CN109735602A - A kind of genomic in situ hybridization method and its application of tree peony - Google Patents
A kind of genomic in situ hybridization method and its application of tree peony Download PDFInfo
- Publication number
- CN109735602A CN109735602A CN201811610043.6A CN201811610043A CN109735602A CN 109735602 A CN109735602 A CN 109735602A CN 201811610043 A CN201811610043 A CN 201811610043A CN 109735602 A CN109735602 A CN 109735602A
- Authority
- CN
- China
- Prior art keywords
- dna
- chromosome
- genomic
- tree peony
- denaturation
- 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.)
- Pending
Links
- 240000005001 Paeonia suffruticosa Species 0.000 title claims abstract description 71
- 235000003889 Paeonia suffruticosa Nutrition 0.000 title claims abstract description 71
- 238000000034 method Methods 0.000 title claims abstract description 53
- 238000007903 genomic in situ hybridization Methods 0.000 title claims abstract description 42
- 210000000349 chromosome Anatomy 0.000 claims abstract description 126
- 238000004925 denaturation Methods 0.000 claims abstract description 55
- 230000036425 denaturation Effects 0.000 claims abstract description 55
- 238000009396 hybridization Methods 0.000 claims abstract description 44
- 239000007788 liquid Substances 0.000 claims abstract description 32
- 238000012545 processing Methods 0.000 claims abstract description 30
- 108020003215 DNA Probes Proteins 0.000 claims abstract description 20
- 239000003298 DNA probe Substances 0.000 claims abstract description 20
- 238000013459 approach Methods 0.000 claims abstract description 7
- 230000002759 chromosomal effect Effects 0.000 claims abstract description 5
- 238000012252 genetic analysis Methods 0.000 claims abstract description 4
- 239000000523 sample Substances 0.000 claims description 56
- 238000002360 preparation method Methods 0.000 claims description 27
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 claims description 26
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 19
- 239000000203 mixture Substances 0.000 claims description 16
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 14
- 238000009835 boiling Methods 0.000 claims description 12
- 238000013519 translation Methods 0.000 claims description 11
- 108090000790 Enzymes Proteins 0.000 claims description 10
- 102000004190 Enzymes Human genes 0.000 claims description 10
- 238000001514 detection method Methods 0.000 claims description 10
- 238000010828 elution Methods 0.000 claims description 10
- 229940088598 enzyme Drugs 0.000 claims description 10
- 102000057297 Pepsin A Human genes 0.000 claims description 8
- 108090000284 Pepsin A Proteins 0.000 claims description 8
- 229940111202 pepsin Drugs 0.000 claims description 8
- 235000019441 ethanol Nutrition 0.000 claims description 7
- 235000011194 food seasoning agent Nutrition 0.000 claims description 7
- 238000002156 mixing Methods 0.000 claims description 6
- 210000001519 tissue Anatomy 0.000 claims description 6
- GDTBXPJZTBHREO-UHFFFAOYSA-N bromine Substances BrBr GDTBXPJZTBHREO-UHFFFAOYSA-N 0.000 claims description 5
- 229910052794 bromium Inorganic materials 0.000 claims description 5
- OOYGSFOGFJDDHP-KMCOLRRFSA-N kanamycin A sulfate Chemical group OS(O)(=O)=O.O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N OOYGSFOGFJDDHP-KMCOLRRFSA-N 0.000 claims description 5
- 108010059892 Cellulase Proteins 0.000 claims description 4
- 229940106157 cellulase Drugs 0.000 claims description 4
- 239000003795 chemical substances by application Substances 0.000 claims description 3
- 230000008569 process Effects 0.000 claims description 3
- 108091092562 ribozyme Proteins 0.000 claims description 3
- 238000001816 cooling Methods 0.000 claims description 2
- 239000003550 marker Substances 0.000 claims description 2
- 238000007781 pre-processing Methods 0.000 claims description 2
- 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 claims 1
- QONQRTHLHBTMGP-UHFFFAOYSA-N digitoxigenin Natural products CC12CCC(C3(CCC(O)CC3CC3)C)C3C11OC1CC2C1=CC(=O)OC1 QONQRTHLHBTMGP-UHFFFAOYSA-N 0.000 claims 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 claims 1
- 239000000941 radioactive substance Substances 0.000 claims 1
- 238000009402 cross-breeding Methods 0.000 abstract description 5
- 108020004414 DNA Proteins 0.000 description 69
- 239000000243 solution Substances 0.000 description 17
- 230000000694 effects Effects 0.000 description 12
- 239000000463 material Substances 0.000 description 12
- 238000011160 research Methods 0.000 description 12
- 241000736199 Paeonia Species 0.000 description 11
- 239000003153 chemical reaction reagent Substances 0.000 description 11
- 238000005516 engineering process Methods 0.000 description 10
- 238000007901 in situ hybridization Methods 0.000 description 10
- 240000003693 Docynia delavayi Species 0.000 description 9
- 239000012153 distilled water Substances 0.000 description 9
- 241000894007 species Species 0.000 description 9
- 241000196324 Embryophyta Species 0.000 description 8
- 229920001213 Polysorbate 20 Polymers 0.000 description 8
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 7
- 230000000052 comparative effect Effects 0.000 description 6
- 239000000843 powder Substances 0.000 description 6
- 235000006484 Paeonia officinalis Nutrition 0.000 description 5
- 102000006382 Ribonucleases Human genes 0.000 description 5
- 108010083644 Ribonucleases Proteins 0.000 description 5
- 229960004756 ethanol Drugs 0.000 description 5
- 239000000284 extract Substances 0.000 description 5
- 238000012216 screening Methods 0.000 description 5
- 238000005406 washing Methods 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 238000009395 breeding Methods 0.000 description 4
- 230000001488 breeding effect Effects 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- 230000007071 enzymatic hydrolysis Effects 0.000 description 4
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 3
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 238000011065 in-situ storage Methods 0.000 description 3
- 230000008774 maternal effect Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 3
- 238000004321 preservation Methods 0.000 description 3
- 239000000376 reactant Substances 0.000 description 3
- 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 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 2
- 235000007516 Chrysanthemum Nutrition 0.000 description 2
- 244000189548 Chrysanthemum x morifolium Species 0.000 description 2
- 102000016911 Deoxyribonucleases Human genes 0.000 description 2
- 108010053770 Deoxyribonucleases Proteins 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
- 229930040373 Paraformaldehyde Natural products 0.000 description 2
- 241001529246 Platymiscium Species 0.000 description 2
- 244000061458 Solanum melongena Species 0.000 description 2
- 150000001412 amines Chemical class 0.000 description 2
- 210000001726 chromosome structure Anatomy 0.000 description 2
- 238000009833 condensation Methods 0.000 description 2
- 230000005494 condensation Effects 0.000 description 2
- 238000002242 deionisation method Methods 0.000 description 2
- 229960000633 dextran sulfate Drugs 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
- 238000004043 dyeing Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 125000002485 formyl group Chemical group [H]C(*)=O 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 210000001672 ovary Anatomy 0.000 description 2
- 229920002866 paraformaldehyde Polymers 0.000 description 2
- 108700022487 rRNA Genes Proteins 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- OCJBOOLMMGQPQU-UHFFFAOYSA-N 1,4-dichlorobenzene Chemical compound ClC1=CC=C(Cl)C=C1 OCJBOOLMMGQPQU-UHFFFAOYSA-N 0.000 description 1
- 241000205585 Aquilegia canadensis Species 0.000 description 1
- 208000002109 Argyria Diseases 0.000 description 1
- WKBOTKDWSSQWDR-UHFFFAOYSA-N Bromine atom Chemical compound [Br] WKBOTKDWSSQWDR-UHFFFAOYSA-N 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 235000016623 Fragaria vesca Nutrition 0.000 description 1
