A molecular marker combination, primer group and application of a cycas revoluta complex

Abstract

This invention belongs to the field of biotechnology and relates to the identification of the *Cycas taiwanensis* complex. Specifically, it relates to an SSR molecular marker combination, primer set, and applications for the *Cycas taiwanensis* complex. Using *Cycas hainanensis* as material, this invention obtained five pairs of SSR molecular marker primers through transcriptome sequencing, SSR site mining, SSR primer design, SSR-PCR reaction system establishment, SSR-PCR amplification, and validity detection. The primers provided by this invention exhibit rich polymorphism, high stability, and clear amplification bands, and can be used for species identification, genetic pedigree analysis, genetic diversity analysis, germplasm resource conservation, and assisted breeding of the *Cycas taiwanensis* complex.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to SSR marker primer pairs for the Taiwan Cycas complex developed based on transcriptome sequences and their applications. Background Technology

[0002] Climate change and geological events have a significant impact on the geographical distribution, speciation, and evolutionary history of species. Historical processes (such as ice ages) have left profound imprints on the genetic structure and population dynamics of extant plants. Cycads are among the oldest gymnosperms and have important scientific, ecological, and economic value. Cycads are prized ornamental plants with beautiful tree shapes, glossy pinnate leaves, and evergreen foliage. They are often used to create large potted plants and are highly popular for their ornamental value. The stems and seeds of cycads are rich in starch and are edible. Cycads also have medicinal value; their seeds are used to treat dysentery, coughs, and hemostasis. Currently, cycads worldwide are threatened by both climate change and human activities, with the genus *Cycas* (…) being particularly vulnerable. Cycas Plants are particularly affected. Cycads ( Cycas The *Cycas* genus is the only cycad species naturally distributed in China, comprising approximately 20 species, most of which are endemic to China. In China, *Cycas* plants are mainly distributed in the southwest and southeast coastal regions, with most species exhibiting narrow distributions. Their survival and reproduction are severely threatened, and they are all listed as Class I National Key Protected Wild Plants. The *Cycas taiwanensis* complex (… Cycas taiwaniana The genus *Cycas* (complex) is a group of cycads distributed in Guangdong, Fujian, and Hainan, China. They are morphologically very similar and difficult to distinguish, and their systematic classification is subject to considerable debate. This group includes *Cycas taiwanensis* (…). 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 semperflorens ( Cycas fairylakea (DY Wang). Due to habitat loss and over-logging, the wild populations of the Cycas Taiwania complex are declining rapidly, and intraspecific genetic diversity is constantly being lost. All six species are critically endangered or endangered and urgently need protection. However, the lack of genetic and differentiation data among species and populations hinders the effective protection and management of this complex.

[0003] Clarifying the species definition and genetic background of complexes is a prerequisite for the rational protection and utilization of this resource. Genetic diversity has crucial application value for conservation biology, providing an important theoretical foundation for the protection and breeding of endangered species. In recent years, with the increasing development of DNA sequencing technology and the continuous reduction of sequencing costs, molecular marker detection technology has become increasingly sophisticated, becoming an effective means of studying the genetic diversity of plant species. SSRs, or Simple Sequence Repeats or Microsatellites, are simple repetitive sequences uniformly distributed in the genome of eukaryotes, consisting of tandem repeat fragments of 2-6 nucleotides. The length polymorphism of tandem repeat sequences mainly stems from the different numbers of repetitions. Markers are randomly distributed in the genome, co-dominant, abundant, and highly polymorphic. Simple repeat sequence markers (SSRs) developed based on transcriptome data have significant advantages over traditional development methods. Traditional SSR marker development has limitations such as cumbersome operation steps, high manpower input, and high economic costs, while transcriptome-derived SSR marker primer pairs provide more comprehensive information, better universality, higher efficiency, and can specifically locate transcriptionally active regions of the genome. This characteristic makes SSR markers exhibit stronger detection sensitivity in genetic diversity research. After the related gene functions are verified, they can be directly applied to molecular marker-assisted breeding systems. More importantly, this method significantly increases the linkage probability between markers and trait-regulating genes, providing an effective tool for elucidating genetic mechanisms. Therefore, enriching the database of primers specific to the *Cycas formosanus* complex is of great significance for its protection. However, the number of microsatellite molecular markers available for use on the *Cycas formosanus* complex is still relatively small. Therefore, the identification of *Cycas formosanus* varieties is still based on geographical nomenclature. To identify the *Cycas formosanus* complex from a genetic perspective, our research group has conducted in-depth research. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention proposes an SSR molecular marker combination, primer set, and application for the Taiwan Cycas complex.

