CN103060430B - Primers for identifying cotton seeds and kit thereof - Google Patents
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Abstract
The invention discloses a primer group for identifying cotton seeds and a kit thereof. The primer group comprises 60 pairs of core primers, and the core primer group disclosed by the invention provides a powerful tool for identifying cotton varieties in production, has the advantages of quickness, accuracy and the like, provides a powerful identifying tool against fake and forged commodities, fake plate and fake commodities and other events cheating and harming farmers, which occur during the future production, provides a reliable method and technical guarantee for handling problem seeds by a local seed management department, and greatly protects cotton breeders, seed production and operation units and production benefits of the majority of cotton farmers. The set of core markers are used for identifying fingerprints of the varieties and the screening of a large number of molecular markers is not required, thus the cost and the time are saved, and the effect of achieving maximum results with little effort is achieved.
Description
Technical field
The present invention relates to a kind of method of identifying cotton variety, belong to technical field of molecular biology.
Background technology
Cotton is the main raw material of textile industry.Cotton in China annual production is 5,000,000 tons of left and right, and the magnitude of value, 75,000,000,000 yuan of left and right, is the second largest farm crop that are only second to grain, and the whole nation has peasant more than 200,000,000 to participate in the production of cotton directly.Textile industry industry chain length, 48% of the fiber process Liang Yizhan world of current China.In recent years, rise in oil price has raised chemical fibre cost, impels textile enterprise to improve and uses cotton ratio, and the ratio of cotton and chemical fibre has reached 1: 1.Along with the recovery of global economy, textiles increase in demand, domestic cotton demand also will keep growing trend.
The strategic position in cotton in Xinjiang base is very important, and its effect is irreplaceable.Xinjiang is high quality cotton, commodity cotton production base and the export base of China's maximum, and cotton always product, per unit area yield, cultivated area surely ranks first in the country for continuous 18 years, and output of cotton accounts for 40% of national ultimate production.Cotton is Economy in Xinjiang mainstay industry, and the raw cotton output value reaches 30,000,000,000 yuan, account for the full boundary plant husbandry output value 65% and output Value of Farming, Forestry, Animal Husbandry 1/3; Cotton income accounts for 35% of whole district's farmers' income, accounts for 60% of main producing region, South Sinkiang farmers' income; Cotton processed output accounts for the 60-80% of full boundary industrial output value; The fiscal revenue of full boundary 15%, the fiscal revenue in Chan Mian county 50% are from cotton and related industries thereof.The Eleventh Five-Year Plan period, cotton in Xinjiang cultivated area is stabilized in more than 2,100 ten thousand mu, and output of cotton remains on ten thousand tons of 250-300.Since 2003, Xinjiang accumulative total recalls more than 1,800 ten thousand tons, cotton, exceeds more than 200 ten thousand tons of the national cotton import volumes same period; 2004 to 2006, the import of Cotton in China year increased to 3,640,000 tons, and import interdependency rises to 38% by 28%; 2006-2008 Xinjiang recalls 3,000,000 tons, cotton every year, has effectively supported the high speed development of China's cotton industry, also for the degree of self-sufficiency that keeps domestic cotton 70% is laid a good foundation.Were it not for the support of cotton in Xinjiang, China will exceed more than 50% the interdependency in international cotton market, and Cotton Industry will be followed the track of an overturned cart of soybean, grease industry.
Cotton in Xinjiang development potentiality is large, and the status in national cotton industry is more important from now on.By optimizing Ecological Layout, Cotton Production has formed the main producing regions such as precocious cotton region, North SinKiang and Zao Zhongshu cotton region, South Sinkiang.New variety application, improving per unit area yield, improve aspect quality and played decisive role.In cultivation, cotton in Xinjiang per unit area yield improves main dependence and increases density, improves total bell number by large group, realizes output and breaks through.Technical, promote short close morning of cultivation technique, under-film drip irrigation technology, technique of balanced fertilizer.Whole district's cotton per unit area yield has reached 119 kilograms for 2009, than nineteen ninety-five growth by 33%, higher than 35 kilograms of average per unit area yields in other area, the whole nation.Wherein, the average per unit area yield of formation reaches 155 kilograms.Cotton in Xinjiang quality better, grade are high, and average yarn count reaches 36, higher 4 than the average yarn count level in the whole nation.
