SSR molecular marker primer combinations for identification of Chrysanthemum indicum and Chrysanthemum indicum germplasm resources

Abstract

The present invention discloses a set of SSR molecular markers for identifying wild chrysanthemum and Shennong fragrant chrysanthemum germplasm resources, consisting of 21 pairs of SSRs numbered SSR01-SSR21. The primer sequences for amplifying these 21 pairs of molecular markers are shown in SEQ ID NO: 1-42, and the present invention belongs to the field of molecular biology technology. Using these 21 pairs of SSR molecular markers, the collected wild chrysanthemum and Shennong fragrant chrysanthemum can be identified and the germplasm genetic diversity analyzed. The present invention uses SSR markers for the first time to jointly detect the genetic diversity of wild chrysanthemum and Shennong fragrant chrysanthemum germplasm. This set of SSR molecular markers has good polymorphism and a high level of genetic diversity. It can be used in the fields of kinship analysis, resource genetic evaluation, identification of interspecific hybrid offspring, positioning of trait genes, and molecular marker-assisted breeding of wild chrysanthemum and Shennong fragrant chrysanthemum.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology, and in particular relates to a group of SSR molecular marker primer combinations for identifying chrysanthemum indicum and Shennong fragrant chrysanthemum germplasm resources and applications thereof. Technical Background

[0002] Chrysanthemum indicum L. is a perennial herbaceous plant in the genus Chrysanthemum, Asteraceae. C. indicum var. aromaticum is a variant of Chrysanthemum indicum. Chrysanthemum indicum has the properties of clearing heat and detoxifying, dispelling wind and dissipating heat. C. indicum var. aromaticum is named for the rich aroma emitted by the entire plant. Both species are widely used in pharmaceuticals, food, and daily necessities. Chrysanthemum indicum is currently widespread across much of China, with diverse habitats resulting in a complex genetic background. C. indicum var. aromaticum is confined to the high-altitude region of Shennongjia, and population genetic studies have yet to be conducted. This complex and ambiguous genetic background hinders the development and utilization of both C. indicum and C. indicum germplasm resources.

[0003] Molecular markers can reflect differences in plant genetics at the DNA level and are widely used in plant genetics and breeding. Simple sequence repeats (SSRs) are widely distributed across eukaryotic genomes. Due to the varying types and repeat counts of the repeating motifs within SSR loci, they exhibit a high degree of polymorphism. Compared to other molecular markers, SSRs offer advantages such as high polymorphic information content, codominance, simplified technology, good reproducibility, and strong specificity, making them considered one of the most reliable molecular markers. Summary of the Invention

[0004] The purpose of the present invention is to overcome the shortcomings of the existing technology and provide a set of SSR molecular marker primer combinations for identifying wild chrysanthemum and Shennong fragrant chrysanthemum germplasm resources.

[0005] In order to achieve the above objectives, the present invention is implemented through the following technical solutions.

[0006] A set of SSR molecular markers for identification of wild chrysanthemum and Shennong fragrant chrysanthemum germplasm resources consists of 21 pairs of SSRs. The primer numbers are SSR01, SSR02, SSR03, SSR04, SSR05, SSR06, SSR07, SSR08, SSR09, SSR10, SSR11, SSR12, SSR13, SSR14, SSR15, SSR16, SSR17, SSR18, SSR19, SSR20, and SSR21. The primer sequences used to amplify these 21 pairs of molecular markers are as follows:

[0007] (1) SSR01 primers for amplifying SSR molecular markers:

[0008] SEQ ID NO:1:SSR01-F:5'-GCACCAGTCACCTTCTTGGT-3'

[0009] SEQ ID NO:2:SSR01-R:5'-GGTGGGAGTGGGAGGTATTT-3'

[0010] (2) SSR02 primers for amplifying SSR molecular markers:

[0011] SEQ ID NO:3:SSR02-F:5'-ACAACGGGCTCTTCAATACG-3'

[0012] SEQ ID NO:4:SSR02-R:5'-CCCAAGACCTAGCCAAAAGA-3'

[0013] (3) SSR03 primer for amplifying SSR molecular markers:

[0014] SEQ ID NO:5:SSR03-F:5'-TTGGTCAAACTGGGCAAAAT-3'

