SSR (Simple Sequence Repeat) molecular marker combination of cycas formosana complex, primer group and application of SSR molecular marker combination and primer group

Abstract

The invention belongs to the technical field of biology, and relates to identification of a cycas formosana complex. The invention particularly relates to an SSR molecular marker combination of a Taiwan cycas complex, a primer group and application. Five pairs of SSR molecular marker primers are obtained through transcriptome sequencing, SSR site excavation, SSR primer design, SSR-PCR reaction system establishment, SSR-PCR amplification and effectiveness detection by taking cycas revoluta as a material. The primer provided by the invention is rich in polymorphism, high in stability and clear in amplification band, and can be used for species identification, genetic lineage analysis, genetic diversity analysis, germplasm resource protection and auxiliary breeding of the cycas formosana complex.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a Taiwan Cycas revoluta complex SSR marker primer pair developed based on transcriptome sequences and an application thereof. Background Art

[0002] Climate change and geological events have a significant impact on the geographical distribution, speciation and evolutionary history of species. Historical processes (such as the Ice Age) have left a deep mark on the genetic structure and population dynamics of existing plants. Cycads are the oldest gymnosperms and have important scientific research, ecological and economic value. Cycads have beautiful tree shapes, shiny pinnate leaves, and are evergreen all year round. They are precious horticultural ornamental plants and are often made into large potted plants. They are deeply loved by people and have important ornamental value. The stems and seeds of cycads are rich in starch and can be eaten. Cycads also have medicinal value, and their seeds have the effects of curing dysentery, relieving cough and stopping bleeding. At present, cycads around the world are under the dual threat of climate change and human activities, among which Cycas ( Cycas ) plants are particularly serious. Cycas ( Cycas ) is the only cycad plant with natural distribution in China, with about 20 species, most of which are endemic to China. China's cycad plants are mainly distributed in the southwest and southeast coast. Most species are narrowly distributed groups. Their survival and reproduction are seriously threatened, and they are all listed as national first-class key protected wild plants. Taiwan Cycad Complex ( Cycas taiwaniana The Taiwan Cycad complex is a group of cycads distributed in Guangdong, Fujian, and Hainan, China. They are very similar in morphology and difficult to distinguish. There are many disputes in their systematic classification. Cycas taiwaniana Carruthers), Hainan Cycas ( Cycas hainanensis CJ Chen), Cycas revoluta ( Cycas changjiangensis N. Liu)、Cycas revoluta( Cycas lingshuigensis GA Fu), Sichuan Cycas ( Cycas szechuanensis Cheng et LK Fu) and Cycas sylvatica ( Cycas fairylakea DY Wang). Due to habitat loss and over-harvesting, the wild population of the Taiwan Cycad complex is decreasing rapidly, and the genetic diversity within the species is constantly being lost. All six species are critically endangered or endangered and are in urgent need of protection. However, the lack of genetic and differentiation data between species and populations has restricted the effective protection and management of the complex.

[0003] Clarifying the species definition and genetic background of the Cycas taiwaniana complex is a prerequisite for the reasonable protection and utilization of this resource. Genetic diversity has crucial application value in conservation biology and provides an important theoretical basis for the protection and breeding of endangered species. In recent years, with the continuous development of DNA sequencing technology and the decreasing sequencing cost, molecular marker detection technology has become increasingly perfect and has become an effective means for studying the genetic diversity of plant species. SSR, namely Simple Sequence Repeat or Microsatellite, is a simple repeat sequence evenly distributed in the eukaryotic genome, consisting of tandem repeat fragments of 2-6 nucleotides. The length polymorphism of tandem repeat sequences mainly stems from the different number of repeats. The markers are randomly distributed in the genome, co-dominant, with high abundance and high polymorphism. The Simple Sequence Repeat markers (SSR) developed based on transcriptome data have significant advantages compared with traditional development methods. The development of traditional SSR markers has limitations such as cumbersome operation steps, large human input, and high economic costs. In contrast, the primer pair information of SSR markers derived from the transcriptome is more comprehensive, has better universality, and higher efficiency. It can also specifically locate the transcriptionally active regions of the genome. This characteristic enables SSR markers to show stronger detection sensitivity in genetic diversity research. After the functions of related genes are verified, they can be directly applied to the molecular marker-assisted breeding system. More importantly, this method significantly increases the linkage probability between markers and trait regulatory genes and provides an effective tool for analyzing genetic mechanisms. Therefore, enriching the specific primer database of the Cycas taiwaniana complex is of great significance for its protection. However, the microsatellite molecular markers available for the Cycas taiwaniana complex are still relatively few. Therefore, the variety identification of the Cycas taiwaniana complex still stays at naming by region. To identify the Cycas taiwaniana complex from a genetic perspective, our research group has conducted in-depth research. Summary of the Invention

[0004] To solve the above technical problems, the present invention provides a combination of SSR molecular markers, a primer set, and their applications for the Cycas taiwaniana complex.

