CN104059992A - Method for identifying seed purity of muskmelon hybrid variety based on EST-SSR (expressed sequence tag-simple sequence repeat) marker - Google Patents
Method for identifying seed purity of muskmelon hybrid variety based on EST-SSR (expressed sequence tag-simple sequence repeat) marker Download PDFInfo
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Abstract
The invention discloses a method for identifying seed purity of a muskmelon variety namely green angel hybrid variety based on an EST-SSR (expressed sequence tag-simple sequence repeat) marker. The method comprises the following steps: using the muskmelon variety namely green angel hybrid variety and parental genome DNA (deoxyribonucleic acid) of the muskmelon variety namely green angel hybrid variety as a template; designing 57 pairs of EST-SSR primers by using Primer 3.0 online primer design through 214 muskmelon EST sequences published by a GeneBank database; obtaining a pair of complementary band type primers of which the hybrid variety band type is parental by screening. By virtue of a field verification test, the primer is good in stability, is relatively matched with results of the field test, and can be used for performing purity identification on the muskmelon hybrid variety. A detection method disclosed by the invention can be used for completing seed purity identification within 3 hours, and has the advantages of high speed, low cost, convenience in operation and the like.
Description
Technical field
The invention belongs to agriculture vegetable breeding and applied technical field, relate to muskmelon cross-fertilize seed Purity method, more particularly a kind of cross-fertilize seed seed purity identification method based on EST-SSR molecular marking technique.
Background technology
Seed is the production means of basic most critical in agriculture production, and the height of its purity is the leading indicator of directly weighing seed quality quality.Seed purity reduces can significantly reduce output and the product quality of crop, makes peasant suffer huge financial loss.Traditional seed purity identification method is to be viewed as foundation with the field shape of plant after planting, and its cycle is long, workload is large, and is subject to environment, seasonal factor impact, causes result to have deviation.Therefore, develop seed purity identification method fast and accurately and become the common problem of paying close attention to of seed R&D institution and enterprise.
Be accompanied by the development of modern agricultural technology, Purity Identification technology progressively develops into by traditional morphological markers qualification the integrated cause determination technology system that integrates morphological markers qualification, biochemical marker qualification and DNA molecular markers for identification.DNA molecular marker is the direct reflection of DNA heritable variation on molecular level, their genetic stabilities, contain much information, be not subject to the impact of internal and external environment, and have whether have nothing to do, detect the outstanding advantages such as rapid and easy and simple to handle with genetic expression, therefore, DNA molecule marker becomes one of detection method of desirable Rapid identification seed purity, and wherein RAPD, RFLP, AFLP, SSR and ISSR mark etc. have application in Hybrid seed purity test.
SSR mark, because it has the advantages such as codominance, polymorphism is high, experimental arrangement is simple, is one of mark the most frequently used in current Identifying Crop Cultivars and Purity Identification research.The SSR mark of exploitation mainly contains genome SSR and EST-SSR two classes at present.With respect to genome SSR, EST-SSR makes full use of existing sequencing data, has saved the step such as library construction and order-checking in SSR primer development process, has reduced cost.In recent years, along with improving constantly and molecular biological further investigation of sequencing technologies, the EST of a large amount of melon crops is sequenced, up to now, world Curcurbitaceae genome plan research group includes watermelon ESTs7856 bar, 126940 of muskmelons, 359105 of cucumber altogether.
EST sequence can be from three large database concept EMBL(European Molecular Bioglogy Laboratory of the world), NCBI(American National biotechnology information center) and DDBJ(Japan DNA database) obtain, recycling related software can design and obtain SSR primer.In est sequence, the content of SSR is higher, and between kind, has good versatility.Feng Jianming etc. are to 978 unigene sequential analyses of watermelon, retrieve altogether 2136 EST-SSR, the frequency of occurrences is 53.4%, the research such as Kong finds that a SSR appears in the every 4.7kb of muskmelon est sequence, 22 EST-SSR primer pair average energies disclose 2.9 allelotrope, average expectation heterozygosity 0.442, has 15 pairs of primers can on cucumber, amplify band.Guan Yuan etc. retrieve 63 SSR from 902 cucumber fruits EST, have designed 37 pairs of primers, and have 14 pairs of primers has polymorphic between cucumber and muskmelon.Hu Jianbin etc. retrieve 784 SSR from derive from 6507 ESTs of ncbi database.Kong etc. be used to come from NCBI cucumber EST sequences Design 54 couples of EST-SSR, analyzed the genetic diversity of 20 parts of cucumber materials, wherein have 13 pairs of primers in muskmelon, to amplify specific fragment, 7 pairs of primers detect polymorphism.The development and application of EST-SSR in Curcurbitaceae melon crop, still in initial period, due to the EST limited amount deriving from watermelon, muskmelon and cucumber, causes utilizable SSR on the low side.
