CN106636424B

CN106636424B - In-situ hybridization probe and method for identifying barley genome by using same

Info

Publication number: CN106636424B
Application number: CN201710021330.2A
Authority: CN
Inventors: 陈光登; 胡德益; 李廷轩; 张锡洲; 王永东; 郑子成; 余海英; 康亮珠
Original assignee: Sichuan Agricultural University
Current assignee: Sichuan Agricultural University
Priority date: 2017-01-11
Filing date: 2017-01-11
Publication date: 2020-03-31
Anticipated expiration: 2037-01-11
Also published as: CN106636424A

Abstract

The invention provides an in situ hybridization probe and a method for identifying a barley chromosome set by using the same, the in situ hybridization probe Oligo-442A01 can be used for marking barley chromosomes, the nucleotide sequence of the probe is shown as SEQ ID NO.3, and the nucleotide is fluorescently marked to construct the probe. Using the probe and (AGG)₅The formed probe combination can effectively identify the barley genome under the condition of not carrying out chromosome degeneration, and is a probe combination method for better identifying and distinguishing the barley genome. The probe and the method provided by the invention can accurately mark and identify the barley chromosome group, have good effects on analyzing the morphology and behavior of the barley chromosome, and have good application prospects in the fields of barley molecular cytology and chromosome engineering breeding.

Description

In-situ hybridization probe and method for identifying barley genome by using same

Technical Field

The invention relates to the field of molecular cytogenetics, in particular to the determination and application of oligonucleotides in a barley chromosome in-situ hybridization (FISH) process, and also relates to the establishment of a non-denaturing fluorescence in-situ hybridization (ND-FISH) reaction system for barley by using the oligonucleotide probe provided by the invention and the application of the oligonucleotide probe to barley genome identification.

Background

The chromosome complex structure of higher organisms is complicated, and a large number of repetitive sequences exist in different chromosome regions, so that the repetitive sequences are generally marked in the field of molecular cytogenetics to identify chromosomes based on the characteristic. The method is characterized in that a probe is prepared by carrying out fluorescent labeling on a repetitive sequence, and the distribution state of a probe signal on a chromosome is analyzed by using a chromosome in situ hybridization method, so that a genome is distinguished from a chromosome group. Furthermore, it is also common practice to analyze chromosome behavior by comparing changes in probe signals on chromosomes. Further, the chromosome in situ hybridization method plays a crucial role in chromosome engineering breeding, and is one of the most effective and direct methods for identifying and distinguishing the genome of a specific species from the chromosome set.

Currently, only Hv01 has been reported as a barley subtelomere probe, which is obtained by amplifying a subtelomere repeat of the barley genome (Schubert et al, the Plant Journal 1998,14: 494). However, the process of amplifying the target sequence by labeling is complex, time-consuming, labor-consuming, and affected by various factors, so that the labeling effect is often not ideal. Especially when a large number of test operations are carried out, the method greatly influences the test progress and effect. Meanwhile, the method for carrying out the chromosome in-situ hybridization experiment by marking and amplifying the target sequence needs to carry out denaturation treatment on the chromosome of the target material, thereby not only increasing the test flow, but also easily generating displacement on the chromosome position on the glass slide in the treatment process. Currently, ND-FISH technology is gradually emerging to replace traditional FISH test methods. The method can directly mark the chromosome of the target material under the condition of invariance by designing a proper oligonucleotide probe, greatly simplifies the experimental steps, shortens the experimental time and ensures the experimental effect. Therefore, providing a nucleotide probe that stably labels the sub-telomeric region of barley would help solve the current dilemma faced in situ hybridization assays to label the sub-telomeric region of barley.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention provides an oligonucleotide probe capable of stably marking a barley genome and application of the probe in combination with other oligonucleotide probes in identification of the barley genome by an ND-FISH method.

