Polymorphic microsatellite molecular marker primer for spirodela polyrhiza and application of polymorphic microsatellite molecular marker primer

Abstract

The invention belongs to the field of plant molecular genetics, and relates to a spirodela polymorphic microsatellite molecular marker primer and application thereof. The kit comprises 10 pairs of microsatellite primers which form 5 groups of duplex PCR systems, and the kit can be applied to population genetic diversity analysis of spirodela polyrhiza. The population genetic diversity analysis comprises the following steps: collecting a sample of spirodela polyrhiza to be identified; extracting spirodela polyrhiza genome DNA from the spirodela polyrhiza sample; taking the extracted DNA as a template, and carrying out PCR (Polymerase Chain Reaction) amplification on the DNA template by using the screened 10 pairs of microsatellite primers; carrying out electrophoresis detection on a PCR product, and then carrying out silver staining; the silver staining result is digitally analyzed, the UPGMA clustering analysis chart is drawn by genetic analysis software, the clustering analysis chart of the spirodela polyrhiza is identified according to the clustering analysis result, and the method has huge popularization and utilization value.

Description

Technical Field

[0001] The present invention belongs to the field of animal molecular genetics, and particularly relates to primers of polymorphic microsatellite molecular markers of Spirodela polyrhiza and their applications. Background Art

[0002] Spirodela polyrhiza is widely distributed in tropical and temperate regions of the world and is distributed in all provinces of China, both in the north and the south. It is commonly found in paddy fields, ponds, lake bays, and ditches. The thallus of Spirodela polyrhiza is flat, often purple on the back, and the thallus is broadly obovate. Spirodela polyrhiza mainly reproduces asexually. When environmental conditions are suitable, one generation can be cloned in 2 days, resulting in relatively low genetic diversity of its population. Spirodela polyrhiza can grow rapidly in sewage containing organic matter and also has strong absorption and utilization abilities for nitrogen, phosphorus, heavy metals, and some specific pollutants, and can be used to treat eutrophic water bodies and various sewage.

[0003] Studies have found that the study of Spirodela polyrhiza genetic diversity can evaluate the degradation degree of this species in a region or a population, and further evaluate the growth status and adaptability of Spirodela polyrhiza in this region, providing reference value for water ecological restoration. Microsatellite DNA, also known as short tandem repeats (STR) and simple sequence repeat (SSR), refers to simple repeat sequences less than 10 nucleotides in the DNA genome, which are widely present in eukaryotic genomes. Most of them are sequences with 2 - 6 bases as the core unit and arranged in tandem repeats. SSR can be used to construct genetic maps, select genetic markers, establish DNA fingerprints, for variety identification, or use marker-assisted selection to accelerate the breeding speed. In addition, SSR markers have other uses, such as for gene mapping, QTL analysis, pedigree analysis, identification of genetic relationships, analysis of genetic distance between populations, and research on evolution and genetic diversity. There is no report on the genetic diversity of Spirodela polyrhiza, and a method for evaluating the genetic diversity of Spirodela polyrhiza is urgently needed. Summary of the Invention

[0004] In order to solve the above technical problems existing in the background art, the present invention provides primers of polymorphic microsatellite molecular markers of Spirodela polyrhiza and their applications.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] On the one hand, the present invention provides primers of polymorphic microsatellite molecular markers of Spirodela polyrhiza, including 10 pairs. The sequences and annealing temperatures of the primers are respectively:

[0007]

[0008]

[0009] Among them, primer pair ZP1 is used in combination with primer pair ZP2; primer pair ZP3 is used in combination with primer pair ZP4; primer ZP5 is used in combination with primer pair ZP6; primer pair ZP7 is used in combination with primer pair ZP8; primer pair ZP9 is used in combination with primer pair ZP10.

[0010] In a second aspect, the present invention also provides the use of primers of Spirodela polyrhiza polymorphic microsatellite molecular markers in identifying and distinguishing the genetic diversity of Spirodela polyrhiza populations. The application method includes the following steps:

[0011] (1) Extract Spirodela polyrhiza genomic DNA;

[0012] (2) Using the Spirodela polyrhiza genomic DNA obtained in step (1) as a template, perform PCR amplification with the above 5 sets of duplex PCR primers to obtain amplification products;

[0013] (3) Electrophorese and silver stain the amplification products obtained in step (2) using polyacrylamide gel;

[0014] (4) Analyze the silver staining results in step (3) through genetic analysis software, measure the genetic distance between each individual, draw a UPGMA cluster analysis diagram based on the genetic distance, distinguish the genetic relationships of different Spirodela polyrhiza populations according to the clustering results, and identify and distinguish the genetic diversity of Spirodela polyrhiza populations.

