CN106636424A - In-situ hybridization probe and method for identifying barley chromosome set by adopting same - Google Patents
In-situ hybridization probe and method for identifying barley chromosome set by adopting same Download PDFInfo
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Abstract
The invention provides an in-situ hybridization probe and a method for identifying a barley chromosome set by adopting the in-situ hybridization probe. The in-situ hybridization probe Oligo-442A01 can be used for marking the barley chromosome, wherein the nucleotide sequence of the in-situ hybridization probe Oligo-442A01 is shown as SEQ ID NO.3, and the nucleotide is subjected to fluorescence labeling, so that the probe is constructed. A probe assembly composed of the probe and (AGG)5 can be used for effectively identifying the barley chromosome set under the condition that chromosome denaturation is not needed, and the method belongs to a good probe assembly method for identifying and distinguishing the barley chromosome set. The probe and the method provided by the invention can be used for accurately marking and identifying the barley chromosome set, have good effects for analyzing the morphology and the behavior of the barley chromosome, and have a good application prospect in the fields of barley molecular cytology and chromosome engineering breeding.
Description
Technical field
The present invention relates to during molecular cytogenetics field, more particularly to barley chromosome in situ hybridization (FISH)
The determination of oligonucleotides and application, the invention further relates to the oligonucleotide probe provided using the present invention carries out non denatured to barley
The foundation of FISH (ND-FISH) reaction system and the application that barley chromosome group is identified.
Background technology
Higher organism chromosome complex structure is miscellaneous, there is substantial amounts of repetitive sequence in coloured differently body region, special based on this
Property, generally it is used to identify chromosome by mark repetitive sequence in molecular cytogenetics field.It is by repetitive sequence
Carry out fluorescent label and prepare probe, using Chromosomal in situ hybridization method the distribution shape of probe signals on chromosome is analyzed
State, so as to distinguish genome and genome.Additionally, being also analysis chromosome by comparing the change of probe signals on chromosome
The common practice of behavior.Further, Chromosomal in situ hybridization method also plays vital in chromosome engineering breeding
Effect, Chromosomal in situ hybridization method be identify, distinguish particular species genome and genome is most effective, direct method it
One.
At present, it has been reported that barley subtelomeric probe only have Hv01, its be by expand barley gene group subtelomeric weight
(Schubert et al.The Plant Journal 1998,14 obtained from complex sequences:494).However, being expanded by marking
Gaining mark sequence flow is complicated, time and effort consuming, and affected by many factors so that mark effect is often undesirable.Especially entering
When row lot of experiments is operated, this method will to a great extent affect test process and effect.Meanwhile, by mark amplification mesh
Mark sequence carries out the method for Chromosomal in situ hybridization experiment to be needed to carry out degenerative treatments to target material chromosome, is not only increased
Experiment process, while easily producing displacement to the chromosome position on slide in processing procedure.Currently, ND-FISH technologies by
Step is risen, and substitutes traditional FISH test methods.It can be by the suitable few conjunction thuja acid probe of design, under conditions of consistency
Directly target material chromosome is marked, experimental procedure has greatly been simplified, has been shortened test period, while ensure that examination
Test effect.Therefore it provides the nucleotide probe in an energy stable labelling barley subtelomeric region will be helpful to solve current original position
The predicament for marking barley subtelomeric region to be faced in cross experiment.
The content of the invention
To overcome the shortcomings of in prior art, the present invention provides a kind of oligonucleotides of energy stable labelling barley chromosome group
Probe and the probe combine the application that other oligonucleotide probes identify barley chromosome group by ND-FISH methods.
