CN104694627B - Two DNA repetitive sequences for identifying sweet potato chromosome and FISH identification method - Google Patents
Two DNA repetitive sequences for identifying sweet potato chromosome and FISH identification method Download PDFInfo
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Abstract
The invention discloses two DNA repetitive sequences for identifying sweet potato chromosomes and a FISH identification method, belonging to the technical field of cytogenetics and molecular biology. The sequence is SEQ NO: 1 and the nucleotide sequence Itf _1 shown in SEQ NO: 2, and (b) a nucleotide sequence Itf _2 shown in figure 2. The method comprises the steps of extracting plasmid DNA, forming Itf _1 and Itf _2 probes by using Itf _1 marked by biotin and Itf _2 marked by digoxin through an incision translation method, and effectively solving the problem that the sweet potato cannot be distinguished and identified through a traditional mode due to the problems of small chromosome, more number, thick cytoplasm, weak dyeing capacity and the like through the steps of chromosome piece preparation, fluorescence in-situ hybridization, signal detection and the like.
Description
Technical field
The invention belongs to cytogenetics and technical field of molecular biology, and in particular to for differentiating sweet potato chromosome
Two repetitive dna sequences and FISH discrimination methods.
Background technology
Sweet potato is Convolvulaceae sweet potato platymiscium, rich in starch, protein, cellulose, beta carotene and multivitamin,
The champion of 13 kinds of optimal vegetables is classified as by the World Health Organization.Widely cultivated in the torrid zone such as sub-, non-, Latin America and subtropical zone,
It is a kind of important grain, vegetables, insutrial crop and novel energy crop.In recent years, sweet potato the energy, healthy food,
The value of feed etc. is increasingly valued by people, and has cultivated many new improved seeds.
The basic research of sweet potato genus plant chromosome is weak, relatively lags behind in other crops such as rice, wheat.Sweet potato genus are planted
Thing chromosome has 2 ×, 4 ×, 6 × tri- kinds of ploidies, cultigen is essentially 6 ×.Its chromosome is smaller, and number is more, and cytoplasm is dense
Thick and colouring power is weak, it is more difficult to obtains clear clean chromosome sectioning, it is difficult to studied from cytogenetics level.At present
Research be concentrated mainly on the observation of chromosome number and meiotic behavior, to the chromosome the Nomenclature Composition and Structure of Complexes of sweet potato polyploid
Research is very few.
Banding technique and hybridization in situ technique (ISH) are to study two kinds of important technologies of chromosome the Nomenclature Composition and Structure of Complexes.
Banding technique is mainly to be distinguished by the position of the normal heterochromatin of coloured differently body.Hybridization in situ technique mainly has genome former
Position hybridization technique (GISH) and fluorescence in situ hybridization technique (FISH), GISH are mainly the difference using DNA homology between species
It is different, blockaded with the genomic DNA of another species with appropriate concentration, in situ hybridization is carried out on target chromosome.FISH is root
According to the special DNA sequence dna of population in known microorganisms different classifications rank, to utilize the specific oligos piece of fluorescence labeling
Duan Zuowei probes, hybridize with DNA molecular in Metagenomics, by fluorescence detecting system detection signal DNA sequence dna in chromosome
Or the target DNA sequence on DNA microsections, and then determine its hybridization site.
Above-mentioned prior art can effectively distinguish that chromosome is larger, less or obvious discrimination species difference
To chromosome, but it is smaller for chromosome, and number is more, sweet potato species similar in form, passes through the aobvious band of routine, in situ hybridization
Etc. technology difference still can not be identified to chromosome well.Equally, GISH method is for sufficiently complex sweet of genetic background
Potato species can not play good recognition effect.
The content of the invention
It is an object of the invention to provide two repetitive dna sequences for differentiating sweet potato chromosome.
It is a further object of the present invention to provide more than one to state the FISH side that two repetitive dna sequences are probe
Method, directly perceived, simplicity, quickly and efficiently differentiate sweet potato chromosome.
