Development and application of SSR molecular marker primer sets based on the whole genome of Coptis chinensis

Abstract

The present invention discloses a group of SSR primer sets developed based on the whole genome of Coptis chinensis, which belongs to the field of molecular marker technology. The primer set consists of 22 pairs of primers, and the sequences of these 22 pairs of primers are shown in SEQ ID NO: 1-44. The present invention also provides the application of the SSR molecular marker primer set in genetic diversity analysis and germplasm genetic pedigree analysis of Coptis chinensis. The primer set can effectively perform genetic diversity analysis and population cluster analysis on Coptis chinensis of different base sources and wild and cultivated Coptis chinensis germplasm resources under different habitat conditions. These 22 pairs of primers have the advantages of rich polymorphism, stable amplification, and good repeatability. They are the first SSR molecular markers developed based on the whole genome of Coptis chinensis and perform population genetic diversity analysis. The present invention lays the foundation for the protection of genetic diversity of Coptis chinensis germplasm resources, kinship research, and core germplasm library screening.

Description

Technical Field

[0001] The present invention belongs to the field of molecular detection technology, and specifically relates to an SSR molecular marker primer set developed based on the whole genome of Coptis chinensis and its application, which is used for genetic diversity analysis of Coptis chinensis populations and identification of germplasm resources. Technical Background

[0002] Coptis chinensis is a perennial herbaceous plant of the genus Coptis Salisb. in the Ranunculaceae family. It is derived from the dried rhizomes of Coptis chinensis Franch., Coptis teeta Wall., and Coptis dletoidea CYCheng et Hsiao. Coptis chinensis var. brevisepala WTWang et Hsiao is a variant of Coptis chinensis. Coptis chinensis is primarily produced in Hubei, Sichuan, Chongqing, Anhui, and Yunnan provinces, as well as in Lixian County, Hunan, Poyang County, Jiangxi, Jian'ou County, Fujian, Liuzhou City, Guangxi, and Yuhang County, Zhejiang.

[0003] Research on Coptis chinensis (Coptis chinensis) has primarily focused on its chemical composition, pharmacological effects, quality standards, and clinical and daily applications. However, molecular biological studies examining the genetic diversity of Coptis chinensis populations are limited. In recent years, due to the widespread harvesting of wild Coptis chinensis resources and the deterioration of the ecological environment, wild Coptis chinensis varieties, such as Yalian and Yunlian, are on the verge of extinction, and wild Coptis chinensis is extremely scarce. Therefore, the development of a suite of molecular markers to preserve the genetic diversity of Coptis chinensis germplasm is urgently needed.

[0004] Simple sequence repeats (SSRs) are simple repetitive sequences consisting of 1 to 6 nucleotides and are widely distributed throughout the genomes of eukaryotes. Although SSRs are distributed at different locations throughout the genome, the sequences at their ends are mostly conserved single-copy sequences. Based on these sequences, a pair of specific primers are designed to amplify the core microsatellite DNA sequence using PCR technology, and electrophoresis analysis can be used to determine its length polymorphism. Due to their advantages such as good codominance, ability to distinguish homozygous and heterozygous genes, high polymorphism, good reproducibility, and excellent stability, SSR markers have been widely used in molecular-assisted breeding, genetic diversity analysis, and germplasm resource identification.

[0005] In order to carry out molecular biology SSR marker-assisted identification of Coptis chinensis plant germplasm resources, promote the breeding of Coptis chinensis varieties, accelerate the development and utilization of germplasm resources, and protect the diversity of Coptis chinensis germplasm resources, it is urgent to develop SSR primers for Coptis chinensis to provide a scientific basis for research such as the evaluation of Coptis chinensis germplasm resources, the cultivation of excellent varieties, and the construction of a core germplasm bank. Summary of the Invention

[0006] Based on the full-length genome of Coptidis rhizome and the abundant SSR information obtained, this paper validates and analyzes the polymorphism of selected SSRs through PCR detection, thereby obtaining universal polymorphic primers. This paper aims to provide a new SSR primer set for analyzing the genetic diversity of Coptidis rhizome and its application, and lays a foundation for screening the Coptidis rhizome core germplasm library, studying phylogenetic relationships, and breeding superior varieties.

