A combination of SSR molecular marker primers for Chrysanthemum chinense and its application

Abstract

The present invention provides a primer combination for SSR molecular markers of Chrysanthemum chinense, which comprises: primers for amplifying cpSSR2, the forward and reverse primers are shown in SEQ ID NOs.1-2; primers for amplifying cpSSR5, the forward and reverse primers are shown in SEQ ID NOs.3-4; primers for amplifying cpSSR11, the forward and reverse primers are shown in SEQ ID NOs.5-6; primers for amplifying cpSSR12, the forward and reverse primers are shown in SEQ ID NOs.7-8; primers for amplifying cpSSR23, the forward and reverse primers are shown in SEQ ID NOs.9-10. The primer combination for SSR molecular markers of Chrysanthemum chinense provided by the present invention can analyze the genetic diversity of Chrysanthemum chinense, and provide reliable marker resources for the identification of different population materials of Chrysanthemum chinense and genetic evolution analysis.

Description

Technical Field

[0001] The present invention belongs to the field of plant biotechnology, and in particular relates to the development and application of SSR molecular markers of the chloroplast genome of Alocasia macrophylla. Background Art

[0002] Chrysosplenium L. is a perennial herbaceous plant in the Saxifragaceae family, comprising approximately 70 species. This genus boasts remarkable medicinal properties and has long been used to treat a variety of ailments. Among them, Chrysosplenium macrophyllus Oliv. (tiger grass, horse-ear grass, dragon grass) is endemic to China and is found in Hubei, Hunan, Jiangxi, Guizhou, Anhui, Zhejiang, Sichuan, Yunnan, Guangdong, Shaanxi, Fujian Provinces, Guangxi Zhuang Autonomous Region, and Chongqing. It is a commonly used Chinese herbal medicine for its heat-clearing and detoxifying properties, tissue-stimulating and astringent properties. The entire plant, with its bitter, astringent, and cold properties, is used to treat infantile convulsions, lung and ear diseases, and other conditions.

[0003] Simple sequence repeats (SSRs) consist of tandemly repeated units of 1 to 6 nucleotides and are widely found in the nuclear, chloroplast, and mitochondrial genomes of eukaryotes. Due to their abundance, high polymorphism, codominance, good reproducibility, and ease of manipulation, they have been widely used in germplasm resource and variety identification and genetic diversity analysis. The chloroplast genome is unisexually inherited, undergoes little genetic recombination, and is more conserved than the nuclear genome. Therefore, SSR markers developed based on chloroplast genome information can be used for phylogenetic studies of higher taxonomic levels, interspecific and intraspecific identification, and the construction of genetic maps and DNA fingerprints. Currently, a large number of chloroplast genomes have been sequenced, and chloroplast SSR markers have been developed and applied. Sha Xiufen et al. analyzed the genetic diversity of 61 Salvia miltiorrhiza species using 10 cpSSR primer pairs and 13 mtSSR primer pairs, and verified the interoperability of the primers across species of the genus Salvia. Li Siqiao et al. used two pairs of cpSSR primers developed to distinguish Zanthoxylum bungeanum, Zanthoxylum tsutsuga, and Zanthoxylum japonicum. Zhang Jingzhen et al. used chloroplast SSR markers to analyze the genetic diversity of 64 Chinese yam germplasm resources and successfully constructed a DNA fingerprint of Chinese yam using two pairs of cpSSR primers. Lou Wenrui et al. initially developed cpSSR markers for the genus Carya and screened six polymorphic primer pairs that can be used to distinguish between different species in the genus Carya.

[0004] Currently, there are very few reports on Chrysopus macrophyllus. Huang Wen et al. used SRAP markers to conduct genetic polymorphism and cluster analysis on 36 accessions from 24 species of Chrysopus genus, including Chrysopus macrophyllus, and constructed DNA fingerprints for 38 accessions. However, no relevant analysis of Chrysopus macrophyllus has been reported. Summary of the Invention

[0005] In order to fill the gap of SSR molecular markers of Chrysanthemum grandiflorum in the field, the present invention developed cpSSR molecular markers based on the chloroplast genome of Chrysanthemum grandiflorum, and screened out five groups of polymorphic cpSSR molecular marker primers. The above-mentioned Chrysanthemum grandiflorum SSR molecular marker primer combination can analyze the genetic diversity of Chrysanthemum grandiflorum, and provide a reliable marker resource for the identification of different population materials of Chrysanthemum grandiflorum and genetic evolution analysis.

