CN110747287A - Haynaldia villosa chromosome specific oligonucleotide probe and application thereof - Google Patents
Haynaldia villosa chromosome specific oligonucleotide probe and application thereof Download PDFInfo
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Abstract
The invention discloses a haynaldia villosa chromosome specific oligonucleotide probe and application thereof. The nucleotide sequence of the probe is shown as SEQ ID NO. 1, and the 5' end is marked by 6-FAM or TAMRA fluorescent group. The oligo-6VSCL56 probe provided by the invention can generate strong and clear signals at the end part and the sub-end part of each chromosome of haynaldia villosa, and belongs to an oligonucleotide probe specific to the chromosomes of haynaldia villosa. The probe is used for fluorescence in situ hybridization, so that on one hand, the time and the labor are saved, the repeatability is good, on the other hand, the cost is lower, and the same effect as the traditional genome in situ hybridization can be achieved. The total genome, the whole chromosome or the whole arm chromosome of the haynaldia villosa in the genetic background of the wheat can be quickly and accurately identified by using the probe.
Description
Technical Field
The invention belongs to the field of chromosome engineering, and particularly relates to a haynaldia villosa chromosome specific oligonucleotide probe and application thereof.
Background
The diploid Haynaldia villosa (2n is 14, VV) is a wild closely related species of wheat, has the excellent properties of powdery mildew resistance, rust disease resistance, yellow mosaic disease resistance, take-all disease resistance, eye spot disease resistance, strong tillering capacity, large spikelet number, drought resistance, high grain protein content and the like, and can provide important gene resources for genetic improvement of common wheat. At present, excellent genes of haynaldia villosa have been transferred into wheat backgrounds by means of chromosome engineering, such as powdery mildew resistance genes Pm21 and Pm55(Zhanget al.,2016), wheat yellow mosaic resistance gene Wss1(Zhao et al.,2013), stem rust resistance gene Sr52(Qiet al.,2011), and grain storage protein and yield-related genes (Zhang et al., 2015; Zhang et al.,2016b), and the like.
Cytogenetic methods are the most important and direct means to identify and track exogenous chromosomes (fragments) in the wheat background. The conventional GISH (genome in situ hybridization) technology generally uses whole genome DNA as a probe, and the method mainly comprises the following steps: firstly, extracting the whole genome DNA of the haynaldia villosa from leaves, then purifying and measuring the concentration of the genome DNA, labeling a probe by using an incised translation method, and carrying out amplification detection on signals after hybridization. The method has the defects of complex probe labeling process, high labeling cost, time consumption and the like, and has great limitation in large-scale application. Oligonucleotide (oligonucleotide) is a generic name of short nucleotides (including DNA and RNA) with a length of generally dozens of bases, and has the characteristics of simple synthesis, strong penetrating power, easy combination with DNA molecules and the like. In recent years, Oligo-FISH has been widely used for plant chromosome analysis due to its simple operation steps, short time, good repeatability of experimental results, and low cost of design and use (Du et al, 2017). Haynaldia villosa is a wild kindred species of wheat and is an important gene resource for improving wheat. Due to the limited number of probes available for the identification of haynaldia villosa, the resolution of the haynaldia villosa FISH karyotype is still insufficient to accurately characterize very small fragment translocation staining systems, making it difficult to characterize the variation between wheat and haynaldia villosa chromosomes. Therefore, development of an identification probe capable of rapidly and accurately identifying the haynaldia villosa chromosome is urgently required.
Disclosure of Invention
The invention aims to provide a haynaldia villosa chromosome specific oligonucleotide probe which can solve the problems of complex probe labeling, high price and the like in the current GISH technology aiming at the defects in the prior art.
Another object of the present invention is to provide the use of the oligonucleotide probe.
The invention also aims to provide a hybridization solution and application thereof.
In order to achieve the purpose, the invention adopts the technical scheme that:
a haynaldia villosa chromosome specific oligonucleotide probe is characterized in that the nucleotide sequence of the probe is shown as SEQ ID NO. 1.
