CN114875169A - SSR molecular marker primer group developed based on coptis chinensis whole genome and application thereof - Google Patents
SSR molecular marker primer group developed based on coptis chinensis whole genome and application thereof Download PDFInfo
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Abstract
The invention discloses a group of SSR primer groups developed based on the coptis chinensis whole genome, belonging to the technical field of molecular markers. The primer group consists of 22 pairs of primers, the sequence of the 22 pairs of primers is shown as SEQ ID NO:1-44, the invention also provides application of the SSR molecular marker primer group in coptis genetic diversity analysis and germplasm resource genetic pedigree analysis, and the primer group can effectively perform genetic diversity analysis and population clustering analysis on coptis chinensis of different origins and wild and cultivated coptis chinensis germplasm resources under different habitat conditions. The 22 pairs of primers have the advantages of rich polymorphism, stable amplification, good repeatability and the like, are SSR molecular markers developed based on the whole genome of the coptis chinensis for the first time and are used for analyzing the population genetic diversity, and the invention lays a foundation for genetic diversity protection, genetic relationship research and core germplasm library screening of the coptis chinensis germplasm resources.
Description
Technical Field
The invention belongs to the technical field of molecular detection, and particularly relates to an SSR molecular marker primer group developed based on a coptis chinensis whole genome and application thereof, which are used for coptis chinensis population genetic diversity analysis and germplasm resource identification.
Technical Field
The Coptidis rhizoma is perennial herb of Coptis (Coptis Salisb.) belonging to Ranunculaceae, and is derived from dried rhizome of Coptis chinensis (Coptis chinensis Franch.), Coptis Teeta Wall, Coptis triangularis (Coptis dletidea C.Y. Cheng et Hsiao) belonging to Ranunculaceae. Coptis brevicaulis var. brevisepala W.T.Wang et Hsiao is a variety of Coptis chinensis. The main producing areas of Coptidis rhizoma include Hubei, Sichuan, Chongqing, Anhui and Yunnan, as well as Hunan' 28583, county, Jiangxi Poyang, Fujian Jianou, Guangxi Liuzhou and Zhejiang Yushao.
The research aiming at the coptis chinensis mainly focuses on the aspects of chemical components, pharmacological action, quality standard, clinical and daily application, and the research aiming at the genetic diversity of the coptis chinensis population in the aspect of molecular biology is less. In recent years, as a large amount of artificial wild coptis resources are mined and the ecological environment is deteriorated, wild coptis deltoidea and wild coptis coerulescens are endangered to be extinct, and the wild coptis is extremely deficient, a set of molecular markers are developed to protect the genetic diversity of coptis germplasm.
Simple Sequence Repeat (SSR) is a simple repeat consisting of 1-6 nucleotides and is widely distributed throughout the genome of eukaryotes. Although SSRs are distributed in different positions of the whole genome, sequences at two ends of the SSRs are mostly conservative single-copy sequences, a pair of specific primers is designed according to the sequences at the two ends, a core microsatellite DNA sequence is amplified by a PCR technology, and the polymorphism of the length of the core microsatellite DNA sequence can be obtained by an electrophoresis analysis technology. SSR markers have been widely used in molecular assisted breeding, genetic diversity analysis, germplasm resource identification and other studies due to their advantages of good codominance, ability to distinguish homozygous genes from heterozygous genes, high polymorphism, good repeatability, good stability and the like.
In order to develop molecular biology SSR markers to assist in identifying coptis plant germplasm resources, promote fine coptis seed breeding, accelerate development and utilization of germplasm resources and protect diversity of coptis germplasm resources, an SSR primer of coptis is urgently needed to be developed, so that scientific bases are provided for researches on coptis germplasm resource evaluation, fine variety cultivation, core germplasm base construction and the like.
Disclosure of Invention
On the basis of the full-length genome of the coptis chinensis, verification and polymorphism analysis are carried out on a selected part of SSRs through PCR detection on the basis of a large amount of obtained SSR information, so that the universal polymorphism primers are obtained. The invention aims to provide a novel SSR primer group for analyzing the genetic diversity of coptis chinensis and application thereof, and lays a foundation for the screening of a coptis chinensis core germplasm bank, the research of genetic relationship and the breeding of excellent varieties.