Summary of the invention
The object of the invention is to disclose a kind of muskmelon cross-fertilize seed seed purity identification method based on EST-SSR molecular marking technique, for achieving the above object, the invention provides following technical scheme:
Present method, by analyzing carrying out DNA extraction, pcr amplification and EST-SSR for examination muskmelon cross-fertilize seed, has been confirmed the complementary banding pattern of codominance that cross-fertilize seed and parents thereof are stable, thereby muskmelon cross-fertilize seed has been carried out to Purity.For the identification of the SSR primer of muskmelon cross-fertilize seed seed purity, comprise primer forward sequence: 5 '-GCACGAGGCTCAACTACC-3 ', reverse primer sequence: 5 '-GAGACCGAAATTGAAGAATAG-3 '.
The method of Rapid identification muskmelon cross-fertilize seed seed purity of the present invention, the method supplies the genomic dna of planting experimentally son (Tianjin Ke Run Vegetable Research Institute) as template taking the green angel of greenhouse melon kind, and the green angel parent of known kind DNA otherness fragment is analyzed.For the sampling of examination muskmelon cross-fertilize seed and DNA extraction method be: to greenhouse melon (from Tianjin Ke Run Vegetable Research Institute) random sampling, 50 strains altogether, get tender leaf part 50～100 mg near root, liquid nitrogen freezing grinds (the biological crusher of Retsch MM400), add CTAB lysis buffer (20 g/L CTAB, 1.4 M NaCl, 0.1 M Tris-HCl, 20 mM Na2EDTA) 500 μ L, 65 DEG C of waters bath with thermostatic control (Neslab) cracking 30min, follow-up DNA extraction purifying is undertaken by resin type genomic dna purification test kit (match Parkson) operation.After DNA extraction, concentration unification is diluted to 50 ± 1 ng/ μ L, Nanodrop ND1000, Thermol).DNA extraction method also can be undertaken by commercial reagent box.
For examination muskmelon cross-fertilize seed PCR method be:
(1) SSR upstream primer sequence:
5’- GCACGAGGCTCAACTACC -3’，
SSR downstream primer sequence: 5 '-GAGACCGAAATTGAAGAATAG-3 '.
(2) adopt above-mentioned SSR primer pair experimental cultivar sample template DNA to carry out pcr amplification, the cumulative volume of pcr amplification reaction is wherein 10 μ L, amplification reaction system is: 2 × GoTaq Master Mixes, 5 μ L, the each 0.5 μ L of upstream and downstream primer 10 μ mol/L, for examination muskmelon cross-fertilize seed genomic dna template (50 ng/ μ L) 1 μ L, distilled water is supplied 10 μ L; PCR response procedures is 95 DEG C of denaturation 2 min, 32 amplification cycles (94 DEG C of 45 sec, 55 DEG C of 45 sec, 72 DEG C, 45 sec); 72 DEG C are extended 7 min, 4 DEG C of preservations;
Carry out gel electrophoresis for examination muskmelon cross-fertilize seed pcr amplification product, non-denaturing polyacrylamide gel concentration is 8%, and gel component is: deionized water 21 mL, 10 × TBE, 3 mL, 40 % PAG 6 mL, TMED 30 μ L, 10% overcurrent acid amides 300 μ L; Setting voltage 250 V, electrophoresis 1 h, powered-down, dyes; Dyeing course: silver dyes 8～10 min, washing rapidly, NaOH colour developing several minutes, until band is clear, washing again;
For examination muskmelon cross-fertilize seed EST-SSR interpretation of result, relatively cross-fertilize seed and parents' thereof key band, to carrying out the complementary band of codominance type analysis for planting experimentally sub-sample, account for the ratio-dependent seed purity of gross sample quantity by cross-fertilize seed quantity in calculating experimental cultivar sample.
