CN114438256B - SSR molecular marker for genetic diversity analysis of maple leaf clematis and application thereof - Google Patents
SSR molecular marker for genetic diversity analysis of maple leaf clematis and application thereof Download PDFInfo
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- 239000003147 molecular marker Substances 0.000 title abstract description 7
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- 108020004414 DNA Proteins 0.000 claims description 179
- 102000053602 DNA Human genes 0.000 claims description 116
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- 238000000034 method Methods 0.000 claims description 29
- 241000208140 Acer Species 0.000 claims description 22
- 238000004806 packaging method and process Methods 0.000 claims description 2
- 238000011160 research Methods 0.000 abstract description 5
- 238000012163 sequencing technique Methods 0.000 abstract description 4
- 108091092878 Microsatellite Proteins 0.000 description 52
- 239000000463 material Substances 0.000 description 14
- 108700028369 Alleles Proteins 0.000 description 9
- 241000196324 Embryophyta Species 0.000 description 9
- 238000012216 screening Methods 0.000 description 9
- 238000012360 testing method Methods 0.000 description 6
- 235000015988 Acer ginnala Nutrition 0.000 description 5
- 241000580956 Acer tataricum subsp. ginnala Species 0.000 description 5
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- 239000003153 chemical reaction reagent Substances 0.000 description 3
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- 238000011156 evaluation Methods 0.000 description 2
- 230000007614 genetic variation Effects 0.000 description 2
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- 229910052664 nepheline Inorganic materials 0.000 description 2
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- 108090000623 proteins and genes Proteins 0.000 description 2
- 241000023308 Acca Species 0.000 description 1
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
- 241001256255 Clematis acerifolia Species 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 241000218201 Ranunculaceae Species 0.000 description 1
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- 238000007621 cluster analysis Methods 0.000 description 1
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- 238000012257 pre-denaturation Methods 0.000 description 1
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- 238000010998 test method Methods 0.000 description 1
- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention discloses an SSR molecular marker for genetic diversity analysis of maple leaf clematis and application thereof. The invention also discloses an SSR primer group for analyzing genetic diversity of the clematis acervata lugens, which consists of a primer pair 1-a primer pair 29. The SSR primer group has any one of the following functions of n 1) -n 5): n 1) analyzing or evaluating genetic diversity of the clematis acervata lugens; n 2) analyzing or evaluating the genetic differentiation degree of the clematis acervata lugens; n 3) analyzing or evaluating genetic relatedness of the clematis acervata lugens; n 4) constructing or preparing a maple leaf clematis genetic map or a fingerprint map; n 5) identifying or distinguishing different geographic populations of maple leaf clematis. The invention utilizes the sequencing result of the simplified genome to splice, screens out SSR molecular markers suitable for the genetic diversity research of the maple leaf clematis, and has important significance for developing the genetic diversity research and the conservation work of the maple leaf clematis.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an SSR molecular marker for genetic diversity analysis of maple leaf clematis and application thereof.
Background
Maple leaf clematis (Clematis acerifolia maxim.) is a plant of the genus clematis of the family Ranunculaceae. The flower pose is elegant in the April in the flowering period, and is mainly distributed in the mountain, the sulcus of the head, and the like in the Beijing area, and sometimes distributed in the Hebei province and Henan province. Because of climate change, economic development and change of self genetic characteristics, the maple leaf clematis population presents the problems of less plant quantity, narrow distribution and slower self-renewal and propagation of the population, and belongs to the first-class protection plants in Beijing city.
Genetic diversity refers to the sum of genetic variations within a population and between populations. Genetic diversity reflects the ability of a plant to resist a hostile environment. The current situation of the plant can be known through genetic diversity evaluation, the future of plant development can be predicted, and the method has an important effect on protecting rare plants. The evaluation of genetic diversity of plants can be carried out using phenotypic markers, biochemical markers and molecular markers. SSR (Simple Sequence Repeats) has abundant marker quantity and covers the whole genome, has extremely abundant polymorphism and codominance characteristics, and is widely applied to genetic diversity research.
In order to protect the maple leaf clematis, the population state is evaluated, the population development trend is judged, a feasible strategy is provided for the maple leaf clematis protection, and the genetic diversity is studied. Because the clematis acervata lugens has no genome sequencing related information, the development of related SSR molecular markers has a certain difficulty.
Disclosure of Invention
The invention aims to provide an SSR molecular marker for genetic diversity analysis of maple leaf clematis and application thereof.
In order to achieve the aim, the invention firstly provides an SSR primer group which can be used for analyzing genetic diversity of maple clematis.
The SSR primer group provided by the invention can be used for genetic diversity analysis of the clematis acervata lugens, and comprises a primer pair 1, a primer pair 2, a primer pair 3, a primer pair 4, a primer pair 5, a primer pair 6, a primer pair 7, a primer pair 8, a primer pair 9, a primer pair 10, a primer pair 11, a primer pair 12, a primer pair 13, a primer pair 14, a primer pair 15, a primer pair 16, a primer pair 17, a primer pair 18, a primer pair 19, a primer pair 20, a primer pair 21, a primer pair 22, a primer pair 23, a primer pair 24, a primer pair 25, a primer pair 26, a primer pair 27, a primer pair 28 and a primer pair 29;
the primer pair 1 consists of a primer 1-F and a primer 1-R; the primer pair 2 consists of a primer 2-F and a primer 2-R; the primer pair 3 consists of a primer 3-F and a primer 3-R; the primer pair 4 consists of a primer 4-F and a primer 4-R; the primer pair 5 consists of a primer 5-F and a primer 5-R; the primer pair 6 consists of a primer 6-F and a primer 6-R; the primer pair 7 consists of a primer 7-F and a primer 7-R; the primer pair 8 consists of a primer 8-F and a primer 8-R; the primer pair 9 consists of a primer 9-F and a primer 9-R; the primer pair 10 consists of a primer 10-F and a primer 10-R; the primer pair 11 consists of a primer 11-F and a primer 11-R; the primer pair 12 consists of a primer 12-F and a primer 12-R; the primer pair 13 consists of a primer 13-F and a primer 13-R; the primer pair 14 consists of a primer 14-F and a primer 14-R; the primer pair 15 consists of a primer 15-F and a primer 15-R; the primer pair 16 consists of a primer 16-F and a primer 16-R; the primer pair 17 consists of a primer 17-F and a primer 17-R; the primer pair 18 consists of a primer 18-F and a primer 18-R; the primer pair 19 consists of a primer 19-F and a primer 19-R; the primer pair 20 consists of a primer 20-F and a primer 20-R; the primer pair 21 consists of a primer 21-F and a primer 21-R; the primer pair 22 consists of a primer 22-F and a primer 22-R; the primer pair 23 consists of a primer 23-F and a primer 23-R; the primer pair 24 consists of a primer 24-F and a primer 24-R; the primer pair 25 consists of a primer 25-F and a primer 25-R; the primer pair 26 consists of a primer 26-F and a primer 26-R; the primer pair 27 consists of a primer 27-F and a primer 27-R; the primer pair 28 consists of a primer 28-F and a primer 28-R; the primer pair 29 consists of a primer 29-F and a primer 29-R;
The primer 1-F is a single-stranded DNA molecule shown in a sequence 1 in a sequence table;
the primer 1-R is a single-stranded DNA molecule shown in a sequence 2 of a sequence table;
the primer 2-F is a single-stranded DNA molecule shown in a sequence 3 of a sequence table;
the primer 2-R is a single-stranded DNA molecule shown in a sequence 4 of a sequence table;
the primer 3-F is a single-stranded DNA molecule shown in a sequence 5 of a sequence table;
the primer 3-R is a single-stranded DNA molecule shown in a sequence 6 of a sequence table;
the primer 4-F is a single-stranded DNA molecule shown in a sequence 7 of a sequence table;
the primer 4-R is a single-stranded DNA molecule shown in a sequence 8 of a sequence table;
the primer 5-F is a single-stranded DNA molecule shown in a sequence 9 of a sequence table;
the primer 5-R is a single-stranded DNA molecule shown in a sequence 10 of a sequence table;
the primer 6-F is a single-stranded DNA molecule shown in a sequence 11 in a sequence table;
the primer 6-R is a single-stranded DNA molecule shown in a sequence 12 of a sequence table;
the primer 7-F is a single-stranded DNA molecule shown in a sequence 13 in a sequence table;
the primer 7-R is a single-stranded DNA molecule shown in a sequence 14 of a sequence table;
the primer 8-F is a single-stranded DNA molecule shown in a sequence 15 of a sequence table;
the primer 8-R is a single-stranded DNA molecule shown in a sequence 16 of a sequence table;
The primer 9-F is a single-stranded DNA molecule shown in a sequence 17 in a sequence table;
the primer 9-R is a single-stranded DNA molecule shown in a sequence 18 of a sequence table;
the primer 10-F is a single-stranded DNA molecule shown in a sequence 19 in a sequence table;
the primer 10-R is a single-stranded DNA molecule shown in a sequence 20 of a sequence table;
the primer 11-F is a single-stranded DNA molecule shown in a sequence 21 of a sequence table;
the primer 11-R is a single-stranded DNA molecule shown in a sequence 22 in a sequence table;
the primer 12-F is a single-stranded DNA molecule shown in a sequence 23 of a sequence table;
the primer 12-R is a single-stranded DNA molecule shown in a sequence 24 of a sequence table;
the primer 13-F is a single-stranded DNA molecule shown in a sequence 25 of a sequence table;
the primer 13-R is a single-stranded DNA molecule shown in a sequence 26 of a sequence table;
the primer 14-F is a single-stranded DNA molecule shown in a sequence 27 of a sequence table;
the primer 14-R is a single-stranded DNA molecule shown in a sequence 28 of a sequence table;
the primer 15-F is a single-stranded DNA molecule shown in a sequence 29 of a sequence table;
the primer 15-R is a single-stranded DNA molecule shown in a sequence 30 of a sequence table;
the primer 16-F is a single-stranded DNA molecule shown in a sequence 31 of a sequence table;
the primer 16-R is a single-stranded DNA molecule shown in a sequence 32 of a sequence table;
The primer 17-F is a single-stranded DNA molecule shown in a sequence 33 of a sequence table;
the primer 17-R is a single-stranded DNA molecule shown in a sequence 34 of a sequence table;
the primer 18-F is a single-stranded DNA molecule shown in a sequence 35 of a sequence table;
the primer 18-R is a single-stranded DNA molecule shown in a sequence 36 of a sequence table;
the primer 19-F is a single-stranded DNA molecule shown in a sequence 37 of a sequence table;
the primer 19-R is a single-stranded DNA molecule shown in a sequence 38 of a sequence table;
the primer 20-F is a single-stranded DNA molecule shown in a sequence 39 of a sequence table;
the primer 20-R is a single-stranded DNA molecule shown in a sequence 40 of a sequence table;
the primer 21-F is a single-stranded DNA molecule shown in a sequence 41 of a sequence table;
the primer 21-R is a single-stranded DNA molecule shown in a sequence 42 of a sequence table;
the primer 22-F is a single-stranded DNA molecule shown in a sequence 43 of a sequence table;
the primer 22-R is a single-stranded DNA molecule shown in a sequence 44 of a sequence table;
the primer 23-F is a single-stranded DNA molecule shown in a sequence 45 of a sequence table;
the primer 23-R is a single-stranded DNA molecule shown in a sequence 46 of a sequence table;
the primer 24-F is a single-stranded DNA molecule shown in a sequence 47 of a sequence table;
the primer 24-R is a single-stranded DNA molecule shown in a sequence 48 of a sequence table;
The primer 25-F is a single-stranded DNA molecule shown in a sequence 49 of a sequence table;
the primer 25-R is a single-stranded DNA molecule shown in a sequence 50 of a sequence table;
the primer 26-F is a single-stranded DNA molecule shown in a sequence 51 of a sequence table;
the primer 26-R is a single-stranded DNA molecule shown in a sequence 52 of a sequence table;
the primer 27-F is a single-stranded DNA molecule shown in a sequence 53 of a sequence table;
the primer 27-R is a single-stranded DNA molecule shown in a sequence 54 of a sequence table;
the primer 28-F is a single-stranded DNA molecule shown in a sequence 55 of a sequence table;
the primer 28-R is a single-stranded DNA molecule shown in a sequence 56 of a sequence table;
the primer 29-F is a single-stranded DNA molecule shown in a sequence 57 in a sequence table;
the primer 29-R is a single-stranded DNA molecule shown in a sequence 58 of a sequence table.
Further, the SSR primer set is composed of the primer pair 1, the primer pair 2, the primer pair 3, the primer pair 4, the primer pair 5, the primer pair 6, the primer pair 7, the primer pair 8, the primer pair 9, the primer pair 10, the primer pair 11, the primer pair 12, the primer pair 13, the primer pair 14, the primer pair 15, the primer pair 16, the primer pair 17, the primer pair 18, the primer pair 19, the primer pair 20, the primer pair 21, the primer pair 22, the primer pair 23, the primer pair 24, the primer pair 25, the primer pair 26, the primer pair 27, the primer pair 28, and the primer pair 29.
Further, the molar ratio of each primer in each primer pair of the SSR primer set is 1:1.
In order to achieve the purpose, the invention also provides a new application of the SSR primer group.
The invention provides application of the SSR primer set in any one of the following m 1) to m 10):
m 1) preparing a product for analyzing or evaluating genetic diversity of the clematis maple leaves;
m 2) preparing a product for analyzing or evaluating the genetic differentiation degree of the clematis maple leaves;
m 3) preparing a product for analyzing or evaluating genetic relativity of the clematis acervata lugens;
m 4) preparing a product for constructing or preparing a genetic map or a fingerprint map of the clematis maple leaves;
m 5) preparing and identifying products of maple leaf clematis in different geographical populations;
m 6) analyzing or evaluating genetic diversity of the clematis acervata lugens;
m 7) analyzing or evaluating the genetic differentiation degree of the clematis maple leaves;
m 8) analyzing or evaluating genetic relatedness of the clematis acervata lugens;
m 9) constructing or preparing a maple leaf clematis genetic map or a fingerprint map;
m 10) identifying or distinguishing different geographic populations of maple leaf clematis.
In order to achieve the above object, the present invention also provides a kit comprising the above SSR primer set;
the kit has any one of the following functions n 1) -n 5):
n 1) analyzing or evaluating genetic diversity of the clematis acervata lugens;
n 2) analyzing or evaluating the genetic differentiation degree of the clematis acervata lugens;
n 3) analyzing or evaluating genetic relatedness of the clematis acervata lugens;
n 4) constructing or preparing a maple leaf clematis genetic map or a fingerprint map;
n 5) identifying or distinguishing different geographic populations of maple leaf clematis.
Further, the kit also includes other reagents for PCR amplification, such as dNTPs, DNA polymerase, buffer and H 2 O, etc.
Further, the other reagents for PCR amplification were Mix (Nanjinozan Biotechnology Co., ltd., cat. No. p 115-02) and H 2 O。
The preparation method of the kit also belongs to the protection scope of the invention. The preparation method of the kit comprises the step of packaging each primer in the SSR primer group separately.
In order to achieve the purpose, the invention also provides a new application of the kit.
The invention provides the use of the above kit in any one of the following m 1) to m 10):
m 1) preparing a product for analyzing or evaluating genetic diversity of the clematis maple leaves;
m 2) preparing a product for analyzing or evaluating the genetic differentiation degree of the clematis maple leaves;
m 3) preparing a product for analyzing or evaluating genetic relativity of the clematis acervata lugens;
m 4) preparing a product for constructing or preparing a genetic map or a fingerprint map of the clematis maple leaves;
m 5) preparing and identifying products of maple leaf clematis in different geographical populations;
m 6) analyzing or evaluating genetic diversity of the clematis acervata lugens;
m 7) analyzing or evaluating the genetic differentiation degree of the clematis maple leaves;
m 8) analyzing or evaluating genetic relatedness of the clematis acervata lugens;
m 9) constructing or preparing a maple leaf clematis genetic map or a fingerprint map;
m 10) identifying or distinguishing different geographic populations of maple leaf clematis.
In order to achieve the above purpose, the invention also provides a method for analyzing or evaluating genetic diversity of maple leaf clematis, a method for analyzing or evaluating genetic differentiation degree of maple leaf clematis, a method for analyzing or evaluating genetic relatedness of maple leaf clematis, a method for constructing or preparing maple leaf clematis genetic map or fingerprint map, or a method for identifying or distinguishing different geographical populations of maple leaf clematis.
The invention provides a method for analyzing or evaluating genetic diversity of maple leaf clematis, a method for analyzing or evaluating genetic differentiation degree of maple leaf clematis, a method for analyzing or evaluating genetic relativity of maple leaf clematis, a method for constructing or preparing maple leaf clematis genetic map or fingerprint map, or a method for identifying or distinguishing different geographical populations of maple leaf clematis, which comprises the step of carrying out PCR amplification on maple leaf genome DNA by adopting the SSR primer group.
In any of the methods described above,
the primer annealing temperature is 62 ℃ and the cycle number is 30 when the primer pair 1 is adopted for PCR amplification;
primer annealing temperature is 58 ℃ and cycle number is 32 when the primer pair 2 is adopted for PCR amplification;
primer annealing temperature is 60 ℃ and cycle number is 30 when the primer pair 3 is adopted for PCR amplification;
primer annealing temperature is 60 ℃ and cycle number is 30 when the primer pair 4 is adopted for PCR amplification;
primer annealing temperature is 58 ℃ and cycle number is 32 when the primer pair 5 is adopted for PCR amplification;
the primer annealing temperature is 58 ℃ and the cycle number is 32 when the primer pair 6 is adopted for PCR amplification;
the primer annealing temperature is 58 ℃ and the cycle number is 32 when the primer pair 7 is adopted for PCR amplification;
primer annealing temperature is 60 ℃ and cycle number is 30 when the primer pair 8 is adopted for PCR amplification;
the primer annealing temperature is 58 ℃ and the cycle number is 32 when the primer pair 9 is adopted for PCR amplification;
primer annealing temperature is 58 ℃ and cycle number is 32 when the primer pair 10 is adopted for PCR amplification;
the primer annealing temperature is 62 ℃ and the cycle number is 30 when the primer pair 11 is adopted for PCR amplification;
the primer annealing temperature is 58 ℃ and the cycle number is 32 when the primer pair 12 is adopted for PCR amplification;
The primer annealing temperature is 58 ℃ and the cycle number is 32 when the primer pair 13 is adopted for PCR amplification;
primer annealing temperature is 60 ℃ and cycle number is 30 when the primer pair 14 is adopted for PCR amplification;
the primer annealing temperature in PCR amplification using the primer set 15 was 58℃and the cycle number was 32.
The primer annealing temperature is 58 ℃ and the cycle number is 32 when the primer pair 16 is adopted for PCR amplification;
the primer annealing temperature is 58 ℃ and the cycle number is 32 when the primer pair 17 is adopted for PCR amplification;
primer annealing temperature is 58 ℃ and cycle number is 32 when the primer pair 18 is adopted for PCR amplification;
the primer annealing temperature is 58 ℃ and the cycle number is 32 when the primer pair 19 is adopted for PCR amplification;
primer annealing temperature is 58 ℃ and cycle number is 32 when the primer pair 20 is adopted for PCR amplification;
primer annealing temperature is 60 ℃ and cycle number is 30 when the primer pair 21 is adopted for PCR amplification;
primer annealing temperature is 58 ℃ and cycle number is 32 when the primer pair 22 is adopted for PCR amplification;
primer annealing temperature is 58 ℃ and cycle number is 32 when the primer pair 23 is adopted for PCR amplification;
primer annealing temperature is 58 ℃ and cycle number is 32 when the primer pair 24 is adopted for PCR amplification;
primer annealing temperature is 58 ℃ and cycle number is 32 when the primer pair 25 is adopted for PCR amplification;
Primer annealing temperature is 58 ℃ and cycle number is 32 when the primer pair 26 is adopted for PCR amplification;
the primer annealing temperature is 58 ℃ and the cycle number is 32 when the primer pair 27 is adopted for PCR amplification;
the primer annealing temperature is 58 ℃ and the cycle number is 32 when the primer pair 28 is adopted for PCR amplification;
the primer annealing temperature in PCR amplification using the primer set 29 was 58℃and the cycle number was 32.
The reaction procedure for PCR amplification is specifically as follows: PCR procedure: firstly, carrying out pre-denaturation treatment at 94 ℃ for 5min; then denaturation at 94 ℃ for 30s, annealing at 58-62 ℃ for 30s and extension at 72 ℃ for 30s, thus performing 30-32 cycles; then extending at 72 ℃ for 7min, and finally preserving at 25 ℃.
The reaction system (total volume 10. Mu.L) for PCR amplification was specifically as follows: DNA 1. Mu.L, pre-sequence primer (concentration 0.3. Mu. Mol/L in system) 0.3. Mu.L, post-sequence primer (concentration 0.3. Mu. Mol/L in system) 0.3. Mu. L, mix 5. Mu. L, H 2 O 3.4μL。
Each PCR amplification reaction system corresponds to a pair of SSR primers and a sample.
The PCR amplification further comprises the steps of gel running and gel dyeing after the completion of the PCR amplification.
In order to achieve the above object, the present invention finally provides a method for developing the above SSR primer set.
The development method of the SSR primer group provided by the invention comprises the following steps:
1) Simplified genome sequencing is carried out on the maple leaf clematis samples of different geographic populations, all the sequencing results of the samples are spliced together, a splicing program is operated, 20% of the sequence with the longest splicing is saved as fasta text format, and each fasta file contains a complete splicing sequence;
2) Each splice sequence was checked using ssrhunter1.3 software under the following conditions: the number of nucleotides constituting the repeating element is at most 6, and the number of repeating times is at least 4, so that candidate SSR molecular markers are obtained;
3) Primer3 (v.0.4.0) (https:// bioinfo. Ut. Ee/Primer3-0.4.0 /) was used to Primer the candidate SSR molecular markers, the Primer design conditions were as follows: the total length of the amplified product is less than or equal to 200bp, the initial position of a pre-sequence primer is less than or equal to 125bp, and the initial position of a post-sequence primer is more than or equal to 175bp, so that candidate SSR primers are obtained;
4) Selecting representative samples from different geographical population maple leaf clematis samples respectively to carry out screening on the candidate SSR primer running gel, wherein the screening standard is as follows: the SSR primer can amplify clear and stable bands in each representative sample, and the SSR primer has at least 1 difference in the amplified bands in different representative samples, so that the SSR primer set is finally obtained.
In the step 1), the number of the maple leaf clematis samples of different geographical populations is 90, and the maple leaf clematis samples are respectively 60 maple leaf clematis from Beijing, 20 maple leaf clematis from Hebei and 10 maple leaf clematis from Henan.
In the 4), the number of the representative samples is 6, namely 2 maple leaf clematis from Beijing, 2 maple leaf clematis from Hebei and 2 maple leaf clematis from Henan.
In any of the above applications or methods, the maple leaf clematis or the maple leaf clematis of different geographical populations is maple leaf clematis from Beijing, maple leaf clematis from Hebei, and maple leaf clematis from Henan. Further, the maple leaf clematis from Beijing can be specifically maple leaf clematis from Fangshan Sandu, wuhe two-tunnel, nan Dan Yangda canyon, jingchao, xiayunling and Xinghuang. The maple leaf clematis from Hebei can be maple leaf clematis from Siback, guzhuang. The maple leaf clematis from Henan can be specifically maple leaf clematis from Yunnan mountain.
The invention utilizes the sequencing result of the simplified genome to splice, screens out SSR molecular markers suitable for the genetic diversity research of the maple leaf clematis, and has important significance for developing the genetic diversity research and the conservation work of the maple leaf clematis.
Drawings
FIG. 1 shows the individual clustering analysis of 90 maple leaf clematis. In the figures, 1 to 90 correspond to the test materials numbered 1 to 90 in Table 1, respectively.
Detailed Description
The following examples facilitate a better understanding of the present invention, but are not intended to limit the same. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores. The quantitative tests in the following examples were all set up in triplicate and the results averaged.
The experimental apparatus used in the following examples is as follows: PCR instrument, gel electrophoresis apparatus, water bath, oven.
Example 1 screening of SSR molecular markers of Acer ginnala Maxim
1. Test materials and methods
1. Test materials
Maple leaf clematis is mainly distributed in Beijing area and sometimes in Hebei and Henan. Based on the results of the field exploration, the following 9 sites were selected for sampling: four back yu (Hebei), kuzhuang (Hebei), fangshan Sandu (Beijing), wuhe two tunnels (Beijing), yun Taishan (Henan), nan Dan Yangda canyon (Beijing), jingxi gudao (Beijing), xiayuling (Beijing) and Xinghuang (Beijing), the sampling standard is that 10 plants are sampled for each population, the distance between single plants is at least 2m, and materials in the gap of the same cliff are avoided. The relevant information and numbers of all test materials are shown in table 1.
TABLE 1 group sampling basic information
| Group numbering | Location of site | Number of samples | Sample numbering |
| p1 | Four-back valley (Hebei) | 10 | 1-10 |
| p2 | Gauzhuang (Hebei) | 10 | 11-20 |
| p3 | House mountain Sandu (Beijing) | 10 | 21-30 |
| p4 | Five-in-two tunnel (Beijing) | 10 | 31-40 |
| p5 | Yun Taishan (Henan) | 10 | 41-50 |
| p6 | South Dan Yangda canyon (Beijing) | 10 | 51-60 |
| p7 | Jingxi ancient road (Beijing) | 10 | 61-70 |
| p8 | Nepheline cloud (Beijing) | 10 | 71-80 |
| p9 | Apricot yellow (Beijing) | 10 | 81-90 |
2. Test method
And (3) respectively extracting the genome DNA of the test material in the step (1) by adopting a CTAB method, and then carrying out PCR amplification by taking the extracted genome DNA as a template.
The reaction system for PCR amplification (total volume 10. Mu.L) is shown in Table 2.
The reaction conditions for PCR amplification are shown in Table 3. The annealing temperature X and the cycle number Y of different primer pairs are different.
TABLE 2 PCR reaction System
TABLE 3 PCR reaction conditions
And (5) after the PCR amplification is finished, running and dyeing.
2. Design and screening of SSR molecular markers of maple leaf clematis
1. Because the related information of the genome sequencing of the clematis acervata lugens is not related at present, the invention uses a method for simplifying the genome sequencing to obtain the basic information of the genome. The specific method comprises the following steps: and (3) carrying out simplified genome sequencing on the samples numbered 1-90 in the step one, splicing all the sequencing results of the samples together, running a splicing program, and storing the sequence with the longest splicing length of 20% into a fasta text format, wherein each fasta file contains a complete splicing sequence.
2. Each splice sequence was checked using ssrhulter 1.3 software under the following conditions: the number of nucleotides constituting the repeating element is at most 6, and the number of repetition is at least 4, so that the candidate SSR molecular marker is obtained and stored as a word document.
3. Primer3 (v.0.4.0) (https:// bioinfo. Ut. Ee/Primer3-0.4.0 /) was used to Primer candidate SSR molecular markers, and the Primer design conditions were as follows: the total length of the amplified product is less than or equal to 200bp, the initial position of the primer of the pre-sequence is less than or equal to 125bp, and the initial position of the primer of the post-sequence is more than or equal to 175bp. 1600 pairs of candidate SSR primers are selected from 20000 sequences.
4. Selecting No. 6 and No. 7 samples from samples No. 1-20 as representative of Hebei distributed maple leaf clematis, selecting No. 41 and No. 48 samples from samples No. 41-50 as representative of Hebei distributed maple leaf clematis, selecting No. 63 and No. 67 samples from samples No. 51-90 as representative of Beijing distributed maple leaf clematis, screening candidate SSR primer running gel based on 6 representative samples, wherein the specific screening criteria are as follows: the SSR primers selected amplified distinct and stable bands in each representative sample and differed in at least 1 of the amplified bands in the 6 representative samples.
And finally, 29 pairs of SSR primers are obtained by screening from 1600 pairs of SSR primers and used for subsequent analysis of genetic diversity, and the sequence information of the 29 pairs of SSR primers is specifically shown in table 4.
Sequence information of SSR primers of tables 4 and 29
3. Annealing temperature and cycle number optimization of PCR amplification of SSR primer
Taking No. 6, no. 7, no. 41, no. 48, no. 63 and No. 67 as samples, respectively carrying out PCR amplification on 29 pairs of SSR primers obtained by screening in the second step under the conditions of different annealing temperatures (58 ℃, 60 ℃, 62 ℃) and different cycle numbers (30 and 32), running and dyeing, and determining the annealing temperature and the cycle number of the PCR amplification suitable for each pair of SSR primers according to the following standard: the amplified bands were clear and stable and the same primer showed at least 1 difference in amplified bands among 6 samples. The annealing temperature and cycle number optimization results of 29 pairs of SSR primer PCR amplification are shown in Table 4.
4. Verification of SSR molecular markers of acer ginnala
And (3) carrying out PCR amplification on the samples numbered 1-90 in the first step by adopting 29 pairs of SSR primers obtained by screening in the second step, wherein a PCR amplification reaction system is shown in a table 2, and PCR amplification reaction conditions are shown in a table 3. Each PCR amplification reaction system corresponds to a pair of SSR primers and a sample. The annealing temperatures and cycle numbers used for PCR amplification of each pair of SSR primers are shown in Table 4.
The results show that: each primer pair can be amplified in all maple leaf clematis samples to obtain polymorphic bands. Wherein,, the primer pair CA63 is amplified in 90 maple leaf clematis samples to obtain 3 types of bands, the primer pair CA64 is amplified in 90 maple leaf clematis samples to obtain 3 types of bands, the primer pair CA75 is amplified in 90 maple leaf clematis samples to obtain 4 types of bands, the primer pair CA85 is amplified in 90 maple leaf clematis samples to obtain 2 types of bands, the primer pair CA122 is amplified in 90 maple leaf clematis samples to obtain 2 types of bands, the primer pair CA142 is amplified in 90 maple leaf clematis samples to obtain 4 types of bands, the primer pair CA143 is amplified in 90 maple leaf clematis samples to obtain 2 types of bands, the primer pair CA255 is amplified in 90 maple leaf clematis samples to obtain 2 types of bands, the primer pair CA142 is amplified in 90 maple leaf clematis samples to obtain 2 types of bands, the primer pair CA143 is amplified in 90 maple leaf clematis samples to obtain 2 types of bands, and the primer pair CA143 is amplified in 90 maple leaf clematis samples to obtain 2 types of bands to obtain 6 types of bands. The primer pair CA356 is amplified in 90 maple leaf clematis samples to obtain 2 types of bands, the primer pair CA425 is amplified in 90 maple leaf clematis samples to obtain 3 types of bands, the primer pair CA454 is amplified in 90 maple leaf clematis samples to obtain 4 types of bands, the primer pair CA487 is amplified in 90 maple leaf clematis samples to obtain 3 types of bands, the primer pair CA506 is amplified in 90 maple leaf clematis samples to obtain 3 types of bands, the primer pair CA551 is amplified in 90 maple leaf clematis samples to obtain 2 types of bands, the primer pair CA554 is amplified in 90 maple leaf clematis samples to obtain 3 types of bands, the primer pair CA620 is amplified in 90 maple leaf clematis samples to obtain 2 types of bands, the primer pair CA635 is amplified in 90 maple leaf clematis samples to obtain 2 types of bands, the primer pair CA720 is amplified in 90 maple leaf clematis samples to obtain 6 types of bands, the primer pair CA782 is amplified in 90 maple leaf clematis samples to obtain 6 types of bands, the primer pair CA830 is amplified in 90 maple leaf clematis samples to obtain 2 types of bands, the primer pair CA997 is amplified in 90 maple leaf clematis samples to obtain 2 types of bands, the primer pair CA1091 is amplified in 90 maple leaf clematis samples to obtain 4 types of bands, the primer pair CA1152 is amplified in 90 maple leaf clematis samples to obtain 3 types of bands, the primer pair CA1324 is amplified in 90 maple leaf clematis samples to obtain 6 types of bands, the primer pair CA1544 is amplified in 90 maple leaf clematis samples to obtain 2 types of bands, and the primer pair CA1547 is amplified in 90 maple leaf clematis samples to obtain 5 types of bands.
Example 2 analysis of genetic diversity of Acer ginnala Maxim Using SSR molecular markers
1. 29 SSR locus genetic diversity parameters
The observed allele (Na), effective allele (Ne), shannon's diversity index (I), observed heterozygosity (Ho), expected heterozygosity (He), and F statistics (Fit, fis, fst) were calculated for 29 SSR sites using the population genetics software POPGENE version 1.32. Polymorphism Information Content (PIC) was calculated using PowerMarker V3.25 software. Gene flow (Num) = (1-Fst)/4 Fst was calculated.
The results are shown in Table 5. As can be seen from Table 5, the observed allele (Na) for 29 SSR sites varied over a range of 2 to 4 with an average value of 2.4483 in 9 populations. The effective allele (Ne) varied from 1.1172 to 2.2839 with an average value of 1.6310. The shannon's diversity index (I) varies from 0.2146 to 1.0092 with an average value of 0.5682; the primer with highest value of shannon's diversity index (I) is CA315, and the primer with lowest value is CA255, CA356, CA620 and CA635. The observed heterozygosity (Ho) varied from 0 to 0.8889, the average value was 0.3169, the primer with the highest observed heterozygosity (Ho) was CA305, and among CA143, CA830, CA1544 and CA551, the observed heterozygosity (Ho) was 0, indicating homozygosity at these 4 sites. The desired heterozygosity (He) varies from 0.1055 to 0.5653 with an average value of 0.3483. The variation range of the Polymorphism Information Content (PIC) is 0.0994 to 0.4888, the primer with the highest value of the Polymorphism Information Content (PIC) is CA315, the primer with the lowest value is CA255, CA356, CA620 and CA635, and the average value is 0.2910; 12 primers with Polymorphism Information Content (PIC) in the range of 0 to 0.25 account for 41.4%; 17 primers with Polymorphism Information Content (PIC) in the range of 0.25 to 0.5 account for 58.6%. F statistic analysis of the 29 pairs of markers revealed that the average value of the inbred coefficients Fis in the subpopulation was-0.3442 and the average value of the inbred coefficients Fit in the population was 0.0851, indicating that the loci were mostly heterozygotes in the subpopulation and more homozygotes in the population. Among the 9 populations, fst varied from 0.064 to 1 at polymorphic sites with an average of 0.3194, indicating 31.94% of allele frequency variation due to inter-population genetic variation, indicating a greater degree of genetic differentiation between the 9 populations. The gene flow (Num) of the polymorphic sites varied from 0 to 3.6573 with an average value of 0.5328.
Tables 5 and 29 for marker genetic diversity parameters
2. Genetic diversity parameter of maple leaf clematis population
The observed allele (Na), effective allele (Ne), shannon's diversity index (I), observed heterozygosity (Ho) and expected heterozygosity (He) were calculated for the 9 maple leaf clematis population using population genetics software POPGENE version 1.32.
As a result, as shown in Table 6, it was found from Table 6 that the observed allele (Na) of 9 groups varied in a range of 1.5517 to 1.7586, the average value was 1.6207, the highest value appeared in group p5, and the lowest value appeared in group p7. The effective allele (Ne) varies from 1.3485 to 1.5176 with an average value of 1.4299, the highest value occurring in population p5 and the lowest value occurring in population p8. The shannon's diversity index (I) varies from 0.2972 to 0.4011. The observed heterozygosity (Ho) was at a maximum of 0.3862 and a minimum of 0.2621. The desired heterozygosity (He) ranges from 0.2087 to 0.2800. The highest values for shannon's diversity index (I), observed heterozygosity (Ho), and desired heterozygosity (He) described above all occur in population p5, and the lowest values occur in population p6.
TABLE 6 genetic diversity parameters of maple leaf clematis population
| Group of people | Na | Ne | I | Ho | He |
| 1 | 1.6207 | 1.4675 | 0.3612 | 0.3207 | 0.2624 |
| 2 | 1.6207 | 1.4275 | 0.3501 | 0.3103 | 0.2505 |
| 3 | 1.5862 | 1.4405 | 0.3530 | 0.3448 | 0.2566 |
| 4 | 1.6207 | 1.4582 | 0.3661 | 0.3138 | 0.2655 |
| 5 | 1.7586 | 1.5176 | 0.4011 | 0.3862 | 0.2800 |
| 6 | 1.5862 | 1.3529 | 0.2972 | 0.2621 | 0.2087 |
| 7 | 1.5517 | 1.4280 | 0.3340 | 0.3034 | 0.2441 |
| 8 | 1.5862 | 1.3485 | 0.3099 | 0.3 | 0.2172 |
| 9 | 1.6552 | 1.4286 | 0.3492 | 0.3103 | 0.2481 |
| mean | 1.6207 | 1.4299 | 0.3469 | 0.3168 | 0.2481 |
Example 3 calculation of the genetic differentiation coefficient Fst between 9 Acer ginnala Maxim populations Using SSR molecular markers
The genetic differentiation coefficient (Fst) is a marker for the degree of differentiation between populations, varying between 0 and 1. Fst is less than 0.05, meaning that there is substantially no genetic differentiation trend between the two populations, fst is moderately differentiated between 0.05 and 0.15, fst is genetically differentiated to a greater extent between 0.15 and 0.25, and Fst is more than 0.25 indicates a greater extent of differentiation. Genetic differentiation coefficients of 9 maple leaf clematis populations were calculated using GenALEx 6.5.
As shown in Table 7, it is understood from Table 7 that the variation of the genetic differentiation coefficient (Fst) between 9 groups of maple leaf clematis was in the range of 0.023 to 0.469. The genetic differentiation coefficient (Fst) of maple clematis distributed in Henan and maple clematis distributed in Hebei and Beijing is greater than 0.25, which indicates that there is a great degree of genetic differentiation, and the maximum genetic differentiation coefficient (Fst) is between the group p8 and the group p5 (fst=0.469). Fst=0.044 between two populations distributed in river north, indicating that there is substantially no genetic differentiation between the two populations; the Fst value of maple leaf clematis distributed in Hebei is smaller than 0.05, which indicates that no differentiation exists between the groups basically; population p2 and population p3 distributed in river north have moderate genetic differentiation (fst=0.054), and population p4 has substantially no genetic differentiation; there was moderate genetic differentiation between the northwest distribution population and the Beijing distribution south Dan Yang population p6, the Beijing archway population p7, the nepheline cloud population p8, and the apricot population p 9.
The 6 populations distributed in Beijing were analyzed. There is substantially no genetic differentiation between p3 and p4, p3 and p8 of maple clematis population distributed in beijing, and there is moderate genetic differentiation between every two of maple clematis population distributed in beijing. The genetic differentiation coefficient between populations p3 and p4 is the smallest and between populations p3 and p6 the largest.
TABLE 7 genetic differentiation coefficient Fst for 9 populations
| Group of people | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 |
| 1 | 0.000 | ||||||||
| 2 | 0.044 | 0.000 | |||||||
| 3 | 0.036 | 0.054 | 0.000 | ||||||
| 4 | 0.023 | 0.032 | 0.039 | 0.000 | |||||
| 5 | 0.458 | 0.451 | 0.465 | 0.459 | 0.000 | ||||
| 6 | 0.108 | 0.067 | 0.104 | 0.080 | 0.448 | 0.000 | |||
| 7 | 0.085 | 0.073 | 0.078 | 0.073 | 0.446 | 0.076 | 0.000 | ||
| 8 | 0.069 | 0.065 | 0.040 | 0.051 | 0.469 | 0.060 | 0.070 | 0.000 | |
| 9 | 0.073 | 0.090 | 0.078 | 0.059 | 0.462 | 0.087 | 0.084 | 0.084 | 0 |
Example 4 Cluster analysis
Clustering analysis was performed on 90 sample materials using NTSYSpc 2.11a software.
As shown in fig. 1, it is clear from fig. 1 that 10 materials of maple leaf clematis group p5 distributed in henna are classified into two types with other materials at the genetic consistency of 0.38, and the materials distributed in beijing and henna are crossed, which means that the difference between the materials in henna and the materials in beijing and henna is large and the difference between the materials in beijing and henna is small. This is consistent with the trend of the previous analysis.
Example 5 analysis of genetic distance and genetic consistency of maple clematis group using SSR molecular markers the genetic distance and genetic consistency of the group was calculated using POPGENE version 1.32.
As shown in Table 8, it is clear from Table 8 that the variation of the genetic distances between 9 populations was 0.0148 to 0.9603, the closest populations were p1 and p4, and the farthest populations were p3 and p5. The genetic distance variation range of 6 groups distributed in Beijing is 0.0207 to 0.0699; the genetic distance between population 3 and population 4 is closest and the genetic distance between population 3 and population 6 is the farthest. The genetic distance between maple leaf clematis distributed in Henan and the population distributed in Beijing and Hebei is more than 0.8, and the genetic distance between Beijing and Hebei population is less than 0.1, which indicates that the genetic difference between Henan and Hebei population is larger.
TABLE 8 genetic identity between populations (upper) and genetic distance (lower)
| Group of people | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 |
| 1 | 0.9690 | 0.9812 | 0.9853 | 0.3959 | 0.9292 | 0.9455 | 0.9544 | 0.9533 | |
| 2 | 0.0315 | 0.9624 | 0.9796 | 0.4128 | 0.9629 | 0.9564 | 0.9581 | 0.9408 | |
| 3 | 0.0189 | 0.0383 | 0.9795 | 0.3828 | 0.9324 | 0.9508 | 0.9781 | 0.9501 | |
| 4 | 0.0148 | 0.0206 | 0.0207 | 0.3975 | 0.9577 | 0.9598 | 0.9734 | 0.9595 | |
| 5 | 0.9266 | 0.8849 | 0.9603 | 0.9226 | 0.4155 | 0.4384 | 0.3916 | 0.3887 | |
| 6 | 0.0734 | 0.0378 | 0.0699 | 0.0432 | 0.8783 | 0.9604 | 0.9618 | 0.9453 | |
| 7 | 0.0561 | 0.0446 | 0.0504 | 0.0410 | 0.8247 | 0.0405 | 0.9578 | 0.9547 | |
| 8 | 0.0467 | 0.0428 | 0.0221 | 0.0270 | 0.9375 | 0.0390 | 0.0431 | 0.9458 | |
| 9 | 0.0479 | 0.0610 | 0.0512 | 0.0414 | 0.9450 | 0.0562 | 0.0464 | 0.0557 |
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that it will be apparent to those skilled in the art that several modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the scope of the invention.
Sequence listing
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<210> 53
<211> 21
<212> DNA
<213> artificial sequence
<400> 53
caatttcgga aacatcgaat c 21
<210> 54
<211> 20
<212> DNA
<213> artificial sequence
<400> 54
ttcgctcaag actcccaagt 20
<210> 55
<211> 20
<212> DNA
<213> artificial sequence
<400> 55
gtgggcgatg gagatagaga 20
<210> 56
<211> 20
<212> DNA
<213> artificial sequence
<400> 56
tcaccaaaac gcatcagaac 20
<210> 57
<211> 20
<212> DNA
<213> artificial sequence
<400> 57
gaagtttaca cgcacgctca 20
<210> 58
<211> 20
<212> DNA
<213> artificial sequence
<400> 58
tgccaaggaa agagaatcca 20
Claims (9)
- An ssr primer set comprising primer pair 1, primer pair 2, primer pair 3, primer pair 4, primer pair 5, primer pair 6, primer pair 7, primer pair 8, primer pair 9, primer pair 10, primer pair 11, primer pair 12, primer pair 13, primer pair 14, primer pair 15, primer pair 16, primer pair 17, primer pair 18, primer pair 19, primer pair 20, primer pair 21, primer pair 22, primer pair 23, primer pair 24, primer pair 25, primer pair 26, primer pair 27, primer pair 28, and primer pair 29;the primer pair 1 consists of a primer 1-F and a primer 1-R; the primer pair 2 consists of a primer 2-F and a primer 2-R; the primer pair 3 consists of a primer 3-F and a primer 3-R; the primer pair 4 consists of a primer 4-F and a primer 4-R; the primer pair 5 consists of a primer 5-F and a primer 5-R; the primer pair 6 consists of a primer 6-F and a primer 6-R; the primer pair 7 consists of a primer 7-F and a primer 7-R; the primer pair 8 consists of a primer 8-F and a primer 8-R; the primer pair 9 consists of a primer 9-F and a primer 9-R; the primer pair 10 consists of a primer 10-F and a primer 10-R; the primer pair 11 consists of a primer 11-F and a primer 11-R; the primer pair 12 consists of a primer 12-F and a primer 12-R; the primer pair 13 consists of a primer 13-F and a primer 13-R; the primer pair 14 consists of a primer 14-F and a primer 14-R; the primer pair 15 consists of a primer 15-F and a primer 15-R; the primer pair 16 consists of a primer 16-F and a primer 16-R; the primer pair 17 consists of a primer 17-F and a primer 17-R; the primer pair 18 consists of a primer 18-F and a primer 18-R; the primer pair 19 consists of a primer 19-F and a primer 19-R; the primer pair 20 consists of a primer 20-F and a primer 20-R; the primer pair 21 consists of a primer 21-F and a primer 21-R; the primer pair 22 consists of a primer 22-F and a primer 22-R; the primer pair 23 consists of a primer 23-F and a primer 23-R; the primer pair 24 consists of a primer 24-F and a primer 24-R; the primer pair 25 consists of a primer 25-F and a primer 25-R; the primer pair 26 consists of a primer 26-F and a primer 26-R; the primer pair 27 consists of a primer 27-F and a primer 27-R; the primer pair 28 consists of a primer 28-F and a primer 28-R; the primer pair 29 consists of a primer 29-F and a primer 29-R;The primer 1-F is a single-stranded DNA molecule shown in a sequence 1 in a sequence table;the primer 1-R is a single-stranded DNA molecule shown in a sequence 2 of a sequence table;the primer 2-F is a single-stranded DNA molecule shown in a sequence 3 of a sequence table;the primer 2-R is a single-stranded DNA molecule shown in a sequence 4 of a sequence table;the primer 3-F is a single-stranded DNA molecule shown in a sequence 5 of a sequence table;the primer 3-R is a single-stranded DNA molecule shown in a sequence 6 of a sequence table;the primer 4-F is a single-stranded DNA molecule shown in a sequence 7 of a sequence table;the primer 4-R is a single-stranded DNA molecule shown in a sequence 8 of a sequence table;the primer 5-F is a single-stranded DNA molecule shown in a sequence 9 of a sequence table;the primer 5-R is a single-stranded DNA molecule shown in a sequence 10 of a sequence table;the primer 6-F is a single-stranded DNA molecule shown in a sequence 11 in a sequence table;the primer 6-R is a single-stranded DNA molecule shown in a sequence 12 of a sequence table;the primer 7-F is a single-stranded DNA molecule shown in a sequence 13 in a sequence table;the primer 7-R is a single-stranded DNA molecule shown in a sequence 14 of a sequence table;the primer 8-F is a single-stranded DNA molecule shown in a sequence 15 of a sequence table;the primer 8-R is a single-stranded DNA molecule shown in a sequence 16 of a sequence table;The primer 9-F is a single-stranded DNA molecule shown in a sequence 17 in a sequence table;the primer 9-R is a single-stranded DNA molecule shown in a sequence 18 of a sequence table;the primer 10-F is a single-stranded DNA molecule shown in a sequence 19 in a sequence table;the primer 10-R is a single-stranded DNA molecule shown in a sequence 20 of a sequence table;the primer 11-F is a single-stranded DNA molecule shown in a sequence 21 of a sequence table;the primer 11-R is a single-stranded DNA molecule shown in a sequence 22 in a sequence table;the primer 12-F is a single-stranded DNA molecule shown in a sequence 23 of a sequence table;the primer 12-R is a single-stranded DNA molecule shown in a sequence 24 of a sequence table;the primer 13-F is a single-stranded DNA molecule shown in a sequence 25 of a sequence table;the primer 13-R is a single-stranded DNA molecule shown in a sequence 26 of a sequence table;the primer 14-F is a single-stranded DNA molecule shown in a sequence 27 of a sequence table;the primer 14-R is a single-stranded DNA molecule shown in a sequence 28 of a sequence table;the primer 15-F is a single-stranded DNA molecule shown in a sequence 29 of a sequence table;the primer 15-R is a single-stranded DNA molecule shown in a sequence 30 of a sequence table;the primer 16-F is a single-stranded DNA molecule shown in a sequence 31 of a sequence table;the primer 16-R is a single-stranded DNA molecule shown in a sequence 32 of a sequence table;The primer 17-F is a single-stranded DNA molecule shown in a sequence 33 of a sequence table;the primer 17-R is a single-stranded DNA molecule shown in a sequence 34 of a sequence table;the primer 18-F is a single-stranded DNA molecule shown in a sequence 35 of a sequence table;the primer 18-R is a single-stranded DNA molecule shown in a sequence 36 of a sequence table;the primer 19-F is a single-stranded DNA molecule shown in a sequence 37 of a sequence table;the primer 19-R is a single-stranded DNA molecule shown in a sequence 38 of a sequence table;the primer 20-F is a single-stranded DNA molecule shown in a sequence 39 of a sequence table;the primer 20-R is a single-stranded DNA molecule shown in a sequence 40 of a sequence table;the primer 21-F is a single-stranded DNA molecule shown in a sequence 41 of a sequence table;the primer 21-R is a single-stranded DNA molecule shown in a sequence 42 of a sequence table;the primer 22-F is a single-stranded DNA molecule shown in a sequence 43 of a sequence table;the primer 22-R is a single-stranded DNA molecule shown in a sequence 44 of a sequence table;the primer 23-F is a single-stranded DNA molecule shown in a sequence 45 of a sequence table;the primer 23-R is a single-stranded DNA molecule shown in a sequence 46 of a sequence table;the primer 24-F is a single-stranded DNA molecule shown in a sequence 47 of a sequence table;the primer 24-R is a single-stranded DNA molecule shown in a sequence 48 of a sequence table;The primer 25-F is a single-stranded DNA molecule shown in a sequence 49 of a sequence table;the primer 25-R is a single-stranded DNA molecule shown in a sequence 50 of a sequence table;the primer 26-F is a single-stranded DNA molecule shown in a sequence 51 of a sequence table;the primer 26-R is a single-stranded DNA molecule shown in a sequence 52 of a sequence table;the primer 27-F is a single-stranded DNA molecule shown in a sequence 53 of a sequence table;the primer 27-R is a single-stranded DNA molecule shown in a sequence 54 of a sequence table;the primer 28-F is a single-stranded DNA molecule shown in a sequence 55 of a sequence table;the primer 28-R is a single-stranded DNA molecule shown in a sequence 56 of a sequence table;the primer 29-F is a single-stranded DNA molecule shown in a sequence 57 in a sequence table;the primer 29-R is a single-stranded DNA molecule shown in a sequence 58 of a sequence table.
- 2. The SSR primer set of claim 1, wherein: the SSR primer set consists of the primer pair 1, the primer pair 2, the primer pair 3, the primer pair 4, the primer pair 5, the primer pair 6, the primer pair 7, the primer pair 8, the primer pair 9, the primer pair 10, the primer pair 11, the primer pair 12, the primer pair 13, the primer pair 14, the primer pair 15, the primer pair 16, the primer pair 17, the primer pair 18, the primer pair 19, the primer pair 20, the primer pair 21, the primer pair 22, the primer pair 23, the primer pair 24, the primer pair 25, the primer pair 26, the primer pair 27, the primer pair 28, and the primer pair 29.
- 3. Use of a SSR primer set according to claim 1 or 2 in any one of the following m 1) -m 10):m 1) preparing a product for analyzing or evaluating genetic diversity of the clematis maple leaves;m 2) preparing a product for analyzing or evaluating the genetic differentiation degree of the clematis maple leaves;m 3) preparing a product for analyzing or evaluating genetic relativity of the clematis acervata lugens;m 4) preparing a product for constructing or preparing a genetic map or a fingerprint map of the clematis maple leaves;m 5) preparing and identifying products of maple leaf clematis in different geographical populations;m 6) analyzing or evaluating genetic diversity of the clematis acervata lugens;m 7) analyzing or evaluating the genetic differentiation degree of the clematis maple leaves;m 8) analyzing or evaluating genetic relatedness of the clematis acervata lugens;m 9) constructing or preparing a maple leaf clematis genetic map or a fingerprint map;m 10) identifying or distinguishing different geographic populations of maple leaf clematis.
- 4. A kit comprising the SSR primer set of claim 1 or 2; the kit has any one of the following functions n 1) -n 5):n 1) analyzing or evaluating genetic diversity of the clematis acervata lugens;n 2) analyzing or evaluating the genetic differentiation degree of the clematis acervata lugens;n 3) analyzing or evaluating genetic relatedness of the clematis acervata lugens;n 4) constructing or preparing a maple leaf clematis genetic map or a fingerprint map;n 5) identifying or distinguishing different geographic populations of maple leaf clematis.
- 5. A method for preparing a kit according to claim 4, comprising the step of individually packaging each of the primers in the SSR primer set according to claim 1 or 2.
- 6. Use of the kit of claim 4 in any one of the following m 1) -m 10):m 1) preparing a product for analyzing or evaluating genetic diversity of the clematis maple leaves;m 2) preparing a product for analyzing or evaluating the genetic differentiation degree of the clematis maple leaves;m 3) preparing a product for analyzing or evaluating genetic relativity of the clematis acervata lugens;m 4) preparing a product for constructing or preparing a genetic map or a fingerprint map of the clematis maple leaves;m 5) preparing and identifying products of maple leaf clematis in different geographical populations;m 6) analyzing or evaluating genetic diversity of the clematis acervata lugens;m 7) analyzing or evaluating the genetic differentiation degree of the clematis maple leaves;m 8) analyzing or evaluating genetic relatedness of the clematis acervata lugens;m 9) constructing or preparing a maple leaf clematis genetic map or a fingerprint map;m 10) identifying or distinguishing different geographic populations of maple leaf clematis.
- 7. A method for analyzing or evaluating genetic diversity of maple leaf clematis, a method for analyzing or evaluating genetic differentiation degree of maple leaf clematis, a method for analyzing or evaluating genetic relatedness of maple leaf clematis, a method for constructing or preparing a genetic map or a fingerprint map of maple leaf clematis, or a method for identifying or distinguishing different geographical populations of maple leaf clematis, comprising the step of performing PCR amplification on maple leaf genomic DNA by using the SSR primer set of claim 1 or 2.
- 8. The method of claim 7, wherein:the primer annealing temperature is 62 ℃ and the cycle number is 30 when the primer pair 1 is adopted for PCR amplification;primer annealing temperature is 58 ℃ and cycle number is 32 when the primer pair 2 is adopted for PCR amplification;primer annealing temperature is 60 ℃ and cycle number is 30 when the primer pair 3 is adopted for PCR amplification;primer annealing temperature is 60 ℃ and cycle number is 30 when the primer pair 4 is adopted for PCR amplification;primer annealing temperature is 58 ℃ and cycle number is 32 when the primer pair 5 is adopted for PCR amplification;the primer annealing temperature is 58 ℃ and the cycle number is 32 when the primer pair 6 is adopted for PCR amplification;the primer annealing temperature is 58 ℃ and the cycle number is 32 when the primer pair 7 is adopted for PCR amplification;primer annealing temperature is 60 ℃ and cycle number is 30 when the primer pair 8 is adopted for PCR amplification;the primer annealing temperature is 58 ℃ and the cycle number is 32 when the primer pair 9 is adopted for PCR amplification;primer annealing temperature is 58 ℃ and cycle number is 32 when the primer pair 10 is adopted for PCR amplification;the primer annealing temperature is 62 ℃ and the cycle number is 30 when the primer pair 11 is adopted for PCR amplification;the primer annealing temperature is 58 ℃ and the cycle number is 32 when the primer pair 12 is adopted for PCR amplification;The primer annealing temperature is 58 ℃ and the cycle number is 32 when the primer pair 13 is adopted for PCR amplification;primer annealing temperature is 60 ℃ and cycle number is 30 when the primer pair 14 is adopted for PCR amplification;primer annealing temperature is 58 ℃ and cycle number is 32 when the primer pair 15 is adopted for PCR amplification;the primer annealing temperature is 58 ℃ and the cycle number is 32 when the primer pair 16 is adopted for PCR amplification;the primer annealing temperature is 58 ℃ and the cycle number is 32 when the primer pair 17 is adopted for PCR amplification;primer annealing temperature is 58 ℃ and cycle number is 32 when the primer pair 18 is adopted for PCR amplification;the primer annealing temperature is 58 ℃ and the cycle number is 32 when the primer pair 19 is adopted for PCR amplification;primer annealing temperature is 58 ℃ and cycle number is 32 when the primer pair 20 is adopted for PCR amplification;primer annealing temperature is 60 ℃ and cycle number is 30 when the primer pair 21 is adopted for PCR amplification;primer annealing temperature is 58 ℃ and cycle number is 32 when the primer pair 22 is adopted for PCR amplification;primer annealing temperature is 58 ℃ and cycle number is 32 when the primer pair 23 is adopted for PCR amplification;primer annealing temperature is 58 ℃ and cycle number is 32 when the primer pair 24 is adopted for PCR amplification;primer annealing temperature is 58 ℃ and cycle number is 32 when the primer pair 25 is adopted for PCR amplification;Primer annealing temperature is 58 ℃ and cycle number is 32 when the primer pair 26 is adopted for PCR amplification;the primer annealing temperature is 58 ℃ and the cycle number is 32 when the primer pair 27 is adopted for PCR amplification;the primer annealing temperature is 58 ℃ and the cycle number is 32 when the primer pair 28 is adopted for PCR amplification;the primer annealing temperature in PCR amplification using the primer set 29 was 58℃and the cycle number was 32.
- 9. The use according to claim 3 or the kit according to claim 4 or the use according to claim 6 or the method according to claim 7 or 8, characterized in that: the maple leaf clematis is maple leaf clematis from Beijing, maple leaf clematis from Hebei and maple leaf clematis from Henan.
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