[0015] SEQ ID NO:6:SSR03-R:5'-GGGGTCGATGGTATTTGGTA-3'

[0016] (4) SSR04 primers for amplifying SSR molecular markers:

[0017] SEQ ID NO:7:SSR04-F:5'-TATCATCCGCCAATTCATCA-3'

[0018] SEQ ID NO:8:SSR04-R:5'-ATGTAGCGCCAGAAGACGTT-3'

[0019] (5) SSR05 primers for amplifying SSR molecular markers:

[0020] SEQ ID NO:9:SSR05-F:5'-TTGATCGTGCAGAAGTGGAG-3'

[0021] SEQ ID NO:10:SSR05-R:5'-CCTAAGCGCATGTTCCTAC-3'

[0022] (6) SSR06 primers for amplifying SSR molecular markers:

[0023] SEQ ID NO:11:SSR06-F:5'-CCCGTTTGTGCTAATGGAGT-3'

[0024] SEQ ID NO:12:SSR06-R:5'-AAGAGCAAACCGAGAAGTGC-3'

[0025] (7) SSR07 primers for amplifying SSR molecular markers:

[0026] SEQ ID NO:13:SSR07-F:5'-ACCCACGGTCAAATCACAAT-3'

[0027] SEQ ID NO:14:SSR07-R:5'-CTCACTTGTGGGAGCCAAAC-3'

[0028] (8) SSR08 primers for amplifying SSR molecular markers:

[0029] SEQ ID NO:15:SSR08-F:5'-CAAAACCTTTCACCCCAATC-3'

[0030] SEQ ID NO:16:SSR08-R:5'-ATCATGTGGTGGTCATGTGG-3'

[0031] (9) SSR09 primers for amplifying SSR molecular markers:

[0032] SEQ ID NO:17:SSR09-F:5'-ACCTCACCCTCCCTCATTCT-3'

[0033] SEQ ID NO:18:SSR09-R:5'-AGCCATACGGTGGCTTTACCA-3'

[0034] (10) SSR10 primers for amplifying SSR molecular markers:

[0035] SEQ ID NO:19:SSR10-F:5'-TTTGTGCAGGAGGGAAGTTT-3'

[0036] SEQ ID NO:20:SSR10-R:5'-CAACCATTATTTGGTCGGCTA-3'

[0037] (11) SSR11 primers for amplifying SSR molecular markers:

[0038] SEQ ID NO:21:SSR11-F:5'-AAAGATGCAGCAACCACCTC-3'

[0039] SEQ ID NO:22:SSR11-R:5'-TCTTTTGTAGGACGGCTTGG-3'

[0040] (12) SSR12 primers for amplifying SSR molecular markers:

[0041] SEQ ID NO:23:SSR12-F:5'-CCCGTGTGACATAATTCTATCTAGC-3'

[0042] SEQ ID NO:24:SSR12-R:5'-CTGCATCTGGATCTGGATTTC-3'

[0043] (13) SSR13 primers for amplifying SSR molecular markers:

[0044] SEQ ID NO:25:SSR13-F:5'-CTTTTCCTCACCACCCAAAA-3'

[0045] SEQ ID NO:26:SSR13-R:5'-AACAATCCACCTGGCAGAAC-3'

[0046] (14) SSR14 primers for amplifying SSR molecular markers:

[0047] SEQ ID NO:27:SSR14-F:5'-TCAAACACCACCCAACAAAA-3'

[0048] SEQ ID NO:28:SSR14-R:5'-ATATTCGGCAACAAGCAACC-3'

[0049] (15) SSR15 primers for amplifying SSR molecular markers:

[0050] SEQ ID NO:29:SSR15-F:5'-GGATTCGTTGGAGAGACGAG-3'

[0051] SEQ ID NO:30:SSR15-R:5'-TTGTTGCGGTTGTGATTGTT-3'

[0052] (16) SSR16 primers for amplifying SSR molecular markers:

[0053] SEQ ID NO:31:SSR16-F:5'-ATTCTCGGCATTTGTTTTGG-3'

[0054] SEQ ID NO:32:SSR16-R:5'-GAACCCCTATGTCCCATCCT-3'