The invention firstly provides a core primer group based on a coptis chinensis SSR marker, which comprises 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, CC179, 22 pairs of primer sequences are shown as follows:
(1) CC3 primer for amplifying SSR molecular marker:
SEQ ID NO:1：CC3-F：5'-TATTGTGGGTGAAGTTATGG-3'
SEQ ID NO:2：CC3-R：5'-GTGTTATTTTGTCGTTTAGTGA-3'
(2) CC6 primer for amplifying SSR molecular marker:
SEQ ID NO:3：CC6-F：5'-GATAAAGGGGACCAAGAC-3'
SEQ ID NO:4：CC6-R：5'-GTAGCGAATGGGCAATA-3'
(3) CC18 primer for amplifying SSR molecular marker:
SEQ ID NO:5：CC18-F：5'-CCGTTCACCCACTACAA-3'
SEQ ID NO:6：CC18-R：5'-AAAGCAAACACCTCCAA-3'
(4) CC27 primer for amplifying SSR molecular marker:
SEQ ID NO:7：CC27-F：5'-CTAAACAAGATTTCGTTACTCC-3'
SEQ ID NO:8：CC27-R：5'-AAGGCTATTCTCAAAGCACTA-3'
(5) CC29 primer for amplifying SSR molecular marker:
SEQ ID NO:9：CC29-F：5'-GGAGACTCACTGCCAACA-3'
SEQ ID NO:10：CC29-R：5'-CGAGAAATGACCAAGGAA-3'
(6) CC38 primer for amplifying SSR molecular marker:
SEQ ID NO:11：CC38-F：5'-GTAACCAAACCCCTTCT-3'
SEQ ID NO:12：CC38-R：5'-CTCGTCCACTAAAATCCA-3'
(7) CC43 primer for amplifying SSR molecular marker:
SEQ ID NO:13：CC43-F：5'-ATTACGAGAAAGGTGGAG-3'
SEQ ID NO:14：CC43-R：5'-GATGTGATGGGAGGAGA-3'
(8) CC49 primer for amplifying SSR molecular marker:
SEQ ID NO:15：CC49-F：5'-TGAAATGTTCGGCTCTT-3'
SEQ ID NO:16：CC49-R：5'-GCCAACGGTGGTCTAAT-3'
(9) CC53 primer for amplifying SSR molecular marker:
SEQ ID NO:17：CC53-F：5'-TCGTTTCCGTCAAGTGC-3'
SEQ ID NO:18：CC53-R：5'-TCAAGATGCCAACCTCC-3'
(10) CC57 primer for amplifying SSR molecular marker:
SEQ ID NO:19：CC57-F：5'-CGGAAGAATAGGCAATG-3'
SEQ ID NO:20：CC57-R：5'-TGTCACTCGCCAGAAAT-3'
(11) CC90 primer for amplifying SSR molecular marker:
SEQ ID NO:21：SSR11-F：5'-ATTGGAGATACCGTGAA-3'
SEQ ID NO:22：SSR11-R：5'-GAAATAGGAGGAATAGTGAT-3'
(12) CC98 primer for amplifying SSR molecular marker:
SEQ ID NO:23：CC98-F：5'-CGGAACCCTAACAAGAT-3'
SEQ ID NO:24：CC98-R：5'-CGATGCCAAATGTATGA-3'
(13) CC103 primer for amplifying SSR molecular marker:
SEQ ID NO:25：CC103-F：5'-TGCGTCACAGTTTCAGT-3'
SEQ ID NO:26：CC103-R：5'-CGTAAGATAGCAGCATAGAG-3'
(14) CC106 primer for amplifying SSR molecular marker:
SEQ ID NO:27：CC106-F：5'-GGGGATTGAGGATGGAA-3'
SEQ ID NO:28：CC106-R：5'-CCCTTGTGGGAACTTTA-3'
(15) CC113 primer for amplifying SSR molecular marker:
SEQ ID NO:29：CC113-F：5'-TTTCTGTTTTCTCCGCTAT-3'
SEQ ID NO:30：CC113-R：5'-AAATGTCCTTGGGTTCA-3'
(16) CC117 primer for amplifying SSR molecular marker:
SEQ ID NO:31：CC117-F：5'-AAATGTAGGAGCAGAGC-3'
SEQ ID NO:32：CC117-R：5'-GAAAGGGAGTTAGAAGAA-3'
(17) CC129 primer for amplifying SSR molecular marker:
SEQ ID NO:33：CC129-F：5'-GCCACATTATTTATTACC-3'
SEQ ID NO:34：CC129-R：5'-TAGTTTACGAGGCTTTC-3'
(18) CC134 primer for amplifying SSR molecular marker:
SEQ ID NO:35：CC134-F：5'-GAGGGTGAAGCCGTTAT-3'
SEQ ID NO:36：CC134-R：5'-TCTGCCAAGGGTGTATC-3'
(19) CC144 primer for amplifying SSR molecular marker:
SEQ ID NO:37：CC144-F：5'-TCGCATTCCAGTAACCA-3'
SEQ ID NO:38：CC144-R：5'-ATCCCTGAAACCATAAGT-3'
(20) CC162 primer for amplifying SSR molecular marker:
SEQ ID NO:39：CC162-F：5'-CCCTGTGGTGAGATTTG-3'
SEQ ID NO:40：CC162-R：5'-TTTACGCTCTTGTCCCT-3'
(21) CC169 primer for amplifying SSR molecular marker:
SEQ ID NO:41：CC169-F：5'-TCTTCCCTCCTCCACAA-3'
SEQ ID NO:42：CC169-R：5'-TCCAGCGAGCCTCTTAT-3'
