CN112877407B - A nondenaturing fluorescence in situ hybridization method for cotton metaphase chromosomes - Google Patents

Abstract

The invention relates to a cotton metaphase chromosome non-denaturing fluorescence in situ hybridization method, which comprises designing an oligonucleotide mixing tank of a single chromosome or a chromosome segment according to published cotton genome sequence information; the target chromosome or the oligonucleotide mixed probe of the chromosome section is obtained through terminal connection specific mark; non-denaturing fluorescent in situ hybridization was performed on cotton somatic mitotic metaphase chromosomes. Compared with the traditional FISH probe hybridization, the method provided by the invention has the advantages that the obtained oligonucleotide probe chromosome has strong specificity and high hybridization signal detection rate; the designed oligonucleotide probe does not need to undergo complex steps of denaturation, repeated elution and the like, reduces chromosome loss, and has the advantages of high speed, simplicity, convenience, rapidness and reliable result; technical support is provided for cotton chromosome DNA rearrangement, chromosome pairing, genetic relationship analysis, heterogenic chromatin analysis, high-throughput identification of genetic resources and the like, and reference is provided for plant repeated sequence research with small chromosomes.

Description

A nondenaturing fluorescence in situ hybridization method for cotton metaphase chromosomes

Technical field

The invention relates to a non-denaturing fluorescence in situ hybridization method of cotton metaphase chromosomes, and belongs to the technical fields of bioinformatics and molecular cytogenetics.

Background technique

Allotetraploid cotton is an important model plant for plant polyploidy research. In research on plant allopolyploidization, chromosome evolution and distant hybrid breeding, chromosome identification is an important basis for chromosome structure analysis. Fluorescence in situ hybridization (FISH) technology based on the principle of complementary base pairing can achieve accurate chromosome positioning of specific nucleic acid probes. It has become one of the key technologies for identifying and identifying genomes and chromosomes, and is widely used in cotton cytogenetic analysis. application. Currently, in research related to FISH-based chromosome identification of Gossypium, probes mainly come from some special repetitive sequences of the genome (such as rDNA sequences), chromosome-specific BAC clones, etc. Probes based on rDNA sequences can only be limited to identifying a few chromosomes containing rDNA sequences; obtaining chromosome-specific BAC clones relies on a large number of BAC library screening efforts. Moreover, in species with high genome repetitive sequence content, the number of BAC clones is limited. The influence of repeated sequences is non-specific and strong, resulting in strong interference from stray signals during FISH analysis, which increases the limitations of technical applications. In addition, using these probes, the FISH process is complex, time-consuming and labor-intensive. Therefore, it is necessary to establish a new method and develop more cotton chromosome-specific probes to facilitate the accurate identification of chromosomes and chromosome segments, understand the structural characteristics of specific chromosomes and chromosome segments, and provide repeatability for plants with small chromosomes. Sequence research provides reference.

Contents of the invention

(1) Technical problems to be solved

In order to solve the above-mentioned problems of the prior art, the present invention provides a non-denaturing fluorescence in situ hybridization (ND-FISH) for cotton chromosomes, aiming to solve the current problems of complex probe labeling and cumbersome procedures for FISH. Clarify the distribution of the tandem repeat sequences represented by the probe on the chromosome, understand the structural characteristics of the chromosome, and establish specific chromosome landmarks to accurately identify specific chromosomes or chromosome segments and conduct comparative analysis among different cotton species.

(2) Technical solutions

In order to achieve the above objectives, the main technical solutions adopted by the present invention include:

A cotton metaphase chromosome non-denaturing fluorescence in situ hybridization (ND-FISH) method, which includes the following steps:

S1. Based on the published cotton genome sequence information, design an oligonucleotide mixed pool of single chromosomes or chromosome segments; the obtained oligonucleotide mixed pool includes chromosome-specific oligonucleotides of upland cotton A04, A06, A09, A10, D02 and D08. Gypsy-RS, the sequence is shown in SEQ ID NO.1-6;

S2. The oligonucleotide mixed pool is specifically labeled by end-ligation to obtain a labeled oligonucleotide mixed pool, that is, an oligonucleotide mixed probe of the target chromosome or chromosome segment;

S3. Use the oligonucleotide mixed probe obtained in S2 to perform non-denaturing fluorescence in situ hybridization (ND-FISH) on cotton somatic cell mitotic metaphase chromosomes.