[0005] The technical solution of this invention is implemented as follows:

[0006] The SSR molecular marker assemblages of the Taiwan Cycas complex include SSR11, SSR33, SSR37, SSR42 and SSR50 molecular markers.

[0007] Primer set used to identify the above-mentioned SSR molecular marker combination of the Taiwan cycad complex:

[0008] SSR11 primers

[0009] SEQ ID No.1:SSR11-F:5'-TGGGACGGTGACACTCTTG-3';

[0010] SEQ ID No.2: SSR11-R: 5'- TGACTTCGCGGTGGTACG -3';

[0011] SSR33 primers

[0012] SEQ ID No.3:SSR33-F:5'-AGCGGATCCCGTTATAGCC-3';

[0013] SEQ ID No.4: SSR33-R: 5'-TTTCGCCAACGGTTTCCTG -3';

[0014] SSR37 primers

[0015] SEQ ID No.5: SSR37-F: 5'-TCCTCGGGTCAATACGTGG-3';

[0016] SEQ ID No.6: SSR37-R: 5'-CGACTTCATGTGGATGCGG-3';

[0017] SSR42 primers

[0018] SEQ ID No.7: SSR42-F: 5'- TGTCAAAGCCGCTCAATCC -3';

[0019] SEQ ID No.8: SSR42-R: 5'- GGAATGGCCAAGAAGTGCC -3';

[0020] SSR50 primers

[0021] SEQ ID No.9: SSR50-F: 5'-ATTCGGTATGCTTTCCAC-3';

[0022] SEQ ID No. 10: SSR50-R:5'-AGGTAATCATTTCCCACA-3'.

[0023] The kit containing the primer set described above also includes detection reagents.

[0024] A method for identifying monophyletic species of the cycad complex in Taiwan, the method being based on the aforementioned SSR molecular marker combination for the cycad complex in Taiwan;

[0025] The steps are as follows:

[0026] (1) Extract total DNA from the cycad complex to be tested in Taiwan;

[0027] (2) Using the total DNA from step (1) as a template, perform PCR amplification using the primer set described in claim 2, and recover the amplification products of each primer set;

[0028] (3) Perform polyacrylamide gel electrophoresis on the PCR products obtained in step (2);

[0029] (4) Genetic diversity parameters are obtained by sorting and analyzing the polymorphic bands amplified in step (3), and then compared with 18 populations by cluster analysis to determine the monocultures and populations of the test cycad complex in Taiwan.

[0030] The PCR amplification system in step (2) above 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.

[0031] The above PCR amplification program is as follows: 95 ℃ pre-denaturation for 4 min; 95 ℃ denaturation for 30 s, 50-56 ℃ annealing for 30 s, 72 ℃ extension for 1 min, run for 35 cycles; 72 ℃ incubation for 10 min.

[0032] The information in step (3) above was organized using the software GenAlex.

[0033] The genetic diversity parameters mentioned above include the number of alleles, the effective number of alleles, observed heterozygosity, expected heterozygosity, and the percentage of polymorphic sites.

[0034] The clustering analyses mentioned above include Bayesian clustering analysis and genetic distance clustering analysis.

[0035] The two monotypic lines mentioned above are Cycas chuanxiensis and Cycas taiwanensis; the populations include FAIRY1, FAIRY2, FAIRY3, FAIRY4, FAIRY5, SZE1, HAI1, HAI2, HAI3, HAI4, HAI5, CHA1, CHA2, CHA3, CHA4, LING1, LING2 and TAI1.

