CN107012200B - Anvil pumpkin ' the method for identifying molecules of big anvil 1 ' cenospecies - Google Patents
Anvil pumpkin ' the method for identifying molecules of big anvil 1 ' cenospecies Download PDFInfo
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- CN107012200B CN107012200B CN201610677462.6A CN201610677462A CN107012200B CN 107012200 B CN107012200 B CN 107012200B CN 201610677462 A CN201610677462 A CN 201610677462A CN 107012200 B CN107012200 B CN 107012200B
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- anvil
- primer
- specific band
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
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Abstract
The invention discloses the SSR primers and method that are used for anvil pumpkin ' big anvil 1 ' Hybridization identification.SSR primer used are as follows: forward primer: 5 '-CAAATTCAGACGCTTCTTTTGG-3 ' and reverse primer: 5 '-AGAATTGAGCAAAAAGGAGATGG-3 ', identification method is the following steps are included: (1) is extracted for trying pumpkin seedling leaves genomic DNA；(2) using pumpkin genomic DNA as template, PCR amplification is carried out using SSR primer；(3) polyacrylamide gel electrophoresis is carried out to amplified production；(4) electrophoresis result is analyzed, according to the specific band of PCR amplification, if male parent and maternal specific band, as real cenospecies occurs simultaneously in filial generation；If only there is the specific band of male parent or female parent in filial generation, as false hybridization.This method has quick, accurate, low cost, simple operation and other advantages.
Description
Technical field
The present invention relates to Molecular Detection field, it is a kind of quickly, detection anvil conveniently, accurate and effective is with pumpkin ' big anvil 1 '
The method for identifying molecules of cenospecies, belongs to molecular biology field.
Background technique
Seed purity is to guarantee that kind merit is able to most spine in the key factor showed and seed quality control
The problem of hand.Influence of traditional field ecology cenospecies method of inspection vulnerable to environmental condition and cultivation step, qualification cycle
Long, at high cost, qualification result accuracy is poor.
With the fast development of marker assisted selection tool, DNA molecular marker is better than traditional identification in terms of many
Method can make up many defects and problem present in conventional identification method, be to carry out quickly, accurately reflecting to seed cenospecies
Fixed developing direction and inevitable choice.Currently, SSR molecular marker technology is at this stage for identifying the common method of cenospecies.
SSR (Simple sequence repeat, SSR) i.e. simple repeated sequence is to be prevalent in eukaryotic gene
One of group repetitive sequence.SSR marker belongs to microsatellite marker, has many good qualities, including polymorphism height, codominance, repetition
Property it is good, quantity is abundant, and easy to operate easy detection wide to genomic coverage, thus become building genetic linkage maps,
Diversity Detection, carry out molecular mark, pedigree analysis, Variety fingerprinting draw, variety detection with
And the ideal tools of objective trait molecular marker screening, it has been widely used in gene mapping and cloning, cultivar identification, crops
The fields such as breeding and Study on Evolution.
Anvil is with ' SF1 ' for female parent with pumpkin ' big anvil 1 ', and ' S26 ' is the pumpkin first cross kind that male parent is bred as.Have
Strong, the high resistance to wilt good with cucumber affinity, Grafted Cucumber Seedling increase sugar-preserved gourd brightness the characteristics of, cucumber in greenhouse production in big face
Product application.In order to guarantee the popularization of superior hybrid crosses and generate maximum economic benefit, filtering out can be to pumpkin ' big anvil 1
Number ' cenospecies carry out precise Identification SSR primer, cause seed production purity not high during the production of hybrid seeds with solution ' big anvil 1 '
Problem provides an accurate, stable, quick, practical ' side for big anvil 1 ' Hybridization identification for seed production and operation enterprise
Method.
Summary of the invention
The object of the present invention is to provide one kind for detecting ' the primer sequence of big anvil 1 ' squash hybridization kind.Mesh of the present invention
Also reside in provide it is a kind of utilize above-mentioned primer carry out the ' method for identifying molecules of big anvil 1 ' squash hybridization kind.
