CN106967793B - Molecular detection method for rapidly identifying cornus wisoniana population - Google Patents
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- 241001477876 Cornus wilsoniana Species 0.000 title claims abstract description 33
- 238000001514 detection method Methods 0.000 title claims abstract description 17
- 108020004414 DNA Proteins 0.000 claims abstract description 40
- 108091092878 Microsatellite Proteins 0.000 claims abstract description 13
- 239000003147 molecular marker Substances 0.000 claims abstract description 11
- 238000012408 PCR amplification Methods 0.000 claims abstract description 10
- 238000007619 statistical method Methods 0.000 claims abstract description 6
- 238000000034 method Methods 0.000 claims description 12
- 241000759832 Cornus walteri Species 0.000 claims description 9
- 238000000137 annealing Methods 0.000 claims description 4
- 238000004925 denaturation Methods 0.000 claims description 2
- 230000036425 denaturation Effects 0.000 claims description 2
- 239000012154 double-distilled water Substances 0.000 claims description 2
- 238000012257 pre-denaturation Methods 0.000 claims description 2
- 102000004190 Enzymes Human genes 0.000 claims 1
- 108090000790 Enzymes Proteins 0.000 claims 1
- 230000001066 destructive effect Effects 0.000 abstract description 3
- 238000005070 sampling Methods 0.000 abstract description 3
- 241000894007 species Species 0.000 abstract description 3
- 241000196324 Embryophyta Species 0.000 description 11
- 239000003921 oil Substances 0.000 description 4
- 239000003550 marker Substances 0.000 description 3
- 241000209020 Cornus Species 0.000 description 2
- 238000007400 DNA extraction Methods 0.000 description 2
- 244000147058 Derris elliptica Species 0.000 description 2
- 208000031226 Hyperlipidaemia Diseases 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 235000013399 edible fruits Nutrition 0.000 description 2
- 239000008157 edible vegetable oil Substances 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 241001647745 Banksia Species 0.000 description 1
- 241000142975 Cornaceae Species 0.000 description 1
- 241000759836 Cornus chinensis Species 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- 235000014123 Pistacia chinensis Nutrition 0.000 description 1
- 240000000432 Pistacia chinensis Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
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- 238000012986 modification Methods 0.000 description 1
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- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000001603 reducing effect Effects 0.000 description 1
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- C12Q2600/156—Polymorphic or mutational markers
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Abstract
The invention discloses a molecular detection method for rapidly identifying cornus wisoniana population, which comprises the steps of respectively carrying out PCR amplification by using DNA of cornus wisoniana as a template and SEQ ID Nos. 1-30 as primer pairs to obtain microsatellite molecular marker data; and then carrying out statistical analysis on the microsatellite molecular marker data, and identifying and classifying the cornus wisoniana population. The molecular detection method for rapidly identifying the cornus wisoniana population quickly obtains genome information on the premise of non-destructive sampling, has the advantages of less workload, high efficiency and accurate, time-saving and labor-saving judgment, and the judgment accuracy is as high as 95%; has important value and application prospect for the rapid molecular detection of species populations.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a molecular detection method for rapidly identifying cornus wisoniana population.
Background
Cornus wilsoniana Wanger, also called Cornus wilsoniana Wanger, Sago oleifera, Pistacia chinensis, Banksia indica and the like, belongs to Cornus of Cornaceae, belongs to the genus of Cornus, and is a special multipurpose local woody oil tree species in China. Cornus walteri fruit oil has been used as edible oil for 100 years and is identified by the national food ministry in 1981, and the Cornus walteri oil is first-grade edible oil; in 12 months in 2013, the Councis cornus chinensis fruit oil is approved by the State health and family planning Commission to be a new food raw material in China. Animal and clinical test results show that cornus wisoniana oil can significantly reduce cholesterol in vivo, prevent and treat hyperlipidemia, especially hyperlipidemia of the elderly, and has weight reducing effect. Cornus wisoniana is an important oil tree species with economic, ecological and social benefits.
However, the population of cornus wisoniana is generally difficult to distinguish from phenotypic characteristics, and especially in the 70-80 s of the last century, artificial wide spread has been carried out in a certain area, and certain difficulty is brought to collection, selection and establishment of a gene bank. Therefore, an accurate method for judging the origin of the population is urgently needed to be solved, and necessary technical support is provided for the breeding of cornus wisoniana.
The plant genome has good stability, is basically not influenced by the environment and is convenient to obtain materials. Therefore, it is fully feasible to trace the origin of plants by detecting their genomic differences.