[0055] (17) SSR17 primers for amplifying SSR molecular markers:

[0056] SEQ ID NO:33:SSR17-F:5'-TTCCCATCATTTTCCCAAAC-3'

[0057] SEQ ID NO:34:SSR17-R:5'-TAACAGCAGCAGGAGCAGAA-3'

[0058] (18) SSR18 primers for amplifying SSR molecular markers:

[0059] SEQ ID NO:35:SSR18-F:5'-CCCGGTTTGAAATCTAGGAA-3'

[0060] SEQ ID NO:36:SSR18-R:5'-TCCCAAATGGTTTCGACAAT-3'

[0061] (19) SSR19 primers for amplifying SSR molecular markers:

[0062] SEQ ID NO:37:SSR19-F:5'-TCGAACCGTTTCACTCTTCC-3'

[0063] SEQ ID NO:38:SSR19-R:5'-AACAATCCAAGCCTGACCAC-3'

[0064] (20) SSR20 primers for amplifying SSR molecular markers:

[0065] SEQ ID NO:39:SSR20-F:5'-TTCCTTCATCCTGGACAACC-3'

[0066] SEQ ID NO:40:SSR20-R:5'-ACCACCAGAACCAGAACCAG-3'

[0067] (21) SSR21 primers for amplifying SSR molecular markers:

[0068] SEQ ID NO:41:SSR21-F:5'-ACCATCCGCAGGTTTACAAG-3'

[0069] SEQ ID NO:42:SSR21-R:5'-CCCACGATGTCGTTCTCTTT-3'

[0070] In another aspect, the present invention provides the use of the above-mentioned SSR molecular marker primer combination in the screening of wild chrysanthemum and Shennong fragrant chrysanthemum or the identification of germplasm resources.

[0071] In another aspect, the present invention provides a method for screening or identifying germplasm resources of wild chrysanthemum and Shennong fragrant chrysanthemum, the method comprising the following steps:

[0072] First, based on the genome information of the closely related species of Shennong chrysanthemum, Juhuanao, and the transcriptome data of Shennong chrysanthemum (http: / / www.amwayabrc.com / ), MISA software (http: / / pgrc.ipk-gatersleben.de / misa / ) was used to screen SSR sites in the nuclear genome and transcriptome, respectively. The search criteria parameters were set to mono-, di-, tri-, tetra-, penta-, and hexanucleotide repeats of at least 10, 6, 5, 5, 5, and 5 times. The obtained SSR sites and flanking conserved sequences were batch imported into Primer3 software (https: / / sourceforge.net / projects / primer3) for primer design. The parameters for primer design were: (1) primer length 18-27 bp; (2) PCR product size 100-500 bp; (3) GC content 40-60%; (4) annealing temperature 55-65°C. The synthetic fluorescent primers are prepared by adding M13 (GTAAAACGACGGCCAGT) as a linker sequence at the 5' end of the positive primer, and the linker sequence is modified with different fluorescent groups (Fam / Hex / Rox / Tamra).

[0073] The specific steps of this method are as follows:

[0074] (1) Extraction of total DNA from the plants to be tested;

[0075] (2) using the total DNA extracted in step (1) as a template, PCR amplification was performed using the screened and designed SSR molecular marker primer combinations;

[0076] (3) detecting the PCR amplification product of step (2) by capillary electrophoresis and collecting data;

[0077] (4) Based on the data obtained in step (3), genetic diversity indicators, clustering and polymorphism information content (PIC) were calculated and analyzed.

[0078] Specifically, the PCR amplification system described in step (2) is: 2ul of DNA template (50ng / μl), 0.5ul of 5μM forward primer, 0.5ul of 5μM reverse primer, 5μl of 2×Es Taq Master Mix, 1.85μl of double-distilled water, and 0.15ul of Fam / Hex / Rox / Tamra, and the total volume of the system is 10μl.

[0079] The PCR amplification program was as follows: 95°C, 2 min; 95°C, 30 sec; 55°C, 30 sec; 72°C, 30 sec; 35 cycles; 72°C, 2 min; and storage at 4°C.