The oligonucleotide probe of the present invention is preferably labeled with a fluorescent label.
The oligonucleotide probe is further preferably labeled at the 5' end with a 6-FAM or TAMRA fluorophore.
The invention provides a method for obtaining a specific oligonucleotide probe of a haynaldia villosa chromosome, which comprises the following steps:
(1) and (3) probe development: tandem repeat analysis was performed using Repatexplor 2(http:// www.repeatexplorer.org.) based on secondary sequencing data of the Haynaldia villosa 6VS chromosome. And designing the tandem repeat sequences with different confidences into oligonucleotide probes of 55 +/-4 bp by utilizing Oligo 7 oligonucleotide design software.
(2) Preparing a probe: the designed oligonucleotide probe was synthesized by TsingKe (Beijing Optimalaceae) Bio Inc., and the oligonucleotide sequence was fluorescently labeled with 6-FAM or TAMRA fluorophore at the 5' end.
(3) And (3) probe verification: after the probe is synthesized, the function of the probe is verified by gradually carrying out FISH experiment. The oligo sequences with attached fluorophores are used as probes to hybridize with the chromosomes of Haynaldia villosa (91C43), Haynaldia villosa [ T.durum-H.villosum ampphipid (AABBVV) ] and China spring (Chinese spring, CS, AABBDD) of triticum aestivum, so as to verify the specificity of the probes on the chromosomes of Haynaldia villosa.
(4) The correct oligonucleotide probe is determined, and the nucleotide sequence of the probe is shown as SEQ ID NO. 1.
The oligonucleotide probe disclosed by the invention is applied to detection of haynaldia villosa chromosomes.
The oligonucleotide probe disclosed by the invention is applied to detecting haynaldia villosa chromosomes in the genetic background of wheat.
A hybridization solution comprising said oligonucleotide probes and buffers at the following final concentrations: 50% dFA (deionized formamide), 10% DS (dextran sulfate), 0.3. mu.g/. mu.L ssDNA (salmon sperm DNA), 2 XSSC, probe 3.3. mu. mol/. mu.L.
The hybridization solution disclosed by the invention is applied to detection of the dasypyrum villosum chromosome.
The hybridization solution disclosed by the invention is applied to detection of haynaldia villosa chromosomes in the genetic background of wheat.
A method of detecting haynaldia villosa genome, whole chromosome or whole arm chromosome in a genetic background of wheat, comprising: making chromosome of wheat, adding hybridization solution of claim 6 or 7 for in situ hybridization, eluting, observing under a fluorescence microscope, taking a picture by an OLYMPUS DP72 CCD camera, and generating strong and clear hybridization signals in the end and sub-end regions of each chromosome of haynaldia villosa if haynaldia villosa genome, whole chromosome or whole arm chromosome is contained in the genetic background of wheat; if the genetic background of wheat does not contain the haynaldia villosa genome, the whole chromosome or the whole arm chromosome, no hybridization signal is generated.
Advantageous effects
(1) The invention provides a haynaldia villosa chromosome specific oligonucleotide probe, the oligonucleotide probe is oligo-6VSCL56, the nucleotide is composed of a base with length of 59bp, and the probe can be constructed by carrying out fluorescence labeling on the nucleotide.
(2) The oligo-6VSCL56 probe provided by the invention can generate strong and clear signals at the end part and the sub-end part of each chromosome of haynaldia villosa, and belongs to an oligonucleotide probe specific to the chromosomes of haynaldia villosa. The probe is used for Fluorescence In Situ Hybridization (FISH), so that on one hand, the time and the labor are saved, the repeatability is good, on the other hand, the cost is lower, and the same effect as the traditional Genome In Situ Hybridization (GISH) can be achieved. The total genome, the whole chromosome or the whole arm chromosome of the haynaldia villosa in the genetic background of the wheat can be quickly and accurately identified by using the probe.