- 240000009088 Fragaria x ananassa Species 0.000 description 1
- 235000011363 Fragaria x ananassa Nutrition 0.000 description 1
- 108010034791 Heterochromatin Proteins 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- 108010076876 Keratins Proteins 0.000 description 1
- 102000011782 Keratins Human genes 0.000 description 1
- 241000234435 Lilium Species 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 229960000583 acetic acid Drugs 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 125000001246 bromo group Chemical class Br* 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 230000032823 cell division Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 210000002230 centromere Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000013611 chromosomal DNA Substances 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 238000013329 compounding Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- 230000002559 cytogenic effect Effects 0.000 description 1
- 230000003412 degenerative effect Effects 0.000 description 1
- 229960000935 dehydrated alcohol Drugs 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000010413 gardening Methods 0.000 description 1
- 238000012214 genetic breeding Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000012362 glacial acetic acid Substances 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 210000004458 heterochromatin Anatomy 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000016507 interphase Effects 0.000 description 1
- 238000009403 interspecific hybridization Methods 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 230000011278 mitosis Effects 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 150000007523 nucleic acids Chemical group 0.000 description 1
- 210000004940 nucleus Anatomy 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 210000000745 plant chromosome Anatomy 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000003252 repetitive effect Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- 238000010025 steaming Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 1
- 229940038773 trisodium citrate Drugs 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to a kind of genomic in situ hybridization method and its application of tree peony.The present invention provides a kind of genomic in situ hybridization method of tree peony, uses three-step approach to carry out denaturation treatment with the hybrid mixed liquid and chromosome to be detected for blockading DNA to comprising DNA probe, by the hybrid mixed liquid and chromosomal hybridation after denaturation；After hybrid mixed liquid and chromosome is are carried out denaturation treatment respectively by the three-step approach, then hybrid mixed liquid and chromosome are mixed and carry out co-variation processing.The present invention is comprehensively adjusted and is optimized by the conditional parameter to genomic in situ hybridization, efficient tree peony genomic in situ hybridization method is obtained, it hybridizes success rate and specificity significantly improves, hybridization signal has higher clarity on chromosome, the chromosome from hybridization parents' sheet can be accurately distinguished, the Germplasm Identification of tree peony and the genetic analysis of crossbreeding can be used in practice.
Description
Technical field
The present invention relates to gene engineering technology fields, and in particular to a kind of genomic in situ hybridization method of tree peony and its answers
With.
Background technique
Tree peony belongs to Paeoniaceae Paeonia sect. Moutan, for China traditional famous flower, growth condition it is with a long history, have compared with
High ornamental and application value.By more than 1600 years artificially breedings, tree peony formd numerous excellent variety.Wherein between subgroup
Distant hybridization has played extremely important effect during breed of variety, currently, peony association, the U.S. is listed male
Red subgroup intermolecular hybrid kind has 426.But the genetic constitution for the successful mechanism of distant hybridization between tree peony subgroup, distant hybrid
Equal researchs also extremely lack.Research between distant hybrid progeny chromosome tree peony subgroup is conducive to disclose the miscellaneous of hybrid generation
Mechanism is handed over, provides certain guidance for the distant hybridization breeding of tree peony.
The identification of chromosome is the element task of chromosome research.The identification of chromosome refers to will not by certain label
Same chromosomal region separates, including genome, individual chromosome and chromosome segment, this is always cytogenetical study
Important content (Kato et al., 2005).
Pressed disc method opens one of the conventional means of the beginning of chromosome karyotype analysis and always karyotyping
(Belling, 1921).Traditional karyotyping is with the basic measurement data of chromosome such as relative length, centromere positions and arm
Ratio etc. is the foundation of chromosome identification, and important role is played in initial research.About Paeonia sect. Moutan plant
Karyotyping, existing forefathers did a large amount of work, and were related to all wild species of tree peony and multiple kinds of Cultivated tree peonies, are
Theoretical basis has been established in this group of plant cytology research, is worked determining wild species classification position, Evolvement and genetic breeding
Deng with important value.(Stebbins, 1938；Li Maoxue etc., 1982；Wang Lianying etc., 1983；Yu Zhaoying etc., 1987；?
Praise equality, 1988；Hong Deyuan etc., 1988；Gong Xun etc., 1991).Traditional karyotyping is for chromosome similar in form or area
The differentiation of Duan Bianyi lacks accuracy, it is therefore desirable to can further display the technology of the internal structure of chromosome to be dyed
Body accurately identifies.
Chromosome banding technique can show the lines of chromosome by certain technology, carry out the knowledge of chromosome internal structure
Not, if chromosome structure morphs, band line can be used to distinguish each chromosome and Preliminary Identification dye it can also happen that variation
The variation of colour solid is a turning point (Casperson et of the chromosome identification from formalness research internally structural research
Al., 1968).Chromosome banding technique includes a variety of band analysis methods such as C band, G band, argentation, N band, R band, T band.Separately
Outside, high resolution banding keeps Chromosomes Banding thin, even more up to 800 with line, and chromosome identification is more accurate, is facing
Bed medicine and plant chromosome identification etc. all have application (Yunis, 1976).Chromosome banding technique also has one in tree peony
The analysis result of fixed application, C band and silver staining band etc. can be used as identify kind and the foundation of kind (open and praise equality, 1990；Yu Ling
Deng 1997；Pei Yanlong, 1993).
Smaller for some chromosomes, species similar in concentrating degree height and form, chromosome is after banding technique is handled
It is limited that the band line with line or generation accordingly cannot be generated, therefore be still difficult to accurately identify all chromosome.Chromosomal in situ
The fluorescence signal site that hybridization technique (in situ hybridization) can be generated by the combination of probe and target DNA carries out
Chromosome accurately identifies.This method uses the nucleic acid sequence of label as probe, is hybridized with the target DNA on chromosome,
Since probe forms in different chromosomes, is distributed, the difference of position and repeat number, accurately identifying for chromosome can be carried out, is answered
With relatively broad.