[0080] The advantages of the present invention are that, based on the genomic information of the closely related species of Chrysanthemum chinense and the transcriptome data of Chrysanthemum chinense, 21 pairs of primers with good polymorphism were selected to comprehensively amplify the DNA of 203 samples of Chrysanthemum indicum and Chrysanthemum indicum. This is the first time that SSR markers have been used to jointly detect the genetic diversity of Chrysanthemum indicum and Chrysanthemum indicum germplasm. This set of SSR molecular markers has good polymorphism and a high level of genetic diversity, laying a foundation for the identification of interspecific hybrid progeny of Chrysanthemum indicum and Chrysanthemum indicum, molecular marker-assisted breeding, genome comparative studies, genetic map construction, QTL mapping of important traits, utilization and preservation of core resources, and molecular-assisted breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] Figure 1 This is a partial amplification capillary electrophoresis detection peak graph of SSR01. The five small graphs from top to bottom in the figure represent the detection peak graphs of SSR01 for a total of five samples, namely HP-CIA14, SND-CIA58, TMY-CIA151, SND-CI90, and HS-CI194.

[0082] Figure 2 This is the genetic clustering relationship diagram of the samples when K=3. DETAILED DESCRIPTION

[0083] The present invention is further described below in conjunction with the accompanying drawings and specific examples. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified. Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art.

[0084] Example 1

[0085] Test materials

[0086] This study used 41 germplasm resources from two wild chrysanthemum populations in Shennongjia, 151 germplasm resources from three Shennong fragrant chrysanthemum populations in Shennongjia, and 11 wild chrysanthemum germplasm resources from Hongshan District, Wuhan, collected and preserved by the Hubei Shennongjia Institute of Traditional Chinese Medicine. Specific information is shown in Table 1 below. All experimental materials were planted in a greenhouse at the Hubei Shennongjia Institute of Traditional Chinese Medicine.

[0087] Table 1 Basic information of 203 germplasm resources

[0088]

[0089]

[0090] Test methods

[0091] 1. Total DNA Extraction

[0092] In this study, the modified CTAB method was used to extract genomic DNA from Shennong Xiangju and Yeju. The specific steps are as follows:

[0093] (1) Weigh 0.03 g of dried leaves and place them in a 2.0 ml EP tube. Add two steel balls and mill in a ball mill at 60 Hz for 2 min. Perform the operation in a clean environment to avoid cross-contamination of the weighed samples.

[0094] (2) Add 800 μl of pre-cooled nuclear fractionation solution to the EP tube, cover and mix for 5 minutes, then centrifuge at 12,000 rpm for 10 minutes. After centrifugation, discard the supernatant and repeat the above steps 2-3 times until the supernatant no longer has a noticeable green color.

[0095] (3) Add 800 μl of preheated CTAB solution to 65°C to the EP tube. Insert the EP tube into the floating plate and place it in a 65°C constant temperature water bath for 40 min. Gently shake the tube every 10 min. After the water bath ends, add 750 μl of chloroform-isoamyl alcohol mixture (volume ratio 24:1) to the EP tube, mix well, and centrifuge at 12,000 rpm for 5 min. Remove the EP tube after centrifugation.

[0096] (4) Use a 1000ul pipette to draw 750ul of the supernatant into a new 2.0ml EP tube, add 750ul of chloroform-isoamyl alcohol mixture (volume ratio 24:1), mix well, and centrifuge at 12000r / min for 5min. After centrifugation, remove the EP tube.

[0097] (5) Use a 1000ul pipette to draw 700ul of the supernatant into a new 1.5ml EP tube, add 600ml of -20℃ pre-cooled isopropanol, and freeze at -20℃ for 30min. After taking it out, let it stand at room temperature for 20min. Then draw 800ul of the supernatant and transfer it to the DNA adsorption column (the adsorption column is placed in the collection tube). Centrifuge at 12000r / min for 1min. After centrifugation, remove the EP tube.

[0098] (6) Discard the waste liquid in the collection tube and add 700 μl of 70% ethanol solution to the adsorption column. Let it stand for 5 minutes to allow the ethanol to fully dissolve the impurities. Centrifuge at 12,000 rpm for 1 minute. Repeat this operation 2-3 times until the ethanol solution in the collection tube has no obvious color.