The DNA molecular marker is one of important methods for rapidly identifying the biological genome difference, wherein the microsatellite marker has the advantages of high polymorphism, good stability, convenient operation and the like, and by detecting the microsatellite marker in a target plant population, not only can individuals with obvious differences in characters be distinguished, but also the similarity on the population level can be effectively judged.
At present, no related report on the rapid identification of cornus wisoniana population molecules exists at home and abroad.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides a molecular detection method for rapidly identifying cornus wisoniana population under non-destructive sampling and a primer pair thereof.
In order to realize the purpose of the invention, the invention adopts the following technical scheme:
a molecular detection method for rapidly identifying cornus wisoniana population comprises the following steps:
(1) taking DNA of cornus wisoniana as a template, respectively taking SEQ ID Nos. 1-2, 3-4, 5-6, 7-8, 9-10, 11-12, 13-14, 15-16, 17-18, 19-20, 21-22, 23-24, 25-26, 27-28 and 29-30 as primer pairs, carrying out PCR amplification to obtain microsatellite molecular marker data;
(2) and carrying out statistical analysis on the microsatellite molecular marker data, and identifying and classifying cornus wisoniana population.
In some embodiments, the reaction procedure of the PCR amplification in step (1) is:
step S1, pre-denaturation at 94 ℃ for 5 min;
step S2, denaturation at 94 ℃ for 30S;
step S3, annealing at 57 ℃ for 30S;
step S4, extension at 72 ℃ for 20S;
step S5, repeating the steps S2 to S4 35 times, and the last step S4 lasts for 10 min.
In some embodiments, the reaction system for PCR amplification in step (1) is: DNA template 1.0. mu.L, primer pair 0.2. mu. L, Mix 10. mu.L, plus ddH2O to 20. mu.L.
The invention also provides a primer pair for rapidly identifying the cornus wisoniana population, and the following technical scheme is adopted:
a primer pair for rapidly identifying Cornus walteri population comprises 1-2 of SEQ ID Nos, 3-4 of SEQ ID Nos, 5-6 of SEQ ID Nos, 7-8 of SEQ ID Nos, 9-10 of SEQ ID Nos, 11-12 of SEQ ID Nos, 13-14 of SEQ ID Nos, 15-16 of SEQ ID Nos, 17-18 of SEQ ID Nos, 19-20 of SEQ ID Nos, 21-22 of SEQ ID Nos, 23-24 of SEQ ID Nos, 25-26 of SEQ ID Nos, 27-28 of SEQ ID Nos and 29-30 of SEQ ID Nos.
According to the method, DNA extraction is carried out on target test plants, DNA amplification is carried out according to 15 microsatellite marker primer pairs with good stability and strong polymorphism of cornus wisoniana, molecular marker data are obtained, and the molecular marker data are analyzed by adopting statistical analysis software, so that the cornus wisoniana population is classified. Compared with the prior art, the invention has the following beneficial effects:
1. the molecular detection method for rapidly identifying the cornus wisoniana population quickly obtains genome information on the premise of non-destructive sampling, has the advantages of less workload, high efficiency and accurate, time-saving and labor-saving judgment, and the judgment accuracy is as high as 95%;
2. the molecular detection method for rapidly identifying the cornus wisoniana population has important value and application prospect for rapid molecular detection of species populations.
Drawings
FIG. 1 is a flow chart of the molecular detection method for rapidly identifying Cornus walteri population of the present invention;
fig. 2 is a two-dimensional graph for distinguishing the population of 20 cornus wisoniana plants in embodiment 1 of the present invention, wherein LC: guangdong Lechang population; JX: jiangxi Ganzhou population.
Detailed Description
The invention is further described below with reference to the drawings and specific examples, which are not described for prior art. Specific examples of the present invention are given below, but the examples are only for the purpose of further describing the present invention in detail and do not limit the claims of the present invention.
Example 1 molecular detection method for rapidly identifying cornus wisoniana population
Referring to fig. 1, a flow chart of a molecular detection method for rapidly identifying cornus wisoniana population is shown, wherein the detection method comprises the following steps:
(1) DNA extraction is carried out on leaf samples of 20 cornus wisoniana plants in 2 populations in the west and the east of Guangdong according to a conventional method in the field;
(2) respectively taking the DNA extracted in the step (1) as a template, and respectively taking SEQ ID Nos. 1-2, 3-4, 5-6, 7-8, 9-10, 11-12, 13-14, 15-16, 17-18, 19-20, 21-22, 23-24, 25-26, 27-28 and 29-30 as primer pairs for PCR amplification to obtain microsatellite molecular marker data;
wherein, the sequences of the primer pairs are as follows:
the reaction system for each PCR amplification (20. mu.L) was:
the reaction procedure for PCR amplification was:
step S1, pre-denaturing the whole genome DNA at 94 ℃ for 5min in sequence;
step S2, denaturing the pre-denatured product at 94 ℃ for 30S to obtain a denatured product;
step S3, annealing the denatured product at 57 ℃ for 30S to obtain an annealed product;
step S4, extending the annealing product at 72 ℃ for 20S;
step S5, repeating the steps S2 to S4 35 times, and the last step S4 lasts for 10 min.