[0006] The technical solution adopted to achieve the above-mentioned purpose of the present invention is:

[0007] A primer combination for SSR molecular markers of Chrysotrichum macrophylla, wherein the SSR molecular markers include five groups: cpSSR2, cpSSR5, cpSSR11, cpSSR12, and cpSSR23, and the nucleotide sequences thereof are shown in SEQ ID NOs. 11 to 15;

[0008] The primer combination includes:

[0009] Primers for amplifying the molecular marker cpSSR2, the forward and reverse primers are shown in SEQ ID NOs. 1-2;

[0010] Primers for amplifying the molecular marker cpSSR5, the forward and reverse primers are shown in SEQ ID NOs. 3-4;

[0011] Primers for amplifying the molecular marker cpSSR11, the forward and reverse primers are shown in SEQ ID NOs. 5-6;

[0012] Primers for amplifying the molecular marker cpSSR12, the forward and reverse primers are shown in SEQ ID NOs. 7-8;

[0013] The primers for amplifying the molecular marker cpSSR23, the forward and reverse primers are shown in SEQ ID NOs. 9-10.

[0014] The SSR molecular marker primer combination of Chrysanthemum spp. can be applied to the genetic diversity analysis of Chrysanthemum spp. population, and the specific steps are as follows:

[0015] (1) Genomic DNA extraction: High-quality total genomic DNA from C. chinensis was extracted using a modified CTAB method. DNA quality and concentration were determined using an ultraviolet spectrophotometer and confirmed by 1% agarose gel electrophoresis.

[0016] (2) PCR reaction system: Five groups were prepared, each with a total reaction volume of 10 μL, including 5 μL of 2×T5 SuperPCR Mix (PAGE), 10 μmol·L -10.4 μL each of forward and reverse primers, 1 μL of total genomic DNA, 3.2 μL of ddH2O;

[0017] The PCR reaction program was as follows: pre-denaturation at 98°C for 2 min; denaturation at 98°C for 10 s, annealing at 58°C for 10 s, and extension at 72°C for 10 s, for a total of 35 cycles; total extension at 72°C for 2 min, followed by storage at 4°C;

[0018] (3) Electrophoresis detection: The amplified products were separated by 4% polyacrylamide gel electrophoresis in 1× TBE buffer at 90W constant voltage for 1.5–2.0 h. The polymorphism of the primers was detected by silver staining, and the electrophoresis images were photographed.

[0019] (4) Data analysis: Based on the electrophoresis images, the band reading method was used to record the reproducible and easily distinguishable bands on the image as "1" and the absence of bands at the same position as "0". The data were then input into Excel to establish the original "01" data matrix;

[0020] The genetic similarity coefficient was calculated using NTSYS software, and the unweighted paired arithmetic mean (UPGMA) method was used for genetic similarity cluster analysis.

[0021] GENDIVE version 3.06 software was used to calculate diversity indicators such as the number of alleles Na, the effective number of alleles Ne, the observed heterozygosity Ho, and the expected heterozygosity He.

[0022] The method for obtaining the SSR molecular marker primer combination of the large-leaf golden waist comprises the following steps:

[0023] (1) Acquisition of the chloroplast genome sequence of the large-leaved golden waist and identification of cpSSR loci

[0024] The chloroplast genome sequence of C. chinensis MK973001 was downloaded from the GenBank public database. The MISAperl script software was used to search for SSR loci distributed in the chloroplast genome to obtain the nucleotide sequences of the primary screening cpSSR molecular markers. The search criteria were as follows: the minimum number of repeats for mononucleotide, dinucleotide, trinucleotide, tetranucleotide, pentanucleotide, and hexanucleotide repeat types was set to 10, 6, 5, 5, 5, and 5, respectively.