[0005] The technical solution of the present invention is realized as follows: The combination of SSR molecular markers for the Cycas taiwaniana complex includes SSR11, SSR33, SSR37, SSR42, and SSR50 molecular markers.

[0006] A primer set for identifying the above combination of SSR molecular markers for the Cycas taiwaniana complex: SSR11 primer SEQ ID No.1: SSR11-F: 5’- TGGGACGGTGACACTCTTG -3’; SEQ ID No.2: SSR11-R: 5'-TGACTTCGCGGTGGTACG-3'; SSR33 primer SEQ ID No.3: SSR33-F: 5'-AGCGGATCCCGTTATAGCC-3'; SEQ ID No.4: SSR33-R: 5'-TTTCGCCAACGGTTTCCTG-3'; SSR37 primer SEQ ID No.5: SSR37-F: 5'-TCCTCGGGTCAATACGTGG-3'; SEQ ID No.6: SSR37-R: 5'-CGACTTCATGTGGATGCGG-3'; SSR42 primer SEQ ID No.7: SSR42-F: 5'-TGTCAAAGCCGCTCAATCC-3'; SEQ ID No.8: SSR42-R: 5'-GGAATGGCCAAGAAGTGCC-3'; SSR50 primer SEQ ID No.9: SSR50-F: 5'-ATTCGGTATGCTTTCCAC-3'; SEQ ID No.10: SSR50-R: 5'-AGGTAATCATTTCCCACA-3'.

[0007] The kit containing the above primer sets further includes detection reagents.

[0008] A method for identifying the monophyletic species of Cycas taiwaniana complex, said method being based on the above SSR molecular marker combination of Cycas taiwaniana complex; The steps are as follows: (1) Extract the total DNA of the Cycas taiwaniana complex to be tested; (2) Using the total DNA in step (1) as a template, perform PCR amplification with the primer sets described in claim 2, and recover the amplification products of each primer set; (3) Perform polyacrylamide gel electrophoresis on the PCR products obtained in step (2); (4) Organize and analyze the polymorphic bands amplified in step (3) to obtain genetic diversity parameters, and then perform cluster analysis and comparison with 18 populations to determine the monophyletic species and populations of the Cycas taiwaniana complex to be tested.

[0009] The PCR amplification system in the above step (2) is: 20 μl, including 5 μl of DNA template, 2 μl of 10× buffer, 0.4 μl of dNTP, 0.6 μl of forward and reverse primers, 0.2 μl of Taq DNA polymerase, and 11.2 μl of sterilized ultrapure water.

[0010] The PCR amplification program is as follows: pre-denaturation at 95 °C for 4 min; denaturation at 95 °C for 30 s, annealing at 50 - 56 °C for 30 s, extension at 72 °C for 1 min, after running 35 cycles; incubation at 72 °C for 10 min.

[0011] In the above step (3), the information is organized using the software GenAlex.

[0012] The above genetic diversity parameters include the number of alleles, effective number of alleles, observed heterozygosity, expected heterozygosity, and percentage of polymorphic loci.

[0013] The above clustering analysis includes Bayesian clustering analysis and genetic distance clustering analysis.

[0014] The above two monophyletic groups are Cycas szechuanensis and Cycas taiwaniana; the populations include FAIRY1, FAIRY2, FAIRY3, FAIRY4, FAIRY5, SZE1, HAI1, HAI2, HAI3, HAI4, HAI5, CHA1, CHA2, CHA3, CHA4, LING1, LING2, and TAI1.

[0015] The present invention has the following beneficial effects: 1. The present invention uses Cycas hainanensis as the material, and through transcriptome sequencing, SSR locus mining and primer design, total DNA extraction, SSR-PCR amplification and effectiveness detection, a total of 5 pairs of SSR molecular marker primers are obtained. The primers provided by the present invention have rich polymorphism, high stability, and clear amplification bands, and can be used for species identification, genetic pedigree analysis, genetic diversity analysis, germplasm resource protection, and assisted breeding of the Cycas taiwaniana complex.

