Cotton metaphase chromosome non-denaturing fluorescence in situ hybridization method

文档序号：527186 发布日期：2021-06-01 浏览：8次中文

阅读说明：本技术 一种棉花中期染色体非变性荧光原位杂交方法 (Cotton metaphase chromosome non-denaturing fluorescence in situ hybridization method ) 是由刘玉玲杨足君彭仁海田艳淼韦洋洋刘震李鹏涛卢全伟李兆国于 2021-03-30 设计创作，主要内容包括：本发明涉及一种棉花中期染色体非变性荧光原位杂交方法,其包括根据公布的棉花基因组序列信息,设计单染色体或染色体区段的寡核苷酸混合池；经末端连接特异标记,获得目标染色体或染色体区段的寡核苷酸混合探针；对棉花体细胞有丝分裂中期染色体进行非变性的荧光原位杂交。本发明的方法相比于传统FISH探针杂交,获得的寡核酸探针染色体特异性强,杂交信号检出率高；所设计的寡核酸探针不需要经过复杂的变性、重复洗脱等步骤,减少染色体丢失,速度快,简便快捷,结果可靠；为棉花染色体DNA重排、染色体配对、亲缘关系分析、异源染色质分析与遗传资源的高通量鉴定等提供技术支持,也为具有小型染色体的植物重复序列研究提供了借鉴。(The invention relates to a cotton metaphase chromosome non-denaturing fluorescence in situ hybridization method, which comprises the steps of designing an oligonucleotide mixing pool of a single chromosome or a chromosome segment according to published cotton genome sequence information; connecting a specific marker through the tail end to obtain an oligonucleotide mixed probe of a target chromosome or a chromosome segment; non-denaturing fluorescent in situ hybridization was performed on cotton somatic mitotic metaphase chromosomes. Compared with the traditional FISH probe hybridization, the method of the invention has the advantages that the obtained oligonucleotide probe has strong chromosome specificity and high hybridization signal detectable rate; the designed oligonucleotide probe does not need to undergo complicated steps of denaturation, repeated elution and the like, reduces chromosome loss, and has high speed, simplicity, convenience, rapidness and reliable result; provides technical support for cotton chromosome DNA rearrangement, chromosome pairing, genetic relationship analysis, heterochromatin analysis, high-throughput identification of genetic resources and the like, and also provides reference for the research of plant repetitive sequences with small chromosomes.)

1. A 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 the published cotton genome sequence information;

s2, connecting the specific mark to the oligonucleotide mixing pool through the end to obtain the marked oligonucleotide mixing pool, namely the oligonucleotide mixing probe of the target chromosome or chromosome segment;

s3, and performing non-denaturing fluorescent in situ hybridization on the cotton somatic mitosis metaphase chromosome by using the oligonucleotide mixed probe obtained in S2.

2. The cotton metaphase chromosome non-denaturing fluorescent in situ hybridization method according to claim 1, wherein in step S1, the whole genome analysis is performed on each chromosome sequence of the sequenced cotton species by using a Tandem Repeats Finder bioinformatics analysis software package on the genome sequence data, the matching parameters are set to 2, 7 and 7, which correspond to matching, mismatching, insertion/deletion, respectively, the minimum matching value of each chromosome is 50 to identify the repeat sequences, and the repeat sequences are classified into three types according to the segment size of the cycle distance; analyzing the distribution of the repetitive sequences in various cotton genome chromosomes by using SPSS software;

screening specific repetitive sequences of various cotton Gypsy transposon germ lines, and establishing a visual database of the distribution of various cotton Gypsy-RS; in the distribution visualization database of various cotton Gypsy-RS, specific oligonucleotide sequences based on cotton chromosome groups, chromosomes or chromosome segments are separated, and a series of Gypsy-RS-based oligonucleotide sequences are designed to form an oligonucleotide mixing pool.

3. The cotton oligonucleotide non-denaturing fluorescent in situ hybridization method of claim 2 wherein said RSs are classified into three categories of <20, 20-60 and > 60.

4. The cotton metaphase chromosome non-denaturing fluorescence in situ hybridization method of claim 2, wherein the SPSS software is SPSS, Chicago, IL version 22.0.

5. The cotton metaphase chromosome non-denaturing fluorescence in situ hybridization method according to claim 1, wherein in step S2, the 5' end of each selected specific Gypsy-RS oligonucleotide sequence in the oligonucleotide mixing pool is linked with a specific label to be labeled as a fluorescence in situ hybridization probe, and non-denaturing fluorescence in situ hybridization verification is performed to make the obtained oligonucleotide sequence have chromosome or chromosome segment specificity.

