CN107630100A - A kind of method of muskmelon cenospecies " beautiful exquisite " Purity Identification based on EST SSR markers - Google Patents
A kind of method of muskmelon cenospecies " beautiful exquisite " Purity Identification based on EST SSR markers Download PDFInfo
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- CN107630100A CN107630100A CN201610556144.4A CN201610556144A CN107630100A CN 107630100 A CN107630100 A CN 107630100A CN 201610556144 A CN201610556144 A CN 201610556144A CN 107630100 A CN107630100 A CN 107630100A
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Abstract
The invention discloses a kind of method of melon variety " beautiful exquisite " cenospecies Purity Identification based on EST SSR markers.This method, by announcing muskmelon est sequence on Gene Bank databases, using 3.0 online design of primers of Primer, designs EST SSR primers using the genomic DNA of melon variety " beautiful exquisite " cenospecies and its parents as template.By screening, the complementary banding pattern primer that 1 pair of cenospecies banding pattern is parents is obtained, it is good through field checking test primer stability, and more coincide with field test results, Purity can be carried out to muskmelon Hybrid.The detection method of the present invention can complete Purity Identification work within 3h, have the advantage such as quick, inexpensive, easy to operate.
Description
Technical field
The invention belongs to the vegetable breeding and applied technical field of agricultural, and in particular to muskmelon cenospecies " beautiful exquisite " purity
Authentication method, it is a kind of cenospecies seed purity identification method based on EST-SSR molecular marking techniques.
Background technology
" beautiful exquisite " is the Melon new varieties that Tianjin Ke Run Vegetable Research Institutes are completely newly cultivated, and plant growing way is vigorous, comprehensive
Close resistance to protrude, based on the climing result of grandson.Fruit development period 30 days, fruit pyriform, 400~500 grams of single fruit weight.Pericarp green,
Fruit face is bright and clean.Pulp aquamarine, soluble solid content 16%, meat is crisp, aromatic flavour, in good taste.
The height of muskmelon seedses purity is the direct leading indicator for weighing seed quality quality.Seed purity reduces can be notable
The yield and product quality of crop are reduced, peasant is suffered huge economic loss.Traditional seed purity identification method be with
After planting the field shape of plant is viewed as foundation, and its cycle length, workload are big, and are easily influenceed by environment, seasonal factor, cause
As a result deviation be present.Therefore, fast and accurately seed purity identification method has turned into seed R＆D institution and enterprise is common for exploitation
The problem of concern.
EST-SSR marks are current crop varieties because it has the advantages that codominance, polymorphism are high, experimental arrangement is simple
One of the most frequently used mark in identification and Purity Identification research.In recent years, the continuous improvement with sequencing technologies and molecule
The further investigation of biology, the EST of a large amount of melon crops are sequenced, and so far, international Curcurbitaceae genome plan research is small
Group includes watermelon ESTs7856 bars, muskmelon 126940, cucumber 359105 altogether.
The content of the invention
It is an object of the invention to disclose a kind of muskmelon cenospecies " beautiful exquisite " based on EST-SSR molecular marking techniques
Seed purity identification method, to achieve the above object, the present invention provide following technical scheme：
This method by analyzing carrying out DNA extractions, PCR amplifications and EST-SSR for examination muskmelon cenospecies " beautiful exquisite ",
Confirm the codominance complementation banding pattern of cenospecies and its parents' stabilization, so as to carry out purity to muskmelon cenospecies " beautiful exquisite " seed
Identification.For identifying the SSR primers of muskmelon cenospecies " beautiful exquisite " seed purity, including primer forward direction sequence：5’-
ATCCAACTCGACCAAG-AAAC-3 ', reverse primer sequences：5’-CAGCTCTACAACAACATCTC-3’.
The method of Rapid identification muskmelon cenospecies seed purity of the present invention, this method is with greenhouse melon kind " the beautiful tinkling of pieces of jade
Jade for asking rain " be template for the genomic DNA for planting experimentally son, to known kind " jade is exquisite " parent's DNA otherness fragments analyze.For examination
Muskmelon cenospecies samples and DNA extraction method is：To greenhouse melon grab sample, 50 plants altogether, take close to the tender leaf portion of root
Divide 50~100mg, liquid nitrogen frozen grinding (Retsch MM400 biologies crusher), add CTAB lysis buffers (20 g/L
CTAB, 1.4M NaCl, 0.1M Tris-HCl, 20mM Na2EDTA) 500 μ L, 65 DEG C of water bath with thermostatic control (Neslab) cracking
30min, follow-up DNA extraction purifications are carried out by resin type genomic DNA purification kit (SBS Genetech) operation.It is dense after DNA extractions
Degree is uniformly diluted to 50 ± 1ng/ μ L, Nanodrop ND1000, Thermol).DNA extraction method can also be by commercial reagent
Box is carried out.
