Ginkgo polymorphic est-ssr molecular marker primer and application thereof

Abstract

The application discloses ginkgo polymorphic EST-SSR molecular marker primers and application thereof, and belongs to the technical field of molecular marker development and application thereof. EST-SSR sites are screened from ginkgo transcriptome data, and 100 pairs of primers are designed and synthesized. Through effectiveness verification and polymorphism screening with 141 ginkgo germplasm genomic DNAs as templates, finally, 20 pairs of specific EST-SSR molecular marker primers with good stability and high polymorphism are obtained. The application further discloses application of the self-developed molecular marker primers in ginkgo genetic diversity analysis and core germplasm construction and the like. The EST-SSR molecular marker primers provided by the application provide theoretical and technical support for ginkgo genetic evolution analysis, kinship identification and good seed selection and the like, and have important value for rational utilization, preservation and protection of ginkgo germplasm resources.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of EST-SSR molecular marker development and its application, and particularly relates to a specific molecular marker primer of ginkgo transcriptome polymorphism EST-SSR, and further relates to application of the molecular marker primer in ginkgo genetic diversity analysis and core germplasm construction. BACKGROUND

[0002] Ginkgo biloba L. is one of the oldest surviving gymnosperms in the world, and is known as a living fossil. As a unique native tree species in China, ginkgo not only contains rich genetic resources, but also is an important economic tree species with multiple application values such as ornamental, medicinal, edible and timber uses, and has broad prospects for development and utilization. At present, the number of ginkgo resources in China accounts for about 90% of the total amount in the world, and is distributed in more than 20 provinces in China. Among them, the vast area of the middle and lower reaches of the Yangtze River and the Nanling Mountains south of the Huaihe River, including Jiangsu, Guangxi, Hunan, Shandong, Zhejiang and Hubei, has the longest history of ginkgo cultivation, and has formed rich germplasm resources. The above areas are the main base for ginkgo seed kernel and leaf production in China. Although ginkgo is widely distributed and has a large number, most of them are artificially cultivated, and natural wild populations are rare. In addition, ginkgo is a single species of Ginkgoaceae and Ginkgo, which leads to the lack of genetic diversity, and has been listed as a grade I endangered species.

[0003] Genetic diversity is the basis and core of biodiversity, which determines the ability of plants to adapt to the environment and maintain the stability of the ecosystem. Understanding the variation rules and influencing factors of genetic diversity is of great significance for the rational use and protection of plant resources. The main factors affecting plant genetic diversity include reproductive system, natural selection, genetic drift, gene mutation, gene flow, environmental change and human disturbance. Therefore, the study of plant genetic diversity can not only understand the level of genetic variation and the stability of genetic structure of the population, but also more targetedly establish protection measures and provide efficient strategies to improve the level of population genetic diversity. As the manifestation of DNA level variation, molecular markers are commonly used in the study of plant genetic diversity. Simple sequence repeat (SSR), as a heritable co-dominant molecular marker, has the advantages of large number, simple development, high polymorphism and good stability, and is widely used in the study of plant genetic diversity analysis, core germplasm construction and kinship identification. SSR can be divided into expressed sequence tag (EST-SSR) and genomic SSR (g-SSR), among which EST-SSR is mainly derived from transcriptome data, and its polymorphism may be directly related to gene expression function, with higher universality and transformation rate. With the development of gene sequencing technology and the reduction of sequencing cost, it is also convenient to develop EST-SSR molecular markers using transcriptome data. The lack of molecular markers is an important factor hindering the study of genetic diversity of Ginkgo biloba, so it is necessary to develop sufficient, stable and efficient SSR molecular markers.

[0004] Core germplasm refers to the genetic diversity represented by part of the original germplasm by retaining part of the germplasm resources. This part of the retained germplasm must meet the two conditions of minimum resource quantity and maximum genetic repetition. In recent years, with the increase of genetic resources, it has brought difficulties to the preservation and utilization of Ginkgo biloba germplasm resources. Core germplasm provides a way to solve the problem of germplasm preservation caused by the large number of Ginkgo biloba resources, and provides a strategy for the rational protection and effective utilization of Ginkgo biloba genetic resources. At present, the construction of Ginkgo biloba core germplasm mainly uses ISSR, AFLP and other dominant marker data, and there is no report on the construction of Ginkgo biloba core germplasm using SSR molecular marker data. SUMMARY

[0005] In view of the above problems, one of the technical problems to be solved by the present application is to develop Ginkgo biloba polymorphic EST-SSR molecular marker primers, and another technical problem to be solved is to apply the polymorphic EST-SSR molecular marker primers to the genetic diversity analysis and core germplasm construction of Ginkgo biloba germplasm resources.

[0006] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0007] Development of Ginkgo biloba L. transcriptome EST-SSR molecular marker primers, comprising the following steps:

[0008] (1) Extraction of genomic DNA;

[0009] The present application has collected 141 Ginkgo biloba L. germplasm resources, of which 101 are different varieties (lines) of Ginkgo biloba L. from 10 main producing areas at home and abroad such as Hunan, Guangxi, Guizhou, Hubei, Jiangsu, Liaoning, Shandong, Shaanxi, Zhejiang and Japan, and the remaining 40 are unknown varieties (lines) of Ginkgo biloba L. from Huanglongxian in Jiangsu.

[0010] (2) Screening of Ginkgo biloba L. transcriptome EST-SSR sites;

[0011] The MISA software is used to search for EST-SSR sites in the Ginkgo biloba L. transcriptome data, and the search criteria are as follows: the number of single nucleotide, di-nucleotide, tri-nucleotide, tetra-nucleotide, penta-nucleotide and hexa-nucleotide repeats is not less than 10, 6, 5, 5, 5 and 5, respectively.

[0012] (3) EST-SSR primer design;

[0013] The Primer 5.0 software is used to design primers for the SSR sites with a sequence length of more than 20 bp.

[0014] (4) EST-SSR effectiveness verification and polymorphic primer screening;

[0015] The SSR-PCR amplification and 2% agarose gel electrophoresis technology are used to screen the EST-SSR primers that can effectively amplify the target bands, and the 8% non-denaturing polyacrylamide gel electrophoresis technology is used to screen the polymorphic primers, wherein the SSR-PCR amplification system is 10 μl, including 2x TSINGKE Master Mix 5 μl; 2 μmol / L of each 1 μl of SSR primer; <1 μg of DNA template; and finally ddH2O is added to 10 μl. The SSR-PCR amplification program is as follows: 96℃ pre-denaturation for 2 min; 96℃ denaturation for 10 s; annealing temperature (T m ) 54-60℃, reannealing time 30 s; 72℃ extension for 1 min, 30-35 cycles; 72℃ extension for 1 min; 4℃ storage.