6. The cotton metaphase chromosome non-denaturing fluorescence in situ hybridization method of claim 1, wherein in step S3, the non-denaturing fluorescence in situ hybridization is performed by adding oligonucleotide mixed probes into 2 × SSC 1 × TE solution as hybridization solution, directly adding the mixture on prepared cotton somatic cell mitosis metaphase chromosome slides, performing hybridization, and after the hybridization, placing the slides directly in 2 × SSC solution, and the plastic film cover slides naturally fall off; drying in the dark, adding DAPI containing anti-fluorescence attenuator, adding cover glass, and observing with fluorescence microscope.

7. The cotton metaphase chromosome non-denaturing fluorescence in situ hybridization method according to claim 6, characterized in that the hybridization is performed by putting in an incubator at 42 ℃ for 1-3 h.

8. The cotton oligonucleotide non-denaturing fluorescent in situ hybridization method of claim 1 wherein the obtained oligonucleotide pool comprises Gossypium hirsutum A04, A06, A09, A10, D02 and D08 chromosome-specific Gypsy-RS, the sequence of which is shown in SEQ ID NO. 1-6.

9. The probe for cotton metaphase chromosome non-denaturing fluorescence in situ hybridization is characterized by comprising Gypsy-RS specific to upland cotton chromosomes A04, A06, A09, A10, D02 and D08, the sequence is shown as SEQ ID NO.1-6, and the 5' end of the probe is connected with a fluorescence labeling group.

10. The probe of claim 9, wherein the fluorescent labeling group comprises FAM, TAMRA, JOE, CY3, or ROX.

Technical Field

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

Background

The alloploid cotton is an important model plant for plant polyploidy research, and in the research of plant allopolyploid, chromosome evolution and distant hybridization breeding, chromosome identification is an important basis for chromosome structure analysis. The Fluorescence In Situ Hybridization (FISH) technology based on the base complementary pairing principle can realize the accurate chromosome positioning of a specific nucleic acid probe, becomes one of key technologies for identifying and recognizing genomes and chromosome groups, and is widely applied to cotton cytogenetics analysis. Currently, in researches related to 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. While rDNA sequence-based probes are limited to recognizing only a few chromosomes that contain rDNA sequences; the acquisition of chromosome specific BAC clone depends on a large amount of BAC library screening work, and in species with high genome repetitive sequence content, the influence of the repetitive sequence in the BAC clone is strong in non-specificity, so that the interference of a mixed signal in the FISH analysis process is strong, and the limitation of technical application is increased. In addition, the FISH procedure is complex, time consuming and laborious with these probes. Therefore, it is necessary to establish a new method to develop more cotton chromosome specific probes to facilitate accurate identification of chromosomes and chromosome segments, to understand the structural characteristics of specific chromosomes and chromosome segments, and to provide a reference for the research of plant repetitive sequences with small chromosomes.

Disclosure of Invention

Technical problem to be solved

In order to solve the above problems in the prior art, the present invention provides a non-denaturing fluorescence in situ hybridization (ND-FISH) for cotton chromosome, which aims to solve the problems of complicated probe labeling and complicated procedures in the current FISH. The distribution of the tandem repeat sequences represented by the probes on the chromosome is determined, the structural characteristics of the chromosome are known, and specific landmarks of the chromosome are established, so that specific chromosomes or chromosome segments are accurately identified, and comparison analysis among different cotton species is performed.

(II) technical scheme

In order to achieve the purpose, the invention adopts the main technical scheme that:

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

s1, designing an oligonucleotide mixing pool of a single chromosome or a chromosome segment according to the published cotton genome sequence information;

s3, and performing non-denaturing fluorescence in situ hybridization (ND-FISH) on the chromosome in the mitosis metaphase of the cotton somatic cell by using the oligonucleotide mixed probe obtained in S2.

The cotton metaphase chromosome non-denaturing fluorescence in situ hybridization method as described above, preferably, in step S1, performing whole genome analysis on each chromosome sequence of the sequenced cotton species by using a Tandem Repeats Finder (TRF) bioinformatics analysis software package on the genome sequence data, setting the matching parameters to be 2, 7 and 7, corresponding to matching, mismatching, insertion/deletion, respectively, the minimum matching value of each chromosome is 50 to identify Repeat Sequences (RS), and dividing the RS into three types according to the segment size of the cycle distance; analyzing the distribution of the repetitive sequences in various cotton genome chromosomes by using SPSS software;

screening various cotton variety Gypsy transposon germline specific repeat sequences (Gypsy-RS), and establishing a visual database of the distribution of various cotton variety Gypsy-RS; in the distribution visualization database of various cotton Gypsy-RS, specific oligonucleotide sequences based on cotton chromosome groups, chromosomes or chromosome segments are separated, and a series of Gypsy-RS-based oligonucleotide sequences are designed to form an oligonucleotide mixing pool.