To achieve these goals, the present invention adopts the following technical scheme that：
For differentiating the repetitive dna sequence of sweet potato chromosome, sequence Itf_1 is SEQ NO：Nucleotide sequence shown in 1,
Itf_2 is SEQ NO：Nucleotide sequence shown in 2.
A kind of fluorescence in-situ hybridization method for differentiating sweet potato chromosome using above-mentioned repetitive dna sequence, extraction of plasmid DNA
Afterwards, by incising shifting method, the Itf_1 with biotin labeling and the Itf_2 with digoxigenin labeled, nucleotide sequence is formed such as
SEQ NO：Itf_1 probes and nucleotide sequence such as SEQ NO shown in 1：Itf_2 probes shown in 2, for sweet potato chromosome
Discriminating, specific steps include：
1) preparation of chromosome piece
Routinely vegetable material culture, when plant to be planted tip of a root length is to 0.5-1cm, the eugonic tip of a root is cut, through 8- hydroxyls
After base quinoline handles 2h at 25 DEG C, 24h is fixed with Ka Nuoshi fixers；Deionized water rinsing soaks 30min, fully washes away solid
Determine liquid；With the cellulose pectase containing 2% cellulase and 1% pectase 1-2h is digested at 37 DEG C；Deionized water rinsing soaks
30min, fully wash away enzyme liquid；The 1-2 tips of a root are taken to break the tip of a root into pieces with forceps tips on clean slide, the hanging Ka Nuoshi that is added dropwise consolidates
Determining liquid makes it fully scatter, and bakes, dries on alcolhol burner；Microscopy, select -20 DEG C of preservations of well dispersed chromosome piece；
2) FISH
1. roasting piece：Before hybridization, the slice, thin piece made is placed in 65 DEG C of baking ovens and dries 30-60min；
2. the preparation of hybridization solution：SsDNA4 μ L and each 3 μ L of Itf_1, Itf_2 DNA probe are taken, is mixed after in 65 DEG C of baking ovens
4 μ L are dried to, μ L of 70% deionized formamide 10, μ L of 50% dextran sulfate 4, the μ L of 20 × SSC 2 is added and mixes, sealed membrane envelope
It is good standby；
3. the preparation of denaturing liquid：μ L of distilled water 20,20 × SSC10 μ L, the μ L of 70% deionized formamide 70 are taken, is mixed, envelope
Membrana oralis is sealed standby；
4. probe is denatured：The chromosome piece in baking oven is taken out, baking oven is warming up to 85 DEG C, above-mentioned denaturing liquid is added on taking-up
Slide on, plus cover glass；2-4min is denatured in 85 DEG C of baking ovens；Get rid of cover glass, immediately successively immerse -20 DEG C 70%,
95%th, each 5min in 100% ice ethanol；Take out slide, more than air drying 30min；
5. hybridize：Hybridization solution is put into boiling water and is denatured 10min, is immediately placed in cooled on ice 10min, previous step is added on and does
On slide after dry, covered, room temperature 5-10min；It is placed in 80-85 DEG C of baking oven and is denatured 2min, is put into culture dish,
It is placed in 37 DEG C of incubators and cultivates 17-21h；
6. eluted after hybridization：Take out incubator in slide, be placed in 2 × SSC room temperature elution 2 on decolorization swinging table ×
5min；It is placed in 42 DEG C of 2 × SSC and elutes 10min；Elute 5min in 2 × SSC on decolorization swinging table again；Decolourize to shake in 1 × TNT
5min is eluted on bed, is dried；The sealed membrane that clip and slide size are coincide, take out TNB room temperatures and melt；Under the conditions of lucifuge, one is taken
Individual 1.5 μ L EP pipes, 100 μ L TNB are added, 1 μ L Bio, 0.5 μ L Dig antibody, mixes, takes 100 μ L to be added on the slide dried
On, sealed membrane is covered, is placed in 37 DEG C of incubators and cultivates 1h；Take out slide, be placed in 1 × TNT on decolorization swinging table elution 3 ×
5min；Dry slide 5-10min；
3) signal detection
Add 13 μ L DAPI, covered, dry, mounting, be placed under fluorescence microscope, mesh is determined according to film-making coordinate
Chromosome is marked, chromosome is taken pictures, then conversion filter successively, the hybridization signal of two probes is clapped respectively
According to；
4) secondary hybridization
1. develop a film：Take the cover glass of hybridization for the first time off, slide is placed in 2 × SSC solution on decolorization swinging table and elutes 2
×5min；It is placed in 2 × SSC solution and elutes 2 × 10min again on decolorization swinging table；Then 70%, 95%, 100% is immersed successively
Ethanol in each 5min of room temperature elution on decolorization swinging table；It is placed in Ka Nuoshi fixers and fixes 5min, takes out slide, dry, fill
Divide exposure 2-3 days；
2. FISH：With 45S, 5S rDNA probes carry out second of FISH, the same step of crossover process
2, then the hybridization signal of two probes is taken pictures respectively；Chromosome and hybridization signal are entered using imaging system analysis software
Row image is synthesized, and image is adjusted with image processing software.