(22) CC179 primer for amplifying SSR molecular markers:
SEQ ID NO:43：CC179-F：5'-AGTGATGTATCGGTTGTC-3'
SEQ ID NO:44：CC179-R：5'-AAATAAGTAAGCGTCCTC-3'。
in another aspect, the invention provides an application of the primer group in genetic diversity analysis and germplasm resource identification of coptis chinensis populations.
A method for analyzing genetic diversity and identifying germplasm resources of a coptis chinensis population comprises the following steps:
(1) extracting the DNA of a coptis sample to be detected;
(2) adding a universal M13 linker sequence to the forward primer of each primer pair of the primer set to obtain an M13 linker forward primer;
(3) performing PCR amplification by using the DNA extracted in the step (1) as a template and using a reverse primer and an M13 adaptor forward primer of each pair of primers of the primer group to obtain a fluorescent PCR amplification product;
(4) performing capillary fluorescence electrophoresis detection on the obtained fluorescence PCR amplification product, reading capillary electrophoresis data by using GeneMarker V2.2.0 software, and counting a band detection result;
(5) and (4) performing genetic diversity analysis and germplasm genetic identification on the coptis chinensis population by using the statistical strip detection result.
Wherein, the specific method for analyzing the population genetic diversity and identifying the germplasm heredity in the step (5) is as follows:
A. recording the size of the amplified fragment of each coptis sample corresponding to each pair of primers according to the detection result of the band in the step (4);
B. based on the amplified fragment size obtained by the comprehensive arrangement, the following genetic diversity parameters, such as the number of alleles (N), were calculated for each primer pair using GenAlex6.5 software a ) Effective allele number (N) e ) And observing the heterozygosity (H) o ) Expected heterozygosity (H) e ) The Shannon information index (I), the allele frequencies of different primers and the like, and after calculating the alleles of the different primers by using GenAlex6.5 software, calculating the Polymorphic Information Content (PIC) by using PIC _ Cale software;
C. and B, according to the sizes of the amplified fragments obtained by comprehensive arrangement in the step A, numbering and reading the amplified fragments from large to small, marking the amplified bands as '1' when the amplified bands appear, marking the amplified bands as '0' when no band appears, establishing an original matrix, and importing an ape process edition package of an R language to draw the N-J tree.
Wherein, the PCR amplification system in the step (3) is as follows: mu.l genomic DNA, 5. mu.l of 2 XTaq PCR Master Mix, 0.05. mu.l M13 adaptor forward primer at a concentration of 10 pmol/. mu.l, 0.24. mu.l reverse primer at a concentration of 10 pmol/. mu.l, 0.15. mu. L M13 fluorescent modifier, 2.56. mu.L ddH 2 O。
Wherein, the reaction procedure of the PCR amplification is as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 30s for 35 cycles; final 72 ℃ terminal extension for 10 min.
The coptis sample is young and tender leaves of coptis.