In view of The above, The present invention has determined primers based on The results of studies of The subtelomeric region of barley chromosome by Schubert et al (The Plant Journal 1998,14: 494):

upstream primer sequence, 5'-CGAAACTCGCATTTTGGCC-3' (HvT01-F)

The downstream primer sequence, 5'-AGAGTTCCCGTAACCGGCCC-3' (HvT01-R), was used to perform PCR amplification reaction on the whole genomic DNA of cultivated barley Baudin and wild barley CN4027, and the products were detected by 2% agarose gel electrophoresis, and were found to have 4 bright bands as shown in FIG. 1. The band about 250bp is selected for recovery and plasmid construction for sequencing, and the result shows that the sequence length is 113-116bp, which indicates that two fragments with extremely high similarity which are connected in front and back exist in the recovered nucleotide. The sequence alignment analysis of the sequencing result Baudin1 by using NCBI database detects that 64 copies exist on a full-length 105111bp sequence HV _ Mba442-A01(GenBank accession number is AC 256248.1). HV _ Mba442-A01 was divided into small fragments and repeatedly aligned with itself in NCBI database to finally determine a relatively conserved nucleotide sequence of 55bp in full length for probe preparation, with the base sequence:

5'-CTACTACTCACTGATTTTGGGTCCCGGGGCGATACGAACGTTCGGGGAACTTCGG-3', designated Oligo-442A01, which was synthesized and fluorescently labeled for probe preparation. The invention provides an application of the oligonucleotide probe in a barley chromosome marking method and a chromosome set identification in an ND-FISH method.

The oligonucleotide probe of the invention can be used for detecting barley material chromosomes.

The invention provides the application of the oligonucleotide probe in the ND-FISH method for identifying barley chromosome group, which uses the oligonucleotide probe and the oligonucleotide probe (AGG)₅Combined probe combination pairThe ND-FISH test is carried out on the wheat chromosome, and the distribution of two probe signal sites of the barley chromosome set is combined, if the barley chromosome set can be distinguished under a fluorescence microscope, the oligonucleotide probe Oligo-442A01 and (AGG) are indicated₅The combination of (a) can be used to identify a barley genome.

The invention provides an oligonucleotide probe for detecting barley material chromosome, namely Oligo-442A01, the probe nucleotide sequence is as follows:

5’-CTACTACTCACTGATTTTGGGTCCCGGGGCGATACGAACGTTCGGGGAACTTCGG-3’。(SEQ IDNO.3)

further, a method of detecting chromosomes in barley material by Oligo-442A01 is provided.

The invention provides an application of an Oligo-442A01 probe in an ND-FISH method.

The invention provides an application of an Oligo-442A01 probe in improvement of barley germplasm resources.

The present invention provides Oligo-442A01 probe binding (AGG)₅The application of the probe in the ND-FISH method for identifying the barley material genome.

The invention provides a method for detecting barley material chromosome by using oligonucleotide probe Oligo-442A01 in ND-FISH method, wherein the oligonucleotide probe Oligo-442A01 is used for carrying out ND-FISH test on the barley material chromosome, if a barley part region shows corresponding signals under a fluorescence microscope, the oligonucleotide probe Oligo-442A01 can be used for detecting barley material chromosome subtelomere region in ND-FISH method.

Wherein the ND-FISH method reaction program is as follows: examining chromosome under optical microscope, selecting slide glass with clear chromosome morphology and good dispersion, dripping 10 μ l (0.35 μ l1OD/ml Oligo-442A01 probe and 9.65 μ l hybridization solution) mixed hybridization solution, covering with cover glass, placing in humid cassette, incubating at 37 deg.C for 2h, washing hybridization solution twice with 2 XSSC buffer solution, ddH₂O wash 1 time, add 10. mu.l of DAPI stain drop wise, cover the slides, observe the chromosomes under an OLYMPUS BX63 fluorescent microscope and take pictures using an OLYMPUS DP80CCD camera. The hybridization solution is as follows: 5mM Tris-HCl,0.5mM EDTA,0.15M NaCl,0.015M sodium citrate.

The invention provides application of the method in barley cytology detection.

