAAGTTGATCCTTAATTATAATTTCTCTCATTCTTCCAAAAACCCTCATATGTATTGGTCCATGAGAGCCCAATCTCAATCCATTAGGGATCAATACCTTTACAGTTTTAAATTAAGTATCATGATTTTGTGAACTGATGTACGCACTTAACTTTATTCATAACTTAAATTCTATSEQ ID NO.7[Pinus sylvestris var.mongolica]Ps70:

[0080] TGAATATATCAAACTGCAATTTCTTCACCAAAAATAAATACTGCGGAAGCAAAGATAGAAATTGAGAAATACTAGCACACACACACACACATAAAATGAGATAGTTATTTTTGGACAAACGCCCACAGAAAAACCCAATGTGGAAAAATTGGTGGAGCAACTAAGGGTTTATTTTATTATGTGGCAACGAATTTTGAGTACAATCTTTGGAAGCCCAACTGACTATCCAATTAGCAAGGTATGACAAAAAATATTTAGAGA

[0081] SEQ ID NO.8[Pinus sylvestris var.mongolica]Ps82:

[0082] AATGATATCAGGAAGCTTTCTCTTAAGAAGAATTGAGGAGTATCTAGATGAGACCATCATCCCATGTTTGTCAAGGTTCACTCAAGTAAGAGATGAACTAGGTGGGGTGGTCTTGGTTGTATCTAATCATGATCTTGTGATTTTTCCCTTACTTGGACTTCACAAGTCAAGGAAGGGGTTATAGGATGTTGTTAGTGATAGAGAGAGAGAGCTTCCCAAATTGGGAAATGTTTTGGTGTGGCTCCACACCAAAAGAGATCTAGAGAGGAGCC

[0083] SEQ ID NO.9[Pinus sylvestris var.mongolica]Ps89:

[0084] TTAAATATATTTTCTCAAATTTTATTATCATATAAATTTAATTAATATAAATAAAAACTTTTACTTGCTACAAAAGCCTCCGGAAAATCATATAACTTTTCTCACTTCGCAAATTGGGTTCCAATATTTTATTTTATTTTATTTTATTTTTTATTTTTTTGAATGTAATACTCAAAATCATAAAACTTTTCCTATTTGAGAAATTGAGTTCCAATATATATATATTTTGTTGGGAATGTAATGAACCTATAGCTGGTTC

[0085] SEQ ID NO.10[Pinus sylvestris var.mongolica]Ps163:

[0086] GAGAGGGTGGGGGAGATAACAACCATTCTACCCAAACACCGAGATAACCATAATTGACTATATATATATATATATATATAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGACGTCCT TAATCCATTACGTCATATCATGCTTAGTATTTGTTTCTTTGTGATGTATATGTCTCCACATATACAAATGTGTATACATGTAAATATATATGTGTGTGTTTTGTAAAATGGGATGTAAGAAGAGGGG

[0087] SEQ ID NO.11[Pinus sylvestris var.mongolica]Ps164:

[0088] CGGGCTTTGAAATGTAGTGTATAACATATCGCGTCCAGGGACAAATCATACTCGGCGTTATCAGTGGTGACACACACACACACACACACATAGATGCACACACACACACACGCACACACACACACACACACACACACACACACACA CACACACACATATATATCGGCAGGTATCAGGTGCGATATGTGATTGCAATATATCGCATTGCAACCCTAATTGTGTCAGGGGCTGATACTTAACATTATGCGATATGGGGTTGCGAT

[0089] During PCR amplification, each SSR site is amplified separately using the SSR primers in Table 2. When PCR amplification is performed on a certain SSR marker, the primers used are the M13 primer and a specific primer pair for the SSR site (SSR marker core primer pair). For the M13 primer, a fluorescent label is added to the 5' end of the M13 primer (sequence is SEQ ID NO.34: 5'-CACGACGTTGTAAAACGAC-3') using the fluorescent labeling dUTP method. In addition, a segment of M13 sequence (M13 sequence is the sequence of M13 primer) is added to the 5' end of the forward primer (F primer) of each SSR site. The fluorescent label is any one of FAM, NED, VIC and ROX.

