CN102876801A - Method for identifying exogenous chromosomes and chromosome segments in plant distant hybrids - Google Patents
Method for identifying exogenous chromosomes and chromosome segments in plant distant hybrids Download PDFInfo
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Abstract
The invention provides a method for identifying exogenous chromosomes and chromosome segments in plant distant hybrids. The method includes the steps of firstly, microscopically separating monosomes or chromosome segments of chromosomes to be tested of a plant to be tested; secondly, using the separated monosomes or chromosome segments in the first step to build a fluorescence probe of the monosomes or chromosome segments; thirdly, using the fluorescence probe built in the second step to perform fluorescence in situ hybridization to chromosomes in metaphase division of the hybrids; and fourthly, analyzing the fluorescence in situ hybridization result obtained in the third step to determine distribution of fluorescence signals of the chromosomes to be tested of the plant to be tested. Exogenous chromosomes and chromosome segments in wheat distant hybrids can be identified by the method, and the method is applicable to identification of exogenous chromosomes and chromosome segments in distant hybrids of other plants.
Description
Technical field
The present invention relates to that plant chromosome is coated with the foundation of dyeing technique and the application during exogenous chromosome and chromosome segment are identified in the Wide_cross in Plant kind.Particularly, the present invention relates to a kind of method of utilizing chromosome painting plant identification distant hybrids exogenous chromosome and chromosome segment.
Background technology
Wheat (Triticum aestirum L.) is one of important in the world food crop (2n=6x=AABBDD), and its cultivated area and output all occupy the cereal crop first place.In China, wheat is only second to paddy rice, occupies very consequence in agricultural.The hereditary basis of Chinese Wheat Cultivars is comparatively narrow, has limited to a great extent the raising of wheat yield and the improvement of quality.The sibling species of wheat is huge, a precious genetic resources storehouse, and heritable variation is very abundant, has the good characteristic that many cultivated wheats do not have, and as disease-resistant, degeneration-resistant, barren-resistant, high nutrition, high protein and large fringe are spent more many etc.Therefore, use the method for distant hybirdization, chromosome engineering, excellent genes in the sibling species is imported in the common wheat genome, utilize the excavation of cellular elements biological assay technology, mark and utilize the gene of disease-resistant gene potential on the sibling species karyomit(e), nutrient efficient gene, adaptability and yielding ability, initiative distant hybirdization new germ plasm genetic resources, can further widen the hereditary basis of wheat, improve its production potential, cultivate the new variety of wheat of many anti-, nutrient efficient, wide suitable, stable yields type, to ensure the grain security of China.
Rye (Secale cereal L.) (2n=2x=14) is also to be to be successfully used to one of nearly edge species of improveing wheat most the earliest.Rye has many common wheats good character can't be obtained, as disease and insect resistance (rust, Powdery Mildew, aphid etc.), resistance (cold-resistant, drought-enduring, barren-resistant, Salt And Alkali Tolerance, dry heat resistance wind etc.), resistant to lodging, ability for tillering strong and contain higher Methionin etc.
[1-2]Particularly carry a large amount of resistant genes such as leaf rust resistance (Lr26), stripe rust resisting (Yr9), anti-stem rust (Sr31), mildew-resistance (Pro8) and anti-plant hopper (Gb2) on the rye 1RS chromosome segment.It is reported that some disease-resistant genes that distribute on the 1R of rye, 2R, 3R, the 6R karyomit(e) are imported in the wheat
[3]
Thinopyrum intermedium (Agropyrom intermedium) (2n=6x=42) has a BYDV (Bailey yellow dwarf virus, barley yellow dwarf) resistance, it has many good economical characters for the wheat utilization simultaneously, to stem rust, bar rust, leaf rust, Powdery Mildew and bunt immunity, high resistance root rot, bushy stunt, anti-leaf blight, and have the rhizome regenerative power, salt resistance ability is strong, is one of desirable resistance source of wheat breeding.Wheat-wheatgrass alien addition line TAI-27 is the 14 pairs of karyomit(e)s of two genomes with Thinopyrum intermedium (claiming again sky blue wheatgrass), appends to respectively one of addition line in the two cover wheat-wheatgrass alien addition lines of setting up in the common wheat
[4], identify through artificial inoculation, show as the high resistance barley yellow dwarf
[5], show the BYDV resistant gene of Thinopyrum intermedium has been transferred among the TAI-27, the anti-source of high-quality is provided for the resistance breeding of wheat
[4]
Long fringe couchgrass (Elytrigia elongate=Thinopyrum elongatum=Agropyron elongatum) (2n=10x=70) has the multiple good characters such as disease-resistant, degeneration-resistant, high protein, is one of nearly edge wild species that are most widely used in the genetic improvement of wheat.The long fringe couchgrass of discovery such as academician of the Chinese Academy of Sciences's Li Zhensheng have good rust-resisting property, so carry out distant hybirdization research take long fringe couchgrass as host system.Effort through more than 20 years, genome that successfully will long fringe couchgrass, karyomit(e), chromosome segment import in the common wheat, formulated the little wheat new germ plasms of laying down such as octoploid of wheat-wheatgrass (littlely lay down 68, littlely lay down 693, littlely lay down 784, littlely lay down 7430, little lay down 763), addition line (little lay down 759), substitution line (the little blue grain of laying down) and translocation line (little lay down 96), bred little lay down No. 4, little lay down No. 5, little lay down No. 6, littlely lay down 54 and the little high yields such as 81 of laying down, disease-resistant high-quality wheat variety [6-7].