[0016] 2. The SSR molecular marker primers developed based on transcriptome data in the present invention, compared with traditional development methods, can develop SSR marker primers in large quantities, and a batch of SSR marker primer pairs with high amplification efficiency and good polymorphism are obtained. SSR markers have accuracy and stability, and are universal among 6 species of the Cycas taiwaniana complex, and can be directly used to identify the genetic diversity of the Cycas taiwaniana complex from different distribution sources, providing a theoretical basis for germplasm resource identification, formulation of protection strategies, and molecular assisted breeding of rare and endangered Cycas plants, and having good application prospects. Brief Description of the Drawings

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0018] Figure 1 It is the distribution of dinucleotide and trinucleotide motif types in SSR.

[0019] Figure 2 It is the agarose electrophoresis band pattern of primers SSR11(a), SSR33(b), SSR37(c), SSR42(d), and SSR50(e) in the tested individuals of 6 species of the Cycas taiwaniana complex.

[0020] Figure 3 It is the Bayesian clustering analysis diagram of 18 populations of the Cycas taiwaniana complex.

[0021] Figure 4 It is the UPGMA clustering diagram of 18 populations of the Cycas taiwaniana complex. Detailed implementation manners

[0022] The following will clearly and completely describe the technical solutions of the present invention in combination with the embodiments of the present invention. Obviously, the described embodiments are only the preferred embodiments of the present invention and are not used to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection scope of the present invention.

[0023] Unless otherwise specified, the test methods used in the following experimental examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, are reagents and materials that can be obtained from commercial channels. Embodiment

[0024] Embodiment 1 Development of SSR primers for Cycas hainanensis The present invention provides SSR sequence search, SSR marker primer design and verification based on transcriptome high-throughput sequencing and combined with bioinformatics methods. The specific implementation manners are as follows: 1.1 Experimental materials The transcriptome sequencing sample is the fresh leaves of Cycas hainanensis transplanted in the South China Botanical Garden. After collection, it is stored in RNAlater preservation solution (Takara Biotechnology Co., Ltd.) and immediately placed in a -80 °C refrigerator for frozen storage after being brought back to the laboratory. The sample RNA is extracted using an RNA kit.

[0025] 1.2. Transcriptome sequencing RNA-Seq transcriptome sequencing was commissioned to Novogene Bioinformatics Technology Co., Ltd. The sequencing reads were assembled using the De Novo assembly method (Grabherr et al., 2011). After assembly, a total of 81,561 unigenes with an average length of 761 bp and an N50 of 983 bp were obtained, which served as the basic data for SSR molecular marker development.

[0026] 1.3 Mining of SSR loci and primer design MISA was used to search for SSR loci, and six types of SSRs were obtained: single-base repeat SSRs (minimum repeat number of 10), dinucleotide repeat SSRs (repeat number of 4 or more), trinucleotide repeat SSRs (repeat number of 4 or more), tetranucleotide repeat SSRs (repeat number of 4 or more), pentanucleotide repeat SSRs (repeat number of 4 or more), and hexanucleotide repeat SSRs (repeat number of 4 or more). Highly polymorphic SSRs were screened, such as long repeat units and compound SSRs. The distribution of dinucleotide and trinucleotide motif types in SSRs is as Figure 1 shown.

[0027] The Primer 5.0 software was used for batch primer design. The design criteria were as follows: primer length was 18 - 25 bp, primer amplification length was 120 bp - 300 bp, GC content was between 40% - 60%, annealing temperature (Tm) was 50 - 60 °C, the Tm values of the upstream and downstream primers were ≤ 2 °C, the appearance of primer dimers, mismatches, and hairpin structures was avoided, and duplicate primer pairs were excluded.