As described above, in the non-denaturing fluorescence in situ hybridization method of cotton metaphase chromosomes, preferably, in step S1, the genome sequence information is analyzed using the Tandem Repeats Finder (TRF) bioinformatics analysis software package, and the sequence of each chromosome of the sequenced cotton species is carried out. For whole-genome analysis, the matching parameters are set to 2, 7, and 7, corresponding to matching, mismatching, and insertion/deletion respectively. The minimum matching value of each chromosome is 50 to identify repeated sequences (Repeats sequence, RS), and fragments according to the periodic distance Size divided RS into three categories; SPSS software was used to analyze the distribution of repeated sequences in the genome chromosomes of various cotton species;

Screen the Gypsy transposon germline-specific repeat sequences (Gypsy-RS) of various cotton species and establish a visual database of Gypsy-RS distribution of various cotton species; in the above visual database of Gypsy-RS distribution of various cotton species, separation is based on cotton chromosomes For specific oligonucleotide sequences of groups, chromosomes or chromosome segments, a series of Gypsy-RS-based oligonucleotide sequences are designed to form an oligonucleotide mixed pool.

As described above, in the non-denaturing fluorescence in situ hybridization method of cotton metaphase chromosomes, preferably, the RS is divided into three categories: <20, 20-60 and >60.

As described above, the non-denaturing fluorescence in situ hybridization method of cotton metaphase chromosomes, preferably, the SPSS software is version 22.0, SPSS, Chicago, IL.

As described above, in the non-denaturing fluorescence in situ hybridization method of cotton metaphase chromosomes, preferably, in step S2, each screened specific Gypsy-RS oligonucleotide sequence in the oligonucleotide mixed pool is connected to a specific label at the 5' end. , labeled into fluorescence in situ hybridization (FISH) probes, and non-denaturing fluorescence in situ hybridization (ND-FISH) verification is carried out, so that the obtained oligonucleotide sequence has chromosome or chromosome segment specificity.

The non-denaturing fluorescence in situ hybridization method of cotton metaphase chromosomes as described above. Preferably, in step S3, the non-denaturing fluorescence in situ hybridization is to add oligonucleotide mixed probes to 2×SSC 1×TE solution as hybridization. The solution is directly added to the prepared cotton somatic cell mitosis metaphase chromosome slide, and hybridization is performed. After hybridization, the slide is directly placed in the 2×SSC solution, and the plastic film cover slip will naturally fall off; dry it in the dark, and add anti- The fluorescence attenuator DAPI is added to a coverslip and observed under a fluorescence microscope.

For the nondenaturing fluorescence in situ hybridization method of cotton metaphase chromosomes as described above, preferably, the hybridization is placed in a 42°C incubator for 1 to 3 hours.

Probes used for non-denaturing fluorescence in situ hybridization of cotton metaphase chromosomes include Gypsy-RS specific for upland cotton A04, A06, A09, A10, D02 and D08 chromosomes. The sequences are shown in SEQ ID NO. 1-6. The probe The 5′ end of the needle is connected to a fluorescent labeling group.

For the probe as described above, preferably, the fluorescent labeling group includes FAM, TAMRA, JOE, CY3 or ROX.

(3) Beneficial effects

The beneficial effects of the present invention are:

The probe provided by the invention performs non-denaturing fluorescence in situ hybridization on cotton somatic cell metaphase chromosomes, establishes a non-denaturing FISH method (ND-FISH) for cotton metaphase chromosomes, and provides methods for cotton chromosome DNA rearrangement, chromosome pairing, genetic relationship analysis, and heterogeneity analysis. It provides technical support for identification of source chromatin, and also provides reference for the study of repeated sequences in plants with small chromosomes.