It is for examination muskmelon cenospecies PCR method：
(1) SSR upstream primer sequences：5 '-ATCCAACTCGACCAAGAAAC-3 ', SSR downstream primer sequence：5’-
CAGCTCTACAACAACATCTC-3’。
(2) performing PCR amplification, pcr amplification reaction therein are entered using above-mentioned SSR primer pairs experimental cultivar sample template DNA
Cumulative volume be 10 μ L, amplification reaction system is：The μ L of Master Mix 5,10 μm of ol/ of upstream and downstream primer
Each 0.5 μ L of L, for trying muskmelon cenospecies genomic DNA template (50ng/ μ L) 1 μ L, distilled water supplies 10 μ L；PCR response procedures
For 95 DEG C of pre-degeneration 2min, 32 amplification cycles (94 DEG C of 45sec, 55 DEG C of 45sec, 72 DEG C of 45sec)；72 DEG C extension 7min, 4 DEG C
Preserve；
(3) gel electrophoresis is carried out for examination muskmelon cenospecies pcr amplification product, non-denaturing polyacrylamide gel concentration is
8%, gel component is：The μ L of deionized water 21mL, 10 × TBE 3mL, 40%PAG 6 mL, TMED 30,10% excessively stream acid amides
300μL；Voltage 250V, electrophoresis 1h are set, power supply is closed, is dyed；Dyeing course：8~10min of silver staining, rapid washing,
NaOH develops the color several minutes, until band is clear, washes again；
(4) for examination muskmelon cenospecies EST-SSR interpretations of result, cenospecies and its key band of parents are compared, to for trying
Seed sample carries out codominance complementation band analysis, and gross sample quantity is accounted for by calculating cenospecies quantity in experimental cultivar sample
Ratio-dependent seed purity.
For the detection method of the present invention in the case where not calculating DNA extraction process and taking, PCR takes 90min, EST-SSR
60-90min is analyzed, so the detection method of the present invention can complete Purity Identification work within 3h, is had quick, low
The advantage such as cost, easy to operate.
In order to more clearly illustrate the assay method of the present invention, the test method of the present invention is done with detailed below
Explanation.
1st, principle
This method is using a kind of its principle of new method for nucleic acid analysis：EST-SSR is using 1~6 base as repetition
Unit, it is the SSR being present in EST.EST (Expressed sequence tag, EST) is in body by mRNA
Outer reverse transcription is into cDNA and after being cloned into plasmid or phage vector construction cDNA library, extensive random picking cDNA clone,
The Express Sequence Tags for being about 150~500bp obtained after its 5 ' end or 3 ' ends are sequenced.Est sequence comes from laboratory
The cDNA library of structure is downloaded from American National Bioinformatics Institute (NCBI) Genbank.Synthesized using related software
Primer, performing PCR amplification and gel electrophoresis are finally entered to research material, is analyzed by specific spectruming belt, so as to calculate cenospecies
Purity.
2nd, design of primers
By the 214 muskmelon est sequences announced on Gene Bank databases, set using 3.0 online primers of Primer
Meter, primer are synthesized by Shanghai life work, and obtaining 1 pair of EST-SSR primer through screening can be used for muskmelon cenospecies Purity Identification.
Primer is synthesized by Shanghai bio-engineering corporation.
The primer sequence table of table 1 is as follows：
| Primer | Sequence (5'to3') |
| TJYLL- forward primers | ATCCAACTCGACCAAGAAAC |
| TJYLL- reverse primers | CAGCTCTACAACAACATCTC |
3rd, sampling and DNA extractions
Carry out grab sample to greenhouse melon, 50 plants altogether, take 50~100mg of tender leaf part close to root, liquid nitrogen is cold
Freeze grinding (Retsch MM400 biologies crusher), add CTAB lysis buffers (20g/L CTAB, 1.4M NaCl, 0.1M
Tris-HCl, 20mM Na2EDTA) 500 μ L, 65 DEG C of waters bath with thermostatic control (Neslab) crack 30min, and follow-up DNA extraction purifications are pressed
Resin type genomic DNA purification kit (SBS Genetech) operation is carried out.Concentration is uniformly diluted to 50 ± 1ng/ μ L after DNA extractions,
Nanodrop ND1000, Thermol).DNA extraction method can also be carried out by commercial reagent box.