[0016] (5) Data analysis of EST-SSR polymorphic primers.

[0017] The bands migrating to the same position are recorded as one gene locus, and the bands on all loci are recorded as "1" for bands and "0" for no bands, to construct a "0, 1" matrix and obtain the EST-SSR molecular marker data. The Popgene 32 software is used to calculate the number of alleles (N a), effective allele number (Effective Number of Allele, N e ) were calculated by Powermarker v.3.25 software.

[0018] The primers developed in the present application include the following 20 pairs of polymorphic primers:

[0019] GBSSR04447-F: 5'-CCGAGAGAAAGATACAGCCG-3'; SEQ ID NO. 1;

[0020] GBSSR04447-R: 5'-CCACAACCCAAAAAGCCTTA-3'; SEQ ID NO. 2;

[0021] GBSSR05243-F: 5'-CAGATTGTCATGCACCCCTA-3'; SEQ ID NO. 3;

[0022] GBSSR05243-R: 5'-TCCTTGGTAACGCCATTCTC-3'; SEQ ID NO. 4;

[0023] GBSSR10422-F: 5'-ATCACGATTTCTTCGTTGGG-3'; SEQ ID NO. 5;

[0024] GBSSR10422-R: 5'-GCTTGAATGTCCTCTGCCTC-3'; SEQ ID NO. 6;

[0025] GBSSR02181-F: 5'-CAGACACATGAACCCCTTCC-3'; SEQ ID NO. 7;

[0026] GBSSR02181-R: 5'-ATGCCCTGGCATATTTTGAT-3'; SEQ ID NO. 8;

[0027] GBSSR16030-F: 5'-AAGCGTCTCGAGAAAGTGGA-3'; SEQ ID NO. 9;

[0028] GBSSR16030-R: 5'-TACGATGCAGGAAAACATGC-3'; SEQ ID NO. 10;

[0029] GBSSR21240-F: 5'-AGGAGAACAGAGGCTGTGGA-3'; SEQ ID NO. 11;

[0030] GBSSR21240-R: 5'-TTTGTTATTTCCGTGGGAGC-3'; SEQ ID NO. 12;

[0031] GBSSR13689-F: 5'-AATTTGGATGTCGCCATTGT-3'; SEQ ID NO. 13;

[0032] GBSSR13689-R: 5'-AAGCCCTGATATGACCATGC-3'; SEQ ID NO. 14;

[0033] GBSSR05999-F: 5'-GTAGGCCTCTCCTCCAATCC-3'; SEQ ID NO. 15;

[0034] GBSSR05999-R: 5'-AATGGACCACATTGGGTGTT-3'; SEQ ID NO. 16;

[0035] GBSSR21429-F: 5'-ACCAATGGCTGTTGATGTGA-3'; SEQ ID NO. 17;

[0036] GBSSR21429-R: 5'-GCAAAAACAACATCCAGGCT-3'; SEQ ID NO. 18;

[0037] GBSSR25769-F: 5'-CACCCCTGGGATTATTGATG-3'; SEQ ID NO. 19;

[0038] GBSSR25769-R: 5'-ATGAGTGGAATGTGGGCTTC-3'; SEQ ID NO. 20;

[0039] GBSSR29168-F: 5'-CCATGTCTCCAAGGTCGATT-3'; SEQ ID NO. 21;

[0040] GBSSR29168-R: 5'-CTTGGCGAATACTGCATTGA-3'; SEQ ID NO. 22;

[0041] GBSSR27072-F: 5'-CAAGCAAATTAGTGCTGCCA-3'; SEQ ID NO. 23;

[0042] GBSSR27072-R: 5'-TGAATGCCTGATGATTTGGA-3'; SEQ ID NO. 24;

[0043] GBSSR27825-F: 5'-ATGGGCGTCGTGGATAGTAG-3'; SEQ ID NO. 25;

[0044] GBSSR27825-R: 5'-TTTTCCCAGATAGGCATTGG-3'; SEQ ID NO. 26;

[0045] GBSSR31776-F: 5'-AGTGCATTTCATTTGCTTCG-3'; SEQ ID NO. 27;

[0046] GBSSR31776-R: 5'-TTTCAGTCTCTGCGGGAGAT-3'; SEQ ID NO. 28;

[0047] GBSSR34776-F: 5'-AGAACGGTGCCAACAATAGG-3'; SEQ ID NO. 29;

[0048] GBSSR34776-R: 5'-TCCCTGATTGCCAAAGTAGG-3'; SEQ ID NO. 30;

[0049] GBSSR31083-F: 5'-AAGTGGAGTTGTGAAACGGG-3'; SEQ ID NO. 31;

[0050] GBSSR31083-R: 5'-TTTAGGCTGGAATGGATTGG-3'; SEQ ID NO. 32;

[0051] GBSSR27860-F: 5'-AGGTGGATGGGCATATTCAG-3'; SEQ ID NO. 33;

[0052] GBSSR27860-R: 5'-CACAGTTGGCAGACGAAAAA-3'; SEQ ID NO. 34;

[0053] GBSSR01538-F: 5'-AGAGATTTTGCGACAGAGC-3'; SEQ ID NO. 35;

[0054] GBSSR01538-R: 5'-GGTAGCAGTTGAACCGTTA-3'; SEQ ID NO. 36;

[0055] GBSSR00112-F: 5'-AGGGAAAAAGTGAAAGAGAGAG-3'; SEQ ID NO. 37;

[0056] GBSSR00112-R: 5'-CTAGTCAAGGCGAGGTTAAAGA-3'; SEQ ID NO. 38;

[0057] GBSSR10214-F: 5'-TTTGGGAGTAGTGTGTTGT-3'; SEQ ID NO. 39;

[0058] GBSSR10214-R: 5'-CTGGATTGCATTTGAAGTC-3'; SEQ ID NO. 40.