To achieve the above object, the present invention adopts the following technical scheme:
(1) genomic DNA of pumpkin seedling is extracted；
(2) using genomic DNA as template, from acquired SSR primer carry out PCR amplification filter out male parent ' S26 ' with
The apparent primer NG22 of specific band difference between maternal ' SF1 '；
(3) using the genomic DNA of anvil squash hybridization kind ' big anvil 1 ' as template, PCR is carried out using SSR primer NG22
Amplification carries out polyacrylamide gel electrophoresis detection to amplified production；
(4) electrophoresis detection result is analyzed: if filial generation while there is male parent and maternal specific band, as very
Positive cenospecies；If only there is the specific band of male parent or female parent in filial generation, as false cenospecies；
Detailed description of the invention
Figure: amplification of the SSR marker NG22 in parent and filial generation, M are pBR322DNA/Msp I, and 1-6 is ' big anvil 1
Number ', 7-8 is male parent, and 9-10 is female parent.
Specific embodiment
In order to more clearly illustrate method of the invention, test method of the invention is made with specifically below
Bright, need to be illustrated herein: reagent used in the present invention is commercially available.
(1) for trying pumpkin material
Material therefor includes anvil squash hybridization kind ' big anvil 1 ' and its male parent ' S26 ' and female parent ' SF1 '.
(2) for the extraction of examination pumpkin genomic DNA
(1) leaf harvest: will be seeded in culturing pot for examination pumpkin material, to three leaves wholeheartedly period, take at the top of plant compared with
For tender blade, saved backup at -80 DEG C.
(2) take a piece of cushaw leaf in 1.5ml centrifuge tube, be fully ground on ice chest, be added 600 μ l preheating 2 ×
CTAB buffer, the beta -mercaptoethanol of 10 μ l, mixes gently, 65 DEG C of water-bath 1h (jiggling once every 10min).
(3) add isometric chloroform, mix gently, 12000rpm is centrifuged 10min, takes supernatant in another new centrifuge tube
In.
(4) ibid.
(5) the product isopropanol for the equal bodies pre-cooling being added, mixes gently, -20 DEG C of preservation 30min, 12000rpm is centrifuged
10min abandons supernatant.
(6) 70% ethanol washings precipitate 2-3 times, are placed in superclean bench and air-dry.
(7) ddH of 50 μ l is added2O dissolution.
(8) detection of DNA mass: 1% agarose gel electrophoresis detection.
(3) screening of SSR molecular marker primer
It is poor to filter out the specific band between male parent and female parent for progress PCR amplification from acquired 56 SSR primers
Different apparent primer, i.e. NG22.
PCR reaction system and program
(1) PCR reaction system
(2) PCR response procedures
PCR response procedures:
94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, 57 DEG C of annealing 30s, 72 DEG C of extension 1min, 30 recycle；72 DEG C are always prolonged
Stretch 10min.It is saved in 4 DEG C of refrigerators.
PCR product detection
(3) 12% native polyacrylamide gel electrophoresis detects PCR amplification result:
Gel component (15ml):
150V electrophoresis 3h closes power supply, and twice to colloid washing, the fixed 10min of 10% ethyl alcohol is washed twice, 1% nitric acid
It is sensitized 5min, twice, 0.2% cma staining 30min, twice, 2% sodium hydroxide develops the color to there is clear purpose for washing for washing
Band, twice, 10% acetic acid stops aobvious 2min for washing, washes preservation of taking pictures twice.
As shown, 1-5: anvil amplifies two band of 107bp and 113bp with pumpkin ' big anvil 1 '；6,7: male parent ' S26 '
Amplify the band of 113bp；8,9: maternal ' SF1 ' amplifies the band of 107bp.Specific band is recycled, is served
The sequencing of marine growth engineering company.The sequence of 113bp band is as shown in SEQ ID NO:3 in cenospecies, 107bp band
Sequence as shown in SEQ ID NO:4, be consistent with the sequence of male parent, maternal amplified production.
。
Claims (2)
1. the SSR primer NG22 that one kind is used for anvil pumpkin ' big anvil 1 ' cenospecies Molecular Identification, which is characterized in that draw upstream
Object: 5 '-CAAATTCAGACGCTTCTTTTGG-3 ', downstream primer: 5 '-AGAATTGAGCAAAAAGGAGATGG-3 ', it is described on
Primer nucleotide sequences are swum as shown in SEQ ID NO:1, the downstream primer nucleotide sequence is as shown in SEQ ID NO:2.