[0028] 1.4 PCR amplification and primer screening The PCR amplification system was 20 μl, including 5 μl of DNA template, 2 μl of 10× buffer, 0.4 μl of dNTP, 0.6 μl of forward and reverse primers, 0.2 μl of Taq DNA polymerase, and 11.2 μl of sterilized ultrapure water; the PCR reaction program was: pre-denaturation at 95 °C for 4 min; denaturation at 95 °C for 30 s, annealing at 50 - 56 °C for 30 s, extension at 72 °C for 1 min, after running 35 cycles; incubation at 72 °C for 10 min. Fifty pairs of primers were randomly synthesized from the SSR loci designed in batch from the transcriptome, and four individuals were randomly selected from each of the six species of the Cycas taiwaniana complex for PCR amplification. The amplified products were subjected to validity identification after 40% polyacrylamide gel electrophoresis and silver staining. Finally, 5 pairs of primers were screened that could amplify successfully in all species of the Cycas taiwaniana complex, showing clear bands ( Figure 2 )).

[0029] The primer set for the SSR molecular marker combination used to identify the Cycas taiwaniana complex includes SSR11, SSR33, SSR37, SSR42, and SSR50 molecular markers.

[0030] SSR11 primer SEQ ID No.1: SSR11-F: 5’- TGGGACGGTGACACTCTTG -3’; SEQ ID No.2: SSR11-R: 5’- TGACTTCGCGGTGGTACG -3’; SSR33 primer SEQ ID No.3: SSR33-F: 5’- AGCGGATCCCGTTATAGCC -3’; SEQ ID No.4: SSR33-R: 5’- TTTCGCCAACGGTTTCCTG -3’; SSR37 primer SEQ ID No.5: SSR37-F: 5’- TCCTCGGGTCAATACGTGG -3’; SEQ ID No.6: SSR37-R: 5’- CGACTTCATGTGGATGCGG -3’; SSR42 primer SEQ ID No.7: SSR42-F: 5’- TGTCAAAGCCGCTCAATCC -3’; SEQ ID No.8: SSR42-R: 5’- GGAATGGCCAAGAAGTGCC -3’; SSR50 primer SEQ ID No.9: SSR50-F: 5’- ATTCGGTATGCTTTCCAC -3’; SEQ ID No.10: SSR50-R: 5’- AGGTAATCATTTCCCACA -3’.

[0031] Example 2 Application of the screened SSR primer sets in the genetic diversity analysis of 18 populations of 6 species of Cycas taiwaniana complex 2.1 Experimental materials For the study on species delimitation and genetic diversity of Cycas taiwaniana complex, based on further investigation of the distribution of all wild populations of Cycas taiwaniana complex, leaf samples of all wild populations of this complex were collected, dried with silica gel, and stored at -20 °C. A total of 6 species, 18 populations, and 180 individuals were selected for DNA extraction.

[0032] 2.2 Total DNA extraction DNA was extracted using the modified CTAB method (Doyle, 1987). The specific steps are as follows: 1) Weigh 1.0 g of fresh and tender leaves and put them into a mortar. Add liquid nitrogen and grind until the leaves are completely ground into powder.

[0033] 2) Transfer the ground powder into a 1.5 ml centrifuge tube, add 900 μl of CTAB, shake well, and place it in a 4°C refrigerator for 30 min. Take it out after 30 min, centrifuge at 5000 r / min for 5 min, and discard the supernatant.

[0034] 3) Add 600 μl of 3×CTAB extraction buffer preheated at 65°C, mix well, and incubate in a 65°C water bath for 1 h, shaking well every 15 min.

[0035] 4) After the water bath, cool it down, centrifuge at 10000 r / min for 10 min. Transfer the supernatant into a new centrifuge tube, add 600 μl of chloroform:isoamyl alcohol (24:1), mix well, and centrifuge at 10000 r / min for 10 min. Repeat this process 2 - 4 times until a very thin protein layer precipitates in the middle.

[0036] 5) Transfer the supernatant into a new centrifuge tube, first add 300 μl of 5M sodium chloride, then add 600 μl of isopropanol, shake well, and precipitate at -20°C for at least 30 min. Centrifuge at 10000 r / min for 10 min, and discard the supernatant.

[0037] 6) Add 75% ethanol to the centrifuge tube and wash twice to make the precipitate detach from the tube wall. Discard the supernatant, add 500 μl of ethanol and wash the precipitate once more.

[0038] 7) Invert it on absorbent paper and air-dry at room temperature, or dry it in an oven at 37°C. Add 100 μl of 1×TE buffer to dissolve.