[0025] (2) cpSSR primer design

[0026] Primer 3 software was used to design primers based on the nucleotide sequences of the cpSSR molecular markers screened in the previous step. The primer design principles were: GC content 40-60%, annealing temperature 57-60°C, primer length 18-23 bp, and expected amplification product length 100-300 bp; the nucleotide sequences of the primary screening cpSSR primers were obtained;

[0027] (3) Secondary screening of cpSSR primers

[0028] First, the improved CTAB method is used to extract high-quality total genomic DNA from Chrysanthemum grandiflorum, and then this is used as a template to construct a PCR reaction system for the initial screening cpSSR primers in the previous step, and a PCR reaction is carried out. After the PCR amplification is completed, gel electrophoresis is used to separate the amplified products, silver staining is used to detect the polymorphism of the primers, and electrophoresis images are photographed and collected; the primers corresponding to the electrophoresis bands with good stability, high polymorphism and high clarity are selected to obtain the Chrysanthemum grandiflorum SSR molecular marker primer combination.

[0029] Due to the adoption of the above technical solution, the present invention has the following beneficial effects:

[0030] (1) The present invention uses the SSR markers developed from the chloroplast genome sequence of C. macrophylla to fill the gap in SSR molecular markers for C. macrophylla.

[0031] (2) The present invention used the developed cpSSR polymorphic marker primers to analyze and evaluate the polymorphism among the large-leaf golden waist populations.

[0032] (3) This invention provides ideas for subsequent scholars to screen more SSR loci of Chrysanthemum chinense, and provides reliable marker resources for the identification of different population materials of Chrysanthemum chinense and population evolution analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is the electrophoresis diagram of the primers for the molecular marker cpSSR11 in Example 1;

[0034] Figure 2 This is the electrophoresis diagram of the primers for the molecular marker cpSSR12 in Example 1;

[0035] Figure 3 The electrophoresis results of 45 samples of Chrysanthemum longifolium after amplification with cpSSR12 primers are shown;

[0036] Figure 4 This is the UPGMA cluster map of 45 Chrysotrichum macrophylla accessions constructed using five pairs of cpSSR polymorphic primers. DETAILED DESCRIPTION

[0037] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is further described below with reference to the embodiments, but the protection scope of the present invention is not limited thereto:

[0038] Example 1 Site identification and primer design and screening of Chrysanthemum macrophylla cpSSR

[0039] (1) Download of the chloroplast genome of the large-leaved golden waist and identification of cpSSR loci

[0040] The chloroplast genome sequence of A. truncatula (MK973001) was downloaded from the GenBank public database (https: / / www.ncbi.nlm.nih.gov) in FASTA format. The MISA Perl script was used to search for SSR loci distributed within the chloroplast genome. The search criteria were: a minimum number of repeats of 10 for mononucleotide, 6 for dinucleotide, 5 for trinucleotide, 5 for tetranucleotide, pentanucleotide, and 5 for hexanucleotide repeat types, respectively. CPSSR screening revealed 30 SSR loci in the chloroplast genome sequence (Table 1), including 23 single-base repeats (6 A bases, 1 C base, and 17 T bases) and 6 double-base repeats (1 AT repeat and 5 TA repeats). T and A repeats accounted for approximately 66.00% of the SSR loci, indicating that single-base repeats of T and A predominate in the SSRs within the A. truncatula chloroplast genome.

[0041] Table 1 Statistics of cpSSR loci in Chrysanthemum glaucum

[0042]

[0043] (2) cpSSR primer design

[0044] Primer 3 software was used to design cpSSR primers. The design principles were: GC content of 40-60%, annealing temperature of 57-60°C, primer length of 18-23 bp, and expected amplification product length of 100-300 bp. A total of 30 pairs of cpSSR primers were designed based on the cpSSR loci. The primer sequences are detailed in Table 2. Primer sequences were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0045] Table 2 Primer information of cpSSR markers of Chrysops macrophylla

[0046]

[0047]

[0048]

