CN112877407B - Cotton metaphase chromosome non-denaturing fluorescence in situ hybridization method - Google Patents
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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
Technical Field
The invention relates to a cotton metaphase chromosome non-denaturing fluorescence in situ hybridization method, belonging to the technical fields of bioinformatics and molecular cytogenetics.
Background
The heterotetraploid cotton is an important model plant for plant polyploidy research, and chromosome identification is an important basis for chromosome structure analysis in plant heteroploidy, chromosome evolution and distant hybridization breeding research. Fluorescent In Situ Hybridization (FISH) technology based on the base complementary pairing principle can realize accurate chromosome positioning of specific nucleic acid probes, has become one of key technologies for identifying and recognizing genome and chromosome set, and is widely applied to cotton cytogenetic analysis. Currently, in related studies of cotton FISH-based chromosome identification, probes are mainly derived from some genome-specific repetitive sequences (such as rDNA sequences), chromosome-specific BAC clones, and the like. Probes based on rDNA sequences can be limited to recognize only a few chromosomes containing rDNA sequences; the acquisition of chromosome specific BAC clones depends on a large number of BAC library screening works, and in species with high genome repetitive sequence content, the influence of repetitive sequences in BAC clones is non-specific and strong, so that the interference of mixed signals in the FISH analysis process is strong, and the limitation of technical application is increased. In addition, FISH procedures are complicated, time-consuming and labor-consuming with these probes. Therefore, a new method is necessary to be established, more cotton chromosome specific probes are developed to facilitate accurate identification of chromosomes and chromosome segments, the structural characteristics of specific chromosomes and chromosome segments are known, and reference is provided for research on plant repeated sequences with small chromosomes.
Disclosure of Invention
First, the technical problem to be solved
In order to solve the problems in the prior art, the invention provides non-denaturing fluorescence in situ hybridization (ND-FISH) for cotton chromosomes, and aims to solve the problems of complex probe marking and complex procedures of the prior FISH. The distribution of the tandem repeat sequences represented by the probes on the chromosome is clarified, the structural characteristics of the chromosome are known, and specific landmarks of the chromosome are established, so that specific chromosomes or chromosome sections are accurately identified, and comparison analysis between different cotton seeds is carried out.
(II) technical scheme
In order to achieve the above purpose, the main technical scheme adopted by the invention comprises the following steps:
a cotton metaphase chromosome non-denaturing fluorescence in situ hybridization (ND-FISH) method comprising the steps of:
s1, designing an oligonucleotide mixing pool of a single chromosome or a chromosome segment according to published cotton genome sequence information; the obtained oligonucleotide mixing pool comprises upland cotton A04, A06, A09, A10, D02 and D08 chromosome specific Gypsy-RS, and the sequence is shown as SEQ ID NO. 1-6;
s2, connecting the oligonucleotide mixing pool with a specific label through the tail end to obtain a labeled oligonucleotide mixing pool, namely an oligonucleotide mixing probe of a target chromosome or chromosome segment;
s3, performing non-denaturing fluorescence in situ hybridization (ND-FISH) on cotton somatic cell mitosis metaphase chromosomes by using the oligonucleotide mixed probe obtained in the S2.
Preferably, in step S1, genome sequence information is subjected to whole genome analysis by using Tandem Repeats Finder (TRF) bioinformatics analysis software package, each chromosome sequence of the sequenced cotton seeds is subjected to whole genome analysis, matching parameters are set to 2, 7 and 7, respectively corresponding to matching, mismatch, insertion/deletion, and the minimum matching value of each chromosome is 50 to identify Repeated Sequences (RSs), and RSs are classified into three types according to the segment sizes of the cycle distances; analyzing the distribution of the repeated sequences in the genome chromosomes of each cotton seed by using SPSS software;
screening the specific repeated sequences (Gypsy-RS) of the germline of each cotton seed Gypsy transposon, and establishing a visual database of the distribution of the Gypsy-RS of each cotton seed; in the various cotton Gypsy-RS distribution visualization databases, specific oligonucleotide sequences based on cotton seed chromosome groups, chromosomes or chromosome segments are separated, and a series of oligonucleotide sequences based on the Gypsy-RS are designed to form an oligonucleotide mixing pool.
As with the cotton metaphase chromosomal non-denaturing fluorescence in situ hybridization method described above, the RS is preferably classified into three categories <20, 20-60 and >60.
As described above, the cotton metaphase chromosomal non-denaturing fluorescence in situ hybridization method is preferably performed using SPSS software, version 22.0, SPSS, chicago, IL.