What the Itf_1 probes were obtained by：According to SEQ NO：Nucleotide sequence design primer amplification shown in 1 is treated
Template is surveyed, the primer is sense primer：5 '-TTCCCGATGCGTGGAGTT-3 ', anti-sense primer：5’-
CTCCATTGCGGTTGTCTTA-3 ', the nucleotide sequence such as SEQ NO marked using PCR methods synthesizing biotinylated：Spy shown in 1
Pin, obtain Itf_1 probes；
What the Itf_2 probes were obtained by：According to SEQ NO：Nucleotide sequence design primer amplification shown in 2 is treated
Template is surveyed, the primer is sense primer：5 '-CCCCACCTTCAACCAACT-3 ', anti-sense primer：5’-
GCAGCGGTTAGGAGGTGA-3 ', the nucleotide sequence such as SEQ NO of digoxigenin labeled are synthesized using PCR methods：Spy shown in 2
Pin, obtain Itf_2 probes.
Preferably, the Ka Nuoshi fixers are formulated by 3 parts of absolute ethyl alcohols and 1 part of glacial acetic acid.
Preferably, the FISH step 4. in, probe is denatured 3min in 85 DEG C of baking ovens.
Compared with prior art, the invention has the characteristics that：
The present invention carries out FISH using two repetitive dna sequences, and the fluorescence signal of probe is strong, is easy to detect,
The problems such as and repeatability is high, efficiently solves sweet potato because chromosome is smaller, number is more, and cytoplasm is dense and colouring power is weak
And it can not carry out differentiating the problem of identification by traditional mode；Plus 45S rDNA and 5S rDNA probes, 4 are only needed altogether
Individual probe, twice hybridization can effectively arrange the effect of sweet potato caryogram, enter for sweet potato Chromosome Identification and structural research, sweet potato
Change research etc. and provide new technology and approach.
Brief description of the drawings
Fig. 1 is Itf_1, Itf_2,45S, and 5S probes are at twice to sweet potato wild diploid species I.trifida (2n=30)
The microscopy picture of chromosome fluorescence in-situ hybridization, A~D are first order fluorescence in situ hybridization result, and wherein A is Itf_1 probe signals,
Totally 8 to (in coloured picture shown in red), and B is Itf_2 probe signals, and totally 4 to (shown in green signal in coloured picture), and C is dyeing
Body, D are composite diagram；A '~D ' is second-order fluorescence in situ hybridization result, and wherein A ' is 45S probe signals, and totally 3 in coloured picture to (showing
It is shown as yellow), B ' is 5S probe signals, and totally 1 in coloured picture to (being shown as light blue), and C ' is chromosome, and D ' is composite diagram.