[0015] Among them, the reaction system for PCR amplification in step (2) is 25 μL: 3 μL of 10×PCR Buffer, 1 μL of 2.5 mmol / L dNTP, 3 μL of 2 mmol / L MgCl2, 1 μL each of the upstream and downstream primers of the two primer pairs, 0.5 μL of 0.5 U / μL Taq enzyme, 2 μL of DNA template, and 11.5 μL of ultrapure water.

[0016] The PCR amplification reaction procedure in step (2) is: pre-denaturation at 94°C for 3 minutes; then denaturation at 94°C for 30 seconds, annealing at 56°C for 30 seconds, extension at 72°C for 45 seconds, for a total of 35 cycles; finally, extension at 72°C for 10 minutes and storage at 4°C.

[0017] In step (3), the PCR products are electrophoresed and silver stained using 12% polyacrylamide gel.

[0018] The genetic analysis software in step (4) can be MEGA software.

[0019] In a third aspect, the present invention also provides a kit for analyzing the genetic diversity of Spirodela polyrhiza populations, which includes at least the above 10 pairs of primers.

[0020] Compared with the prior art, the advantages and beneficial effects of the present invention include:

[0021] The present invention can rapidly evaluate the genetic diversity of Spirodela polyrhiza. This method uses a dual PCR system developed for analyzing the genetic diversity of Spirodela polyrhiza. Compared with the traditional PCR system for analyzing the genetic diversity of species by microsatellites, this system can save about 50% of reagents and time. This method first proposes a method for evaluating the genetic relationship of Spirodela polyrhiza, providing technical support for analyzing the relationship between Spirodela polyrhiza in different regions and populations; it helps to quickly and accurately identify Spirodela polyrhiza of different families. The present invention can rapidly identify the genetic relationship of Spirodela polyrhiza, and then judge the growth status of Spirodela polyrhiza, providing a theoretical basis and reference value for evaluating the disaster of Spirodela polyrhiza flooding and its effective utilization in water ecological restoration. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a UPGMA cluster analysis diagram of Spirodela polyrhiza in the embodiment, where the numbers 1-18 correspond to the 1-18 samples in the embodiment;

[0023] Figure 2 It is the silver staining result of dual PCR amplification in the embodiment. DETAILED DESCRIPTION OF THE INVENTION

[0024] The reagents used in the embodiment include:

[0025] Buffer LP1, buffer LP2, buffer LP3, wash buffer PW, adsorption column CB3, and elution buffer TE are all from the Tiangen new plant genomic DNA extraction kit of Tiangen Biochemical Technology Co., Ltd.

[0026] (1) Selection of Spirodela polyrhiza samples:

[0027] Randomly select 6 Spirodela polyrhiza plants in the Xuanwu Lake waters in Nanjing (the samples are labeled 1-6), randomly select 6 Spirodela polyrhiza plants in the Xili Lake waters in Shenzhen (the samples are labeled 7-12), and randomly select 6 Spirodela polyrhiza plants in the Tangxun Lake waters in Wuhan (the samples are labeled 13-18).

[0028] (2) Extraction of Spirodela polyrhiza genomic DNA:

[0029] The genomic DNA of Spirodela polyrhiza is extracted using the Tiangen new plant genomic DNA extraction kit (Tiangen Biochemical Technology Co., Ltd., Beijing). The specific operation process is as follows:

[0030] 2.1. Use forceps to take about 100 mg of the dry plant tissue of the Spirodela polyrhiza sample obtained in step (1) into a 2 mL centrifuge tube (to avoid cross - contamination, the forceps for taking plant tissue again need to be burned on an alcohol lamp). Add two quartz sands, place it on a shaker for pulverization, shake for 1 minute in both forward and reverse directions until the plant tissue becomes powdery. After centrifuging for 30 s, add 500 μL of buffer LP1 and 6 μL of RNase A (10 mg / mL), vortex for 1 minute (check if it adheres to the wall, if so, add another 100 μL of buffer LP1), incubate in a dry bath at 65 °C with shaking at 300 rpm for 10 minutes, and then place it in an ice bath for 2 minutes.