In recent years, along with the fast development that cotton in Xinjiang is produced, the harm of disease and pest day increases the weight of.Cotton in Xinjiang production requirement seed selection is applicable to the development of local Cotton Production, and the Productive Potential of meeting the market requirement is large, yield stability good, adaptable new variety.Kind research and development lag behind, and affect cotton core competitiveness and improve.Further widen hereditary basis, strengthen output and resistance breeding, the Cotton new variety of the different fiber types of seed selection, to realize fiber type production diversification significant.
What cotton in Xinjiang was annual at present uses kind of demand huge, reaches every year more than 100,000 tons.Under so huge sowing quantity demand, in production, fake and inferior puppet is emitted, deck OEM seed happens occasionally, bring massive losses often to breeding man, Seed enterprises and cotton grower, the situation of this confusion is also unfavorable for the supervision of seed management department simultaneously, for ensureing the normal development of cotton in Xinjiang production and the standardized administration of Seed Market, set up a set of effective, simple and quick detection technique and seem particularly necessary.
The fast development of DNA fingerprinting technology, provides good solution route for what cultivar identification, purity detecting, the research of kind phylogentic relationship and classification and Product patent were weighed just when maintenance etc.DNA fingerprinting refers to the DNA electrophoretogram that can differentiate difference between biont, and it is to be based upon on the basis of DNA molecular marker.This electrophoretogram rich polymorphism, has individual specificity and the environmental stability of height, as people's fingerprint, thereby is called as " finger printing ".Finger printing is the powerful of differential variety, strain (containing hybrid strain, self-mating system), has the advantages such as quick, accurate.Therefore fingerprint pattern technology is very suitable for the qualification work of variety source.This technology is applied in various Crop Germplasm Resources and Purity research, and is bringing into play more and more important effect.Under such market environment overall background, the foundation of carrying out cotton in Xinjiang kind fingerprint pattern technology has vital role with application to addressing the above problem just.
The DNA fingerprinting state of the art
As everyone knows, Crop Germplasm Resources is as one of national grand strategy resource, it is the important foundation of high crop yield stable yields, will occupy more and more consequence (Tanksley and McCouch, 1997) in following life science and research of agricultural science field.At present, China's seed is produced and operating unit also manages not too specification, defective seed, even false plant sneak into repeatedly market and for the production of, cause grain drop in production, also greatly damaged Product patent owner's interests and peasant's economic interests, therefore carry out cultivar resources identification and seem particularly important simultaneously.In early days, people adopt morphological method to identify kind more, i.e. the surface characteristic of plant, as plant height, spike length, grain look, dry granular heavily etc.The method is easy, economical, but due to many Morphologic Characters qualification cycles long, affected by environment large (Liu Xun is first-class, 1998, hubei agricultural science, (1): 33-35; (3): 27-32; Liang Mingshan etc., 2001), and along with the centralization that utilizes of breeding parent, cultivar identification is difficulty more and more, and traditional morphological method is more and more not suitable with the needs of cultivar identification and purity check.