Fig. 2 is Itf_1, Itf_2,45S, and 5S probes are at twice to sweet potato hexaploid wild species I.trifida (6n=90)
The microscopy picture of chromosome fluorescence in-situ hybridization, A~D are first order fluorescence in situ hybridization result, and wherein A is Itf_1 probe signals,
Totally 24 to (in coloured picture shown in red), and B is Itf_2 probe signals, and totally 6 to (shown in green signal in coloured picture), and C is dyeing
Body, D are composite diagram；A '~D ' is second-order fluorescence in situ hybridization result, and wherein A ' is 45S probe signals, and totally 9 in coloured picture to (showing
It is shown as yellow), B ' is 5S probe signals, and totally 3 pairs, C ' is chromosome, and D ' is composite diagram.
Fig. 3 is Itf_1, Itf_2,45S, and 5S probes are at twice to cultigen sweet potato Xushen21 well (6n=90) chromosome fluorescence
The microscopy picture of in situ hybridization, A~D are first order fluorescence in situ hybridization result, and wherein A is Itf_1 probe signals, and totally 24 to (color
It is shown in red in figure), B is Itf_2 probe signals, and totally 6 to (shown in green signal in coloured picture), and C is chromosome, and D is conjunction
Cheng Tu；A '~D ' is second-order fluorescence in situ hybridization result, and wherein A ' is 45S probe signals, and totally 9 to (being shown as yellow in coloured picture
Color), B ' is 5S probe signals, and totally 3 pairs, C ' is chromosome, and D ' is composite diagram.
Embodiment
The invention will be further described with reference to the accompanying drawings and examples.Experimental method in following embodiments, such as nothing
Specified otherwise, it is conventional method；Experiment material used, unless otherwise specified, is purchased from routine biochemistry in following embodiments
Reagent shop.
Material source
Sweet potato wild diploid species I.trifida, by sweet potato research institute of Chinese Academy of Agricultural Sciences Xuzhou sweet potato research center
There is provided.
Sweet potato hexaploid wild species I.trifida, by sweet potato research institute of Chinese Academy of Agricultural Sciences Xuzhou sweet potato research center
There is provided.
Cultigen sweet potato (Xushen21 well, Ipomoea batatas cv.Xushu No.18), it is sweet by the Chinese Academy of Agricultural Sciences
Potato research institute Xuzhou sweet potato research center provides.
45S rDNA probes, the plasmid containing 45S rDNA is extracted from Escherichia coli, using digoxin random priming
It is marked as probe.
5S rDNA probes, the plasmid containing 5S rDNA is extracted from Escherichia coli, using biotin random priming mark
Remember into probe.
Embodiment 1：The preparation of Itf_1, Itf_2 probe
1) extraction of genomic DNA：CTAB methods (Doyle J J, Doyle J L.A rapid DNA isolation
procedure for small quantities of fresh leaf tissue.Phytochemistry Bulletin,
1987,19:11-15)；
2) high-flux sequence：Sequencing throughput is 10G, carries out the retrieval of repeated fragment, and pass through similarity segments cluster point
Analysis obtains repeated fragment；
3) design of primers (software primer5)：
Itf_F1：5 '-TTCCCGATGCGTGGAGTT-3 ' (sense primer, SEQ ID NO：3)
Itf_R1：5 '-CTCCATTGCGGTTGTCTTA-3 ' (anti-sense primer, SEQ ID NO：4)