[0031] 2.2. Add 150 μL of buffer LP2, vortex for 1 minute to mix well, centrifuge at 12000 rpm for 5 minutes, and transfer the supernatant (take 150 μL each time, take it twice) to a new 2 mL centrifuge tube.

[0032] 2.3. Add 450 μL of buffer LP3 (1.5 times the volume of the supernatant), vortex for 15 s.

[0033] 2.4. Place the adsorption column CB3 into the collection tube, pour all the product obtained in 2.3 into the adsorption column CB3, centrifuge at 12000 rpm for 1 minute in a centrifuge, and retain the adsorption column CB3 in the collection tube.

[0034] 2.5. Add 600 μL of wash buffer PW to the adsorption column CB3 retained in the collection tube in 2.4 to wash the DNA, centrifuge at 12000 rpm for 1 minute in a centrifuge, and retain the adsorption column CB3 in the collection tube.

[0035] 2.6. Repeat the process of washing with wash buffer PW in 2.5 (if the filter membrane of the adsorption column CB3 shows green, then wash it once with 500 μL of absolute ethanol, and the washing process is the same as that with wash buffer PW) until the filter membrane of the adsorption column CB3 shows colorless.

[0036] 2.7. After centrifuging the collection tube processed in 2.6 at 12000 rpm for 2 minutes, open the lid of the adsorption column CB3 and place it horizontally to dry. It can be directly placed at room temperature for 30 minutes or placed in an oven at 32 °C for 10 minutes to completely dry the residual wash buffer in the adsorption column CB3. (It is best when the adsorption membrane is slightly wrinkled and almost no alcohol smell can be smelled when getting close.)

[0037] 2.8. Collect the Spirodela polyrhiza genomic DNA in the adsorption column CB3 into a centrifuge tube through the elution buffer (TE) (ensure that its pH value is within the range of 7.0 - 8.5), and obtain 18 Spirodela polyrhiza genomic DNAs.

[0038] Before amplification, the following steps are also required to detect and preserve the 18 Spirodela polyrhiza genomic DNAs obtained above:

[0039] a. Detect whether the extraction is successful by 1% agarose gel electrophoresis, and determine its concentration by a micro-spectrophotometer.

[0040] b. Dilute the Spirodela polyrhiza genomic DNA 5-fold according to the experimental requirements as a working solution and store it at 4°C in the refrigerator for standby. The remaining mother liquor is stored at -20°C in the refrigerator for long-term preservation.

[0041] (3) Amplification reaction and electrophoresis detection of Spirodela polyrhiza PCR products:

[0042] 3.1 Obtaining microsatellite primers

[0043] Use microsatellite search software to search for microsatellite fragments in the Spirodela polyrhiza genome and the transcriptome obtained by the applicant's previous sequencing. Select 500 microsatellite molecular marker sequences with a repeat unit of more than 3 bases for the fragments containing microsatellites. Use Primer 6 software and conventional design methods to design microsatellite primers, and a total of 500 pairs of primers are designed and sent to the primer company for synthesis.

[0044] 3.2 Microsatellite primer screening

[0045] Randomly select 6 out of the 18 Spirodela polyrhiza genomic DNAs obtained in step 2.8 as templates to preliminarily screen the microsatellite primers synthesized in step 3.1, and select microsatellite molecular marker sequences with high polymorphism, few deletions, and stable amplified bands;

[0046] The reaction system for PCR amplification is 25 μL: 3 μL of 10×PCR Buffer, 1 μL of 2.5 mmol / L dNTP, 3 μL of 2 mmol / L MgCl2, 1 μL of each upstream and downstream primer, 0.5 μL of 0.5 U / μL Taq enzyme, 2 μL of DNA template, and 13.5 μL of ultrapure water.

[0047] The PCR amplification reaction program is: pre-denaturation at 94°C for 3 minutes; then denaturation at 94°C for 30 seconds, annealing at 56°C for 30 seconds, extension at 72°C for 45 seconds, for a total of 35 cycles; finally, extension at 72°C for 10 minutes and storage at 4°C.

[0048] 3.3 Detection of primer universality at polymorphic sites

[0049] Based on the microsatellite molecular markers with high polymorphism, few deletions, and stable amplified bands obtained in step 3.2, use the 18 Spirodela polyrhiza genomic DNAs obtained in step 2.8 to perform PCR amplification again, and the amplified products obtained are used for the next step to evaluate the microsatellite markers.