After by distant hybirdization exogenous chromosome or chromosome segment successfully being imported to general wheat background, need to identify accurately it, could effectively be used.Used at present C to divide the methods such as band, biochemical marker, molecule marker, genomic in situ hybridization (Genome in situ hybridization, GISH), polychrome genomic in situ hybridization (multicolor GISH) to come exogenous genetic material in the analysis and identification Wheat Background.C divides with method there not being specificity banding pattern or not ideal with the karyomit(e) that line is few, of light color, fragment is little, and it is very responsive to envrionment conditions that C divides band simultaneously, uses different reagent, the different material of selection to tend to draw different results.The biochemical marker number is fewer, and some isozyme genes is loss of activity during evolution, and this has brought difficulty for the chromatinic evaluation of external source.The PCR molecule marker has easily and fast advantage, but only effective to following the tracks of specific karyomit(e) or fragment in the genetic marker of some chromosomal regions.The genomic in situ hybridization technology utilizes the different plant species genome of gene infiltration as probe, for identifying that the external source chromatin in the wheat allodidiploid is a kind of very useful technology
[8]For example, with biotin labeled H. villosa chromosome DNA as probe, DNA blockades with the common wheat genome, utilizes the GISH technology can successfully detect the cluster hair wheat chromatin that imports in the wheat, and clearly shows the definite position of translocation chromosome breaking point
[9]Equally, the GISH technology can successfully detect long fringe couchgrass karyomit(e) in the wheat genetic background or the fragment of transposition
[10]Use a plurality of different genes groups as probe, the polychrome genomic in situ hybridization is for identifying that simultaneously two or more genomes provide another possibility in the polyploid.For example, GISH and polychrome-GISH technology is successfully applied to the cytogenetics composition of five double diploid systems that identify that wheat and Thinopyrum intermedium hybridization obtain
[11]Equally, GISH and polychrome-GISH technology be successfully applied to also that evaluation wheat-long fringe couchgrass is additional, the molecular cytology feature of replacement and translocation line
[12]But genomic in situ hybridization has certain limitation, as: can't determine exogenous chromosome or chromosome segment belong to which homology group, the substitution line specific karyomit(e) that replaces, position and the breakpoint that translocation line occurs with GISH merely.In addition, when exogenous chromosome and chromosome of wheat sibship are nearer, be difficult to obtain more satisfactory crossbreeding effect.
Comparatively speaking, chromosome painting (chromosome painting) can better be identified the exogenous chromosome in the cross-fertilize seed.Chromosome painting is about to whole chromosome, certain bar chromosome arm (long-armed or galianconism) or the DNA of certain fragment of karyomit(e) and is prepared into probe, then use the method for fluorescence in situ hybridization, with probe hybridization to metacinesis phase karyomit(e), utilize the signal distributions of fluorescence microscope fluorescein on karyomit(e), thereby analyze and study chromosomal restructuring and distortion
[13]In the human and animal, chromosome painting is comparatively ripe, and this technology has very high specificity and resolving power, can be used to detect human and animal's chromosome aberration
[14], sex identification
[15], microclone
[16]Deng.Take differential from karyomit(e) or chromosome segment DOP-PCR (Degenerate-Oligonucleotide-Primed PCR) amplified production as probe, many scholars are to wheat (Triticum aestivum)
[17], broad bean (Vicia faba)
[18], barley (Horeum vulgare)
[18], dragon spruce (Picea abies)
[18], petunia (Petunia hybrida)
[19], rye (Secale cereale)
[20]Also carry out the chromosome painting experiment Deng metacinesis phase karyomit(e), whether blockaded but the result shows, identical hybridization signal on all karyomit(e)s, all occurred.Schubert etc. think that this may owing to compare with Mammals karyomit(e), regular swivel base, transposition event occur between the plant chromosome cause having widely consistence between the plant coloured differently body genome sequence
[19]But, utilize some plant specific staining body DOP-PCR amplified productions that contain special sequence as probe, carry out the chromosome painting experiment, but obtained success.Such as, utilize differential from petunia (Petunia hybrid) B chromosome as probe, petunia metacinesis phase karyomit(e) is coated with dyes, the result shows that the hybridization signal on the B chromosome is more much better than than the brightness of A group chromosome hybridization signal
[21]Equally, utilize differential from garden sorrel (Rumex acetosa) Y chromosome DOP-PCR amplified production be that probe carries out fluorescence in situ hybridization (FISH), Y chromosome is owing to contain special tumor-necrosis factor glycoproteins, it is easy to make a distinction with euchromosome
[22]But chromosome painting never succeeds for the detection of the euchromosome (that is, A chromosome) of plant.