Hybridization in situ technique is divided into fluorescence in situ hybridization (Fluorescence in situ hybridization, FISH)
With genomic in situ hybridization (Genome in situ hybridization, GISH).In plant FISH experiment, using most
Probe be clone rDNA and other main tandem repetitive sequences.According to the presence or absence of hybridization signal, quantity and position
Difference, all or part of chromosome of many plants accurately identified, and accurate caryogram is thereby established.FISH technology exists
Have application in Chinese herbaceous peony group Chinese herbaceous peony platymiscium, according to its distribution of signal site on chromosome, can recognize that chromosome structure becomes
Different (Zhang et al., 1998；), carry out positioning, the building research of rRNA gene physical map of rDNA on chromosome
(Zhang et al., 1999), the hybridization signal of rRNA gene and kernel number research in SAT- chromosome, interphase nucleus
(Uchino et al., 2000), and the research such as the site rRNA positioning between different populations in planting (Luo et al., 2006), to Chinese herbaceous peony
The Study on Evolution of medicine platymiscium has biggish meaning.The hybridization signal of FISH technology is the site on chromosome, and needs to carry out
The screening of probe with separate, carry out distant hybrid progeny in cannot easily distinguish the dyeing from different parental gene groups
Body.The hybridization that whole chromosome is carried out using total genomic dna as probe, can recognize plant hybrid, allopolyploid and recombination
Breeding lines in separate sources chromosome and chromosome segment, this technology is genomic in situ hybridization technology
(GISH)。
GISH technology uses the total genomic dna from a species as label probe, with total gene of another species
Group DNA is blockaded with debita spissitudo, and in situ hybridization, DNA probe and child chromosome group are carried out in child chromosome
Chromosomal hybridation derived from the species shows hybridization signal, so that the genome of separate sources be distinguished.This technology
It is simple and easy to do, do not have to separation probe, the total genomic dna that need to only extract plant is marked as probe, can be with whole dyeing
Body hybridization, and hybridization site can be observed in cell division any period, can quickly and accurately carry out target chromosome
The detection of (segment) and genome, the identification and the Origin of Species for being widely used in plant heterochromatin are developed in research, in wind
Hybrid generation's chromosome identification etc. is carried out in the gardening plants such as son, composite family, strawberry, honeysuckle, lily is applied (Abd El-
Twab&Kondo, 2004；Barba-Gonzalez et al., 2006；Budylin et al., 2014；Jang et al.,
2015；Miyashita&Hoshino, 2015；Liu et al., 2016).The genome of tree peony larger (13-16Gb) and heterozygosis
Property it is high so that application of the GISH detection method in tree peony is restricted, still applied in tree peony without GISH technology at present
Report.
Summary of the invention
The technical issues of to solve in the prior art, the purpose of the present invention is to provide a kind of genome of tree peony is miscellaneous in situ
Friendship method, for the mode of inheritance of the parental chromosomes during precise Identification tree peony interspecific hybridization and its in offspring chromosome group
In composition.
The present invention provides a kind of genomic in situ hybridization method of tree peony, uses three-step approach to comprising DNA probe and envelope
Hinder DNA hybrid mixed liquid and chromosome to be detected carry out denaturation treatment, by after denaturation hybrid mixed liquid and chromosome it is miscellaneous
It hands over；
After the hybrid mixed liquid and the chromosome to be detected is are carried out denaturation treatment respectively by the three-step approach, then will
Hybrid mixed liquid and chromosome mixing after denaturation, carry out total denaturation treatment.It is denaturalized respectively using chromosome and hybrid mixed liquid
The three-step approach degenerative process of co-variation again afterwards can guarantee DNA probe, blockade DNA and the double-strand of chromosomal DNA is sufficiently opened,
It creates favorable conditions for hybridization, substantially increases the success rate and specificity of hybridization.
Wherein, the denaturation of the hybrid mixed liquid is by hybrid mixed liquid in 95~98 DEG C of 10~15min of denaturation；
The denaturation of the chromosome to be detected is 70~75 DEG C of water-baths in formamide solution by chromosome slide to be detected
It is denaturalized 3~5min；
The co-variation is that will mix through the hybrid mixed liquid of denaturation treatment with chromosome to be detected, is denaturalized in 80~85 DEG C
10~15min.
Blockading DNA can be by the non-specific hybridization site (homologous sequence) in hybridization blocker chromosome, in tree peony
In genomic in situ hybridization, excess in a certain range of the DNA relative to DNA probe is blockaded, is advantageously implemented and more preferably seals
Effect is hindered, guarantees the specificity of hybridization.
In the present invention, the hybrid mixed liquid middle probe DNA is 1:10~1:50 with the mass ratio for blockading DNA.
Preferably, the hybrid mixed liquid middle probe DNA and the mass ratio for blockading DNA are 1:25~1:40.
Further, suitable DNA probe and blockade DNA clip size and preparation condition can guarantee hybridization spy
Anisotropic and success rate.
In the present invention, the clip size of the DNA probe is 200~500bp；
The label of the DNA probe is using radioactivity or the label of on-radiation；It is preferred that being marked using digoxin.
Preferably, DNA probe of the present invention is prepared as the total genomic dna and DIG-Nick of tree peony
Translation Mix mixing, is protected from light 60~70min in 15 DEG C.
Specifically, the dosage of the DIG-Nick Translation Mix is 4~6 μ L/ μ g total genomic dnas.
In the present invention, the clip size for blockading DNA is 200~500bp.
Preferably, it is described blockade DNA be prepared as boiling the total genomic dna of tree peony in boiling water bath processing 150~
300min。
Genomic in situ hybridization method provided by the invention can be with the mitosis of distant hybrid progeny between tree peony subgroup
The chromosome of mid-term be target to be measured, using the complete genome DNA of a parent species as DNA probe, another parent species it is complete
Genomic DNA is to blockade DNA, carries out genomic in situ hybridization.