Based on object above, the present invention is according to Schubert et al. (The Plant Journal 1998,14:494)
Primer is determined to the result of study in barley chromosome subtelomeric area：
Upstream primer sequence, 5 '-CGAAACTCGCATTTTGGCC-3 ' are (HvT01-F)
Downstream primer sequence, 5 '-AGAGTTCCCGTAACCGGCCC-3 ' (HvT01-R), using the primer pair Cultivate berley
Baudin, Wild Barley CN4027 complete genome DNAs carry out pcr amplification reaction, are detected by 2% agarose gel electrophoresis, send out
There are as shown in Figure 14 compared with bright wisp band in existing its product.Choose its 250bp or so bands and reclaim and build plasmid and be sequenced,
As a result show, its sequence length is 113-116bp, illustrate there are two similitude poles connected in front and back in reclaimed nucleotides
High fragment.Sequence alignment analysis are carried out to sequencing result Baudin 1 using ncbi database, detects it in one section of total length
To there are 64 copies in 105111bp sequences HV_Mba442-A01 (GenBank accession number is AC256248.1).By HV_
Mba442-A01 is divided into after small fragment and comparing with its own in ncbi database repeatedly, it is final determine one section it is relatively conservative complete
The nucleotides of a length of 55bp is prepared for probe, and its base sequence is：
5 '-CTACTACTCACTGATTTTGGGTCCCGGGGCGATACGAACGTTCGGGGAACTTCGG- 3 ', are named as
Oligo-442A01, synthesizes the sequence and fluorescence labeling is carried out to it for preparing probe.The invention provides above-mentioned few nucleosides
Acid probe is in ND-FISH methods to barley chromosome labeling method and the application of genome identification.
The oligonucleotide probe of the present invention can be used to detect barley material chromosome.
The invention provides the application that above-mentioned oligonucleotide probe is identified barley chromosome group in ND-FISH methods,
It is with above-mentioned oligonucleotide probe and oligonucleotide probe (AGG)5The probe combinations of composition carry out ND-FISH to barley chromosome
Test, two kinds of probe signals site distribution situations of comprehensive barley chromosome group, if can dye barley under fluorescence microscope
Body group differentiation is opened, and illustrates oligonucleotide probe Oligo-442A01 and (AGG)5Combination can be used to identify barley chromosome group.
The invention provides the oligonucleotide probe of detection barley material chromosome, i.e. Oligo-442A01, probe nucleosides
Acid sequence is：
5’-CTACTACTCACTGATTTTGGGTCCCGGGGCGATACGAACGTTCGGGGAACTTCGG-3’。(SEQ ID
NO.3)
Further, there is provided methods of the Oligo-442A01 in detection barley material chromosome.
The invention provides application of the Oligo-442A01 probes in ND-FISH methods.
The invention provides application of the Oligo-442A01 probes in Barley Genetic Germplasm improvement.
The invention provides Oligo-442A01 probes combine (AGG)5Probe identifies barley material in ND-FISH methods
Genomic application.
The invention provides a kind of oligonucleotide probe Oligo-442A01 detects that barley material contaminates in ND-FISH methods
The method of colour solid, carries out ND-FISH tests to barley material chromosome, if in fluorescence with oligonucleotide probe Oligo-442A01
Barley subregion shows corresponding signal under microscope, illustrates that oligonucleotide probe Oligo-442A01 can be used for ND-
Barley material chromosome subtelomeric region is detected in FISH methods.
Wherein, the ND-FISH methods response procedures are：Chromosome is checked under light microscope, the dyeing bodily form is chosen
Clear, the well dispersed slide of state, is added dropwise 10 μ l (0.35 μ l1OD/ml Oligo-442A01 probes and 9.65 μ l hybridization solutions)
Bulk crossing liquid, lid cover glass, be placed in humidity magazine in 37 DEG C incubation 2h, 2 × SSC buffer solution for cleaning hybridization solution twice,
ddH2O is washed 1 time, 10 μ l DAPI dye liquors of dropwise addition, lid cover glass, in OLYMPUS BX63 fluorescence microscopy Microscopic observations chromosome simultaneously
Photo is shot using OLYMPUS DP80CCD cameras.Above-mentioned hybridization solution is：5mM Tris-HCl,0.5mM EDTA,0.15M
NaCl, 0.015M sodium citrate.
The invention provides application of the said method in Barley Cells detection.
The invention provides a kind of oligonucleotide probe Oligo-442A01 and oligonucleotide probe (AGG)5Combination is in ND-
The genomic application of barley material is identified in FISH methods, with oligonucleotide probe Oligo-442A01 and oligonucleotide probe
(AGG)5By a certain percentage concentration combination is carried out ND-FISH and is tested to barley material chromosome, and the two of comprehensive barley chromosome group
Probe signals site distribution situation is planted, if can open barley chromosome group differentiation under fluorescence microscope, illustrates that oligonucleotides is visited
Pin Oligo-442A01 and (AGG)5Combination can be used to identify barley chromosome group.