[0007] The present invention first provides a core primer set based on SSR markers of Coptis chinensis, which includes the following 22 primer pairs: CC3, CC6, CC18, CC27, CC29, CC38, CC43, CC49, CC53, CC57, CC90, CC98, CC103, CC106, CC113, CC117, CC129, CC134, CCSSR144, CC162, CC169, and CC179. The sequences of the 22 primer pairs are as follows:

[0008] (1) CC3 primer for amplifying SSR molecular markers:

[0009] SEQ ID NO:1:CC3-F:5'-TATTGTGGGTGAAGTTATGG-3'

[0010] SEQ ID NO:2:CC3-R:5'-GTGTTATTTTGTCGTTTAGTGA-3'

[0011] (2) CC6 primer for amplifying SSR molecular markers:

[0012] SEQ ID NO:3:CC6-F:5'-GATAAAGGGGACCAAGAC-3'

[0013] SEQ ID NO:4:CC6-R:5'-GTAGCGAATGGGCAATA-3'

[0014] (3) CC18 primers for amplifying SSR molecular markers:

[0015] SEQ ID NO:5:CC18-F:5'-CCGTTCACCCACTACAA-3'

[0016] SEQ ID NO:6:CC18-R:5'-AAAGAAACACCTCCAA-3'

[0017] (4) CC27 primers for amplifying SSR molecular markers:

[0018] SEQ ID NO:7:CC27-F:5'-CTAAACAAGATTTCGTTACTCC-3'

[0019] SEQ ID NO:8:CC27-R:5'-AAGGCTATTCTCAAAGCACTA-3'

[0020] (5) CC29 primer for amplifying SSR molecular markers:

[0021] SEQ ID NO:9:CC29-F:5'-GGAGACTCACTGCCAACA-3'

[0022] SEQ ID NO:10:CC29-R:5'-CGAGAAATGACCAAGGAA-3'

[0023] (6) CC38 primers for amplifying SSR molecular markers:

[0024] SEQ ID NO:11:CC38-F:5'-GTAACCAAACCCCTCT-3'

[0025] SEQ ID NO:12:CC38-R:5'-CTCGTCCACTAAAATCCA-3'

[0026] (7) CC43 primers for amplifying SSR molecular markers:

[0027] SEQ ID NO:13:CC43-F:5'-ATTACGAGAAAGGTGGAG-3'

[0028] SEQ ID NO:14:CC43-R:5'-GATGTGATGGGAGGAGA-3'

[0029] (8) CC49 primers for amplifying SSR molecular markers:

[0030] SEQ ID NO:15:CC49-F:5'-TGAAATGTTCGGCTCTT-3'

[0031] SEQ ID NO:16:CC49-R:5'-GCCAACGGTGGTCTAAT-3'

[0032] (9) CC53 primers for amplifying SSR molecular markers:

[0033] SEQ ID NO:17:CC53-F:5'-TCGTTTCCGTCAAGTGC-3'

[0034] SEQ ID NO:18:CC53-R:5'-TCAAGATGCCAACCTCC-3'

[0035] (10) CC57 primers for amplifying SSR molecular markers:

[0036] SEQ ID NO:19:CC57-F:5'-CGGAAGAATAGGCAATG-3'

[0037] SEQ ID NO:20:CC57-R:5'-TGTCACTCGCCAGAAAT-3'

[0038] (11) CC90 primers for amplifying SSR molecular markers:

[0039] SEQ ID NO:21:SSR11-F:5'-ATTGGAGATACCGTGAA-3'

[0040] SEQ ID NO:22:SSR11-R:5'-GAAATAGGAGGAATAGTGAT-3'

[0041] (12) CC98 primers for amplifying SSR molecular markers:

[0042] SEQ ID NO:23:CC98-F:5'-CGGAACCCTAACAAGAT-3'

[0043] SEQ ID NO:24:CC98-R:5'-CGATGCCAAATGTATGA-3'

[0044] (13) CC103 primers for amplifying SSR molecular markers:

[0045] SEQ ID NO:25:CC103-F:5'-TGCGTCACAGTTTCAGT-3'

[0046] SEQ ID NO:26:CC103-R:5'-CGTAAGATAGCAGCATAGAG-3'

[0047] (14) CC106 primers for amplifying SSR molecular markers:

[0048] SEQ ID NO:27:CC106-F:5'-GGGGATTGAGGATGGAA-3'

[0049] SEQ ID NO:28:CC106-R:5'-CCCTTGTGGGAACTTTA-3'

[0050] (15) CC113 primers for amplifying SSR molecular markers:

[0051] SEQ ID NO:29: CC113-F:5'-TTTCTGTTTTTCTCCGCTAT-3'

[0052] SEQ ID NO: 30: CC113-R: 5'-AAATGTCCTTGGGTTCA-3'

[0053] (16) CC117 primers for amplifying SSR molecular markers:

[0054] SEQ ID NO:31:CC117-F:5'-AAATGTAGGAGCAGAGC-3'