Preferably, in the present invention, the genome blockaded DNA and derive from the tree peony of the same race with tree peony hybrid parent to be measured
DNA。
Tree peony genomic in situ hybridization method of the present invention includes the following steps:
(1) preparation and pretreatment of chromosome slide sample: the tissue of tree peony to be detected is pre-processed, is fixed and enzyme
Xie Hou using the slide for going the hypotonic flame seasoning of wall to prepare tree peony chromosome to be measured, then digest to chromosome slide pre-
Processing；
(2) probe marks and blockades DNA preparation: by the total genomic dna of tree peony and DIG-Nick Translation
Mix mixing, is protected from light 60~70min in 15 DEG C, obtains DNA probe；
The total genomic dna of tree peony is boiled into 150~300min of processing in boiling water bath, obtains blockading DNA；
(3) denaturation of chromosome and probe: by the pretreated chromosome slide of step (1) preparation in 70% formyl
70~75 DEG C of 3~5min of denaturation, are dehydrated step by step in the ethyl alcohol of pre-cooling, dry in amine；
It will be comprising DNA probe and blockading the hybrid mixed liquid of DNA in 95~98 DEG C of 10~15min of denaturation；
Hybrid mixed liquid through denaturation treatment is added to the chromosome slide through denaturation treatment, is denaturalized in 80~85 DEG C
10min；
(4) hybridize
The hybrid mixed liquid through denaturation treatment that step (3) is obtained hybridizes with the mixture of chromosome to be detected in 37 DEG C
12-16h；
(5) elution and signal detection: the hybrid product that step (4) obtains adds fluorescent marker after elution, Seal treatment
Anti digoxin antibody processing, after washed, add anti-fluorescence decay agent, mounting.
Wherein, elution described in above-mentioned steps (5) includes miscellaneous using 30%~50% deionized formamide solution elution
The step of handing over product.
Wherein, the preparation and pretreatment of above-mentioned steps (1) the chromosome slide sample, specifically comprises the following steps:
1. using paracide-α-bromine, how mixed liquor pre-processes the tissue of tree peony to be detected, 4 DEG C of processing 10~
After 12h, fixed using Kano fixer；
2. the mixed enzyme solution comprising 2% cellulase and 0.5% pectase is added, after 37 DEG C of 70~90min of enzymolysis processing,
Using going the hypotonic flame seasoning film-making of wall；
3. RNA enzyme is added, 37 DEG C of 1~2h of processing；After washed, pepsin, 37 DEG C of 30~40min of processing are added.
Above-mentioned steps 1. in, through the fixed peony tissue to be detected of Kano fixer can in 70% alcohol it is -20 DEG C long-term
It saves.
Above-mentioned steps are 2. specifically: the mixed enzyme solution comprising 2% cellulase and 0.5% pectase, 37 DEG C of enzymatic hydrolysis are added
70~90min；Distilled water washes away enzyme solution, and using the hypotonic flame seasoning film-making of wall is gone, it is good to select Chromosome spread under microscope
Good chromosome slide.
Above-mentioned steps are 3. specifically: and the chromosome slide for 2. selecting step is after 60 DEG C of drying 12h, addition RNA enzyme, and 37
DEG C processing 1~2h；After washed, pepsin, 37 DEG C of 30~40min of processing are added；After washed, 4% deionization first is added
Amide, the fixed 10min of room temperature, prevents chromosome from falling off.
Further, the present invention provides the genomic in situ hybridization method and hybridizes in the Germplasm Identification of tree peony and tree peony
Application in the genetic analysis of breeding.
The beneficial effects of the present invention are,
The present invention is comprehensively adjusted and is optimized by the conditional parameter to genomic in situ hybridization, has been obtained efficient
Tree peony genomic in situ hybridization method carries out tree peony genomic in situ hybridization using method provided by the invention, and success rate is significant
It improves, and the specificity hybridized significantly improves, hybridization signal has higher clarity on chromosome, can accurately distinguish and
From in the chromosome of hybridization parents' sheet, it can be used for the Germplasm Identification of tree peony and the genetic analysis of crossbreeding in practice.
Detailed description of the invention
Fig. 1 is the tree peony genomic in situ hybridization result picture of the embodiment of the present invention 2, wherein A is chromosome mid-term figure；B
For DIG hybridization signal effect picture；C is idiogram.
Fig. 2 is the tree peony genomic in situ hybridization result picture of the embodiment of the present invention 3, wherein A is chromosome mid-term figure；B
For DIG hybridization signal effect picture；C is idiogram.
Fig. 3 is the tree peony genomic in situ hybridization result picture of the embodiment of the present invention 4, wherein A is chromosome mid-term figure；B
For DIG hybridization signal effect picture；C is idiogram.
Specific embodiment
The preferred embodiment of the present invention is described in detail below in conjunction with embodiment.It will be appreciated that following real
Providing merely to play the purpose of explanation for example is applied, is not used to limit the scope of the present invention.The skill of this field
Art personnel without departing from the spirit and purpose of the present invention, can carry out various modifications and replace to the present invention.
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
The material source of tree peony to be measured is in the tender son of children of Paeonia × lemoinei ' High Noon ' in following embodiments
Room, ' High Noon ' Distant hybridization variety between tree peony subgroup is P.delavayi (chrysanthemum) and P. × suffruticosa
(P.delavayi is meat floral disc subgroup to filial generation, and P. × suffruticosa is keratin floral disc subgroup.)；Maternal DNA material
Material derives from P.delavayi (chrysanthemum)；Male parent DNA material source is in two kind ' Luoyang of P. × suffruticosa
Hong ' and ' Hoki '.
Various reagents formula used in following embodiments and the preparation method is as follows:
(1) preparation of detection reagent
Anti-DIG-Rhodamine: 200 μ g of freeze-dried powder is dissolved in 1ml ddH2In O, multigelation is avoided, is distributed into 10
Part, it is kept in dark place 2 months in 4 DEG C, it can also -20 DEG C of preservations.
(2) preparation of closed reagent
BSA dry powder 5g is weighed, (effect is more preferable) is prepared with 100ml distilled water or 4 × SSC-Tween 20 and dispenses, -20 DEG C
It saves.
(3) compounding medicine in hybridization solution
50%DS (dextran sulfate): it weighs 0.5g dry powder and is dissolved in the sterile ddH of 1ml2In O (65 DEG C of dissolutions), dress up small
Pipe, -20 DEG C of preservations.
10%SDS (dodecyl sodium sulfate): weighing 2g SDS, is dissolved in the sterile ddH of 18ml2In O, hydrotropy is heated, is added
Dense HCl, adjusting pH is 7.2, and water is added to be settled to 20ml packing.
(4) buffer
10 × PBS: NaCl 80g, KCl 2.0g, Na are weighed2HPO4 14.4g、KH2PO42.4g adds distilled water to dissolve, and adjusts
PH 7.5 is saved, 1000ml, high-temperature sterilization are settled to.
20 × SSC: weighing trisodium citrate 88.4g and be dissolved in distilled water, and NaCl 175.4g is added after being filtered with filter paper,
PH 7.0 is adjusted, 1000ml, high-temperature sterilization are settled to.
1 × PBS:10 × PBS and distilled water ratio are 1:9 preparation.
2 × SSC:20 × SSC and distilled water ratio are 1:9 preparation.
4 × SSC:20 × SSC and distilled water ratio are 1:4 preparation.
(5) pretreating reagent
100mg/ μ l RNase solution A: 10mg powder is dissolved in 1ml ddH2Be made into stoste in O, when use by stoste in
2 × SSC is diluted use according to the proportion of 1:99.