4th, PCR reacts
Using SSR upstream primer sequences：5 '-ATCCAACTCGACCAAGAAAC-3 ', SSR downstream primer sequence：5’-
CAGCTCTACAACAACATCTC-3’。
SSR primer pair experimental cultivar sample templates DNA enters performing PCR amplification (instrument is AB Veriti PCR), therein
The cumulative volume of pcr amplification reaction is 10 μ L, and amplification reaction system is：The μ L of Master Mix 5, upstream is with
Primer each 0.5 μ L of 10 μm of ol/L are swum, for trying muskmelon cenospecies genomic DNA template (50ng/ μ L) 1 μ L, distilled water supplies 10 μ L.
PCR response procedures are 95 DEG C of pre-degeneration 2min, 32 amplification cycles (94 DEG C of 45sec, 55 DEG C of 45sec, 72 DEG C of 45 sec)；72℃
Extend 7min, 4 DEG C of preservations.
5th, native polyacrylamide gel electrophoresis and silver staining
For examination muskmelon cenospecies pcr amplification product carry out gel electrophoresis (instrument is BIO-RAD electrophoresis apparatuses), non denatured poly- third
Acrylamide gel strength is 8%, and gel component is：The μ of deionized water 21mL, 10 × TBE 3mL, 40%PAG 6mL, TMED 30
L, the μ L of 10% excessively stream acid amides 300；The μ L of loading 1~2, voltage 250V, electrophoresis 1h are set, close power supply, dyed；
Dyeing course (instrument is ORBITAL decolorization swinging tables)：The silver nitrate that 6mL 10% is added in 600mL ultra-pure waters is molten
Liquid, 8~10min of silver staining；Washed rapidly in 600mL ultra-pure waters；9g sodium hydroxide powders are added in 600 mL ultra-pure waters, it is molten
3mL formaldehyde is added after solution, is developed the color several minutes, until band is clear；Washed again in 600mL ultra-pure waters.
6th, EST-SSR spectral band analysis
By analyzing EST-SSR results, compare the cenospecies of test sample and its key band of parents, to for examination hybridization
Kind carries out Purity.The SSR primers obtained with screening, female parent show as 1 key band, and male parent shows as being different from female parent
1 key band, homozygous cenospecies is to show as the set of this 2 key bands, that is, codominant complementary banding pattern；
It is non-it is homozygous can not show as codominant complementary banding pattern, maternal key band banding pattern may be shown as, that is, occur it is maternal from
Hand over.
7th, Purity
By the ratio-dependent seed purity for calculating cenospecies in experimental cultivar sample:
Seed purity=cenospecies quantity/test sample quantity * 100%.
Brief description of the drawings:
Fig. 1 is the electrophoresis that 1 pair of SSR primer that screening obtains is used for muskmelon cenospecies " beautiful exquisite " and its parents' amplified production
Analyze collection of illustrative plates.1~6 maternal banding pattern, 7~12 cenospecies banding patterns, 13~18 male parent banding patterns are followed successively by from left to right.
Embodiment
In order to more clearly illustrate the method for the present invention, the test method of the present invention is made with specifically below
Bright, need to be illustrated herein is：Primer sequence of the present invention is shown in Table 1.
Embodiment 1
(1) reagent：The production of Promega companiesMaster Mix solution；The EST- of Shanghai life work synthesis
SSR primers；
(2) EST-SSR primer sequences table is as follows：
| Primer | Sequence (5'to3') |
| TJYLL- forward primers | ATCCAACTCGACCAAGAAAC |
| TJYLL- reverse primers | CAGCTCTACAACAACATCTC |
(3) sampling and DNA extractions
160 plants of experimental cultivar seedling, liquid nitrogen frozen grinding (Retsch MM400 biologies crusher), it is slow to add CTAB cracking
Fliud flushing (20g/L CTAB, 1.4M NaCl, 0.1M Tris-HCl, 20mM Na2EDTA) 500 μ L, 65 DEG C of waters bath with thermostatic control
(Neslab) 30min is cracked, follow-up DNA extraction purifications are carried out by resin type genomic DNA purification kit (SBS Genetech) operation.
Concentration is uniformly diluted to 50 ± 1ng/ μ L, Nanodrop ND1000, Thermol after DNA extractions).DNA extraction method can also
Carried out by commercial reagent box.
