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Ribosome DNA oligonucleotide mixed probe sleeve based on reference genome of chrysanthemum related species plant and development method

An oligonucleotide probe and reference genome technology, applied in the field of molecular cytogenetics research, can solve the problems of cumbersome probe preparation method and process, signal interference, difficult identification, etc., and achieve outstanding practicability and scientificity, repeatability High-performance, easy-to-detect effects

Pending Publication Date: 2022-01-21
NANJING AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to aim at the current analysis of the heterologous 5S rDNA and 45S rDNA used in the traditional fluorescence in situ hybridization technique in the molecular cytogenetics research of Chrysanthemum, the probe preparation method and process are cumbersome and the cost is high , and the signal is easily interfered by heterologous fragments, and it is difficult to identify the problem. Provide 5S rDNA and 45S rDNA oligonucleotide probe development methods and probe sets based on the reference genome data of Chrysanthemum and its related genera, so as to clarify The differences of various species under Chrysanthemum are outstanding in practicability and science

Method used

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  • Ribosome DNA oligonucleotide mixed probe sleeve based on reference genome of chrysanthemum related species plant and development method
  • Ribosome DNA oligonucleotide mixed probe sleeve based on reference genome of chrysanthemum related species plant and development method
  • Ribosome DNA oligonucleotide mixed probe sleeve based on reference genome of chrysanthemum related species plant and development method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1 Chrysanthemum 5S rDNA and 45S rDNA oligonucleotide probe development

[0053] Using the downloaded Arabidopsis rDNA data, local Blast comparisons were performed on the existing Chrysanthemum Brain, Chamomile, Artemisia annua and Sunflower. The consensus sequence was obtained by comparing 5S, 5.8S, 18S and 28S rDNA sequences respectively, the results are as follows figure 1 shown. The consensus sequence lengths of 5S, 5.8S, 18S and 28S rDNA were 123bp, 164bp, 952bp and 3402bp, respectively. The coding sequence of 5S rDNA showed high similarity in Chrysanthemum chinensis, Chamomile, and Artemisia annua.

[0054] In order to obtain representative 5S rDNA probes, the obtained consensus sequences were used for multiple sequence alignment to design 5S rDNA probes. The comparison results showed that the coding sequence of 5S rDNA was relatively conserved in Chrysanthemum chinensis, Chamomile and Artemisia annua. The synthetic 5SrDNA oligonucleotide probe set ...

Embodiment 2

[0070] Embodiment 2 prepares Chrysanthemum 5S rDNA and 45S rDNA oligonucleotide probe cover hybridization solution

[0071] The oligonucleotide probe set hybridization solution configuration system is as follows:

[0072]

[0073] Further, in the oligonucleotide mixed probe set, the concentration of each probe is JH 5S rDNA-1 (0.55ng / μL); JH 5S rDNA-2 (0.55ng / μL); JH 5.8S rDNA-1 ( 0.55ng / μL); JH 5.8S rDNA-2 (0.55ng / μL); JH 18S rDNA-1 (0.55ng / μL); JH 18S rDNA-2 (0.55ng / μL); JH 18S rDNA-3 (0.55 ng / μL); JH 28S rDNA-1 (0.55ng / μL); JH 28S rDNA-2 (0.55ng / μL); JH 28S rDNA-3 (0.55ng / μL); JH 28S rDNA-4 (0.55ng / μL) μL); JH 28S rDNA-5 (0.55ng / μL). The amount of each probe added was 0.5 μL.

[0074] Mix the above ingredients in a 1.5mL centrifuge tube, shake and centrifuge, place in a heating block at 105°C for denaturation for 13 minutes, take it out, and place it in -20°C ice alcohol for 10 minutes to prepare the oligonucleotide probe set hybridization solution .

Embodiment 3

[0075] Example 3 5S rDNA and 45S rDNA oligonucleotide probe set signal detection and high-resolution chromosome fluorescence in situ hybridization of Chrysanthemum plants

[0076] Experimental material: Chrysanthemum brain (C.nankinense) chromosome.

[0077] experiment method:

[0078] (1) Root tip treatment of Chrysanthemum plants: Propagate the experimental materials in MS medium respectively, and take Chrysanthemum plant tissue culture seedlings that have been subcultured 4-5 times in a constant temperature culture room at 25°C to remove residual exogenous substances in the body. Hormone, select the Chrysanthemum tissue culture seedlings after subculture to observe rooting, when 4-5 root systems with a length of 2-3cm appear in the tissue culture seedlings, take out the tissue culture seedlings from the MS medium and wash them thoroughly. Immerse the root system in a petri dish containing 0.2 μmol / L amafonophos-methyl solution for 4 hours, rinse it with clean water, cut of...

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Abstract

The invention provides a ribosome DNA oligonucleotide mixed probe sleeve based on a reference genome of a chrysanthemum related species plant, and discloses a development method of the ribosome DNA oligonucleotide mixed probe sleeve based on the reference genome of the chrysanthemum related species plant. The method specifically comprises the following steps: carrying out local Blast comparison on the existing chrysanthemum nankingense, wild chrysanthemum, artemisia scoparia and sunflower by utilizing downloaded arabidopsis thaliana rDNA data to obtain a 5S rDNA and 45S rDNA (5.8 S, 18S and 28S) consensus sequence; using the data to develop a 5S rDNA probe and a 45S rDNA probe in Oligo7 software; the synthesized 5S rDNA oligonucleotide probe sleeve and the synthesized 45SrDNA oligonucleotide probe sleeve respectively comprise 2 oligonucleotide probes and 10 oligonucleotide probes. The oligonucleotide probe development method and the probe sleeve developed by the invention can be effectively used for constructing a chromosome analysis karyotype map of a chrysanthemum plant, detection is carried out through FISH analysis by virtue of the assembled oligonucleotide probe sleeve, tedious procedures and heterologous fragments of cloning, recombination and probe marking are avoided, the procedure of FISH analysis is greatly simplified, and the experiment efficiency is improved.

Description

technical field [0001] The invention belongs to the field of molecular cytogenetics research, and discloses a ribosomal DNA oligonucleotide mixed probe set based on the reference genome of chrysanthemum close relatives. Background technique [0002] Chrysanthemum plants are mainly distributed in Europe and Asia, have high ornamental value, and are considered to be important ornamental horticultural crops in the Chamomile family. As the core germplasm resource of chrysanthemum, the cytological research of Chrysanthemum has not been fully clarified, which is very important for the exploration of chrysanthemum. origin Problems and chrysanthemum genetics and breeding research have produced huge obstacles. [0003] Ribosomal DNA (rDNA), a highly conserved tandem repeat sequence, within a genus of plant variation is conserved under intense pressure of natural selection. In higher eukaryotes, rDNA is divided into two categories: the 45S rDNA sequence that accounts for the majorit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/6841G16B20/20C12N15/11
CPCC12Q1/6895C12Q1/6841G16B20/20C12Q2527/125C12Q2563/107
Inventor 王海滨何俊陈发棣林思思房伟民陈素梅宋爱萍
Owner NANJING AGRICULTURAL UNIVERSITY
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