2. using method for identifying molecules of the primer detection anvil with squash hybridization kind ' big anvil 1 ' described in claim 1, feature
Include the following steps:
(1) genomic DNA of pumpkin seedling is extracted；
(2) using genomic DNA as template, using the NG22 primer of claim 1 into PCR amplification；
(3) polyacrylamide gel electrophoresis detection is carried out to pcr amplification product；
(4) electrophoresis detection result is analyzed: if filial generation while there is male parent and maternal specific band, as really
Cenospecies；If only there is the specific band of male parent or female parent in filial generation, as false cenospecies, produced by SSR molecular marker primer
Specific band size it is as follows: primer NG22 to specific band size caused by male parent be 113bp, to produced by female parent
Specific band size be 107bp.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610677462.6A CN107012200B (en) | 2016-08-17 | 2016-08-17 | Anvil pumpkin ' the method for identifying molecules of big anvil 1 ' cenospecies |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610677462.6A CN107012200B (en) | 2016-08-17 | 2016-08-17 | Anvil pumpkin ' the method for identifying molecules of big anvil 1 ' cenospecies |
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| Publication Number | Publication Date |
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| CN107012200A CN107012200A (en) | 2017-08-04 |
| CN107012200B true CN107012200B (en) | 2019-09-10 |
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| CN201610677462.6A Expired - Fee Related CN107012200B (en) | 2016-08-17 | 2016-08-17 | Anvil pumpkin ' the method for identifying molecules of big anvil 1 ' cenospecies |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009029771A2 (en) * | 2007-08-29 | 2009-03-05 | Monsanto Technology Llc | Methods and compositions for breeding for preferred traits |
| CN103088127A (en) * | 2013-01-05 | 2013-05-08 | 广东省农业科学院蔬菜研究所 | Primers and method for purity identification of hybrid seeds of Chinese pumpkin 'Guangmi NO.1' |
| WO2014200348A1 (en) * | 2013-06-14 | 2014-12-18 | Keygene N.V. | Directed strategies for improving phenotypic traits |
| CN104694642A (en) * | 2015-02-25 | 2015-06-10 | 华中农业大学 | Pumpkin SSR labeled primers applied to multiplex PCR reaction and application of pumpkin SSR labeled primers |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8609928B2 (en) * | 2010-07-15 | 2013-12-17 | Vilmorin & Cie | Squash leaf curl virus (SLCV) resistance in cucurbits |
-
2016
- 2016-08-17 CN CN201610677462.6A patent/CN107012200B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009029771A2 (en) * | 2007-08-29 | 2009-03-05 | Monsanto Technology Llc | Methods and compositions for breeding for preferred traits |
| CN103088127A (en) * | 2013-01-05 | 2013-05-08 | 广东省农业科学院蔬菜研究所 | Primers and method for purity identification of hybrid seeds of Chinese pumpkin 'Guangmi NO.1' |
| WO2014200348A1 (en) * | 2013-06-14 | 2014-12-18 | Keygene N.V. | Directed strategies for improving phenotypic traits |
| CN104694642A (en) * | 2015-02-25 | 2015-06-10 | 华中农业大学 | Pumpkin SSR labeled primers applied to multiplex PCR reaction and application of pumpkin SSR labeled primers |
Non-Patent Citations (3)
| Title |
|---|
| Development of microsatellite markers from an enriched genomic library of pumpkin (Cucurbita moschata L.);Watcharawongpaiboon, N. 等;《Songklanakarin J. Sci. Technol.》;20071031;第29卷(第5期);1217-1223 |
| 利用SSR标记技术鉴定南瓜杂交种纯度的研究;韩小霞;《分子植物育种》;20140115;第12卷(第1期);112-117 |
| 南瓜属EST-SSR标记的开发及在杂种纯度鉴定中的应用;张国裕等;《华北农学报》;20111231;第26卷(第6期);97-101 |
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| CN107012200A (en) | 2017-08-04 |
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Granted publication date: 20190910 Termination date: 20200817 |