As described above for the cotton metaphase chromosome non-denaturing fluorescence in situ hybridization method, preferably, the RSs are classified into three categories of <20, 20-60 and > 60.

The cotton metaphase chromosome non-denaturing fluorescence in situ hybridization method as described above, preferably, the SPSS software is 22.0 edition, SPSS, Chicago, IL.

In the method for non-denaturing fluorescent in situ hybridization of cotton metaphase chromosome as described above, preferably, in step S2, the specific Gypsy-RS oligonucleotide sequence selected from each oligonucleotide pool is linked to a specific label at the 5' end, and labeled as a Fluorescent In Situ Hybridization (FISH) probe, and non-denaturing fluorescent in situ hybridization (ND-FISH) verification is performed, so that the obtained oligonucleotide sequence has chromosome or chromosome segment specificity.

The method for non-denaturing fluorescent in situ hybridization of cotton metaphase chromosome as described above, preferably, in step S3, the non-denaturing fluorescent in situ hybridization is to add oligonucleotide mixed probes to 2 × SSC 1 × TE solution as hybridization solution, directly add them on the prepared cotton somatic cell mitosis metaphase chromosome slide for hybridization, after the hybridization, the slide is directly put in 2 × SSC solution, and the plastic film cover slip naturally falls off; drying in the dark, adding DAPI containing anti-fluorescence attenuator, adding cover glass, and observing with fluorescence microscope.

According to the cotton metaphase chromosome non-denaturing fluorescence in situ hybridization method, preferably, the hybridization is carried out in a 42 ℃ incubator for 1-3 h.

The cotton metaphase chromosome non-denaturing fluorescence in situ hybridization method preferably obtains an oligonucleotide mixed pool comprising Gossypium hirsutum A04, A06, A09, A10, D02 and D08 chromosome-specific Gypsy-RS, and the sequence is shown as SEQ ID NO. 1-6.

The probe for cotton metaphase chromosome non-denaturing fluorescence in situ hybridization comprises Gossypium hirsutum 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 fluorescence labeling group.

Preferably, the fluorescent label group comprises FAM, TAMRA, JOE, CY3 or ROX.

(III) advantageous effects

The invention has the beneficial effects that:

the probe provided by the invention performs non-denaturing fluorescence in situ hybridization on cotton somatic metaphase chromosomes, establishes a cotton metaphase chromosome non-denaturing FISH method (ND-FISH), provides technical support for cotton chromosome DNA rearrangement, chromosome pairing, genetic relationship analysis, heterologous chromatin identification and the like, and provides reference for the research of plant repetitive sequences 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 the research requirement, and can also cover partial segments of the chromosome, or a plurality of regions of the chromosome, or different regions of different chromosomes; the designed non-denatured oligonucleotide probe is short and 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 successfully designed, data is permanently stored, enough probe quantity can be obtained after each synthesis, the stability of the probe is strong in the using process, the using cost is low, and the probe is suitable for high-throughput identification of a large number of samples and is convenient for communication and use among different researchers.

Compared with the traditional FISH probe hybridization, the designed oligonucleotide probe does not need complicated steps of denaturation, repeated elution and the like, reduces chromosome loss, has high speed, is simple, convenient and quick, and has reliable result, and the whole hybridization process is 1-3 hours.

Drawings

FIG. 1 shows the ND-FISH verification results using upland cotton metaphase chromosome slides as targets and fluorescent dye-labeled oligonucleotide primers designed based on upland cotton genome tandem repeat sequences as probes; wherein 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 signal; d is the oligonucleotide sequence A10-23, 2 pairs of green signals; e is the oligonucleotide sequence D02-608, 1 pair of red signal; f is the oligonucleotide sequence D08-43, 1 pair green signal.

Detailed Description

For a better understanding of the present invention, reference will now be made in detail to the present invention by way of examples, which are illustrated in the accompanying drawings, and not by way of specific emphasis, of the present invention.

Example 1

The method is designed according to a tandem repeat sequence of a tetraploid upland cotton (AD1) genome sequence, and comprises the following specific steps: genome sequence data of tetraploid upland cotton was analyzed for each chromosome sequence using a Tandem Repeat Finder (TRF) bioinformatics analysis software package, matching parameters were set to 2, 7 corresponding to match, mismatch, insert/delete, respectively, the minimum match value of each chromosome was 50 to identify Repeat Sequences (RS), RSs were classified into three categories (<20, 20-60 and >60) according to the segment size of the cycle distance. The distribution of RS in various cotton genomic chromosomes was analyzed using SPSS software (version 22.0, SPSS, Chicago, IL). Screening various cotton variety Gypsy transposon germline specific repeat sequences (Gypsy-RS), and establishing a visual database of the distribution of various cotton variety Gypsy-RS. In a Gypsy-RS distribution visualization database, Gypsy-RS specific to chromosomes based on Gossypium hirsutum A04, A06, A09, A10, D02 and D08 is isolated, and oligonucleotide sequences based on the Gypsy-RS corresponding to the chromosomes are designed.