The present invention provides an oligonucleotide probe Oligo-442A01 and an oligonucleotide probe (AGG)₅Use of oligonucleotide probe Oligo-442A01 in combination with oligonucleotide probe (AGG) for identifying a genomic barley material in a ND-FISH method₅Performing ND-FISH test on barley material chromosome according to certain proportion concentration combination, synthesizing two probe signal site distribution conditions of barley chromosome set, if the barley chromosome set can be distinguished under fluorescence microscope, indicating oligonucleotide probe Oligo-442A01 and (AGG)₅The combination of (a) can be used to identify a barley genome.

Wherein the ND-FISH method reaction program is as follows: examining chromosome under optical microscope, selecting slide glass with clear chromosome morphology and good dispersion, dripping 10ul (0.35 ul 1OD/ml Oligo-442A01 probe, 0.35 ul 1OD/ml (AGG))₅Probe and 9.30. mu.l hybridization solution), cover with a glass slide, incubate in a moist cassette at 37 ℃ for 2h, wash hybridization solution twice with 2 XSSC buffer, ddH₂O wash 1 time, add 10. mu.l of DAPI stain drop wise, cover the slides, observe the chromosomes under an OLYMPUS BX63 fluorescent microscope and take pictures using an OLYMPUS DP80CCD camera. The hybridization solution is as follows: 5mM Tris-HCl,0.5mM EDTA,0.15M NaCl,0.015M sodium citrate.

The oligonucleotide probe for marking the barley chromosome in the ND-FISH method developed by the invention directly detects the barley chromosome, and the result shows that the probe can be obviously marked in the subtelomere region of the barley chromosome. The method has the characteristics of high sensitivity and good detection effect, overcomes the defects of complex flow and large interference on the marking effect caused by the traditional plasmid marking method, and is a good oligonucleotide probe for detecting the subtelomere region of the barley chromosome. Meanwhile, the application of the ND-FISH method for identifying the barley genome has the characteristics of short time consumption, good sensitivity, good detection effect and easy differentiation of the genome, and is a better method for identifying the barley genome. By using the oligonucleotide probe and the identification method provided by the invention, the subsequent research operation is more convenient and effective.

Drawings

FIG. 1 is a PCR amplification gel electrophoresis detection chart of cultivated barley Baudin and wild barley CN4027, which is sequentially marked with Marker, Baudin and CN4027 from left to right. In the figure, 4 brighter bands exist between 100bp and 500bp, and bands of about 250bp are recovery bands.

FIG. 2 shows the comparison of the amplified sequences of Baudin of cultivated barley and CN4027 of wild barley. For each material, 24 colony coated plates with better growth vigor are selected from the coated plates, and 3 colony strips are selected from the coated plates for sequencing, wherein Baudin1, Baudin9 and Baudin21 respectively represent the 1 st, 9 th and 21 st colony strips of the material Baudin, and CN 402711, CN 402717 and CN 402719 respectively represent the 11 th, 17 th and 19 th colony strips of the material CN 4027.

FIG. 3 is a graph showing the effect of oligonucleotide probe Oligo-442A01 on the labeling of the chromosomes of cultivated barley Baudin (left) and wild barley CN4027 (right) in ND-FISH system, wherein the gray region is the DAPI-labeled barley chromosome and the subtelomere highlight region is the Oligo-442A01 signal point.

FIG. 4 shows oligonucleotide probes Oligo-442A01 and (AGG)₅The signal profiles of the chromosomes after in situ hybridization were performed on cultivated barley Baudin (left) and wild barley CN4027 (right) in the ND-FISH system. Wherein the subtelomere highlight region is Oligo-442A01 signal point, and the juxtaglomer highlight region is (AGG)₅A signal point. As can be seen from the figure, the oligonucleotide probes Oligo-442A01 and (AGG)₅The signal combinations have significant differences in signal distribution across the various genomes of barley material and similar signal distribution sites for the same genome in two barley materials that are relatively distantly related. Illustrative oligonucleotide probes Oligo-442A01 and (AGG)₅The combination of (a) can effectively distinguish barley genomes.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.

Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The technical means used in the examples are conventional means well known to those skilled in the art. The barley material used in the present invention: the cultivar Baudin and the wild variety CN4027 are from Sichuan university resource college. The biochemical reagents used in the present invention are all commercially available. The invention comprises the following reagents and formula:

DNA extraction kit: TaKaRa MiniBEST Plant DNA Extraction Kit (TaKaRa). PCR amplification kit: EmeraldAmp MAX PCR Master Mix (TaKaRa). cDNA recovery kit: TaKaRa MiniBESTAgarose Gel DNA Extraction Kit Ver.4.0 (TaKaRa). Plasmid: pMD^TM19-T Vector cloning kit (TaKaRa). Competent cells: agrobacterium tumefaciens LBA4404 Electro-cells (TaKaRa). Agarose gel: gDNA was checked using 0.5% agarose gel and cDNA was checked using 2.0% agarose gel. The specific formulation is shown in table 1.

TABLE 1

LB medium (pH 7.0): 1% (W/V) tryptone, 0.5% (W/V) Yeast Extract, 1% (W/V) NaCl.

2 XSSC buffer: 0.3M NaCl,0.03M sodium citrate.

70% formamide denaturant: 70% formamide, dissolved in2 XSSC buffer.

Hybridization solution: 5mM Tris-HCl,0.5mM EDTA,0.15M NaCl,0.015M sodium citrate.

Example 1 analysis of barley sub-telomere repeat sequences and determination of oligonucleotide Probe sequences

1. Extraction of barley genomic DNA

Selecting Baudin and CN4027 young leaves to be detected, extracting DNA by a column method (TaKaRa MiniBEST plant DNA Extraction Kit), wherein the Extraction steps are as follows:

a) 150mg of fresh young leaves were ground into fine powder with liquid nitrogen, and 500. mu.l of Buffer HS I and 10. mu.l of 50 XTDTT Buffer were rapidly added thereto and mixed, 10. mu.l of RNase A (10mg/ml) was then added thereto, followed by shaking thoroughly and mixing, and incubation in a 56 ℃ water bath for 10 minutes.

b) Add 62.5. mu.l of Buffer KAC and mix well. The mixture was left on ice for 5 minutes and centrifuged at 12,000rpm for 5 minutes. And taking the supernatant, adding buffer GB with the same volume as the supernatant, and fully and uniformly mixing.

c) Spin Column was set in the Collection Tube and the solution was transferred to the Spin Column. Mu.l of Buffer WA WAs added to the Spin Column, centrifuged at 12,000rpm for 1 minute, and the filtrate WAs discarded.

d) Mu.l of Buffer WB was added to Spin Column, centrifuged at 12,000rpm for 1 min, and the filtrate was discarded.

e) Spin Column was mounted on the Collection Tube and centrifuged at 12,000rpm for 2 minutes.

f) Spin Column was placed on a new 1.5ml centrifuge tube, and 40. mu.l of precipitation Buffer preheated to 65 ℃ was added to the center of the Spin Column membrane and allowed to stand at room temperature for 5 minutes.

g) DNA was eluted by centrifugation at 12,000rpm for 2 minutes.

h) DNA concentration and quality were checked by 0.5% agarose gel electrophoresis.

2. PCR amplification

According to the literature (Schubert I, Shi F, fungi J, et al, an effective screening for quantitative reactions and transposition of barley chromosomes added to common heat. the Plant Journal,1998,14:494.) the results of a study of barley chromosome subtelomere marker probes are referred to by published primers HvT01, the nucleotide sequence of which is as follows:

upstream primer sequence, 5'-CGAAACTCGCATTTTGGCC-3' (HvT 01-F);

downstream primer sequence, 5'-AGAGTTCCCGTAACCGGCCC-3' (HvT 01-R). The PCR amplification reaction is carried out on Baudin and CN4027 by using the primer.

And (3) PCR reaction system:

PCR reaction procedure:

the amplification results are shown in FIG. 1.