[0099] (7) Place the empty adsorption column in a centrifuge and centrifuge at 12,000 rpm for 1 minute. Remove the column and transfer it to a 1.5 ml EP tube. Open the cap of the column and air-dry it in a fume hood for 30 minutes until the ethanol smell is gone.

[0100] (8) Use a pipette to draw up 40 μl of sterile ultrapure water and drop it vertically into the middle of the filter layer of the adsorption column. Let it stand for 5 minutes and then centrifuge at 12,000 rpm for 1 minute. Repeat this operation twice. Write the number on the EP tube cap and store the extracted DNA solution at -20°C.

[0101] (9) 2 μL of DNA was used for 1.0% agarose gel electrophoresis and 1 μL of DNA was used for concentration measurement using a NanoDrop spectrophotometer.

[0102] 2. SSR molecular markers and primer combinations

[0103] 2.1. Based on the genome information of the closely related species of Shennong chrysanthemum, Juhuanao, and the transcriptome data of Shennong chrysanthemum (http: / / www.amwayabrc.com / ), MISA software (http: / / pgrc.ipk-gatersleben.de / misa / ) was used to screen SSR loci in the nuclear genome and transcriptome, respectively. The search criteria parameters were set as mono-, di-, tri-, tetra-, penta-, and hexanucleotide repeats of at least 10, 6, 5, 5, 5, and 5 times. The obtained SSR loci and flanking conserved sequences were batch imported into Primer3 software (https: / / sourceforge.net / projects / primer3) for primer design. The parameters set for primer design were: (1) primer length 18-27 bp; (2) PCR product size 100-500 bp; (3) GC content 40-60%; (4) annealing temperature 55-65°C.

[0104] The synthetic fluorescent primers are prepared by adding M13 (GTAAAACGACGGCCAGT) as a linker sequence at the 5' end of the positive primer, and the linker sequence is modified with different fluorescent groups (Fam / Hex / Rox / Tamra).

[0105] 2.2. Primers were designed based on SSR molecular markers. The primers are shown in Table 2 below.

[0106] Table 2 SSR molecular marker primers

[0107]

[0108]

[0109] 3. Sample Amplification PCR amplification of the samples was performed using the primers listed in Table 2.

[0110] The PCR amplification system was as follows: 2ul DNA template (50ng / μl), 0.5ul 5μM forward primer, 0.5ul reverse primer, 5μl 2×EsTaq Master Mix, 1.85μl double-distilled water, 0.15ul Fam / Hex / Rox / Tamra, and the total volume of the system was 10μl.

[0111] The PCR amplification program was as follows: 95°C, 2 min; 95°C, 30 sec; 55°C, 30 sec; 72°C, 30 sec; 35 cycles; 72°C, 2 min; and storage at 4°C.

[0112] 4. Detection

[0113] Amplified products were detected by 1.0% agarose gel electrophoresis. After amplification, the PCR products were diluted 10-fold. Deionized formamide (HIDI) and the LIZ500 molecular weight internal standard were mixed at a volume ratio of 100:1. 9 μL of this mixture and 1 μL of the PCR product were added to a 96-well sample plate. Finally, samples from four 96-well plates containing four different fluorescent groups were evenly mixed in a single loading plate. The mixed PCR fluorescent products were detected by fluorescence capillary electrophoresis using an ABI3730XL DNA sequencer, and the raw data were banded using GeneMarker V2.2.0 software.

[0114] 5. Data Analysis

[0115] Genetic diversity parameters of population samples, including the number of alleles (N), were calculated using POPGENE version 1.3.2. a ), effective number of alleles (Ne ), observed heterozygosity (H o ), expected heterozygosity (H e ), Shannon's information index (I), polymorphic information content (PIC) was calculated using PIC-Cale software, cluster analysis was performed by calculating SM distance using UPGMA (unweighted pair group method with arithmetic) using R language, and population structure analysis was performed using STRCTURE software.

[0116] Test results

[0117] 1. The polymorphism of the SSR molecular marker primers screened and designed is good. Figure 1 Shown is the capillary electrophoresis detection peak diagram of some samples after amplification of one of the SSR01 molecular markers. It can be seen from the figure that the amplification effect of the SSR molecular marker primer is good, with a stable baseline, sharp peak shape and good polymorphism.