In the cotton metaphase chromosome non-denaturing fluorescence in situ hybridization method described above, preferably, in step S2, each selected specific Gypsy-RS oligonucleotide sequence in the oligonucleotide mixing pool is connected with a specific label at the 5' end, labeled as a Fluorescence In Situ Hybridization (FISH) probe, and non-denaturing fluorescence in situ hybridization (ND-FISH) verification is performed, so that the obtained oligonucleotide sequence has chromosome or chromosome segment specificity.
In the method for in situ hybridization of cotton metaphase chromosome non-denaturing fluorescence, preferably, in step S3, the non-denaturing fluorescence in situ hybridization is to add the oligonucleotide mixed probe into 2×ssc 1×te solution as hybridization solution, directly add the hybridization solution onto prepared cotton somatic cell mitosis metaphase chromosome slide, and after hybridization, directly place the slide in 2×ssc solution, and naturally drop the plastic film cover plate; air-drying in dark, adding DAPI containing anti-fluorescence attenuator, adding cover glass, and observing with fluorescence microscope.
As described above, the cotton metaphase chromosome non-denaturing fluorescence in situ hybridization method is preferably carried out by placing the cotton metaphase chromosome non-denaturing fluorescence in a 42 ℃ incubator for hybridization for 1-3 hours.
The probe for cotton metaphase chromosome non-denaturing fluorescence in situ hybridization comprises upland cotton A04, A06, A09, A10, D02 and D08 chromosome specific Gypsy-RS, the sequence is shown as SEQ ID NO.1-6, and the 5' end of the probe is connected with a fluorescent marker group.
The probe as described above, preferably the fluorescent labelling group comprises FAM, TAMRA, JOE, CY3 or ROX.
(III) beneficial effects
The beneficial effects of the invention are as follows:
the probe provided by the invention carries out non-denatured fluorescence in situ hybridization on cotton somatic cell metaphase chromosomes, establishes a cotton metaphase chromosome non-denatured FISH method (ND-FISH), provides technical support for cotton chromosome DNA rearrangement, chromosome pairing, genetic relationship analysis, heterologous chromatin identification and the like, and also provides reference for plant repeated sequence research with small chromosomes.
Compared with the traditional FISH probe, the oligonucleotide probe designed by the invention is derived from a genome sequence, and the chromosome or chromosome segment specificity of the probe is basically ensured through strict related data processing in the design process; the probe is flexible in design, can be designed to cover the whole chromosome according to research requirements, and can also cover partial fragments of the chromosome, or a plurality of areas of the chromosome, or different areas of different chromosomes; the designed non-denatured oligonucleotide probe is short and is in a specific single-stranded state, can be completely hybridized with a target chromosome, and can accurately reflect the chromosome structure; once the probe is designed successfully, the data is permanently stored, enough probe quantity can be obtained after each synthesis, the stability of the probe in the use process is strong, the use cost is low, the probe is suitable for high-flux identification of a large number of samples, and the probe is convenient for different researchers to use in a communication way.
Compared with the traditional FISH (FISH-based hybridization), the designed oligonucleotide probe does not need to undergo complex steps of denaturation, repeated elution and the like, reduces chromosome loss, is high in speed, is simple, convenient and rapid in the whole hybridization process for 1-3 hours, and is reliable in result.
Drawings
FIG. 1 shows ND-FISH verification results by taking upland cotton metaphase chromosome production as a target and taking a fluorescent dye-labeled oligonucleotide primer designed based on a upland cotton genome tandem repeat as a probe; wherein A is the red signal of oligonucleotide sequence A04-222,2; b is the green signal of the oligonucleotide sequence A06-39,2; c is the green signal of the oligonucleotide sequence A09-75,2; d is the green signal of the oligonucleotide sequence A10-23,2; e is the red signal of oligonucleotide sequence D02-608,1; f is the green signal of the oligonucleotide sequence D08-43,1.
Detailed Description
For a better understanding of the present invention, reference will now be made to the following detailed description of the invention, taken in conjunction with the accompanying drawings, in which it is not explicitly stated that the prior art is employed.
Example 1
The method is designed according to the tandem repeat sequence of the tetraploid upland cotton (AD 1) genome sequence, and comprises the following specific steps: genomic sequence data of tetraploid upland cotton was analyzed for each chromosomal sequence using Tandem Repeats Finder (TRF) bioinformatics analysis software package, the matching parameters were set to 2, 7, respectively corresponding to match, mismatch, insert/delete, and the minimum match value for each chromosome was 50 to identify Repeated Sequences (RSs), and RSs were classified into three categories according to the segment size of the cycle distance (< 20, 20-60, and > 60). The distribution of RS in the genome chromosomes of each cotton species was analyzed using SPSS software (version 22.0, SPSS, chicago, IL). Screening the specific repeated sequences (Gypsy-RS) of the germ line of each cotton seed, and establishing a visual database of the Gypsy-RS distribution of each cotton seed. In a Gypsy-RS distribution visualization database, a Gypsy-RS specific based on the upland cotton A04, A06, A09, A10, D02 and D08 chromosomes is separated, and an oligonucleotide sequence based on the Gypsy-RS corresponding to the chromosome is designed.