With Plant Genome in situ hybridization (GISH) Comparatively speaking, plant chromosome is coated with and dyes experiment and has a not available advantage of GISH: (1) plant chromosome is coated with and dyes experiment and can distinguish the trickle structural changes of exogenous chromosome in the Wide_cross in Plant kind, such as disappearance, repeat, transposition, inversion etc., for example, in this research TAI-27 addition chromosome is carried out micro-separation and DOP-PCR amplification, TAI-27 metacinesis phase karyomit(e) is coated with as probe with it and dyes experiment, the result shows in TAI-27 except additional a pair of Thinopyrum intermedium karyomit(e), chromosome painting experiment can also identify dyad displacement has occured, and wherein minimum dyad owing to terminal deletion causes (this result is that Plant Genome in situ hybridization is shown not out); (2) utilize plant chromosome to be coated with and dye experiment, can carry out micro-separation to specific karyomit(e), and then understand the genetic information that specific karyomit(e) contains, as previously mentioned B chromosome, Y chromosome, the rye 1R karyomit(e) that contains a lot of characteristic genes that this institute relates to, the additional Thinopyrum intermedium karyomit(e) of TAI-27, the additional long fringe couchgrass karyomit(e) (galianconism of 7E) of 7ES/CS, it is carried out successful micro-separation and DOP-PCR amplification, for understanding its genetic information that contains and laying a good foundation for cloning resistance related gene.
Summary of the invention
The invention provides a kind of method of utilizing exogenous chromosome and chromosome segment in the chromosome painting plant identification distant hybrids.
Particularly, method of the present invention comprises the steps:
1) chromosomal monosome or chromosome segment to be detected in the measuring plants are treated in micro-separation;
2) utilizing step 1) monosome or the chromosome segment that separate make up monosome or chromosome segment fluorescent probe;
3) utilizing step 2) monosome or the chromosome segment fluorescent probe that make up carry out fluorescence in situ hybridization to cross-fertilize seed metacinesis phase karyomit(e); With
4) analytical procedure 3) the fluorescence in situ hybridization result that obtains, determine to treat that chromosomal fluorescent signal to be detected in the measuring plants distributes.
In one embodiment of the invention, step 1) by preparation Wide_cross in Plant kind tip of a root metacinesis phase, utilize the monosome microseparation separate chromosomal monosome to be detected or chromosome segment or with monosome or the chromosome segment of at least part of homology of karyomit(e) to be detected.Determine and to identify according to GISH result with the chromosomal of exogenous chromosome homology to be measured, concretely, utilize the genomic dna structure fluorescent probe in exogenous chromosome to be measured source that the distant hybridization kind is carried out fluorescence in situ hybridization, at microscopically, observe fluorescent signal distribution situation on karyomit(e), thus the karyomit(e) of at least part of homology of exogenous chromosome definite and to be detected." at least part of homology " refers generally at least 20% homology, and those skilled in the art can easily determine needed homology according to practical application.
In one embodiment of the invention, step 2) by to step 1) monosome or the chromosome segment that separate carry out the DOP-PCR amplification, and by the nick translation method monosome of described separation or chromosome segment DOP-PCR amplified production are carried out fluorescent probe and make up.Step 2 wherein) to differential from monosome carry out two-wheeled DOP-PCR amplification.
Method of the present invention is applicable to the various plants kind, for example, the monocotyledonss such as wheat, rye, Thinopyrum intermedium, long fringe couchgrass, paddy rice, corn also are applicable to the dicotyledonss such as cotton, soybean simultaneously, preferably be applicable to monocotyledons, more preferably be applicable to grass.
In a specific embodiments of the present invention, the invention provides a kind of plant chromosome that utilizes and be coated with the method that dyeing technique is identified exogenous chromosome in the wheat distance edge hybrid kind, it is characterized in that:
1) chromosomal monosome or chromosome segment to be detected in the micro-separation wheat edge species far away is such as the Thinopyrum intermedium karyomit(e) that adds among the additional long fringe couchgrass karyomit(e) of the 1R karyomit(e) of rye, 7ES/CS, the TAI-27 etc.;
2) utilizing step 1) monosome or the chromosome segment that separate make up monosome or chromosome segment fluorescent probe;
3) utilizing step 2) monosome or the chromosome segment fluorescent probe that make up carry out fluorescence in situ hybridization to cross-fertilize seed metacinesis phase karyomit(e); With
4) analytical procedure 3) the fluorescence in situ hybridization result that obtains, determine to treat that chromosomal fluorescent signal to be detected described in the measuring plants distributes.
In a specific embodiments of the present invention, described karyomit(e) to be detected and chromosome segment are selected from rye 1R karyomit(e), Thinopyrum intermedium-wheat addition line TAI-27 addition chromosome, long fringe couchgrass-wheat addition line 7ES/CS addition chromosome.
Method of the present invention is applicable to detect the euchromosome in the plant, special B chromosome and sex chromosome, especially, method of the present invention is first successfully with the euchromosome in the high-sensitivity detection plant, having overcome the undesirable problem of plant euchromosome detection effect in the prior art, is a kind of technological breakthrough.Specifically, method of the present invention has following some technological breakthrough: the 1) improvement of Chromosome Technique, method of the present invention has adopted more advanced Chromosome Technique, and laughing gas (N is adopted in tip of a root pre-treatment
2O) and the chemical reagent such as unconventional chemical reagent colchicine, 8-hydroxy-quinoline, santochlor, major advantage is embodied in toxicological harmless, dispersion effect is good; 2) fluorescent probe structure and fluorescence in situ hybridization technique have also embodied the characteristics such as quick, efficient, that signal is strong with comparing with class methods; 3) monosome is coated with the breakthrough of dyeing technique, compare with the animal chromosome painting, plant chromosome chromosome painting particularly plant euchromosome is coated with dyeing technique development and is in the stage that falls behind very much, main manifestations is clear, the poor specificity of poor TV signal, be difficult to reach the requirement of practical application, and adopt method of the present invention, the fluorescent in situ result shows that the fluorescent hybridization signal is very clear, specificity is very strong, can well be applied to the evaluation of exogenous chromosome in the distant hybrids, this has good pushing effect to the development that plant chromosome is coated with dyeing technique.
