CN105907869A - SSR primer and method for rootstock pumpkin "No. 2 Huang Chenggen" hybrid seed purity identification - Google Patents
SSR primer and method for rootstock pumpkin "No. 2 Huang Chenggen" hybrid seed purity identification Download PDFInfo
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- 235000015136 pumpkin Nutrition 0.000 title abstract 4
- 238000012408 PCR amplification Methods 0.000 claims abstract description 8
- 230000003321 amplification Effects 0.000 claims abstract description 4
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 4
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- 241000196324 Embryophyta Species 0.000 claims abstract description 3
- 101000958312 Homo sapiens Lymphocyte antigen 6 complex locus protein G6f Proteins 0.000 claims description 14
- 102100038226 Lymphocyte antigen 6 complex locus protein G6f Human genes 0.000 claims description 14
- 230000008774 maternal effect Effects 0.000 claims description 12
- 239000003550 marker Substances 0.000 claims description 8
- 235000009852 Cucurbita pepo Nutrition 0.000 claims description 6
- 238000009396 hybridization Methods 0.000 claims description 6
- 238000001502 gel electrophoresis Methods 0.000 claims description 5
- 235000020354 squash Nutrition 0.000 claims description 5
- 230000004087 circulation Effects 0.000 claims description 3
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- 238000004925 denaturation Methods 0.000 claims description 3
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- 102000004190 Enzymes Human genes 0.000 claims description 2
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- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 2
- 230000001186 cumulative effect Effects 0.000 claims 1
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- 108020004414 DNA Proteins 0.000 description 9
- 238000004519 manufacturing process Methods 0.000 description 6
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 208000002109 Argyria Diseases 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
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- 239000006228 supernatant Substances 0.000 description 3
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 240000008067 Cucumis sativus Species 0.000 description 2
- 235000010799 Cucumis sativus var sativus Nutrition 0.000 description 2
- 241000219122 Cucurbita Species 0.000 description 2
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- 239000000463 material Substances 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 235000013311 vegetables Nutrition 0.000 description 2
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- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
- 208000027877 Disorders of Sex Development Diseases 0.000 description 1
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- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
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- 238000000605 extraction Methods 0.000 description 1
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- 201000005611 hermaphroditism Diseases 0.000 description 1
- 230000010196 hermaphroditism Effects 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical compound O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 description 1
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Abstract
The invention discloses an SSR primer and method for rootstock pumpkin "No. 2 Huang Chenggen" hybrid seed purity identification. The SSR primer includes a forward primer: 5'-GGCATTTCTGAGAACAGCTT-3' and a reverse primer 5'-ACGTTAGTTATGCTATTTTGTAGGC-3'. The identification method includes: 1) extracting the genomic DNA of rootstock pumpkin seedlings; 2) using the rootstock pumpkin genomic DNA as the template, and using the SSR primer to conduct PCR amplification; 3) carrying out polyacrylamide gel electrophoresis on the amplification product; and 4) analyzing the electrophoresis result to conclude that a single plant having the specific bands of male parent and female parent simultaneously is a true hybrid, and the offspring only having the female parent characteristic band without male parent characteristic band is a false hybrid or self-bred seed. The primer and the method provided by the invention can effectively and rapidly distinguish "No. 2 Huang Chenggen" hybrid seeds and the female parent seeds and male parent seeds thereof, and accurately detect the hybrid seed purity. The method has the advantages of rapidity, accuracy, low cost, and simple operation, etc.
Description
Technical field:
The invention belongs to Molecular Detection field, be specifically related to a kind of for anvil squash hybridization kind " yellow really root 2 " seed
The primer of Purity and method.
Background technology:
The height of seed quality is related to agricultural production security, and one of important indicator weighing seed quality is exactly pure
Degree, therefore carries out purity detecting particularly important during agricultural production and quality monitoring to seed.With morphology Marker Identification
The purity of kind and the true and false, time-consumingly grow, be subject to seasonal restrictions, polymorphism poor.Along with the extensive application of DNA molecular marker technology, make
Must identify that from genomic level purity has become the important directions of Purity Identification.The most conventional molecular marking technique bag
Include AFLP (Amplified fragment length polymorphism), RAPD (Random amplified
polymorphic DNA)、SCAR(Sequence Characterized Amplified Region)、ISSR(Inter
simple sequence repeat)、DAF(DNA amplified fingerprint)、SSR(Simple sequence
Repeat) etc..SSR molecular marker technology is abundant with its marker number, polymorphism high, in codominant inheritance, reproducible, be prone to
The plurality of advantages such as operation, reliable results become quick, stable, Purity method accurately, can be by Parent complementation band
The cenospecies of type and its parents, possibly even the cenospecies that some allos pollen cause is distinguished.Based on These characteristics,
One of SSR marker preferable molecular marker progressively becoming vegetable crop cultivar identification.