[0059] The application of ginkgo transcriptome EST-SSR molecular marker primers in genetic diversity analysis of ginkgo germplasm resources includes the following steps:

[0060] (1) Based on the above-mentioned EST-SSR primers, EST-SSR molecular marker data is obtained;

[0061] (2) Based on the EST-SSR molecular marker data, bioinformatics software is used to analyze the genetic diversity of ginkgo germplasm resources;

[0062] The genetic diversity of the above-mentioned 141 ginkgo germplasm resources is analyzed, and the Popgene 32 software is used to calculate the average expected heterozygosity or genetic diversity index (Average Expective Heterozygosity, H e , which refers to the overall (species level) genetic diversity index), Shannon diversity index (I), gene differentiation coefficient (Gene Differentiation Coefficient, G st ), and gene flow (Gene Flow, N m ), and the genetic diversity of the ginkgo population is analyzed, including: the number of alleles (N a ), the number of effective alleles (N e ), the observed heterozygosity (H o ), and the population genetic diversity index (H e ) (which refers to the population (population level) genetic diversity index);

[0063] Molecular variance analysis was performed by using GenAlE_v6.502 software, and UPGMA cluster analysis was performed by using NTSYS software.

[0064] The application also provides application of the ginkgo transcriptome EST-SSR molecular marker primer in construction of ginkgo core germplasm.

[0065] The method comprises the following steps:

[0066] (1) obtaining EST-SSR molecular marker data based on the EST-SSR polymorphism primer in claim 6; (2) constructing ginkgo core germplasm based on the EST-SSR molecular marker data.

[0067] The core germplasm of the above 141 original ginkgo germplasm resources is constructed, and any combination of two grouping principles (grouping according to production place, completely random grouping), four in-group sampling ratios (simple ratio, logarithmic ratio, square root ratio and genetic diversity ratio), two sampling methods (site priority cluster sampling method and random cluster sampling method), and six overall sampling ratios (5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%) is performed, so that 72 sampling schemes are formed, and the best sampling strategy is screened as follows: sampling is performed according to the scheme that the production place is grouped, the in-group is sampled according to the genetic diversity ratio and the site priority cluster method, and the overall sampling ratio is 25%. The representativeness of the core germplasm is evaluated through five indexes such as the number of alleles, the number of effective alleles, the Shannon diversity index (I), the genetic diversity index (H e ) and the percentage of polymorphic loci (PPL). Finally, the principal component analysis is used to determine the core germplasm.

[0068] From the above technical scheme, the application provides ginkgo transcriptome EST-SSR molecular marker primers and application thereof in genetic diversity analysis of ginkgo germplasm resources and construction of core germplasm. The shortage of the number of SSR molecular markers is a key factor limiting the analysis and evaluation of ginkgo genetic resources, so it is necessary to develop a sufficient number of molecular markers. Genetic diversity is the core of biological diversity, and the study of genetic diversity is of great significance to understanding the origin and evolution of species, breeding and resource protection. The core germplasm can represent the maximum genetic diversity of the original germplasm, and has practical significance for the rational preservation, utilization and evaluation of resources. The determination of the sampling strategy is the key to the construction of the core germplasm, including four steps of determining the grouping principle, the sampling ratio in the group, the sampling method and the overall sampling ratio. The grouping principle is usually divided into random grouping and grouping according to the place of production; the sampling ratio in the group is mainly divided into simple ratio, square root ratio, logarithmic ratio and genetic diversity ratio; the sampling method is divided into site priority clustering sampling method and random clustering sampling method; and the overall sampling ratio of the core germplasm of most species is 5%-40%. The genetic structure of the species itself and the type of data information will lead to significant differences in the core germplasm, so after obtaining the EST-SSR molecular marker data, the sampling strategy needs to be screened to determine the best sampling scheme.

[0069] Compared with the prior art, the application can achieve the following beneficial effects:

[0070] (1) The number of EST-SSR molecular markers is rich, the polymorphism is high, and the stability is good, the amplification experiment operation of the EST-SSR molecular marker primer in the application is simple, and the band is clear and easy to distinguish. It is suitable for the construction of ginkgo genetic diversity analysis and core germplasm;

[0071] (2) The material used in the application is from 10 main ginkgo producing areas at home and abroad, the varieties (lines) are many, and the coverage is wide, which can more comprehensively reveal the genetic diversity level of ginkgo germplasm resources;

[0072] (3) As a perennial tall tree, ginkgo often needs long-term field investigation, so as to form complete, stable and objective phenotype data, which brings great difficulty to the construction of ginkgo core germplasm. Compared with the traditional method of constructing core germplasm by using phenotype data, EST-SSR, as a co-dominant marker directly reflecting the DNA molecular variation level of species, has the advantage of being not affected by plant environment and development stage, which can effectively save the cost of manpower and material resources. The ginkgo core germplasm constructed based on the EST-SSR molecular marker data can represent the maximum genetic diversity of the original germplasm with good representation and effectively remove redundant materials. BRIEF DESCRIPTION OF DRAWINGS

[0073] Figure 1Amplification map for verifying the effectiveness of part of EST-SSR primers in 'Zhongnanlin 2' of the application. Lanes 1-14 represent primers GBSSR21429, GBSSR22445, GBSSR24634, GBSSR25270, GBSSR25769, GBSSR27072, GBSSR27845, GBSSR27860, GBSSR29168, GBSSR31083, GBSSR31176, GBSSR32986, GBSSR34776, GBSSR38701, respectively; M is 500 bp DNA Marker.

[0074] Figure 2 Amplification map for screening polymorphism of part of EST-SSR primers (GBSSR01538, GBSSR21240, GBSSR00112, GBSSR04447) in part of ginkgo germplasm resources; M is 500 bp DNA Marker.

[0075] Figure 3 Cluster map of 11 ginkgo populations;

[0076] Figure 3 JS (Jiangsu), SD (Shandong), HN (Hunan), HB (Hubei), GX (Guangxi), GZ (Guizhou), SX (Shaanxi), ZJ (Zhejiang), LN (Liaoning), RB (Japan), HLX (Huanglongxian).

[0077] Figure 4 Cluster map of 141 ginkgo germplasm resources.

[0078] Figure 5 Principal component analysis of ginkgo core germplasm and original germplasm; 1 represents original germplasm, and 2 represents core germplasm. DETAILED DESCRIPTION

[0079] The application is further illustrated below in conjunction with examples, but is not limited to the application.