[0050] Among them, the reaction system for PCR amplification is 25 μL: 3 μL of 10×PCR Buffer, 1 μL of 2.5 mmol / L dNTP, 3 μL of 2 mmol / L MgCl2, 1 μL each of the upstream and downstream primers, 0.5 μL of 0.5 U / μL Taq enzyme, 2 μL of DNA template, and 13.5 μL of ultrapure water.

[0051] The PCR amplification reaction program is as follows: pre-denaturation at 94°C for 3 minutes; then denaturation at 94°C for 30 seconds, annealing at 56°C for 30 seconds, extension at 72°C for 45 seconds, for a total of 35 cycles; finally, extension at 72°C for 10 minutes and storage at 4°C.

[0052] 3.4 Dual PCR amplification

[0053] After the amplification of the genomic DNA of 18 Spirodela polyrhiza based on Step 3.3, microsatellite molecular markers with good polymorphism, few deletions, and stability can still be amplified for further screening. The specific screening method is as follows: randomly combine the microsatellite primers screened in Step 3.3 in pairs to form a dual PCR primer set, use the dual PCR amplification system to amplify the DNA of the above 18 samples, and screen out a dual PCR system for microsatellite molecular markers that can stably amplify with good polymorphism, few deletions, and stable amplified bands for all 18 samples. Finally, the following microsatellite molecular markers are selected:

[0054] The microsatellite molecular marker corresponding to primer pair ZP1 is:

[0055] tagttctccacatgtacgaagtgtattgcagacgggacttagcagttggtctccttactcccc tgcaa cgaggaaaggtagt cccctgaaatgcatccaagacttcagtcaatgtttgtgtgacagaacgttgtccaatgacttaaacaaatagatagatagatagatagatagatagagatagagagagggagggacagagagagagagaggacagaagagtaccactgtgtcccttattcatgatgttaaaaaaacctctttcaagaaaa actcaccagcctccactc ctggagaagtagtcacagtcgagagagatagccagggc

[0056] The microsatellite molecular marker corresponding to primer pair ZP2 is:

[0057] cacattcctgcgcaacggcttcacacggtagtcagctactccaagtctcaagcacttgaccacgaga gaaacttcatctttagctgacatcactgtgaatccaattatcaaaggagaagtcaactaaaagtcaaacaaaagtagataggcagatagatagatagatagattgagagagagagagaggaagagagagagagagagagagagagacgattgacttactgactattgga atatgccttagttcctcca tccccattatgtatattttttttttttttgaaagaagatagagagccactcgaaaacagtggccc

[0058] The microsatellite molecular marker corresponding to primer pair ZP3 is:

[0059] aattagcgggagaaagtcagaggcttcgcttaccagggagagaaaaatgtac acggagaagacaacca gacc aaactggactaccgcgtagaggaatacagtccgcggcctactcgcttcggcatccgagagagagatagatagatagatagacagacagacagacagacagacagagacaaagggattactgagatgctcataagaaagcatttatctgatgcacgggagctttacccatggcggcggcggtgaaggaggataagagacccagaagaacgccgcaaggcagtgataccgccgtcgcc atcgtgccctttcggg

[0060] The microsatellite molecular marker corresponding to primer pair ZP4 is:

[0061] agggagggaggggagggagggagggaggg aggggagggagggtgagacg gggcgggcgagacagggagggagggagggcggagagtgaggcgggggagggagggcgtgcgagacggtgtggggagggagggaggggggagagtgaggcgggggagggagggagggagggaggggagggagggaggggagggagggagggaggggagggagggagggcgggagagagagagagagagagagagagagagagagagaggctggaatgacgtggctgccacatcgttccactctt agcc ggctgccacgtggactctca

[0062] The microsatellite molecular marker corresponding to primer pair ZP5 is:

[0063] tctataagaggggaagggacccccacaaaccctagccgtcatttgagaaaaaaagaaggagtctcctccatctc gagtgatctgctcacggaaggc cggggaaggagtccaaccgccgtcgccgccgcctaccgcgcgccgcacgctcgtgccgccgccgccgccgcgagctgaccgct gccactccttgcctcgcca cgctgatatccctcgcatgttgctccagccgccgccgctgccgccgccgcctcagccgccaccacccgccgccgcgcgggacgcagctccgccatcctccggccatccccctccacg