Compared with the prior art, the technical scheme of the invention has the following beneficial effects:
the invention adopts genome technology and SSR molecular marker technology, designs SSR primers by utilizing coptis genome, and has various types of sample germplasm base sources for genetic diversity analysis, including coptis chinensis, Yunnan coptis chinensis and coptis bresepalata; the sampling range is wide, and the sampling range comprises a plurality of provinces such as Anhui, Yunnan, Hubei, Chongqing and Sichuan; the sampling germplasm comprises wild germplasm and cultivation germplasm, and the primer group designed by the invention has high accuracy, specificity and stability through verification, and lays a foundation for genetic diversity protection, genetic relationship research and core germplasm library screening of coptis germplasm resources.
Drawings
FIG. 1 is a N-J tree clustering diagram of genetic diversity of Coptidis rhizoma.
FIG. 2 is a capillary fluorescence electrophoresis detection peak image obtained by amplifying a part of coptis material by using a primer CC57, in FIG. 2, sample1 shows an amplification pattern of sample AES1, sample2 shows an amplification pattern of sample AES2, sample3 shows an amplification pattern of sample AWSX1, sample4 shows an amplification pattern of sample ALTX1, and sample5 shows an amplification pattern of sample DHS 5.
Detailed Description
The SSR primer set of the present invention and its specific application are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Utilizing the existing coptis chinensis whole genome sequence, and using MISA software (http:// pgrc. ipk-gatersleen. de/MISA /) to scan and screen SSR loci to obtain a large amount of SSR loci; primer design was performed on the sites using Primer5.0 software.
In the present invention, the criteria for the screening are: mononucleotides are repeated at least 10 times, dinucleotides are repeated at least 6 times, and trinucleotides, tetranucleotides, pentanucleotides, and hexanucleotides are repeated at least 5 times to be recognized as SSR sites. The invention utilizes Primer5.0 software to carry out primer design on the sites, and the parameters are set as follows: the length of the primer is 18-27 bp; the annealing temperature is 55-65 ℃; the length of the PCR product ranges from 100bp to 300 bp; the GC content is between 40% and 60%, and finally 180 pairs of SSR primers are successfully designed. The amplification efficiency and polymorphism of the primers were studied using 8 samples, and the criteria for screening were: the amplification efficiency is higher than 80%, and the number of alleles is more than or equal to 3. Finally, 22 primers which stably amplified a clear polymorphic band were selected from 180 pairs of primers, whose sequences are shown in Table 1 and synthesized by Competition Biotechnology engineering (Shanghai) Co., Ltd.
TABLE 1 Coptidis rhizoma 22 vs. SSR primer information
Example 2
The SSR primer group synthesized based on the embodiment 1 can be applied to the analysis of genetic diversity and the identification of germplasm resources of coptis chinensis populations, and the analysis method comprises the following steps:
A. extracting genome DNA of a coptis sample to be detected;
and (5) verifying the effectiveness of the developed SSR marker by using 65 coptis chinensis samples. The coptis sample information is shown in table 2. The samples are young leaves of coptis chinensis, and only 1 single plant is selected for collecting samples in each sample.
TABLE 2 rhizoma Coptidis sample information
| Rhizoma coptidis germplasm | Province part | Wild or cultivated | Sample labeling | Number of samples |
| Coptidis rhizoma (Coptis Brevipedunculata (Franch.) Brevipedt.) | (Anhui) | Wild plant | DHS1-10 | 10 |
| Yunnan coptis root | Yunnan province | Wild plant | CGS1-7 | 7 |
| Yunnan coptis root | Yunnan province | Wild plant | CJP1-8 | 8 |
| Coptis chinensis | (Hubei) | Wild plant | AES1-10 | 10 |
| Coptis chinensis | (Hubei) | Cultivation of plants | ABK1-11 | 11 |
| Coptis chinensis | Chongqing | Cultivation of plants | ALTX1-10 | 10 |
| Coptis chinensis | Sichuan | Cultivation of plants | AWSX1-9 | 9 |
Selecting 65 parts of healthy tender leaves of the material, extracting DNA of the tender leaves by adopting an improved CTAB method, detecting the concentration and purity of the DNA of the material to be tested by using a Nanodrop nucleic acid determinator and 1% agarose gel electrophoresis, and storing qualified DNA samples at the temperature of-20 ℃ for later use.