Wherein, the ND-FISH methods response procedures are：Chromosome is checked under light microscope, the dyeing bodily form is chosen
Clear, the well dispersed slide of state, is added dropwise 10ul (0.35 μ l 1OD/ml Oligo-442A01 probes, 0.35 μ l 1OD/ml
(AGG)5Probe and 9.30 μ l hybridization solutions) bulk crossing liquid, lid cover glass, be placed in humidity magazine in 37 DEG C incubation 2h, 2 ×
SSC buffer solution for cleaning hybridization solution twice, ddH2O is washed 1 time, and 10 μ l DAPI dye liquors, lid cover glass, in OLYMPUS BX63 is added dropwise
Fluorescence microscopy Microscopic observation chromosome simultaneously shoots photo using OLYMPUS DP80CCD cameras.Above-mentioned hybridization solution is：5mM
Tris-HCl, 0.5mM EDTA, 0.15M NaCl, 0.015M sodium citrates.
Barley chromosome is marked oligonucleotide probe in the ND-FISH methods that the present invention is developed, it is directly right
Barley chromosome detected, as a result shows that the probe can significantly be marked on barley chromosome subtelomeric region.The method is not
Only there is the characteristics of sensitivity is high, Detection results are preferable, while also overcoming the flow process that traditional plasmid markers method is brought
Complicated, mark effect is disturbed larger deficiency, is a kind of oligonucleotides in good detection barley chromosome subtelomeric region
Probe.Meanwhile, the present invention provide ND-FISH methods identification barley chromosome group application have take short, sensitivity it is good, inspection
The characteristics of effect is good, genome is easily distinguishable is surveyed, is a kind of method for preferably identifying barley chromosome group.Using the present invention
The oligonucleotide probe and authentication method of offer, will make follow-up study operation more convenient effectively.
Description of the drawings
Fig. 1 is that Cultivate berley Baudin, Wild Barley CN4027PCR expand detected through gel electrophoresis figure, from left to right successively
For Marker, Baudin, CN4027.In figure between 100bp-500bp exist 4 compared with bright wisp band, 250bp or so band for reclaim
Band.
Fig. 2 is Cultivate berley Baudin, Wild Barley CN4027 extension increasing sequence comparing results.Each material is chosen from coated plate
The preferable bacterium colony coated plate of 24 plants of growing ways, then 3 bacterium colony bands sequencings are selected from coated plate, Baudin1, Baudin9, Baudin21 point
Not Biao Shi the 1st, 9,21 articles of bacterium colony bands of material Baudin, CN4027 11, CN4027 17, CN4027 19 represent respectively material
The 11st, 17,19 articles of bacterium colony bands of CN4027.
Fig. 3 be oligonucleotide probe Oligo-442A01 in ND-FISH systems to Cultivate berley Baudin (left side), wild
The mark design sketch of barley CN4027 (right side) chromosome, its grey area is the barley chromosome of DAPI marks, and subtelomeric is high
Bright area is Oligo-442A01 signaling points.
Fig. 4 is oligonucleotide probe Oligo-442A01 and (AGG)5To Cultivate berley Baudin in ND-FISH systems
(left side), Wild Barley CN4027 (right side) carry out the signal distribution plots of in situ hybridization after stain colour solid.Wherein subtelomeric highlight regions are
Oligo-442A01 signaling points, nearly centromere highlight regions are (AGG)5Signaling point.Can find from figure, oligonucleotide probe
Oligo-442A01 and (AGG)5Signal combines signal distributions in each genome of barley material and there is significant difference, and
Same genome signal distributions site in two affiliations barley material relatively far away from is similar.Illustrate that oligonucleotides is visited
Pin Oligo-442A01 and (AGG)5Combination can efficiently differentiate barley chromosome group.
Specific embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.Without departing substantially from spirit of the invention
In the case of essence, the modification made to the inventive method, step or condition or replacement belong to the scope of the present invention.
If not specializing, the conventional meanses that technological means used is well known to those skilled in the art in embodiment.
The conventional meanses that technological means used is well known to those skilled in the art in embodiment.Barley material used in the present invention
Material：Cultivar Baudin, wild varieties CN4027, are all from Sichuan Agricultural University Resource Institute.Biochemistry used herein
Reagent is commercially available.The each reagent of the present invention and formula are as follows：
DNA extraction kit：TaKaRa MiniBEST Plant DNA Extraction Kit(TaKaRa).PCR expands
Increase kit：EmeraldAmp MAX PCR Master Mix(TaKaRa).CDNA QIAquick Gel Extraction Kits：TaKaRa MiniBEST
Agarose Gel DNA Extraction Kit Ver.4.0(TaKaRa).Plasmid：pMDTM19-T Vector Cloning
Kit(TaKaRa).Competent cell：Agrobacterium tumefaciens LBA4404 Electro-Cells
(TaKaRa).Ago-Gel：GDNA, 2.0% Ago-Gel inspection cDNA are checked using 0.5% Ago-Gel.Its tool
Body formula such as table 1.