[0118] 2. Genetic Analysis and Clustering

[0119] All samples were amplified using this set of SSR molecular markers, and the genetic diversity parameters of the primers were calculated as shown in Table 3 ;

[0120] Table 3: Genetic diversity of 21 pairs of SSR markers

[0121]

[0122]

[0123] The results showed that 5 to 16 alleles were amplified at a single SSR locus, and the effective alleles (N e ) ranged from 1.278 to 4.632, and the mean PIC information index of these SSR loci was 0.529. The corresponding diversity levels of other genetic parameters were also high, indicating that these 21 primer pairs had high polymorphism and could be used for population genetic analysis of the experimental materials.

[0124] The NJ tree results showed that Hongshan Chrysanthemum (HS-CI) clustered into a single branch, while most of the Shennong Xiangju samples and suspected Chrysanthemum samples clustered separately. Only a few Shennong Xiangju individuals and a few suspected Chrysanthemum individuals showed inter-population sample mixing. The results of STRUCTURE analysis showed that when K = 2, Hongshan Chrysanthemum (HS-CI) had obvious genetic differentiation from other population samples; when K = 3 ( Figure 2), the suspected wild chrysanthemum samples (SND-CI and TMY-CI) from Shennongding and Tianmenya were further separated from other Shennong fragrant chrysanthemum samples; in the Shennongjia area, the genetic differences between Shennong fragrant chrysanthemum and wild chrysanthemum were large, and the genetic differences between Shennong fragrant chrysanthemums in different regions were small.

[0125] This study used SSR markers to detect the genetic diversity of wild chrysanthemum and Shennong fragrant chrysanthemum, and the experimental results were accurate and reliable. Unlike traditional polyacrylamide gel electrophoresis detection technology, this study used capillary electrophoresis detection technology, with a detection error within 1-2bp. Compared with traditional technologies, this method greatly improved detection efficiency and accuracy. In addition, the use of multiple fluorescent markers, such as Fam / Hex / Rox / Tamra mixed plate loading, further improved capillary electrophoresis detection efficiency and significantly reduced detection costs. The identification and evaluation of genetic diversity using comprehensive molecular markers ensured greater representativeness and coverage of the genome, and can well represent the genetic diversity of wild chrysanthemum and Shennong fragrant chrysanthemum germplasm resources. Experiments confirmed that this batch of SSR molecular markers has good polymorphism and can be used in the genetic evaluation of wild chrysanthemum and Shennong fragrant chrysanthemum resources, identification of interspecific hybrid offspring, and molecular marker-assisted breeding. Sequence Listing <110> Hubei University of Chinese Medicine <120> SSR molecular marker primer combinations for identification of Chrysanthemum indicum and Chrysanthemum indicum germplasm resources <160> 42 <170> SIPOSequenceListing 1.