CN103205500B - A kind of quick analysis and the multi-color fluorescence in situ hybridization method of qualification Semen Tritici aestivi exogenous chromosome - Google Patents
A kind of quick analysis and the multi-color fluorescence in situ hybridization method of qualification Semen Tritici aestivi exogenous chromosome Download PDFInfo
- Publication number
- CN103205500B CN103205500B CN201310127485.6A CN201310127485A CN103205500B CN 103205500 B CN103205500 B CN 103205500B CN 201310127485 A CN201310127485 A CN 201310127485A CN 103205500 B CN103205500 B CN 103205500B
- Authority
- CN
- China
- Prior art keywords
- chromosome
- genome
- situ hybridization
- wheat
- fluorescence
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 210000000349 chromosome Anatomy 0.000 title claims abstract description 123
- 238000000034 method Methods 0.000 title claims abstract description 71
- 238000007901 in situ hybridization Methods 0.000 title claims abstract description 40
- 210000000582 semen Anatomy 0.000 title claims abstract description 38
- 238000004458 analytical method Methods 0.000 title claims abstract description 29
- 238000012797 qualification Methods 0.000 title claims description 15
- 241000209140 Triticum Species 0.000 claims abstract description 33
- 235000021307 Triticum Nutrition 0.000 claims abstract description 33
- 238000002372 labelling Methods 0.000 claims abstract description 27
- 239000007850 fluorescent dye Substances 0.000 claims abstract description 24
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 23
- 230000031864 metaphase Effects 0.000 claims abstract description 15
- 230000011278 mitosis Effects 0.000 claims abstract description 10
- 238000013519 translation Methods 0.000 claims abstract description 9
- 241000190150 Bipolaris sorokiniana Species 0.000 claims abstract description 6
- 230000032823 cell division Effects 0.000 claims abstract description 6
- 230000000442 meristematic effect Effects 0.000 claims abstract description 5
- 238000009396 hybridization Methods 0.000 claims description 35
- 235000007238 Secale cereale Nutrition 0.000 claims description 33
- 241000209056 Secale Species 0.000 claims description 17
- 244000082988 Secale cereale Species 0.000 claims description 16
- 241000209126 Thinopyrum ponticum Species 0.000 claims description 11
- 230000008569 process Effects 0.000 claims description 9
- 230000009261 transgenic effect Effects 0.000 claims description 8
- 241001522110 Aegilops tauschii Species 0.000 claims description 6
- 239000012634 fragment Substances 0.000 claims description 6
- 241001329985 Triticeae Species 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- 239000001913 cellulose Substances 0.000 claims description 4
- 229920002678 cellulose Polymers 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 4
- 238000012545 processing Methods 0.000 claims description 4
- 210000001519 tissue Anatomy 0.000 claims description 3
- 108010043121 Green Fluorescent Proteins Proteins 0.000 claims description 2
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 claims description 2
- 244000040347 Eragrostis elongata Species 0.000 claims 1
- 230000000295 complement effect Effects 0.000 claims 1
- 230000002194 synthesizing effect Effects 0.000 claims 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-N acetic acid Substances CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 23
- 239000000523 sample Substances 0.000 description 23
- 241000196324 Embryophyta Species 0.000 description 16
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 11
- 230000005945 translocation Effects 0.000 description 10
- 239000000203 mixture Substances 0.000 description 9
- 244000098338 Triticum aestivum Species 0.000 description 8
- 238000011160 research Methods 0.000 description 8
- 210000000745 plant chromosome Anatomy 0.000 description 7
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- 239000000463 material Substances 0.000 description 6
- 238000000386 microscopy Methods 0.000 description 6
- 230000003252 repetitive effect Effects 0.000 description 6
- 241000894007 species Species 0.000 description 6
- 238000009395 breeding Methods 0.000 description 5
- 230000001488 breeding effect Effects 0.000 description 5
- 210000004027 cell Anatomy 0.000 description 5
- 238000011161 development Methods 0.000 description 5
- 230000018109 developmental process Effects 0.000 description 5
- 229960004756 ethanol Drugs 0.000 description 5
- 101150076489 B gene Proteins 0.000 description 4
- 101100245267 Caenorhabditis elegans pas-1 gene Proteins 0.000 description 4
- 241000209125 Thinopyrum elongatum Species 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 230000006872 improvement Effects 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 230000017105 transposition Effects 0.000 description 4
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 3
- 229960002685 biotin Drugs 0.000 description 3
- 235000020958 biotin Nutrition 0.000 description 3
- 239000011616 biotin Substances 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000000284 extract Substances 0.000 description 3
- 238000011010 flushing procedure Methods 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 241000209148 Aegilops speltoides Species 0.000 description 2
- 108010059892 Cellulase Proteins 0.000 description 2
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 2
- LTMHDMANZUZIPE-AMTYYWEZSA-N Digoxin Natural products O([C@H]1[C@H](C)O[C@H](O[C@@H]2C[C@@H]3[C@@](C)([C@@H]4[C@H]([C@]5(O)[C@](C)([C@H](O)C4)[C@H](C4=CC(=O)OC4)CC5)CC3)CC2)C[C@@H]1O)[C@H]1O[C@H](C)[C@@H](O[C@H]2O[C@@H](C)[C@H](O)[C@@H](O)C2)[C@@H](O)C1 LTMHDMANZUZIPE-AMTYYWEZSA-N 0.000 description 2
- 241000508725 Elymus repens Species 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 241001673118 Thinopyrum intermedium Species 0.000 description 2
- 239000002250 absorbent Substances 0.000 description 2
- 230000002745 absorbent Effects 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 229940106157 cellulase Drugs 0.000 description 2
- 235000013339 cereals Nutrition 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000024321 chromosome segregation Effects 0.000 description 2
- 230000007850 degeneration Effects 0.000 description 2
- LTMHDMANZUZIPE-PUGKRICDSA-N digoxin Chemical compound C1[C@H](O)[C@H](O)[C@@H](C)O[C@H]1O[C@@H]1[C@@H](C)O[C@@H](O[C@@H]2[C@H](O[C@@H](O[C@@H]3C[C@@H]4[C@]([C@@H]5[C@H]([C@]6(CC[C@@H]([C@@]6(C)[C@H](O)C5)C=5COC(=O)C=5)O)CC4)(C)CC3)C[C@@H]2O)C)C[C@@H]1O LTMHDMANZUZIPE-PUGKRICDSA-N 0.000 description 2
- 229960005156 digoxin Drugs 0.000 description 2
- LTMHDMANZUZIPE-UHFFFAOYSA-N digoxine Natural products C1C(O)C(O)C(C)OC1OC1C(C)OC(OC2C(OC(OC3CC4C(C5C(C6(CCC(C6(C)C(O)C5)C=5COC(=O)C=5)O)CC4)(C)CC3)CC2O)C)CC1O LTMHDMANZUZIPE-UHFFFAOYSA-N 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 238000007903 genomic in situ hybridization Methods 0.000 description 2