B. Adding a universal M13 joint sequence 'tgtaaaacgacggccagt' to a forward primer of each primer shown in the sequence table 1 to obtain an M13 joint forward primer;
C. and B, taking the genomic DNA of the sample to be detected extracted in the step A as a template, and carrying out PCR amplification by using a reverse primer and an M13 joint forward primer to obtain a fluorescent PCR amplification product: the PCR amplification system is as follows: mu.l genomic DNA, 5. mu.L of 2 XTaq PCR Master Mix, 0.05. mu.L of forward primer at a concentration of 10 pmol/. mu.L, 0.24. mu.L of reverse primer at a concentration of 10 pmol/. mu.L, 0.15. mu. L M13 fluorescent modifier (R)OX or HEX or FAM or TAMRA), 2.56. mu. LddH 2 And O. The reaction procedure for PCR amplification was: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 30s, for 35 cycles; final 72 ℃ terminal extension for 10 min.
D. And D, performing capillary fluorescence electrophoresis detection on the fluorescence PCR amplification product obtained in the step C by using a DNA sequencer ABI3730XL, reading the size of the sample fragment and the number of alleles by using GeneMarker v2.2.0, and counting the band detection result.
E. The amplified fragment sizes obtained by the comprehensive arrangement were used to calculate the following genetic diversity parameters, such as the number of alleles (N), for each primer pair using GenAlex6.5 software a ) Effective allele number (N) e ) And observing the heterozygosity (H) o ) Expected heterozygosity (H) e ) The Shannon information index (I), the allele frequencies of different primers and the like, and after calculating the alleles of the different primers by using GenAlex6.5 software, calculating the Polymorphic Information Content (PIC) by using PIC _ Cale software; according to the size of the amplified fragments obtained by comprehensive arrangement, numbering and reading the amplified fragments from large to small, marking the amplified bands as '1' when the amplified bands appear and marking the amplified bands as '0' when no amplified bands appear, establishing an original matrix, importing an ape program package of an R language to draw an N-J tree, and performing cluster analysis on 65 coptis sample individuals.
FIG. 2 shows the result of capillary fluorescence electrophoresis detection of a part of Coptidis rhizoma material amplified by the primer CC 57.
The amplification results of 65 parts of coptis material with 22 pairs of SSR primers are shown in Table 3.
TABLE 322 statistics of amplification information of Coptidis rhizoma materials with SSR polymorphic primers
As can be seen from Table 3, 259 polymorphic bands were co-amplified by 22 pairs of primers, each pair having an average allele number of 11.77 and an effective allele number ranging from 1.346 to 3.373. The Shannon information diversity index, the expected heterozygosity and the polymorphic information content index are indexes reflecting primer polymorphism, the higher the value is, the higher the polymorphism is, the average values of the three indexes are respectively 0.854, 0.470 and 0.764, which shows that the genetic diversity among coptis chinensis materials is very rich, and the SSR primer group developed by the invention can be applied to the analysis of the genetic diversity of coptis chinensis and the mass spectrum analysis. From the perspective of each primer, the primers CC3, CC38, CC53 and CC98 have higher Shannon information diversity index, expected heterozygosity and polymorphic information content index, which shows that the primers have better amplification effect, higher genetic diversity value and higher identification efficiency.
In order to further verify the application of the primer group in the genetic diversity of the coptis chinensis, an original matrix is established for the amplified fragment sizes of all the population samples, an ape edit package in the R language is imported to draw an N-J tree, and 65 coptis sample individuals are subjected to cluster analysis, wherein the result is shown in figure 1. The results of the N-J tree show that different genetic distances can classify all coptis samples into five major categories: I. II, III, IV and V. The first major class (I) comprises 15 samples, which are divided into two branches, and respectively covers all the wild Yunnan coptis chinensis from Yunnan; the second major class (II) consists of 10 samples, encompassing all wild coptidis brevifolia from anhui; the third major class (III) consists of 10 samples, covering all wild coptis from north of huo; the fourth major group (IV) comprises 11 samples, which are divided into two branches, and covers all the cultivated Coptis chinensis from Hubei; the fifth major group (V) consisted of 19 samples, which were divided into three branches, covering all the cultivated Coptis chinensis from Chongqing and Sichuan.