For measuring method of the present invention can be more clearly described, below test method of the present invention is done with detailed explanation.
, principle:
A kind of its principle of novel method for nucleic acid analysis of present method application is: EST-SSR is taking 1～6 base as repeating unit, is the SSR being present in EST.EST (Expressed sequence tag; expressed sequence tag) be by mRNA in vitro reverse transcription become cDNA and be cloned into plasmid or phage vector construction cDNA library after; extensive random picking cDNA clone, the Express Sequence Tags that is about 150～500bp obtaining after its 5 ' end or 3 ' end are checked order.Est sequence from laboratory build cDNA library or from the Genbank at American National bioinformation center (NCBI), download.Adopt related software synthetic primer, finally research material is carried out to pcr amplification and gel electrophoresis, analyze by specific spectruming belt, thereby calculate the purity of cross-fertilize seed.
2, design of primers
By 214 muskmelon est sequences announcing on Gene Bank database, utilize the online design of primers of Primer 3.0, primer is synthetic by the raw work in Shanghai, has obtained 1 pair of EST-SSR primer can be used for muskmelon cross-fertilize seed Purity Identification through screening.Primer is synthesized by Shanghai bio-engineering corporation.
Table 1 primer sequence table is as follows:
| Primer title | Sequence (5'to3') |
| SSR forward primer | GCACGAGGCTCAACTACC |
| SSR reverse primer | GAGACCGAAATTGAAGAATAG |
3, sampling and DNA extraction
Greenhouse melon is carried out to random sampling, 50 strains altogether, get the tender leaf part 50～100mg near root, liquid nitrogen freezing grinds (the biological crusher of Retsch MM400), adds CTAB lysis buffer (20 g/L CTAB, 1.4 M NaCl, 0.1 M Tris-HCl, 20 mM Na2EDTA) 500 μ L, 65 DEG C of waters bath with thermostatic control (Neslab) cracking 30min, follow-up DNA extraction purifying is undertaken by resin type genomic dna purification test kit (match Parkson) operation.After DNA extraction, concentration unification is diluted to 50 ± 1 ng/ μ L, Nanodrop ND1000, Thermol).DNA extraction method also can be undertaken by commercial reagent box.
, PCR reaction
Adopt (1) SSR upstream primer sequence:
5’- GCACGAGGCTCAACTACC -3’，
SSR downstream primer sequence: 5 '-GAGACCGAAATTGAAGAATAG-3 '.
SSR primer pair experimental cultivar sample template DNA carries out pcr amplification (instrument is AB Veriti PCR), the cumulative volume of pcr amplification reaction is wherein 10 μ L, amplification reaction system is: 2 × GoTaq Master Mixes, 5 μ L, the each 0.5 μ L of upstream and downstream primer 10 μ mol/L, for examination muskmelon cross-fertilize seed genomic dna template (50 ng/ μ L) 1 μ L, distilled water is supplied 10 μ L.PCR response procedures is 95 DEG C of denaturation 2 min, 32 amplification cycles (94 DEG C of 45 sec, 55 DEG C of 45 sec, 72 DEG C of 45 sec); 72 DEG C are extended 7 min, 4 DEG C of preservations.
5, native polyacrylamide gel electrophoresis and silver dye
Carry out gel electrophoresis (instrument is BIO-RAD electrophoresis apparatus) for examination muskmelon cross-fertilize seed pcr amplification product, non-denaturing polyacrylamide gel concentration is 8%, and gel component is: deionized water 21 mL, 10 × TBE, 3 mL, 40 % PAG 6 mL, TMED 30 μ L, 10% overcurrent acid amides 300 μ L; Loading 1～2 μ L, setting voltage 250 V, electrophoresis 1 h, powered-down, dyes;
Dyeing course (instrument is ORBITAL decolorization swinging table): add the silver nitrate solution of 6 mL 10 % in 600 mL ultrapure waters, silver dyes 8～10 min; Washing rapidly in 600 mL ultrapure waters; In 600 mL ultrapure waters, add 9 g sodium hydroxide powder, after dissolving, add 3 mL formaldehyde, develop the color several minutes, until band is clear; Washing again in 600 mL ultrapure waters.