Fructus Cucurbitae moschatae is Cucurbitaceae Cucurbita vegetable crop, belongs to the different flowering plant of hermaphroditism.String powder during the production of hybrid seeds and
Mechanical admixture after results causes improved seeds to mix, and the selfed seed that maternal emasculation is the most thoroughly formed in addition, is to cause seed to be lost
Pass the main cause that purity declines.Meanwhile, along with Fructus Cucurbitae moschatae Cultivars number increase and breeding material hereditary basis day by day
Narrow so that interracial hereditary difference is more and more less, cause the difficulty of variety authentication and Purity to continue to increase, therefore
Set up a set of accurately, quickly, stable, be not to ensure that breeding is timely by the Hybrid seed purity test technical system of such environmental effects
Sell and safe handling, specification Seed Market and promote that China seed industry takes part in international competition in the urgent need to.
Anvil Fructus Cucurbitae moschatae " yellow really root 2 " is with S4236 as female parent, and XR is the anvil Fructus Cucurbitae moschatae first cross kind that male parent is bred as.
" yellow really root 2 " has strong with Fructus Cucumidis sativi affinity, and high resistance to wilt good, Grafted Cucumber Seedling increases the feature of sugar-preserved gourd brightness, setting
Execute large-area applications in cucumber production.In order to ensure the popularization of superior hybrid crosses and produce maximum economic benefit, filter out
Anvil Fructus Cucurbitae moschatae " yellow really root 2 " hybrid seed purity is carried out SSR primer and the method for precise Identification, to solve " yellow really root 2
Number " during the production of hybrid seeds, cause the problem that seed production purity is the highest, for seed production and operation enterprise provide one accurately, stable,
Quickly, the method that practical " yellow really root 2 " hybrid seed purity is identified.
Summary of the invention:
It is an object of the invention to provide the SSR for anvil Fructus Cucurbitae moschatae half-blood " yellow really root 2 " Purity Identification
Primer NG32 and one quickly, the method for precise Identification " yellow really root 2 " seed purity.
For achieving the above object, the technical solution adopted in the present invention is as follows:
A kind of for the anvil SSR primer NG32 of squash hybridization kind " yellow really root 2 " Purity Identification, the following institute of sequence
Show:
NG32-forward primer: 5 '-GGCATTTCTGAGAACAGCTT-3 ' (SEQ ID NO:1),
NG32-reverse primer: 5 '-ACGTTAGTTATGCTATTTTGTAGGC-3 ' (SEQ ID NO:2).
A kind of for anvil Fructus Cucurbitae moschatae " the yellow really root 2 " method that hybrid seed purity is identified, specifically comprise the following steps that
1) " yellow really root 2 " and male parent, maternal seedling genomic DNA are extracted respectively；
2) with the genomic DNA of said extracted as template, use SSR primer that NG32 is carried out PCR amplification；
3) product of amplification is carried out polyacrylamide gel electrophoresis；
4) Gel electrophoresis results is analyzed, there is male parent, the individual plant of maternal specific band is real the most simultaneously
Cenospecies, only maternal characteristic bands and offspring without male parent characteristic bands be pseudostationary or selfed seed, calculating seed purity.
25 μ l reaction systems of PCR amplification are: genomic DNA 1.0 μ l, 25mmol L-1MgCl22.5 μ l,
2.0mmol·L-1DNTP 2 μ l, NG32 primer 10 μm ol L-1It is respectively 0.5 μ l, Taq enzyme 0.2U 0.1 μ l, 10 × PCR
Buffer 2.5 μ l, ddH20 complements to 25 μ l.
The program of PCR amplification is: after 94 DEG C of denaturations 5min, 94 DEG C of degeneration 30s, 57 DEG C of annealing 30s, and 72 DEG C extend 30s,
After 35 circulations, 72 DEG C keep 10min, are subsequently placed in 4 DEG C of preservations.