[0049] (3) Polymorphic primer screening

[0050] DNA from 12 accessions of Chrysotrichum spp. was used as template. The cp-SSR reaction system consisted of a 10 μL volume containing 5 μL of 2×T5 Super PCR Mix (PAGE), 0.4 μL of each forward and reverse primer (10 μmol L⁻¹), 1 μL of genomic DNA, and 3.2 μL of ddH₂O. The PCR reaction procedure was 98°C pre-denaturation for 2 min, followed by 35 cycles of denaturation at 98°C for 10 s, annealing at 58°C for 10 s, and extension at 72°C for 10 s. The final extension at 72°C was 2 min, followed by storage at 4°C. The primers used are listed in Table 2. After PCR amplification, the amplified products were separated by 4% denaturing polyacrylamide gel electrophoresis in 1×TBE buffer at 90 W constant voltage for 1.5–2.0 h. Primer polymorphisms were detected by silver staining, and electrophoresis images were photographed. The results showed that all 30 primer pairs could amplify bands in 12 samples of Chrysanthemi chinensis. Among them, 5 primer pairs amplified electrophoresis bands with good stability, high polymorphism and high clarity in 12 samples of Chrysanthemi chinensis. The primer numbers were cpSSR2, cpSSR5, cpSSR11, cpSSR12 and cpSSR23 (shown in bold in Table 2). Figure 1 and Figure 2 , Figure 1 The electrophoresis diagram of cpSSR11 primers is shown in Figure 2. Figure 2 This is the electrophoresis diagram of the cpSSR12 primer. Due to space considerations, the present invention does not show all the electrophoresis diagrams in the accompanying drawings. Figure 1 and Figure 2 Medium, M: Maker; 1-12: 12 portions of large-leaf golden waist.

[0051] Example 2 Application of 5 pairs of polymorphic cpSSR primers in the study of genetic diversity of Chrysopogon japonicus population

[0052] (1) Genomic DNA extraction:

[0053] A total of 45 samples of C. macrophylla were collected from different locations. Detailed information is shown in Table 3. Genomic DNA from leaf tissues was extracted using a modified CTAB method. DNA quality and concentration were determined using an ultraviolet spectrophotometer and confirmed by 1% agarose gel electrophoresis. DNA concentration was uniformly diluted to 80 ng / μL and stored in a -20°C refrigerator for later use.

[0054] Table 3. Information on the sampling locations of Chrysanthemum chinense

[0055]

[0056]

[0057] (2) cp-SSR reaction system: The total volume of the cp-SSR reaction system was 10 μL, including 5 μL of 2×T5Super PCR Mix (PAGE), forward and reverse primers (10 μmol·L -1 ) 0.4 μL each, 1 μL genomic DNA, 3.2 μL ddH2O. The PCR reaction procedure was 98°C pre-denaturation for 2 min; 98°C denaturation for 10 s, 58°C annealing for 10 s, 72°C extension for 10 s, for a total of 35 cycles; 72°C total extension for 2 min, and then storage at 4°C. The primers used were 5 pairs of polymorphic primers (see bold in Table 2). (3) Electrophoresis detection: The amplified products were separated by 4% polyacrylamide gel electrophoresis in 1×TBE buffer at 90W constant voltage for 1.5-2.0 h. The polymorphism of the primers was detected by silver staining, and the electrophoresis images were photographed. Figure 3 The results are from the amplification of 45 samples of Chrysotrichum serrata using the cpSSR12 primer. Due to space considerations, not all electrophoresis diagrams are shown in the accompanying drawings.

[0058] (4) Data analysis: Based on the electrophoresis images, the band reading method was used manually, and reproducible and easily distinguishable bands on the image were recorded as "1". The absence of bands at the same position was recorded as "0". The data were input into Excel to establish the original "01" data matrix. The genetic similarity coefficient was calculated using NTSYS software, and the unweighted paired arithmetic mean method (UPGMA) was used for genetic similarity cluster analysis. The diversity indicators such as the number of alleles (Na), the effective number of alleles (Ne), the observed heterozygosity (Ho), and the expected heterozygosity (He) were calculated using GENDIVE version 3.06 software. The calculation results are shown in Table 4.

[0059] Table 4 Primer polymorphism information analysis table

[0060]

[0061] In the table: Na: allele; Ne: effective number of alleles; Ho: observed heterozygosity; He: expected heterozygosity

[0062] As can be seen from Table 4, the allele variation range of the five primer pairs is between 3 and 6, with an average of 4.4. The effective number of alleles (Ne) is between 1.015 and 1.195, with an average of 1.079. The observed heterozygosity (Ho) is between 0 and 0.267, with an average of 0.061. The expected heterozygosity (He) is between 0.034 and 0.254, with an average of 0.172.