3. Determination of oligonucleotide probe nucleotide sequence and probe preparation

In FIG. 1, 4 obvious bands exist, about 250bp bands are selected for recovery, and after plasmid construction, the bands are sent for sequencing, and the result is shown in FIG. 2. The sequencing results showed that the base sequence was 113-116bp long, indicating that the discontinuous bands present in FIG. 1 are most likely consecutive repeats. The B1 sequence was introduced into the NCBI database for sequence alignment analysis, and 64 copies of the sequence were detected on a 105111bp full-length sequence HV _ Mba442-A01(GenBank accession AC 256248.1). The HV _ Mba442-A01 was divided into small fragments and repeatedly aligned with itself in the NCBI database to finally determine a relatively conserved full-length 55bp nucleotide for probe preparation, which had 75 copies in HV _ Mba442-A01 and the base sequences were:

5'-CTACTACTCACTGATTTTGGGTCCCGGGGCGATACGAACGTTCGGGGAACTTCGG-3', named Oligo-442A01, was synthesized and labeled with a fluorescent labeling substance to prepare a probe.

The oligonucleotide probes of the invention are expected to be readily and rapidly used to identify sub-telomeric regions of barley chromosomes.

Example 2 method of labeling barley chromosome with oligonucleotide Probe Oligo-442A01 in ND-FISH method

1. Chromosome preparation of barley root tip cells

Selecting root tip with root length of 1-2cm after germination in N₂Treatment in O for 4h, inactivation with acetic acid, ddH₂Cleaning, cutting meristem region, performing enzymolysis at 37 deg.C in enzyme mixture (cellulase: pectinase: 1) for 37min, removing enzyme solution, and sequentially adding ddH₂Washing with anhydrous ethanol for 2 times. Add 20. mu.l of acetic acid per 1 tip and stir well until the cells are in suspension, and drop 10. mu.l of suspension onto each slide. Checking chromosome under optical microscope, and selecting carrier with clear chromosome form and good dispersionAnd (4) sliding the glass, and keeping the glass in a refrigerator at 4 ℃ for later use after marking.

2. Chromosome in situ hybridization

Taking out the glass slide, naturally drying, and mixing the hybridization solution according to 0.35 μ l of 1 OD. multidot.ml per glass slide^-1Oligo-442A01 was mixed with 9.65. mu.l of the hybridization solution, the mixed hybridization solution was repeatedly applied to the pipette tip, 10. mu.l of the mixed hybridization solution was pipetted onto a dry slide, and the slide was incubated in a wet cassette at 37 ℃ for 2 h. Taking out, washing with 2 times SSC 2 times in dark condition, and washing with ddH₂O washing for 5min for 1 time. After air-drying, 10. mu.l of DAPI was added dropwise to each slide, and the slide was covered with a cover glass to observe the chromosome under an OLYMPUS BX63 fluorescent microscope and to take a photograph using an OLYMPUS DP80CCD camera. The hybridization solution is as follows: 5mM Tris-HCl,0.5mM EDTA,0.15M NaCl,0.015M sodium citrate.

As shown in FIG. 3, significant signal points exist in each sub-telomeric region of barley chromosome, which indicates that the oligonucleotide probe Oligo-442A01 developed by the invention has significant marking effect on the sub-telomeric region of barley in ND-FISH test, and can be used for rapidly and accurately detecting the sub-telomeric region of barley chromosome.

EXAMPLE 3 oligonucleotide probes Oligo-442A01 and (AGG)₅Establishment and application of barley genome identification method in ND-FISH method

The slides prepared in example 2 were selected and air dried naturally, and the hybridization solution was mixed in an amount of 0.35. mu.l/slide and 1 OD. multidot.ml^-1Oligo-442A01、0.35μl 1OD·ml^-1(AGG)₅Mixing with 9.30. mu.l of hybridization solution, repeatedly beating the mixed hybridization solution with a pipette, dripping 10. mu.l of mixed hybridization solution on a dry glass slide, and incubating the glass slide in a moist cassette at 37 ℃ for 2 h. Taking out, washing with 2 times SSC 2 times in dark condition, and washing with ddH₂O washing for 5min for 1 time. After air-drying, 10. mu.l of DAPI was added dropwise to each slide, and the slide was covered with a cover glass to observe the chromosome under an OLYMPUS BX63 fluorescent microscope and to take a photograph using an OLYMPUS DP80CCD camera. The hybridization solution is as follows: 5mM Tris-HCl,0.5mM EDTA,0.15M NaCl,0.015M sodium citrate.