, EST-SSR spectral band analysis
By analyzing EST-SSR result, the relatively cross-fertilize seed of test sample and parents' key band thereof, to carrying out Purity for examination cross-fertilize seed.The SSR primer obtaining with screening, female parent shows as 1 key band, and male parent shows as and is different from 1 maternal key band, and the cross-fertilize seed of isozygotying shows as the set of these 2 key bands, namely codominant complementary banding pattern; Non-isozygoty can not show as codominant complementary banding pattern, may show as maternal key band banding pattern, there is maternal selfing.
, Purity
By calculating the ratio-dependent seed purity of cross-fertilize seed in experimental cultivar sample:
Seed purity=cross-fertilize seed quantity/test sample quantity * 100%.
Detection method of the present invention is not being calculated DNA extraction process in the situation that consuming time, PCR 90min consuming time, EST-SSR analyzes 60-90min, so detection method of the present invention can complete Purity Identification work within 3h, have fast, low cost, the advantage such as easy to operate, it relatively sees the following form:
| Relatively | Seed purity | Time | Workload | Seasonal factor | Polymorphism | Cost | Stability |
| Traditional Purity Identification | 98.1% | 3 months | Greatly | The impact of the condition such as temperature, humidity is large | Morphologic difference | High | Poor |
| Purity Identification of the present invention | 98.0% | 3 hours | Little | Not affected by seasonal factor | Difference on DNA level | Low | Good |
The positively effect that muskmelon cross-fertilize seed seed purity identification method based on EST-SSR molecular marking technique disclosed by the invention compared with prior art had is:
(1) method of the present invention, taking the green angel cross-fertilize seed of melon variety and parents' thereof genomic dna as template, by 214 muskmelon est sequences announcing on Gene Bank database, is utilized the online design of primers of Primer 3.0, has designed 57 pairs of EST-SSR primers.Through screening, obtain 1 pair of cross-fertilize seed banding pattern the complementary banding pattern primer that is parents, through field, proof test primer has good stability, and comparatively identical with field test results, can carry out Purity to muskmelon Hybrid.
(2) detection method of the present invention is not being calculated DNA extraction process in the situation that consuming time, PCR 90min consuming time, EST-SSR analyzes 60-90min, so detection method of the present invention can complete Purity Identification work within 3h, has fast, low cost, the advantage such as easy to operate.
(3) the present invention focuses on the banding pattern complementation of male parent, female parent and F1 generation in the screening of primer, the easy observation and analysis of electrophoretic band.
Brief description of the drawings:
Fig. 1 is that 1 pair of SSR primer of screening acquisition is for the electrophoretic analysis collection of illustrative plates of muskmelon cross-fertilize seed and parents' amplified production thereof.Be followed successively by from left to right female parent 1～3, cross-fertilize seed 1～3, male parent 1～3;
Fig. 2 is the electrophoretic analysis collection of illustrative plates of 10 strain individual plants and parents' amplified production thereof; Be followed successively by from left to right female parent 1～3, cross-fertilize seed 4～13, male parent 14～16;
Fig. 3 is the electrophoretic analysis collection of illustrative plates of 10 strain individual plants and parents' amplified production thereof; Be followed successively by from left to right female parent 1～3, cross-fertilize seed 4～13, male parent 14～16;
Fig. 4 is the electrophoretic analysis collection of illustrative plates of 10 strain individual plants and parents' amplified production thereof; Be followed successively by from left to right female parent 1～3, cross-fertilize seed 4～13, male parent 14～16;
Fig. 5 is the electrophoretic analysis collection of illustrative plates of 10 strain individual plants and parents' amplified production thereof; Be followed successively by from left to right female parent 1～3, cross-fertilize seed 4～13, male parent 14～16;
Fig. 6 is the electrophoretic analysis collection of illustrative plates of 10 strain individual plants and parents' amplified production thereof; Be followed successively by from left to right female parent 1～3, cross-fertilize seed 4～13, male parent 14～16.