Although one or more in the above-mentioned technology have obtained application in relevant research, but the experimental program of several like this technology completes of integrated application, and be applied in the evaluation of exogenous chromosome in the Wide_cross in Plant kind or chromosome segment and do not see the correlative study report.The present invention utilizes this cover research approach successfully with the external source euchromosome in the high-sensitivity detection distant hybirdization plant first, overcome the undesirable problem of plant euchromosome detection effect in the prior art, be a kind of technological breakthrough, embodied this research method and had preferably advance, novelty and practicality.
Description of drawings
From the detailed description below in conjunction with accompanying drawing, above-mentioned feature and advantage of the present invention will be more obvious, wherein:
Fig. 1. the chromosomal micro-separation of rye 1R and utilize the 1R chromosome probe to identify karyomit(e) and the chromosome segment in rye source at Wheat-rye addition line 1R/CS and wheat-rye translocation line 1RS/1BL:
The A. chromosomal micro-sepn process of rye 1R (left side: rye cell metacinesis phase, arrow shows 1R karyomit(e); Right: the rye cell metacinesis phase after 1R karyomit(e) is separated);
B. rye 1R karyomit(e) second is taken turns DOP-PCR amplification electrophorogram (M:Marker; CK: negative control; 1: the 1R monosome amplified production of separation);
C. utilize 1R monosome fluorescent probe that 1R/CS (left figure), 1RS/1BL (right figure) metacinesis phase karyomit(e) are carried out fluorescence in situ hybridization result (arrow shows the fluorescent signal designation of chromosome, and the result shows to only have on 1R monosome and the 1RS chromosome segment fluorescent signal).
Fig. 2 .TAI-27 adds the chromosomal micro-separation of Thinopyrum intermedium and the chromosomal chromosome painting of the additional Thinopyrum intermedium of TAI-27 is identified:
A.TAI-27 adds the chromosomal micro-sepn process of Thinopyrum intermedium, and (left side: cell metacinesis phase, arrow are shown additional Thinopyrum intermedium karyomit(e); Right: the cell metacinesis phase after additional microchromosome is separated);
The additional Thinopyrum intermedium karyomit(e) second of B.TAI-27 is taken turns DOP-PCR amplification electrophorogram (M:Marker; CK: contrast; 1, the attach list karyomit(e) of 2,3,4 representative separation);
C. left figure shows genomic in situ hybridization (GISH) result who carries out take the Thinopyrum intermedium genome as probe, right figure demonstration utilizes the additional Thinopyrum intermedium monosome fluorescent probe of TAI-27 that TAI-27 metacinesis phase karyomit(e) is carried out the fluorescence in situ hybridization result, and (arrow shows the fluorescent signal designation of chromosome, compare with genomic in situ hybridization, the chromosome painting result shows in TAI-27, a pair of exogenous chromosome has been replaced the dyad of wheat, and wheat has added a pair of external source microchromosome, and this generation to microchromosome is owing to additional dyad end fracture produces);
D. utilize the additional Thinopyrum intermedium chromosome fluorescence probe of TAI-27 that middle couchgrass metacinesis phase karyomit(e) is carried out fluorescence in situ hybridization result (result shows that similar fluorescent signal all appears in zone, the nearly kinetochore of most karyomit(e)s).
Fig. 3 .7ES/CS adds the chromosomal micro-separation of long fringe couchgrass and the chromosomal chromosome painting of the additional long fringe couchgrass of 7ES/CS is identified:
A.7ES/CS (left side: cell metacinesis phase, arrow are shown additional long fringe couchgrass karyomit(e) in the additional chromosomal micro-sepn process of long fringe couchgrass; Right: the cell metacinesis phase after additional microchromosome is separated);
B.7ES/CS additional long fringe couchgrass karyomit(e) second is taken turns DOP-PCR amplification electrophorogram (M:Marker; 1, the attach list karyomit(e) of 2,3,4 representative separation);
C. utilize the additional long fringe couchgrass chromosome fluorescence probe of 7ES/CS that 7ES/CS metacinesis phase karyomit(e) is carried out fluorescence in situ hybridization result (arrow shows the fluorescent signal designation of chromosome, and the result shows to only have on the additional dyad of wheat fluorescent signal)
Embodiment
Describe the present invention in detail below with reference to embodiment and accompanying drawing.What those having ordinary skill in the art will appreciate that is, following embodiment is illustrational purpose, and it should not be interpreted as limitation of the present invention by any way.Protection scope of the present invention is limited by accompanying claim.
In addition, specify unless those skilled in the art be also to be understood that in addition, used reagent is commercially available other reagent of analytical pure level among the following embodiment.
The chromosomal micro-separation of embodiment 1. rye 1R reaches dyes evaluation to addition chromosome and being coated with of chromosome segment in Wheat-rye addition line 1R/CS and wheat-rye translocation line 1RS/1BL rye source
1. rye is divided the preparation of phase mitosis metaphase
Used rye (Secale cereal) king II is available from USDA herbage and herding research department (USDA, ARS, FRRL, UT, USA).