The resulting oligonucleotide sequences include:

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, in which 5' base was fluorescently labeled (D08-43-FAM; A04-222-TAMRA; A06-399-TAMRA; A09-75-FAM; D02-608-2-TAMRA) to obtain a probe for non-denaturing fluorescent in situ hybridization (ND-FISH) analysis.

The obtained fluorescence labeling probe is subjected to a non-denaturing fluorescence in situ hybridization (ND-FISH) test to verify the chromosome distribution of the probe. The specific test process comprises the following steps: (1) probe dilution: each 2 OD labeled probes were centrifuged (8000 rpm, 2min), and 200. mu.L of 1 XTE (pH8.0) was added as a mother solution and stored at-20 ℃. (2) Working fluid: the mother solution was diluted 10-fold with 2 XSSC 1 XTE (pH7.0) to prepare a hybridization solution for the probe. (wherein, the preparation method of the used solution 2 XSSC 1 XTE is that firstly 20 XSSC mother solution is prepared, 175.3g NaCl, 88.2g sodium citrate and ddH are added₂Adjusting the pH value to 7.0 by 10mol/L NaOH when the volume of the solution is between O and 1000 mL; 10 × TE mother liquor: 1mol/L Tris-HCl with the pH value of 8.05 mL and 0.5mol/L EDTA with the pH value of 8.01 mL are added with water to 50mL, and the pH value is adjusted to 8.0; the mother liquor is respectively treated with ddH₂O dilution to 4 XSSC and 2 XTE; 4 XSSC and 2 XTE were mixed in equal volumes, and the pH was adjusted to 7.0 to obtain 2 XSSC 1 XTE (pH 7.0)). (3) Hybridization solution: each 1-2. mu.L of the working solution was added to 6.0. mu.L of 2 XSSC 1 XTE (pH7.0) to obtain a hybridization solution. Directly adding on the prepared chromosome glass of the cotton somatic cell in mitosis metaphase on land, and covering with a plastic film cover plate. Placing in a 42 ℃ incubator for hybridization for 1-3 h. (4) After hybridization, the slide was directly placed in 2 XSSC solution and the plastic cover slip was naturally released. Drying in the dark, adding DAPI containing anti-fluorescence attenuator, adding cover glass, observing with fluorescence microscope, and obtaining the result shown in figure 1, wherein A is oligonucleotide sequence A04-222, and the sequence is corrected from left to rightIntegrated map of DAPI-stained chromosome map, A04-222 signal, integrated map, and results showing 2 pairs of red signals; similarly, B is the oligonucleotide sequence A06-39, 2 pairs of green signal; c is oligonucleotide sequence A09-75, 2 pairs of green signal, D is oligonucleotide sequence A10-23, 2 pairs of green signal; the above results indicate that A, B, C, D four probes each have 1 pair of signals on the homologous chromosomes of the corresponding parts of the a subgenomic and D subgenomic chromosomes, but the a subgenomic chromosome and the D subgenomic chromosome show a difference in signal intensity, i.e., the signal on the a subgenomic chromosome is slightly stronger. E is the oligonucleotide sequence D02-608, 1 pair of red signal; f is the oligonucleotide sequence D08-43, 1 pair green signal, indicating that E, F probe has only 1 pair signal in the corresponding D subgenome. The result shows that the oligonucleotide hybrid probe designed by the invention can obtain clear hybridization signals by carrying out non-denaturing fluorescence in situ hybridization on cotton somatic mitosis metaphase chromosomes, can be used as an ideal probe for chromosome recognition, and provides technical support for cotton chromosome DNA rearrangement, chromosome pairing, heterochromatin identification and the like. The chromosome specific probes are used for hybridizing cotton seeds related to the A genome, the D genome and the AD genome of the cotton diploid, and visual evidence is provided for genetic relationship analysis of cotton according to chromosome distribution and signal site difference of hybridization signals.

The FISH analysis in the prior art is carried out by adopting the probe designed in the prior art, and the interference is strong, so that no fluorescence is displayed. The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in other forms, and any person skilled in the art can change or modify the technical content disclosed above into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present invention are within the protection scope of the technical solution of the present invention.

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