Compared with traditional FISH probes, the oligonucleotide probes designed in the present invention are derived from genome sequences. After strict relevant data processing during the design process, the chromosome or chromosome segment specificity of the probes is basically guaranteed; The design is flexible and can be designed to cover the entire chromosome, partial fragments of the chromosome, multiple regions of the chromosome, or different regions of different chromosomes according to research needs; the designed non-denaturing oligonucleotide probe is shorter , and is in a specific single-stranded state, can completely hybridize with the target chromosome, and can accurately reflect the chromosome structure; once the probe is successfully designed, the data is permanently saved, and a sufficient amount of probe can be obtained after each synthesis. During the use of the probe It has strong stability and low cost of use. It is suitable for high-throughput identification of a large number of samples and facilitates communication and use between different researchers.

Compared with traditional FISH probe hybridization, the designed oligonucleotide probe does not need to go through complex denaturation, repeated elution and other steps, reducing chromosome loss, and is fast. The entire hybridization process takes 1-3 hours, which is simple and fast, and the results are reliable.

Description of the drawings

Figure 1 shows the results of ND-FISH verification using upland cotton metaphase chromosome preparation as the target and fluorescent dye-labeled oligonucleotide primers designed based on the tandem repeat sequence of the upland cotton genome as probes; where A is the oligonucleotide sequence A04 -222, 2 pairs of red signals; B is the oligonucleotide sequence A06-39, 2 pairs of green signals; C is the oligonucleotide sequence A09-75, 2 pairs of green signals; D is the oligonucleotide sequence A10-23 , 2 pairs of green signals; E is the oligonucleotide sequence D02-608, 1 pair of red signals; F is the oligonucleotide sequence D08-43, 1 pair of green signals.

Detailed ways

In order to better explain the present invention and facilitate understanding, the present invention will be described in detail below through specific embodiments in conjunction with the accompanying drawings, and the prior art will be used if not explicitly emphasized.

Example 1

Based on the tandem repeat sequence design of the tetraploid upland cotton (AD1) genome sequence, the specific steps are as follows: Use the Tandem Repeats Finder (TRF) bioinformatics analysis software package to analyze the genome sequence data of the tetraploid upland cotton and match each chromosome sequence. The parameters are set to 2, 7, and 7, corresponding to matching, mismatching, and insertion/deletion respectively. The minimum matching value of each chromosome is 50 to identify repeated sequences (Repeats sequence, RS). The RS is divided into three parts according to the fragment size of the periodic distance. categories (<20, 20-60 and >60). SPSS software (version 22.0, SPSS, Chicago, IL) was used to analyze the distribution of RS in the genome chromosomes of various cotton species. Screen the Gypsy transposon germline-specific repeat sequences (Gypsy-RS) of various cotton species and establish a visual database of Gypsy-RS distribution of various cotton species. In the Gypsy-RS distribution visualization database, specific Gypsy-RS based on chromosomes A04, A06, A09, A10, D02 and D08 of Gossypium hirsutum were isolated, and oligonucleotide sequences based on Gypsy-RS for corresponding chromosomes were designed.

The resulting oligonucleotide sequences include:

A04-222 (SEQ ID NO.1): 5'-GCTCAACTCATTTCTCGCAATATGAGTTGAATTTTGAAAACAGAA-3'

A06-399 (SEQ ID NO.2): 5'-CGTTTAACAAAATCAATTCACAATTTCTTTCTTTCTTTAAAACATTTCC-3'

A09-75 (SEQ ID NO.3): 5'-TTGAAAAACAAAAATTGAAAATACCTCAACGTGTCTTGAGGTTCA-3'

A10-23 (SEQ ID NO.4): 5'-AACACTTCAATTTGCAGCACTTT-3'

D02-608 (SEQ ID NO.5): 5'-AATTTCAATAATCTTCTGATATGGATCCTCTTCACTTCACGGTTCTA-3'

D08-43 (SEQ ID NO.6): 5'-TTGAATTTTGAAAACAGAAATTGAAATTACCTCAACG-3'

Send the designed sequence to the company for oligonucleotide sequence synthesis, and fluorescently label the 5' end base during synthesis (D08-43-FAM; A04-222-TAMRA; A06-399-TAMRA; A09-75-FAM ; D02-608-2-TAMRA) to obtain probes for nondenaturing fluorescence in situ hybridization (ND-FISH) analysis.