Table 1
LB culture mediums (pH 7.0)：1% (W/V) tryptone, 0.5% (W/V) Yeast Extract, 1% (W/V)
NaCl。
2 × SSC buffer solutions：0.3M NaCl, 0.03M sodium citrate.
70% formamide denaturation agent：70% formamide, in being dissolved in 2 × SSC buffer solutions.
Hybridization solution：5mM Tris-HCl, 0.5mM EDTA, 0.15M NaCl, 0.015M sodium citrates.
The analysis of the barley subtelomeric repetitive sequence of embodiment 1 and the determination of sequence oligonucleotide probe
1st, the extraction of barley gene group DNA
Detected materials Baudin, CN4027 young leaflet tablet is chosen, using column method (TaKaRa MiniBEST Plant
DNA Extraction Kit) DNA is extracted, extraction step is as follows：
A) the fresh young leaflet tablets of 150mg are taken, liquid nitrogen grinding is rapidly added the Buffer HS I and 10 μ l of 500 μ l into fine powder
50 × DTT Buffer mix, be subsequently adding the RNase A (10mg/ml) of 10 μ l, fully vibration is mixed, in 56 DEG C of water-baths
Incubate 10 minutes.
B) the Buffer KAC of 62.5 μ l are added, is fully mixed.Place on ice 5 minutes, 12,000rpm centrifugations 5 minutes.Take
Supernatant, adds and the isopyknic BufferGB of supernatant, fully mixes.
C) Spin Column are placed in into Collection Tube, solution is moved in Spin Column.By 500 μ l's
Buffer WA are added into Spin Column, 12,000rpm centrifugations 1 minute, abandon filtrate.
D) the Buffer WB of 700 μ l are added into Spin Column, filtrate is abandoned in 12,000rpm centrifugations 1 minute.
E) Spin Column are placed on Collection Tube, 12,000rpm centrifugations 2 minutes.
F) Spin Column are placed on the centrifuge tube of new 1.5ml, are added in the centre of Spin Column films
65 DEG C of 40 μ l Elution Buffer are preheated to, 5 minutes are stored at room temperature.
G) 12,000rpm is centrifuged 2 minutes eluted dnas.
H) 0.5% agarose gel electrophoresis detection DNA concentration and quality.
2nd, PCR amplifications
According to document (Schubert I, Shi F, Fuchs J, et al.An efficient screening for
terminal deletions and translocations of barley chromosomes added to common
wheat.The Plant Journal,1998,14:494.) to the result of study of barley chromosome subtelomeric label probe, ginseng
The primer HvT01 of its announcement is examined, its nucleotide sequence is as follows：
Upstream primer sequence, 5 '-CGAAACTCGCATTTTGGCC-3 ' are (HvT01-F)；
Downstream primer sequence, 5 '-AGAGTTCCCGTAACCGGCCC-3 ' are (HvT01-R).Using primer pair Baudin,
CN4027 carries out pcr amplification reaction.
PCR reaction systems：
PCR response procedures：
Amplification such as Fig. 1.
3rd, prepared by the determination of oligonucleotide probe nucleotide sequence and probe
There are 4 obvious bands in Fig. 1, select 250bp or so band to reclaim, build sample presentation sequencing after plasmid, as a result such as
Fig. 2.Sequencing result shows that base sequence is 113-116bp length, illustrates that broken strip band present in Fig. 1 is very likely continuous phase
The repetitive sequence for connecing.B1 sequences are imported in ncbi database carries out sequence alignment analysis, detects that it is in one section of total length
There are 64 copies in 105111bp sequences HV_Mba442-A01 (GenBank accession number is AC256248.1).By HV_
Mba442-A01 is divided into after small fragment and comparing with its own in ncbi database repeatedly, it is final determine one section it is relatively conservative complete
For probe preparation, there are 75 copies in it to the nucleotides of a length of 55bp, base sequence is in HV_Mba442-A01：
5 '-CTACTACTCACTGATTTTGGGTCCCGGGGCGATACGAACGTTCGGGGAACTTCGG- 3 ', are ordered
Entitled Oligo-442A01, synthesizes the sequence and it is carried out and fluorescence labeling material is marked for making probe.