[0064] The microsatellite molecular marker corresponding to primer pair ZP6 is:

[0065] aagtgggcactttcctctcttgacccaccatgggctctataagaggggaagggacccccccacaaaccctagctgtcatttgagaaaaaaggagtttcctct cgagtggctgctcaaggaaggtg agggaaggggtccaaccgccgacgtcgccgccgccgccgccgcactgccgtgccgccgctgcgccgccacctccgccgc caccccttgccccgac ccagta tcgacttcgcgcgcgcctgctgcgccacgccacctgcgcgcccagctgctgcagccgccgccgcgatcgccgccgccgccccatcgcgccgc

[0066] The microsatellite molecular marker corresponding to primer pair ZP7 is:

[0067] aaaaatcg gggaagcggatgacgaagaggggggctggaggaaaatgtaggaatggcaaaggggaggatcgcggaggggacgcgtggagcaagatcgggaggaggagctgcagatgcggggtagcggtgacgatgcgaggacatcggtagtggcggcggcggcggcggtggcggcggcatcaatagtgaagttcttgtggccggggcaaggagtggcggcggttgtcacggcggcagtggtggcgccacggaggcgtgcgacaacggcgactgcagttggactcc ttctttggcctt ccgtgagca gccactcgag

[0068] The microsatellite molecular marker corresponding to primer pair ZP8 is:

[0069] ccctttctcttccttctctcgctattcccttctccctctctccctctttcgctctccgtcgcgaggagagattcactcctctcctggcacgcg cgcgagtgcgagcgtaa gggggaggaggaggaaaagttcttcctcctccacgacgtaaggaggaggaggaggaaaagctcttcctctcctcgcgacggaaattgcttctgtcgcgaggcgaccgcaaggaggacaccattctcctctctccctctctcttcgtgacgtacgtcgcaaggagaggcgggaggag ttctccactt ctcctcctcttctt tcttc

[0070] The microsatellite molecular marker corresponding to primer pair ZP9 is:

[0071] cccctctcggcc cattatgggctctataagaggagaaggg acccccccccccccccacaaaccctagctgtcatttgaagaaagaaaggagtgatctccctcaaaattggctgctcatggaaggccgcggaaggagtccaaccgtcgtcaccgccgccgccgccgcctctgcacgcatccgtgctgccgccaccgccgccgtgacgaccgctgtcactccttgccccggccacgggt tcttctccactgtcatcccgacgtcgccatcctcaagccgccgccgccactgctgtcctcaagccgccgccgccgcct

[0072] The microsatellite molecular marker corresponding to primer pair ZP10 is:

[0073] ggcc cggggttgacggaaaatggtaga gagagagaaggggtggaggtccgtggaggggaagagaggtggaagccgggctcgaggagggggaatgaacggaggatggcggtgctgcgatccggcaatggcgcggcggcggcagcggcctgaggcggcggcggcggcggccccgaggctggcgcggcgcaggaaggcggcaggatgata tatagcgaccaag cgcaaggga ggcggcggtctcggcggcagggcacggctgcggcatcgtggtagtgcggcagcgatggcggcagcggaggcggtggttggtctccttcc

[0074] The reaction system for PCR amplification is 25 μL: 3 μL of 10×PCR Buffer, 1 μL of 2.5 mmol / L dNTP, 3 μL of 2 mmol / L MgCl2, 1 μL each of the upstream and downstream primers of the two primer pairs, 0.5 μL of 0.5 U / μL Taq enzyme, 2 μL of DNA template, and 11.5 μL of ultrapure water.

[0075] The PCR amplification reaction program is: pre-denaturation at 94°C for 3 minutes; then denaturation at 94°C for 30 seconds, annealing at 56°C for 30 seconds, extension at 72°C for 45 seconds, for a total of 35 cycles; finally, extension at 72°C for 10 minutes and preservation at 4°C.

[0076] 3.5 Detection and identification

[0077] Electrophoretically separate the above-mentioned Spirodela polyrhiza PCR products using a 12% non-denaturing polyacrylamide gel, and take a photo after silver staining.

[0078] Finally, 10 pairs of better microsatellite marker primers were screened out. Among them, primer pair ZP1 is used in combination with primer pair ZP2; primer pair ZP3 is used in combination with primer pair ZP4; primer ZP5 is used in combination with primer pair ZP6; primer pair ZP7 is used in combination with primer pair ZP8; primer pair ZP9 is used in combination with primer pair ZP10. The primer information is shown in Table 1.