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <400> 1 gcaccagtca ccttcttggt 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <400> 2 ggtgggagtg ggaggtattt 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <400> 3 acaacgggct cttcaatacg 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <400> 4 cccaagacct agccaaaaga 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <400> 5 ttggtcaaac tgggcaaaat 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <400> 6 ggggtcgatg gtatttggta 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <400> 7 tatcatccgc caattcatca 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <400> 8 atgtagcgcc agaagacgtt 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <400> 9 ttgatcgtgc agaagtggag 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <400> 10 cctaagcgca tgttccctac 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <400> 11 cccgtttgtg ctaatggagt 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <400> 12 aagagcaaac cgagaagtgc 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <400> 13 acccacggtc aaatcacaat 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <400> 14 ctcacttgtg ggagccaaac 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <400> 15 caaaaccttt caccccaatc 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <400> 16 atcatgtggt ggtcatgtgg 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <400> 17 acctcaccct ccctcattct 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <400> 18 agccatacgt ggctttacca 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <400> 19 tttgtgcagg agggaagttt 20 <210> 20 <211> twenty one <212> DNA <213> Artificial Sequence <400> 20 caaccattat ttggtcggct a 21 <210> twenty one <211> 20 <212> DNA <213> Artificial Sequence <400> twenty one aaagatgcag caaccacctc 20 <210> twenty two <211> 20 <212> DNA <213> Artificial Sequence <400> twenty two tcttttgtag gacggcttgg 20 <210> twenty three <211> 25 <212> DNA <213> Artificial Sequence <400> twenty three cccgtgtgac ataattctat ctagc 25 <210> twenty four <211> twenty one <212> DNA <213> Artificial Sequence <400> twenty four ctgcatctgg atctggattt c 21 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <400> 25 cttttcctca ccacccaaaa 20 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <400> 26 aacaatccac ctggcagaac 20 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <400> 27 tcaaacacca cccaacaaaa 20 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <400> 28 atattcggca acaagcaacc 20 <210> 29 <211> 20 <212> DNA <213> Artificial Sequence <400> 29 ggattcgttg gagagacgag 20 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <400> 30 ttgttgcggt tgtgattgtt 20 <210> 31 <211> 20 <212> DNA <213> Artificial Sequence <400> 31 attctcggca tttgttttgg 20 <210> 32 <211> 20 <212> DNA <213> Artificial Sequence <400> 32 gaacccctat gtcccatcct 20 <210> 33 <211> 20 <212> DNA <213> Artificial Sequence <400> 33 ttcccatcat tttcccaaac 20 <210> 34 <211> 20 <212> DNA <213> Artificial Sequence <400> 34 taacagcagc aggagcagaa 20 <210> 35 <211> 20 <212> DNA <213> Artificial Sequence <400> 35 cccggtttga aatctaggaa 20 <210> 36 <211> 20 <212> DNA <213> Artificial Sequence <400> 36 tcccaaatgg tttcgacaat 20 <210> 37 <211> 20 <212> DNA <213> Artificial Sequence <400> 37 tcgaaccgtt tcactcttcc 20 <210> 38 <211> 20 <212> DNA <213> Artificial Sequence <400> 38 aacaatccaa gcctgaccac 20 <210> 39 <211> 20 <212> DNA <213> Artificial Sequence <400> 39 ttccttcatc ctggacaacc 20 <210> 40 <211> 20 <212> DNA <213> Artificial Sequence <400> 40 accaccagaa ccagaaccag 20 <210> 41 <211> 20 <212> DNA <213> Artificial Sequence <400> 41 accatccgca ggtttacaag 20 <210> 42 <211> 20 <212> DNA <213> Artificial Sequence <400> 42 cccacgatgt cgttctcttt 20