[0055] SEQ ID NO:32:CC117-R:5'-GAAAGGGAGTTAGAAGAA-3'

[0056] (17) CC129 primers for amplifying SSR molecular markers:

[0057] SEQ ID NO:33:CC129-F:5'-GCCACATTATTTATTACC-3'

[0058] SEQ ID NO:34:CC129-R:5'-TAGTTTACGAGGCTTC-3'

[0059] (18) CC134 primers for amplifying SSR molecular markers:

[0060] SEQ ID NO:35:CC134-F:5'-GAGGGTGAAGCCGTTAT-3'

[0061] SEQ ID NO: 36: CC134-R: 5'-TCTGCCAAGGGTGTATC-3'

[0062] (19) CC144 primers for amplifying SSR molecular markers:

[0063] SEQ ID NO:37:CC144-F:5'-TCGATTCCAGTAACCA-3'

[0064] SEQ ID NO:38:CC144-R:5'-ATCCCTGAAACCATAAGT-3'

[0065] (20) CC162 primers for amplifying SSR molecular markers:

[0066] SEQ ID NO: 39: CC162-F: 5'-CCCTGTGTGAGATTTG-3'

[0067] SEQ ID NO:40:CC162-R:5'-TTTACGCTCTTGTCCCT-3'

[0068] (21) CC169 primers for amplifying SSR molecular markers:

[0069] SEQ ID NO:41:CC169-F:5'-TCTTCCCTCCTCCACAA-3'

[0070] SEQ ID NO:42:CC169-R:5'-TCCAGCGAGCCTCTTAT-3'

[0071] (22) CC179 primers for amplifying SSR molecular markers:

[0072] SEQ ID NO:43:CC179-F:5'-AGTGATGTATCGGTTGTC-3'

[0073] SEQ ID NO:44: CC179-R:5'-AAATAAGTAAGCGTCCTC-3'.

[0074] In another aspect, the present invention provides the use of the above primer set in the analysis of genetic diversity of Coptis chinensis populations and identification of germplasm resources.

[0075] A method for analyzing the genetic diversity of a Coptis chinensis population and identifying its germplasm resources comprises the following steps:

[0076] (1) extracting DNA from the Rhizoma Coptidis sample to be tested;

[0077] (2) adding a universal M13 adapter sequence to the forward primer of each pair of primers in the primer set to obtain an M13 adapter forward primer;

[0078] (3) using the DNA extracted in step (1) as a template, performing PCR amplification using the reverse primer of each pair of primers in the primer set and the M13 adapter forward primer to obtain a fluorescent PCR amplification product;

[0079] (4) The obtained fluorescent PCR amplification products were detected by capillary fluorescence electrophoresis, and the capillary electrophoresis data were read using GeneMarker V2.2.0 software to count the band detection results;

[0080] (5) The results of statistical band detection were used to analyze the genetic diversity of the Coptis chinensis population and conduct genetic identification of germplasm.

[0081] The specific methods for population genetic diversity analysis and germplasm genetic identification in step (5) are as follows:

[0082] A. Based on the band detection results in step (4), record the size of the amplified fragment of each Rhizoma Coptidis sample corresponding to each primer pair;

[0083] B. Based on the size of the amplified fragments obtained through comprehensive analysis, the following genetic diversity parameters of each primer pair were calculated using GenAlex 6.5 software, such as the number of alleles (N a ), effective number of alleles (N e ), observed heterozygosity (H o ), expected heterozygosity (H e ), Shannon information index (I) and allele frequencies of different primers, etc. After calculating the allele frequencies of different primers using GenAlex6.5 software, the polymorphism information content (PIC) was calculated using PIC_Cale software;

[0084] C. Based on the sizes of the amplified fragments obtained in step A, the amplified fragments were numbered and read from large to small, with the presence of an amplified band being marked as "1" and the absence of an amplified band being marked as "0". An original matrix was established and imported into the ape programming package in R language to draw the NJ tree.

[0085] The PCR amplification system in step (3) is as follows: 2 μL genomic DNA, 5 μL 2× Taq PCR Master Mix, 0.05 μL M13 adapter forward primer at a concentration of 10 pmol / μL, 0.24 μL reverse primer at a concentration of 10 pmol / μL, 0.15 μL M13 fluorescent modification group, and 2.56 μL ddH2O.

[0086] The PCR amplification reaction procedure is as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s, for a total of 35 cycles; and finally, terminal extension at 72°C for 10 min.

[0087] The Coptidis rhizome sample is young leaves of Coptidis rhizome.