In recent years, along with the development of biotechnology, a series of DNA molecular marker technology have been born, as restriction fragment length polymorphism (restfiction fragment length polymorphism, be called for short RFLP), randomly amplified polymorphic DNA (random amplified polymorphic DNA, be called for short RAPD), amplified fragment length polymorphism (amplified fragment length polymorphism, be called for short AFLP), SSLP (simple sequence length polymorphism, be called for short SSLP), random amplification microsatellite polymorphism (random amplified micro-satellite polymorphism, be called for short RAMP), sequence tagged site (sequence-tagged sites, be called for short STS), DNA cloning fingerprint (DNA amplified fingerprinting, DAF), amplicon length polymorphism (amplicon length polymorphism, be called for short ALP), the sub-polymorphism of specific amplified (specific ampiliconpolymorphism, be called for short SAP), single strand conformation polymorphism (singlestrand conformation polymorphi sm, be called for short SSCP) and single nucleotide polymorphism (single nucleotide polymorphisms, be called for short SNPs) etc. (Li Meifang and Zhou Kaida, 2001, Zou Yuping and Ge Song, 2003, Wang Zhonghua, 2005).
Compared with above-mentioned morphological markers, DNA molecular marker has following multiple advantage: (1) directly, with the form performance of hereditary material DNA, all can detect at different tissues and the developmental stage of organism, be subject to the impact of season and environment less; (2) quantity is many, distribution is wide, spreads all over whole genome; (3) polymorphism is high, naturally exists many allelic variations, does not need the special special genetic stocks of creating; (4) show as " neutrality ", do not affect the expression of objective trait, with bad proterties without inevitable chain; (5) there are many molecule markers to show as codominance (codominance), can identify homozygous genotype and the heterozygous genes type of crop varieties or strain, for breeding utilization provides great facility (Jia Jizeng, 1996).Genetic marker is mainly divided into: the four kind types such as morphological markers, cytological marker, biochemical marker, molecule marker.
Morphological markers morphological markers is a kind of formalness feature, as of short stem, abnormal leaf, male sterile etc.Detecting heritable variation from morphology or phenotypic character is the most direct also the most simple and easy to do method.Generalized Morphological mark comprises the relevant marks such as pigment, physiological property, reproductive characteristic disease and insect resistance.Owing to existing the complicated middle-chain such as genetic expression, regulation and control, ontogeny between phenotype and genotype, how to reflect that according to the difference in phenotype the difference in genotype just becomes the key point of morphological method detection heritable variation.In cotton, conventionally phenotypic character used comprises that plant height, cotyledonary node are high, stem fine hair, fruit branch number, plant type, bell-shaped, leaf, the size of bell, blade split scarce etc.Morphological markers shortcoming is comparatively small amt, and genetic expression is less stable sometimes, is subject to the aobvious recessive impact of envrionment conditions and gene.
Cytological marker cytogenetical study is found, karyotype (chromosome number, size, satellite have or not, centromere position etc.) and banding pattern (C band, N band, G band etc.) are analyzed karyomit(e) and the relative position that can measure gene place, also can carry out by chromosomal substitution etc. the location of gene.The variation (as disappearance, transposition, inversion, repeat etc.) that chromosome number object changes (as monomer, nullisomic, trisome, limbs) and chromosome structure also all respectively has its specific cytologic characteristic, also can be used as a kind of cell marking.Obviously, the number of cytological marker is also very limited.For the identical species of chromosome number, be difficult to distinguish with cytological marker.
Biochemical marker biochemical marker is also protein labeling, mainly comprises allozyme and isoenzyme mark.This kind phenotypic marker is difficult for affected by environment, can well reflect genetic diversity.But its detection means complexity, limited amount, can not meet the needs of marker assisted selection.
The molecular marking technique growing up after the eighties 20th century of molecule marker is taking DNA polymorphism as basic genetic marker.Molecule marker has that genetic polymorphism is high, codominance, information completely, have in a large number and be uniformly distributed, select neutrality, stability, favorable reproducibility, contain much information in genome, analysis efficiency is high, detection means simple and fast, exploitation and the feature such as use cost is low.Therefore, molecular marking technique has been widely used in the aspects such as Crop Genetic Breeding, the discriminating of plant sibship, origin of species evolution, analysis of genetic diversity, genomic mapping, the assignment of genes gene mapping, gene pool structure since occurring.Existing tens of kinds of DNA marker technology now, and technology itself is also constantly improved and is developed.
The feature producing according to molecule marker, can be divided into it five classes conventionally.