1% pepsin solution: 10mg powder is dissolved in 1ml ddH by 10mg powder2It is made into stoste in O, when use will
Stoste is diluted use according to the proportion of 1:99 in 2 × SSC.
Paracide-α bromine how mixed liquor: weigh paracide crystallization about 20g in brown reagent bottle, 45 DEG C of steaming be added
How simultaneously distilled water 500ml shakes about 3min, after being stored at room temperature overnight, 2~3 drop α bromines shake well is added, can be used after standing,
Room temperature preservation.
The preparation of (6) 4 × SSC-Tween20 solution
In every 4 × SSC of 100ml solution plus 0.2ml Tween 20, in other solution for needing to be added Tween20
The ratio of Tween20 is same as above.
(7) operation instructions of DIG-Nick Translation Mix (No.11745816910)
Specific steps: it takes 1 μ g total genomic dna to add distilled water to 16 μ l, 4 μ l DIG-Nick Translation is added
Mix, of short duration centrifugation after mixing, is protected from light in 15 DEG C, and the detection of probe fragment size is carried out in reaction process.(by reactant
It is placed on ice, takes 3 μ l reactants that loading buffer is added, after 95 DEG C of denaturation 3min, be placed in 3min on ice, 1.5%
Agarose gel electrophoresis, probe length should between 200~500bp, if length be greater than 500bp, can be by reactant in 15
DEG C again detection probe length again after incubating.) after probe length reaches requirement, 1 μ l 0.5M is added in every 20 μ l system
EDTA (pH8.0) terminates reaction in 65 DEG C of reaction 10min.
The conditional filtering of 1 tree peony genomic in situ hybridization of embodiment
To obtain preferable results of hybridization, the present embodiment is respectively to the preparation of chromosome slide sample, chromosome slide mark
This pretreatment, DNA extract, probe label and blockades the condition of elution step after DNA preparation and hybridization and carries out screening and excellent
Change, determines the optimum condition parameter of above-mentioned steps.
1, the preparation of chromosome slide sample
(1) using paracide-α-bromine, how mixed liquor carries out in advance the young tender ovary of P. × lemoinei ' High Noon '
Processing, 4 DEG C of pretreatments 0~for 24 hours, 4 DEG C of fixations for 24 hours, screen suitable pretreated duration in the fixer of Kano.The result shows that
Pre-process link in, the convenient time be 10-12h, be lower than 10h, chromosome condensation is longer, be higher than 12h chromosome condensation compared with
It is short, and be easy to generate toxic action to cell, it is unfavorable for film-making.
(2) enzymatic hydrolysis film-making is carried out to tree peony material, mixed enzyme solution (2% is added in the material previously treated that step (1) obtains
Cellulase and 0.5% pectase), under conditions of 37 DEG C, pH 4.0-4.5,50~100min of enzymatic hydrolysis, screen suitable enzyme
Solve the time.The result shows that convenient enzymolysis time is 70-90min, is lower than 70min, and dissociation is not thorough, high in enzymolysis step
In 90min, tissue is excessively loose, is unfavorable for the elution of enzyme solution, and be easily lost a large amount of cells.
(3) using the slide for going the hypotonic flame seasoning of wall to prepare tree peony chromosome to be measured.
2, the pretreatment of chromosome slide sample
The processing of RNase and pepsin are carried out to the sufficiently dry chromosome slide that above-mentioned steps 1 obtain.It is added dropwise not
RNase (100mg/ μ l) containing DNase, 37 DEG C of processing 30min~120min, is washed 3 times, each 5min with 2 × SSC；It is added dropwise
1% pepsin, 37 DEG C of processing 10~40min, 1 × PBS are washed 2 times, each 5min.It is transferred in 4% paraformaldehyde fixed
10min.2 × SSC is washed 3 times, each 5min.The result shows that RNase processing 1h, pepsin 30min after stain colour solid
Background is cleaner, be lower than this duration, there are cell residue object influence subsequent experimental, be higher than this duration, be easy to cause chromosome from
It falls off on slide.
3, DNA is extracted, probe marks and blockade DNA preparation
DNA is extracted: the blade or bud of the parent tree peony of Paeonia × lemoinei ' High Noon ' is taken, using CTAB method
(Ai Delai Plant Genome rapidly extracting kit, DN14) extracts total genomic dna, with total gene of maternal P.delavayi
For group DNA as probe material, the total genomic dna of male parent P. × suffruticosa, which is used as, blockades material.
Probe label: it is marked using DIG-Nick Translation Mix (Roche, No.11745816910)
The total genomic dna of P.delavayi.It is protected from light 30-120min in 15 DEG C, carries out screening (the screening ladder of probe label time
Degree is 10min).The detection that probe fragment size is carried out in reaction process controls probe length between 200~500bp.
The result shows that probe marks the time in 60-70min, probe length is between 200~500bp；Lower than at this time
Long, probe length is greater than 500bp, is higher than this duration, and probe length is less than 200bp, is unfavorable for hybridizing.
Blockade the preparation of DNA: by P. × suffruticosa ' Luoyang Hong ' and P. × suffruticosa
The total genomic dna of ' Hoki ' carries out the sieve for blockading DNA preparation time respectively at boiling 30~300min of processing in boiling water bath
Choosing, screening gradient are 30min, and the detection of probe fragment size is carried out in reaction process, control blockade DNA length 200~
Between 500bp.
The result shows that blockading preparation time in 150-300min, DNA length is blockaded between 200~500bp；It is lower than
150min, the length for blockading DNA are integrally greater than 500bp, are higher than 300min, and the length for blockading DNA is integrally less than 200bp, not
Conducive to hybridization.
4, it is eluted after hybridizing
Slide is taken out after hybridization, cover plate is removed with 2 × SSC drift, in 20%~50% deionized formamide solution (0.1
× SSC prepare) in 42 DEG C wash 2 times, each 5min；It is washed 2 times with 0.1 × SSC in 42 DEG C, each 5min；With 2 × SSC
In 42 DEG C of washing 5min.In 4 × SSC-Tween 20, room temperature washing 5min.Screen the deionization formyl for eluting the first step
The concentration of amine aqueous solution, the results showed that, it can be obtained preferably using the slide after the washing hybridization of 30%-50% deionized formamide
It elutes effect to be not easy to elute probe and blockade DNA when the solution concentration of deionized formamide is lower than 30%, when being higher than 50% pair
The signal hybridized can have an impact.