[0090] For example, when performing PCR on the Ps8 site, three primers need to be added to the PCR reaction system. The sequence of the first primer is 5'-CACGACGTTGTAAAACGACTCAGAAATATGCCAGGGCTAC-3', that is, the sequence of the M13 primer is added to the 5' end of the F primer of the Ps8 site in Table 1; the sequence of the second primer is 5'-TGAAAATGGTGCACAAGAGG-3', that is, the R primer of the Ps8 site in Table 1; the third primer is the fluorescently labeled M13 primer, that is, 5'-fluorescently labeled-CACGACGTTGTAAAACGAC-3'.

[0091] The numbers and sequences of the SSR marker core primer pairs finally used are shown in Table 2.

[0092] Table 2

[0093]

[0094]

[0095] A 10 μL PCR reaction system was used. In each PCR reaction system, the content of template DNA (genomic DNA of Pinus sylvestris var. mongolica) was 5 ng, the concentration of the forward primer with M13 sequence was 0.5 μM, the concentration of the reverse primer was 0.5 μM, the concentration of M13 primer (with fluorescent label) was 0.5 μM, the concentration of dNTPs was 150 μM, the concentration of MgCl2 was 2.0 μM, and it contained 1×PCR buffer and 0.25 U Taq DNA polymerase. The PCR amplification reaction was carried out on a Veritas cycler (brand: Applied Biosystems). The PCR amplification program was: 94℃ pre-denaturation for 4 min; 94℃ denaturation for 30 s, 50-55℃ annealing for 30 s, 72℃ extension for 30 s, a total of 31 cycles; and finally 72℃ extension for 10 min. The PCR products were detected by ABI3730xl DNA analyzer (Applies Biosystems), and the alleles were read and counted using Gene-Marker 2.2.0 software (SoftGenetics).

[0096] 3. Construction of core collection and representativeness test

[0097] Based on phenotypic traits and SSR markers, the core germplasm was extracted using Power Core 1.0 software, adopting the strategy of maximizing phenotypic variation and allele number (M strategy) and the principles of phenotypic extreme value priority and rare allele priority.

[0098] After extracting the core germplasm, the representativeness and validity of the core germplasm were tested by calculating the mean difference percentage (MD), variance difference percentage (VD), range conformity rate (CR) and coefficient of variation change rate (VR), as well as the difference test of phenotypic traits and genetic diversity parameters and principal coordinate analysis. Among them, MD < 20% and CR > 80% are the minimum standards. The smaller the MD, the better, and the larger the other parameters, the better.

[0099] The phenotypic diversity of 517 31-year-old original germplasm of Pinus sylvestris was analyzed. The phenotypic diversity parameters and genetic diversity parameters are shown in Table 3 and Table 4, respectively.

[0100] Table 3 Phenotypic diversity parameters of 517 original accessions and 46 core accessions of Pinus sylvestris var. mongolica

[0101]

[0102] Table 4 Genetic diversity parameters of 517 original germplasms and 46 core germplasms of Pinus sylvestris var. mongolica

[0103]

[0104] From the data in the table, we can see that for the original germplasm population, the average coefficient of variation of each trait is 29.62%, and the diversity index H' is 1.688 (Table 3). Therefore, there is a rich phenotypic variation in Pinus sylvestris germplasm, which provides a basis for core germplasm construction and improved variety breeding.

[0105] A total of 92 alleles were detected from 517 original germplasms of Pinus sylvestris var. mongolica using 11 pairs of SSR primers (Table 4). e ), Shannon-Wiener diversity index (I), observed heterozygosity (H o ), expected heterozygosity (H e ) and the average values ​​of polymorphism information content (PIC) were 3.464, 1.216, 0.518, 0.566 and 0.522, respectively, indicating that the germplasm resources of Pinus sylvestris var. mongolica have rich genetic diversity.