1) N
2O processes the tip of a root and pricks a hole with tweezers at the lid of 1.5ml centrifuge tube, uncap, and spraying water in the centrifuge tube to tube wall with spray bottle has a layer of water drops, and the tip of a root that cuts 1-2cm is put into pipe, puts into N behind the cover lid
2Process 1-3h in the O air chamber, pressure is 10ATM (1.01Mpa);
2) tip of a root is fixing with fixing 10 minutes (being no more than 1h) (can directly add 90% acetic acid in original 1.5ml pipe with holes, or shift the tip of a root and fix to new pipe) of the tip of a root on 90% Acetic Acid Glacil; The tip of a root after fixing changes over to be put into-20 ℃ in 70% the ethanol and can preserve for many years;
3) washing washes the tip of a root with water and (changed H 2-3 time in 10 minutes
2O), be put on ice;
4) shift the tip of a root to dried filter paper, rotate gently the dope that the tip of a root is removed tip of a root surface;
Enzymolysis downcuts the long vegetative point of 2mm and partly changes (the enzymolysis solution preparation a: plastic culture dish is put on the balance of (2-3 the tip of a root/pipe) in the ice-cold enzyme liquid of 20 μ L over to, add 1 * citrate buffer solution (10mM Trisodium Citrate+10mM EDTA to the inside, pH 5.5) 4.85g, taking-up places on ice, take by weighing 50mg polygalacturonase Y-23 (available from Beijing Hua Lvyuan Science and Technology Ltd., 1%w/w) and 100mg cellulase Onozuka R-10 (available from Beijing Hua Lvyuan Science and Technology Ltd., 2%w/w) add in the damping fluid, with rifle head pressure-vaccum hydrotropy, the enzyme dissolving installs in the 0.5ml thin-walled PCR pipe rear dividing with skimmer, it is once freezing rapidly to put into dry ice,-20 ℃ of preservations), put into 37 ℃ water-bath 30-50 minute;
5) taking out centrifuge tube puts on ice, add 500 μ L, 70% ethanol, mixing, outwell ethanol after, add 70% new ethanol of 500 μ L, pour out again ethanol, stay 40 μ L ethanol, smash the tip of a root to pieces with dissecting needle, several seconds of vortex or with finger attack tube wall suspension cell several times, centrifugal 10-20 second on little whizzer, rotating speed is no more than 2000rcf, carefully is inverted the PCR pipe and flows to end fully in filter paper or thieving paper to ethanol;
6) put centrifuge tube on ice, add 30 μ L anhydrous acetic acids, vortex or with rifle head pressure-vaccum mixing gently, inhale 5-8 μ L and drip sheet, slide glass has been placed in the moisture preservation box, covers moisture preservation box behind the sheet, places and can utilize optical microphotograph endoscope objective lens 20 * lower karyomit(e) of observing after 5 minutes.
2. the chromosomal micro-separation of rye 1R
Be the fine glass stick of 1.0mm with diameter at first, under spirit lamp, utilizing Leitz to draw the manual diameter that is drawn into of pin instrument is fine glass needle about 1 μ m.The glass needle that (Olympus1M) draws by hand under inverted microscope carries out all around paddling, gently scraping by Leitz micrurgy instrument (code-No.93314 is available from German Lycra company) to the karyomit(e) of identification.To be stained with 1R karyomit(e) (with the microchromosome of satellite, can be in achromophil situation recognition and verification, Figure 1A is shown in the left arrow; Figure 1A, right be the image of 1R karyomit(e) after separated) needle point fracture 10 μ L Proteinase Ks (Proteinase K be housed in advance, recombinant PCR Grade 03115887001Roche, 50ng/ μ L, by 1 * Taq dna polymerase buffer liquid preparation) in the 0.2mlEppendof pipe of reaction buffer, room temperature is (centrifugation rate 4000rpm) centrifugal 30s at a high speed, and-20 ℃ of placements are stand-by.
3. rye 1R karyomit(e) DOP-PCR (Degenerate-Oligonucleotide-Primed PCR) amplification
At first will contain the differential that obtains by embodiment 1.2 and be put in 37 ℃ from chromosomal PCR pipe and carry out the reaction of enzymolysis Deproteinization, process 10min inactivating protein enzyme K for 90 ℃ behind the 2h, as the template of first round DOP-PCR reaction.
(N represents A with DOP-PCR primer (5 '-CCGACTCGAGNNNNNNATGTGG-3 '), G, T or C), water, damping fluid (be with by the DOP-PCR amplification kit, the DOP-PCR amplification kit is available from the Beijing Quanshijin Biotechnology Co., Ltd) under ultraviolet lamp, shine 30min, operation all operates at ultra-clean operator's console.
First round DOP-PCR amplification reaction system:
Response procedures: 94 ℃ of 5min; 94 ℃ of 1min, 30 ℃ of 1.5min, 72 ℃ of 3min, 5 circulations; 94 ℃ of 1min, 55 ℃ of 1min, 72 ℃ of 1.5min, 25 circulations; 72 ℃ of 10min; 4 ℃ of preservations.
Second takes turns the DOP-PCR amplified reaction: as template, reaction system is the same to get 1/10 (5 μ L) of first round PCR product.Response procedures: 95 ℃ of 5min; 94 ℃ of 1min, 55 ℃ of 1min, 72 ℃ of 1.5min, 30 circulations; 72 ℃ of 10min; 4 ℃ of preservations.Amplification is seen Figure 1B.