[0080] Example 1: Development of ginkgo transcriptome EST-SSR molecular marker primers

[0081] 1. Materials and methods

[0082] 1.1 Materials

[0083] Based on the ginkgo transcriptome database of the research group, the SSR site screening and analysis are carried out. 141 ginkgo germplasm resources are collected for polymorphic EST-SSR primer screening, wherein 101 ginkgo germplasm resources are known varieties (lines), and the remaining 40 ginkgo germplasm resources are unknown varieties, which are named according to Huanglongxian 1-40. 11 ginkgo varieties (lines) from Hunan are planted in the ginkgo test demonstration base of Xiling working area in Damiaokou forest farm, Dong'an county, Hunan province, and the remaining 90 varieties (lines) of ginkgo are planted in the national ginkgo breeding base in Pizhou, Jiangsu province, and 40 unknown varieties (lines) of ginkgo are planted in Huanglongxian village, Jiangning district, Nanjing, Jiangsu province. In July-August, the green tender leaves at the top of the branches are selected, dried and stored in silica gel bags, and immediately after returning to the laboratory, the silica gel is poured out, and the leaves are stored in a-80 DEG C ultra-low temperature refrigerator. The information of the 141 ginkgo germplasm resources is shown in table 1.

[0084] Table 1 141 ginkgo germplasm resource information

[0085]

[0086]

[0087]

[0088] 1.2 Method

[0089] 1.2.1 Transcriptome EST-SSR site screening

[0090] MISA software (https: / / webblast.ipkgatersleben.de / misa / index.php?action=1) is used for EST-SSR site search, and the search standard is: the repetition number of single nucleotide, di-nucleotide, tri-nucleotide, tetra-nucleotide, penta-nucleotide and hexa-nucleotide is not less than 10, 6, 5, 5, 5 and 5 respectively. The number of ESI-SSR sites and base composition statistics are carried out by EXCEL software.

[0091] 1.2.2 EST-SSR primer design

[0092] Primer 5.0 software is used for primer design of the SSR site with sequence length >20bp, and 100 pairs of SSR primers are randomly selected and synthesized by Beijing Qikexin Biotechnology Co., Ltd.

[0093] 1.2.3 Extraction of genomic DNA

[0094] The genomic DNA of Ginkgo biloba was extracted by the plant genomic DNA extraction kit of Beijing Tsingke Biological Technology Co. Ltd. The quality and integrity of the DNA were identified by 1% agarose gel electrophoresis, and the concentration of the DNA was detected by ultraviolet spectrophotometer. The DNA was diluted to 40 ng / ul and stored in a -20 °C refrigerator for standby.

[0095] 1.2.4 Verification of EST-SSR effectiveness and screening of polymorphic primers

[0096] The effectiveness of the EST-SSR primers was verified by the SSR-PCR amplification technology, and the genomic DNA of 'Zhongnanlin 2' was used as the template. The polymorphic EST-SSR primers were screened by 8% non-denaturing polyacrylamide gel electrophoresis technology, and the genomic DNA of the above 141 ginkgo germplasm resources was used as the template. The optimal annealing temperature (Tm) of the primers was determined by the gradient PCR gene amplifier. m The SSR-PCR amplification system was 10 μl, including 2x TSINGKE MasterMix 5 μl, 2 μmol / L SSR primer 1 μl, <1 μg DNA template, and finally ddH2O was added to 10 μl. The SSR-PCR amplification program was as follows: 96 °C pre-denaturation for 2 min; 96 °C denaturation for 10 s; annealing temperature 55 °C, reannealing time 30 s; 72 °C extension for 1 min, 30-35 cycles; 72 °C extension for 1 min; 4 °C storage.

[0097] 1.2.5 Data processing and analysis of EST-SSR polymorphic primers

[0098] The bands migrated to the same position were recorded as one genetic locus, and the bands on all loci were recorded as "1", and the bands were recorded as "0". The "0, 1" matrix was constructed to obtain the EST-SSR molecular marker data. The allele number (N a ), the effective allele number (N e ) were calculated by Popgene 32 software. The polymorphic site percentage (PPL) and the polymorphic information content (PIC) of the primers were calculated by Powermarker v.3.25 software.

[0099] 2 Test results

[0100] 2.1 Distribution characteristics of EST-SSR loci in ginkgo transcriptome

[0101] The Ginkgo transcriptome was assembled, and the results (Table 2) yielded 43,073 unigenes with a total length of 115,099,549.6 bp. A search revealed 173,160 EST-SSR loci. These loci were distributed across 15,176 unigenes, with an SSR frequency of 35.23%. Of these, 10,147 unigenes contained more than one EST-SSR locus, and 29,416 unigenes existed as complexes. On average, there was one EST-SSR locus every 6,647.01 bp (approximately 6.65 kb).

[0102] Table 2. Distribution characteristics of SSR sites in the Ginkgo transcriptome.

[0103]

[0104]

[0105] 2.2 Validation of Ginkgo transcriptome EST-SSR and screening of polymorphic primers

[0106] The validity verification results show that ( Figure 1 ), 50 pairs of EST-SSR primers were able to effectively amplify bands of the expected size with clear backgrounds, accounting for 50% of the synthesized primers. Using genomic DNA from 141 Ginkgo germplasm resources, polymorphic primer bands were screened from these 50 pairs of primers, and the results ( Figure 2 A total of 20 pairs of polymorphic primers were obtained, accounting for 40% of the total number of primers that could effectively amplify bands. Table 3 shows the amplification results of the 20 pairs of EST-SSR primers in 141 Ginkgo germplasm resources. A total of 85 alleles were amplified, with 2-8 alleles detected at each locus, averaging 4.250 alleles. GBSSR27860 had the most alleles with 8, while GBSSR27072 and GBSSR34776 had the fewest with 2 alleles each. The average number of effective alleles and the average percentage of polymorphic loci were 2.291 and 0.951, respectively. The average polymorphism information content (PIC) was 0.589, indicating that the EST-SSR primers had a high level of polymorphism (PIC>0.500) and were suitable for Ginkgo genetic diversity analysis and the construction of core germplasm. Information on the 20 pairs of EST-SSR molecular marker primers is shown in Table 4.

[0107] Table 3. Amplification results of 20 EST-SSR primer pairs

[0108]

[0109]

[0110] Table 4 Information on 20 EST-SSR primer pairs

[0111]

[0112]

[0113] Conclusions

[0114] The present application shows that the development of EST-SSR molecular markers from the transcriptome of Ginkgo biloba is effective, with high frequency, multiple SSR sites, rich types, great polymorphism potential and availability. Through EST-SSR primer effectiveness verification and polymorphism screening, 20 pairs of EST-SSR molecular marker primers with good stability and high polymorphism were successfully developed, providing stable and reliable molecular marker materials for Ginkgo biloba genetic diversity analysis, core germplasm construction, etc.