The oligonucleotide probe of the expected present invention can easily and fast be used to identify barley chromosome subtelomeric region.
The oligonucleotide probe Oligo-442A01 of embodiment 2 is in ND-FISH methods to the method for barley chromosome mark
1st, barley root-tip cells chromosome sectioning
The long 1-2cm tips of a root of root are in N after selection germination24h, acetic acid inactivation, ddH are processed in O2O is cleaned, and cuts meristematic zone in 37
DEG C enzyme mixation (cellulase：Pectase=1:1) enzymolysis 37min in, to absorb and use ddH successively after enzyme liquid2O, absolute ethyl alcohol are each
Wash 2 times.Every 1 tip of a root adds 20 μ l acetic acid to be stirred well to cell and suspension is presented, and 10 μ l suspension are dripped on every slide.
Chromosome is checked under light microscope, clear, the well dispersed slide of chromosome morphology is chosen, and carries out 4 DEG C of refrigerators of mark
In save backup.
2nd, Chromosomal in situ hybridization
Take out slide to dry naturally, bulk crossing liquid presses every μ l 1ODml of slide 0.35-1Oligo-442A01
With the proportional arrangement of 9.65 μ l hybridization solutions, bulk crossing liquid is beaten repeatedly with liquid-transfering gun, draw 10 μ l bulk crossings drops in
On dry slide, lid cover glass 37 DEG C of incubation 2h in moistening magazine.After taking-up under the conditions of lucifuge 2 × SSC wash 2 times,
ddH2O is washed 1 time, each 5min.Naturally every slide is added dropwise 10 μ l DAPI after drying, and lid cover glass is in OLYMPUS BX63
Fluorescence microscopy Microscopic observation chromosome simultaneously shoots photo using OLYMPUS DP80CCD cameras.Above-mentioned hybridization solution is：5mM
Tris-HCl, 0.5mM EDTA, 0.15M NaCl, 0.015M sodium citrates.
As a result such as Fig. 3, there is significant signaling point in each chromosome subtelomeric region of barley, show the widow of present invention exploitation
Nucleotide probe Oligo-442A01 ND-FISH test in barley subtelomeric region have significantly mark effect, can
Fast and accurately it is used to detect barley chromosome subtelomeric region.
The oligonucleotide probe Oligo-442A01 of embodiment 3 and (AGG)5To barley chromosome group in ND-FISH methods
The foundation and application of authentication method
Choose the slide made in embodiment 2 to dry naturally, bulk crossing liquid presses every μ l 1OD of slide 0.35
ml-1 Oligo-442A01、0.35μl 1OD·ml-1(AGG)5With the proportional arrangement of 9.30 μ l hybridization solutions, will be mixed with liquid-transfering gun
Hybridization solution beats repeatedly, draws 10 μ l bulk crossings drops on dry slide, and lid cover glass is 37 DEG C in moistening magazine
Incubation 2h.2 × SSC washes 2 times, ddH under the conditions of lucifuge after taking-up2O is washed 1 time, each 5min.Naturally every slide drop after drying
Plus 10 μ l DAPI, lid cover glass is in OLYMPUS BX63 fluorescence microscopy Microscopic observation chromosomes and uses OLYMPUS DP80CCD
Camera shoots photo.Above-mentioned hybridization solution is：5mM Tris-HCl, 0.5mM EDTA, 0.15M NaCl, 0.015M sodium citrates.