- 238000011065 in-situ storage Methods 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- -1 0.47g Chemical compound 0.000 description 1
- PXFBZOLANLWPMH-UHFFFAOYSA-N 16-Epiaffinine Natural products C1C(C2=CC=CC=C2N2)=C2C(=O)CC2C(=CC)CN(C)C1C2CO PXFBZOLANLWPMH-UHFFFAOYSA-N 0.000 description 1
- OALHHIHQOFIMEF-UHFFFAOYSA-N 3',6'-dihydroxy-2',4',5',7'-tetraiodo-3h-spiro[2-benzofuran-1,9'-xanthene]-3-one Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC(I)=C(O)C(I)=C1OC1=C(I)C(O)=C(I)C=C21 OALHHIHQOFIMEF-UHFFFAOYSA-N 0.000 description 1
- 102000004594 DNA Polymerase I Human genes 0.000 description 1
- 108010017826 DNA Polymerase I Proteins 0.000 description 1
- 108020003215 DNA Probes Proteins 0.000 description 1
- 239000003298 DNA probe Substances 0.000 description 1
- SHIBSTMRCDJXLN-UHFFFAOYSA-N Digoxigenin Natural products C1CC(C2C(C3(C)CCC(O)CC3CC2)CC2O)(O)C2(C)C1C1=CC(=O)OC1 SHIBSTMRCDJXLN-UHFFFAOYSA-N 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- NYHBQMYGNKIUIF-UUOKFMHZSA-N Guanosine Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O NYHBQMYGNKIUIF-UUOKFMHZSA-N 0.000 description 1
- 241000209510 Liliopsida Species 0.000 description 1
- 208000020584 Polyploidy Diseases 0.000 description 1
- 239000007984 Tris EDTA buffer Substances 0.000 description 1
- 235000019714 Triticale Nutrition 0.000 description 1
- 241000209147 Triticum urartu Species 0.000 description 1
- 230000009418 agronomic effect Effects 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000010009 beating Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000007979 citrate buffer Substances 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 238000009402 cross-breeding Methods 0.000 description 1
- 229960000935 dehydrated alcohol Drugs 0.000 description 1
- QONQRTHLHBTMGP-UHFFFAOYSA-N digitoxigenin Natural products CC12CCC(C3(CCC(O)CC3CC3)C)C3C11OC1CC2C1=CC(=O)OC1 QONQRTHLHBTMGP-UHFFFAOYSA-N 0.000 description 1
- SHIBSTMRCDJXLN-KCZCNTNESA-N digoxigenin Chemical compound C1([C@@H]2[C@@]3([C@@](CC2)(O)[C@H]2[C@@H]([C@@]4(C)CC[C@H](O)C[C@H]4CC2)C[C@H]3O)C)=CC(=O)OC1 SHIBSTMRCDJXLN-KCZCNTNESA-N 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 235000013312 flour Nutrition 0.000 description 1
- 239000006260 foam Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 238000001823 molecular biology technique Methods 0.000 description 1
- 239000003147 molecular marker Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000007894 restriction fragment length polymorphism technique Methods 0.000 description 1
- 239000002342 ribonucleoside Substances 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 230000007226 seed germination Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 239000003039 volatile agent Substances 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 241000228158 x Triticosecale Species 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention provides a kind of quick analysis and identify the multi-color fluorescence in situ hybridization method of Semen Tritici aestivi exogenous chromosome, described method comprises the steps: 1) pass through N2O gas pretreatment vigorous the organizing of enzymolysis Helminthosporium sativum meristematic zone cell division obtain the division phases specimen that image is clear, Chromosome spread is good；2) fluorescent probe of nick translation method labeling wheat race basal gene group is utilized；3) utilize step 2) fluorescent probe that builds is to utilizing step 1) mitosis metaphase split coil method chromosome carry out fluorescence in situ hybridization；4) analytical procedure 3) the fluorescence in situ hybridization result that obtains.The method utilizing the present invention to provide not only can be used to identify exogenous chromosome and chromosome segment in wheat distance edge hybrid kind, it is also possible to is applied in the appraisal to exogenous chromosome in other crops distant hybridses and chromosome segment.
Description
Technical field
The invention belongs to Celluar and Molecular Biology research field.The method that the present invention relates to the multi-color fluorescence in situ hybridization of a kind of quick analysis and qualification Semen Tritici aestivi exogenous chromosome, the invention still further relates to a kind of method that plant chromosome specimen to being obtained carries out repeating hybridization analysis, the invention still further relates to a kind of method preparing plant chromosome division phases specimen, it is possible to obtain the division phases specimen that image is clear, Chromosome spread is good.
Background technology
Semen Tritici aestivi is one of the most important cereal crops, closely related with socio-economic development, grain security supply and human nutrition health.Genetic improvement is the key link improving yield and quality of wheat, and the improvement of current wheat breed mainly uses conventional cross-breeding approach, but traditional breeding way exists that cycle length, character improvement be notable and the shortcoming such as narrow range.Hereditary basis is narrow has become as key factor that the Main Agronomic Characters such as restriction crop varieties unit yield, resistance, adaptability promote further (leaf make the country prosperous etal.2011).Therefore, the hereditary basis widening kind has become the urgent needs of crop breeding development.
Distant hybridization is the important channel that species are formed, it it is one of the key factor of biological evolution, distant hybridization can break the boundary between kind of (or section, genus), the hereditary material between different plant species is made to exchange or combine, the beneficial characteristics that two or more species accumulate through long-term evolution is combined, double and natural selection through chromosome set is natural again, form the new species that vitality is higher.Exogenous gene is imported by distant hybridization, significant to modern wheat flour quality.
The GENE SOURCES of common wheat is wider, and in Tribe Triticeae, more than 300 species great majority can hybridize with common wheat, and 15 kinds (including 5 kinds of Triticum) of existing 5 genus displaced disease-resistant gene to common wheat.China the most by Semen Tritici aestivi and Thinopyrum intermedium, E. elongata distant hybridization success in the 1950's, has the most been bred as a collection of kind and has promoted on producing.Such as " the dragon wheat No. 1 ", " imperial wheat No. 2 ", the spring wheat varieties such as " Neoma Foams 6 " that are bred as by Thinopyrum intermedium.Meanwhile, 1 to No. 7 high-quality of octoploid of wheat-wheatgrass " in Yuan ", disease-resistant of incubation, it has also become good breeding parent.Being bred as a collection of wheat breed by Thinopyrum ponticum, wherein " No.6 Xiaoyan " Annual planting area once reached 66.67 ten thousand hm2, and used up to 20 years (Dong Yu treasure 2001) on producing.