[0036] The present invention has the following beneficial effects:

[0037] 1. This invention uses *Cycas hainanensis* as material and obtains five pairs of SSR molecular marker primers through transcriptome sequencing, SSR site mining and primer design, total DNA extraction, SSR-PCR amplification and validity detection. The primers provided by this invention are highly polymorphic, stable, and produce clear amplification bands, and can be used for species identification, genetic pedigree analysis, genetic diversity analysis, germplasm resource conservation, and assisted breeding of *Cycas hainanensis* complex.

[0038] 2. This invention utilizes SSR molecular marker primers developed based on transcriptome data. Compared to traditional methods, this allows for the large-scale development of SSR marker primers, yielding a batch of SSR marker primer pairs with high amplification efficiency and good polymorphism. The SSR markers exhibit accuracy and stability, and are universally applicable across the six species of the *Cycas formosanus* complex. They can be directly used to identify the genetic diversity of *Cycas formosanus* complexes from different distribution areas, providing a theoretical basis for the identification of germplasm resources, the formulation of conservation strategies, and molecular-assisted breeding of rare and endangered cycads, demonstrating promising application prospects. Attached Figure Description

[0039] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0040] Figure 1 The distribution of dinucleotide and trinucleotide motif types in SSR.

[0041] Figure 2 Agarose gel electrophoresis bands of primers SSR11(a), SSR33(b), SSR37(c), SSR42(d), and SSR50(e) in six species of individuals from the Cycas retusa complex in Taiwan.

[0042] Figure 3 Bayesian cluster analysis diagram of 18 populations of Cycas taiwanensis complex.

[0043] Figure 4 UPGMA clustering diagram of 18 populations of the Cycas taiwanensis complex. Detailed Implementation

[0044] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0045] Unless otherwise specified, the experimental methods used in the following experimental examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified. Example

[0046] Example 1: Development of SSR primers for Cycas hainanensis

[0047] The present invention provides a method for SSR sequence finding and SSR marker primer design and validation based on high-throughput transcriptome sequencing and combined with bioinformatics methods. The specific implementation is as follows:

[0048] 1.1 Experimental Materials

[0049] Transcriptome sequencing samples were fresh leaves transplanted from *Cycas hainanensis* at the South China Botanical Garden. After collection, they were stored in RNAlater preservation solution (Takara Biotechnology Co., Ltd.) and immediately frozen at -80 ℃ upon arrival at the laboratory. RNA was extracted from the samples using an RNA kit.

[0050] 1.2. Transcriptome Sequencing

[0051] RNA-Seq transcriptome sequencing was commissioned to Novogene. Sequencing reads were obtained using the de novo assembly method (Grabherr et al., 2011), resulting in 81,561 unigenes with an average length of 761 bp and an N50 of 983 bp, which served as the basis for SSR molecular marker development.

[0052] 1.3 SSR locus mining and primer design

[0053] SSR site identification was performed using MISA, resulting in six types of SSRs: single-base repeat SSRs (minimum repeat count of 10), double-base repeat SSRs (4 or more repeat counts), triple-base repeat SSRs (4 or more repeat counts), tetra-base repeat SSRs (4 or more repeat counts), penta-base repeat SSRs (4 or more repeat counts), and hexa-base repeat SSRs (4 or more repeat counts). High-polymorphism SSRs, such as long repeat units and complex SSRs, were screened. The distribution of dinucleotide and trinucleotide motif types in SSRs is shown below. Figure 1 As shown.

[0054] Primer batch design was performed using Primer 5.0 software. The design criteria were as follows: primer length 18-25bp, primer amplification length 120bp-300bp, GC content between 40% and 60%, annealing temperature (Tm) 50-60℃, and upstream and downstream primer Tm values ​​≤2℃. Primer dimers, mismatches, and hairpin structures were avoided, and duplicate primer pairs were removed.

[0055] 1.4 PCR amplification and primer screening

[0056] The PCR amplification system consisted of 20 μl of PCR amplification material, including 5 μl of DNA template, 2 μl of 10× buffer, 0.4 μl of dNTPs, 0.6 μl of forward and reverse primers, 0.2 μl of Taq DNA polymerase, and 11.2 μl of sterile ultrapure water. The PCR reaction program was as follows: 95℃ pre-denaturation for 4 min; 95℃ denaturation for 30 s, 50-56℃ annealing for 30 s, 72℃ extension for 1 min, for 35 cycles; and 72℃ incubation for 10 min. Fifty primer pairs were randomly synthesized from SSR loci obtained through batch design from the transcriptome. Four individuals from each of the six species of *Cycas taiwanensis* complex were randomly selected for PCR amplification. The amplification products were evaluated for effectiveness after 40% polyacrylamide gel electrophoresis and silver staining. Five primer pairs were ultimately selected that successfully amplified all species of *Cycas taiwanensis* complex, showing clear bands. Figure 2 ).