Itf_F2:：5 '-CCCCACCTTCAACCAACT-3 ' (sense primer, SEQ ID NO：5)
Itf_R2：5 '-GCAGCGGTTAGGAGGTGA-3 ' (anti-sense primer, SEQ ID NO：6)
4) PCR is expanded：95 DEG C of denaturation 5min, (95 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C of extension 1min) carry out 30 altogether
Secondary circulation；50 μ L reaction systems：The μ L of template solution 2, the μ L of sense primer 2, the μ L of 2 μ L, PCR mix of anti-sense primer 25, distilled water 19
μL；
5) recovery of PCR primer：AxyPrep DNA gel QIAquick Gel Extraction Kits；
6) clone of repeated fragment：TAKARA carriers：PMD18T, conventional cloning methods；
7) a small amount of extractions of plasmid：
1. the single positive colony of picking (adds 5-10mL LB in LB fluid nutrient mediums (containing Amp) in 25mL conical flasks
Fluid nutrient medium), 37 DEG C of concussion and cultivate 12h；
2. drawing bacterium solution in 2mL centrifuge tubes, 13200rpm, 2min, supernatant is abandoned；
3. plus 100 μ L ice bath Solution I, vortex oscillation, thalline is fully suspended；
4. plus 200 μ L Solution II, it is gentle to mix 6 times, gently mix to the transparent shape of solution (within 5min)；
5. plus 150 μ L Solution III, it is gentle to mix 6 times；
6. 7min is stood on ice；
7. plus isometric chloroform albumen, gently overturn and mix 10min, 13200rpm, 10min, take supernatant；
8. adding the absolute ethyl alcohol of diploid product into supernatant, mixing of turning upside down, 13200rpm, 10min, supernatant is abandoned；
9. plus the ethanol of 1mL 75%, turn upside down and washed once, 13200rpm, 3min, abandon supernatant；
10. 13200rpm, 1min, sucked away raffinate, abandon it；Super-clean bench or room temperature are dried, appropriate TE solution dissolving, 4 DEG C
Preserve；
5) preparation (incising shifting method) of probe：25 μ L systems：2.5 μ L 10xBuffer, 1.5 μ L dNTP, 1.5 μ L lifes
Thing element/digoxin, 500-750ng DNAs, 0.8 μ L DNA pol, 1 μ L DNase I, water complement to 25 μ L；15 in PCR instrument
DEG C reaction 1.75h, add 1 μ L 0.5mol/L EDTA terminating reactions, -20 DEG C preservation, 1% agarose gel electrophoresis analysis PCR
Product, sequencing obtains the Itf_1 sequences that length is 409bp, such as SEQ ID NO：Shown in 1, and the Itf_2 that length is 409bp
Sequence, such as SEQ ID NO：Shown in 2.
Embodiment 2：Itf_1, Itf_2,45S, 5S probe dye to sweet potato wild diploid species I.trifida (2n=30)
Body FISH
1) preparation of chromosome piece
Routinely vegetable material culture, when plant to be planted tip of a root length is to 0.5-1cm, the eugonic tip of a root is cut, through 8- hydroxyls
After base quinoline handles 2h at 25 DEG C, Ka Nuoshi fixer (absolute ethyl alcohols：Glacial acetic acid=3:1) fixed 24h；Deionized water rinsing
30min is soaked, fully washes away fixer；With the cellulose pectase containing 2% cellulase and 1% pectase 2h is digested at 37 DEG C
(can appropriate regulating time according to sample kind difference)；Deionized water rinsing soaks 30min, fully washes away enzyme liquid；Take the 1-2 tips of a root
In on clean slide, the tip of a root is broken into pieces with forceps tips, the hanging Ka Nuoshi fixers that are added dropwise make it fully scatter, and one is baked on alcolhol burner
Under, dry；Microscopy, select -20 DEG C of preservations of well dispersed chromosome piece.