(4) PCR reacts
Pcr amplification primer thing as described in upper table for examination seedling DNA profiling and its parent DNA to entering performing PCR amplification (instrument
For AB Veriti PCR), the cumulative volume of pcr amplification reaction therein is 10 μ L, and amplification reaction system is：
Master Mix 5 each 0.5 μ L of μ L, 10 μm of ol/L of upstream and downstream primer, experimental cultivar female parent gene group DNA profiling (50ng/
μ L) 1 μ L, distilled water supplies 10 μ L.PCR response procedures are 95 DEG C of pre-degeneration 2min, and 32 amplification cycles (94 DEG C of 45sec, 55
DEG C 45sec, 72 DEG C of 45sec)；72 DEG C of extension 7min, 4 DEG C of preservations.
(5) gel electrophoresis and silver staining
The μ L of experimental cultivar female parent PCR primer loading 1~2, voltage 250V, electrophoresis 1h are set, close power supply, dyed；
Dyeing course：8~10min of silver staining, rapid washing, NaOH develop the color several minutes, until band is clear, washed again；
(6) EST-SSR interpretations of result：160 plants for having that 3 plants of electrophoresis banding pattern is consistent with maternal banding pattern in examination seedling, be non-
Cenospecies.
(7) Purity
By the ratio-dependent seed purity for calculating cenospecies in experimental cultivar sample.
Seed purity=157/160*100%=98.1%.
Embodiment 2
(1) reagent：The production of Promega companiesMaster Mix solution；The EST- of Shanghai life work synthesis
SSR primers；
(2) EST-SSR primer sequences table is as follows：
| Primer | Sequence (5'to3') |
| TJYLL- forward primers | ATCCAACTCGACCAAGAAAC |
| TJYLL- reverse primers | CAGCTCTACAACAACATCTC |
(3) sampling and DNA profiling extraction
138 plants of experimental cultivar seedling, liquid nitrogen frozen grinding (Retsch MM400 biologies crusher), it is slow to add CTAB cracking
Fliud flushing (20g/L CTAB, 1.4M NaCl, 0.1M Tris-HCl, 20mM Na2EDTA) 500 μ L, 65 DEG C of waters bath with thermostatic control
(Neslab) 30min is cracked, follow-up DNA extraction purifications are carried out by resin type genomic DNA purification kit (SBS Genetech) operation.
Concentration is uniformly diluted to 50 ± 1ng/ μ L, Nanodrop ND1000, Thermol after DNA extractions).DNA extraction method can also
Carried out by commercial reagent box.
(4) PCR reacts
Pcr amplification primer thing as described in upper table for examination seedling DNA profiling and its parent DNA to entering performing PCR amplification (instrument
For AB Veriti PCR), the cumulative volume of pcr amplification reaction therein is 10 μ L, and amplification reaction system is：
Master Mix 5 each 0.5 μ L of μ L, 10 μm of ol/L of upstream and downstream primer, experimental cultivar male parent gene group DNA profiling (50ng/
μ L) 1 μ L, distilled water supplies 10 μ L.PCR response procedures are 95 DEG C of pre-degeneration 2min, and 32 amplification cycles (94 DEG C of 45sec, 55
DEG C 45sec, 72 DEG C of 45sec)；72 DEG C of extension 7min, 4 DEG C of preservations.
(5) gel electrophoresis and silver staining
The μ L of experimental cultivar male parent PCR primer loading 1~2, voltage 250V, electrophoresis 1h are set, close power supply, dyed；
Dyeing course：8~10min of silver staining, rapid washing, NaOH develop the color several minutes, until band is clear, washed again；
(6) EST-SSR interpretations of result：138 plants for having that 2 plants of electrophoresis banding pattern is consistent with maternal banding pattern in examination seedling, be non-
Cenospecies.
(7) Purity
By the ratio-dependent seed purity for calculating cenospecies in experimental cultivar sample.
Seed purity=136/138*100%=98.5%.
Embodiment 3
(1) reagent：The production of Promega companiesMaster Mix solution；The EST- of Shanghai life work synthesis
SSR primers；
(2) EST-SSR primer sequences table is as follows：
| Primer | Sequence (5'to3') |
| TJYLL- forward primers | ATCCAACTCGACCAAGAAAC |
| TJYLL- reverse primers | CAGCTCTACAACAACATCTC |
(3) sampling and DNA extractions
Experimental cultivar takes cenospecies individual plant 92, liquid nitrogen frozen grinding (Retsch MM400 biologies crusher), adds CTAB
Lysis buffer (20g/L CTAB, 1.4MNaCl, 0.1M Tris-HCl, 20mM Na2EDTA) 500 μ L, 65 DEG C of waters bath with thermostatic control
(Neslab) crack 30min, follow-up DNA extraction purifications by resin type genomic DNA purification kit (SBS Genetech) operate into
OK.Concentration is uniformly diluted to 50 ± 1ng/ μ L, Nanodrop ND1000, Thermol after DNA extractions).DNA extraction method also may be used
To be carried out by commercial reagent box.