The resulting oligonucleotide sequences included:
A04-222(SEQ ID NO.1)：5'-GCTCAACTCATTTCTCGCAATATGAGTTGAATTTTGAAAACAGAA-3'
A06-399(SEQ ID NO.2)：5'-CGTTTAACAAAATCAATTCACAATTTCTTTCTTCTTTAAAACATTTCC-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'-AATTTCAATAATCTCTGATATGGATCCTCTCTTCACTTCACGGTTCTA-3'
D08-43(SEQ ID NO.6)：5'-TTGAATTTTGAAAACAGAAATTGAAATTACCTCAACG-3'
the designed sequence was sent to the company for oligonucleotide sequence synthesis, and fluorescent labeling (D08-43-FAM; A04-222-TAMRA; A06-399-TAMRA; A09-75-FAM; D02-608-2-TAMRA) was performed on the 5' base during synthesis to obtain a probe for non-denaturing fluorescence in situ hybridization (ND-FISH) analysis.
The obtained fluorescent-labeled probe is subjected to non-denaturing fluorescence in situ hybridization (ND-FISH) test, and chromosome distribution of the probe is verified. The specific test flow comprises: (1) probe dilution: every 2 OD labeled probes, centrifuged (8000 rpm, 2 min), 200. Mu.L of 1 XTE (pH 8.0) was added as a mother solution and stored at-20 ℃. (2) working solution: the mother liquor was diluted 10-fold with 2 XSSC 1 XSTE (pH 7.0), and a hybridization solution for the probe was prepared. (wherein, the preparation method of the solution 2 XSSC 1 XSSC TE comprises preparing 20 XSSC mother liquor of 175.3g NaCl,88.2g sodium citrate and adding ddH 2 O to 1000mL, and regulating the pH value to 7.0 by 10mol/L NaOH; 10×te mother liquor: 1mol/L Tris-HCl pH8.0 5mL,0.5mol/L EDTA pH8.0 1mL, adding water to 50mL, and adjusting pH to 8.0; the mother liquor is respectively ddH 2 O was diluted to 4 x SSC and 2 x TE; equal volumes of 4 XSSC and 2 XSSC were mixed, and the pH was adjusted to 7.0 to obtain 2 XSSC 1 XSSC (pH 7.0)). (3) hybridization solution: 1-2. Mu.L of each probe was added to 6.0. Mu.L of 2 XSSC 1 XSSC TE (pH 7.0) as a hybridization solution. Directly adding onto a prepared upland cotton somatic cell mitosis metaphase chromosome slide, and covering with a plastic film cover plate. Placing the mixture in a constant temperature box at 42 ℃ for hybridization for 1-3h. (4) After hybridization, the slide glass is directly placed in a 2 XSSC solution, and the plastic film cover plate naturally falls off. Drying in the dark, adding DAPI containing an anti-fluorescence attenuator, adding a cover glass, and observing by a fluorescence microscope, wherein A is an oligonucleotide sequence A04-222, and a corrected integration map, a DAPI-lined chromosome map, an A04-222 signal and an integration map are sequentially displayed from left to right, and the result shows that 2 pairs of red signals are displayed; likewise, B is the green signal of oligonucleotide sequences A06-39,2; c is the green signal of the oligonucleotide sequence A09-75,2, and D is the green signal of the oligonucleotide sequence A10-23,2; the above results indicate that A, B, C, D four probes are in the A subgenomic group and the D subgenomic group1 pair of signals are present on each of the corresponding partial homologous chromosomes of (a), but the difference in signal intensity is shown on the a subgenomic chromosome and the D subgenomic chromosome, i.e. the signal is slightly stronger on the a subgenomic chromosome. E is the red signal of oligonucleotide sequence D02-608,1; f is the green signal of oligonucleotide sequence D08-43,1, indicating that the E, F probe has 1 pair of signals only in the corresponding D subgenomic group. The result shows that the oligonucleotide mixed probe designed by the invention can obtain clear hybridization signals through non-denatured fluorescence in-situ hybridization of cotton somatic cell mitosis metaphase chromosomes, can be used as an ideal probe for chromosome recognition, and provides technical support for cotton chromosome DNA rearrangement, chromosome pairing, heterogenic chromatin identification and the like. The chromosome specific probes are used for hybridization in cotton diploid A genome, cotton diploid D genome and AD genome related cotton seeds, and visual evidence is provided for genetic relationship analysis of cotton according to chromosome distribution and signal locus difference of hybridization signals.