Drawings
FIG. 1 is a graph showing the results of fluorescence in situ hybridization
A is chromosome of tricholoma villosum (h.villosa,91C43,2n ═ 14, VV) root tip cell metaphase, DAPI counter-stained, blue.
B and C are hybridization effect graphs of oligo probe oligo-6VSCL56 (red) combined with Haynaldia villosa genomic DNA (dv gDNA) (green) probe in metaphase of mitosis of Haynaldia villosa (H.villosa,91C43,2n ═ 14, VV) root tip cells;
d is chromosome of meissypium harderium (t.durum-h.villosa amphloid, 2n 42, AABBVV) in metaphase of root tip cells, DAPI over-stained, blue.
E and F are hybridization effect graphs of probe oligo-6VSCL56 (red) binding to Haynaldia villosa genomic DNA (dv gDNA) (green) in metaphase of mitosis of durum wheat-Haynaldia villosa diploid (T.durum-H.villosa amphloid, 2n 42, AABBVV) root tip cells;
g is chromosome of common wheat in China Spring (CS, AABBDD) root tip cell mitosis metaphase, and is blue in DAPI counterstain.
H and I are hybridization effect graphs of probe oligo-6VSCL56 (red) in the metaphase of mitosis of Chinese Spring (CS, AABBDD) root tip cells of common wheat.
Detailed Description
Example 1 fluorescence in situ hybridization verification experiment of Haynaldia villosa chromosome
(1) And preparing the haynaldia villosa root tip material:
a. putting the haynaldia villosa seeds into a culture dish paved with filter paper, adding water until the seeds are submerged, and putting the mixture into a constant-temperature incubator at 25 ℃ for culturing for about 18 hours;
b. pouring water after the seeds germinate and reveal white, keeping the filter paper moist, refrigerating at 4 ℃, and transferring the culture dish to a constant-temperature incubator at 25 ℃ for growth after 18 hours;
c. adding 20 μmoL/L Amifostine (APM) solution when the root length is about 1.5-2.0 cm, and treating in 25 deg.C incubator for about 2.5 hr;
d. after APM solution treatment, seeds are washed clean, roots are cut in sequence, the cut roots are placed into a 0.5mL centrifuge tube (with a hole) with a small amount of water, and laughing gas (N)2O) (pressure 1MPa) for about 1 hour;
e. after the laughing gas treatment is finished, adding pre-cooled 90% glacial acetic acid solution into the centrifugal tube for fixation, after 5-10min, putting the centrifugal tube into the centrifugal tube filled with 70% glacial ethanol solution, and then storing the centrifugal tube in a refrigerator at the temperature of-20 ℃ for later use.
(2) And tabletting:
a. preparing a root tip: taking out the root, drying with filter paper, and placing into the container containing ddH2Soaking in O culture dish for 5min, and removing ethanol;
b. repeating step (a);
c. taking out the roots in the step (b), cutting off root crowns by using a blade, putting the milky white meristem tissues into a 1.5mL centrifuge tube containing enzyme solution, and then putting the centrifuge tube into a water bath kettle at 37 ℃ for enzymolysis for about 50 min;
d. after the water bath was completed, the enzyme solution was aspirated, and 400. mu.L of ddH was added to the centrifuge tube2O, washing for 5min, and repeatingSecondly;
e. will ddH2O suction, then add 50. mu.L ddH into the centrifuge tube2O, stirring uniformly and oscillating by using a dissecting needle, centrifuging at 8000rpm for 1min, and repeating once again;
f. will ddH2Sucking out O, adding 50 μ L100% anhydrous ethanol into the centrifuge tube, oscillating, centrifuging at 8000rpm for 1min, sucking out 100% anhydrous ethanol;
g. repeating the step (g), standing the centrifuge tube for drying;
h. after drying, adding 40 mu L of 3:1 (glacial acetic acid: methanol) into a centrifuge tube, and uniformly mixing by oscillation;
i. placing the centrifugal tube on ice and standing for 3min to precipitate impurities;
j. sucking the upper suspension liquid by using a liquid transfer gun, preparing a dropping sheet, and dropping 8-10 mu L of suspension liquid on each glass slide;
k. standing, and observing with microscope after the diffusion is uniform, wherein the obtained product can be used for subsequent in situ hybridization experiments.