Embodiment
For method of the present invention can be more clearly described, below test method of the present invention is done with detailed explanation, need be illustrated at this: the present invention's reagent used all has commercially available, and primer sequence of the present invention is in table 1.
embodiment 1
(1) reagent: the GoTaq Master Mixes solution that Promega company produces; The synthetic EST-SSR primer of the raw work in Shanghai;
(2) EST-SSR primer sequence table is as follows:
| Primer title | Sequence (5'to3') |
| SSR forward primer | GCACGAGGCTCAACTACC |
| SSR reverse primer | GAGACCGAAATTGAAGAATAG |
(3) sampling and DNA extraction
Experimental cultivar is got maternal seedling (Tianjin Ke Run Vegetable Research Institute), and liquid nitrogen freezing grinds (the biological crusher of Retsch MM400), adds CTAB lysis buffer (20g/L CTAB, 1.4M NaCl, 0.1M Tris-HCl, 20mM Na
2eDTA) 500 μ L, 65 DEG C of waters bath with thermostatic control (Neslab) cracking 30min, follow-up DNA extraction purifying is undertaken by resin type genomic dna purification test kit (match Parkson) operation.After DNA extraction, concentration unification is diluted to 50 ± 1 ng/ μ L, Nanodrop ND1000, Thermol).DNA extraction method also can be undertaken by commercial reagent box.
(4) PCR reaction
By the pcr amplification primer described in upper table, the maternal seedling template DNA of experimental cultivar is carried out to pcr amplification (instrument is AB Veriti PCR), the cumulative volume of pcr amplification reaction is wherein 10 μ L, amplification reaction system is: 2 × GoTaq Master Mixes, 5 μ L, the each 0.5 μ L of upstream and downstream primer 10 μ mol/L, experimental cultivar female parent gene group DNA profiling (50 ng/ μ L) 1 μ L, distilled water is supplied 10 μ L.PCR response procedures is 95 DEG C of denaturation 2 min, 32 amplification cycles (94 DEG C of 45 sec, 55 DEG C of 45 sec, 72 DEG C of 45 sec); 72 DEG C are extended 7 min, 4 DEG C of preservations.
(5) gel electrophoresis and silver dye
The maternal PCR product of experimental cultivar loading 1～2 μ L, setting voltage 250V, electrophoresis 1h, powered-down, dyes; Dyeing course: silver dyes 8～10 min, washing rapidly, NaOH colour developing several minutes, until band is clear, washing again;
(6) EST-SSR interpretation of result: the maternal banding pattern of experimental cultivar is 1 key band.
embodiment 2
(1) reagent: the GoTaq Master Mixes solution that Promega company produces; The synthetic EST-SSR primer of the raw work in Shanghai;
(2) EST-SSR primer sequence table is as follows:
| Primer title | Sequence (5'to3') |
| SSR forward primer | GCACGAGGCTCAACTACC |
| SSR reverse primer | GAGACCGAAATTGAAGAATAG |
(3) sampling and DNA extraction
Experimental cultivar is got male parent seedling (Tianjin Ke Run Vegetable Research Institute), liquid nitrogen freezing grinds (the biological crusher of Retsch MM400), add CTAB lysis buffer (20 g/L CTAB, 1.4 M NaCl, 0.1 M Tris-HCl, 20 mM Na2EDTA) 500 μ L, 65 DEG C of waters bath with thermostatic control (Neslab) cracking 30min, follow-up DNA extraction purifying is undertaken by resin type genomic dna purification test kit (match Parkson) operation.After DNA extraction, concentration unification is diluted to 50 ± 1 ng/ μ L, Nanodrop ND1000, Thermol).DNA extraction method also can be undertaken by commercial reagent box.
(4) PCR reaction
By the pcr amplification primer described in upper table, experimental cultivar male parent seedling template DNA is carried out to pcr amplification (instrument is AB Veriti PCR), the cumulative volume of pcr amplification reaction is wherein 10 μ L, amplification reaction system is: 2 × GoTaq Master Mixes, 5 μ L, the each 0.5 μ L of upstream and downstream primer 10 μ mol/L, experimental cultivar male parent gene group DNA profiling (50 ng/ μ L) 1 μ L, distilled water is supplied 10 μ L.PCR response procedures is 95 DEG C of denaturation 2 min, 32 amplification cycles (94 DEG C of 45 sec, 55 DEG C of 45 sec, 72 DEG C of 45 sec); 72 DEG C are extended 7 min, 4 DEG C of preservations.