Chromosome engineering had once played very important effect in the polyploid crop breeding histories such as Semen Tritici aestivi.20 century 70s, Europe is found that the 1RS/1BL translocation line material of rye (Secale cereale L.) in the Semen Tritici aestivi filial generation with rye (Secale cereale L.), and improve for wheat breed, cultivate some row high yields, disease-resistant, the dwarf varietiy of wheats such as love woods, and in the world the rapid spread in each Semen Tritici aestivi main producing region, every country is according to the breeding objective of oneself subsequently, the thousands of 1RS/1BL kind of transformation, produces for world wheat and is made that outstanding contribution.1BL/1RS transposition is widely distributed in world's wheat breed, by chromosome translocation specific between planting and replacement, the galianconism (1RS) of chromosomes of rye 1R is present in the chromosome set of a large amount of common wheat, and many genes to economical character with important function and resistant gene thus proceed to (appoint swallow and Wang Tao 2006) Wheat volatiles from rye (Secale cereale L.).
Traditional chromosome engineering exists that efficiency is low, cycle length, recombinant chromosome are difficult to accurate and effective and follow the tracks of and Rapid identification, and be difficult to series of problems such as being connected mutually with modern molecular biology technique, seriously limits developing further of chromosome engineering research.So analysis chromosome set achievement rapidly and efficiently is particularly important.Progress along with genome walking technology, the development of especially DNA hybridization in situ technique, useful instrument is provided for the qualification of alien chromosome lines, the identification of translocation breakpoint, the size of transposition fragment, position and breakpoint can be identified exactly, carry out wheat breed improvement for the useful gene of autotelic transfer and lay a good foundation.
Genomic in situ hybridization (GenomicinsituhybridizationGISH) is a kind of hybridization in situ technique grown up late 1980s.It uses from the total genomic dna of species as label probe, blockades with suitable concentration with the total genomic dna of another species, carries out in situ hybridization on target chromosome.Blockading between DNA and labeled DNA probe, the DNA that blockades is preferential and General Sequences hybridizes, and remaining specific sequence is mainly labeled probe and is hybridized.Mukai (MukaiandGill1991) etc. plants Trotociurartu total genomic dna with biotin labeling diploid AA genome ancestors, digoxigenin labeled diploid DD genome ancestors plant Aegilopssquarrosa total genomic dna, the BB genome ancestors that unlabelled total genomic dna is possible plant Aegilopsspeltoides, hexaploid common wheat (Triticumaestivum) metaphase chromosome is carried out in situ hybridization, only just A, B and D genome is shown as yellow, brown and orange with two kinds of fluoresceins simultaneously.Hereafter Sanchez-Moran (Sanchez-Moranetal.1999) etc. utilize this technology different colours show simultaneously wheat-rye derive kind in A, B, D and R genome.(the Hanetal.2003 such as Korea Spro；nullHanetal.2004) digoxin and biotin labeled genome GISH is utilized to identify the chromosome composition of partial amphidiploid and analyze the transposition between chromosome，But the probes label methods used is mostly indirect method，I.e. use the non-fluorescence material such as label probe such as biotin or digoxin，Fluorescent material is brought into again by affine connection or immunoreation，There is hybridization signal amplification，Although indirect method FISH can amplify hybridization signal，Increase hybridization sensitivity，But to hatch through loaded down with trivial details eluting and antibody after Za Jiao and amplify hybridization signal，Waste time and energy，And in elution process, easily cause the problems such as chromosome loss deformation，This experiment is by direct method FISH，I.e. when nick-translation method label probe，It is directly incorporated into fluorescein ribonucleoside triphosphote label，Just can microscopy through simple flushing after hybridization，Greatly simplifie in situ hybridization program，Keep the optimal morphology of chromosome，Mutual transposition to identifying between exogenous chromosome composition and analysis and chromosome can well be observed.
Summary of the invention
It is an object of the present invention to provide a kind of quick analysis and identify the multi-color fluorescence in situ hybridization method of Semen Tritici aestivi exogenous chromosome.
Specifically, the analysis of the present invention and the method for qualification Semen Tritici aestivi exogenous chromosome comprise the steps:
1) N is passed through2O gas pretreatment vigorous the organizing of enzymolysis Helminthosporium sativum meristematic zone cell division obtain the division phases specimen that image is clear, Chromosome spread is good；
2) nick translation method labelling labeling wheat race basal gene group fluorescent probe is utilized；
3) utilize step 2) fluorescent probe that builds is to utilizing step 1) mitosis metaphase split coil method chromosome carry out fluorescence in situ hybridization；
4) analytical procedure 3) the fluorescence in situ hybridization result that obtains.
Wherein step 1) comprise the steps: (a) N2O gas pretreatment: take suitable Helminthosporium sativum, is placed in moistening centrifuge tube, puts into N2O air chamber processes 0.5-5 hour, N2The pressure of O is 10ATM (1.01Mpa)；B () is by N2The tip of a root of O gas pretreatment is fixed；C () enzymolysis processing: washed with water by the tip of a root fixed, cut off root tips separate living tissue, by pectase and cellulose treatment 30min-2h.
Wherein step 2) the Tribe Triticeae basal gene group of labelling selected from AA genome, DD genome, RR rye (Secale cereale L.) genome, 10X E. elongata genome, or transgenic purpose fragment vector.
In above-mentioned steps, the step of most critical is step 3) mixed proportion of fluorescent probe of nick translation method labelling used by hybridization, the mass ratio of AA genome and DD genome can not be less than 1: 4.Such as, split coil method mitosis metaphase of little wheat of laying down (couchgrass and common wheat filial generation) and Triticale hexaploide Lart. (rye (Secale cereale L.) and common wheat filial generation) is carried out fluorescence in situ hybridization；nullProbe mixed proportion therein should be that the number according to different materials (exogenous chromosome is derived from rye (Secale cereale L.) or couchgrass) exogenous chromosome carrys out mixed probe，AA genome and DD genomic homology are higher，So AA genome and DD genome have crisscrossing，The color of chromosome is inconsistent (during probe labelling with the color of probe labelling，AA genome is green，DD genome is red，After having hybridized，AA genome is green，DD genome is red)，So probe ratio (AA genome and the ratio of DD genome) can not be less than 1: 4，BB genome is as blockading，Certain homology is had with AA genome and DD genome，But and BB genome (oneself is) homology own is the highest，So separating four chromosome sets with an a high proportion of BB genome of blockading to beneficially one time hybridization region of blockading，Blockading of BB genome is relatively up to 160-200 times than (mass ratio).