(3) And hybridization:
a. preparing a hybridization solution:
b. and (3) denaturation of the hybridization solution: the amount of hybridization solution (17.5. mu.L/piece) included 5. mu.L/piece of oligonucleotide probe; dFA (deionized formamide), 7.5. mu.L/disc; 20 XSSC, 1.5. mu.L/tablet; ssDNA (salmon sperm DNA), 0.5. mu.L/slide; 50% DS (dextran sulfate), 3. mu.L/tablet. Mixing the above hybridization solution, centrifuging, placing into a heating block at 105 deg.C for denaturation for 13min, immediately placing into ice-alcohol bath at-20 deg.C for at least 10min to prevent renaturation of hybridization solution;
c. modification of tabletting: treating the glass slide prepared in the step (2) for 3min under an ultraviolet crosslinking instrument so as to enhance the adhesive force of the chromosome on the glass slide;
d. then alkali denaturation:
① 70% ethanol (500mL) +0.15mol/L sodium hydroxide (3g), 4min, denaturation;
② dehydrating with 70% ethanol at room temperature for 5 min;
③ dehydrating with 95% ethanol at room temperature for 5 min;
④ dehydrating with 100% ethanol at room temperature for 5 min;
after gradient dehydration, air-dried for standby.
e. In situ hybridization: dropping the denatured hybridization solution on the denatured glass slide, covering the glass slide, placing the glass slide in a light-proof wet box with moderate humidity after the hybridization solution is completely diffused, and hybridizing for at least 6h in a 37 ℃ incubator;
f. elution after hybridization: the slide was taken out of the wet box and the coverslip was spun off, room temperature, ddH2O，10min；
g. After washing, the cells were air-dried, and Vecta shield solution (H1200), 7. mu.L/sheet, coverslipped, observed under a fluorescence microscope (OLYMPUS BX53), and photographed with a CCD camera of type OLYMPUS DP 72. The results are shown in FIGS. A-C.
As can be seen from FIGS. A-C, the probe oligo-6VSCL56 developed by the applicant produced strong and clear hybridization signals in the apical and subterminal regions of each chromosome of Haynaldia villosa (91C 43).
Referring to the above method, hybridization was performed in the mitotic middle stage of the durum wheat-haynaldia amphidiploid (2n ═ 42, AABBVV) root tip cells using probe oligo-6VSCL56 (red) in combination with haynaldia genomic dna (dv gdna) (green), and the hybridization effect was as shown in fig. D-F. As can be seen, the probe produced hybridization signals only in the terminal and subterminal regions of each chromosome of Haynaldia villosa, but did not produce hybridization signals in both chromosomes of the A and B genomes.
By referring to the above method, hybridization of China spring (China spring, CS, AABBDD) of Triticum aestivum was carried out using the probe oligo-6VSCL56 (red), and the hybridization effect was shown in FIG. G-I. As can be seen from the figure, no hybridization signal was detected on any of the 42 chromosomes in spring China. The probe is further proved to be a haynaldia villosa chromosome specific oligonucleotide probe.
Reference to the literature
Zhang R,Sun B,Chen J,et al.Pm55,a developmental-stage and tissue-specific powdery mildew resistance gene introgressed from Dasypyrum villosuminto common wheat[J].Theor.Appl.Genet.2016b,129,1975-1984.
Zhao R H,Wang H Y,Xiao J,et al.Introduction of 4VS Chromosomerecombinants using the CS ph1b mutant and mapping of the wheat yellow mosaicvirus resistance gene from Haynaldia villosa[J].Theoretical and AppliedGenetics,2013,126:2921-2930.