Claims

1. An application of an SSR molecular marker primer combination in the identification of wild chrysanthemum and Shennong fragrant chrysanthemum germplasm resources, characterized in that: The SSR molecular markers include SSR01, SSR02, SSR03, SSR04, SSR05, SSR06, SSR07, SSR08, SSR09, SSR10, SSR11, SSR12, SSR13, SSR14, SSR15, SSR16, SSR17, SSR18, SSR19, SSR20 and SSR21, The primer combination is: (1) SSR01 primers for amplifying SSR molecular markers: SSR01-F: 5'-GCACCAGTCACCTTCTTGGT-3' SSR01-R: 5'-GGTGGGAGTGGGAGGTATTT-3' (2) SSR02 primers for amplifying SSR molecular markers: SSR02-F:5'-ACAACGGGCTCTTCAATACG-3' SSR02-R: 5'-CCCAAGACCTAGCCAAAAGA-3' (3) SSR03 primers for amplifying SSR molecular markers: SSR03-F:5'-TTGGTCAAACTGGGCAAAAT-3' SSR03-R: 5'-GGGGTCGATGGTATTTGGTA-3' (4) SSR04 primers for amplifying SSR molecular markers: SSR04-F:5'-TATCATCCGCCAATTCATCA-3' SSR04-R: 5'-ATGTAGCGCCAGAAGACGTT-3' (5) SSR05 primers for amplifying SSR molecular markers: SSR05-F: 5'-TTGATCGTGCAGAAGTGGAG-3' SSR05-R: 5'-CCTAAGCGCATGTTCCTAC-3' (6) SSR06 primers for amplifying SSR molecular markers: SSR06-F: 5'-CCCGTTTGTGCTAATGGAGT-3' SSR06-R: 5'-AAGAGCAAACCGAGAAGTGC-3' (7) SSR07 primers for amplifying SSR molecular markers: SSR07-F:5'-ACCCACGGTCAAATCACAAT-3' SSR07-R: 5'-CTCACTTGTGGGAGCCAAAC-3' (8) SSR08 primers for amplifying SSR molecular markers: SSR08-F: 5'-CAAAACCTTTCACCCCAATC-3' SSR08-R: 5'-ATCATGTGGTGGTCATGTGG-3' (9) SSR09 primers for amplifying SSR molecular markers: SSR09-F: 5'-ACCTCACCCTCCCTCATTCT-3' SSR09-R: 5'-AGCCATACGTGGCTTTTACCA-3' (10) SSR10 primers for amplifying SSR molecular markers: SSR10-F: 5'-TTTGTGCAGGAGGGAAGTTT-3' SSR10-R: 5'-CAACCATTATTTGGTCGGCTA-3' (11) SSR11 primers for amplifying SSR molecular markers: SSR11-F: 5'-AAAGATGCAGCAACCACCTC-3' SSR11-R: 5'-TCTTTTGTAGGACGGCTTGG-3' (12) SSR12 primers for amplifying SSR molecular markers: SSR12-F: 5'-CCCGTGTGACATAATTCTATCTAGC-3' SSR12-R: 5'-CTGCATCTGGATCTGGATTTC-3' (13) SSR13 primers for amplifying SSR molecular markers: SSR13-F: 5'-CTTTTCCTCACCACCCAAAA-3' SSR13-R: 5'-AACAATCCACCTGGCAGAAC-3' (14) SSR14 primers for amplifying SSR molecular markers: SSR14-F: 5'-TCAAACACCACCCAACAAAA-3' SSR14-R: 5'-ATATTCGGCAACAAGCAACC-3' (15) SSR15 primers for amplifying SSR molecular markers: SSR15-F: 5'-GGATTCGTTGGAGAGACGAG-3' SSR15-R: 5'-TTGTTGCGGTTGTGATTGTT-3' (16) SSR16 primers for amplifying SSR molecular markers: SSR16-F: 5'-ATTCTCGGCATTTGTTTTGG-3' SSR16-R: 5'-GAACCCCTATGTCCCATCCT-3' (17) SSR17 primers for amplifying SSR molecular markers: SSR17-F: 5'-TTCCCATCATTTTCCCAAAC-3' SSR17-R: 5'-TAACAGCAGCAGGAGCAGAA-3' (18) SSR18 primers for amplifying SSR molecular markers: SSR18-F: 5'-CCCGGTTTGAAATCTAGGAA-3' SSR18-R: 5'-TCCCAAATGGTTTCGACAAT-3' (19) SSR19 primers for amplifying SSR molecular markers: SSR19-F: 5'-TCGAACCGTTTCACTCTTCC-3' SSR19-R: 5'-AACAATCCAAGCCTGACCAC-3' (20) SSR20 primers for amplifying SSR molecular markers: SSR20-F: 5'-TTCCTTCATCCTGGACAACC-3' SSR20-R: 5'-ACCACCAGAACCAGAACCAG-3' (21) SSR21 primers for amplifying SSR molecular markers: SSR21-F:5'-ACCATCCGCAGGTTTACAAG-3' SSR21-R: 5'-CCCACGATGTCGTTCTCTTT-3'.

2. A method for identifying germplasm resources of wild chrysanthemum and Shennong fragrant chrysanthemum, characterized in that The following steps are involved: (1) Extraction of total DNA from the plants to be tested; (2) using the total DNA extracted in step (1) as a template, and performing PCR amplification using the SSR molecular marker primer combination described in claim 1; (3) detecting the PCR amplification product of step (2) by capillary electrophoresis and collecting data; (4) Based on the data obtained in step (3), genetic diversity indicators, clustering and polymorphism information content (PIC) were calculated and analyzed.

3. The method according to claim 2, characterized in that The PCR amplification procedure in step (2) is: 95°C, 2 min; 95°C, 30 sec; 55°C, 30 sec; 72°C, 30 sec; 35 cycles; 72°C, 2 min; and storage at 4°C.

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