Embodiment 2
The present embodiment provides a kind of genomic in situ hybridization methods of tree peony, the specific steps are as follows:
(1) preparation of chromosome slide sample:
Using paracide-α-bromine, how mixed liquor locates the young tender ovary of P. × lemoinei ' High Noon ' in advance
Reason, so that the chromosome of material shortens and accumulate more mid-term phase cells, 4 DEG C of processing 10h；
It is added Kano fixer (dehydrated alcohol: glacial acetic acid=3:1), for 24 hours, mixed enzyme solution (2% fiber is added in 4 DEG C of fixations
Plain enzyme and 0.5% pectase, pH 4.0-4.5), 37 DEG C of enzymatic hydrolysis 70min；It is to be measured male using going the hypotonic flame seasoning of wall to prepare
The slide of red chromosome.
(2) pretreatment of chromosome slide sample
The RNase (100mg/ μ l) for being free of DNase is added dropwise on the sufficiently dry chromosome slide that step (1) obtains,
37 DEG C of processing 1h, are washed 3 times, each 5min with 2 × SSC；1% pepsin, 37 DEG C of processing 30min, 1 × PBS washings 2 are added dropwise
It is secondary, each 5min.It is transferred in 4% paraformaldehyde and fixes 10min.2 × SSC is washed 3 times, each 5min.
(3) probe marks and blockades DNA preparation
CTAB method extracts the total genomic dna of the parent of Paeonia × lemoinei ' High Noon '.
Using the total genomic dna of DIG-Nick Translation Mix label P.delavayi, label reaction is 15
DEG C it is protected from light 60min, probe length is between 200~500bp.
By the total genomic dna of P. × suffruticosa ' Luoyang Hong ' respectively at boiling processing in boiling water bath
150min blockades DNA length between 200~500bp.
(4) denaturation of chromosome and probe
Pretreated chromosome slide prepared by step 2 75 DEG C of water-bath denaturation in 70% deionized formamide
3min is dehydrated 3min in 70%, 90%, 100% ethyl alcohol that -20 DEG C are pre-chilled step by step, is air-dried.
Sequentially add reagent shown in table 1, preparing hybrid mixed liquor, wherein the dosage for blockading DNA is 2 μ l, mixed in vortex
It is mixed in clutch, is placed in 95 DEG C of denaturation 10min in PCR instrument.
By above-mentioned hybrid mixed drop on the chromosome slide through denaturation treatment, piece, 85 DEG C of denaturation 10min are covered.
1 hybrid mixed liquid ingredient of table and concentration
(5) hybridize
Hybrid mixed liquid after co-variation is hybridized into 12h under dark condition in 37 DEG C of incubators with chromosome slide.
(6) elution and signal detection
Slide is taken out after hybridization, removes cover plate with 2 × SSC drift, (0.1 × SSC matches in 30% deionized formamide solution
System) in 42 DEG C wash 2 times, each 5min.It is washed 2 times with 0.1 × SSC in 42 DEG C, each 5min.It is washed with 2 × SSC in 42 DEG C
Wash 5min.In 4 × SSC-Tween 20, room temperature washing 5min.
5%BSA (4 × SSC-Tween20 preparation) confining liquid of 40 μ l is added dropwise on slide glass, 37 DEG C of processing 30min are added
The Anti-DIG-Rhodamine antibody (2 μ g/ml are dissolved in the PBS buffer solution containing 5%BSA) of 30 μ l, 37 DEG C of Incubation in dark 1h.With
1 × PBS-Tween 20,1 × PBS are washed 3 times respectively at 37 DEG C, each 5min.The anti-fluorescence decay agent containing DAPI is added dropwise
Vectashield mounting, in Olympus BX-51 progress fluorescence microscope, (chromosome of tree peony is multiple with DAPI after being protected from light
Contaminate it is blue, hybridization signal be in aubergine), taken pictures using Cytovision software, Photoshop CS6 software carries out figure
As processing.Result using the genomic in situ hybridization of the method progress of the present embodiment is as shown in Figure 1.
Embodiment 3
The present embodiment provides a kind of in-situ hybridization method of tree peony genome, the difference with the method for embodiment 2 is only that
The source that step (3) used blockade DNA is different, and step (3) is specific as follows:
(3) probe marks and blockades DNA preparation
CTAB method extracts the total genomic dna of the parent of Paeonia × lemoinei ' High Noon '.
Using the total genomic dna of DIG-Nick Translation Mix label P.delavayi, label reaction is 15
DEG C it is protected from light 60min, probe length is between 200~500bp.
By the total genomic dna of P. × suffruticosa ' Hoki ' respectively at boiling processing 150min in boiling water bath, seal
DNA length is hindered between 200~500bp.
Result using the tree peony genomic in situ hybridization to be measured of the progress of method described in the present embodiment is as shown in Figure 2.
Embodiment 4
The present embodiment provides a kind of in-situ hybridization method of tree peony genome, the difference with the method for embodiment 2 is only that
Step (4) is different, and step (4) is specific as follows:
Pretreated chromosome slide prepared by step 2 75 DEG C of water-bath denaturation in 70% deionized formamide
3min is dehydrated 3min in 70%, 90%, 100% ethyl alcohol that -20 DEG C are pre-chilled step by step, is air-dried.
Sequentially add reagent shown in table 1, preparing hybrid mixed liquor, wherein the dosage for blockading DNA is 3 μ l, mixed in vortex
It is mixed in clutch, is placed in 98 DEG C of denaturation 10min in PCR instrument.
By above-mentioned hybrid mixed drop on the chromosome slide through denaturation treatment, piece, 85 DEG C of denaturation 10min are covered.
The genomic in situ hybridization result of embodiment 4 is as shown in Figure 3.
The results of hybridization of comprehensive analysis embodiment 2~4 it is found that the maternal P.delavayi using tree peony to be measured genome
DNA is as probe can ' chromosome of High Noon ' be divided into two groups by filial generation.' High Noon ' is diploid, chromosome number
It is 10, two group chromosome proportions are 1:1.In Fig. 1, Fig. 2 and Fig. 3, a group chromosome of aubergine is derived from
P.delavayi, a blue group chromosome are from P. × suffruticosa.
Different cultivars when probe is consistent with the ratio blockaded, under the same parent kind of tree peony to be measured
(' Luoyang Hong ' blockades DNA with ' Hoki's '), can reach and preferably blockades effect, and then obtains preferable hybridization and tie
Fruit can distinguish two group chromosomes (Fig. 1 is compared with Fig. 2), increase the range of choice for blockading material.
When blockade DNA material it is consistent when, DNA probe with blockade the ratio of DNA there is large effect to results of hybridization,
In blockade the results of hybridization when mass ratio of DNA and DNA probe is 75:2 and be substantially better than results of hybridization when mass ratio is 25:1
(Fig. 1 is compared with Fig. 3).
Comparative example 1
This comparative example provides a kind of in-situ hybridization method of tree peony genome, and the difference with the method for embodiment 2 is only that
Step (4) is different, and step (4) is specific as follows:
Sequentially add reagent shown in table 1, preparing hybrid mixed liquor, wherein the dosage for blockading DNA is 2 μ l, mixed in vortex
It is mixed in clutch, by above-mentioned hybrid mixed drop on pretreated chromosome slide prepared by step (2), covers piece, 85 DEG C
It is denaturalized 10min.