[0088] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0089] The prior art does not have an SSR primer set developed for multiple genetic resources of Coptis chinensis. The present invention applies genomic technology and SSR molecular marker technology, and uses the Coptis chinensis genome to design SSR primers. The sampled germplasm source species for genetic diversity analysis are diverse, including Coptis chinensis, Coptis yunnanensis, and Coptis chinensis short-calyx; the sampling range is wide, including Anhui, Yunnan, Hubei, Chongqing, Sichuan and other provinces; the sampled germplasm includes wild germplasm and cultivated germplasm. It has been verified that the primer set designed by the present invention has high accuracy, specificity and strong stability, which lays a foundation for the protection of genetic diversity of Coptis chinensis germplasm resources, kinship research, and core germplasm bank screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0090] Figure 1 This is the NJ tree cluster diagram of the genetic diversity of Coptis chinensis.

[0091] Figure 2 This is the capillary fluorescence electrophoresis peak diagram obtained by amplifying some Rhizoma Coptidis materials with primer CC57. Figure 2 Here, sample1 represents the amplification map of sample AES1, sample2 represents the amplification map of sample AES2, sample3 represents the amplification map of sample AWSX1, sample4 represents the amplification map of sample ALTX1, and sample5 represents the amplification map of sample DHS5. DETAILED DESCRIPTION

[0092] The SSR primer set of the present invention and its specific applications are described in detail below with reference to the examples, but they should not be understood as limiting the scope of protection of the present invention.

[0093] Example 1

[0094] The existing whole genome sequence of Coptis chinensis was used to scan and screen SSR loci using MISA software (http: / / pgrc.ipk-gatersleben.de / misa / ) to obtain a large number of SSR loci; primers were designed for the loci using Primer 5.0 software.

[0095] In the present invention, the screening criteria are: a mononucleotide is repeated at least 10 times, a dinucleotide is repeated at least 6 times, and a trinucleotide, tetranucleotide, pentanucleotide and hexanucleotide are repeated at least 5 times to be identified as an SSR site. The present invention uses Primer5.0 software to design primers for these sites, and the parameters are set as follows: primer length 18-27bp; annealing temperature is 55-65°C; PCR product length range is between 100bp and 300bp; GC content is between 40% and 60%, and finally 180 pairs of SSR primers are successfully designed. 8 samples were used to study the amplification efficiency and polymorphism of the primers, and the screening criteria are: amplification efficiency higher than 80%, and the number of alleles is ≥3. Finally, 22 pairs of primers that can stably amplify clear and polymorphic bands were screened out from the 180 pairs of primers. The primer sequences are shown in Table 1 and were commissioned to be synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0096] Table 1 Information of 22 pairs of SSR primers in Coptis chinensis

[0097]

[0098]

[0099] Example 2

[0100] The SSR primer set synthesized based on Example 1 can be applied to the genetic diversity analysis of Coptidis rhizome population and the identification of germplasm resources. The analysis method comprises the following steps:

[0101] A. Extract genomic DNA of the Rhizoma Coptidis sample to be tested;

[0102] The developed SSR markers were validated using 65 samples of Coptis chinensis. Information on the Coptis chinensis samples is shown in Table 2. The samples were young leaves of Coptis chinensis, and only one individual plant was sampled for each sample.

[0103] Table 2 Information of Coptis chinensis samples

[0104] Coptis chinensis germplasm province Wild or cultivated Sample number Number of samples Coptis chinensis Anhui wild DHS1-10 10 Yunnan Coptis chinensis Yunnan wild CGS1-7 7 Yunnan Coptis chinensis Yunnan wild CJP1-8 8 Coptis chinensis Hubei wild AES1-10 10 Coptis chinensis Hubei cultivation ABK1-11 11 Coptis chinensis Chongqing cultivation ALTX1-10 10 Coptis chinensis Sichuan cultivation AWSX1-9 9

[0105] Healthy young leaves of the above 65 materials were selected, and their DNA was extracted using the improved CTAB method. The DNA concentration and purity of the test materials were detected using a Nanodrop nucleic acid analyzer and 1% agarose gel electrophoresis. The qualified DNA samples were stored at -20°C for future use.

[0106] B. Add the universal M13 adapter sequence "tgtaaaacgacggccagt" to the forward primer of each primer shown in Sequence Table 1 to obtain the M13 adapter forward primer;

[0107] C. Using the genomic DNA of the test sample extracted in step A as a template, perform PCR amplification with a reverse primer and an M13 adapter forward primer to obtain a fluorescent PCR amplification product. The PCR amplification system is as follows: 2 μL of genomic DNA, 5 μL of 2× Taq PCR Master Mix, 0.05 μL of a 10 pmol / μL forward primer, 0.24 μL of a 10 pmol / μL reverse primer, 0.15 μL of an M13 fluorescent modifier (ROX, HEX, FAM, or TAMRA), and 2.56 μL of ddH2O. The PCR amplification reaction procedure is as follows: initial denaturation at 94°C for 5 min; 35 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s; and a final end extension at 72°C for 10 min.