Described gel electrophoresis is 8% native polyacrylamide gel electrophoresis, and silver staining detects.
The method of the present invention can hybrid seed be maternal with it, male parent seed zone separates by " yellow really root 2 ", quickly detects
Go out the purity of hybrid seed.This method has quick, accurate, low cost, simple operation and other advantages, it is possible to substitute conventional hybridization kind
The method of sub-Purity, has higher commercial application value.
Accompanying drawing explanation
Figure be Fructus Cucurbitae moschatae " really yellow root 2 " Purity Identification PCR primer polyacrylamide gel electrophoresis collection of illustrative plates (M:
PBR322DNA/MspI molecular weight standard；1,2,3,4,5,6: cenospecies " yellow really root 2 "；, 7,8: male parent " XR "；9,10: female
This " S4236 "；
Detailed description of the invention
Below in conjunction with embodiment, the present invention is described in further detail, but is not limited thereto.
Embodiment 1
The anvil foundation of Fructus Cucurbitae moschatae " yellow really root 2 " hybrid seed purity detection method.
1, the SSR primer of Purity is screened.
From the Fructus Cucurbitae moschatae SSR primer announced between parent's (maternal " S4236 ", male parent " XR "), screen, select and show altogether
The primer NG32 of sex differernce labelling band, sequence is as follows:
NG32-forward primer: 5 '-GGCATTTCTGAGAACAGCTT-3 ' (SEQ ID NO:1)
NG32-reverse primer: 5 '-ACGTTAGTTATGCTATTTTGTAGGC-3 ' (SEQ ID NO:2)
This marker bands is clear, reproducible.Primer NG32 can produce male parent specific marker NG32 123 He of 123bp
The maternal specific marker NG32 89 of 89bp.
2, utilize SSR primer NG32 that " yellow really root 2 " hybrid seed is carried out Purity.
(1) extraction of Fructus Cucurbitae moschatae genomic DNA
Experiment material is anvil Fructus Cucurbitae moschatae " yellow really root 2 " and male parent (XR), maternal (S4236) cotyledon DNA.
Step is as follows:
1. liquid nitrogen is with pestle in the centrifuge tube of 2ml, is rapidly added 2%CTAB when liquid nitrogen evaporates dry soon and extracts slow
Rushing liquid 700 μ l, mixing is placed on 65 DEG C of water-bath middle temperature bath 60min (shaking once every 5min).
2. stand to room temperature 12000rpm at 4 DEG C and be centrifuged 10min, supernatant is transferred to new 2ml centrifuge tube.
3. isopyknic phenol is added: chloroform: isoamyl alcohol (25:24:1), reverse mixing, stand 3-5 minute, at 4 DEG C
12000rpm is centrifuged 10min, is proceeded to by supernatant in new 1.5ml centrifuge tube.
4. add the isopropanol of 2/3 volume pre-cooling, slowly mix, be placed at-20 DEG C placement 30min.
5. at 4 DEG C, 12000rpm is centrifuged 10min, abandons supernatant, adds 70% washing with alcohol DNA of 200～300 μ l pre-coolings
Precipitation (twice), micro-dry.
6. add 30 μ l sterilized water to dissolve.
(2) PCR amplification: use the PCR amplification system of 25 μ l
PCR amplification program is as follows:
94 DEG C of denaturations 5min；
94 DEG C of degeneration 30s, 57 DEG C of annealing 30s, 72 DEG C extend 40s, 35 circulations；
72 DEG C extend 7min；
4 DEG C of preservations.
(3) gel electrophoresis
Take 5 μ l PCR primer and add 1 μ l 6x loading buffer mixing, take 1 μ l and be splined on the non denatured poly-third of 8%
Acrylamide gel electrophoresis, 150V voltage stabilizing 2.5h, electrophoresis carries out 0.1%AgNO3 silver staining 20min after terminating；With 2% after silver staining
NaOH, 0.4% formaldehyde, 0.04%Na2CO3 develop the color, after colour developing on lamp box photographic analysis.
(4) amplification
Anvil Fructus Cucurbitae moschatae " yellow really root 2 " cenospecies amplifies 123bp and 89bp two band；Male parent amplifies the bar of 123bp
Band；Female parent amplifies the band (see figure) of 89bp.