2) FISH
1. roasting piece：Before hybridization, the slice, thin piece made is placed in 65 DEG C of baking ovens and dries 30-60min；
2. the preparation of hybridization solution：SsDNA4 μ L and each 3 μ L of Itf_1, Itf_2 DNA probe are taken, is mixed after in 65 DEG C of baking ovens
4 μ L are dried to, μ L of 70% deionized formamide 10, μ L of 50% dextran sulfate 4, the μ L of 20 × SSC 2 is added and mixes, sealed membrane envelope
It is good standby；
3. the preparation of denaturing liquid：μ L of distilled water 20,20 × SSC10 μ L, the μ L of 70% deionized formamide 70 are taken, is mixed, envelope
Membrana oralis is sealed standby；
4. probe is denatured：The chromosome piece in baking oven is taken out, baking oven is warming up to 85 DEG C, above-mentioned denaturing liquid is added on taking-up
Slide on, plus cover glass；2-3min is denatured in 85% baking oven；Get rid of cover glass, immediately successively immerse -20 DEG C 70%,
95%th, each 5min in 100% ice ethanol；Take out slide, more than air drying 30min；
5. hybridize：Hybridization solution is put into boiling water and is denatured 10min, is immediately placed in cooled on ice 10min, previous step is added on and does
On slide after dry, covered (20 × 40mm), room temperature 5-10min；It is placed in 80-85 DEG C of baking oven and is denatured 2min, puts
Enter in culture dish, be placed in 37 DEG C of incubators and cultivate 17-21h；
6. eluted after hybridization：Take out incubator in slide, be placed in 2 × SSC room temperature elution 2 on decolorization swinging table ×
5min；It is placed in 10min in 42 DEG C of 2 × SSC；Elute 5min in 2 × SSC on decolorization swinging table again；In 1 × TNT on decolorization swinging table
5min is eluted, is dried；The sealed membrane that clip and slide size are coincide, take out TNB room temperatures and melt (following operation lucifuge progress)；Take
One 1.5 μ L EP pipes, add 100 μ LTNB, antibody (1 μ L Bio, 0.5 μ L Dig), mix, take 100 μ L to be added on the glass dried
On piece, sealed membrane is covered, is placed in 37 DEG C of incubators and cultivates 1h；Take out slide, be placed in 1 × TNT elute 5min (3 times, decolourize
Shaking table)；Dry slide 5-10min；
3) signal detection
Add 13 μ L DAPI, covered, dry, mounting, be placed under OLYMPUS BX63 fluorescence microscopes, according to system
Piece coordinate determines target chromosome, and chromosome is taken pictures, then conversion filter successively, respectively to the hybridization of two probes
Signal is taken pictures；
4) secondary hybridization
1. develop a film：Take the cover glass of hybridization for the first time off, slide is placed in 2 × SSC solution on decolorization swinging table and elutes 2
×5min；It is placed in 2 × SSC solution and elutes 2 × 10min again on decolorization swinging table；Then 70%, 95%, 100% is immersed successively
Ethanol in each 5min of room temperature elution on decolorization swinging table；It is placed in Ka Nuoshi fixers and fixes 5min, takes out slide, dry, fill
Divide exposure 2-3 days；
2. FISH：With 45S, 5S rDNA probes carry out second of FISH, the same step of crossover process
2, then the hybridization signal of two probes is taken pictures respectively；
Chromosome and hybridization signal are carried out using Scop-pro 7.0C Configuration imaging systems analysis softwares
Image is synthesized, and image is adjusted with the softwares of Adobe Photoshop 7.0.
As shown in figure 1, A~D is first order fluorescence in situ hybridization result, wherein A is Itf_1 probe signals, and totally 8 to (coloured picture
In it is shown in red), B is Itf_2 probe signals, and totally 4 to (shown in green signal in coloured picture), and C is chromosome, and D is synthesis
Figure；A '~D ' is second-order fluorescence in situ hybridization result, and wherein A ' is 45S probe signals, and totally 3 to (being shown as yellow) in coloured picture,
B ' is 5S probe signals, and totally 1 in coloured picture to (being shown as light blue), and C ' is chromosome, and D ' is composite diagram.
Embodiment 3：Itf_1, Itf_2,45S, 5S probe dye to sweet potato hexaploid wild species I.trifida (6n=90)
Body FISH
As described in Example 2, microscopy image is as shown in Figure 2 for specific steps.A~D is first order fluorescence in situ hybridization result, its
Middle A is Itf_1 probe signals, and totally 24 to (in coloured picture shown in red), and B is Itf_2 probe signals, and totally 6 in coloured picture to (showing
For green), C is chromosome, and D is composite diagram；A '~D ' is second-order fluorescence in situ hybridization result, and wherein A ' is 45S probes
Signal, totally 24 to (being shown as yellow) in coloured picture, and B ' is 5S probe signals, and totally 3 pairs, C ' is chromosome, and D ' is composite diagram.
