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Fluorescence in-situ hybridization probe for identifying group B chromosomes of triticum aestivum as well as design method and application of fluorescence in-situ hybridization probe

A fluorescence in situ hybridization and chromosome technology, applied in the field of molecular cytogenetics, can solve the problems of insufficient signal, clear, wrong identification, inaccuracy, etc., to achieve good application prospects, save time, and improve experimental efficiency.

Active Publication Date: 2021-11-26
GUIZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this study only used one probe for identification on group B chromosomes, it can be seen from the signal photos that the signal is negligible, and the signal is not obvious and clear, which may lead to identification errors or inaccurate situations

Method used

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  • Fluorescence in-situ hybridization probe for identifying group B chromosomes of triticum aestivum as well as design method and application of fluorescence in-situ hybridization probe
  • Fluorescence in-situ hybridization probe for identifying group B chromosomes of triticum aestivum as well as design method and application of fluorescence in-situ hybridization probe
  • Fluorescence in-situ hybridization probe for identifying group B chromosomes of triticum aestivum as well as design method and application of fluorescence in-situ hybridization probe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] 1. Development of oligonucleotide probes

[0069] (1) Download the wheat genome data from the NCBI database, and use MISA software to analyze the SSR repeat sequence of the above data, and obtain different types of wheat SSR repeat sequences.

[0070] (2) Select the SSR with a large number of repetitions as the candidate sequence. The obtained candidate SSR sequences were compared and verified by the online visual comparison website B2DSC (http: / / mcgb.uestc.edu.cn / b2dsc), and the SSR sequences with the appropriate size (20-60bp) of wheat fragments were selected.

[0071] (3) Obtain the SSR sequence shown in the sequence listing.

[0072] 2. Probe Preparation

[0073] The SSR sequence designed in 1 was sent to Thermo Fisher Scientific (Shanghai) Co., Ltd. to synthesize an oligonucleotide probe, and its 5' end was fluorescently labeled with 6-FAM.

Embodiment 2

[0075] 1. Wheat root tip chromosome preparation

[0076] (1) Put the common wheat seeds into a petri dish and soak them in distilled water for 6 hours, put them on filter paper to germinate, and wash them twice a day with distilled water.

[0077] (2) When the root grows to 1.5-2cm, pour off the excess water in the culture dish, cut off the root tip and put it into a 0.5mL centrifuge tube that has been punched in the lid and sprayed with distilled water to wet the tube wall, and then put the centrifuge tube into filled with N 2 O gas in a closed metal tank for 2h.

[0078] (3) Take out the centrifuge tube, add 90% acetic acid to fix it for 5 minutes, and rinse it with distilled water 3 times.

[0079] (4) Cut the roots cleaned with distilled water and take the root tip meristematic zone, put them into 1% pectinase and 2% cellulase solution, and put them in a water bath at 37°C for 50 minutes for enzymatic hydrolysis.

[0080] (5) The enzymatically hydrolyzed root tip was wa...

Embodiment 3

[0084] 1. FISH test

[0085] (1) Prepare a hybridization solution system, each slide contains probe 0.8 μL, ssDNA 4 μL, 2×SSC+1×TE 5.2 μL.

[0086] (2) Put the slides with clear chromosomes into the ultraviolet cross-linking instrument for 2-3 times of cross-linking.

[0087] (3) 10 μL of hybridization solution was dropped on each slide, covered with a cover glass and placed in a humid aluminum box for hybridization at 42°C for 2 hours.

[0088] (4) Rinse the coverslips with 2×SSC and dry them, add 20 μL DAPI dropwise to each slide, and cover the coverslips.

[0089] Further, the DAPI solution is obtained by diluting the DAPI stock solution (VECTOR, USA) 3 times with 1×PBS.

[0090] (5) Slides were placed under an Olympus BX60 fluorescence microscope and photographed with a Cellsens Standard camera system.

[0091] 2. Result identification

[0092] Probe first (ATGTTG) 4 Do the first FISH, and observe the photographs, such as figure 1 shown; following signal elution on t...

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Abstract

The invention belongs to the field of molecular cell genetics, and particularly relates to a fluorescence in-situ hybridization probe for identifying group B chromosomes of triticum aestivum as well as a design method and application of the fluorescence in-situ hybridization probe. An oligonucleotide probe is designed on the basis of a genome SSR sequence of the triticum aestivum, and compared with a probe set consisting of multiple probes, clear karyotypes of group B chromosomes can be obtained by using the probe to perform fluorescence in-situ hybridization (FISH), so that not only is the experiment input cost reduced, but also the time is saved, and the experiment efficiency is improved. The probe disclosed by the invention can be used for rapidly and accurately identifying the group B chromosome in the wheat genetic background, and has a good application prospect in the field of wheat genetic breeding.

Description

technical field [0001] The invention belongs to the field of molecular cytogenetics, and in particular relates to a fluorescent in situ hybridization probe for identifying common wheat group B chromosomes and a design method and application thereof. Background technique [0002] Common wheat (scientific name: Triticum aestivum L), belonging to the genus Triticum Poaceae, is one of the most important food crops in the world. However, due to the loss or failure of a large number of genes controlling beneficial traits during the long-term evolution process, a high-efficiency, Accurate method for the identification of genetic material in wheat. [0003] Fluorescence in situ hybridization (FISH) is a new in situ hybridization method formed by replacing isotope labels with fluorescent labels. At present, in the molecular cytogenetics research of common wheat, when using the existing fluorescence in situ hybridization technology for analysis, it is usually necessary to use multipl...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/6841
CPCC12Q1/6895C12Q1/6841C12Q2600/156C12Q2563/107C12Q2525/151Y02P60/21
Inventor 熊兴伟杨锐杨志芬张素勤耿广东
Owner GUIZHOU UNIV
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