The first kind: taking Southern hybridization technique as basis, as restriction fragment length polymorphism (RFLP).
Equations of The Second Kind: taking round pcr as basis, as randomly amplified polymorphic DNA (RAPD), sequence tagged site (STS), expressed sequence tag (EST), sequence signature amplified fragments (SCAR), enzyme are cut amplification polymorphism (CAP), amplified fragment length polymorphism (AFLP).
The 3rd class: taking tumor-necrosis factor glycoproteins as basis, as simple sequence repeats repetition (ISSR), single nucleotide polymorphism (SNP) between (SSR), simple sequence.
The 4th class: taking single strand conformation polymorphism as basis, as strand material polymorphism (SSCP).
The 5th class: hybridization in situ technique (ISH), comprises fluorescence in situ hybridization (FISH), in situ hybridization (GISH) etc.
The technology of the present invention applicable cases
SSR (simple sequence repeat), simple sequence repeats, and claims again microsatellite DNA (microsatellite DNA), is the s-generation molecule marker growing up on PCR basis in recent years.SSR is ubiquitous tumor-necrosis factor glycoproteins in a kind of eukaryotic gene group, and tumor-necrosis factor glycoproteins is generally by 1-6 based composition, and multiplicity is alterable height between different plant species or same species different genotype.These tumor-necrosis factor glycoproteins two ends are conservative single-copy sequence mostly, therefore can design special primer according to conserved sequence, by round pcr, middle core tumor-necrosis factor glycoproteins is increased out, utilize Polyacrylamide Gel Electrophoresis technology can obtain its length polymorphism (Zhang Lirong etc., 2002).SSR has all genetics advantages of RFLP, and has avoided the radioisotopic shortcoming of use in RFLP technology, higher than the repeatability of RAPD and stability again, thereby has become the study hotspot in genetic marker at present.
But, how to obtain suitable primer sets, can mention the ability of Seed Identification, the increase workload of exceeding is again this area technical problem urgently to be resolved hurrily.
Summary of the invention
The present invention is directed to the existing authentication method cycle long, differentiate and provide powerful, the present invention to adopt following technical scheme for the production of cotton variety:
One aspect of the present invention relates to a kind of method of identifying cotton variety, it is characterized in that comprising the steps:
Testing sample cotton total DNA extraction and purifying;
The mensuration total DNA purity of cotton and concentration;
Adopt the primer in primer sets respectively the total DNA of cotton to be carried out to pcr amplification reaction;
Amplified production is carried out to gel electrophoresis simultaneously, and obtain gel electrophoresis images;
Gel electrophoresis images and standard spectrogram are compared to determine cotton type.
Described primer sets comprises following 60 pairs of primers:
One preferred embodiment in, other included primer of described primer sets is no more than 15 pairs, is preferably no more than 10 pairs, is further preferably no more than 5 pairs, does not further preferably contain other primer pair.
In a preferred embodiment of the present invention, preferably cotton in Xinjiang seed of described cotton seeds.
In a preferred embodiment of the present invention, also comprise the standard spectrogram that adopts above-mentioned steps to build cotton in Xinjiang seed.
The production that method of the present invention is cotton variety is differentiated provides powerful; there is the advantages such as quick, accurate; also the strong discriminating instrument of the providing of harmful farming part of cheating the farmers such as fake and forged commodity, deck counterfeit for occurring in producing from now on; provide reliable method and technical guarantee for local seed administrative authority processes the dispute of problem seed, greatly protected cotton breeding person, seed production and operation unit and numerous cotton growers' production interests.Once there is personation seed production dispute problem, no matter be that 60 pairs of SSR core marks that seed management department, production unit, operating unit or peasant household can set up by the technology of the present invention are differentiated fast and accurately, reach the object of production application.Produce in addition upper new authorization kind and also can overlap core mark by this and carry out the finger printing protection of kind, without a large amount of screenings of carrying out again molecule marker, the cost-saving and time, reach the effect of getting twice the result with half the effort.