[0115] Example 2: Genetic diversity analysis of Ginkgo biloba germplasm resources

[0116] 1. Materials and methods

[0117] 1.1 Materials

[0118] The test material was 141 Ginkgo biloba germplasm resources, same as in Example 1.

[0119] 1.2 Methods

[0120] The 20 pairs of polymorphic EST-SSR molecular marker data obtained above were input into Popgene 32 software to calculate the average expected heterozygosity or genetic diversity index (H e ), Shannon diversity index (I), genetic differentiation coefficient (G st ), and gene flow (N m ). Molecular variance analysis was performed using GenAlE_v6.502 software, and UPGMA clustering analysis was performed using NTSYS software.

[0121] 2. Test results

[0122] 2.1 Genetic diversity analysis of Ginkgo biloba germplasm resources

[0123] The results of genetic diversity analysis (Table 5) showed that the 141 Ginkgo biloba germplasm resources had a high level of genetic diversity. The genetic diversity index (H e ) was between 0.328 and 0.847, with an average of 0.520, indicating that the Ginkgo biloba population had not been subjected to high-intensity selection and had rich polymorphism. The Shannon diversity index (I) was between 0.563 and 1.957, with an average of 0.923. The average genetic differentiation coefficient (G st) was 0.112, indicating that genetic variation mainly existed within the Ginkgo population, and only 11.2% of variation occurred within the population. The average gene flow (N m ) was 1.980, indicating that there was a certain degree of gene exchange between Ginkgo populations, and the degree of genetic differentiation between populations was low.

[0124] Table 5 Genetic diversity of 141 Ginkgo germplasm resources

[0125]

[0126]

[0127] 2.2 Analysis of genetic diversity of different Ginkgo populations

[0128] The genetic diversity of 11 Ginkgo populations was analyzed using 20 pairs of EST-SSR primers. The results (Table 6) showed that the levels of genetic diversity of different populations were different. Among them, the Ginkgo population in Guizhou had the largest number of alleles (N a ), effective number of alleles (N e ), observed heterozygosity (H o ), and genetic diversity index (H e ), which were 3.211, 2.554, 0.941, and 0.580, respectively, indicating the highest level of genetic diversity. The Ginkgo population in Huanglongxian had the smallest number of alleles (N a ), effective number of alleles (N e ), observed heterozygosity (H o ), genetic diversity index (H e ), and Shannon diversity index (I), which were 2.250, 1.390, 0.252, 0.431, and 0.431, respectively, indicating the lowest level of genetic diversity. The genetic diversity level of the Ginkgo population in Huanglongxian was significantly different from that of other populations, which might be related to the single planting or introduction of Ginkgo resources varieties (lines) in the local area. The ranking of the genetic diversity levels of the 11 populations was: Guizhou > Hubei > Guangxi > Hunan > Jiangsu > Shandong > Zhejiang > Shaanxi > Japan > Liaoning > Huanglongxian. Except for the Ginkgo population in Huanglongxian, the Ginkgo populations in Southwest China (Guizhou, Hubei, Guangxi, and Hunan) had the richest genetic diversity, followed by the populations in East China (Jiangsu, Shandong, and Zhejiang), and the populations in Northwest China (Shaanxi and Liaoning) had the lowest genetic diversity, which might be related to the unsuitable climate for Ginkgo growth in Northwest China. The results of molecular variance analysis (Table 7) showed that the genetic variation of Ginkgo populations mainly occurred within the populations (86%), and only 14% of the genetic variation occurred between the populations, indicating that the degree of genetic differentiation between populations was small.

[0129] Table 6 Genetic diversity analysis of different Ginkgo populations

[0130]

[0131] Table 7 Analysis of molecular variance of Ginkgo populations

[0132]

[0133] 2.3 Cluster analysis

[0134] In order to understand the genetic relationship among Ginkgo germplasm resources, UPGMA cluster analysis of populations and individuals was carried out using 20 pairs of EST-SST primers. Figure 3 ), 11 populations could be divided into 2 groups and 3 subgroups at genetic distance 0.100. The first group included Jiangsu, Shandong, Hunan, Hubei, Guangxi, Guizhou, Shaanxi, Liaoning and Japan populations, in which Japan population was clustered into a subgroup, indicating that the genetic relationship between Japan population and other populations was farthest, while Jiangsu and Shandong populations were clustered into a group, indicating that their genetic relationship was closest. The second group only included Huanglongxian population, indicating that it had the farthest genetic relationship with other populations. Figure 4 ), 141 Ginkgo germplasm resources could be divided into 3 groups, in which most of Ginkgo from Huanglongxian were clustered into a group, and had the closest genetic relationship with TaiXing 4 (42), Xiajin Ginkgo (68) and TaiXing 00590 (37). The rest of Ginkgo germplasm resources were not strictly clustered according to geographical origin, which might be related to the fact that different varieties (lines) of Ginkgo broke through geographical restrictions and produced more frequent gene exchange under long-term artificial selection, such as grafting, hybrid breeding, introduction to different places, etc.

[0135] 3. Conclusion

[0136] The 20 pairs of EST-SSR molecular marker primers developed based on Ginkgo transcriptome have a high polymorphism level (PIC=0.589>0.500), and are suitable for Ginkgo genetic diversity analysis and core germplasm construction, etc.

[0137] Further, the genetic diversity analysis of 141 Ginkgo germplasm resources by the above-mentioned EST-SSR molecular markers showed that Ginkgo has rich genetic diversity (H e =0.520>0.500), the genetic diversity level of each population is different, and there is a certain gene exchange among populations, resulting in a low degree of genetic differentiation.

[0138] Example 3: Construction of Ginkgo core germplasm

[0139] 1. Materials and methods

[0140] 1.1 Materials

[0141] The test material is the same as that in Example 2, which is 141 Ginkgo germplasm resources.

[0142] 1.2 Method

[0143] 1.2.1 Construction of the core collection

[0144] In order to compare the differences of different sampling methods and obtain the best sampling strategy, 2 grouping principles (grouping by origin, completely random and ungrouping), 4 sampling proportions in each group (simple proportion, logarithmic proportion, square root proportion and genetic diversity proportion), 2 sampling methods (site-preferred cluster sampling method, random cluster sampling method) and 6 overall sampling proportions (5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%) were combined randomly, forming 72 sampling schemes. The genetic distance was calculated based on the SM similarity coefficient.