[0039] 8) Detect the integrity of DNA by 1.5% agarose gel electrophoresis. That is, mix 2 μl of DNA sample with 2 μl of buffer, add the sample into the wells of 1.5% agarose gel using a pipette, electrophorese at 230 V for 30 min, stain with 0.5% EB solution for 20 min, and detect the DNA molecular weight size under ultraviolet light in a gel imaging system. Use a NanoDrop ND2000 spectrophotometer to detect the DNA concentration and purity. The A260 / A280 detection values of all sample DNAs are between 1.8 - 2.

[0040] 9) Store it in a -20°C refrigerator.

[0041] 2.3 Data processing Import the microsatellite fragment length information data into the software GenAlex, v. 6.5 (Peakall and Smouse, 2012) for collation and output the format suitable for subsequent analysis by other software; use the software GenAlex, v. 6.5 to calculate genetic diversity parameters, including the number of alleles ( N A ), the effective number of alleles ( A E ), the observed heterozygosity ( H O ), the expected heterozygosity ( H E ), and the percentage of polymorphic loci ( PPB ); use the software STRUCTURE, v. 2.2 (Pritchard et al., 2000) for Bayesian clustering analysis of population structure, with the number of MCMC iterations being 1×10 5 , the iteration length being 1×10 5 generations, the K value being set to 1 - 18, and each K value being run 20 times, and the results are as Figure 3 shown; based on the unweighted pair group mean analysis (UPGMA), use the software NTSYS-pc, v. 1.4 (Jensen, 1989) to perform clustering analysis on the genetic distances among 18 populations of Cycas taiwaniana complex, and the results are as Figure 4 shown.

[0042] 2.4 Genetic diversity analysis Five pairs of SSR molecular marker primer combinations with high polymorphism were obtained through the above steps. Using these five pairs of primers, PCR amplification was performed on 180 individuals from 18 populations of the Cycas taiwaniana complex, and genetic diversity analysis was carried out. The genetic diversity of the five microsatellite loci in the Cycas taiwaniana complex is shown in Table 1. A total of 9 alleles were detected, and the number of alleles at each microsatellite locus ranged from 1.30 to 2.31, with an average of 1.79. The genetic diversity of 180 individuals from 18 populations of the Cycas taiwaniana complex at the microsatellite locus level is shown in Table 2. Populations SZE1, HAI1, HAI2, HAI3, HAI4, HAI5, CHA1, CHA2, CHA3, CHA4, LING1, and LING2 had relatively high genetic diversity, with the percentage of polymorphic loci all being 100%. Populations FAIRY2, FAIRY3, FAIRY4, and FAIRY5 had relatively low genetic diversity, with the percentages of polymorphic loci being 34.12%, 66.87%, 66.87%, and 66.87% respectively.

[0043] Table 1 Amplification of the Cycas taiwaniana complex with five pairs of primers Table 2 Genetic diversity indices of 18 populations of the Cycas taiwaniana complex 2.5 Cluster analysis The genetic structure of 18 populations of the Cycas taiwaniana complex was analyzed using the STRUCTURE software. As Figure 3 can be seen, the Cycas taiwaniana populations can be divided into two groups. Six populations of Cycas fairylakea and Cycas sichuanensis are grouped into one group, and the other 12 populations of the four other species are grouped into one group. The UPGMA cluster analysis is as Figure 4 shown. When the genetic distance is 0.37, the 18 populations of the Cycas taiwaniana complex can be clustered into two major branches. Cycas fairylakea (FAIRY1, FAIRY2, FAIRY3, FAIRY4) and Cycas sichuanensis (SZE1) are clustered into one branch, and Cycas rumphii (LING1, LING2), Cycas hainanensis (HAI1 - 5), Cycas taiwaniana (TAI1), and Cycas changii (CHA1 - 4) are clustered into one branch.

[0044] 2.6 Species delimitation of the Cycas taiwaniana complex Both the population genetic structure and the cluster analysis results obtained from the SSR marker primers developed based on the transcriptome sequences of Cycas hainanensis support that it is most appropriate to divide the Cycas taiwaniana complex into two monophyletic species: Cycas sichuanensis ( Cycas szechuanensis W. C.Cheng et L. K. Fu in Acta Phytotax. Sin, 13(4): 81 - 82, 22 1975) and Cycas taiwaniana ( Cycas taiwanianaCarruthers in Acta Phytotax. Sin. 21(2): 209-210, 1893), Cycas fairylakea ( Cycas fairylakea D. Y. Wang in Cycads in China, 54-57, 1996) should be merged into Cycas szechuanensis, while Cycas hainanensis ( Cycas hainanensis C. J. Chen in Acta Phytotax. Sin, 13(4): 82, 1975), Cycas pectinata ( Cycas lingshuigensis G. A. Fu in Bulletin of Botanical Research, 24(4): 387-388, 2004) and Cycas changii ( Cycas changjiangensis N. Liu in Acta Phytotax. Sin, 36(6): 552-554, 1998) should be classified into Cycas taiwaniana.