From the cluster analysis result shown in fig. 1, all the wild Yunnan coptis chinensis samples (CGS1-7, CJP1-8) and the wild Brachystemma calycinum samples (DHS1-10) have more significant difference in genetic distance compared with the wild coptis chinensis samples (AES1-10) and the cultivated coptis chinensis samples; the wild coptis chinensis sample (AES1-10) has a large genetic distance compared with all the cultivated coptis chinensis samples; the genetic distance among all the samples of the cultivated coptis is small, in all the samples of the cultivated coptis, except that the cultivated coptis (ABK1-11) from Hubei is gathered into one, the cultivated coptis from Chongqing (ALTX1-10) and Sichuan (AWSX1-9) are relatively mixed, and the genetic difference is small, which shows that the germplasm of the cultivated coptis is relatively mixed and has no genetic differentiation on obvious geographical distance due to frequent introduction and domestication in recent years.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
<110> Hubei university of traditional Chinese medicine
<120> SSR molecular marker primer group developed based on coptis chinensis whole genome and application thereof
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<211> 17
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 11
gtaaccaaac cccttct 17
<210> 12
<211> 18
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 12
ctcgtccact aaaatcca 18
<210> 13
<211> 18
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 13
attacgagaa aggtggag 18
<210> 14
<211> 17
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 14
gatgtgatgg gaggaga 17
<210> 15
<211> 17
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 15
tgaaatgttc ggctctt 17
<210> 16
<211> 17
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 16
gccaacggtg gtctaat 17
<210> 17
<211> 17
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 17
tcgtttccgt caagtgc 17
<210> 18
<211> 17
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 18
tcaagatgcc aacctcc 17
<210> 19
<211> 17
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 19
cggaagaata ggcaatg 17
<210> 20
<211> 17
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 20
tgtcactcgc cagaaat 17
<210> 21
<211> 17
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 21
attggagata ccgtgaa 17
<210> 22
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 22
gaaataggag gaatagtgat 20
<210> 23
<211> 17
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 23
cggaacccta acaagat 17
<210> 24
<211> 17
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 24
cgatgccaaa tgtatga 17
<210> 25
<211> 17
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 25
tgcgtcacag tttcagt 17
<210> 26
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 26
cgtaagatag cagcatagag 20
<210> 27
<211> 17
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 27
ggggattgag gatggaa 17
<210> 28
<211> 17
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 28
cccttgtggg aacttta 17
<210> 29
<211> 19
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 29
tttctgtttt ctccgctat 19
<210> 30
<211> 17
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 30
aaatgtcctt gggttca 17
<210> 31
<211> 17
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 31
aaatgtagga gcagagc 17
<210> 32
<211> 18
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 32
gaaagggagt tagaagaa 18
<210> 33
<211> 18
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 33
gccacattat ttattacc 18
<210> 34
<211> 17
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 34
tagtttacga ggctttc 17
<210> 35
<211> 17
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 35
gagggtgaag ccgttat 17
<210> 36
<211> 17
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 36
tctgccaagg gtgtatc 17
<210> 37
<211> 17
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 37
tcgcattcca gtaacca 17
<210> 38
<211> 18
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 38
atccctgaaa ccataagt 18
<210> 39
<211> 17
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 39
ccctgtggtg agatttg 17
<210> 40
<211> 17
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 40
tttacgctct tgtccct 17