[0079] Table 1 Basic information of 10 pairs of Spirodela polyrhiza microsatellite primers

[0080]

[0081]

[0082] (4) Genetic structure analysis

[0083] Digitally process the silver staining results of the duplex PCR amplification obtained in step (3) (read the PCR products using Gene Marker software). Experimental results (the silver staining results are as Figure 2 shown), and at the same time construct a UPGMA cluster analysis diagram through the software MEGA. The results are as Figure 1 shown. These 18 strains of Spirodela polyrhiza are divided into three branches. Among them, 1-6 are clustered into one branch; 7-12 are clustered into one branch; 13-18 are clustered into one branch. The results of this cluster analysis diagram are consistent with the analysis results of the Gene Marker software (1-6 are clustered into one branch, indicating that the Spirodela polyrhiza in the Nanjing water area is relatively closely related to the Spirodela polyrhiza in other water areas; 7-12 are clustered into one branch, indicating that the Spirodela polyrhiza in the Shenzhen water area is relatively closely related to the Spirodela polyrhiza in other water areas; 13-18 are clustered into one branch, indicating that the Spirodela polyrhiza in the Wuhan water area is relatively closely related to the Spirodela polyrhiza in other water areas. This technology can accurately determine the genetic relationship of Spirodela polyrhiza). It shows that the 10 pairs of Spirodela polyrhiza microsatellite primers provided by the present invention can evaluate the genetic relationship of Spirodela polyrhiza, providing a new technical method and means for studying the genetic diversity and genetic relationship analysis of Spirodela polyrhiza.

Claims

1. Primers for polymorphic microsatellite molecular markers of Spirodendrum truncatum, characterized in that: The primers have 10 pairs, forming 5 groups, for double PCR reaction. The information of the primers is as follows:

2. The primer of the polymorphic microsatellite molecular marker of Spirodela according to claim 1, characterized in that: The primer pair ZP1 is used in combination with the primer pair ZP2; the primer pair ZP3 is used in combination with the primer pair ZP4; the primer pair ZP5 is used in combination with the primer pair ZP6; the primer pair ZP7 is used in combination with the primer pair ZP8; and the primer pair ZP9 is used in combination with the primer pair ZP10.

3. Use of the primers according to claim 1 or 2 in identifying and distinguishing the genetic diversity of Spirodendrum truncatum populations.

4. The application method of the primer according to claim 1 or 2, characterized in that: The following steps are involved: (1) Extracting genomic DNA from different populations of Spirodendrum aviculare; (2) using the Spirodela genomic DNA obtained in step (1) as a template, and performing PCR amplification using the five sets of dual PCR primer pairs described in claim 1 to obtain an amplified product; (3) electrophoresis and silver staining of the amplified product obtained in step (2) using polyacrylamide gel; (4) The silver staining results of step (3) are analyzed using genetic analysis software to measure the genetic distance between each individual, a UPGMA cluster analysis diagram is drawn based on the genetic distance, and the genetic diversity of the Spirodendrum truncatum population is identified and differentiated based on the clustering results.

5. The application method according to claim 4, characterized in that: The reaction system for PCR amplification in step (2) is 25 μL: 3 μL of 10×PCR Buffer, 1 μL of 2.5 mmol / L dNTP, 3 μL of 2 mmol / L MgCl2, 1 μL of each of the upstream and downstream primers of the two pairs of primers, 0.5 μL of 0.5 U / μL Taq enzyme, 2 μL of DNA template, and 11.5 μL of ultrapure water.

6. The application method according to claim 4, characterized in that: The PCR amplification reaction procedure in step (2) is: pre-denaturation at 94°C for 3 minutes; then denaturation at 94°C for 30 seconds, annealing at 56°C for 30 seconds, and extension at 72°C for 45 seconds, for a total of 35 cycles; finally extension at 72°C for 10 minutes and storage at 4°C.

7. The application method according to claim 4, characterized in that: In step (3), the PCR product was electrophoresed using 12% polyacrylamide gel and silver stained.

8. The application method according to claim 4, characterized in that: The genetic analysis software described in step (4) is MEGA software.

9. A kit for analyzing the genetic diversity of Spirodendrum truncatum population, characterized in that: The kit at least comprises the primers according to claim 1 or 2.

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