[0145] 1.2.2 Evaluation of the core collection

[0146] The representativeness of the core collection was evaluated by 5 indexes including the number of alleles, the number of effective alleles, Shannon diversity index (I), genetic diversity index (H e ) and percentage of polymorphic loci (PPL). Finally, principal component analysis was used to determine the core collection.

[0147] 1.2.2.1 Grouping principle

[0148] The 141 Ginkgo germplasm resources were grouped according to the origin, which could be divided into 11 groups: 5 from Guangxi, 8 from Guizhou, 4 from Hubei, 41 from Jiangsu, 3 from Liaoning, 20 from Shandong, 3 from Shaanxi, 4 from Zhejiang, 11 from Hunan, 2 from Japan and 40 from Huanglongxian in Jiangsu.

[0149] 1.2.2.2 Sampling proportion in each group

[0150] (1) Simple proportion: the proportion of the number of samples in each group to the number of core collection was the same as the proportion of the number of resources in each group to the number of original germplasm. It was calculated by the following formula:

[0151] N i = p x N io

[0152] In the formula, Ni represents the number of samples in the i th group; p represents the sampling proportion; N io represents the number of resources in each group.

[0153] (2) Logarithmic proportion: the proportion of the number of samples in each group to the number of core collection was the same as the proportion of the sum of the logarithmic values of the number of resources in each group to the logarithmic values of the number of original germplasm. It was calculated by the following formula:

[0154]

[0155] Wherein, Ni represents the number of samples in the ith group; n represents the number of groups; ρ represents the sampling ratio; and N represents the number of original germplasm. io Wherein, Ni represents the number of samples in the ith group; n represents the number of groups; ρ represents the sampling ratio; and N represents the number of original germplasm.

[0156] (3) Square root ratio: the ratio of the number of samples in each group to the number of core germplasm is the same as the ratio of the square root of the number of resources in each group to the square root of the number of original germplasm. It is calculated by the following formula:

[0157]

[0158] Wherein, Ni represents the number of samples in the ith group; n represents the number of groups; ρ represents the sampling ratio; and N represents the number of original germplasm.

[0159] (4) Genetic diversity ratio: the ratio of the number of samples in each group to the number of core germplasm is the same as the ratio of the square root of the number of resources in each group to the genetic diversity index of the number of original germplasm. It is calculated by the following formula:

[0160]

[0161] Wherein, Ni represents the number of samples in the ith group; n represents the number of groups; ρ represents the sampling ratio; and N represents the number of original germplasm. i Wherein, Ni represents the number of samples in the ith group; n represents the number of groups; ρ represents the sampling ratio; and N represents the number of original germplasm.

[0162] 1.2.2.3 Sampling method

[0163] (1) Site priority clustering sampling method: in the 2 germplasm resources of the lowest branch of the clustering diagram, the germplasm with more number of alleles is preferentially selected into the next round of clustering. If they have the same alleles, the germplasm with smaller allele frequency is selected. If both are the same, 1 germplasm is randomly selected. The selected germplasm continues to cluster and is removed, and according to the above method, the clustering is performed until the number of germplasm meeting the requirements is reached.

[0164] (2) Random clustering sampling method: in the 2 germplasm resources of the lowest branch of the clustering diagram, 1 germplasm is randomly selected into the next round of clustering, and the selected germplasm continues to cluster and is removed, and according to the above method, the clustering is performed until the number of germplasm meeting the requirements is reached.

[0165] 2 Test results

[0166] 2.1 Comparative analysis of different group sampling ratio strategies

[0167] The core germplasm constructed by the above 4 different group sampling ratios and the random ungrouping method was compared and analyzed, and the results showed (Table 8) that, except in the square root ratio condition, the average genetic diversity index (H eThe average Shannon diversity index (I) and average gene diversity index (H) of the core germplasm constructed under the other three ratio conditions are smaller than those of random clustering sampling. e The values ​​of H and H were both higher than those obtained by random clustering, indicating that the core germplasm constructed by the locus-priority clustering method has richer genetic diversity and better results. Among the locus-priority clustering methods, the core germplasm constructed using the locus-priority clustering method had the richest genetic diversity (I = 0.924, H...). e =0.520); followed by the square root proportion (I = 0.910, H = 0.520); e =0.514) and logarithmic proportion (I = 0.909, H e =0.513); the genetic diversity index of core germplasm constructed with simple proportions is the lowest (I = 0.905, H = 0.513); e =0.509). This indicates that using the genetic diversity ratio is the most effective method for constructing core germplasm. In random clustering, the core germplasm constructed using the genetic diversity ratio also exhibited the richest genetic diversity (I = 0.886, H = 0.509). e =0.478); while the genetic diversity index of the core germplasm constructed randomly without grouping was the lowest (I = 0.857, H = 0.478); e =0.440). This indicates that grouping by origin is more effective than not grouping when constructing core Ginkgo germplasm.

[0168] Table 8 Comparison of different sampling strategies

[0169]

[0170]

[0171] 2.2 Comparative Analysis of Different Population Sampling Proportions

[0172] A comparison of the genetic diversity indices of the core germplasm constructed using the eight different overall sampling proportions revealed (Table 9) that, except for an overall proportion of 5%, the average genetic diversity index (H0) of the core germplasm constructed using the site-priority clustering method was significantly higher. e The average Shannon diversity index (I) and average gene diversity index (H) of the core germplasm constructed under the other seven proportion conditions are smaller than those of random clustering sampling. e) were all larger than that of random clustering sampling method, which indicated that the genetic diversity of the core collection constructed by the site-preferential clustering method was more abundant. This was consistent with the results of the comparative analysis of the sampling proportion strategies in different groups. When the overall sampling proportion was 35%, the average Shannon diversity index (I) of the core collection constructed by the site-preferential clustering method was the largest, which was 0.940; the second was 25% and 40%, and the average Shannon diversity index (I) was 0.939; when the overall sampling proportion was 15%, 20% and 25%, the average gene diversity index (H e ) of the core collection constructed by the site-preferential clustering method reached the maximum, which was 0.523. In summary, when the overall sampling proportion was 25%, the genetic diversity of the core collection was the most abundant.