4. rye 1R karyomit(e) second is taken turns the fluorescent probe structure of DOP-PCR amplified production
1) following component is added in the centrifuge tube (being the needed volume of system of a large amount of marks in the bracket) on ice
Place 15 ℃ of reaction 2h in the PCR instrument (MJ Research PTC-200, the U.S.).(mark-dNTP is Alexa Fluor-488-dUTP (green glow), available from Invitrogen company; Dna polymerase i is available from Invitrogen company, and concentration is 10U/ μ L, can directly use);
2) add 5 * TAE (0.2mol/L Tris-acetic acid, 0.005mol/L EDTA) pH 5.2+140ng/ μ L salmon sperm DNA 175 μ L (350 μ L), vortex is transferred to the 1.5ml centrifuge tube with solution, places on ice;
3) add 90% ethanol-10% sodium acetate (3M, pH 5.2) 0.5ml (1ml), the vortex mixing is put into pipe-20 ℃ of 2h-and is spent the night, precipitation DNA;
4) 15,000 leave heart 20-30min, remove supernatant, 100% ethanol is washed precipitation, and ethanol is removed totally as much as possible;
5) lucifuge, the air-dry dna probe of 30min;
6) precipitation is dissolved in 20 μ L (50 μ L), 2 * SSC, 1 * TE damping fluid (pH 7.0 for 10mmol/LTris-HCl, 1mmol/L EDTA), concentration and probe concentration is about 100ng/ μ L;
7) lucifuge-20 ℃ saves backup.
5. utilizing the rye 1R monosome DOP-PCR amplified production probe that builds is that the additional chromosomes of rye of 1R/CS carries out the chromosome painting evaluation to the wheat-rye annex, and rye chromosomal segments among the wheat-rye translocation line 1RS/1BL is identified
1) method with program 1 prepares Wheat-rye addition line 1R/CS and China spring-rye translocation line 1RS/1BL (available from USDA herbage and herding research department, USDA, ARS, FRRL, UT, USA) Metaphase Chromosome sample.
2) sample to be hybridized (film-making) is put into 0.125J/cm in the UV-crosslinked instrument (code-No.CL-1000 is available from U.S. UVP company)
2Crosslinked.
3) with 2 * SSC (0.3mol/LNaCl, 0.03mol/L Trisodium Citrate), 1 * TE damping fluid (pH 8.0 for 10mmol/L Tris-HCl, 1mmol/L EDTA) dilution probe, different probe dilution ratios is different, general 1: 5-1: 10 dilutions.
4) film-making is placed on ice, adds 5-6 μ L hybridization solution, cover plastic coverslip, put into little iron basin (the iron basin is lined with thieving paper, and water squirts), cover with plastics box, put into boiling water 5min, then put into 55 ℃ of hybridization of wet box of preheating and spend the night.
5) film-making is taken out from wet box, put into 2 * SSC and make the cover glass landing, with thieving paper the slice, thin piece back side is dried, drip a upper anti-quencher (H-1200 who contains DAPI, available from U.S. Vector Labs company), covering the cover glass of 24 * 50mm, is that available fluorescent microscope (Zeiss Universal microscope) carries out microscopy, photograph (Magnafier CCD camera) after several minutes.
6. results and discussions
Result of study is seen Fig. 1.
Shown in Figure 1A, left figure: rye cell metacinesis phase, arrow shows 1R karyomit(e): have satellite, minimum, can clearly recognize in the not dyeing situation; Right figure: the rye cell metacinesis phase after 1R karyomit(e) is separated), under the micrurgy instrument, by the glass needle adhesion method rye 1R monosome has been carried out successful separation.
Monosome after the separation is after processing through Proteinase K, carry out two-wheeled DOP-PCR amplification, its amplification as shown in Figure 1B, the amplification electrophorogram is the disperse shape, molecular weight ranges shows the amplification that monosome DNA succeeds between 250bp-2000bp.
Shown in Fig. 1 C, the very special hybridization of the rye 1R monosome fluorescent probe of micro-separation is on China spring-rye addition line 1R/CS 1R karyomit(e) among (the left figure of Fig. 1 C) and the China spring-rye translocation line 1RS/1BL on the 1RS chromosome segment (the right figure of Fig. 1 C).This research utilizes the rye 1R monosome DOP-PCR amplified production of micro-separation as fluorescent probe first, exogenous chromosome or chromosome segment among China spring-rye addition line 1R/CS and the China spring-rye translocation line 1RS/1BL are identified, result indicating signal is very clear, shows that the method for utilizing the present invention to adopt has successfully made up rye 1R monosome fluorescent probe and identifying China spring-rye addition line and China spring-rye translocation line exogenous chromosome or chromosome segment success.
The micro-separation of embodiment 2.TAI-27 addition chromosome and the chromosome painting of TAI-27 addition chromosome identified
Wheat-wheatgrass alien addition line TAI-27 is the 14 pairs of karyomit(e)s of two genomes with Thinopyrum intermedium (claiming again sky blue wheatgrass), appends to respectively one of addition line in the two cover wheat-wheatgrass alien addition lines of setting up in the common wheat, available from Northeast Normal University.
1.TAI-27 divide the preparation of phase mitosis metaphase
With embodiment 1.1.
2.TAI-27 in the micro-separation of additional Thinopyrum intermedium microchromosome
Be the fine glass stick of 1.0mm with diameter at first, under spirit lamp, utilizing Leitz to draw the manual diameter that is drawn into of pin instrument is fine glass needle about 1 μ m.The glass needle that (Olympus1M) draws by hand under inverted microscope carries out all around paddling, gently scraping by Leitz micrurgy instrument (code-No.93314 is available from German Lycra company) to the karyomit(e) of identification.Be stained with among the TAI-27 additional Thinopyrum intermedium microchromosome (minimum karyomit(e) in the division mutually, can be in achromophil situation recognition and verification, the left figure of Fig. 2 A is shown in the arrow; Fig. 2 A, right figure is the image of 1R karyomit(e) after separated) needle point fracture in 10 μ L Proteinase K (Proteinase K, recombinant PCR Grade 03115887001 Roche, 50ng/ μ L, by 1 * Taq dna polymerase buffer liquid preparation) in the 0.2ml Eppendof pipe of reaction buffer, (4000rpm) centrifugal 30s at a high speed ,-20 ℃ of placements are stand-by.
3.TAI-27 in additional Thinopyrum intermedium microchromosome DOP-PCR amplification
With embodiment 1.3.Amplification is seen Fig. 2 B.
4.TAI-27 in additional Thinopyrum intermedium microchromosome second fluorescent probe of taking turns the DOP-PCR amplified production make up
With embodiment 1.4.
5. utilize Thinopyrum intermedium microchromosome DOP-PCR amplified production probe additional among the TAI-27 that builds that the chromosome painting that the TAI-27 addition chromosome carries out is identified
With embodiment 1.5.
6.TAI-27 genomic in situ hybridization (GISH)
With reference to the method [23] of Han et al., take Thinopyrum intermedium (available from Northeast Normal University) genomic dna as probe,, for the DNA that blockades TAI-27 is identified with the China spring genomic dna, the results are shown in Figure the left figure of 2C.
7. utilizing Thinopyrum intermedium microchromosome DOP-PCR amplified production probe additional among the TAI-27 that builds that middle couchgrass metacinesis phase karyomit(e) is carried out homologous chromosomes identifies
1) method with embodiment 1.1 prepares Thinopyrum intermedium (deriving from Northeast Normal University) Metaphase Chromosome sample.
2) sample to be hybridized (film-making) is put into UV-crosslinked instrument (code-No.CL-1000) 0.125J/cm
2Crosslinked.
3) with 2 * SSC, 1 * TE damping fluid dilution probe, different probe dilution ratios is different, general 1: 5-1: 10 dilutions.
4) film-making is placed on ice, adds 5-6 μ L hybridization solution, cover plastic coverslip, put into little iron basin (the iron basin is lined with thieving paper, and water squirts), cover with plastics box, put into boiling water 5min, then put into 55 ℃ of hybridization of wet box of preheating and spend the night.
5) film-making is taken out from wet box, put into 2 * SSC and make the cover glass landing, with thieving paper the slice, thin piece back side is dried, drip a upper anti-quencher that contains DAPI, cover the cover glass of 24 * 50mm, be that available fluorescent microscope (Zeiss Universal microscope) carries out microscopy, photograph (Magnafier CCD camera) after several minutes, the later stage carries out image with Photoshop 7.0 and processes.
8. results and discussions
Result of study is seen Fig. 2.
Shown in Fig. 2 A, left figure: TAI-27 cell metacinesis phase, arrow are shown additional Thinopyrum intermedium karyomit(e): minimum, can clearly recognize in the not dyeing situation; Right figure: the TAI-27 cell metacinesis phase after additional Thinopyrum intermedium karyomit(e) is separated), under the micrurgy instrument, by the glass needle adhesion method the additional Thinopyrum intermedium karyomit(e) of TAI-27 has been carried out successful separation.
Monosome after the separation is after processing through Proteinase K, carry out two-wheeled DOP-PCR amplification, its amplification is shown in Fig. 2 B, and the amplification electrophorogram is the disperse shape, molecular weight ranges shows the amplification that monosome DNA succeeds between 250bp-2000bp.
Shown in Fig. 2 C, (the left figure of Fig. 2 C) compares with genomic in situ hybridization (GISH), chromosome painting result (the right figure of Fig. 2 C) take the monosome DNA that separates as probe shows, in TAI-27, a pair of exogenous chromosome has been replaced the dyad of wheat, and wheat has added a pair of external source microchromosome (shown in two arrows in Fig. 2 C right figure left side), and this generation to microchromosome is owing to additional dyad end fracture produces.From experimental result, the chromosome painting experiment is than the easier slight change that identifies chromosome structure of genomic in situ hybridization experiment, such as disappearance, fracture etc.
Shown in Fig. 2 D, utilize TAI-27 attach list chromosome fluorescence probe that middle couchgrass metacinesis phase karyomit(e) is carried out fluorescence in situ hybridization, the result shows that all karyomit(e)s of Thinopyrum intermedium karyomit(e) are positioned at centric position and very strong special signal all occurs, shows that itself and the additional microchromosome of TAI-27 have homology.This experimental result has reflected the evaluation that utilizes chromosome painting experiment can be advantageously applied to homologous chromosomes in the cross-fertilize seed or chromosome segment.