[0057] The primer set used to identify the SSR molecular marker combination of the Taiwan Cycas complex includes SSR11, SSR33, SSR37, SSR42 and SSR50 molecular markers.

[0058] SSR11 primers

[0059] SEQ ID No.1:SSR11-F:5'-TGGGACGGTGACACTCTTG-3';

[0060] SEQ ID No.2: SSR11-R: 5'- TGACTTCGCGGTGGTACG -3';

[0061] SSR33 primers

[0062] SEQ ID No.3:SSR33-F:5'-AGCGGATCCCGTTATAGCC-3';

[0063] SEQ ID No.4: SSR33-R: 5'-TTTCGCCAACGGTTTCCTG -3';

[0064] SSR37 primers

[0065] SEQ ID No.5: SSR37-F: 5'-TCCTCGGGTCAATACGTGG-3';

[0066] SEQ ID No.6: SSR37-R: 5'-CGACTTCATGTGGATGCGG-3';

[0067] SSR42 primers

[0068] SEQ ID No.7: SSR42-F: 5'- TGTCAAAGCCGCTCAATCC -3';

[0069] SEQ ID No.8: SSR42-R: 5'- GGAATGGCCAAGAAGTGCC -3';

[0070] SSR50 primers

[0071] SEQ ID No.9: SSR50-F: 5'-ATTCGGTATGCTTTCCAC-3';

[0072] SEQ ID No. 10: SSR50-R:5'-AGGTAATCATTTCCCACA-3'.

[0073] Example 2: The selected SSR primer set was used for genetic diversity analysis of 18 populations from 6 species of Cycas fortatus complex.

[0074] 2.1 Experimental Materials

[0075] The specimens for the species delineation and genetic diversity study of the Cycas taiwanensis complex were obtained by collecting leaf samples from all wild populations of the complex, drying them in silica gel, and storing them at -20 ℃, based on a further investigation of the distribution of all wild populations of the Cycas taiwanensis complex. A total of 180 individuals from 6 species and 18 populations were selected for DNA extraction.

[0076] 2.2 Total DNA Extraction

[0077] DNA was extracted using a modified CTAB method (Doyle, 1987), and the specific steps are as follows:

[0078] 1) Weigh 1.0g of fresh, tender leaves, put them in a mortar, add liquid nitrogen and grind until all the leaves are ground into powder.

[0079] 2) Transfer the ground powder into a 1.5 ml centrifuge tube, add 900 μl of CTAB, vortex until homogeneous, and place in a 4°C refrigerator for 30 min. After 30 min, remove the tube, centrifuge at 5000 r / min for 5 min, and discard the supernatant.

[0080] 3) Add 600 μl of 3×CTAB extract preheated at 65℃, mix well, and incubate in a water bath at 65℃ for 1 h, shaking once every 15 min.

[0081] 4) After the water bath is complete, cool the mixture and centrifuge at 10,000 r / min for 10 min. Transfer the supernatant to a new centrifuge tube, add 600 μl of chloroform:isoamyl alcohol (24:1), mix well, and centrifuge at 10,000 r / min for 10 min. Repeat 2-4 times until a very thin protein layer precipitates in the middle.

[0082] 5) Transfer the supernatant to a new centrifuge tube, add 300 μl of 5M sodium chloride, then add 600 μl of isopropanol, shake well, and incubate at -20°C for at least 30 min to precipitate. Centrifuge at 10000 r / min for 10 min and discard the supernatant.

[0083] 6) Add 75% ethanol to the centrifuge tube and wash twice to remove the precipitate from the tube wall. Discard the supernatant and add 500 μl of ethanol to wash the precipitate again.