(4) PCR reacts
Pcr amplification primer thing as described in upper table enters performing PCR amplification to experimental cultivar cenospecies individual plant template DNA, and (instrument is
AB Veriti PCR), the cumulative volume of pcr amplification reaction therein is 10 μ L, and amplification reaction system is：
Each 0.5 μ L of μ L, 10 μm of ol/L of upstream and downstream primer of Master Mix 5, experimental cultivar cenospecies genomic DNA template
(50ng/ μ L) 1 μ L, distilled water supply 10 μ L.PCR response procedures are 95 DEG C of pre-degeneration 2min, 32 (94 DEG C 45 of amplification cycles
Sec, 55 DEG C of 45sec, 72 DEG C of 45sec)；72 DEG C of extension 7min, 4 DEG C of preservations.
(5) gel electrophoresis and silver staining
The μ L of experimental cultivar cenospecies PCR primer loading 1~2, voltage 250V, electrophoresis 1h are set, close power supply, contaminated
Color；Dyeing course：8~10min of silver staining, rapid washing, NaOH develop the color several minutes, until band is clear, washed again；
(6) EST-SSR interpretations of result：Experimental cultivar cenospecies banding pattern is different from 1 of female parent for maternal 1 with male parent,
The set of this 2 key bands, that is, codominant complementary banding pattern.
(7) Purity
By the ratio-dependent seed purity for calculating cenospecies in experimental cultivar sample.
Seed purity=91/92*100%=98.9%.
Sequence table
<110>Agriculture In Tianjin quality standard and detection technique research institute
<120>The method of muskmelon cenospecies " beautiful exquisite " Purity Identification based on EST-SSR marks
<160> 2
<210> 1
<211> 20 bp
<212> DNA
<213>Artificial sequence
<400> 1
ATCCAACTCG ACCAAGAAAC 20
<210> 2
<211> 20 bp
<212> DNA
<213>Artificial sequence
<400> 2
CAGCTCTACA ACAACATCTC 20
Claims (2)
1. 1 pair of SSR primer for identifying muskmelon cenospecies " beautiful exquisite " seed purity, it is characterised in that including sense primer
Sequence：5 '-ATCCAACTCGACCAAGAAAC-3 ', downstream primer sequence：5’-CAGCTCTACAACAACATCTC-3’.
2. one kind uses primer described in claim 1, muskmelon cenospecies " beautiful exquisite " seed purity mirror based on EST-SSR marks
Fixed method, it is characterised in that comprise the following steps：
(1) performing PCR amplification is entered using the SSR primer pair experimental cultivar sample templates DNA described in claim 1, PCR therein expands
The cumulative volume for increasing reaction is 10 μ L, and amplification reaction system is：The μ L of Master Mix 5, upstream and downstream primer
Each 0.5 μ L of 10 μm of ol/L, for trying muskmelon cenospecies genomic DNA template (50ng/ μ L) 1 μ L, distilled water supplies 10 μ L；PCR is anti-
It is 95 DEG C of pre-degeneration 2min to answer program, 32 amplification cycles (94 DEG C of 45sec, 55 DEG C of 45sec, 72 DEG C of 45sec)；72 DEG C of extensions
7min, 4 DEG C of preservations；
(2) gel electrophoresis is carried out to pcr amplification product, non-denaturing polyacrylamide gel concentration is 8%, and gel component is：Go
Ionized water 21mL, 10 × TBE 3mL, 40%PAG 6mL, TMED 30 μ L, the μ L of 10% excessively stream acid amides 300；Voltage 250V is set,
Electrophoresis 30min, power supply is closed, is dyed；Dyeing course：8~10min of silver staining, rapid washing, NaOH develop the color several minutes, directly
It is clear to band, wash again；
(3) by analyzing EST-SSR results, cenospecies and its key band of parents are compared, to being total to for planting experimentally subsample
Dominant complementary band analysis, by calculate cenospecies quantity in experimental cultivar sample account for gross sample quantity ratio-dependent seed it is pure
Degree.
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Cited By (1)
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| CN109439783A (en) * | 2018-07-17 | 2019-03-08 | 江苏绿港现代农业发展有限公司 | A kind of SSR primer and method for sweet 25 Purities of muskmelon cenospecies gold |
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