Results of hybridization shows that probe cannot be combined sufficiently with chromosome, be caused since chromosome and probe denaturation are insufficient
Crossbreeding effect is unobvious or cannot hybridize.
Comparative example 2
This comparative example provides a kind of in-situ hybridization method of tree peony genome, and the difference with the method for embodiment 2 is only that
Step (4) is different, and step (4) is specific as follows:
Pretreated chromosome slide prepared by step 2 75 DEG C of water-bath denaturation in 70% deionized formamide
3min is dehydrated 3min in 70%, 90%, 100% ethyl alcohol that -20 DEG C are pre-chilled step by step, is air-dried.
Sequentially add reagent shown in table 1, preparing hybrid mixed liquor, wherein the dosage for blockading DNA is 2 μ l, mixed in vortex
It is mixed in clutch, is placed in 95 DEG C of denaturation 10min in PCR instrument.
Results of hybridization show due to chromosome and probe denaturation it is insufficient, prevent probe with chromosome from sufficiently being combined,
Cause crossbreeding effect unobvious or cannot hybridize.
Comparative example 3
This comparative example provides a kind of in-situ hybridization method of tree peony genome, and the difference with the method for embodiment 2 is only that
Step (4) is different, and step (4) is specific as follows:
Pretreated chromosome slide prepared by step 2 80 DEG C of water-bath denaturation in 70% deionized formamide
1min is dehydrated 3min in 70%, 90%, 100% ethyl alcohol that -20 DEG C are pre-chilled step by step, is air-dried.
Sequentially add reagent shown in table 1, preparing hybrid mixed liquor, wherein the dosage for blockading DNA is 2 μ l, mixed in vortex
It is mixed in clutch, is placed in 95 DEG C of denaturation 15min in PCR instrument.
By above-mentioned hybrid mixed drop on the chromosome slide through denaturation treatment, piece, 85 DEG C of denaturation 10min are covered.
Results of hybridization show due to chromosome and probe denaturation it is insufficient, prevent probe with chromosome from sufficiently being combined,
Cause crossbreeding effect unobvious or cannot hybridize.
In conclusion tree peony GISH method provided by the invention is capable of the chromosome of effective district molecule separate sources in,
It can be used for carrying out the authenticity of filial generation and the composition research of chromosome in tree peony hybridization.
Although above having used general explanation, specific embodiment and test, the present invention is made to retouch in detail
It states, but on the basis of the present invention, it can be made some modifications or improvements, this is apparent to those skilled in the art
's.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to claimed
Range.
Claims (10)
1. a kind of genomic in situ hybridization method of tree peony, which is characterized in that using three-step approach to comprising DNA probe and blockading
The hybrid mixed liquid of DNA and chromosome to be detected carry out denaturation treatment, by the hybrid mixed liquid and chromosomal hybridation after denaturation；
After the hybrid mixed liquid and the chromosome to be detected is are carried out denaturation treatment respectively by the three-step approach, then will denaturation
Hybrid mixed liquid and chromosome mixing afterwards, carry out total denaturation treatment；
The denaturation of the hybrid mixed liquid is by hybrid mixed liquid in 95~98 DEG C of 10~15min of denaturation；
The denaturation of the chromosome to be detected is 70~75 DEG C of water-bath denaturation 3 in formamide solution by chromosome slide to be detected
~5min；
The co-variation be will be mixed through the hybrid mixed liquid of denaturation treatment with chromosome to be detected, in 80~85 DEG C denaturation 10~
15min。
2. genomic in situ hybridization method according to claim 1, which is characterized in that the hybrid mixed liquid middle probe
DNA is 1:10~1:50 with the mass ratio for blockading DNA；
Preferably, the hybrid mixed liquid middle probe DNA and the mass ratio for blockading DNA are 1:25~1:40.
3. genomic in situ hybridization method according to claim 1 or 2, which is characterized in that the segment of the DNA probe is big
Small is 200~500bp；The DNA probe is marked using radioactivity or non-radioactive substance；It is preferred that using digoxigenin labeled.
4. described in any item genomic in situ hybridization methods according to claim 1~3, which is characterized in that the DNA probe
It is prepared as mixing the total genomic dna of tree peony with DIG-Nick Translation Mix, it is protected from light 60 in 15 DEG C~
70min；
Preferably, the dosage of the DIG-Nick Translation Mix is 4~6 μ L/ μ g total genomic dnas.
5. genomic in situ hybridization method according to any one of claims 1 to 4, which is characterized in that described to blockade DNA's
Clip size is 200~500bp；
Preferably, it is described blockade DNA be prepared as boiling the total genomic dna of tree peony in boiling water bath processing 150~
300min。
6. described in any item genomic in situ hybridization methods according to claim 1~5, which is characterized in that described to blockade DNA
Derived from the genomic DNA of the tree peony of the same race with the hybrid parent of tree peony to be detected.
7. described in any item genomic in situ hybridization methods according to claim 1~6, which comprises the steps of:
(1) preparation and pretreatment of chromosome slide sample: the tissue of tree peony to be detected is pre-processed, fixed and digested
Afterwards, using the chromosome slide for going the hypotonic flame seasoning of wall to prepare tree peony to be detected, then chromosome slide digest pre-
Processing；
(2) probe marks and blockades DNA preparation: the total genomic dna of tree peony and DIG-Nick Translation Mix are mixed
It closes, is protected from light 60~70min in 15 DEG C, obtains DNA probe；
The total genomic dna of tree peony is boiled into 150~300min of processing in boiling water bath, obtains blockading DNA；
(3) denaturation of chromosome and probe: by the pretreated chromosome slide of step (1) preparation in 70% formamide
70~75 DEG C of 3~5min of denaturation, are dehydrated step by step in the ethyl alcohol of pre-cooling, dry；
It will be comprising DNA probe and blockading the hybrid mixed liquid of DNA in 95~98 DEG C of 10~15min of denaturation；
Hybrid mixed liquid through denaturation treatment is added to the chromosome slide through denaturation treatment, in 80~85 DEG C of denaturation 10min；
(4) hybridize
The hybrid mixed liquid through denaturation treatment that step (3) is obtained hybridizes 12- in 37 DEG C with the mixture of chromosome to be detected
16h；
(5) elution and signal detection: the hybrid product that step (4) obtains adds the anti-of fluorescent marker after elution, Seal treatment
DigiTAb processing, after washed, adds anti-fluorescence decay agent, mounting.
8. genomic in situ hybridization method according to claim 7, which is characterized in that described in step (5) elution include
The step of hybrid product is eluted using 30%~50% deionized formamide solution.