The probes designed in the prior art are adopted to perform the FISH analysis in the prior art, and no fluorescence is displayed due to strong interference. The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, and any person skilled in the art may make modifications or alterations to the above disclosed technical content to equivalent embodiments. However, any simple modification, equivalent variation and variation of the above embodiments according to the technical substance 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. The cotton metaphase chromosome non-denaturing fluorescence in situ hybridization method is characterized by comprising the following steps:
s1, designing an oligonucleotide mixing pool of a single chromosome or a chromosome segment according to published cotton genome sequence information; the obtained oligonucleotide mixing pool comprises specific Gypsy-RS of upland cotton A04, A06, A09, A10, D02 and D08 chromosomes, and the sequences are shown as SEQ ID NO. 1-6;
s2, connecting the oligonucleotide mixing pool with a specific label through the tail end to obtain a labeled oligonucleotide mixing pool, namely an oligonucleotide mixing probe of a target chromosome or chromosome segment;
s3, performing non-denaturing fluorescence in situ hybridization on the cotton somatic cell mitosis metaphase chromosome by using the oligonucleotide mixed probe obtained in the S2.
2. The cotton metaphase chromosome non-denaturing fluorescence in situ hybridization method according to claim 1, wherein in step S1, genome sequence information is analyzed by Tandem Repeats Finder bioinformatics analysis software package, whole genome analysis is performed on each chromosome sequence of the sequenced cotton seed, matching parameters are set to 2, 7, respectively corresponding to matching, mismatch, insertion/deletion, and minimum matching value of each chromosome is 50 to identify repeated sequences, and the repeated sequences are classified into three types according to fragment size of cycle distance; analyzing the distribution of the repeated sequences in the genome chromosomes of each cotton seed by using SPSS software;
screening the specific repeated sequences of the Gypsy transposon germ line of each cotton seed, and establishing a visual database of the Gypsy-RS distribution of each cotton seed; in the various cotton Gypsy-RS distribution visualization databases, specific oligonucleotide sequences based on cotton seed chromosome groups, chromosomes or chromosome segments are separated, and a series of oligonucleotide sequences based on the Gypsy-RS are designed to form an oligonucleotide mixing pool.
3. The cotton metaphase chromosomal non-denaturing fluorescent in situ hybridization method of claim 2, wherein the repeat sequences are classified into three categories <20, 20-60 and >60.
4. The cotton metaphase chromosomal non-denaturing fluorescent in situ hybridization method of claim 2, wherein the SPSS software is version 22.0, SPSS, chicago, IL.
5. The cotton metaphase chromosome non-denaturing fluorescent in situ hybridization method according to claim 2, wherein in step S2, the oligonucleotide sequences of the Gypsy-RS in the oligonucleotide mixing pool are linked at the 5' end with a specific label, labeled as fluorescent in situ hybridization probes, and non-denaturing fluorescent in situ hybridization verification is performed, so that the obtained oligonucleotide sequences have chromosome or chromosome segment specificity.
6. The method for in situ hybridization of cotton metaphase chromosome non-denaturing fluorescence according to claim 1, wherein in step S3, the non-denaturing fluorescence in situ hybridization is to add the oligonucleotide mixed probe into 2 XSSC 1 XSTE solution as hybridization solution respectively, directly add the hybridization solution onto prepared cotton somatic cell metaphase chromosome slide, after hybridization, directly put the slide into 2 XSSC solution, and the plastic film cover plate naturally falls off; air-drying in dark, adding DAPI containing anti-fluorescence attenuator, adding cover glass, and observing with fluorescence microscope.
7. The method for cotton metaphase chromosome non-denaturing fluorescence in situ hybridization according to claim 6, wherein hybridization is carried out in a 42℃incubator for 1-3 hours.
8. The probe for cotton metaphase chromosome non-denaturing fluorescence in situ hybridization is characterized by comprising upland cotton A04, A06, A09, A10, D02 and D08 chromosome specific Gypsy-RS, wherein the sequence is shown as SEQ ID NO.1-6, and the 5' end of the probe is connected with a fluorescent marking group.
9. The probe of claim 8, wherein the fluorescent labeling group comprises FAM, TAMRA, JOE, CY3 or ROX.
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