A further object of the present invention is to provide a kind of method that plant chromosome specimen to being obtained carries out repeating hybridization analysis.
Specifically, the present invention plant chromosome specimen obtained is carried out repeat hybridization analysis method comprise the steps:
1) N is passed through2O gas pretreatment enzymolysis of plants Meristernatic zone or vigorous the organizing of bud point meristematic zone cell division obtain the division phases specimen that image is clear, Chromosome spread is good；
2) nick translation method mark fluorescent probe is utilized；
3) utilize step 2) fluorescent probe that builds is to utilizing step 1) mitosis metaphase split coil method chromosome carry out fluorescence in situ hybridization；
4) rinsing through simple, on the basis of keeping chromosome morphology, the multiple hybridization probe of Reusability carries out one or many hybridization, carries out qualification and the analysis of exogenous chromosome.
In above-mentioned steps, the step of most critical is step 4) in, rinse through simple, the multiple hybridization probe of Reusability on the basis of keeping chromosome morphology, carry out one or many hybridization successively, carry out qualification and the analysis of exogenous chromosome.After having hybridized for the first time; the anti-quencher containing DAPI is had on chromosome specimen; relatively thickness; have only to 2 × SSC (protein on protection chromosome; preventing chromosome from deforming) eluting just can carry out follow-up hybridization (such as the most successively under room temperature; second time hybridization, hybridization etc. for the third time), it is to avoid the problem such as chromosome loss deformation in loaded down with trivial details eluting before and elution process.
In one embodiment of the invention, the present invention provides a kind of quick analysis and identifies the multi-color fluorescence in situ hybridization method of Semen Tritici aestivi exogenous chromosome, it is characterised in that:
1) N is passed through2O gas pretreatment enzymolysis Gansu Province spring 23, the Meristernatic zone cell of octoploid of wheat-wheatgrass XY784 obtains the division phases specimen that image is clear, Chromosome spread is good；
2) nick translation method labelling A genome, D genome, R genome and Thinopyrum ponticum fluorescent probe are utilized；
3) utilize step 2) fluorescent probe that builds is to utilizing step 1) mitosis metaphase split coil method chromosome carry out fluorescence in situ hybridization；
4) analytical procedure 3) the fluorescence in situ hybridization result that obtains.
In one embodiment of the invention, described chromosome to be detected and chromosome segment are selected from Gansu Province spring 23, the transgenic line of octoploid of wheat-wheatgrass XY784 and Pwy86.
Although one or more in above-mentioned technology are applied in relevant research, but the experimental program that the most several technological maheup of integrated application is complete, and it is applied in Wide_cross in Plant kind the qualification of exogenous chromosome or chromosome segment has not seen that correlational study is reported.The present invention utilizes this set research approach successfully with the external source autosome in high-sensitivity detection distant hybridization plant first, overcome the problem that in prior art, plant autosome Detection results is undesirable, it is a kind of technological break-through, embodies this research method and there is preferable advance, innovation and practicality.
In sum, the present invention provides following:
1. quick analysis and a multi-color fluorescence in situ hybridization method for qualification exogenous chromosome, it comprises the steps:
1) N is passed through2O gas pretreatment vigorous the organizing of enzymolysis of plants Meristernatic zone cell division obtain the division phases specimen that image is clear, Chromosome spread is good；
2) fluorescent probe of nick translation method labeling wheat race basal gene group is utilized；
3) utilize step 2) fluorescent probe that builds is to utilizing step 1) mitosis metaphase split coil method chromosome carry out fluorescence in situ hybridization；
4) analytical procedure 3) the fluorescence in situ hybridization result that obtains.
2. the 1st described method, wherein step 1) comprise the steps: (a) N2O gas pretreatment: take suitable Helminthosporium sativum, is placed in moistening centrifuge tube, puts into N2O air chamber processes 0.5-5 hour, N2The pressure of O is 10ATM (1.01Mpa)；B () is by N2The tip of a root of O gas pretreatment is fixed；C () enzymolysis processing: washed with water by the tip of a root fixed, cut off root tips separate living tissue, by pectase and cellulose treatment 30min-2h.
3. the 1st described method, wherein step 2) in the Tribe Triticeae basal gene group of labelling selected from AA genome, DD genome, RR rye (Secale cereale L.) genome, 10X E. elongata genome or transgenic purpose fragment vector.
4. the 1st described method, wherein the mass ratio of the probe mixing of diploid Urartu AA genome and diploid aegilops tauschii D D genome can not be less than 1: 4.
5. the 1st described method, wherein diploid intend this inferior you torr BB genome mass ratio of blockading be 160-200 times.
6. the 1st described method, wherein carries out repeating hybridization analysis to the chromosome of wheat specimen obtained, and the most often step repeats hybridization analysis eluting is with eluting under 2 × SSC room temperature.
7. the method carrying out the plant chromosome specimen obtained repeating hybridization analysis, described method comprises the steps:
1) N is passed through2O gas pretreatment enzymolysis of plants Meristernatic zone or vigorous the organizing of bud point meristematic zone cell division obtain the division phases specimen that image is clear, Chromosome spread is good；
2) nick translation method mark fluorescent probe is utilized；
3) utilize step 2) fluorescent probe that builds is to utilizing step 1) mitosis metaphase split coil method chromosome carry out fluorescence in situ hybridization；
4) rinsing through simple, on the basis of keeping chromosome morphology, the multiple hybridization probe of Reusability carries out one or many hybridization, carries out qualification and the analysis of exogenous chromosome.
8. the 7th described method, wherein step 4) simple flushing be with eluting under 2 × SSC room temperature.
9. the method preparing plant chromosome division phases specimen, described method comprises the steps:
(a)N2O gas pretreatment: take the suitable tip of a root of described plant or bud point, be placed in moistening centrifuge tube, put into N2O air chamber processes 0.5-5 hour, N2The pressure of O is 10ATM (1.01Mpa)；B () is by N2The tip of a root or the bud point of O gas pretreatment are fixed；C () enzymolysis processing: the tip of a root fixed or bud point are washed with water, cut off root tips or shoot tip meristem, by pectase and cellulose treatment 30min-2h.
10. the 9th described method, wherein said plant is monocotyledon.