The results are shown in FIG. 4, from which it can be seen that the oligonucleotide probe Oligo-442A provided by the present invention01 combine (AGG)₅The probe combination of (a) can effectively identify barley genome. In the figure, the subtelomere region shows the signal of Oligo probe Oligo-442A01, and the subtelomere region shows the signal of Oligo probe (AGG)₅A signal. It is characterized in that: chromosome 1H is short and short, Oligo-442A01 signal is strong in long arm and short Arm (AGG)₅The signal is distributed near the centromere, and the signal intensity is weaker; the presence of significant Oligo-442A01 signal in the short arm of chromosome 2H, and the absence of Oligo-442A01 signal in the long arm, (AGG)₅The signal is distributed near the centromere, and the signal intensity is weaker; 3H chromosome long-arm Oligo-442A01 Signal Strength (AGG)₅The signal is distributed near the centromere and has stronger signal intensity; 4H chromosome long and short Oligo-442A01 signals strongly (AGG)₅The signal distribution range is wide, the signal distribution range is mainly located near centromere and in the short arm region, and the signal intensity is strongest in all chromosome groups; the 5H chromosome short arm has obvious Oligo-442A01 signal (AGG)₅The signal is distributed near the centromere; 6H chromosome long and short Oligo-442A01 signals strongly (AGG)₅The signal is distributed near the centromere, and the signal intensity is moderate; Oligo-442A01 signal was present in both the long and short arms of chromosome 7H, but the signal intensity was the weakest, (AGG)₅The signal is concentrated near the centromere, and the signal intensity is moderate.

While the invention has been described in detail in the foregoing by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that certain modifications and improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

SEQUENCE LISTING

<110> Sichuan university of agriculture

<120> in situ hybridization probe and method for identifying barley genome by using same

<130>KHP171110047.2TQ

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<170>PatentIn version 3.5

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<211>19

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<213> Artificial sequence

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cgaaactcgc attttggcc 19

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<211>20

<212>DNA

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agagttcccg taaccggccc 20

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ctactactca ctgattttgg gtcccggggc gatacgaacg ttcggggaac ttcgg 55

Claims

1. The oligonucleotide probe for detecting barley chromosomes is characterized in that the nucleotide sequence is shown as SEQ ID NO. 3.

2. The oligonucleotide probe of claim 1, wherein the oligonucleotide probe is labeled with a fluorescent label.

3. Use of the oligonucleotide probe of claim 1 or 2 for detecting the chromosomal morphology of barley.

4. Use of the oligonucleotide probe of claim 1 or 2 for improving barley germplasm resources.

5. A method for detecting barley chromosomes using fluorescence in situ hybridization, wherein the oligonucleotide probe of claim 2 is used to perform ND-FISH detection on barley material.

6. A method for identifying barley genome by fluorescence in situ hybridization, comprising using the oligonucleotide probe of claim 2 and (AGG)₅The formed probe combination is used for carrying out ND-FISH method detection on the barley material.

7. The method of claim 5 or 6, wherein the chromosome is examined or identified under an optical microscope, a slide with a clear and well-dispersed chromosome morphology is selected, the slide is dehydrated and aired after denaturation, the mixed hybridization solution is dripped on the slide, the slide is covered with a cover slip, the slide is incubated in a humid dark, and the chromosome is observed under a fluorescence microscope after the cover slip is dripped.

8. The method of claim 7, wherein the mixed hybridization solution consists of 0.35 μ l of 1OD/ml Oligo-442A01 probe per slide and 9.65 μ l of hybridization solution, or

Each slide contained 0.35. mu.l of 1OD/ml Oligo-442A01 probe, 0.35. mu.l of 1OD/ml (AGG)₅Probe and 9.30. mu.l hybridization solution; the nucleotide sequence of the Oligo-442A01 probe is shown in SEQ ID NO. 3.

9. Use of the method of any one of claims 5 to 8 for mapping the subtelomere region of the barley chromosome.