[0106] Based on the phenotypic traits and SSR loci data, the phenotypic diversity parameters and genetic diversity parameters of 46 core germplasms selected from 517 original germplasms with a sampling ratio of 8.90% are shown in Tables 3 and 4, respectively. From the data in the table, it can be seen that the mean difference percentage (MD), variance difference percentage (VD), range conformity rate (CR) and coefficient of variation change rate (VR) of the core germplasm are 1.42%, 46.33%, 100.00% and 135.34%, respectively. The core germplasm retains the extreme values ​​and phenotypic grades of all 8 traits and all 95 alleles, effectively retaining phenotypic variation and genetic diversity, and the phenotypic coefficient of variation, phenotypic diversity index (H'), effective number of alleles (N) of the core germplasm are 1.42%, 46.33%, 100.00% and 135.34%, respectively. e ), Shannon-Wiener diversity index (I), expected heterozygosity (H e ) and polymorphism information content (PIC) were significantly higher than those of the original germplasm. This indicates that the combination of the M strategy with the sampling strategy of phenotypic extremes and rare allele priority fully removed redundant germplasm and effectively preserved the phenotypic variation and genetic diversity of the original germplasm. This study provides a scientific basis for parental configuration, molecular association analysis of specific traits, and genomic selection in high-generation breeding of Pinus sylvestris var. mongolica.

[0107] Example 3 Construction of SSR fingerprint of Pinus sylvestris germplasm

[0108] The method of "leaf DNA extraction and SSR amplification" in Example 2 is used to extract the genomic DNA of the Larix gmelinii to be tested, and the Larix gmelinii to be tested is typed according to the composition of the alleles corresponding to each SSR marker in the genomic DNA (i.e., the Larix gmelinii to be tested is typed using SSR markers). Based on the SSR typing results, an SSR fingerprint map of the Larix gmelinii to be tested is drawn, and various quality-specific molecular identity information is characterized by the least alleles.

[0109] In this example, 11 pairs of SSR primers were used to construct the SSR fingerprint of 46 core germplasms of Pinus sylvestris var. mongolica ( Figure 1 ), each core germplasm material has unique molecular identity information. Among them, 21 germplasms have specific alleles, that is, 21 germplasms can be accurately identified with only a single allele, and 40 alleles can cooperate with each other to identify all 46 germplasms. It can be seen that the 11 SSR markers used in this example have a high resolution and can efficiently and conveniently distinguish and identify different germplasms.

[0110] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the claims of this patent application.

Claims

1. A group of SSR markers of Pinus sylvestris var. mongolica, characterized in that: The SSR markers include Ps8, Ps10, Ps23, Ps36, Ps61, Ps63, Ps70, Ps82, Ps89, Ps163 and Ps164; The nucleotide sequence of Ps8 is shown in SEQ ID NO.1; The nucleotide sequence of Ps10 is shown in SEQ ID NO.2; The nucleotide sequence of Ps23 is shown in SEQ ID NO.3; The nucleotide sequence of Ps36 is shown in SEQ ID NO.4; The nucleotide sequence of Ps61 is shown in SEQ ID NO.5; The nucleotide sequence of Ps63 is shown in SEQ ID NO.6; The nucleotide sequence of Ps70 is shown in SEQ ID NO.7; The nucleotide sequence of Ps82 is shown in SEQ ID NO.8; The nucleotide sequence of Ps89 is shown in SEQ ID NO.9; The nucleotide sequence of Ps163 is shown in SEQ ID NO.10; The nucleotide sequence of Ps164 is shown in SEQ ID NO.

11.

2. A primer set for specific amplification of the Pinus sylvestris SSR marker according to claim 1, characterized in that: The primer set contains 11 pairs of SSR marker core primer pairs and M13 primers; wherein: The SSR marker core primer pair for specific amplification of Ps8 consists of primers whose sequences are shown in SEQ ID NO.12 and SEQ ID NO.13, respectively; The SSR marker core primer pair for specific amplification of Ps10 consists of primers whose sequences are shown in SEQ ID NO.14 and SEQ ID NO.15, respectively; The SSR marker core primer pair for specific amplification of Ps23 consists of primers whose sequences are shown in SEQ ID NO.16 and SEQ ID NO.17, respectively; The SSR marker core primer pair for specific amplification of Ps36 consists of primers whose sequences are shown in SEQ ID NO.18 and SEQ ID NO.19, respectively; The SSR marker core primer pair for specific amplification of Ps61 consists of primers whose sequences are shown in SEQ ID NO.20 and SEQ ID NO.21, respectively; The SSR marker core primer pair for specific amplification of Ps63 consists of primers whose sequences are shown in SEQ ID NO.22 and SEQ ID NO.23, respectively; The SSR marker core primer pair for specific amplification of Ps70 consists of primers whose sequences are shown in SEQ ID NO.24 and SEQ ID NO.25, respectively; The SSR marker core primer pair for specific amplification of Ps82 consists of primers whose sequences are shown in SEQ ID NO.26 and SEQ ID NO.27, respectively; The SSR marker core primer pair for specific amplification of Ps89 consists of primers whose sequences are shown in SEQ ID NO.28 and SEQ ID NO.29, respectively; The SSR marker core primer pair for specific amplification of Ps163 consists of primers whose sequences are shown in SEQ ID NO.30 and SEQ ID NO.31, respectively; The SSR marker core primer pair for specific amplification of Ps164 consists of primers whose sequences are shown in SEQ ID NO.32 and SEQ ID NO.33, respectively; The primers shown in SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16, SEQ ID NO.18, SEQ ID NO.20, SEQ ID NO.22, SEQ ID NO.24, SEQ ID NO.26, SEQ ID NO.28, SEQ ID NO.30 and SEQ ID NO.32 are forward primers with M13 sequence; SEQ ID NO.13, SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO.19, SEQ ID NO.21, SEQ ID NO.23, SEQ ID NO.25, SEQ ID NO.27, SEQ ID NO.29, SEQ ID NO.31 and SEQ ID NO.33 are reverse primers; The sequence of the M13 primer is shown in SEQ ID NO.34, and a fluorescent marker is connected to the M13 primer.