Accompanying drawing is sketched
Fig. 1 .1RS/1BL translocation line chromosome forms.Arrow show 1RS chromosome.Figure Green represents rye (Secale cereale L.) R, and yellow represents Semen Tritici aestivi A genome, and brown represents Semen Tritici aestivi 1 B gene group, and redness represents Semen Tritici aestivi D genome.
Fig. 2. octoploid of wheat-wheatgrass chromosome forms.Arrow and asterisk show Semen Tritici aestivi A-D group two to translocation chromosome.Green represents Thinopyrum ponticum, and yellow represents Semen Tritici aestivi A genome, and brown represents Semen Tritici aestivi 1 B gene group, and redness represents Semen Tritici aestivi D genome.
Fig. 3. three in situ hybridization A: the fluorescence in situ hybridization detection of transgenic signal of chromosome of wheat specimen, arrow show the insertion point of genes of interest；B: multi-color fluorescence in situ hybridization, the chromosome of wheat group at detection transgenic loci place, green represents Semen Tritici aestivi A chromosome group, and brown represents Semen Tritici aestivi B chromosome group, and redness represents Semen Tritici aestivi D chromosome set, and arrow show genes of interest and inserts the A chromosome group of Semen Tritici aestivi；C: repetitive sequence pSC119.2 and the concrete chromosome at pAS1 testing goal gene place, green shows repetitive sequence pSC119.2 signal, and redness shows repetitive sequence pAS1 signal, and arrow show genes of interest and inserts the 1A chromosome of Semen Tritici aestivi.
Detailed description of the invention
It is further elucidated with the present invention by the following examples.However, it should be understood that the purpose that described embodiment is merely illustrative, it is not intended to limit scope and spirit of the present invention.
It addition, it should also be realized by those skilled in the art that unless specifically stated otherwise, reagent used in following embodiment is the reagent of commercially available analytical pure rank.
The qualification of embodiment 1. Gansu Province spring 23 spring wheat material
1. the preparation of Gansu Province spring 23 spring wheat split coil method mitosis metaphase
Gansu Province spring 23 spring wheat used, the germ plasm resource (can be found in Yang Wen hero 2005) preserved from the general laboratory of South Korea of Inst. of Genetics and Development Biology, CAS.
1)N2O processes the tip of a root
When the tip of a root grows to 2-3cm, cutting, be placed in moistening centrifuge tube, put into process 1-3h in N2O air chamber after closeing the lid, pressure is 10ATM (1.01Mpa)；
2) tip of a root is fixed
90% acetic acid fixes 5-10 minute (less than 1h), distillation washing 2 times.The tip of a root after Gu Ding can proceed to put into-20 DEG C in the ethanol of 70% and can preserve for many years；
3) enzymolysis
nullWith filter paper, the water on the tip of a root is somewhat blotted，Root apical meristem is cut，Put into 20 μ L mixed liquors (the enzymolysis solution preparation a: plastic culture dish is put on balance of pectase and cellulase，1 × citrate buffer solution (10mM sodium citrate+10mMEDTA is added to the inside，pH5.5)9.7g，Taking-up is placed on ice，Weigh 0.1g pectase Y-23 (purchased from JapanYakult company of Japan，1%w/w) with 0.2g cellulase OnozukaR-10 (purchased from JapanYakult company of Japan，2%w/w) add in buffer，With rifle head pressure-vaccum hydrotropy，Enzyme is dispensed in 0.5ml thin-walled PCR pipe with liquor separator after dissolving，-20 DEG C of preservations) in，37 DEG C of water-baths 40-60 minute，
4) sheet is dripped
2 times are washed with 70% ethanol, some ethanol is stayed when washing second time, about 200ul is in centrifuge tube, with dissecting needle, the tip of a root is smashed to pieces, centrifugal about 10 seconds of low speed (＜ 2000 turns), ethanol is fallen dry, ice acetic acid (each tip of a root 20-40 μ l, depending on plant root tip size) suspension root-tip cells.
5) microscopy
Being placed in moistening box by clean microscope slide, every microscope slide drips 6-7 μ l root-tip cells suspension, covers lid, microscopy after 5 minutes, by chromosome sectioning Saving specimen in refrigerator standby.
2.AA group, the probe labelling of DD group and RR group
1) CTAB method extracts plant genome DNA
Take plant (rye (Secale cereale L.), Thinopyrum ponticum, Triticum tauschii, Urartu and speltoides, the blade that (these seeds are purchased from GermanyIPK institute) children is tender, reference literature (Kidwelletal.1992) extracts the DNA of plant, makes the concentration of DNA be finally reached 200ng/ul, then label probe.Wherein speltoides to blockade, concentration 2000ng/ul to be reached.
2) following component is added on ice in centrifuge tube:
After adding well, repeatedly blowing and beating mixing with rifle head, it is impossible to vortex, 15 DEG C are incubated 2 hours.It is placed in 15 DEG C of reaction 2h in metal bath instrument (H2O3-PRO, China).(-the dNTP of labelling is AlexaFluor-488-dUTP (green glow), Texas-5-dCTP (HONGGUANG) is purchased from Invitrogen company；DNA polymerase i is purchased from Invitrogen company, and concentration is 10U/ μ L, can be used directly, 10xNicktranslationbufferfor100mlTris6.05g, MgCl2, 0.47g, HCl adjust pH7.8)；Wherein rye (Secale cereale L.), E. elongata and Urartu are with green fluorescent label (AlexaFluor-488-dUTP (green glow)), and Triticum tauschii red fluorescence is marked (Texas -5-dCTP), speltoides blockades (i.e., speltoides genomic DNA high pressure is interrupted, 115 DEG C, 8 minutes, it was about 200-300bp that genomic DNA is broken into fragment), before blockading, genomic fragment needs to be broken into 200-300bp, and such effect of blockading is best.
3) 13000 leaving the heart 30-40 minute, abandon supernatant, wash primary sedimentation with 70% ethanol and dehydrated alcohol respectively, lucifuge is dried.
4) add 10 μ l2 × SSC, 1 × TE buffer (pH7.0), make the final concentration of 200ng/ul of probe.
3. utilize labelling good genomic probe that Gansu Province spring 23 spring wheat carries out the qualification of fluorescence in situ hybridization
1) Gansu Province spring 23 spring wheat metaphase chromosome specimen is prepared by the method for program 1.
2) specimen to be hybridized (film-making) is put into 0.125J/cm2 crosslinking in UV-crosslinked instrument (code-No.CL-1000, purchased from UVP company of the U.S.).