(3) And statistically analyzing, identifying and classifying cornus wisoniana population.
The non-commercial GenAlEx 6.503 software is adopted to analyze the microsatellite molecular marker data, and the basic operation principle of statistical analysis is as follows: according to the principle that genome information (the size of the DNA fragment marked by the microsatellite in the embodiment) with the same type of property has similarity, namely the similarity of the marks is high when the similarity of the plants is high, software calculates the similarity of the marks on the mark information between the plants according to the characteristics of the microsatellite mark of each plant, and classifies according to the similarity; and obtaining the species group discrimination and classification of the plants.
The statistical analysis results of 20 plant leaf samples of the Jiangxi and Guangdong 2 populations are shown in FIG. 2, and the results show that 7 plants in the 20 populations are from the Guangdong population and 13 plants are from the Jiangxi population.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Sequence listing
Less than 110 and more than Guangdong province forestry scientific research institute
Molecular detection method for rapidly identifying cornus wisoniana population less than 120%
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Claims (7)
1. A molecular detection method for rapidly identifying cornus wisoniana population is characterized by comprising the following steps:
(1) taking DNA of cornus wisoniana as a template, and respectively taking SEQ ID Nos. 1-2, 3-4, 5-6, 7-8, 9-10, 11-12, 13-14, 15-16, 17-18, 19-20, 21-22, 23-24, 25-26, 27-28 and 29-30 as primer pairs for PCR amplification to obtain microsatellite molecular marker data;
(2) and carrying out statistical analysis on the microsatellite molecular marker data, and identifying and classifying cornus wisoniana population.
2. The method for rapidly identifying Cornus walteri populations as claimed in claim 1, wherein the reaction procedure of the PCR amplification in the step (1) is as follows:
step S1, pre-denaturation at 94 ℃ for 5 min;
step S2, denaturation at 94 ℃ for 30S;
step S3, annealing at 57 ℃ for 30S;
step S4, extension at 72 ℃ for 20S;
step S5, repeating the steps S2 to S4 35 times, and the last step S4 lasts for 10 min.
3. The method for rapidly identifying Cornus walteri populations as claimed in claim 1, wherein the PCR amplification reaction in the step (1)The method comprises the following steps: DNA template 1.0. mu.L, primer pair 0.2. mu. L, Mix 10. mu.L, plus ddH2O to 20. mu.L.
4. The method for rapidly identifying Cornus walteri populations as claimed in claim 3, wherein said Mix comprises Taq enzyme, dNTPs and buffer.
5. Method for the rapid identification of Cornus walteri populations according to any one of claims 1 to 4, characterized in that the software used for said statistical analysis is the non-commercial GenAlEx 6.503 software.
6. The method for rapidly identifying Cornus walteri populations as claimed in any one of claims 1 to 4, wherein the DNA is extracted from leaves.
7. The primer pair for rapidly identifying the cornus wisoniana population is characterized by comprising 1-2 parts of SEQ ID No, 3-4 parts of SEQ ID No, 5-6 parts of SEQ ID No, 7-8 parts of SEQ ID No, 9-10 parts of SEQ ID No, 11-12 parts of SEQ ID No, 13-14 parts of SEQ ID No, 15-16 parts of SEQ ID No, 17-18 parts of SEQ ID No, 19-20 parts of SEQ ID No, 21-22 parts of SEQ ID No, 23-24 parts of SEQ ID No, 25-26 parts of SEQ ID No, 27-28 parts of SEQ ID No and 29-30 parts of SEQ ID No.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101225443A (en) * | 2007-12-21 | 2008-07-23 | 南京大学 | Molecule detecting method for rapidly identifying salt-tolerant iris species |
| CN103255223A (en) * | 2013-05-22 | 2013-08-21 | 青岛农业大学 | Primer and method for authenticating frankliniella occidentalis by using expressed sequence tag (EST) microsatellite markers |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101225443A (en) * | 2007-12-21 | 2008-07-23 | 南京大学 | Molecule detecting method for rapidly identifying salt-tolerant iris species |
| CN103255223A (en) * | 2013-05-22 | 2013-08-21 | 青岛农业大学 | Primer and method for authenticating frankliniella occidentalis by using expressed sequence tag (EST) microsatellite markers |
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