[0084] 7) Invert the container onto absorbent paper to air dry at room temperature, or dry it in a 37°C oven. Dissolve the contents in 100 μl of 1×TE buffer.

[0085] 8) DNA integrity was assessed by 1.5% agarose gel electrophoresis. 2 μl of DNA sample was mixed with 2 μl of buffer, and the sample was pipetted into the wells of a 1.5% agarose gel. Electrophoresis was performed at 230 V for 30 min, followed by staining with 0.5% EB solution for 20 min. The molecular weight of the DNA was then determined under UV light in a gel imaging system. DNA concentration and purity were measured using a NanoDrop ND2000 spectrophotometer. The A260 / A280 ratio of all DNA samples was between 1.8 and 2.

[0086] 9) Store in a refrigerator at -20℃.

[0087] 2.3 Data Processing

[0088] Microsatellite fragment length information data was imported into GenAlex, v. 6.5 (Peakall and Smouse, 2012) for processing, and output in a format suitable for subsequent analysis in other software. GenAlex, v. 6.5 was then used to calculate genetic diversity parameters, including the number of alleles. N A ), effective number of alleles A E ), Observed heterozygosity H O), expected heterozygosity H E Percentage of polymorphic sites PPB The population structure was analyzed using Bayesian clustering with STRUCTURE, v. 2.2 (Pritchard et al., 2000). The number of iterations for MCMC was 1 × 10⁻⁶. 5 The iteration length is 1×10 5 For example, K is set to a value from 1 to 18, and the operation is performed 20 times for each K value. The result is as follows. Figure 3 As shown; based on the unweighted pair group mean analysis (UPGMA), cluster analysis of genetic distances among 18 populations of the *Cycas taiwanensis* complex was performed using NTSYS-pc, v. 1.4 (Jensen, 1989) software. The results are as follows. Figure 4 As shown.

[0089] 2.4 Genetic diversity analysis

[0090] Five pairs of highly polymorphic SSR molecular marker primers were obtained through the above steps. These five primer pairs were used to perform PCR amplification on 180 individuals from 18 populations of the *Cycas formosanus* complex, and genetic diversity analysis was conducted. The genetic diversity of the five microsatellite loci in the *Cycas formosanus* complex is shown in Table 1. A total of nine alleles were detected, with the number of alleles at each microsatellite locus ranging from 1.30 to 2.31, with an average of 1.79. Table 2 shows the genetic diversity at the microsatellite locus level of 180 individuals from 18 populations of the Cycas taiwanensis complex. Populations SZE1, HAI1, HAI2, HAI3, HAI4, HAI5, CHA1, CHA2, CHA3, CHA4, LING1, and LING2 exhibited high genetic diversity, with 100% of their polymorphic loci. Populations FAIRY2, FAIRY3, FAIRY4, and FAIRY5 showed lower genetic diversity, with polymorphic loci percentages of 34.12%, 66.87%, 66.87%, and 66.87%, respectively.

[0091] Table 1. Amplification of the cycad complex in Taiwan using 5 primer pairs

[0092]

[0093] Table 2 Genetic diversity indices of 18 populations of the Cycas taiwanensis complex

[0094]

[0095] 2.5 Cluster Analysis

[0096] The genetic structure of 18 populations of the *Cycas taiwanensis* complex was analyzed using STRUCTURE software. Figure 3 It can be seen that the populations of *Cycas taiwanensis* can be divided into two groups: the six populations of *Cycas xianhuensis* and *Cycas chuanxiongensis* form one group, while the twelve populations of the remaining four species form another group. UPGMA cluster analysis is as follows: Figure 4 As shown, when the genetic distance is 0.37, the 18 populations of the Taiwan Cycas complex can cluster into two major clades: Fairy1, Fairy2, Fairy3, Fairy4 and SZE1 cluster into one clade, while Ling1, Ling2, HAI1-5, TAI1 and CHA1-4 cluster into another clade.