3) film-making is placed on ice, take out the good probe of a small amount of labelling by rye (Secale cereale L.): AA group: BB group: DD group=1: 1: 4: 4 or Thinopyrum ponticum: AA group: BB group: DD group=1: 1.5: 4: 4 dilution proportion, the hybridization solution 6 μ l diluted is added on every microscope slide, covered, 90 DEG C of degeneration 5 minutes, 55 DEG C hybridize more than 6 hours.
4) film-making is taken out from wet box, put into and 2 × SSC makes coverslip landing, with absorbent paper, the slice, thin piece back side is dried, drip a upper anti-quencher (H-1200 containing DAPI, purchased from VectorLabs company of the U.S.), cover the coverslip of 24 × 50mm, after several minutes, i.e. can use fluorescence microscope (OlympusBX61) to carry out microscopy, photograph (MagnafierCCDcamera).
4. result and discussion
Result of study is shown in Fig. 1.
As it is shown in figure 1, be shown that 1RS/1BL translocation line chromosome composition.Arrow show 1RS chromosome.Figure Green represents rye (Secale cereale L.) R, article two, green the short arm of a chromosome, being from rye (Secale cereale L.) 1RS, yellow represents Semen Tritici aestivi A genome, has 14 chromosomes, brown represents Semen Tritici aestivi 1 B gene group, having 14 chromosomes, redness represents Semen Tritici aestivi D genome, has 14 chromosomes, whole cell has 42 chromosomes, is the chromosome composition of 1RS/1BL translocation line.
This research utilizes two fluorescent probes of four genomes first, Gansu Province spring 23 spring wheat is carried out genome Chromosome Identification, just can clearly be analyzed by an in situ hybridization and separate four chromosome sets, and four genomic chromosome compositions are cheer and bright, show that the method utilizing the present invention to use successfully detects and identifies existence and the structure of exogenous chromosome, this experiment terminates simply to need 3 day time to identifying from sending out seed, the most rapidly and efficiently and also sensitive.
The chromosome composition analysis of embodiment 2. octoploid of wheat-wheatgrass XY784
1. the preparation of octoploid of wheat-wheatgrass XY784 split coil method mitosis metaphase
Octoploid of wheat-wheatgrass XY784 used, the germ plasm resource (Zhang Xueyong etal.1995) preserved from Inst. of Genetics and Development Biology, CAS's Li Zhensheng laboratory.
1) N2O processes the tip of a root with embodiment 1.1
2) tip of a root is fixing with embodiment 1.2
3) enzymolysis is with embodiment 1.3
4) sheet is dripped with embodiment 1.4
5) microscopy is with embodiment 1.5
2.AA group, the probe labelling of DD group and Thinopyrum ponticum group
1) CTAB method extracts plant genome DNA with embodiment 2.1
2) probe labelling is with embodiment 2.2
3) with embodiment 2.3
4) with embodiment 2.4
3. utilize labelling good genomic probe that octoploid of wheat-wheatgrass XY784 carries out the qualification of fluorescence in situ hybridization
1) octoploid of wheat-wheatgrass XY784 metaphase chromosome specimen is prepared by the method for program 1.
2) with embodiment 3.2
3) film-making is placed on ice, take out the good probe of a small amount of labelling by Thinopyrum ponticum: AA group: BB group: DD group=1: 1: 4: 4 or Thinopyrum ponticum: AA group: BB group: DD group=1: 1.5: 4: 4 dilution proportion, the hybridization solution 6 μ l diluted is added on every microscope slide, covered, 90 DEG C of degeneration 5 minutes, 55 DEG C hybridize more than 6 hours.
4) with embodiment 3.4
4. result and discussion
Result of study is shown in Fig. 2.
As it is shown in figure 1, be shown that octoploid of wheat-wheatgrass chromosome composition.Green represents Thinopyrum ponticum, chromosome number is 14, yellow represents Semen Tritici aestivi A genome, chromosome number is 12, fewer than normal A group chromosome 2, and brown represents Semen Tritici aestivi 1 B gene group, chromosome number is 14, redness represents Semen Tritici aestivi D genome, and chromosome number is 16, more than normal D group chromosome 2.Arrow and asterisk show Semen Tritici aestivi A-D group two to translocation chromosome.Whole cell has 56 chromosomes, is an octoploid of wheat-wheatgrass.
This research utilizes two fluorescent probes of four genomes first, octoploid of wheat-wheatgrass XY784 is carried out genome Chromosome Identification, just can clearly be analyzed by an in situ hybridization and separate four chromosome sets, and four genomic chromosome compositions are cheer and bright, also analyze A-D group two to translocation chromosome simultaneously, identify for later economical character and molecular marker provides basis, show that the method utilizing the present invention to use successfully detects and identifies existence and the composition of exogenous chromosome, this experiment is from sending out seed to identifying that terminating is to need 3 day time, the most rapidly and efficiently and sensitive.
Prepared by the metaphase chromosome of the different plant of embodiment 3.
The present invention is further described, it will be appreciated by those skilled in the art that the present invention is not limited to these specific embodiments referring to specific embodiment.
1. seed germination
(1) seed is placed in the culture dish being lined with moistening filter paper, 21-23 DEG C of calorstat is sprouted.
2. prepared by plant chromosome specimen
1) with embodiment 1.1.
2) with embodiment 1.2.
3) with embodiment 1.3.
4) with embodiment 1.4.
5) with embodiment 1.5.
3. in situ hybridization
1) Metaphase Chromosomes specimen is prepared by the method for program 1.
2) with embodiment 3.2
3) with embodiment 3.3
4) when carrying out second time in situ hybridization, the chromosome specimen hybridized is put into the anti-quencher washed away in 2 × SSC containing DAPI, with absorbent paper, the slice, thin piece back side is dried, carry out secondary in situ hybridization, subsequent step is with embodiment 3.4, and in situ hybridization is ibid for the third time.
3. result and discussion
Result of study is shown in Fig. 3.
As it is shown on figure 3, three in situ hybridization figure A: fluorescence in situ hybridization detection of transgenic signal of the same specimen of chromosome of wheat, arrow show the insertion point of genes of interest；Figure B: multi-color fluorescence in situ hybridization, the chromosome of wheat group at detection transgenic loci place, green represents Semen Tritici aestivi A chromosome group, and brown represents Semen Tritici aestivi B chromosome group, redness represents Semen Tritici aestivi D chromosome set, and arrow show genes of interest and inserts the A chromosome group of Semen Tritici aestivi；Figure C: repetitive sequence pSC119.2 and the concrete chromosome at pAS1 testing goal gene place, green shows repetitive sequence pSC119.2 signal, and redness shows repetitive sequence pAS1 signal, and arrow show genes of interest and inserts the 1A chromosome of Semen Tritici aestivi.
