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Multicolor fluorescence in situ hybridization (MFISH) method for quickly analyzing and identifying alien chromosome of wheat

A fluorescence in situ hybridization and rapid analysis technology, applied in the field of cell biology and molecular biology research, can solve the problems of restricting the development of chromosome engineering, long cycle, difficult to accurately and effectively track and quickly identify recombinant chromosomes, etc.

Active Publication Date: 2013-07-17
INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Traditional chromosome engineering has a series of problems such as low efficiency, long cycle, difficulty in accurate and effective tracking and rapid identification of recombinant chromosomes, and difficulty in connecting with modern molecular biology techniques, which seriously limit the further development of chromosome engineering research

Method used

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  • Multicolor fluorescence in situ hybridization (MFISH) method for quickly analyzing and identifying alien chromosome of wheat
  • Multicolor fluorescence in situ hybridization (MFISH) method for quickly analyzing and identifying alien chromosome of wheat
  • Multicolor fluorescence in situ hybridization (MFISH) method for quickly analyzing and identifying alien chromosome of wheat

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Embodiment 1. Identification of Longchun 23 spring wheat material

[0057] 1. Preparation of Longchun 23 Spring Wheat Mitotic Metaphase

[0058] The Longchun 23 spring wheat used came from the germplasm resources preserved in the Han Fangpu Laboratory of the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences (see Yang Wenxiong 2005).

[0059] 1)N 2 O treatment of root tip

[0060] When the root tip grows to 2-3cm, cut it off, put it in a wet centrifuge tube, cover it and put it in a N2O air chamber for 1-3h, the pressure is 10ATM (1.01Mpa);

[0061] 2) root tip fixation

[0062] Fix with 90% acetic acid for 5-10 minutes (no more than 1 hour), and wash with distilled water twice. The fixed root tips can be transferred to 70% ethanol and stored at -20°C for many years;

[0063] 3) Enzymolysis

[0064] Blot the water on the root tip with filter paper, cut off the root tip meristem, and put in 20 μL of a mixture of pectinase and cellulase (en...

Embodiment 2

[0087] Example 2. Chromosomal composition analysis of octoploid Tritite XY784

[0088] 1. Preparation of mitotic metaphases of octoploid Trititum XY784

[0089] The octoploid Trititti XY784 used came from the germplasm resources preserved in the Li Zhensheng Laboratory of the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences (Zhang Xueyong et al.1995).

[0090] 1) N2O treatment root tip with embodiment 1.1

[0091] 2) Root tip fixation Same as Example 1.2

[0092] 3) Enzymolysis Same as Example 1.3

[0093] 4) drop sheet is the same as embodiment 1.4

[0094] 5) Microscopic examination is the same as in Example 1.5

[0095] 2. Probe labeling of AA group, DD group and decaploid E. elongatum group

[0096] 1) CTAB method extracts plant genomic DNA with embodiment 2.1

[0097] 2) Probe labeling is the same as in Example 2.2

[0098] 3) with embodiment 2.3

[0099] 4) with embodiment 2.4

[0100] 3. Fluorescence in situ hybridization identificat...

Embodiment 3

[0109] Example 3. Preparation of metaphase chromosomes of different plants

[0110] The present invention will be further described below with reference to specific examples, but those skilled in the art should understand that the present invention is not limited to these specific examples.

[0111] 1. Seed Germination

[0112] (1) Put the seeds in a petri dish lined with moist filter paper, and germinate in an incubator at 21-23°C.

[0113] 2. Plant chromosome specimen preparation

[0114] 1) With embodiment 1.1.

[0115] 2) With embodiment 1.2.

[0116] 3) With embodiment 1.3.

[0117] 4) With embodiment 1.4.

[0118] 5) With embodiment 1.5.

[0119] 3. In Situ Hybridization

[0120] 1) Use the method of procedure 1 to prepare mitotic metaphase chromosome samples.

[0121] 2) with embodiment 3.2

[0122] 3) Same as embodiment 3.3

[0123] 4) When performing the second in situ hybridization, put the hybridized chromosome specimen into 2×SSC to wash off the antifade ...

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Abstract

The invention provides a multicolor fluorescence in situ hybridization (MFISH) method for quickly analyzing and identifying alien chromosome of wheat. The method comprises the following steps: (1) preprocessing actively dividing tissues in meristematic zone of wheat root tip by N2O and conducting enzymolysis on the actively dividing tissues to obtain a metaphase specimen with clear images and uniform distribution of chromosomes; (2) marking a fluorescent probe of fundamental genome of wheat by nick translation method; (3) conducting fluorescence in situ hybridization on chromosomes at mitosis metaphase obtained in step (1) by using the constructed fluorescent probe in step (2); and (4) analyzing the fluorescence in situ hybridization results obtained in step (3). According to the method provided by the invention, not only can alien chromosomes and chromosome segments in distant hybrid wheat be identified, but also alien chromosomes and chromosome segments of distant hybrids of other crops can be identified.

Description

technical field [0001] The invention belongs to the field of cell biology and molecular biology research. The present invention relates to a method for rapid analysis and identification of multi-color fluorescence in situ hybridization of exogenous chromosomes in wheat, and also relates to a method for repeated hybridization analysis of obtained plant chromosome specimens, and also relates to a method for preparing The method for plant chromosome metaphase specimens can obtain metaphase specimens with clear images and well-dispersed chromosomes. Background technique [0002] Wheat is one of the most important food crops in the world, which is closely related to social and economic development, food security supply and human nutrition and health. Genetic improvement is the main link to improve wheat yield and quality. At present, conventional hybrid breeding is still the main method of wheat variety improvement, but traditional breeding methods have disadvantages such as lon...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 韩方普吕振玲
Owner INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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