[0108] D. The fluorescent PCR amplification product obtained in step C was detected by capillary fluorescence electrophoresis using a DNA sequencer ABI3730XL. The sample fragment size and the number of alleles were read using GeneMarker v2.2.0, and the band detection results were counted.

[0109] E. The sizes of the amplified fragments were comprehensively sorted and the following genetic diversity parameters of each primer pair were calculated using GenAlex 6.5 software, such as the number of alleles (N a ), effective number of alleles (N e ), observed heterozygosity (H o ), expected heterozygosity (H e ), Shannon information index (I) and allele frequencies of different primers, etc. After calculating the allele frequencies of different primers using GenAlex6.5 software, the polymorphic information content (PIC) was calculated using PIC_Cale software. According to the sizes of the amplified fragments obtained through comprehensive sorting, the amplified fragments were numbered and read from large to small. The presence of amplified bands was recorded as "1" and the absence of bands was recorded as "0". The original matrix was established and the ape programming package of R language was imported to draw the NJ tree, and cluster analysis was performed on the 65 Coptidis rhizome samples.

[0110] Figure 2 The results of capillary fluorescence electrophoresis were obtained by amplifying some Coptis chinensis materials using primer CC57.

[0111] The amplification results of 22 pairs of SSR primers on 65 Coptidis accessions are shown in Table 3.

[0112] Table 3 Statistics of amplification information of 22 pairs of SSR polymorphic primers on Coptis chinensis materials

[0113]

[0114]

[0115] As shown in Table 3, 22 pairs of primers amplified a total of 259 polymorphic bands, with an average number of alleles per primer pair of 11.77 and an effective number of alleles ranging from 1.346 to 3.373. The Shannon information diversity index, expected heterozygosity, and polymorphic information content index are all indicators reflecting primer polymorphism. The larger the value, the higher the polymorphism. The average values of the three indicators are 0.854, 0.470, and 0.764, respectively, indicating that the genetic diversity among the Rhizoma Coptidis materials is very rich, and the SSR primer set developed by the present invention can be applied to the genetic diversity analysis and germplasm pedigree analysis of Rhizoma Coptidis. From the perspective of each primer, primers CC3, CC38, CC53, and CC98 have higher Shannon information diversity index, expected heterozygosity, and polymorphic information content index, indicating that their amplification effect is good, the genetic diversity value is high, and there is a higher identification efficiency.

[0116] To further verify the application of this primer set in the genetic diversity of Coptis chinensis, an original matrix of the amplified fragment sizes of all population samples was established, and the ape programming package of R language was imported to draw the NJ tree. Cluster analysis was performed on 65 Coptis chinensis samples. The results are shown in Figure 2. Figure 1 The NJ tree results showed that all Coptidis samples could be divided into five categories based on different genetic distances: I, II, III, IV, and V. The first category (I) included 15 samples, which were divided into two branches, covering all wild Coptidis yunnanensis from Yunnan; the second category (II) included 10 samples, covering all wild Coptidis short-calyx from Anhui; the third category (III) included 10 samples, covering all wild Coptidis from Hubei; the fourth category (IV) included 11 samples, which were divided into two branches, covering all cultivated Coptidis from Hubei; and the fifth category (V) included 19 samples, which were divided into three branches, covering all cultivated Coptidis from Chongqing and Sichuan.

[0117] from Figure 1 The results of cluster analysis showed that all wild Yunnan Coptis chinensis samples (CGS1-7, CJP1-8) and wild short-calyx Coptis chinensis samples (DHS1-10) had significantly different genetic distances compared with wild Coptis chinensis samples (AES1-10) and cultivated Coptis chinensis samples; the genetic distance between wild Coptis chinensis samples (AES1-10) was larger than that between all cultivated Coptis chinensis samples; the genetic distance between all cultivated Coptis chinensis samples was smaller. Among all cultivated Coptis chinensis samples, except for cultivated Coptis chinensis from Hubei (ABK1-11) which clustered into one branch, cultivated Coptis chinensis from Chongqing (ALTX1-10) and Sichuan (AWSX1-9) were relatively mixed and had relatively small genetic differences. This suggests that due to frequent introduction and domestication in recent years, the cultivated Coptis chinensis germplasm is relatively mixed and there is no obvious genetic differentiation based on geographical distance.