The obtained fluorescently labeled probe was subjected to nondenaturing fluorescence in situ hybridization (ND-FISH) test to verify the chromosomal distribution of the probe. The specific test process includes: (1) Probe dilution: for every 2 OD labeled probes, centrifuge (8000 rpm, 2 min), add 200 μL 1×TE (pH8.0) as the mother solution, and store at -20°C. (2) Working solution: Use 2×SSC 1×TE (pH7.0) to dilute the mother solution 10 times to prepare the probe hybridization working solution. (Among them, the preparation method of the solution 2×SSC 1×TE is: first prepare 20×SSC mother solution: 175.3g NaCl, 88.2g sodium citrate, add ddH ₂ O to 1000mL, and adjust the pH value to 7.0 with 10mol/L NaOH ; 10×TE mother liquor: 1mol/L Tris-HCl pH 8.0 5ml, 0.5mol/L EDTA pH 8.0 1ml, add water to 50mL, adjust pH to 8.0; the above mother liquor is diluted with ddH ₂ O to 4×SSC and 2×TE; mix 4×SSC and 2×TE in equal volumes and adjust the pH to 7.0 to obtain 2×SSC 1×TE (pH 7.0)). (3) Hybridization solution: Add 1-2 μL working solution of each probe into 6.0 μL 2×SSC 1×TE (pH value 7.0) to form the hybridization solution. Add it directly to the prepared upland cotton somatic cell mitosis metaphase chromosome slide, and cover it with a plastic cover slip. Place in a 42°C incubator for hybridization for 1-3 hours. (4) After hybridization, place the slide directly in the 2×SSC solution, and the plastic cover slip will fall off naturally. Dry in the dark, add DAPI containing anti-fluorescence attenuator, add a cover slip, and observe with a fluorescence microscope. The results are shown in Figure 1, where A is the oligonucleotide sequence A04-222, as shown in the figure, from left to right. The corrected integration map, DAPI-stained chromosome map, A04-222 signal, and integration map are shown in order. The results show that there are 2 pairs of red signals; similarly, B is the oligonucleotide sequence A06-39, and 2 pairs of green signals; C is the oligonucleotide sequence A09-75, with 2 pairs of green signals, and D is the oligonucleotide sequence A10-23, with 2 pairs of green signals; the above results show that the four probes A, B, C, and D are in the A subgenome and The corresponding homologous chromosomes of the D subgenome each have one pair of signals, but the signal intensity on the chromosomes of the A subgenome and the chromosome of the D subgenome shows a difference, that is, the signal on the chromosome of the A subgenome is slightly stronger. E is the oligonucleotide sequence D02-608, with one pair of red signals; F is the oligonucleotide sequence D08-43, with one pair of green signals, indicating that the E and F probes only have one pair of signals in the corresponding D subgenome. The results show that the oligonucleotide hybrid probe designed in the present invention can obtain clear hybridization signals through non-denaturing fluorescence in situ hybridization of cotton somatic cell mitotic metaphase chromosomes, and can be used as an ideal probe for chromosome identification. Provide technical support for cotton chromosome DNA rearrangement, chromosome pairing, heterologous chromatin identification, etc. These chromosome-specific probes are used to hybridize the cotton diploid A genome, D genome, and AD genome-related cotton species of the genus Gossypium. Based on the chromosome distribution and signal site differences of the hybridization signal, it will also provide intuitive analysis of the genetic relationship of the genus Gossypium. evidence.

However, when using probes designed with existing technology to perform FISH analysis using existing technology, there is no fluorescence display due to strong interference. The above are only preferred embodiments of the present invention and are not intended to limit the present invention in other forms. Any person skilled in the art can make changes or modifications to equivalent embodiments with equivalent changes using the technical contents disclosed above. . However, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the protection scope of the technical solution of the present invention.

