Method for quickly establishing metaphase chromosome karyotype of cucumber through genomic in-situ hybridization

A technology of chromosome karyotype and in situ hybridization, which is applied in the direction of biochemical equipment and methods, microbe determination/inspection, etc., can solve the problems of accelerating the progress of cucumber cytogenetics research, achieve easy distinction, increase concentration, probe clear signal effect

Inactive Publication Date: 2014-08-27
NANJING AGRICULTURAL UNIVERSITY
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Problems solved by technology

[0007] The purpose of the present invention is to use its own genome in situ hybridization to quickly construct the method of cucumber metaphase chromosome karyotype. It is to solve the shortcomings of previous cucumber mitotic metaphase chromosome karyotype construction methods, such as tediousness, and to invent a kind o

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  • Method for quickly establishing metaphase chromosome karyotype of cucumber through genomic in-situ hybridization
  • Method for quickly establishing metaphase chromosome karyotype of cucumber through genomic in-situ hybridization
  • Method for quickly establishing metaphase chromosome karyotype of cucumber through genomic in-situ hybridization

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Embodiment Construction

[0025] The implementation procedure of the method for quickly constructing the cucumber metaphase chromosome karyotype by in situ hybridization of the cucumber genome of the present invention comprises:

[0026] (1) Genomic DNA extraction: Take cucumber young leaves, and use CTAB method to extract cucumber genomic DNA. The specific method refers to the method of Murray et al. (1980). Extract high-concentration cucumber genomic DNA for later use.

[0027] (2) Preparation of probes: Genomic DNA probes were labeled using the nick translation method. According to the instructions of the probe labeling kit, use Digoxigenin-11-dUTP (purchased from Roche Company) to label the cucumber genomic DNA with a probe, and set aside.

[0028] (3) Material preparation: Take appropriate root tips from cucumber plants or germinated seeds, pretreat them in 0.002M 8-hydroxyquinoline solution at 25°C for 3 hours to enrich mid-stage dividing cells, and then put them into volume ratio Fix in 3:1 E:...

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Abstract

The invention discloses a method for quickly establishing a metaphase chromosome karyotype of a cucumber through a genomic in-situ hybridization technology. The method includes the steps that genome DNA of the cucumber is extracted and marked as a probe; enzymolysis is conducted on an extracted root tip of the cucumber, a sheet is prepared through a flame drying method, and then a metaphase chromosome sheet with the good dispersity is acquired; the genome DNA serving as the probe is degenerated and then hybridized to the degenerated metaphase chromosome sheet, under the hybridization condition with the high preciseness, clear and differentiable fluorescence signals are produced through a chromosome tandem repeated sequences, and then the metaphase chromosome karyotype of the cucumber is established. The genome DNA of the cucumber serves as the probe, the metaphase chromosome karyotype of the cucumber is quickly established through one-time GISH, and then testing efficiency is improved. Through the method, based on the different signal distribution modes of the genome DNA probe of the cucumber on chromosomes, the metaphase chromosome karyotype of the cucumber can be quickly established, and a novel method and reference are provided for analytical investigation of karyotypes of species.

Description

1. Technical field [0001] The invention discloses a method for rapidly constructing a cucumber mitotic metaphase chromosome karyotype by utilizing the in situ hybridization technology of the cucumber's own genome, which is beneficial to carrying out karyotype analysis and research on cucumbers and other species, and belongs to the field of plant biotechnology. 2. Technical background [0002] Cucumber (Cucumis sativus L., 2n=2x=14) is one of the most important vegetable crops in the world. Cucumber has some special traits, such as diversification of sex phenotype, paternal inheritance of mitochondria, etc.; at the same time, the genome of cucumber is relatively small (about 370Mb), and the number of chromosomes is only 14, which makes cucumber a molecular cytogenetic trait in recent years. One of the model species for scientific research. However, the chromosomes in the cucumber metaphase are small, the dyeing ability is weak, and the morphology of the chromosomes is very s...

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6841C12Q2543/10
Inventor 娄群峰张云霞张振涛杨树琼王筠竹陈劲枫
Owner NANJING AGRICULTURAL UNIVERSITY
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