Brief description of the drawings
Caption: Fig. 1-5th, the finger printing of cotton variety: N47 and N48 represent respectively in new land 47 and No. 48, cotton No. 1 of N0 Representative Cultivars army, early No. 19, the new land of B19 Representative Cultivars.The finger printing of No. 47 in the new land of Fig. 1 Representative Cultivars, wherein for D2000 DNA Ladder Marker, (D2000 plus DNA Ladder is mixed by the PCR product of preparing separately the 1st article (leftmost side) band, have 8 DNA fragmentations, for ease of observing after electrophoresis, specificity is strengthened 750bp band, its concentration is about 100ng/5 μ l, and the DNA concentration of other band is about 50ng/5 μ l.2-49 band primer order is respectively in table 1:
Table 1
Fig. 2-4 the like.Because the technology of the present invention the primer is 60 pairs, therefore on a pictures, be difficult to complete reaction and out (can only have 48 pairs), therefore remaining 12 pairs of primers show on Fig. 5, total four strains on Fig. 2, be respectively in new land in No. 47, new land No. 48, the cotton No. 1 He Xin land of army early No. 19, each kind have 12 pairs of primers.Primer order is as shown in table 2.All pictorial informations all carry out fingerprint amplification with this primer order.
Table 2
Embodiment
Collect Xinjiang Upland Cotton cultivar quantity and amount to 4 (by the end of the kinds of self-fertile in 2010 authorization), kind comprises in new land 47, No. 48, cotton No. 1 of army, early No. 19, new land.Cultivar origin is in each breed breeding unit, and breeding man and bar state germ plasm resource are preserved storehouse.
Experimental technique
cotton DNA extracting solution preparation work
(1) 10M Hcl solution (bottled analytical pure concentrated hydrochloric acid is 10M)
(2) 10M NaOH solution 100ml 40.0 × 10 × 0.1=40 (g)
(3) 3M NaAc solution 50ml 82.03 × 3 × 0.05=12.3045 (g) anhydrous Na Ac PH5.2
(4) 1.0M Tris-Hcl solution 500ml (with 10M Hcl tune PH to 8.0) 121.14 × 1.0 × 0.5=60.57 (g)
(5) 0.5M EDTA solution 50ml (note: indissoluble) 372.24 × 0.5 × 0.05=9.306 (g), 9.306gEDTA-Na2 is dissolved in distilled water, with 10M NaOH tune PH to 8.0, is settled to 50ml, sterilizing room temperature preservation.
(6) TE damping fluid (PH8.0): 1.0M Tris-Hcl solution 2.5ml and 0.5M EDTA solution 0.5ml distilled water are settled to 250ml, after constant volume, sterilizing is preserved.
(7) chloroform: primary isoamyl alcohol=24: 1 solution preparation 200ml: 192ml chloroform+8ml primary isoamyl alcohol
(8) 70% or 75% alcohol.
(9) DNA extraction liquid configures in table 2
Table 3 cotton DNA extract recipe (50ml)
Table?1?Nuclear?extraction?solution?component(50ml)
cotton total DNA extraction and purifying
Adopt CTAB method to extract cotton genomic dna, i.e. a small amount of method DNA extraction.
(1) add 2% β-dredge base ethanol in CTAB extracting solution, fully shake up the water preheating that is placed in 65 DEG C.
(2) gather 0.5-1g blade, put into immediately frappe 2ml centrifuge tube, add liquid nitrogen, be ground into powder fast, add in the CTAB extracting solution of 600ul preheating, firmly turn upside down and mix rapidly, 65 DEG C of water-baths 30 minutes (timing from last sample is put into), slowly put upside down 2-3 time during this time.
(3) take out centrifuge tube, add chloroform one primary isoamyl alcohol (24: 1) of equal-volume (600ul), after cover lid, slowly turn upside down centrifuge tube after 10 minutes, with chloroform one primary isoamyl alcohol (24: 1) equilibrium centrifugation pipe, 20 DEG C of 8000rp/m, centrifugal 15min.
(4) get supernatant, in 1.5ml centrifuge tube, add isopyknic chloroform one primary isoamyl alcohol (24: 1), slowly turn upside down centrifuge tube after 10 minutes, with chloroform one primary isoamyl alcohol (24: 1) equilibrium centrifugation pipe, 10000rp/m20 DEG C, centrifugal 10min.
(5) get supernatant, add the Virahol of 0.60 (0.3ml) times volume-20 DEG C precooling, slowly put upside down centrifuge tube 10 times above (DNA is flocks), be statically placed in-20 DEG C more than refrigerator 30min.
(6) after leaving standstill well, the centrifugal 7min of 12000rpm; The supernatant that inclines, cleans 2-3 time with 70% alcohol, and dehydrated alcohol is washed once, wind 30min on Bechtop.
(7) add 200 μ lTE, the dissolving of spending the night.
the purification step of DNA:
(8) add RNA enzyme (10g (l), 37 DEG C of water-baths 1 hour of 5 μ l (1: 100 volume).
(9) add isopyknic chloroform one primary isoamyl alcohol (24: 1), slowly put upside down centrifuge tube 30-50 time, 13000rp/m, centrifugal 10min.
(10) get supernatant, add the NaAc (pH=5.4) of the 3mol/l of 0.1 times of volume, after mixing, slowly add the ice-cold dehydrated alcohol of 2 times of volumes, after static 5min, flocks hook goes out, and forwards in the centrifuge tube of 1.5ml.
(11) add 70% and 100% alcohol respectively to wash once, air-dry.
(12) be dissolved in the TE solution of 200ul, the refrigerator that is placed in-20 DEG C saves backup.
the mensuration of the total DNA purity of cotton and concentration
Use the long ultraviolet/visible light scanning spectrophotometer of the Nanodrop-ND-1000 all-wave of being produced by genome company to detect DNA purity and concentration.Read A260 and A280 light absorption value.DNA concentration (ng/pl)=extension rate × 50 × A260, can judge DNA purity according to A260/A280, if ratio at 1.6-2.0, proves that DNA purity is better.Be greater than 2.0, prove that RNA pollutes; Be less than 1.6, prove the pollution such as protein, phenol, detected result is good, for follow-up test.
With sky, Beijing, root 100bp DNA Ladder compares, and will further determine its concentration and purity by 1% agarose gel electrophoresis, detects the integrity of DNA simultaneously.
DNA mother liquor dilution: according to mother liquid concentration, with ddH2O be 20ng/ul working fluid by mother liquor dilution one by one, after mixing for pcr amplification.
pcr amplification reaction
(1) primer source
According to forefathers (Xiao, 2009) documents and materials, screening is uniformly distributed 26 chromosomal SSR marks of cotton, the about 4140cM of genetic map size, select to be positioned chromosomal SSR mark taking every 5cM size as spacing and amount to 806 pairs, primer type is abundant, the primer that comprises the 13 large classes such as BNL, CER, CIR, COT, CGR, DPL, DC, GH, JESPR, SHIN, TMB, HAU, NAU, the information such as primer sequence are all from (http://www.ncbi.nlm.nih.gov/ and http://www.cottondb.org).Primer is synthetic by Shanghai Sheng Gong biotechnology company limited.
(2) agents useful for same
React Tag enzyme used, dNTP, 10 × PCR buffer, PAGE glue Maker purchased from Beijing Tian Gen biochemical technology company limited, Acr (acrylamide), Bis-Acr (methene acrylamide), PVP40, DNA Ladder, TEMED are purchased from green source of students Science and Technology Ltd., and other conventional reagent such as AgNO3, NaOH, Na2CO3 are produced by Beijing dicyclo chemical reagent factory.
(3) key instrument
PCR instrument: BIOMETRA-TGRADIENT, GeneAmp PCR Systen 9700; Eppendorf high speed freezing centrifuge; 61 instrument companies of Beijing produce DYY-6C type electrophoresis system (electrophoresis apparatus and electrophoresis chamber); Shaking table etc.
(4) PCR reaction system and amplification program (in table 4 table 5)
Table 4 PCR reaction system
Table 5 pcr amplification program
electrophoresis solution preparation
This experiment electrophoresis gel used is 8% non-denaturing polyacrylamide gel, specifically joins method as follows:
(1) 8%PAGE glue: acrylamide (Acrylamide) Acr 39g, Bis-Acr 1g, 5 × TEB100ml, adding distil water, to 500ml, filters 40 DEG C of preservations.
(2) 10% ammonium persulphates (AP): 1gAP, deionized water is settled to 10ml.-20 DEG C of preservations after dissolving.
(3) sample-loading buffer: 0.25g tetrabromophenol sulfonphthalein+0.25g dimethylbenzene cyanogen+40g sucrose, deionized water is settled to 100ml.
(4) 5 × TBE:Tris 54g, boric acid 27.5g, 0.5M EDTA (PH:8.0) 3.72g, deionized water is settled to 1000ml.
When use, dilute 10 times (5 times)
(5) AgNO3 solution: 0.5g AgNO3+750ul formaldehyde+500ml water
gel preparation and electrophoresis process
(1) with clear water, sheet glass, adhesive tape and comb soaked and with gauze scrub repeatedly, then use clear water and distilled water flushing, naturally drying for subsequent use.
(2) take out cleaned sheet glass, assembling on request, and with the 8%PAGE gel having configured (the every groove of about 10ml) back cover, after the solid underseal of gelling is good, be fixed on electrophoresis chamber.
(3) in Erlenmeyer flask or beaker, pour the 8%PAGE having configured into and add rapidly AP and TEMED preparation gel (the non-sex change glue preparation of polyacrylamide sex change glue preparation: 8%PAGE 20ml+10% Ammonium Persulfate 98.5 400ul+TEMED 30ul), and rapid encapsulating, when encapsulating, avoid occurring bubble, after filling, plug comb.Place 15-20min, prepare electrophoresis.
(4) before electrophoresis, in pcr amplification product, add 2 μ l sample-loading buffers.Pull out comb, electrophoretic buffer (1 × TBE) is added respectively in anodal electrophoresis chamber and negative pole electrophoresis chamber.Damping fluid must exceed edge on short sheet glass.Broken glue and the bubble of removing glue face deposition, each point sample hole adds 4 μ l samples, electrophoresis 70-80min under 180V constant voltage, waiting blue indicator just to run out of glue can stop.After electrophoresis finishes, carefully separate two sheet glass, remove sheet glass cull around, and film is carried out to mark, slowly import slight wobble in silver-colored dye liquor.
rapid silver staining
(1) with the AgNO3 15min-20min that dyes, when dying, silver need add a cover lucifuge;
(2) with distilled water (or deionized water) rinsing 20 seconds;
(3) develop: in 500ml water, add 10gNaOH+0.3g Na2CO3+750ul formaldehyde, until there is band to occur (about 10-15min);
(4) outwell nitrite ion, use distilled water (or deionized water) gently rinsing is washed 2-3 time.
(5) preservative film is tiled desktop, is placed on developed film on film, places after smooth the doubling of glue another side film is wrapped, and carries out after mark, can be placed on and on lamp box, add up banding pattern and photograph.(this glue can be preserved and place for some time)
interpretation of result:
The result showing from Fig. 1-5 can find out in the image of each kind, there is the band of a little features, by comparing these bands, just can determine the true and false and the kind of kind to be measured.Adopt method of the present invention to identify other cotton variety, also can draw similar result.
Should be understood that; above-mentioned embodiment is only exemplary explanation, instead of limitation of the present invention, for those of ordinary skills; can be improved according to the above description or convert, and all these improvement and conversion all should belong to the protection domain of claims of the present invention.
Claims (4)
1. identify a method for cotton variety, it is characterized in that comprising the steps:
Testing sample cotton total DNA extraction and purifying;
The mensuration total DNA purity of cotton and concentration;
Adopt the primer in primer sets respectively the total DNA of cotton to be carried out to pcr amplification reaction;
Amplified production is carried out to gel electrophoresis simultaneously, and obtain gel electrophoresis images;
Gel electrophoresis images and standard spectrogram are compared to determine cotton type; Cotton seeds is cotton in Xinjiang seed;
Described primer sets comprises following 60 pairs of primers, and described primer sets does not contain other primer pair:
。
2. the method for qualification cotton variety claimed in claim 1, is characterized in that described cotton seeds is Xinjiang Upland Cotton seed.
3. the method for qualification cotton variety according to claim 2, characterized by further comprising the step that builds cotton in Xinjiang seed standard spectrogram.
4. according to the method for the qualification cotton variety described in claim 1-3 any one, described qualification refers to purity and/or the true and false of qualification cotton variety.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101429541A (en) * | 2007-11-09 | 2009-05-13 | 中国农业科学院棉花研究所 | SSR numerator fingerprint identification method |
| CN101560556A (en) * | 2009-01-16 | 2009-10-21 | 中国农业科学院棉花研究所 | Method for detecting DNA fingerprint purity in cotton crossbreeds |
| CN101696450A (en) * | 2009-09-29 | 2010-04-21 | 中国农业科学院棉花研究所 | Method for distinguishing conventional cotton and hybrid cotton in assistant mode and special kit thereof |
| CN101824484A (en) * | 2010-06-09 | 2010-09-08 | 中国农业科学院棉花研究所 | DNA fingerprint detection method for authenticity identification of cotton variety |
| CN101921866A (en) * | 2010-09-03 | 2010-12-22 | 山东农业大学 | Method for identifying cotton variety by utilizing SSR core primers |
| CN101982048A (en) * | 2010-09-13 | 2011-03-02 | 中国农业科学院棉花研究所 | Method for identifying aphid-resistant transgenic cotton |
| CN102146448A (en) * | 2011-01-13 | 2011-08-10 | 河南科技学院 | DNA finger prints of 30 main cultivars of upland cotton |
-
2012
- 2012-03-22 CN CN201210076961.1A patent/CN103060430B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101429541A (en) * | 2007-11-09 | 2009-05-13 | 中国农业科学院棉花研究所 | SSR numerator fingerprint identification method |
| CN101560556A (en) * | 2009-01-16 | 2009-10-21 | 中国农业科学院棉花研究所 | Method for detecting DNA fingerprint purity in cotton crossbreeds |
| CN101696450A (en) * | 2009-09-29 | 2010-04-21 | 中国农业科学院棉花研究所 | Method for distinguishing conventional cotton and hybrid cotton in assistant mode and special kit thereof |
| CN101824484A (en) * | 2010-06-09 | 2010-09-08 | 中国农业科学院棉花研究所 | DNA fingerprint detection method for authenticity identification of cotton variety |
| CN101921866A (en) * | 2010-09-03 | 2010-12-22 | 山东农业大学 | Method for identifying cotton variety by utilizing SSR core primers |
| CN101982048A (en) * | 2010-09-13 | 2011-03-02 | 中国农业科学院棉花研究所 | Method for identifying aphid-resistant transgenic cotton |
| CN102146448A (en) * | 2011-01-13 | 2011-08-10 | 河南科技学院 | DNA finger prints of 30 main cultivars of upland cotton |
Non-Patent Citations (2)
| Title |
|---|
| 第75-157页."New SSR Markers for Use in Cotton （Gossypium spp.）Improvement".《The Journal of Cotton Science》.2009,第13卷(第2期), |
| 第75-157页."New SSR Markers for Use in Cotton (Gossypium spp.)Improvement".《The Journal of Cotton Science》.2009,第13卷(第2期), * |
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