(5) gel electrophoresis and silver dye
Experimental cultivar male parent PCR product loading 1～2 μ L, setting voltage 250 V, electrophoresis 1 h, powered-down, dyes; Dyeing course: silver dyes 8～10 min, washing rapidly, NaOH colour developing several minutes, until band is clear, washing again;
(6) EST-SSR interpretation of result: experimental cultivar male parent banding pattern is to be different from 1 maternal key band.
embodiment 3
(1) reagent: the GoTaq Master Mixes solution that Promega company produces; The synthetic EST-SSR primer of the raw work in Shanghai;
(2) EST-SSR primer sequence table is as follows:
| Primer title | Sequence (5'to3') |
| SSR forward primer | GCACGAGGCTCAACTACC |
| SSR reverse primer | GAGACCGAAATTGAAGAATAG |
(3) sampling and DNA extraction
Experimental cultivar is got cross-fertilize seed individual plant 50(Tianjin Ke Run Vegetable Research Institute), liquid nitrogen freezing grinds (the biological crusher of Retsch MM400), add CTAB lysis buffer (20 g/L CTAB, 1.4 M NaCl, 0.1 M Tris-HCl, 20 mM Na2EDTA) 500 μ L, 65 DEG C of waters bath with thermostatic control (Neslab) cracking 30min, follow-up DNA extraction purifying is undertaken by resin type genomic dna purification test kit (match Parkson) operation.After DNA extraction, concentration unification is diluted to 50 ± 1 ng/ μ L, Nanodrop ND1000, Thermol).DNA extraction method also can be undertaken by commercial reagent box.
(4) PCR reaction
By the pcr amplification primer described in upper table, experimental cultivar cross-fertilize seed individual plant template DNA is carried out to pcr amplification (instrument is AB Veriti PCR), the cumulative volume of pcr amplification reaction is wherein 10 μ L, amplification reaction system is: 2 × GoTaq Master Mixes, 5 μ L, the each 0.5 μ L of upstream and downstream primer 10 μ mol/L, experimental cultivar cross-fertilize seed genomic dna template (50 ng/ μ L) 1 μ L, distilled water is supplied 10 μ L.PCR response procedures is 95 DEG C of denaturation 2 min, 32 amplification cycles (94 DEG C of 45 sec, 55 DEG C of 45 sec, 72 DEG C of 45 sec); 72 DEG C are extended 7 min, 4 DEG C of preservations.
(5) gel electrophoresis and silver dye
Experimental cultivar cross-fertilize seed PCR product loading 1～2 μ L, setting voltage 250 V, electrophoresis 1 h, powered-down, dyes; Dyeing course: silver dyes 8～10 min, washing rapidly, NaOH colour developing several minutes, until band is clear, washing again;
(6) EST-SSR interpretation of result: experimental cultivar cross-fertilize seed banding pattern is maternal 1 and is different from maternal 1, the set of these 2 key bands, namely codominant complementary banding pattern with male parent.
(7) Purity
By calculating the ratio-dependent seed purity of cross-fertilize seed in experimental cultivar sample.
Seed purity=49/50*100%=98.0%.
Embodiment 4
authentication method:field planting Purity
authentication step:
1. presoaking and germinating
(1) time: on March 18th, 2014
(2) method: choose at random 120, green angel's seed and carry out presoaking and germinating, 50～55 DEG C of temperature, are stirred to soaking at room temperature 4h, draining away the water is placed in the thermostat container of 35 DEG C with wet towel parcel and carries out vernalization, and after 24h, 50% above seed can be sowed after showing money or valuables one carries unintentionally.
2. sowing
(1) time: on March 19th, 2014
(2) method: germination plantation is lain against in the dish of cave, earthing, water permeable, the insulation of lid plastics film, general 2～3d can be emerged.