<210> 41
<211> 17
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 41
tcttccctcc tccacaa 17
<210> 42
<211> 17
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 42
tccagcgagc ctcttat 17
<210> 43
<211> 18
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 43
agtgatgtat cggttgtc 18
<210> 44
<211> 18
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 44
aaataagtaa gcgtcctc 18
Claims (6)
1. A group of SSR molecular marker primer groups developed based on the golden thread whole genome is characterized in that the primer group consists of 22 pairs of primers, the SSR molecular markers aimed by the 22 pairs of primers are respectively CC3, CC6, CC18, CC27, CC29, CC38, CC43, CC49, CC53, CC57, CC90, CC98, CC103, CC106, CC113, CC117, CC129, CC134, CC144, CC162, CC169 and CC179, and the primer sequences are as follows:
(1) CC3 primer for amplifying SSR molecular marker:
SEQ ID NO:1：CC3-F：5'-TATTGTGGGTGAAGTTATGG-3'
SEQ ID NO:2：CC3-R：5'-GTGTTATTTTGTCGTTTAGTGA-3'
(2) CC6 primer for amplifying SSR molecular marker:
SEQ ID NO:3：CC6-F：5'-GATAAAGGGGACCAAGAC-3'
SEQ ID NO:4：CC6-R：5'-GTAGCGAATGGGCAATA-3'
(3) CC18 primer for amplifying SSR molecular marker:
SEQ ID NO:5：CC18-F：5'-CCGTTCACCCACTACAA-3'
SEQ ID NO:6：CC18-R：5'-AAAGCAAACACCTCCAA-3'
(4) CC27 primer for amplifying SSR molecular marker:
SEQ ID NO:7：CC27-F：5'-CTAAACAAGATTTCGTTACTCC-3'
SEQ ID NO:8：CC27-R：5'-AAGGCTATTCTCAAAGCACTA-3'
(5) CC29 primer for amplifying SSR molecular marker:
SEQ ID NO:9：CC29-F：5'-GGAGACTCACTGCCAACA-3'
SEQ ID NO:10：CC29-R：5'-CGAGAAATGACCAAGGAA-3'
(6) CC38 primer for amplifying SSR molecular marker:
SEQ ID NO:11：CC38-F：5'-GTAACCAAACCCCTTCT-3'
SEQ ID NO:12：CC38-R：5'-CTCGTCCACTAAAATCCA-3'
(7) CC43 primer for amplifying SSR molecular marker:
SEQ ID NO:13：CC43-F：5'-ATTACGAGAAAGGTGGAG-3'
SEQ ID NO:14：CC43-R：5'-GATGTGATGGGAGGAGA-3'
(8) CC49 primer for amplifying SSR molecular marker:
SEQ ID NO:15：CC49-F：5'-TGAAATGTTCGGCTCTT-3'
SEQ ID NO:16：CC49-R：5'-GCCAACGGTGGTCTAAT-3'
(9) CC53 primer for amplifying SSR molecular marker:
SEQ ID NO:17：CC53-F：5'-TCGTTTCCGTCAAGTGC-3'
SEQ ID NO:18：CC53-R：5'-TCAAGATGCCAACCTCC-3'
(10) CC57 primer for amplifying SSR molecular marker:
SEQ ID NO:19：CC57-F：5'-CGGAAGAATAGGCAATG-3'
SEQ ID NO:20：CC57-R：5'-TGTCACTCGCCAGAAAT-3'
(11) CC90 primer for amplifying SSR molecular marker:
SEQ ID NO:21：SSR11-F：5'-ATTGGAGATACCGTGAA-3'
SEQ ID NO:22：SSR11-R：5'-GAAATAGGAGGAATAGTGAT-3'
(12) CC98 primer for amplifying SSR molecular marker:
SEQ ID NO:23：CC98-F：5'-CGGAACCCTAACAAGAT-3'
SEQ ID NO:24：CC98-R：5'-CGATGCCAAATGTATGA-3'
(13) CC103 primer for amplifying SSR molecular marker:
SEQ ID NO:25：CC103-F：5'-TGCGTCACAGTTTCAGT-3'
SEQ ID NO:26：CC103-R：5'-CGTAAGATAGCAGCATAGAG-3'
(14) CC106 primer for amplifying SSR molecular marker:
SEQ ID NO:27：CC106-F：5'-GGGGATTGAGGATGGAA-3'
SEQ ID NO:28：CC106-R：5'-CCCTTGTGGGAACTTTA-3'
(15) CC113 primer for amplifying SSR molecular marker:
SEQ ID NO:29：CC113-F：5'-TTTCTGTTTTCTCCGCTAT-3'
SEQ ID NO:30：CC113-R：5'-AAATGTCCTTGGGTTCA-3'
(16) CC117 primer for amplifying SSR molecular marker:
SEQ ID NO:31：CC117-F：5'-AAATGTAGGAGCAGAGC-3'
SEQ ID NO:32：CC117-R：5'-GAAAGGGAGTTAGAAGAA-3'
(17) CC129 primer for amplifying SSR molecular marker:
SEQ ID NO:33：CC129-F：5'-GCCACATTATTTATTACC-3'
SEQ ID NO:34：CC129-R：5'-TAGTTTACGAGGCTTTC-3'
(18) CC134 primer for amplifying SSR molecular marker:
SEQ ID NO:35：CC134-F：5'-GAGGGTGAAGCCGTTAT-3'
SEQ ID NO:36：CC134-R：5'-TCTGCCAAGGGTGTATC-3'
(19) CC144 primer for amplifying SSR molecular marker:
SEQ ID NO:37：CC144-F：5'-TCGCATTCCAGTAACCA-3'
SEQ ID NO:38：CC144-R：5'-ATCCCTGAAACCATAAGT-3'
(20) CC162 primer for amplifying SSR molecular marker:
SEQ ID NO:39：CC162-F：5'-CCCTGTGGTGAGATTTG-3'
SEQ ID NO:40：CC162-R：5'-TTTACGCTCTTGTCCCT-3'
(21) CC169 primer for amplifying SSR molecular marker:
SEQ ID NO:41：CC169-F：5'-TCTTCCCTCCTCCACAA-3'
SEQ ID NO:42：CC169-R：5'-TCCAGCGAGCCTCTTAT-3'
(22) CC179 primer for amplifying SSR molecular markers:
SEQ ID NO:43：CC179-F：5'-AGTGATGTATCGGTTGTC-3'
SEQ ID NO:44：CC179-R：5'-AAATAAGTAAGCGTCCTC-3'。
2. the primer set of claim 1, wherein the primer set is used for genetic diversity analysis and germplasm resource identification of coptis chinensis population.
3. A method for analyzing genetic diversity and identifying germplasm resources of a coptis chinensis population is characterized by comprising the following steps:
(1) extracting the DNA of a coptis sample to be detected;
(2) adding a universal M13 linker sequence to the forward primer of each primer pair of the primer set of claim 1 to obtain an M13 linker forward primer;
(3) performing PCR amplification by using the DNA extracted in the step (1) as a template and using a reverse primer and an M13 adaptor forward primer of each pair of primers of the primer group in claim 1 to obtain a fluorescent PCR amplification product;
(4) performing capillary fluorescence electrophoresis detection on the fluorescence PCR amplification product obtained in the step (3), reading capillary electrophoresis data by using GeneMarkerV2.2.0 software, and counting a band detection result;
(5) and (4) performing genetic diversity analysis and germplasm genetic identification on the coptis chinensis population by using the statistical strip detection result in the step (4).
4. The method for genetic diversity analysis and germplasm resource identification of a Coptis chinensis Franch population according to claim 3, wherein the PCR amplification system is as follows: mu.l of DNA template, 5. mu.l of 2 XTaq PCR Master Mix, 0.05. mu.l of M13 adaptor forward primer at a concentration of 10 pmol/. mu.l, 0.24. mu.l of reverse primer at a concentration of 10 pmol/. mu.l, 0.15. mu. L M13 fluorescent modifier, 2.56. mu.L ddH 2 O。
5. The method for genetic diversity analysis and germplasm resource identification of a Coptis chinensis Franch group according to claim 3, wherein the reaction procedure of PCR amplification is: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 30s for 35 cycles; final 72 ℃ terminal extension for 10 min.
6. The method for genetic diversity analysis and germplasm resource identification of a Coptis chinensis Franch population according to claim 3, wherein: the coptis sample is young and tender leaves of coptis.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116463453A (en) * | 2023-05-19 | 2023-07-21 | 中国医学科学院药用植物研究所 | SSR primer group based on Isatis tinctoria whole genome development and application thereof |
| CN116516042A (en) * | 2023-05-18 | 2023-08-01 | 河北省农林科学院经济作物研究所 | SSR molecular marker related to fructus forsythiae germplasm resource identification, corresponding primer and application thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116516042A (en) * | 2023-05-18 | 2023-08-01 | 河北省农林科学院经济作物研究所 | SSR molecular marker related to fructus forsythiae germplasm resource identification, corresponding primer and application thereof |
| CN116516042B (en) * | 2023-05-18 | 2024-04-26 | 河北省农林科学院经济作物研究所 | SSR molecular marker related to fructus forsythiae germplasm resource identification, corresponding primer and application thereof |
| CN116463453A (en) * | 2023-05-19 | 2023-07-21 | 中国医学科学院药用植物研究所 | SSR primer group based on Isatis tinctoria whole genome development and application thereof |
| CN116463453B (en) * | 2023-05-19 | 2024-04-02 | 中国医学科学院药用植物研究所 | SSR primer group based on Isatis tinctoria whole genome development and application thereof |
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