[0118] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention. <110> Hubei University of Chinese Medicine <120> Development and application of SSR molecular marker primer sets based on the whole genome of Coptis chinensis <160> 44 <170> SIPOSequenceListing 1.0 <210> 1 <211> 20 <212> DNA <213> Artificial sequence <400> 1 tattgtgggt gaagttatgg 20 <210> 2 <211> twenty two <212> DNA <213> Artificial sequence <400> 2 gtgttatttt gtcgtttagt ga 22 <210> 3 <211> 18 <212> DNA <213> Artificial sequence <400> 3 gataaagggg accaagac 18 <210> 4 <211> 17 <212> DNA <213> Artificial sequence <400> 4 gtagcgaatg ggcaata 17 <210> 5 <211> 17 <212> DNA <213> Artificial sequence <400> 5 ccgttcaccc actacaa 17 <210> 6 <211> 17 <212> DNA <213> Artificial sequence <400> 6 aaagcaaaca cctccaa 17 <210> 7 <211> twenty two <212> DNA <213> Artificial sequence <400> 7 ctaaacaaga tttcgttact cc 22 <210> 8 <211> twenty one <212> DNA <213> Artificial sequence <400> 8 aaggctattc tcaaagcact a 21 <210> 9 <211> 18 <212> DNA <213> Artificial sequence <400> 9 ggagactcac tgccaaca 18 <210> 10 <211> 18 <212> DNA <213> Artificial sequence <400> 10 cgagaaatga ccaaggaa 18 <210> 11 <211> 17 <212> DNA <213> Artificial sequence <400> 11 gtaaccaaac cccttct 17 <210> 12 <211> 18 <212> DNA <213> Artificial sequence <400> 12 ctcgtccact aaaatcca 18 <210> 13 <211> 18 <212> DNA <213> Artificial sequence <400> 13 attacgagaa aggtggag 18 <210> 14 <211> 17 <212> DNA <213> Artificial sequence <400> 14 gatgtgatgg gaggaga 17 <210> 15 <211> 17 <212> DNA <213> Artificial sequence <400> 15 tgaaatgttc ggctctt 17 <210> 16 <211> 17 <212> DNA <213> Artificial sequence <400> 16 gccaacggtg gtctaat 17 <210> 17 <211> 17 <212> DNA <213> Artificial sequence <400> 17 tcgtttccgt caagtgc 17 <210> 18 <211> 17 <212> DNA <213> Artificial sequence <400> 18 tcaagatgcc aacctcc 17 <210> 19 <211> 17 <212> DNA <213> Artificial sequence <400> 19 cggaagaata ggcaatg 17 <210> 20 <211> 17 <212> DNA <213> Artificial sequence <400> 20 tgtcactcgc cagaaat 17 <210> twenty one <211> 17 <212> DNA <213> Artificial sequence <400> twenty one attggagata ccgtgaa 17 <210> twenty two <211> 20 <212> DNA <213> Artificial sequence <400> twenty two gaaataggag gaatagtgat 20 <210> twenty three <211> 17 <212> DNA <213> Artificial sequence <400> twenty three cggaacccta acaagat 17 <210> twenty four <211> 17 <212> DNA <213> Artificial sequence <400> twenty four cgatgccaaa tgtatga 17 <210> 25 <211> 17 <212> DNA <213> Artificial sequence <400> 25 tgcgtcacag tttcagt 17 <210> 26 <211> 20 <212> DNA <213> Artificial sequence <400> 26 cgtaagatag cagcatagag 20 <210> 27 <211> 17 <212> DNA <213> Artificial sequence <400> 27 ggggattgag gatggaa 17 <210> 28 <211> 17 <212> DNA <213> Artificial sequence <400> 28 cccttgtggg aacttta 17 <210> 29 <211> 19 <212> DNA <213> Artificial sequence <400> 29 tttctgtttt ctccgctat 19 <210> 30 <211> 17 <212> DNA <213> Artificial sequence <400> 30 aaatgtcctt gggttca 17 <210> 31 <211> 17 <212> DNA <213> Artificial sequence <400> 31 aaatgtagga gcagagc 17 <210> 32 <211> 18 <212> DNA <213> Artificial sequence <400> 32 gaaagggagt tagaagaa 18 <210> 33 <211> 18 <212> DNA <213> Artificial sequence <400> 33 gccacattat ttattacc 18 <210> 34 <211> 17 <212> DNA <213> Artificial sequence <400> 34 tagtttacga ggctttc 17 <210> 35 <211> 17 <212> DNA <213> Artificial sequence <400> 35 gagggtgaag ccgttat 17 <210> 36 <211> 17 <212> DNA <213> Artificial sequence <400> 36 tctgccaagg gtgtatc 17 <210> 37 <211> 17 <212> DNA <213> Artificial sequence <400> 37 tcgcattcca gtaacca 17 <210> 38 <211> 18 <212> DNA <213> Artificial sequence <400> 38 atccctgaaa ccataagt 18 <210> 39 <211> 17 <212> DNA <213> Artificial sequence <400> 39 ccctgtggtg agatttg 17 <210> 40 <211> 17 <212> DNA <213> Artificial sequence <400> 40 tttacgctct tgtccct 17 <210> 41 <211> 17 <212> DNA <213> Artificial sequence <400> 41 tcttccctcc tccacaa 17 <210> 42 <211> 17 <212> DNA <213> Artificial sequence <400> 42 tccagcgagc ctcttat 17 <210> 43 <211> 18 <212> DNA <213> Artificial sequence <400> 43 agtgatgtat cggttgtc 18 <210> 44 <211> 18 <212> DNA <213> Artificial sequence <400> 44 aaataagtaa gcgtcctc 18