[0045] This application detects all species of the Cycas taiwaniana complex. The Cycas taiwaniana complex includes 6 species. We screened 5 SSR markers from the transcriptome of one of the species (Cycas hainanensis) and detected all populations of these 6 species. From the morphological characteristics, the trunks of Cycas szechuanensis and Cycas fairylakea are cylindrical, and the megasporophylls are both pinnately divided at the edges. From the geographical distribution, Cycas szechuanensis and Cycas fairylakea are located in mainland China, while Cycas hainanensis, Cycas pectinata and Cycas changii are only distributed in Hainan Island, China.

Claims

1. The SSR molecular marker combination of Taiwan Cycas complex is characterized by: Including SSR11, SSR33, SSR37, SSR42 and SSR50 molecular markers.

2. A primer set for identifying the SSR molecular marker combination of the Taiwan Cycas complex according to claim 1, characterized in that: The primer pair sequence of SSR11 is shown in SEQ ID No.1 and SEQ ID No.2, the primer pair sequence of SSR33 is shown in SEQ ID No.3 and SEQ ID No.4, the primer pair sequence of SSR37 is shown in SEQ ID No.5 and SEQ ID No.6, the primer pair sequence of SSR42 is shown in SEQ ID No.7 and SEQ ID No.8, and the primer pair sequence of SSR50 is shown in SEQ ID No.9 and SEQ ID No.

10.

3. A kit comprising the primer set according to claim 2, characterized in that: Also included are detection reagents.

4. A method for identifying monophyletic species of the Taiwan Cycas complex, characterized by: The method is based on the Taiwan Cycas complex SSR molecular marker combination described in claim 1; Here are the steps: (1) Extracting total DNA from the Taiwan Cycas complex to be tested; (2) using the total DNA from step (1) as a template, performing PCR amplification using the primer set described in claim 2, and recovering the amplification products of each primer set; (3) performing polyacrylamide gel electrophoresis and silver staining on the PCR product obtained in step (2); (4) The polymorphic bands amplified in step (3) were sorted and analyzed to obtain genetic diversity parameters, which were then compared with the 18 populations through cluster analysis to determine the single lineage and population of the Taiwan Cycas complex to be tested.

5. The method for identifying monophyletic species of the Taiwan Cycas complex according to claim 4, characterized in that: The PCR amplification system in step (2) is: 20 μl, including 5 μl of DNA template, 2 μl of 10× buffer, 0.4 μl of dNTP, 0.6 μl of forward and reverse primers, 0.2 μl of Taq DNA polymerase and 11.2 μl of sterile ultrapure water.

6. The method for identifying monophyletic species of the Taiwan Cycas complex according to claim 5, characterized in that: The PCR amplification program was as follows: pre-denaturation at 95°C for 4 min; denaturation at 95°C for 30 s, annealing at 50-56°C for 30 s, extension at 72°C for 1 min, and after 35 cycles; insulation at 72°C for 10 min.

7. The method for identifying monophyletic species of the Taiwan Cycas complex according to claim 4, characterized in that: The information in step (3) is collated using the software GenAlex.

8. The method for identifying monophyletic species of the Taiwan Cycas complex according to claim 7, characterized in that: The genetic diversity parameters include the number of alleles, the effective number of alleles, the observed heterozygosity, the expected heterozygosity and the percentage of polymorphic sites.

9. The method for identifying monophyletic species of the Taiwan Cycas complex according to claim 8, characterized in that: The cluster analysis includes Bayesian cluster analysis and genetic distance cluster analysis.

10. The method for identifying monophyletic species of the Taiwan Cycas complex according to claim 8, characterized in that: The single species lineages are Sichuan Cycas and Taiwan Cycas; the populations include FAIRY1, FAIRY2, FAIRY3, FAIRY4, FAIRY5, SZE1, HAI1, HAI2, HAI3, HAI4, HAI5, CHA1, CHA2, CHA3, CHA4, LING1, LING2 and TAI1.

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