Embodiment 4：Itf_1, Itf_2,45S, 5S probe are former to cultigen sweet potato Xushen21 well (6n=90) chromosome fluorescence
Position hybridization
As described in Example 2, microscopy image is as shown in Figure 3 for specific steps.A~D is first order fluorescence in situ hybridization result, its
Middle A is Itf_1 probe signals, and totally 24 to (in coloured picture shown in red), and B is Itf_2 probe signals, and totally 6 in coloured picture to (showing
For green), C is chromosome, and D is composite diagram；A '~D ' is second-order fluorescence in situ hybridization result, and wherein A ' is 45S probes
Signal, totally 9 to (being shown as yellow) in coloured picture, and B ' is 5S probe signals, and totally 3 pairs, C ' is chromosome, and D ' is composite diagram.
Claims (5)
1. two repetitive dna sequences for differentiating sweet potato chromosome, it is characterised in that sequence 1 is SEQ NO：Core shown in 1
Nucleotide sequence, sequence 2 are SEQ NO：Nucleotide sequence shown in 2.
2. a kind of fluorescence in-situ hybridization method for differentiating sweet potato chromosome using repetitive dna sequence described in claim 1, its feature
It is, after extraction of plasmid DNA, by incising shifting method, the Itf_1 with biotin labeling and the Itf_2 with digoxigenin labeled, shape
Into nucleotide sequence such as SEQ NO：Itf_1 probes and nucleotide sequence such as SEQ NO shown in 1：Itf_2 probes shown in 2, use
In the discriminating to sweet potato chromosome, specific steps include：
1) preparation of chromosome piece
Routinely vegetable material culture, when plant to be planted tip of a root length is to 0.5-1cm, the eugonic tip of a root is cut, through 8- hydroxyl quinolines
After quinoline handles 2h at 25 DEG C, 24h is fixed with Ka Nuoshi fixers；Deionized water rinsing soaks 30min, fully washes away fixation
Liquid；With the cellulose pectase containing 2% cellulase and 1% pectase 1-2h is digested at 37 DEG C；Deionized water rinsing soaks
30min, fully wash away enzyme liquid；The 1-2 tips of a root are taken to break the tip of a root into pieces with forceps tips on clean slide, the hanging Ka Nuoshi that is added dropwise consolidates
Determining liquid makes it fully scatter, and bakes, dries on alcolhol burner；Microscopy, select -20 DEG C of preservations of well dispersed chromosome piece；
2) FISH
1. roasting piece：Before hybridization, the slice, thin piece made is placed in 65 DEG C of baking ovens and dries 30-60min；
2. the preparation of hybridization solution：SsDNA4 μ L and each 3 μ L of Itf_1, Itf_2 DNA probe are taken, mixes and is dried to after in 65 DEG C of baking ovens
4 μ L, add μ L of 70% deionized formamide 10, μ L of 50% dextran sulfate 4, the μ L of 20 × SSC 2 and mix, sealed membrane is sealed standby
With；
3. the preparation of denaturing liquid：μ L of distilled water 20,20 × SSC10 μ L, the μ L of 70% deionized formamide 70 are taken, is mixed, sealed membrane
Seal standby；
4. probe is denatured：The chromosome piece in baking oven is taken out, baking oven is warming up to 85 DEG C, above-mentioned denaturing liquid is added on to the load of taking-up
On slide, plus cover glass；2-4min is denatured in 85 DEG C of baking ovens；Get rid of cover glass, immediately successively immerse -20 DEG C 70%, 95%,
Each 5min in 100% ice ethanol；Take out slide, more than air drying 30min；
5. hybridize：Hybridization solution is put into boiling water and is denatured 10min, is immediately placed in cooled on ice 10min, after being added on previous step drying
Slide on, covered, room temperature 5-10min；It is placed in 80-85 DEG C of baking oven and is denatured 2min, be put into culture dish, is placed in