[0063] Cluster analysis was performed on 45 accessions of Chrysanthemum chinense using 5 pairs of polymorphic cpSSR primers. The UPGMA results showed that (e.g. Figure 4As shown in the figure, 45 samples of Chrysanthemum glaucum could be divided into five clusters at a genetic similarity coefficient of 0.8. Cluster I contained 18 samples, primarily from Zhijin County, Nanjiang County, Xuan'en County, Yingshan County, Tongshan County, and Lin'an City. Cluster II contained 19 samples, primarily from Hongya County, Guidong County, Yifeng County, Lichuan City, Zhuxi County, Pan'an County, and Wugang City. Cluster III included only samples from Jianning County, while clusters IV and V both originated from Badong County. The study showed that cluster analysis could group native species from the same source together, indicating that these five primer pairs can be used for genetic diversity analysis and phylogenetic relationship studies of Chrysanthemum glaucum resources.

[0064] The above description is only a preferred embodiment of the present invention and is 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 in the scope of protection of the present invention. Sequence Listing <110> South-Central University for Nationalities <120> A SSR molecular marker primer combination and application of Chrysanthemum macrophylla <160> 15 <170> SIPOSequenceListing 1.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <400> 1 ccggacgggg aatctagaat 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <400> 2 tcacatacct cgacttccca 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <400> 3 ttccgcaatt atggcttccg 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <400> 4 tggttctcgt cggcttacat 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <400> 5 ttgattcccg cgtgttacac 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <400> 6 tttctcaagg gcaagggtct 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <400> 7 ctcagccatc tctcccactt 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <400> 8 tggacgaaat cagcgaggta 20 <210> 9 <211> twenty two <212> DNA <213> Artificial Sequence <400> 9 gctactataa ccttcccgac ca 22 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <400> 10 ggctccggtg fathergagg <210> 11 <211> 263 <212> DNA <213> Liquids (Chrysosplenium macrophyllum) <400> 11 ccggacgggg aatctagaat ataattgttt agtaattggg tggacgatac tttgtataca gactcacgag agtctctgaa tgctcaagca ttctagattg tagctttttt tatatata tttatatata tatatttta ggtgcaaaaa taaagatttt atttttggta accctagtaa agaatgggct gttttatgat tgtttctttg aaacaatcag gagagggtaa ggattagatc aaatgggaag tcgaggtatg tga <210> 12 <211> 215 <212> DNA <213> Liquids (Chrysosplenium macrophyllum) <400> 12 ttccgcaatt atggcttccg tggggtttct fatherttgg atatgctatt tcggggtcaa 120. 120. 120. 120. 120. 120. 120. 120. 120. 120. 120. 120. 120 180. 180. 180. 180. 180. 180. 180. 180. 180. 180 aagaaaactt tgtgtatgta agccgacgag aacca 215 <210> 13 <211> 184 <212> DNA <213> Chrysosplenium macrophyllum <400> 13 ttgattcccg cgtgttacac ttttgaccaa aaaagtttta tcaagtccaa aaattttttt 60 tttttataaa aattatgaaa actttctcga attgaatcct ttcgatttct atatcgaaga 120 tatacttacg aagttgttcc aacttattca ttggcactaa ccctagaccc ttgcccttga 180 gaaa 184 <210> 14 <211> 163 <212> DNA <213> Chrysosplenium macrophyllum <400> 14 ctcagccatc tctcccactt ttaaaaggat aattactatg ttatccttac gcataaagta 60 aggattgaaa aaatatcttc tctctttaat tctaattata tagatatata taacttttac 120 aataaaaaaa aaaaaagaaa gaatacctcg ctgatttcgt cca 163 <210> 15 <211> 187 <212> DNA <213> Chrysosplenium macrophyllum <400> 15 gctactataa ccttcccgac cactattttt tttaaggcat ttcgccgcta aatcaaaaat 60 tcattgatac caagatacct ggtcaaaaaa aaaaacaaga gtaaatatga atagataaaa 120 gaatagtggt tccgtcgttt ctatggttac ttcttaaacg gtgaggtcct ctctatacac 180 cggagcc 187

Claims

1. A SSR molecular marker primer combination for Chrysanthemum macrophylla, Features: The SSR molecular markers include five groups: cpSSR2, cpSSR5, cpSSR11, cpSSR12, and cpSSR23, and their nucleotide sequences are shown in SEQ ID NOs. 11 to 15; The primer combination comprises: Primers for amplifying molecular marker cpSSR2, the nucleotide sequences of the forward and reverse primers are shown in SEQ ID NOs. 1-2; Primers for amplifying molecular marker cpSSR5, the nucleotide sequences of the forward and reverse primers are shown in SEQ ID NOs. 3-4; Primers for amplifying molecular marker cpSSR11, the nucleotide sequences of the forward and reverse primers are shown in SEQ ID NOs. 5-6; Primers for amplifying molecular marker cpSSR12, the nucleotide sequences of the forward and reverse primers are shown in SEQ ID NOs. 7 to 8; The nucleotide sequences of the forward and reverse primers for amplifying the molecular marker cpSSR23 are shown in SEQ ID NOs. 9-10.