This research specimen same to chromosome of wheat first carries out three in situ hybridizations and is analyzed, just can microscopy through simple flushing after hybridization, and the multiple hybridization probe of Reusability on the basis of chromosome morphology can kept, greatly simplifie in situ hybridization program, be highly suitable for qualification and the analysis of exogenous chromosome.
Should be appreciated that, although with reference to the embodiment that it is exemplary, the present invention carried out particularly shown and described, but it will be apparent to an ordinarily skilled person in the art that, under conditions of without departing substantially from by the spirit and scope of the present invention defined in appended claims, the change of various forms and details can be carried out wherein, the combination in any of various embodiment can be carried out.
List of references:
1.HanFP, FedakG, BenabdelmounaA, ArmstrongK, OuelletT (2003) CharacterizationofsixwheatxThinopyrumintermediumderivati vesbyGISH, RFLP, andmulticolorGISH.Genome46:490-495
HanFP, LiuB, FedakG, LiuZH (2004) Genomicconstitutionandvariationinfivepartialamphiploidso fwheat-ThinopyrumintermediumasrevealedbyGISH, multicolorGISHandseedstorageproteinanalysis.Theoreticala ndAppliedGenetics109:1070-1076
KidwellK, OsbornT, BeckmanJ (1992) SimpleplantDNAisolationprocedures.Plantgenomes:methodsfo rgeneticandphysicalmapping.:1-13MukaiY, GillB (1991) Detectionofbarleychromatinaddedtowheatbygenomicinsituhyb ridization.Genome34:448-452
Sanchez-MoranE, BenaventeE, OrellanaJ (1999) SimultaneousidentificationofA, B, DandRgenomesbygenomicinsituhybridizationinwheat-ryederiv atives.Heredity83:249-252
1BL/1RS chromosome translocation in Ren Yan, Wang Tao (2006) Semen Tritici aestivi.
Dong Yuchen (2001) wheat distance edge hybrid breeding .21 century wheat genetic future breeding stratray wheat genetic breeding international symposium collected works
Leaf is made the country prosperous, Chen Ming, Du Lipu, Xu Huijun (2011) wheat transgenic method and commentary thereof. heredity 33:422-430
Zhang Xueyong, Dong Yushen, Yang Xinming (1995) Semen Tritici aestivi and long fringe weir wheat straw, middle weir wheat straw hybrid and the cytogenetical study of derivative offspring thereof. Acta Genetica Sinica 22:217-222
The brief introduction of Yang Wenxiong (2005) New Spring Wheat Variety Longchun 23. agriculture of gansu science and technology: F002-F002.
Claims (5)
1. quick analysis and a multi-color fluorescence in situ hybridization method for qualification Semen Tritici aestivi exogenous chromosome, it comprises the steps:
1) N is passed through2O gas pretreatment vigorous the organizing of enzymolysis Helminthosporium sativum meristematic zone cell division obtain the division phases specimen that image is clear, Chromosome spread is good；
2) fluorescent probe of nick translation method labeling wheat race basal gene group is utilized；
3) utilize step 2) fluorescent probe that builds is to utilizing step 1) mitosis metaphase split coil method chromosome carry out fluorescence in situ hybridization；
4) analytical procedure 3) the fluorescence in situ hybridization result that obtains,
Wherein, step 2) in the Tribe Triticeae basal gene group of labelling be AA genome, DD genome, RR rye (Secale cereale L.) genome or Thinopyrum ponticum genome, or transgenic purpose fragment vector；And the labelling of described fluorescent probe is carried out by mixing the base combining fluorescein when synthesizing the complementary strand of described Tribe Triticeae basal gene group, wherein rye (Secale cereale L.), E. elongata and Urartu green fluorescent label AlexaFluor-488-dUTP labelling, Triticum tauschii red fluorescence is marked Texas-5-dCTP labelling；Step 3) in fluorescent probe used be RR rye (Secale cereale L.) genome, AA genome and the fluorescent probe of DD genome of labelling, or the Thinopyrum ponticum genome of labelling, AA genome and the fluorescent probe of DD genome, and the mixing quality ratio of the fluorescent probe of the AA genome of wherein said labelling and DD genome is no less than 1∶4；Further, step 3) in situ hybridization during blockade with BB genome, and it is 160-200 times for the mass ratio blockaded.
2. the method described in claim 1, wherein step 1) comprise the steps: (a) N2O gas pretreatment: take suitable Helminthosporium sativum, is placed in moistening centrifuge tube, puts into N2O air chamber processes 0.5-5 hour, N2The pressure of O is 10ATM (1.01Mpa)；B () is by N2The tip of a root of O gas pretreatment is fixed；C () enzymolysis processing: washed with water by the tip of a root fixed, cut off root tips separate living tissue, by pectase and cellulose treatment 30min-2h.
3. the method described in claim 1, wherein said AA genome is diploid Urartu AA genome, and described DD genome is diploid aegilops tauschii D D genome.
4. the method described in claim 1, wherein step 3) in situ hybridization during with diploid intend this inferior you torr BB genome blockade.