3. The primer set according to claim 2, characterized in that The fluorescent label connected to the M13 primer is any one of FAM, NED, VIC and ROX.

4. A method for constructing a core germplasm of Pinus sylvestris var. mongolica by combining SSR markers and stable phenotypes, characterized in that: The method comprises: using the SSR markers as claimed in claim 1 to type the original germplasm of Pinus sylvestris var. mongolica that is located in the same site and is 27 to 41 years old and of the same age, to obtain SSR typing results; Measuring the phenotypic traits of the original germplasm of Pinus sylvestris var. mongolica to obtain the phenotypic trait measurement results; screening the core germplasm from the original germplasm of Pinus sylvestris var. mongolica according to the phenotypic trait measurement results and the SSR typing results; Among them, the original germplasm of Pinus sylvestris var. mongolica constitutes the Pinus sylvestris var. mongolica provenance experimental forest, and the Pinus sylvestris var. mongolica provenance experimental forest is an experimental forest adopting a completely randomized block design.

5. The method according to claim 4, characterized in that When typing the original germplasm of Pinus sylvestris, the genomic DNA of the original germplasm of Pinus sylvestris is extracted from individual plants as a PCR template. For each SSR site in the genomic DNA, PCR is performed using the SSR marker core primer pair and M13 primer in the primer set as described in claim 2 that specifically amplify the SSR site.

6. The method according to claim 5, characterized in that When performing PCR on the SSR site, the volume of the PCR reaction system was 10 μL, and the PCR reaction system contained 1×PCR buffer and 0.25UTaq DNA polymerase; in the PCR reaction system, the concentration of the forward primer with the M13 sequence, the concentration of the reverse primer and the concentration of the M13 primer were all 0.5 μM, the concentration of dNTPs was 150 μM, and the concentration of MgCl2 was 2.0 μM.

7. The method according to claim 6, characterized in that The PCR reaction program was as follows: pre-denaturation at 94°C for 4 min; denaturation at 94°C for 30 s, annealing at 50-55°C for 30 s, and extension at 72°C for 30 s, for a total of 31 cycles; and finally extension at 72°C for 10 min.

8. The method according to claim 4, characterized in that The phenotypic traits are divided into growth traits and morphological traits, wherein the growth traits include breast diameter, tree height, height under branches, crown width and single plant volume, and the morphological traits include crown shape, trunk shape and branching characteristics.

9. Application of SSR markers of Pinus sylvestris var. mongolica, characterized in that: The SSR marker of Pinus sylvestris var. mongolica as described in claim 1 and / or the primer set as described in claim 2 are used for genetic diversity analysis, variety identification, fingerprint map construction, molecular-assisted breeding and / or construction of Pinus sylvestris var. mongolica core germplasm of Pinus sylvestris var. mongolica germplasm resources.

10. The use according to claim 9, characterized in that: When used for fingerprint construction, the genomic DNA of the tested Larix gmelinii individual is extracted, and the SSR sites in the genomic DNA are PCR-treated respectively using the primer set as described in claim 2 to obtain SSR typing results, and a fingerprint is constructed based on the SSR typing results.

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