Testing result is shown in Fig. 4, and as can be seen from Figure, the oligonucleotide probe Oligo-442A01 that the present invention is provided is combined
(AGG)5Probe combinations can effectively recognize barley chromosome group.Figure Central Asia Telomere regions highlight bar is oligonucleotide probe
Oligo-442A01 signals, nearly centromere region highlight bar is oligonucleotide probe (AGG)5Signal.Its feature is：1H chromosomes
It is short and small, it is long-armed stronger with galianconism Oligo-442A01 signals, (AGG)5Signal distributions near centromere, and its signal strength signal intensity compared with
It is weak；There is obvious Oligo-442A01 signals in 2H the short arm of a chromosome, and it is long-armed on there is no Oligo-442A01 signals,
(AGG)5Signal distributions are near centromere, and its signal strength signal intensity is weaker；3H chromosome long arm Oligo-442A01 signals are stronger,
(AGG)5Signal distributions are near centromere, and its signal strength signal intensity is stronger；4H chromosome long arms are believed with galianconism Oligo-442A01
It is number relatively strong, (AGG)5Signal distributions scope is wider, is predominantly located at centromere nearby and galianconism region, and its signal strength signal intensity is all
It is most strong in genome；There is obvious Oligo-442A01 signals in 5H the short arm of a chromosome, (AGG)5Signal distributions in
Near silk grain；6H chromosome long arms are stronger with galianconism Oligo-442A01 signals, (AGG)5Signal distributions near centromere, its
Signal strength signal intensity is more moderate；There is Oligo-442A01 signals in 7H chromosome long arms, but its signal strength signal intensity is most weak with galianconism,
(AGG)5Signal is concentrated near centromere, and its signal strength signal intensity is moderate.
Although having used general explanation, specific embodiment and test above, the present invention is described in detail,
But on the basis of the present invention, some modifications can be made to it or be improved, this is to those skilled in the art apparent
's.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to claimed
Scope.
SEQUENCE LISTING
<110>Sichuan Agricultural University
<120>A kind of in situ hybridization probe and its method to the identification of barley chromosome group
<130> KHP171110047.2TQ
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 19
<212> DNA
<213>Artificial sequence
<400> 1
cgaaactcgc attttggcc 19
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence
<400> 2
agagttcccg taaccggccc 20
<210> 3
<211> 52
<212> DNA
<213>Barley
<400> 3
ctactactca ctgattttgg gtcccggggc gatacgaacg ttcggggaac ttcgg 55
Claims (10)
1. it is used to detect the oligonucleotide probe of barley chromosome, it is characterised in that its nucleotide sequence such as SEQ ID NO.3 institutes
Show.
2. oligonucleotide probe as claimed in claim 1, it is characterised in that the oligonucleotides is visited with fluorescent label
Pin.
3. application of the oligonucleotide probe described in claim 1 or 2 in detection barley chromosome form.
4. application of the oligonucleotide probe described in claim 1 or 2 in Barley Genetic Germplasm improvement.
5. a kind of method of employing fluorescence in situ hybridization detection barley chromosome, it is characterised in that using described in claim 2
Oligonucleotide probe carries out ND-FISH detections to barley material, if detected on barley material chromosome under fluorescence microscope
Go out signal, then illustrate that the method can be used to detect barley chromosome.
6. a kind of method that employing FISH identifies barley chromosome group, it is characterised in that using described in claim 2
Oligonucleotide probe and (AGG)5The probe combinations of composition carry out ND-FISH method detections to barley material, if detection knot
Fruit shows that two-by-two chromosome nonhomologous signal distributions have differences, then show that the application of present invention offer can be applicable to differentiate barley dye
Colour solid group.
7. the method as shown in claim 5 or 6, it is characterised in that chromosome is checked or identified under light microscope, is chosen
Clear, the well dispersed slide of chromosome morphology, after degenerative treatments dehydration dry, be added dropwise bulk crossing liquid on slide,
Lid cover glass, is placed in the dark of humidity and is incubated, and cleaning is added dropwise dye liquor lid cover glass, in fluorescence microscopy Microscopic observation chromosome.
8. the method as shown in claim 7, it is characterised in that the bulk crossing liquid consists of every slide containing 0.35 μ l
1OD/ml Oligo-442A01 probes and 9.65 μ l hybridization solutions, or
Every slide contains 0.35 μ l 1OD/ml Oligo-442A01 probes, 0.35 μ l 1OD/ml (AGG)5Probe and 9.30 μ l
Hybridization solution.
9. application of the arbitrary described method of claim 5-8 in Barley Cells detection.
10. application of the arbitrary described method of claim 5-8 in chromosome engineering breeding.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110358859A (en) * | 2019-07-26 | 2019-10-22 | 南京农业大学 | A kind of oligonucleotide probe kit and usage thereof for detecting chromosomes of rye |
| CN110358859B (en) * | 2019-07-26 | 2022-06-10 | 南京农业大学 | Oligonucleotide probe kit for detecting rye chromosome and use method thereof |
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| CN106636424B (en) | 2020-03-31 |
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