Qi L L,Pumphrey M O,Friebe B,et al.Anovel Robertsonian translocationevent leads to transfer of a stem rust resistance gene(Sr52)effective againstrace Ug99 from Dasypyrum villosum into bread wheat[J].Theor Appl Genet,2011,123:159-167.
Du P,Zhuang LF,Wang,YZ,et al.Development of oligonucleotides andmultiplex probes for quick and accurate identification of wheat andThinopyrum bessarabicum chromosomes[J].Genome,2017,60:93-103.
Zhang,R,Hou,F,Feng,Y,Zhang,W,Zhang,M,Chen,P,2015.Characterization ofa Triticum aestivum–Dasypyrum villosum T2VS·2DL translocation lineexpressing a longer spike and more kernels traits.Theor.Appl.Genet.128,2415-2425.
Zhang,R,Sun,B,Chen,J,Cao,A,Xing,L,Feng,Y,Lan,C,Chen,P,2016b.Pm55,adevelopmental-stage and tissue-specific powdery mildew resistance geneintrogressed from Dasypyrum villosum into common wheat.Theor.Appl.Genet.129,1975-1984.
Sequence listing
<110> Nanjing university of agriculture
<120> dasypyrum villosum chromosome specific oligonucleotide probe and application thereof
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<170>SIPOSequenceListing 1.0
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<212>DNA
<213> Artificial Sequence (Artificial Sequence)
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ggggacgatt tcgatggccc gtgaccaccg gtacacggct atttggacgt cgtcaaaact 60
cgtcgtttcg gccatttctg gccgttttcg tgggctatag cacactgttt tggggtcc 118
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<213> Artificial Sequence (Artificial Sequence)
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cggtacacgg ctatttggac gtcgtcaaaa ctcgtcgttt cggccatttc tggccgttt 59
Claims (10)
1. A haynaldia villosa chromosome specific oligonucleotide probe is characterized in that the nucleotide sequence of the probe is shown as SEQ ID NO. 1.
2. The oligonucleotide probe of claim 1, wherein the oligonucleotide probe is labeled with a fluorescent label.
3. The oligonucleotide probe of claim 2, wherein the 5' end of the oligonucleotide probe is labeled with a 6-FAM or TAMRA fluorophore.
4. Use of the oligonucleotide probe of any one of claims 1 to 3 for detecting haynaldia villosa chromosomes.
5. Use of the oligonucleotide probe of any one of claims 1 to 3 for detecting haynaldia villosa chromosomes in a wheat genetic background.
6. A hybridization solution comprising the oligonucleotide probe according to any one of claims 1 to 3 and a buffer.
7. The hybridization solution according to claim 6, wherein the hybridization solution is composed of the oligonucleotide probe according to claim 1 and a buffer; the final concentrations of the probes and buffers used in the hybridization solution were: the oligonucleotide probe of claim 1, 3.3 μmol/μ L, 50% dFA, 0.3 μ g/μ L ssDNA, 2 XSSC, 10% DS.
8. Use of the hybridization solution according to claim 6 for detecting haynaldia villosa chromosomes.
9. Use of the hybridization solution according to claim 6 for detecting haynaldia villosa chromosomes in a wheat genetic background.
10. A method for detecting haynaldia villosa genome, whole chromosome or whole arm chromosome in wheat genetic background, which is characterized by comprising the following steps: making chromosome of wheat, adding hybridization solution of claim 6 or 7 for in situ hybridization, eluting, observing under a fluorescence microscope, taking images by an OLYMPUS DP72 CCD camera, and generating strong and clear hybridization signals in the end and sub-end regions of each chromosome of haynaldia villosa if haynaldia villosa genome, whole chromosome or whole arm chromosome is contained in the genetic background of wheat; if the genetic background of wheat does not contain the haynaldia villosa genome, the whole chromosome or the whole arm chromosome, no hybridization signal is generated.
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