Claims

1. A method for analyzing the genetic diversity of a coptis chinensis population and identifying its germplasm resources, characterized in that The following steps are involved: (1) extracting DNA from the Rhizoma Coptidis sample to be tested; (2) Adding a universal M13 adapter sequence to the forward primer of each pair of primers in the SSR molecular marker primer set to obtain an M13 adapter forward primer; (3) using the DNA extracted in step (1) as a template, performing PCR amplification using the reverse primer of each pair of primers in the SSR molecular marker primer set and the M13 adapter forward primer to obtain a fluorescent PCR amplification product; (4) performing capillary fluorescence electrophoresis on the fluorescent PCR amplification product obtained in step (3), reading the capillary electrophoresis data using GeneMarker V2.2.0 software, and statistically analyzing the band detection results; (5) using the statistical band detection results in step (4) to perform genetic diversity analysis of the Rhizoma Coptidis population and genetic identification of the germplasm, The SSR molecular marker primer set consists of 22 pairs of primers. The SSR molecular markers targeted by these 22 pairs of primers are CC3, CC6, CC18, CC27, CC29, CC38, CC43, CC49, CC53, CC57, CC90, CC98, CC103, CC106, CC113, CC117, CC129, CC134, CC144, CC162, CC169, and CC179, respectively. The primer sequences are as follows: CC3 primers for amplifying SSR molecular markers: SEQ ID NO:1:CC3-F:5'-TATTGTGGGTGAAGTTATGG-3' SEQ ID NO:2:CC3-R:5'-GTGTTATTTTGTCGTTTAGTGA-3' CC6 primers for amplifying SSR molecular markers: SEQ ID NO:3:CC6-F:5'-GATAAAGGGGACCAAGAC-3' SEQ ID NO:4:CC6-R:5'-GTAGCGAATGGGCAATA-3' CC18 primers for amplifying SSR molecular markers: SEQ ID NO:5:CC18-F:5'-CCGTTCACCCACTACAA-3' SEQ ID NO:6:CC18-R:5'-AAAGAAACACCTCCAA-3' CC27 primers for amplifying SSR molecular markers: SEQ ID NO:7:CC27-F:5'-CTAAACAAGATTTCGTTACTCC-3' SEQ ID NO:8:CC27-R:5'-AAGGCTATTCTCAAAGCACTA-3' CC29 primers for amplifying SSR molecular markers: SEQ ID NO:9:CC29-F:5'-GGAGACTCACTGCCAACA-3' SEQ ID NO:10:CC29-R:5'-CGAGAAATGACCAAGGAA-3' CC38 primers for amplifying SSR molecular markers: SEQ ID NO:11:CC38-F:5'-GTAACCAAACCCCTCT-3' SEQ ID NO:12:CC38-R:5'-CTCGTCCACTAAAATCCA-3' CC43 primers for amplifying SSR molecular markers: SEQ ID NO:13:CC43-F:5'-ATTACGAGAAAGGTGGAG-3' SEQ ID NO:14:CC43-R:5'-GATGTGATGGGAGGAGA-3' CC49 primers for amplifying SSR molecular markers: SEQ ID NO:15:CC49-F:5'-TGAAATGTTCGGCTCTT-3' SEQ ID NO:16:CC49-R:5'-GCCAACGGTGGTCTAAT-3' CC53 primers for amplifying SSR molecular markers: SEQ ID NO:17:CC53-F:5'-TCGTTTCCGTCAAGTGC-3' SEQ ID NO:18:CC53-R:5'-TCAAGATGCCAACCTCC-3' CC57 primers for amplifying SSR molecular markers: SEQ ID NO:19:CC57-F:5'-CGGAAGAATAGGCAATG-3' SEQ ID NO:20:CC57-R:5'-TGTCACTCGCCAGAAAT-3' CC90 primers for amplifying SSR molecular markers: SEQ ID NO:21:SSR11-F:5'-ATTGGAGATACCGTGAA-3' SEQ ID NO:22:SSR11-R:5'-GAAATAGGAGGAATAGTGAT-3' CC98 primers for amplifying SSR molecular markers: SEQ ID NO:23:CC98-F:5'-CGGAACCCTAACAAGAT-3' SEQ ID NO:24:CC98-R:5'-CGATGCCAAATGTATGA-3' CC103 primers for amplifying SSR molecular markers: SEQ ID NO:25:CC103-F:5'-TGCGTCACAGTTTCAGT-3' SEQ ID NO:26:CC103-R:5'-CGTAAGATAGCAGCATAGAG-3' CC106 primers for amplifying SSR molecular markers: SEQ ID NO:27:CC106-F:5'-GGGGATTGAGGATGGAA-3' SEQ ID NO:28:CC106-R:5'-CCCTTGTGGGAACTTTA-3' CC113 primers for amplifying SSR molecular markers: SEQ ID NO:29: CC113-F:5'-TTTCTGTTTTTCTCCGCTAT-3' SEQ ID NO: 30: CC113-R: 5'-AAATGTCCTTGGGTTCA-3' CC117 primers for amplifying SSR molecular markers: SEQ ID NO:31:CC117-F:5'-AAATGTAGGAGCAGAGC-3' SEQ ID NO:32:CC117-R:5'-GAAAGGGAGTTAGAAGAA-3' CC129 primers for amplifying SSR molecular markers: SEQ ID NO:33:CC129-F:5'-GCCACATTATTTATTACC-3' SEQ ID NO:34:CC129-R:5'-TAGTTTACGAGGCTTC-3' CC134 primers for amplifying SSR molecular markers: SEQ ID NO:35:CC134-F:5'-GAGGGTGAAGCCGTTAT-3' SEQ ID NO: 36: CC134-R: 5'-TCTGCCAAGGGTGTATC-3' CC144 primers for amplifying SSR molecular markers: SEQ ID NO:37:CC144-F:5'-TCGATTCCAGTAACCA-3' SEQ ID NO:38:CC144-R:5'-ATCCCTGAAACCATAAGT-3' CC162 primers for amplifying SSR molecular markers: SEQ ID NO: 39: CC162-F: 5'-CCCTGTGTGAGATTTG-3' SEQ ID NO:40:CC162-R:5'-TTTACGCTCTTGTCCCT-3' CC169 primers for amplifying SSR molecular markers: SEQ ID NO:41:CC169-F:5'-TCTTCCCTCCTCCACAA-3' SEQ ID NO:42:CC169-R:5'-TCCAGCGAGCCTCTTAT-3' CC179 primers for amplifying SSR molecular markers: SEQ ID NO:43:CC179-F:5'-AGTGATGTATCGGTTGTC-3' SEQ ID NO:44: CC179-R:5'-AAATAAGTAAGCGTCCTC-3', The germplasm resources include wild Coptis chinensis from Yunnan, wild Coptis chinensis from Anhui, wild Coptis chinensis from Hubei, cultivated Coptis chinensis from Hubei, and cultivated Coptis chinensis from Chongqing and Sichuan.

2. The method for analyzing the genetic diversity of Coptidis rhizome populations and identifying germplasm resources according to claim 1, characterized in that: The PCR amplification system was as follows: 2 μL DNA template, 5 μL 2×Taq PCR Master Mix, 0.05 μL M13 adapter forward primer at a concentration of 10 pmol / μL, 0.24 μL reverse primer at a concentration of 10 pmol / μL, 0.15 μL M13 fluorescent modification group, and 2.56 μL ddH2O.

3. The method for analyzing the genetic diversity of Coptidis rhizome populations and identifying germplasm resources according to claim 1, characterized in that: The PCR amplification reaction procedure was as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s, for a total of 35 cycles; and finally, terminal extension at 72°C for 10 min.

4. The method for analyzing the genetic diversity of Coptidis rhizome populations and identifying germplasm resources according to claim 1, wherein: The Coptidis rhizome sample is young leaves of Coptidis rhizome.

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