Embodiment 3.7ES/CS adds the chromosomal micro-separation of long fringe couchgrass and the chromosomal chromosome painting of the additional long fringe couchgrass of 7ES/CS is identified
The material wheat that adopts-long fringe couchgrass alien addition line 7ES is available from Israel Ci Man Wei Science Institute (Weizmann Institute of Sciences, Rehovot, Israel).
1.7ES/CS divide the preparation of phase mitosis metaphase
With embodiment 1.1.
2.7ES/CS in the micro-separation of additional long fringe couchgrass microchromosome
Be the fine glass stick of 1.0mm with diameter at first, under spirit lamp, utilizing Leitz to draw the manual diameter that is drawn into of pin instrument is fine glass needle about 1 μ m.The glass needle that (Olympus1M) draws by hand under inverted microscope carries out all around paddling, gently scraping by Leitz micrurgy instrument (code-No.93314 is available from German Lycra company) to the karyomit(e) of identification.Be stained with among the 7ES/CS additional long fringe couchgrass microchromosome (karyomit(e) of minimum in the division mutually, can be in achromophil situation recognition and verification, the left figure of Fig. 3 A is shown in the arrow; Fig. 3 A, right figure is the image of 7ES/CS karyomit(e) after separated) needle point fracture in 10 μ L Proteinase K (Proteinase K, recombinant PCR Grade 03115887001 Roche, 50ng/ μ L, by 1 * Taq dna polymerase buffer liquid preparation) in the 0.2ml Eppendof pipe of reaction buffer, (4000rpm) centrifugal 30s at a high speed ,-20 ℃ of placements are stand-by.
3.7ES/CS in additional long fringe couchgrass microchromosome DOP-PCR increase
With embodiment 1.3.Amplification is seen Fig. 3 B.
4.7E/CSS in additional long fringe couchgrass microchromosome second take turns the fluorescent probe structure of DOP-PCR amplified production
With embodiment 1.4.
5. utilize long fringe couchgrass microchromosome DOP-PCR amplified production probe additional among the 7ES/CS that builds that the chromosome painting that the 7ES/CS addition chromosome carries out is identified
With embodiment 1.5.
6 results and discussions
Result of study is seen Fig. 3.
As shown in Figure 3A, left figure: 7ES/CS cell metacinesis phase, arrow are shown additional long fringe couchgrass karyomit(e): minimum, can clearly recognize in the not dyeing situation; Right figure: the 7ES/CS cell metacinesis phase after additional long fringe couchgrass karyomit(e) is separated), under the micrurgy instrument, by the glass needle adhesion method the additional long fringe couchgrass karyomit(e) of 7ES/CS has been carried out successful separation.
Monosome after the separation is after processing through Proteinase K, carry out two-wheeled DOP-PCR amplification, its amplification is shown in Fig. 3 B, and the amplification electrophorogram is the disperse shape, molecular weight ranges shows the amplification that monosome DNA succeeds between 250bp-2000bp.
Shown in Fig. 3 C, the very special hybridization of the additional long fringe couchgrass monosome fluorescent probe of the 7ES/CS of micro-separation is on the long fringe couchgrass karyomit(e) that 7ES/CS adds, signal is very clear, shows to utilize the chromosome painting experiment can be advantageously applied in the additional chromosomal evaluation of long fringe couchgrass of 7ES/CS.
Should be appreciated that, although with reference to its exemplary embodiment, the present invention is shown particularly and describe, but will be understood by those skilled in the art that, under the condition that does not deviate from by the defined the spirit and scope of the present invention of accompanying claim, the variation of various forms and details can be carried out therein, the arbitrary combination of various embodiments can be carried out.
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Claims (8)
1. method of utilizing chromosome painting plant identification distant hybrids exogenous chromosome and chromosome segment, it comprises the steps:
1) chromosomal monosome or the chromosome segment to be detected in the measuring plants treated in micro-separation;
2) utilizing step 1) monosome or the chromosome segment that separate make up monosome or chromosome segment fluorescent probe;
3) utilizing step 2) monosome or the chromosome segment fluorescent probe that make up carry out fluorescence in situ hybridization to cross-fertilize seed metacinesis phase karyomit(e); With
4) analytical procedure 3) the fluorescence in situ hybridization result that obtains, determine to treat that chromosomal fluorescent signal to be detected described in the measuring plants distributes.
2. method claimed in claim 1, wherein step 1) by preparation Wide_cross in Plant kind tip of a root metacinesis phase, utilize the monosome microseparation to separate chromosomal monosome or chromosome segment to be detected.
3. method claimed in claim 1, step 2 wherein) by to step 1) monosome or the chromosome segment that separate carry out the DOP-PCR amplification, and by the nick translation method monosome of described separation or chromosome segment DOP-PCR amplified production are carried out fluorescent probe and make up.
4. method claimed in claim 3, wherein step 2) to differential from monosome carry out two-wheeled DOP-PCR amplification.
5. method claimed in claim 1, wherein said plant is monocotyledons or dicotyledons.
6. method claimed in claim 5, wherein said monocotyledons is grass.
7. method claimed in claim 5, wherein said plant is selected from wheat, rye, Thinopyrum intermedium-wheat addition line TAI-27, long fringe couchgrass-wheat addition line 7ES/CS.
8. method claimed in claim 7, wherein said karyomit(e) to be detected and chromosome segment are selected from rye 1R karyomit(e), Thinopyrum intermedium-wheat addition line TAI-27 addition chromosome, long fringe couchgrass-wheat addition line 7ES/CS addition chromosome.
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