5. the method described in claim 1, wherein carries out repeating hybridization analysis to the chromosome of wheat specimen obtained, and the most often step repeats hybridization analysis eluting is with eluting under 2 × SSC room temperature.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310127485.6A CN103205500B (en) | 2013-04-12 | 2013-04-12 | A kind of quick analysis and the multi-color fluorescence in situ hybridization method of qualification Semen Tritici aestivi exogenous chromosome |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310127485.6A CN103205500B (en) | 2013-04-12 | 2013-04-12 | A kind of quick analysis and the multi-color fluorescence in situ hybridization method of qualification Semen Tritici aestivi exogenous chromosome |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103205500A CN103205500A (en) | 2013-07-17 |
| CN103205500B true CN103205500B (en) | 2016-08-03 |
Family
ID=48752904
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310127485.6A Active CN103205500B (en) | 2013-04-12 | 2013-04-12 | A kind of quick analysis and the multi-color fluorescence in situ hybridization method of qualification Semen Tritici aestivi exogenous chromosome |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103205500B (en) |
Families Citing this family (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103993095B (en) * | 2014-06-10 | 2016-02-10 | 中国科学院西北高原生物研究所 | A kind of Psathyrostachys plant cell chromosome division phases sample preparation method |
| CN105567860A (en) * | 2016-03-15 | 2016-05-11 | 南京晓庄学院 | Ammopiptanthus mongolicus chromosome fluorescence in-situ hybridization method |
| CN105648092A (en) * | 2016-03-15 | 2016-06-08 | 南京晓庄学院 | Method for fluorescence in situ hybridization of ammopiptanthus nanus chromosome |
| CN106350593A (en) * | 2016-09-19 | 2017-01-25 | 长江师范学院 | Dripping piece preparation method of plant chromosome |
| CN106399221A (en) * | 2016-09-19 | 2017-02-15 | 长江师范学院 | Microseparation method for monosome of plant |
| CN106932256A (en) * | 2017-01-22 | 2017-07-07 | 河南师范大学 | A kind of method for preparing asparagus root tip cell chromosome division phases sample |
| CN107488712A (en) * | 2017-08-07 | 2017-12-19 | 山东农业大学 | A kind of quick analysis and the polychrome in-situ hybridization method of detection Tribe Triticeae chromosome configuration |
| CN109825553B (en) * | 2019-03-06 | 2019-12-20 | 南京林业大学 | Method for identifying all chromosomes of poplar by using oligonucleotide probe |
| CN110358859B (en) * | 2019-07-26 | 2022-06-10 | 南京农业大学 | Oligonucleotide probe kit for detecting rye chromosome and use method thereof |
| CN112697556B (en) * | 2020-12-25 | 2024-02-23 | 四川省农业科学院园艺研究所 | Method for preparing citrus plant root tip chromosome tablet |
| CN112921074A (en) * | 2021-02-03 | 2021-06-08 | 鲲羽生物科技(江门)有限公司 | In-situ hybridization method for simultaneously detecting multiple loci of genome through combined colors |
| CN113699264B (en) * | 2021-08-12 | 2023-06-16 | 贵州大学 | Fluorescent in-situ hybridization probe for identifying common wheat B group chromosome and design method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102876801A (en) * | 2012-10-22 | 2013-01-16 | 中国科学院遗传与发育生物学研究所 | Method for identifying exogenous chromosomes and chromosome segments in plant distant hybrids |
-
2013
- 2013-04-12 CN CN201310127485.6A patent/CN103205500B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102876801A (en) * | 2012-10-22 | 2013-01-16 | 中国科学院遗传与发育生物学研究所 | Method for identifying exogenous chromosomes and chromosome segments in plant distant hybrids |
Non-Patent Citations (8)
| Title |
|---|
| 八倍体小偃麦远中2染色体组构成的研究;辛文利等;《哈尔滨师范大学自然科学学报》;19981231;第14卷(第2期);第80-85页 * |
| 基于小麦与黑麦1BL/1RS易位的小麦染色体易位研究;杨春玲等;《安徽农业科学》;20091231;第37卷(第4期);第1502-1504页 * |
| 基因组原位杂交中封阻DNA制备方法研究;顾爱侠等;《河北农业大学学报》;20100930;第33卷(第5期);第77-80页 * |
| 基因组原位杂交技术在植物研究中的应用;关鹤等;《分子植物育种》;20061231;第4卷(第3（S）期);第99-105页 * |
| 应用基因组原位杂交鉴定蓝粒小麦及其诱变后代;杨国华等;《遗传学报》;20021231;第29卷(第3期);第255-259页 * |
| 玉米与四倍体多年生玉米代换种质的选育及其基因组原位杂交鉴定;唐祈林等;《遗传学报》;20040430;第31卷(第4期);第340-344页 * |
| 麦类作物体细胞基因组原位杂交（GISH）效果影响因素的分析;林小虎等;《实验生物学报》;20050430;第38卷(第2期);第126-132页 * |
| 麦类作物基因组原位杂交技术要点分析;张延明等;《哈尔滨师范大学自然科学学报》;20041231;第20卷(第5期);第74-76页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103205500A (en) | 2013-07-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103205500B (en) | A kind of quick analysis and the multi-color fluorescence in situ hybridization method of qualification Semen Tritici aestivi exogenous chromosome | |
| CN113774161B (en) | KASP molecular marker of peanut pod and kernel size major QTL and application thereof | |
| CN103952402A (en) | SNP (single nucleotide polymorphism) site related to characters of plant root system and application thereof | |
| CN107475390B (en) | Development and application of decaploid elytrigia elongata series repeat sequence specific probe | |
| CN107400715B (en) | Development and application of special molecular marker and probe of decaploid elytrigia elongata | |
| CN103798132B (en) | A kind of method of scale initiative wheat alien translocation line | |
| CN108660247B (en) | Primer SmemboI-2 for identifying purity of solanum torvum third eggplant based on SNP marker and application | |
| CN1281751C (en) | DNA mark for inspecting purity of seeds and method thereof | |
| CN106755507B (en) | Molecular detection method for distinguishing chromosomes of cultivated rye and wild rye | |
| CN108676903B (en) | Primer SmemboI-1 for identifying purity of solanum torvum third eggplant based on SNP marker and application | |
| CN108004236B (en) | Corn stalk rot disease-resistant molecular breeding method and application thereof | |
| CN110878375A (en) | Specific molecular marker for detecting rice cold-resistant gene CTB4a and application thereof | |
| Li et al. | A set of sampling, preparation, and staining techniques for studying meiosis in cucumber | |
| CN113736907B (en) | SNP locus combination for detecting tomato gray leaf spot resistance and application thereof | |
| CN102747080B (en) | Molecular marker of pot shattering resistance trait major gene locus of rapes and application | |
| CN109234372B (en) | Direct PCR method and application thereof in cabbage hybrid genetic breeding | |
| CN111004857B (en) | Molecular marker primer of soybean branch number major QTL locus and application thereof | |
| CN113249514B (en) | Cucumber female line character related SCAR marker and application thereof | |
| CN111778348B (en) | Flight mass spectrum molecular marker Cap91 related to sweet pepper nuclear male sterility and application thereof | |
| CN113462807B (en) | SNP molecular marker and application thereof in improving barley salt tolerance | |
| CN114292949B (en) | Method for rapidly screening rose germplasm resources of rosa rosea resistant long tube aphid | |
| Adamus et al. | Experiments of backcrossing of interspecific F1 A. cepa× A. roylei hybrids with Allium cepa | |
| CN112662798B (en) | InDel molecular marker for identifying seed purity of muskmelon floral bovine variety and application thereof | |
| Li et al. | Molecular cytology identification of 22 sugarcane germplasm clones from Sri Lanka | |
| CN111004858B (en) | Molecular marker primer of soybean single pod number major QTL (quantitative trait locus) locus and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB03 | Change of inventor or designer information |
Inventor after: Han Fangpu Inventor after: Yuan Jing Inventor after: Shi Qinghua Inventor after: Wang Long Inventor before: Han Fangpu Inventor before: Lv Zhenling |
|
| COR | Change of bibliographic data | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |