A set of SSR markers of Horsfieldia tetratepala and their applications

Abstract

The present invention discloses a group of SSR markers of Phoebe yunnanensis and applications thereof. There are a total of 20 SSR markers of Phoebe yunnanensis; the present invention also provides 20 pairs of SSR marker core primer pairs and fluorescently labeled M13 primers for specific amplification and SSR typing of Phoebe yunnanensis genomic DNA. Based on the above SSR markers, the present invention proposes a method for analyzing the genetic diversity of Phoebe yunnanensis germplasm and a method for constructing a fingerprint map. The present invention establishes an SSR molecular marker technology system for Phoebe yunnanensis for the first time, providing a scientific basis and technical support for the protection of genetic resources and oil development of this extremely small population of endangered species, and has important application value.

Description

Technical Field

[0001] The present invention relates to the technical field of forest genetic diversity protection, specifically a group of SSR markers of Phoebe yunnanensis and applications thereof. Background Art

[0002] Yunnan Fengchu Nan ( Horsfieldiatetratepala CY Wu) belongs to the genus Myristicaceae ( Horsfieldia ), an evergreen tree, is a typical endemic representative species of the tropical seasonal rainforests in Yunnan Province, my country. It grows in dense forests in valleys and slopes at altitudes between 300 and 1000 meters. Yunnan Fengchui Phoebe is highly ornamental due to its unique morphology and brightly colored flowers. Its straight trunk and red to reddish-brown heartwood have straight, easily workable grains and medium-strength wood, making it suitable for manufacturing boxboard or lightweight building panels. Furthermore, its seed kernels contain up to 57.0% oil, primarily lauric acid and myristic acid, which exhibit exceptional anticoagulant and viscosity-enhancing properties, making it an ideal raw material for industrial specialty lubricants widely used in military, aviation, and machinery applications. Therefore, a comprehensive assessment of the genetic diversity, genetic structure, and population dynamics of Yunnan Fengchui Phoebe is urgently needed to develop targeted germplasm resource conservation and utilization strategies, thereby achieving both the rescue and conservation of its extremely small population and the high-value development and utilization of its seed kernel oils.

[0003] Existing research on Phoebe yunnanensis primarily focuses on its distribution, morphological characteristics, chemical composition of branches and leaves, and fatty acids in seeds. However, there is a lack of systematic coverage of the overall diversity of its genetic resources. This research gap has become a major bottleneck restricting the conservation and efficient utilization of Phoebe yunnanensis's genetic resources. Summary of the Invention

[0004] To this end, the technical problem to be solved by the present invention is to provide a group of SSR markers of Phoebe yunnanensis and their applications, and use SSR molecular markers to perform SSR typing, genetic diversity evaluation and fingerprint map construction on Phoebe yunnanensis, providing a scientific basis and technical support for the genetic resource protection and oil development of this extremely small population endangered species, and has important application value.

[0005] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0006] A group of Yunnan wind blowing nanmu HorsfieldiatetratepalaThe SSR markers of CY Wu include HT005, HT007, HT011, HT012, HT015, HT018, HT023, HT029, HT031, HT036, HT039, HT041, HT046, HT048, HT057, HT062, HT064, HT073, HT079, and HT095; the nucleotide sequences of the above SSR markers are shown in SEQ ID NO.1 to SEQ ID NO.20, respectively. These SSR markers are highly polymorphic and have good reproducibility, and can accurately detect and reflect the allelic composition of the corresponding SSR sites in the genome of Phoebe yunnanensis; the combination of these SSR markers can be used for genetic diversity evaluation, DNA fingerprint construction, and variety identification of Phoebe yunnanensis germplasm resources.

[0007] A primer set for specifically amplifying the SSR marker of Psoralea corylifolia, wherein the SSR marker of Psoralea corylifolia is the above-mentioned SSR marker; the primer set comprises 20 pairs of SSR marker core primer pairs and M13 primers with fluorescent labels; the sequences of the SSR marker core primer pairs are shown in SEQ ID NO.22 to SEQ ID NO.61, and in each pair of SSR marker core primer pairs, the even-numbered ones are forward primers, and the odd-numbered ones are reverse primers, and the 5' ends of the forward primers are all connected with M13 sequences, which are identical to the sequences of the M13 primers; the sequences of the M13 primers are shown in SEQ ID NO.21. Using these primer sets, each SSR site in the genome of Psoralea corylifolia can be efficiently and accurately PCR amplified, and the PCR amplification products are fluorescent, so that different PCR amplification products can be accurately distinguished based on their fluorescent signals, greatly improving the sensitivity and accuracy of the detection.

[0008] In the above primer set, the fluorescent label on the M13 primer is any one of FAM, NED, VIC and ROX.

[0009] The analysis method of genetic diversity of P. yunnanensis population was carried out according to the following steps:

[0010] S1. Extract genomic DNA from each individual in the Yunnan Fengchui Nan population;

[0011] S2. Obtain the genotype of the SSR loci corresponding to the above-mentioned SSR markers in the genomic DNA of each strain of Phoebe yunnanensis to obtain the SSR typing results;

[0012] S3. Calculate the genetic diversity parameters of the Yunnan Fengchui population based on the SSR typing results.

[0013] In the above-mentioned analysis method, in step S2, the above-mentioned primer set is used to PCR amplify the genomic DNA of each strain of Psoralea corylifolia, and the length polymorphism of the PCR amplification product is detected to obtain the genotype of the genomic DNA of each strain of Psoralea corylifolia at the said SSR site.

[0014] In the above analysis method, in step S2, for each of the SSR sites in the genomic DNA of each of the Psoralea corylifolia plants, a PCR reaction system is prepared using the SSR marker core primer pair corresponding to the SSR site to perform PCR amplification;

[0015] Each PCR reaction system contained: 5 ng of genomic DNA of P. yunnanensis as a DNA template, 0.5 µM M13 primer, 0.5 µM forward primer, 0.5 µM reverse primer, 150 µM dNTPs, 2.0 µM MgCl2, 0.25 U Taq DNA polymerase, and 10 µL 1× PCR buffer; the volume of each PCR reaction system was 10 µL.

[0016] In the above analysis method, in step S2, the reaction procedure for PCR amplification is: pre-denaturation at 94°C for 4 minutes; denaturation at 94°C for 30 seconds, annealing at 50-55°C for 30 seconds, and extension at 72°C for 30 seconds, for a total of 31 cycles; and final extension at 72°C for 10 minutes.

[0017] In the above analysis method, in step S3, the genetic diversity parameters include the number of alleles, observed heterozygosity, expected heterozygosity, Shannon-Wiener diversity index, and fixation index. These indices can be calculated based on the SSR typing results and can comprehensively quantify and reflect the genetic structure and diversity level of the Fengchuian population in southern Yunnan.

[0018] The method for constructing the fingerprint map of the Yunnan Fengchuenye germplasm is carried out as follows: using a method for detecting the length polymorphism of the PCR product after PCR amplification, the genotypes of the SSR sites corresponding to the SSR markers HT005, HT011, HT036, HT039, HT046, HT048, HT062 and HT079 in the Yunnan Fengchuenye genomic DNA to be detected are obtained, and the fingerprint map of the Yunnan Fengchuenye to be detected is constructed according to the genotypes of the SSR sites; when performing PCR amplification, corresponding SSR marker core primer pairs are used for the SSR sites of the Yunnan Fengchuenye genomic DNA to be detected, and PCR reaction systems are prepared for each of the SSR sites and PCR amplification is performed; the SSR marker core primer pairs are the above-mentioned SSR marker core primer pairs used for specific amplification of HT005, HT011, HT036, HT039, HT046, HT048, HT062 and HT079. The combination of these eight SSR markers can construct a fingerprint with unique molecular identity information, which can quickly distinguish and identify different Yunnan Fengchuennan germplasms, providing a scientific and intuitive molecular identification tool for the comparison, classification and management of germplasm resources.

[0019] Application of the SSR marker of Psoralea corylifolia in Yunnan, wherein the application is to use the above SSR marker for any of the following:

[0020] (1) Variety identification of Phoebe yunnanensis germplasm;

[0021] (2) Molecular marker-assisted breeding of Phoebe yunnanensis;

[0022] (3) Construction of the core germplasm of Phoebe chinensis in southern Yunnan.

[0023] The technical solution of the present invention achieves the following beneficial technical effects:

[0024] 1. The present invention has developed 20 SSR markers for Phoebe yunnanensis for the first time and established an SSR molecular marker technology system for Phoebe yunnanensis. Experiments have shown that the SSR sites corresponding to these 20 SSR markers in the genomic DNA of Phoebe yunnanensis are highly polymorphic. According to the allele composition at these SSR sites, Phoebe yunnanensis individuals or groups with similar genetic backgrounds can be effectively distinguished. At the same time, these 20 SSR sites have good repeatability, that is, when the SSR sites in the genomic DNA of the same individual are PCR amplified, the length of the products obtained by multiple amplifications is stable, and the SSR typing results can be stably reproduced, providing technical support for the evaluation of Phoebe yunnanensis germplasm resources, fingerprint map construction, and molecular marker-assisted breeding, thus laying the foundation for the effective protection of this extremely small population of endangered species and the development and utilization of oil resources.

[0025] 2. Using the 20 SSR markers of Phoebe yunnanensis provided in the present invention, SSR typing of Phoebe yunnanensis germplasm can be accurately performed and genetic diversity evaluation can be performed; based on the SSR typing results, the present invention provides a combination of SSR markers for SSR fingerprint mapping: HT005, HT011, HT062, HT036, HT039, HT046, HT048 and HT079. Using these 8 SSR markers, SSR fingerprint mapping of all 34 Phoebe yunnanensis germplasms in three populations in Yingjiang, Ruili and Mangshi areas of Yunnan Province can be completed. The SSR markers provided in the present invention help to accurately grasp the current status of the genetic resources of Phoebe yunnanensis, and provide a scientific basis for the precise identification, rescue protection and rational exploration and utilization of Phoebe yunnanensis germplasm resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Genetic structure diagram of three Yunnan Fengchui Machilus populations used in the examples of the present invention;

[0027] Figure 2 The fingerprint maps of 34 Phoebe yunnanensis germplasms constructed based on 34 alleles at 8 SSR loci in Example 3 of the present invention. DETAILED DESCRIPTION

[0028] Example 1

[0029] In this example, based on simplified genome sequencing technology, a group of SSR markers with good repeatability and high polymorphism were developed for P. yunnanensis, including HT005, HT007, HT011, HT012, HT015, HT018, HT023, HT029, HT031, HT036, HT039, HT041, HT046, HT048, HT057, HT062, HT064, HT073, HT079 and HT095.

[0030] The individuals of Phoebe yunnanensis used in this example are accession YJ06 from the Yingjiang population of Phoebe yunnanensis, grown in Yingjiang County, Yunnan Province. This biological material is publicly available from the Institute of Tropical Forestry, Chinese Academy of Forestry. This material was used solely for the purpose of repeating the experiments described in this example and is not to be used for any other purpose.

[0031] The specific method for developing SSR markers is further explained below.

[0032] First, total DNA from the leaves of P. yunnanensis YJ06 was extracted using the CTAB method. This DNA served as the genomic DNA of YJ06. The genomic DNA was fragmented into 350-bp fragments using ultrasound. Paired-end libraries were constructed using the NEBNext Ultra™ DNA Library Construction Kit (New England BioLabs, Cat. No. E7370S) according to the provided protocol. Sequencing was then performed on an Illumina HiSeq X Ten platform in PE150 mode. A total of 19.2 Gb of data was obtained. Data quality control was performed using NGS QC Toolkit 2.3.2 software. Sequences were assembled using SPAdes 3.6.1. Genome-wide microsatellite analysis (GMATA) software was then used with conditions set to 2- to 5-base units and replicated at least eight times. A total of 43,293 SSR loci were identified. From these, SSR loci with high polymorphism and good stability were selected for primer design.

[0033] Through screening, the inventors ultimately identified 20 SSR loci and designed primers targeting these 20 SSR loci. Table 1 shows detailed information on the 20 SSR loci and the primers corresponding to each locus in P. yunnanensis. The "allele size" in the table refers to the actual measured fragment size obtained by amplifying the SSR loci in genomic DNA collected from 34 P. yunnanensis strains from three populations using the corresponding primer pairs.

[0034] Table 1 Detailed information of 20 SSR loci in Psoralea corylifolia

[0035]

[0036] The DNA sequences corresponding to the SSR loci together constitute a set of SSR markers for P. yunnanensis. The DNA sequences corresponding to the SSR loci are shown in SEQ ID NO. 1 to SEQ ID NO. 20 in the sequence listing.

[0037] Example 2

[0038] In this example, the genetic diversity of P. yunnanensis was evaluated based on the 20 SSR markers obtained in Example 1.

[0039] 1. Genomic DNA Extraction and Amplification of SSR Loci in Genomic DNA

[0040] Fresh young leaves of Phoebe yunnanensis were collected individually from wild populations of Phoebe yunnanensis in Yingjiang, Ruili, and Mangshi, Yunnan. A total of 34 Phoebe yunnanensis individuals were found in three populations across the three locations. All 34 fresh young leaves were quickly dried using silica gel and preserved.

[0041] For dried leaves, total DNA was extracted from individual leaves using the CTAB method to obtain genomic DNA. The concentration and quality of the extracted genomic DNA were detected by 1.0% agarose gel electrophoresis and NanoDrop-2000 ultra-micro spectrophotometer (Thermo Fisher Scientific), and finally diluted to 50 ng·μL. -1 The diluted genomic DNA needs to be stored at -20℃.

[0042] Primers were designed based on the 20 SSR markers obtained in Example 1, and PCR amplification was performed on the corresponding SSR loci in the diluted genomic DNA. During PCR amplification, a PCR reaction system was prepared for each of the 20 SSR loci in the genomic DNA of each germplasm to amplify the SSR loci.

[0043] After PCR amplification, the length of the PCR product at each SSR locus is examined. The number of different PCR product lengths for a particular SSR locus indicates the number of alleles present at that SSR locus. For an individual, the composition of alleles at each SSR locus (i.e., genotype) is the SSR typing result for that individual.

[0044] PCR amplification of each SSR locus uses an M13 primer and a primer pair specific for that SSR locus (SSR marker core primer pair). For the M13 primer, a fluorescent marker is added to the 5' end of the M13 primer (SEQ ID NO. 21: 5'-CACGACGTTGTAAAACGAC-3') using fluorescent dUTP. Furthermore, an M13 sequence (the M13 sequence is the sequence of the M13 primer) is added to the 5' end of the forward primer (F primer) for each SSR locus. Fluorescent markers can be any of FAM, NED, VIC, and ROX.

[0045] For example, when performing PCR on the HT005 locus, three primers are required in the PCR reaction system. The first primer sequence is 5'-CACGACGTTGTAAAACGACGACCCATCACTCCATTCACA-3', which is the sequence of the M13 primer added to the 5' end of the F primer for the HT005 locus in Table 1. The second primer sequence is 5'-AGTAGCCTGACTGCGGTTTT-3', which is the R primer for the HT005 locus in Table 1. The third primer is the fluorescently labeled M13 primer, i.e., 5'-fluorescent-labeled-CACGACGTTGTAAAACGAC-3'.

[0046] Table 2 shows the numbers and sequences of the SSR marker core primer pairs added to the reaction system for PCR amplification of each SSR locus. Even-numbered primers are forward primers, and odd-numbered primers are reverse primers.

[0047] Table 2

[0048]

[0049] PCR amplification was performed using a 10 μL PCR reaction system containing 5 ng DNA, 0.5 μM forward primer (with an M13 sequence at the 5' end), 0.5 μM reverse primer, 0.5 μM fluorescently labeled M13 primer, 150 μM dNTPs, 2.0 μM MgCl₂, 10 μL 1× PCR buffer, and 0.25 U Taq DNA polymerase. PCR amplification was performed on a Veriti thermal cycler (Applied Biosystems). The PCR amplification program was as follows: initial denaturation at 94°C for 4 min; 31 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s; and a final extension at 72°C for 10 min.

[0050] In some other embodiments, the annealing temperature during PCR amplification may also be other values within the range of 50-55°C.

[0051] The PCR amplification products were detected using an ABI3730xl DNA analyzer (Applies Biosystems), and the alleles were read and counted using Gene-Marker2.2.0 software (SoftGenetics LLC) to obtain the allelic composition of each SSR locus (i.e., SSR typing results).

[0052] 2. Population genetic diversity and genetic structure

[0053] Based on the SSR data set (SSR typing results), the number of alleles (alleles, N a ), effective number of alleles ( N e heterozygosity observed, H o ), Shannon-Wiener diversity index ( I), expected heterozygosity (heterozygosityexpected, H e ) and fixation index ( F ), Hardy-Weinberg equilibrium (HWE) chi-square test and AMOVA analysis were performed. Allele abundance was calculated using HP-RARE 1.0 software ( A r ) and private allele abundance ( A p BOTTLENECK 1.2.02 software was used to detect bottleneck effects in the population. STRUCTURE 2.3.3 software was used to analyze and plot the genetic structure of the population.

[0054] The results showed that 20 core primer pairs of SSR markers amplified a total of 208 alleles from the genomic DNA of 34 accessions of P. yunnanensis (the number of alleles at each site is shown in Table 3). In other words, a total of 208 alleles were detected at all SSR loci in the genomic DNA of 34 accessions of P. yunnanensis. The number of effective alleles at each SSR locus was ( N e ), Shannon-Wiener diversity index ( I ), observed heterozygosity ( H o ), expected heterozygosity ( H e ), and fixed index ( F ) were 5.48, 1.85, 0.49, 0.77, and 0.37, respectively, indicating that the 34 accessions exhibited rich polymorphism at the 20 SSR loci. N a ), effective number of alleles ( N e ), observed heterozygosity ( H o ), expected heterozygosity ( H e ), allele abundance ( A r ), Shannon-Wiener diversity index ( I ) and fixed index ( F ) were only 6.23, 4.31, 0.50, 0.70, 5.62, 1.51, and 0.32, indicating low population genetic diversity. Private allele abundance ( A p ) has a high average value (1.64), indicating that the genetic exchange between populations is not sufficient (see Table 4 for relevant data).

[0055] Table 3 Horizontal genetic diversity of Phoebe sylvestris

[0056]

[0057] Table 4 Genetic diversity of Phoebe yunnanensis populations

[0058]

[0059] The results of the chi-square test for Hardy-Weinberg equilibrium (HWE) for these populations are shown in Table 5. "ns" indicates no significant difference, meaning no deviation from Hardy-Weinberg equilibrium. "*," "**," and "***" indicate significant deviations from Hardy-Weinberg equilibrium at the 0.05, 0.01, and 0.001 levels, respectively. The test results showed that 31 of the 60 tests deviated from Hardy-Weinberg equilibrium, with the Ruili population having the most deviated sites and the Yingjiang population having the fewest. These results suggest that significant genetic drift has occurred in P. fengchuenensis, causing changes in gene and genotype frequencies within the population during genetic transmission, rather than maintaining a constant state. This may have led to a further decline in its genetic diversity and adaptability, resulting in the current extremely small and fragmented population distribution.

[0060] Table 5 Chi-square test of Hardy-Weinberg equilibrium (HWE) for the Psoralea corylifolia population in southern Yunnan

[0061]

[0062] AMOVA analysis revealed the genetic structure and degree of genetic differentiation of the population. Table 6 shows the results of AMOVA analysis within and between the three populations in this example. The AMOVA analysis results show that although the genetic variation of Yunnan Fengchui Nan mainly exists within the population (97.13%), the genetic differentiation between the populations still reached a significant level ( Phi PT =0.029, P =0.009). This indicates that there are obstacles to gene exchange among extremely small populations, which is not conducive to the genetic stability of the species.

[0063] Table 6 Molecular variation analysis of Phoebe sylvestris population in southern Yunnan

[0064]

[0065] like Figure 1 STRUCTURE analysis results of the three Yunnan Fengchui Machilus populations in this example. Figure 1 It can be seen that in k =3 (the number of subpopulations in the group is 3) KThe value (the ratio of the standard deviation of the difference in the average log-likelihood values under different subpopulation numbers to the difference in the average log-likelihood values) is the largest, indicating that the three Yunnan Fengchuinan populations are each divided into a subpopulation. It can be seen that there are significant differences among the three populations, among which the genetic composition of individuals in the Ruili population is more complex than that in the Yingjiang and Mangshi populations.

[0066] Example 3

[0067] In this example, the same method as in Example 2 was used to extract genomic DNA from the Psoralea corylifolia to be tested. SSR typing was performed on the Psoralea corylifolia to be tested using a method of detecting length polymorphism of the PCR amplification product after PCR amplification to obtain the allelic composition of the genomic DNA of the Psoralea corylifolia to be tested at the aforementioned 20 SSR loci (i.e., the genotypes at the aforementioned 20 SSR loci in the genomic DNA of the Psoralea corylifolia to be tested were obtained). An SSR fingerprint of the Psoralea corylifolia to be tested was drawn based on the SSR typing results.

[0068] Furthermore, in order to achieve the goal of efficiently distinguishing the Yunnan Fengchuinan germplasm to be tested, after completing SSR typing using the above 20 SSR loci (SSR markers), some combinations of SSR loci were selected from them, in order to use the least alleles to characterize the specific molecular identity information of various germplasms.

[0069] like Figure 2 This is the fingerprint of 34 P. yunnanensis germplasms constructed based on SSR markers in this example. As can be seen from the figure, the use of three pairs of SSR marker core primer pairs targeting HT005, HT011, and HT062 can accurately identify 23 germplasms. Together with HT036, HT039, HT046, HT048, and HT079, the eight pairs of SSR marker core primer pairs can be used to identify all 34 P. yunnanensis germplasms. It can be seen that the combination of the eight SSR markers HT005, HT011, HT062, HT036, HT039, HT046, HT048, and HT079 used in this example has a high resolution and can efficiently and conveniently distinguish and identify different germplasms.

[0070] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the claims of this patent application.

Claims

1. A group of Yunnan wind-blown nanmu Horsfieldia tetratepala The SSR marker of CY Wu is characterized by: The SSR markers include HT005, HT007, HT011, HT012, HT015, HT018, HT023, HT029, HT031, HT036, HT039, HT041, HT046, HT048, HT057, HT062, HT064, HT073, HT079 and HT095; wherein: The nucleotide sequence of HT005 is shown in SEQ ID NO.1; The nucleotide sequence of HT007 is shown in SEQ ID NO.2; The nucleotide sequence of HT011 is shown in SEQ ID NO.3; The nucleotide sequence of HT012 is shown in SEQ ID NO.4; The nucleotide sequence of HT015 is shown in SEQ ID NO.5; The nucleotide sequence of HT018 is shown in SEQ ID NO.6; The nucleotide sequence of HT023 is shown in SEQ ID NO.7; The nucleotide sequence of HT029 is shown in SEQ ID NO.8; The nucleotide sequence of HT031 is shown in SEQ ID NO.9; The nucleotide sequence of HT036 is shown in SEQ ID NO.10; The nucleotide sequence of HT039 is shown in SEQ ID NO.11; The nucleotide sequence of HT041 is shown in SEQ ID NO.12; The nucleotide sequence of HT046 is shown in SEQ ID NO.13; The nucleotide sequence of HT048 is shown in SEQ ID NO.14; The nucleotide sequence of HT057 is shown in SEQ ID NO.15; The nucleotide sequence of HT062 is shown in SEQ ID NO.16; The nucleotide sequence of HT064 is shown in SEQ ID NO.17; The nucleotide sequence of HT073 is shown in SEQ ID NO.18; The nucleotide sequence of HT079 is shown in SEQ ID NO.19; The nucleotide sequence of HT095 is shown in SEQ ID NO.

20.

2. A primer set for specific amplification of SSR markers of Psoralea corylifolia, characterized in that: The SSR marker of Psoralea corylifolia is the SSR marker according to claim 1; the primer set comprises 20 pairs of SSR marker core primer pairs and M13 primers with fluorescent labels; wherein: The SSR marker core primer pair used for specific amplification of HT005 consists of primers with sequences shown in SEQ ID NO. 22 and SEQ ID NO. 23, respectively; The SSR marker core primer pair used for specific amplification of HT007 consists of primers with sequences shown as SEQ ID NO. 24 and SEQ ID NO. 25, respectively; The SSR marker core primer pair used for specific amplification of HT011 consists of primers with sequences shown as SEQ ID NO. 26 and SEQ ID NO. 27, respectively; The SSR marker core primer pair used for specific amplification of HT012 consists of primers with sequences shown in SEQ ID NO. 28 and SEQ ID NO. 29, respectively; The SSR marker core primer pair used for specific amplification of HT015 consists of primers with sequences shown as SEQ ID NO.30 and SEQ ID NO.31, respectively; The SSR marker core primer pair used for specific amplification of HT018 consists of primers with sequences shown as SEQ ID NO.32 and SEQ ID NO.33, respectively; The core primer pair for the SSR marker used for specific amplification of HT023 consists of primers with sequences shown in SEQ ID NO. 34 and SEQ ID NO. 35, respectively; The SSR marker core primer pair for specific amplification of HT029 consists of primers with sequences shown as SEQ ID NO. 36 and SEQ ID NO. 37, respectively; The SSR marker core primer pair used for specific amplification of HT031 consists of primers with sequences shown in SEQ ID NO. 38 and SEQ ID NO. 39, respectively; The SSR marker core primer pair used for specific amplification of HT036 consists of primers with sequences shown as SEQ ID NO.40 and SEQ ID NO.41, respectively; The SSR marker core primer pair for specific amplification of HT039 consists of primers with sequences shown in SEQ ID NO.42 and SEQ ID NO.43, respectively; The SSR marker core primer pair used for specific amplification of HT041 consists of primers with sequences shown in SEQ ID NO. 44 and SEQ ID NO. 45, respectively; The SSR marker core primer pair used for specific amplification of HT046 consists of primers with sequences shown as SEQ ID NO.46 and SEQ ID NO.47, respectively; The SSR marker core primer pair used for specific amplification of HT048 consists of primers with sequences shown as SEQ ID NO.48 and SEQ ID NO.49, respectively; The SSR marker core primer pair used for specific amplification of HT057 consists of primers with sequences shown as SEQ ID NO.50 and SEQ ID NO.51, respectively; The SSR marker core primer pair used for specific amplification of HT062 consists of primers with sequences shown as SEQ ID NO.52 and SEQ ID NO.53, respectively; The SSR marker core primer pair used for specific amplification of HT064 consists of primers with sequences shown as SEQ ID NO.54 and SEQ ID NO.55, respectively; The SSR marker core primer pair used for specific amplification of HT073 consists of primers with sequences shown as SEQ ID NO.56 and SEQ ID NO.57, respectively; The SSR marker core primer pair used for specific amplification of HT079 consists of primers with sequences shown as SEQ ID NO.58 and SEQ ID NO.59, respectively; The SSR marker core primer pair used for specific amplification of HT095 consists of primers with sequences shown as SEQ ID NO.60 and SEQ ID NO.61, respectively; In each pair of SSR marker core primers, the even-numbered ones are forward primers and the odd-numbered ones are reverse primers. The 5' end of each forward primer is connected to an M13 sequence, which is identical to the sequence of the M13 primer; the sequence of the M13 primer is shown in SEQ ID NO.

21.

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

4. A method for analyzing the genetic diversity of Phoebe yunnanensis population, characterized in that: Follow these steps: S1. Extract genomic DNA from each individual in the Yunnan Fengchui Nan population; S2. Obtain the genotype of the SSR locus corresponding to the SSR marker of claim 1 in the genomic DNA of each strain of Phoebe yunnanensis, and obtain the SSR typing result; S3. Calculate the genetic diversity parameters of the Yunnan Fengchui population based on the SSR typing results.

5. The analysis method according to claim 4, characterized in that In step S2, the primer set as described in claim 2 is used to perform PCR amplification on the genomic DNA of each individual strain of Pseudostellaria yunnanensis, and the length polymorphism of the PCR amplification product is detected to obtain the genotype of the genomic DNA of each strain of Pseudostellaria yunnanensis at the SSR site.

6. The analysis method according to claim 5, characterized in that In step S2, for each of the SSR sites in the genomic DNA of each of the Psoralea corylifolia plants, a PCR reaction system is prepared using the SSR marker core primer pair corresponding to the SSR site in the primer set of claim 2 to perform PCR amplification; Each PCR reaction system contained: 5 ng of genomic DNA of P. yunnanensis as a DNA template, 0.5 µM M13 primer, 0.5 µM forward primer, 0.5 µM reverse primer, 150 µM dNTPs, 2.0 µM MgCl2, 0.25 U Taq DNA polymerase, and 10 µL 1× PCR buffer; the volume of each PCR reaction system was 10 µL.

7. The analysis method according to claim 5, characterized in that In step S2, the reaction procedure for PCR amplification is: pre-denaturation at 94°C for 4 minutes; denaturation at 94°C for 30 seconds, annealing at 50-55°C for 30 seconds, and extension at 72°C for 30 seconds, for a total of 31 cycles; and final extension at 72°C for 10 minutes.

8. The analysis method according to claim 4, characterized in that In step S3, the genetic diversity parameters include the number of alleles, observed heterozygosity, expected heterozygosity, Shannon-Wiener diversity index and fixation index.

9. A method for constructing a fingerprint of Phoebe yunnanensis germplasm, characterized in that: The method is as follows: using a method of detecting length polymorphism of PCR products after PCR amplification, the genotypes of the SSR loci corresponding to the SSR markers HT005, HT011, HT036, HT039, HT046, HT048, HT062 and HT079 in the genomic DNA of the to-be-detected Phoebe yunnanensis are obtained, and a fingerprint of the to-be-detected Phoebe yunnanensis is constructed according to the genotypes of the SSR loci; When performing PCR amplification, for the SSR sites of the genomic DNA of Psoralea corylifolia to be detected, corresponding SSR marker core primer pairs are used to prepare PCR reaction systems for each of the SSR sites and perform PCR amplification; The SSR marker core primer pair is an SSR marker core primer pair in the primer set according to claim 2, which is used for specifically amplifying HT005, HT011, HT036, HT039, HT046, HT048, HT062 and HT079.

10. Application of SSR markers of Psoralea corylifolia, characterized in that: The application is to use the SSR marker according to claim 1 for any of the following: (1) Variety identification of Phoebe yunnanensis germplasm; (2) Molecular marker-assisted breeding of Phoebe yunnanensis.

Images