[0173] Table 9 Comparison of different overall sampling proportions

[0174]

[0175] 2.3 Determination of the optimal sampling scheme and evaluation of the core collection

[0176] The principle of the construction of the core collection is to represent the maximum genetic diversity of the original resources with the least genetic resources. Based on the above analysis results, it was considered that the sampling scheme of grouping according to the production area, sampling in groups according to the genetic diversity proportion and the site-preferential clustering method, and the overall sampling proportion of 25% could construct the core collection of Ginkgo biloba, which could replace the original germplasm with the least resource quantity and the maximum genetic diversity. The final 33 Ginkgo biloba core collections are shown in Table 10. Among them, there are 4 from Guangxi, 4 from Guizhou, 3 from Hubei, 3 from Jiangsu, 3 from Liaoning, 3 from Shandong, 3 from Shaanxi, 3 from Zhejiang, 3 from Hunan, 2 from Japan, 2 from Huanglongxian in Jiangsu. The remaining germplasm constitutes the reserved germplasm.

[0177] Table 10 Core collection of Ginkgo biloba

[0178]

[0179] The evaluation of the core collection of Ginkgo biloba was carried out, and the genetic diversity of the core collection, the reserved germplasm and the original germplasm was compared and analyzed. The results showed that the resource quantity of the core collection was 33, and the retention rate was 23.24%; the number of alleles, the number of effective alleles, the percentage of polymorphic loci (PPL), the gene diversity index (H eThe retention rates of Shannon-Wiener index (H) and Shannon diversity index (I) were 100.00%, 100.57%, 100.00%, 97.31% and 96.75% respectively, indicating that the core germplasm can better represent the genetic diversity of the original germplasm. The resource quantity of the reserved germplasm is 108, and the retention rate is 76.60%; the allele number (Na), the effective allele number (Ne), the percentage of polymorphic loci (PPL), the genetic diversity index (H e The retention rates of Shannon-Wiener index (H) and Shannon diversity index (I) were 52.02%, 89.08%, 90.56%, 89.23% and 71.40% respectively, and the genetic diversity parameters were lower than those of the core germplasm. The principal component analysis results of the core germplasm and the original germplasm are shown in Figure 5 The core germplasm is basically distributed in the entire coordinate graph, indicating that the core germplasm has good representativeness.

[0180] Table 11 Genetic diversity evaluation of the core germplasm

[0181]

[0182] 3. Conclusion

[0183] The present application firstly uses EST-SSR molecular marker data to construct the core germplasm of Ginkgo biloba, and the results show that the 33 Ginkgo biloba core germplasms constructed by using the sampling scheme of grouping according to the production place, clustering in the group according to the genetic diversity proportion and site priority, and the overall sampling proportion of 25%, can replace the original germplasm with the least resource quantity and the maximum genetic diversity. SEQUENCE LISTING <110> Central South University of Forestry and Technology <120> Polymorphic EST-SSR molecular marker primer of Ginkgo biloba and application thereof <160> 40 <170> SIPOSequenceListing 1.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <400> 1 ccgagagaaa gatacagccg 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <400> 2 ccacaaccca aaaagcctta 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <400> 3 cagattgtca tgcaccccta 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <400> 4 tccttggtaa cgccattctc 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <400> 5 atcacgattt cttcgttggg 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <400> 6 gcttgaatgt cctctgcctc 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <400> 7 cagacacatg aaccccttcc 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <400> 8 atgccctggc atattttgat 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <400> 9 aagcgtctcg agaaagtgga 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <400> 10 tacgatgcag gaaaacatgc 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <400> 11 aggagaacag aggctgtgga 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <400> 12 tttgttattt ccgtgggagc 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <400> 13 aatttggatg tcgccattgt 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <400> 14 aagccctgat atgaccatgc 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <400> 15 gtaggcctct cctccaatcc 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <400> 16 aatggaccac attgggtgtt 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <400> 17 accaatggct gttgatgtga 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <400> 18 gcaaaaacaa catccaggct 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <400> 19 cacccctggg attattgatg 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <400> 20 atgagtggaa tgtgggcttc 20 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <400> 21 ccatgtctcc aaggtcgatt 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <400> 22 cttggcgaat actgcattga 20 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <400> 23 caagcaaatt agtgctgcca 20 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <400> 24 tgaatgcctg atgatttgga 20 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <400> 25 atgggcgtcg tggatagtag 20 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <400> 26 ttttcccagat aggcattgg 20 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <400> 27 agtgcatttc atttgcttcg 20 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <400> 28 tttcagtctc tgcgggagat 20 <210> 29 <211> 20 <212> DNA <213> Artificial Sequence <400> 29 agaacggtgc caacaatagg 20 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <400> 30 tccctgattg ccaaagtagg 20 <210> 31 <211> 20 <212> DNA <213> Artificial Sequence <400> 31 aagtggagtt gtgaaacggg 20 <210> 32 <211> 20 <212> DNA <213> Artificial Sequence <400> 32 tttaggctgg aatggattgg 20 <210> 33 <211> 20 <212> DNA <213> Artificial Sequence <400> 33 aggtggatgg gcatattcag 20 <210> 34 <211> 20 <212> DNA <213> Artificial Sequence <400> 34 cacagttggc agacgaaaaa 20 <210> 35 <211> 19 <212> DNA <213> Artificial Sequence <400> 35 agagattttg cgacagagc 19 <210> 36 <211> 19 <212> DNA <213> Artificial Sequence <400> 36 ggtagcagtt gaaccgtta 19 <210> 37 <211> 22 <212> DNA <213> Artificial Sequence <400> 37 agggaaaaag tgaaagagag ag 22 <210> 38 <211> 22 <212> DNA <213> Artificial Sequence <400> 38 TCTAGAGGATCCGAGGTAAAGA 22 <210> 39 <211> 19 <212> DNA <213> Artificial Sequence <400> 39 TTTGGGAGTAGTGTGTGTGT 19 <210> 40 <211> 19 <212> DNA <213> Artificial Sequence <400> 40 CTGGATTGCATTTCAGAGTC 19