[0097] 2.6 Species Delineation of the Cycas Taiwania Complex

[0098] The population genetic structure and cluster analysis results obtained from SSR marker primers developed based on the transcriptome sequence of *Cycas hainanensis* both support that the *Cycas taiwanensis* complex is most appropriately divided into two monophyletic species: *Cycas chuanxiensis* (…). Cycas szechuanensis WCCheng et LK Fu in Acta Phytotax. Sin, 13(4): 81-82, 22 1975) and Taiwan Cycas ( Cycas taiwaniana Carruthers in Acta Phytotax. Sin. 21(2): 209-210, 1893), Cycas davidii ( Cycas fairylakea DY Wang in Cycads in China, 54-57, 1996) should be merged into the Sichuan Cycads, while the Hainan Cycads ( Cycas hainanensis CJ Chenin Acta Phytotax. Sin, 13(4): 82,1975), Cycas revoluta ( Cycas lingshuigensis GA Fuin Bulletin of Botanical Research, 24(4):387-388, 2004) and Cycas revoluta ( Cycas changjiangensis N. Liuin in Acta Phytotax.Sin, 36(6):552-554,1998) should be classified as Taiwan Cycas.

[0099] This application investigated all species within the *Cycas taiwanensis* complex. The *Cycas taiwanensis* complex comprises six species. We screened five SSR markers from the transcriptome of one species (*Cycas hainanensis*) and then analyzed all populations of these six species. Morphologically, *Cycas szechuanensis* and *Cycas xianhuensis* have cylindrical trunks and pectinate megasporophytes. Geographically, *Cycas szechuanensis* and *Cycas xianhuensis* are located in mainland China, while *Cycas hainanensis*, *Cycas beaded*, and *Cycas cucurbita* are found only on Hainan Island, China.

Claims

1. A primer set for identifying a combination of SSR molecular markers of the Cycas taiwaniana complex, characterized by: The primer pair sequence of the molecular marker SSR11 is shown as SEQ ID No. 1 and SEQ ID No. 2, the primer pair sequence of the molecular marker SSR33 is shown as SEQ ID No. 3 and SEQ ID No. 4, the primer pair sequence of the molecular marker SSR37 is shown as SEQ ID No. 5 and SEQ ID No. 6, the primer pair sequence of the molecular marker SSR42 is shown as SEQ ID No. 7 and SEQ ID No. 8, and the primer pair sequence of the molecular marker SSR50 is shown as SEQ ID No. 9 and SEQ ID No.

10.

2. A kit comprising the primer set of claim 1, characterized in that: Also included are detection reagents.

3. A method for identifying a monophyletic species of the Chamaecyparis formosana complex, characterized by, The steps are as follows: (1) extracting total DNA of the to-be-tested Cycas formosana complex; (2) using the total DNA of step (1) as a template, performing PCR amplification by using the primer set of claim 2, and recovering the amplification products of each primer set; (3) performing polyacrylamide gel electrophoresis and silver staining on the PCR products obtained in step (2); (4) arranging and analyzing the polymorphic bands amplified in step (3) to obtain genetic diversity parameters, and then performing cluster analysis with 18 populations to determine the single line and population of the to-be-tested Cycas formosana complex.

4. The method for identifying a single lineage species of the Chamaeropsidium complex according to claim 3, 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 sterilized ultrapure water.

5. The method for identifying a single lineage species of the Chamaeropsidium complex according to claim 4, characterized in that, The PCR amplification program is: 95 ℃ pre-denaturation for 4 min; 95 ℃ denaturation for 30 s, 50-56 ℃ annealing for 30 s, 72 ℃ extension for 1 min, running for 35 cycles; and 72 ℃ incubation for 10 min.

6. The method of identifying a single lineage species of the Chamaeropsidium complex according to claim 5, characterized in that: The arrangement and analysis of the polymorphic bands amplified in step (3) in step (4) are performed by using software GenAlex.

7. The method of identifying a single lineage species of the Chamaeropsidium complex according to claim 6, characterized in that: The genetic diversity parameters include the number of alleles, the number of effective alleles, the observed heterozygosity, the expected heterozygosity and the percentage of polymorphic sites.

8. The method of identifying a single lineage species of the Chamaeropsidium complex according to claim 7, characterized in that: The cluster analysis includes Bayesian cluster analysis and genetic distance cluster analysis.

9. The method of identifying a single lineage species of the Chamaeropsidium complex according to claim 8, characterized in that: The single line is Cycas szechuanensis and Cycas formosana.

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