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Claims (9)

1. A nondenaturing fluorescence in situ hybridization method for cotton metaphase chromosomes, characterized in that it includes the following steps: S1. Based on the published cotton genome sequence information, design an oligonucleotide mixed pool for single chromosomes or chromosome segments; the obtained oligonucleotide mixed pool includes specific oligonucleotides for upland cotton A04, A06, A09, A10, D02 and D08 chromosomes. Gypsy-RS, the sequence is shown in SEQ ID NO.1-6; S2. The oligonucleotide mixed pool is specifically labeled by end-ligation to obtain a labeled oligonucleotide mixed pool, that is, an oligonucleotide mixed probe of the target chromosome or chromosome segment; S3. Use the oligonucleotide mixed probe obtained in S2 to conduct non-denaturing fluorescence in situ hybridization of cotton somatic cell mitotic metaphase chromosomes. 2. The non-denaturing fluorescence in situ hybridization method of cotton metaphase chromosomes as claimed in claim 1, characterized in that, in step S1, the genome sequence information is analyzed using the Tandem Repeats Finder biological information analysis software package, and each of the sequenced cotton species is analyzed. Whole-genome analysis of chromosome sequences was carried out. The matching parameters were set to 2, 7, and 7, corresponding to matches, mismatches, and insertions/deletions respectively. The minimum matching value of each chromosome was 50 to identify repeated sequences. Repeats were classified according to the segment size of the periodic distance. The sequences were divided into three categories; SPSS software was used to analyze the distribution of repeated sequences in the genome chromosomes of various cotton species; Screen the Gypsy transposon germline-specific repeat sequences of various cotton species and establish a visual database of Gypsy-RS distribution of various cotton species; in the above Gypsy-RS distribution visual database of various cotton species, the separation is based on the cotton species chromosome group, chromosome or chromosome Based on the specific oligonucleotide sequence of the segment, a series of oligonucleotide sequences based on Gypsy-RS are designed to form an oligonucleotide mixed pool. 3. The non-denaturing fluorescence in situ hybridization method of cotton metaphase chromosomes according to claim 2, characterized in that the repeated sequences are divided into three categories: <20, 20-60 and >60. 4. The non-denaturing fluorescence in situ hybridization method of cotton metaphase chromosomes according to claim 2, characterized in that the SPSS software is version 22.0, SPSS, Chicago, IL. 5. The non-denaturing fluorescence in situ hybridization method of cotton metaphase chromosomes as claimed in claim 2, characterized in that, in step S2, the oligonucleotide sequence of Gypsy-RS in the oligonucleotide mixing pool is at 5′ The end is connected to a specific label, labeled into a fluorescent in situ hybridization probe, and non-denaturing fluorescence in situ hybridization verification is carried out, so that the obtained oligonucleotide sequence has chromosome or chromosome segment specificity. 6. The non-denaturing fluorescence in situ hybridization method of cotton metaphase chromosomes as claimed in claim 1, characterized in that, in step S3, the non-denaturing fluorescence in situ hybridization is to add oligonucleotide mixed probes to 2×SSC respectively. 1×TE solution is used as the hybridization solution, and is directly added to the prepared cotton somatic cell mitosis metaphase chromosome slides for hybridization. After hybridization, the slides are directly placed in the 2×SSC solution, and the plastic film coverslips will naturally fall off; protect from light. Dry, add DAPI containing anti-fluorescence attenuation agent, add a coverslip, and observe with a fluorescence microscope. 7. The nondenaturing fluorescence in situ hybridization method of cotton metaphase chromosomes according to claim 6, characterized in that the hybridization is placed in a 42°C incubator for 1 to 3 hours. 8. A probe for non-denaturing fluorescence in situ hybridization of cotton metaphase chromosomes, characterized in that it includes Gypsy-RS specific to the chromosomes of Gossypium hirsutum A04, A06, A09, A10, D02 and D08, with a sequence such as SEQ ID NO.1 As shown in -6, the 5' end of the probe is connected to a fluorescent labeling group. 9. The probe of claim 8, wherein the fluorescent labeling group includes FAM, TAMRA, JOE, CY3 or ROX.