Claims

1. Ginkgo transcriptome EST-SSR molecular marker primers, characterized in that, Thanks for watching 20-year-olds: GBSSR04447-F:5′-CCGAGAGAAGATACAGCCG-3′; SEQ ID NO.1; GBSSR04447-R:5′-CCACAACCCAAAAAGCCTTA-3′; SEQ ID NO.2; GBSSR05243-F:5′-CAGATTGTCATGCACCCCTA-3′; SEQ ID NO.3; GBSSR05243-R:5′- TCCTTGGTAACGCCATTCTC-3′; SEQ ID NO.4; GBSSR10422-F:5′-ATCACGATTTCTTCGTTGGG-3′; SEQ ID NO.5; GBSSR10422-R:5′-GCTTGAATGTCCTCTGCCTC-3′; SEQ ID NO.6; GBSSR02181-F:5′- CAGACACATGAACCCCTTCC-3′; SEQ ID NO.7; GBSSR02181-R:5′-ATGCCCTGGCATATTTTGAT-3′; SEQ ID NO.8; GBSSR16030- F:5′- AAGCGTCTCGAGAAAGTGGA-3′; SEQ ID NO.9; GBSSR16030- R:5′-TACGATGCAGGAAAACATGC-3′; SEQ ID NO.10; GBSSR21240 -F:5'- AGGAACAGAGGCTGTGGA-3'; SEQ ID NO.11; GBSSR21240 -R:5′- TTTGTTATTTCCGTGGGAGC-3′; SEQ ID NO.12; GBSSR13689-F:5′- AATTTGGATGTCGCCATTGT-3′; SEQ ID NO.13; GBSSR13689-R:5′- AAGCCCTGATATGACCATGC-3′; SEQ ID NO.14; GBSSR05999-F:5′- GTAGGCCTCTCCTCCAATCC-3′; SEQ ID NO.15; GBSSR05999-R:5′- AATGGACCACATTGGGTGTT-3′; SEQ ID NO.16; GBSSR21429-F:5′-ACCAATGGCTGTTGATGTGA-3′; SEQ ID NO.17; GBSSR21429-R:5′-GCAAAACAACATCCAGGCT-3′; SEQ ID NO.18; GBSSR25769-F:5'- CACCCCTGGGATTATTGATG-3'; SEQ ID NO.19; GBSSR25769-R:5'- ATGAGTGGAATGTGGGCTTC-3'; SEQ ID NO.20; GBSSR29168-F:5'- CCATGTCTCCAAGGTCGATT-3'; SEQ ID NO.21; GBSSR29168-R:5'- CTTGGCGAATACTGCATTGA-3'; SEQ ID NO.22; GBSSR27072-F:5'- CAAGCAAATTAGTGCTGCCA-3'; SEQ ID NO.23; GBSSR27072-R:5'- TGAATGCCTGATGATTTGGA-3'; SEQ ID NO.24; GBSSR27825-F:5'- ATGGGGCGTCGTGGATAGTAG-3'; SEQ ID NO.25; GBSSR27825-R:5'- TTTTCCCAGATAGGCATTGG-3'; SEQ ID NO.26; GBSSR31776-F:5'- AGTGCATTTCATTTGCTTCG-3'; SEQ ID NO.27; GBSSR31776-R:5'-TTTCAGTCTCTGCGGGAGAT-3'; SEQ ID NO.28; GBSSR34776-F:5'- AGAACGGTGCCAACAATAGG-3'; SEQ ID NO.29; GBSSR34776-R:5'-TCCCTGATTGCCAAAGTAGG-3'; SEQ ID NO.30; GBSSR31083-F:5'- AAGTGGAGTTGTGAAACGGG-3'; SEQ ID NO.31; GBSSR31083-R:5'-TTTAGGCTGGAATGGATTGG-3'; SEQ ID NO.32; GBSSR27860-F:5'- AGGTGGATGGGCATATTCAG-3'; SEQ ID NO.33; GBSSR27860-R:5'- CACAGTTGGCAGACGAAAAA-3'; SEQ ID NO.34; GBSSR01538-F:5'- AGAGATTTTGCGACAGAGC-3'; SEQ ID NO.35; GBSSR01538-R:5'-GGTAGCAGTTGAACCGTTA-3'; SEQ ID NO.36; GBSSR00112-F:5'- AGGGAAAAAGTGAAAGAGAGAG-3'; SEQ ID NO.37; GBSSR00112-R:5'- CTAGTCAAGGCGAGGTTAAAGA-3'; SEQ ID NO.38; GBSSR10214-F:5'- TTTGGGAGTAGTGTGTTGT-3'; SEQ ID NO.39; GBSSR10214-R:5'-CTGGATTGCATTTGAAGTC-3'; SEQ ID NO.

40.

2. The application of the Ginkgo transcriptome EST-SSR molecular marker primers as described in claim 1 in the analysis of Ginkgo genetic diversity.

3. The application as described in claim 2, characterized in that, Specifically, the steps include the following: (1) Based on the Ginkgo transcriptome EST-SSR molecular marker primers described in claim 1, obtain EST-SSR molecular marker data; (2) Based on the above EST-SSR molecular marker data, genetic diversity analysis of Ginkgo biloba was performed using bioinformatics software.

4. The application as described in claim 3, characterized in that, include: 1) Input the obtained 20 pairs of polymorphic EST-SSR molecular marker data into the software to calculate the average expected heterozygosity, also known as the gene diversity index, Shannon diversity index, gene differentiation coefficient, and gene flow. 2) Further genetic diversity analysis was conducted on the Ginkgo population, including: number of alleles, effective number of alleles, observed heterozygosity, and population genetic diversity index; 3) Perform molecular variance analysis using software; 4) Use software to perform UPGMA cluster analysis.

5. The application of the Ginkgo transcriptome EST-SSR molecular marker primers as described in claim 1 in the construction of Ginkgo core germplasm.

6. The application as described in claim 5, characterized in that, Includes the following steps: (1) Based on the Ginkgo transcriptome EST-SSR molecular marker primers described in claim 1, obtain EST-SSR molecular marker data; (2) Based on the above EST-SSR molecular marker data, the core germplasm of Ginkgo biloba was constructed.

7. The application as described in claim 6, characterized in that, The method for constructing the core germplasm of Ginkgo biloba is as follows: grouping according to the place of origin, sampling within the group according to the proportion of genetic diversity and the site-priority clustering method, and the overall sampling ratio is 25%.

8. The application as described in claim 7, characterized in that, The representativeness of core germplasm was evaluated using five indicators: number of alleles, effective number of alleles, Shannon diversity index, gene diversity index, and percentage of polymorphic sites. Finally, principal component analysis was used to determine the core germplasm.

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