2. Application of the SSR molecular marker primer combination of Chrysanthemum chinense as described in claim 1 in genetic diversity analysis of Chrysanthemum chinense population.

3. Application of the SSR molecular marker primer combination of Chrysanthemum chinense according to claim 2 in genetic diversity analysis of Chrysanthemum chinense population, Features: The specific steps are as follows: (1) Genomic DNA extraction: The high-quality total genomic DNA of C. chinensis was extracted by the modified CTAB method. The DNA quality and concentration were determined by an ultraviolet spectrophotometer and confirmed by 1% agarose gel electrophoresis. (2) PCR reaction system: Five groups were prepared, each with a total reaction volume of 10 μL, including 5 μL of 2×T5 SuperPCR Mix (PAGE), 10 μmol·L -1 0.4 μL each of forward and reverse primers, 1 μL of total genomic DNA, ddHO 2 O 3.2 μL; The PCR reaction program was as follows: pre-denaturation at 98°C for 2 min; denaturation at 98°C for 10 s, annealing at 58°C for 10 s, and extension at 72°C for 10 s, for a total of 35 cycles; total extension at 72°C for 2 min and storage at 4°C; (3) Electrophoresis detection: The amplified products were separated by 4% polyacrylamide gel electrophoresis, the electrophoresis buffer was 1×TBE, and the electrophoresis was carried out at 90W constant voltage for 1.5 to 2.0 h. The polymorphism of the primers was detected by silver staining, and the electrophoresis images were collected by taking photos; (4) Data analysis: Based on the electrophoresis images, the bands that are reproducible and easily distinguishable on the image are recorded as "1" by manual reading, and the absence of bands at the same position is recorded as "0". The data are then input into Excel to establish the original "01" data matrix; The genetic similarity coefficient was calculated using NTSYS software, and the unweighted paired arithmetic mean method (UPGMA) was used for genetic similarity cluster analysis. GENDIVE version 3.06 software was used to calculate diversity indicators such as the number of alleles Na, the effective number of alleles Ne, the observed heterozygosity Ho and the expected heterozygosity He.

4. A method for obtaining the SSR molecular marker primer combination of Chrysanthemum macrophylla described in claim 1, Features: The acquisition method includes the following steps: (1) Acquisition of Chloroplast Genome Sequence and Identification of cpSSR Loci of Chrysanthemum chinense The chloroplast genome sequence MK973001 of C. chinensis was downloaded from the GenBank public database. The SSR loci distributed in the chloroplast genome were searched using the MISA perl script software to obtain the nucleotide sequences of the primary screening cpSSR molecular markers. The search criteria were as follows: the minimum number of repeats for mononucleotide, dinucleotide, trinucleotide, tetranucleotide, pentanucleotide and hexanucleotide repeat types was set to 10, 6, 5, 5, 5 and 5, respectively. (2) cpSSR primer design Primer 3 software was used to design primers for the nucleotide sequence of the cpSSR molecular markers screened in the previous step. The primer design principles were: GC content 40-60%, annealing temperature 57-60°C, primer length 18-23 bp, and expected amplification product length 100-300 bp; the nucleotide sequence of the primary screening cpSSR primers was obtained; (3) Secondary screening of cpSSR primers First, the improved CTAB method is used to extract high-quality total genomic DNA of Chrysanthemum grandiflorum, and then this is used as a template to construct a PCR reaction system for the initial screening cpSSR primers in the previous step, and a PCR reaction is carried out. After the PCR amplification is completed, gel electrophoresis is used to separate the amplified products, silver staining is used to detect the polymorphism of the primers, and the electrophoresis image is collected by photographing; the primers corresponding to the electrophoresis bands with good stability, high polymorphism and high clarity are selected to obtain the Chrysanthemum grandiflorum SSR molecular marker primer combination.

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