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Method for performing genome in-situ hybridization in chromosome suspension

An in situ hybridization and chromosome technology, applied in the field of cell biology, can solve the problems of weak fluorescent labeling signal and inability to hybridize successfully, and achieve the effect of simple hybridization process, high success rate and good effect

Inactive Publication Date: 2019-05-10
FUJIAN AGRI & FORESTRY UNIV
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Problems solved by technology

In 2005, Ma et al. used heat denaturation method to initially conduct FISH in barley chromosome suspension, but only a weak fluorescent marker signal could be seen.
However, this technique is limited to probes with simple repeats, and other long probes or genomic probes cannot hybridize successfully
However, successful genomic in situ hybridization in chromosomal suspensions has not been reported

Method used

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Examples

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Effect test

Embodiment 1

[0025] Embodiment one, a kind of method that carries out intergenus genome in situ hybridization in chromosomal suspension, comprises the following steps:

[0026] 1. Select the stem section with good root point of the offspring of the hybrid of sugarcane and Banmao pachyrhiza. After washing, use perlite particles as the substrate and cover the surface with perlite particles at 26°C. Cultivate and stimulate the roots in the dark, and keep it moist during the period.

[0027] 2. When the root grows to 1-3 cm, perform cell cycle synchronization on the root tip to obtain a large number of metaphase cells and a large number of metaphase chromosomes. Cell cycle synchronization step: in an incubator at 25°C, place the cane stem vertically in a 500 mL plastic box, and treat it with 2.0 mM hydroxyurea (Hydroxyurea, HU) solution for 18 h; ddH 2 O treatment for 5 h; treatment in 2.5 μM Amiprophos-methyl (APM) solution for 3 h; the liquid must be submerged in the root tip and protected f...

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Abstract

The invention relates to a method for performing genome in-situ hybridization in a chromosome suspension (Genome in situ hybridization in suspension, GISHIS). The method comprises the steps of cell cycle synchronization, preparation of the chromosome suspension, preparation of a genomic probe, denaturation of samples and genomic probe and genome in-situ hybridization in the suspension. Dripping piece microscopic examination is carried out, if a green fluorescence signal is generated in the suspension, a saccharum arundinaceum chromosome is determined, so that it is indicated that green fluorescence is successfully marked onto the saccharum arundinaceum chromosome. A flow cytometry is utilized to achieve the separation of the saccharum arundinaceum chromosome specially marked with the greenfluorescence signal in the chromosome suspension and perform the related sequencing study, thereby providing a new, effective and feasible method for studying a complex polyploid saccharum arundinaceum genome. The method is applied to the genome in-situ hybridization in the chromosome suspension of intergeneric hybridization offspring in various crops.

Description

technical field [0001] The invention relates to a method for in-situ hybridization of intergenus genomes of crops, in particular to a method for in-situ hybridization of intergenus genomes in chromosome suspension, and belongs to the technical field of cell biology. Background technique [0002] Wild sugarcane species have rich genetic diversity and contain a large number of excellent traits that can be used in sugarcane genetics and breeding. Banmao is one of the closely related wild species in sugarcane genetic improvement and breeding. Sugarcane breeding scientists hope to introduce the wild species of Banmao and other closely related genera into the sugarcane background through distant hybridization to broaden the genetics of sugarcane. Base. Through the unremitting efforts of sugarcane breeders, a large number of hybrid offspring of sugarcane and P. chinensis have been obtained. The offspring contain clone materials ranging from one to dozens of P. chinensis chromosome...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6841
Inventor 邓祖湖杨善李雪停吴子莺钱旺余凡徐良年
Owner FUJIAN AGRI & FORESTRY UNIV
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