9. genomic in situ hybridization method according to claim 7 or 8, which is characterized in that the chromosome slide sample
Preparation and pretreatment include the following steps:
(1) using paracide-α-bromine, how mixed liquor pre-processes the tissue of tree peony to be detected, 4 DEG C of 10~12h of processing
Afterwards, it is fixed using Kano fixer；
(2) be added include 2% cellulase and 0.5% pectase mixed enzyme solution, after 37 DEG C of 70~90min of enzymolysis processing, adopt
Spend the hypotonic flame seasoning film-making of wall；
(3) RNA enzyme, 37 DEG C of 1~2h of processing are added；After washed, pepsin, 37 DEG C of 30~40min of processing are added.
10. genomic in situ hybridization method according to any one of claims 1 to 9 is educated in the Germplasm Identification of tree peony and tree peony hybridization
Application in the genetic analysis of kind.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811610043.6A CN109735602A (en) | 2018-12-27 | 2018-12-27 | A kind of genomic in situ hybridization method and its application of tree peony |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811610043.6A CN109735602A (en) | 2018-12-27 | 2018-12-27 | A kind of genomic in situ hybridization method and its application of tree peony |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109735602A true CN109735602A (en) | 2019-05-10 |
Family
ID=66360133
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811610043.6A Pending CN109735602A (en) | 2018-12-27 | 2018-12-27 | A kind of genomic in situ hybridization method and its application of tree peony |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109735602A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114231603A (en) * | 2022-01-06 | 2022-03-25 | 南京海关动植物与食品检测中心 | Primer, reagent, identification method and kit for identifying paeonia rockii |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102787166A (en) * | 2012-07-16 | 2012-11-21 | 北京林业大学 | Fluorescence in situ hybridization method of prunus mume chromosome |
| CN102960237A (en) * | 2012-11-22 | 2013-03-13 | 河南省农业科学院 | Method for obtaining, breeding and storing peanut interspecies hybridization variety, and identifying molecular cytology |
| CN104004849A (en) * | 2014-06-09 | 2014-08-27 | 南京农业大学 | Method for quickly establishing metaphase chromosome karyotype of cucumber through genomic in-situ hybridization |
| CN104073568A (en) * | 2014-07-18 | 2014-10-01 | 西南大学 | Fluorescence in situ hybridization method for metaphase chromosome of mulberry |
| CN104745695A (en) * | 2015-03-23 | 2015-07-01 | 湖北大学 | Method for interspecific hybrid genome in-situ hybridization of crambe cordifolia |
-
2018
- 2018-12-27 CN CN201811610043.6A patent/CN109735602A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102787166A (en) * | 2012-07-16 | 2012-11-21 | 北京林业大学 | Fluorescence in situ hybridization method of prunus mume chromosome |
| CN102960237A (en) * | 2012-11-22 | 2013-03-13 | 河南省农业科学院 | Method for obtaining, breeding and storing peanut interspecies hybridization variety, and identifying molecular cytology |
| CN104004849A (en) * | 2014-06-09 | 2014-08-27 | 南京农业大学 | Method for quickly establishing metaphase chromosome karyotype of cucumber through genomic in-situ hybridization |
| CN104073568A (en) * | 2014-07-18 | 2014-10-01 | 西南大学 | Fluorescence in situ hybridization method for metaphase chromosome of mulberry |
| CN104745695A (en) * | 2015-03-23 | 2015-07-01 | 湖北大学 | Method for interspecific hybrid genome in-situ hybridization of crambe cordifolia |
Non-Patent Citations (2)
| Title |
|---|
| 李梅芳等: "《水稻生物技术育种》", 31 August 2001, 北京：中国农业科技出版社 * |
| 赵娜: "牡丹远缘杂交后代及亲本形态与核型研究", 《中国优秀博硕士学位论文全文数据库(硕士) 农业科技辑(月刊)》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114231603A (en) * | 2022-01-06 | 2022-03-25 | 南京海关动植物与食品检测中心 | Primer, reagent, identification method and kit for identifying paeonia rockii |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104711361B (en) | The method of the red peaceful hybrid seed purity of Rapid identification new water melon breed and the primer and kit of use | |
| Waminal et al. | Karyotype analysis of Panax ginseng CA Meyer, 1843 (Araliaceae) based on rDNA loci and DAPI band distribution | |
| CN103205500A (en) | Multicolor fluorescence in situ hybridization (MFISH) method for quickly analyzing and identifying alien chromosome of wheat | |
| CN109576284A (en) | One multi-functional myb transcription factor gene and application thereof | |
| CN109879944B (en) | EAR1 protein related to plant drought resistance and coding gene and application thereof | |
| CN105039353B (en) | A kind of capsicum pollens development related gene CaMS1 and its application | |
| CN100540665C (en) | Regulate the gene of plant branching, contain the carrier of this gene,, and utilize this microorganism to regulate the method for plant branching by this carrier microorganism transformed | |
| CN109735602A (en) | A kind of genomic in situ hybridization method and its application of tree peony | |
| Bach Holm | The C and Q banding patterns of the chromosomes of Lilium longiflorum (Thunb.) | |
| Parra-Nunez et al. | How to perform an accurate analysis of metaphase I chromosome configurations in autopolyploids of Arabidopsis thaliana | |
| CN109694921A (en) | A kind of method that quick detection group planting mycorrhizal fungi successfully infects host | |
| CN103348009A (en) | Method for preparing fertility-lowered plant | |
| CN109456984B (en) | Rice glume development gene AH1 and application thereof | |
| CN102643829A (en) | Rice yield gene GY6 clone and application thereof | |
| CN107022628A (en) | A kind of molecular labeling for identifying the early evening true hybrid of flower lichee filial generation | |
| CN100389209C (en) | Primer for detecting seed purity and its method | |
| CN108642062B (en) | Mung bean weevil resistance gene VrPGIP allele VrPGIP1-ACC41Molecular marker and application | |
| CN108913699B (en) | Application of protein TaNAC2 in regulation and control of plant seed germination | |
| CN111534524A (en) | Rice field abortive cytoplasmic male sterility restoring gene RF3 and application thereof | |
| CN110423751A (en) | For doing sth. in advance method, the kit, mutated-genotype of Rice Flowering time | |
| CN112154910A (en) | Rapid positioning and cloning method of neutral mutant bridged plant spontaneous mutant gene | |
| CN105039398B (en) | A method of preparing male sterile pepper | |
| CN109897886A (en) | A kind of rice plumule Miho Dockyard NA template preparation method and PCR method for PCR amplification | |
| CN105925694B (en) | A kind of reiterated DNA sequences of black mustard genome and its application | |
| Zheng et al. | Karyotype analysis of autotetraploidy in Brassica rapa ssp. chinensis |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190510 |
|
| RJ01 | Rejection of invention patent application after publication |