Reclaim specific band, serve the order-checking of Hai Shenggong company.The sequence of 123bp band such as SEQ ID NO in hybrid seed:
Shown in 3, the sequence of 89bp band, as shown in SEQ ID NO:4, is consistent with the sequence of male parent, maternal amplified production.
Claims (4)
1. it is used for identifying a pair SSR primer NG32 of anvil Fructus Cucurbitae moschatae " yellow really root 2 " hybrid seed purity, it is characterised in that include
Forward primer: 5 '-GGCATTTCTGAGAACAGCTT-3 ', reverse primer: 5 '-ACGTTAGTTATGCTATTTTGTAGGC-3 '.
2., for an anvil method for squash hybridization kind " yellow really root 2 " Purity Identification, comprise the steps:
1) Fructus Cucurbitae moschatae seedling genomic DNA is extracted；
2) with Fructus Cucurbitae moschatae genomic DNA as template, the SSR primer NG32 of claim 1 is used to carry out PCR amplification；
3) product of amplification is carried out polyacrylamide gel electrophoresis；
4) Gel electrophoresis results is analyzed, there is male parent, the individual plant of maternal specific band is real miscellaneous the most simultaneously
Hand over kind, only maternal characteristic strip and offspring without male parent characteristic strip be pseudostationary or selfed seed, calculating seed purity.
Anvil squash hybridization offspring's Purity method based on SSR marker the most according to claim 2, its feature exists
In: the reaction cumulative volume of described PCR amplification is 25 μ L: genomic DNA 1.0 μ l, 25mmol L-1MgCl22.5 μ l,
2.0mmol·L-1DNTP 2 μ l, NG32 primer 10 μm ol L-1It is respectively 0.5 μ l, Taq enzyme 0.2U 0.1 μ l, 10 × PCR
Buffer 2.5 μ l, ddH20 complements to 25 μ l；PCR amplification program is: 94 DEG C of denaturations 5min；94 DEG C of degeneration 30s, 57 DEG C are moved back
Fire 30s, 72 DEG C extend 30s, 30 circulations；72 DEG C extend 10min, 4 DEG C of preservations.
The Purity method of anvil squash hybridization seed based on SSR marker the most according to claim 2, its feature exists
In: SSR primer produces the male parent specific mark NG32 123 of 123bp in male parent, produces the maternal specific mark of 89bp at female parent
NG32 89。
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| CN106755387A (en) * | 2016-12-14 | 2017-05-31 | 李兴盛 | A kind of utilization molecular labeling Rapid identification cucumber stock is made a concerted effort the method for two purity |
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| CN107805672A (en) * | 2016-09-09 | 2018-03-16 | 宁波市农业科学研究院 | One kind identification giant pumpkin × Cucurbita moschata hybrid stock variety authenticity method |
| CN108085404A (en) * | 2016-11-21 | 2018-05-29 | 北京市农林科学院 | Giant pumpkin female trait molecular marker and the primer pair of identification giant pumpkin female character by force by force |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107805672A (en) * | 2016-09-09 | 2018-03-16 | 宁波市农业科学研究院 | One kind identification giant pumpkin × Cucurbita moschata hybrid stock variety authenticity method |
| CN107805672B (en) * | 2016-09-09 | 2021-03-12 | 宁波市农业科学研究院 | Method for identifying authenticity of Indian pumpkin and Chinese pumpkin hybrid stock varieties |
| CN108085404A (en) * | 2016-11-21 | 2018-05-29 | 北京市农林科学院 | Giant pumpkin female trait molecular marker and the primer pair of identification giant pumpkin female character by force by force |
| CN108085404B (en) * | 2016-11-21 | 2020-10-27 | 北京市农林科学院 | Strong female-like molecular marker of pumpkin indicum and primer pair for identifying strong female-like character of pumpkin indicum |
| CN106755387A (en) * | 2016-12-14 | 2017-05-31 | 李兴盛 | A kind of utilization molecular labeling Rapid identification cucumber stock is made a concerted effort the method for two purity |
| CN107312868A (en) * | 2017-08-30 | 2017-11-03 | 福建省农业科学院作物研究所 | The SSR primer sets developed based on american pumpkin transcript profile sequence and its application |
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| CN105907869B (en) | 2019-04-12 |
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