17-21h is cultivated in 37 DEG C of incubators；
6. eluted after hybridization：The slide in incubator is taken out, is placed in 2 × SSC 2 × 5min of room temperature elution on decolorization swinging table；Put
10min is eluted in 42 DEG C of 2 × SSC；Elute 5min in 2 × SSC on decolorization swinging table again；Eluted in 1 × TNT on decolorization swinging table
5min, dry；The sealed membrane that clip and slide size are coincide, take out TNB room temperatures and melt；Under the conditions of lucifuge, take 1.5 μ L's
EP is managed, and is added 100 μ L TNB, 1 μ L Bio, 0.5 μ LDig antibody, is mixed, take 100 μ L to be added on the slide dried, cover sealing
Film, it is placed in 37 DEG C of incubators and cultivates 1h；Slide is taken out, is placed in 1 × TNT 3 × 5min of elution on decolorization swinging table；Dry slide
5-10min；
3) signal detection
Add 13 μ L DAPI, covered, dry, mounting, be placed under fluorescence microscope, determine that target contaminates according to film-making coordinate
Colour solid, chromosome is taken pictures, then conversion filter successively, the hybridization signal of two probes taken pictures respectively；
4) secondary hybridization
1. develop a film：Take the cover glass of hybridization for the first time off, by slide be placed in 2 × SSC solution on decolorization swinging table elution 2 ×
5min；It is placed in 2 × SSC solution and elutes 2 × 10min again on decolorization swinging table；Then 70%, 95%, 100% is immersed successively
Each 5min of room temperature elution on decolorization swinging table in ethanol；It is placed in Ka Nuoshi fixers and fixes 5min, takes out slide, dry, fully
Exposure 2-3 days；
2. FISH：With 45S, 5S rDNA probes carry out second of FISH, crossover process with step 2, then
The hybridization signal of two probes is taken pictures respectively；Chromosome and hybridization signal are carried out by figure using imaging system analysis software
As synthesis, image is adjusted with image processing software.
3. the fluorescence in-situ hybridization method according to claim 2 for differentiating sweet potato chromosome, it is characterised in that the Itf_
What 1 probe was obtained by：According to SEQ NO：Nucleotide sequence design primer shown in 1 expands template to be measured, the primer
For sense primer：5 '-TTCCCGATGCGTGGAGTT-3 ', anti-sense primer：5 '-CTCCATTGCGGTTGTCTTA-3 ', use
The nucleotide sequence such as SEQ NO of PCR methods synthesizing biotinylated mark：Probe shown in 1, obtain Itf_1 probes；
What the Itf_2 probes were obtained by：According to SEQ NO：Nucleotide sequence design primer shown in 2 expands mould to be measured
Plate, the primer are sense primer：5 '-CCCCACCTTCAACCAACT-3 ', anti-sense primer：5’-
GCAGCGGTTAGGAGGTGA-3 ', the nucleotide sequence such as SEQ NO of digoxigenin labeled are synthesized using PCR methods：Spy shown in 2
Pin, obtain Itf_2 probes.
4. the fluorescence in-situ hybridization method of the discriminating sweet potato chromosome according to Claims 2 or 3, it is characterised in that described
Ka Nuoshi fixers are formulated by 3 parts of absolute ethyl alcohols and 1 part of glacial acetic acid.
5. the fluorescence in-situ hybridization method of the discriminating sweet potato chromosome according to Claims 2 or 3, it is characterised in that fluorescence
In situ hybridization step 4. in, probe is denatured 3min in 85 DEG C of baking ovens.
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| DNA分子标记在甘薯遗传育种研究中的应用;汤佳立等;《江苏农业科学》;20090815(第04期);4-8 * |
| 甘薯栽培种及其近缘野生种的DAPI核型及rDNA-FISH分析;安婷婷 等;《西北植物学报》;20121231;第32卷(第4期);682-687 * |
| 荧光原位杂交技术分析栽培种甘薯(Ipomoea batatas cv.XushuNo.18)染色体;汤佳立等;《遗传》;20100228(第02期);177-182 * |
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