3. field planting
(1) time: on April 15th, 2014
(2) method: seedling is taken out and planted in greenhouse from the dish of cave, do not fall apart and stick together when field planting, guarantee and soil close contact, water flood after field planting, do not leak informaton, with comparatively high temps management, promote slow seedling.
4. training is pruned
(1) time: on April 25th, 2014
(2) method: hang to protection climing cultivation and adopt two climing trainings; pinching when seedling 4-5 sheet leaf; every internode grows two stalwartnesses of selecting and remain after side shoot, son that growing way is suitable is climing; the climing climing excision of first, second grandson of sending of son; the above climing melon that stays of grandson sending of climing three joint of son; every strip is climing can stay 2 ~ 3, melon continuously, stays that melon grandson is climing stays 2 leaf pinching.
qualification result:
1 time: on June 20th, 2014
2 methods: show the characteristic of this kind completely after muskmelon annesl time, add up, show inconsistent person with this kind fruit properties (fruit properties, fruit colour) and be considered as hybrid strain.The total strain number-hybrid strain of purity %=(number)/total strain number × 100%
3 results: through Field observation investigation, in 108 strain muskmelons, there are two strain fruits green in vain, inconsistent with this kind fruit feature (green), be considered as hybrid strain, therefore, green angel's purity is calculated as follows:
Purity %=(108-2)/108 × 100%=98.1%.
SEQUENCE LISTING
<110> Tianjin Kerun Agricultural Science & Technology Co., Ltd.; Agriculture In Tianjin quality standard and detection technique institute
The method of mono-kind of the <120> muskmelon cross-fertilize seed Purity Identification based on EST-SSR mark
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 18
<212> DNA
<213> artificial sequence
<400> 1
gcacgaggct caactacc 18
<210> 2
<211> 21
<212> DNA
<213> artificial sequence
<400> 2
gagaccgaaa ttgaagaata g 21
Claims (3)
1. for the identification of 1 pair of SSR primer of muskmelon cross-fertilize seed seed purity, it is characterized in that comprising upstream primer sequence: 5 '-GCACGAGGCTCAACTACC-3 ', downstream primer sequence: 5 '-GAGACCGAAATTGAAGAATAG-3 '.
2. a primer described in employing claim 1, the method for the muskmelon cross-fertilize seed Seed Identification purity based on EST-SSR mark, is characterized in that being undertaken by following step:
(1) adopt upstream primer sequence claimed in claim 1:
5 '-GCACGAGGCTCAACTACC-3 ', downstream primer sequence:
5 '-GAGACCGAAATTGAAGAATAG-3 ' primer pair experimental cultivar sample template DNA carries out pcr amplification, the cumulative volume of pcr amplification reaction is wherein 10 μ L, amplification reaction system is: 2 × GoTaq Master Mixes, 5 μ L, the each 0. 5 μ L of upstream and downstream primer 10 μ mol/L, for examination muskmelon cross-fertilize seed genomic dna template, 50 ng/ μ L, 1 μ L, distilled water is supplied 10 μ L; PCR response procedures is 95 DEG C of denaturation 2 min, 32 amplification cycles, 94 DEG C of 45 sec, 55 DEG C of 45 sec, 72 DEG C of 45 sec; 72 DEG C are extended 7 min, 4 DEG C of preservations;
(2) pcr amplification product is carried out to gel electrophoresis, non-denaturing polyacrylamide gel concentration is 8%, and gel component is: deionized water 21 mL, 10 × TBE, 3 mL, 40 % PAG 6 mL, TMED 30 μ L, 10% overcurrent acid amides 300 μ L; Setting voltage 250 V, electrophoresis 30 min, powered-down, dyes; Dyeing course: silver dyes 8～10 min, washing rapidly, NaOH colour developing several minutes, until band is clear, washing again;
(3), by analysis EST-SSR result, relatively cross-fertilize seed and parents' thereof key band, to carrying out the complementary band of codominance type analysis for planting experimentally sub-sample, accounts for the ratio-dependent seed purity of gross sample quantity by cross-fertilize seed quantity in calculating experimental cultivar sample.
3. the method for the muskmelon cross-fertilize seed Seed Identification purity based on EST-SSR mark claimed in claim 2, wherein said muskmelon refers to: watermelon, cucumber.
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