Centromere mark realizing fluorescence in situ hybridization with asparagus chromosome and applications of centromere mark

A technique of fluorescence in situ hybridization and chromosomes, applied in the field of molecular cytogenetics, can solve the problems of small chromosomes, difficulties in handling cells, and difficulty in obtaining division equality

Inactive Publication Date: 2016-09-21
HENAN NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, researches on A. chinensis mainly focus on nutritional components, molecular markers, etc., and research on its cytology is relatively lagging behind. Chromosomes are small, so it is difficult to obtain clear and well-dispersed cleavage phases
The research on the centromere markers of A. chinensis has not been reported yet

Method used

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  • Centromere mark realizing fluorescence in situ hybridization with asparagus chromosome and applications of centromere mark
  • Centromere mark realizing fluorescence in situ hybridization with asparagus chromosome and applications of centromere mark

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Embodiment 1

[0022] Such as figure 1 As shown, the acquisition of the centromere marker sequence of A. chinensis, specifically:

[0023] 1. The source of the centromere marker sequence of A. chinensis, which was discovered when the sequence assembled by high-throughput sequencing was verified. Illumina HiSeq2000 sequencing technology was used to sequence the genomes of male and female A. chinensis. The sequencing results obtained a total of 17 Gb sequences (12 times coverage). After assembly, a total of 163,406 scaffolds were obtained, with a length of 400 Mbp. 30% of the Diabolic genome. 20 scaffolds were randomly selected, and sequence-specific primers were designed to amplify the sequence from the genome of A. chinensis and sequenced to verify the reliability of the sequencing and assembly data. The centromere marker sequence used in the present invention is amplified by designing primers for the scaffold5783 sequence.

[0024] 2. The PCR amplification of the centromere marker sequen...

Embodiment 2

[0027] The centromere marker of Chromosome fluorescence in situ hybridization of A. chinensis, the centromere marker is Texas red marker, and its nucleotide sequence is shown in SEQ ID No.1,

[0028] Among them, the primer pair F: 5'-CAACCCCCACTTATCCT, R: 5'-CGGCAATATACACAGATAC was used to carry out PCR amplification with Genomic DNA of A. dNTP, 100-200 ng of genomic DNA, including 0.1 μmol / L of upstream and downstream primers.

Embodiment 3

[0030] A method for centromere marking of Chromosome fluorescent in situ hybridization comprising the following steps:

[0031] Step S1: Preparation of root apical metaphase chromosomes, specifically laying 2-3 layers of filter paper in a petri dish, soaking it in water, putting the seeds of Cypress chinensis, and culturing at 25°C for 5-7 days until the root length is 1.5-2 cm. Cut the root tip into N 2 Treat in an O gas chamber for 1-3 h with a pressure of 10 atm (1.01 Mpa). The root tips were then fixed on ice with 90% acetic acid for 10 min. After the root tip was washed with water, the 2 mm-long part of the root tip growth point was cut and transferred to 20 μL of ice-cold enzyme solution (1% pectolyase Y-23 and 2% Onozuka R-10), and placed in a water bath at 37 °C for 2 h . After the enzymatic hydrolysis, resuspend twice with 70% ethanol, pour off the ethanol, keep 40 μL of ethanol, smash the root tip with a dissecting needle, vortex for a few seconds or flick the tub...

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Abstract

The invention relates to a centromere mark realizing fluorescence in situ hybridization with the asparagus chromosome and applications of the centromere mark, wherein the centromere mark is a Texas red mark, and the nucleotide sequence of the centromere mark is shown as SEQ ID No.1. The marked sequence is taken as a probe to be subjected to fluorescence in situ hybridization with the metaphase chromosome of the root tip of the asparagus, and a concentrated, clear and bright hybridization signal is generated at the centromere position of the chromosome. The developed centromere mark can be applied to the genetic mapping of the centromere position of the asparagus genetic map, and a foundation is laid for the cellular experiments including the karyotype analysis, pachytene stage FISH and fiber-FISH of asparagus.

Description

technical field [0001] The invention belongs to the field of molecular cytogenetics, and in particular relates to a centromere marker which can be used for fluorescence in situ hybridization of Chromosome chromosome and its application. Background technique [0002] DNA fluorescence in situ hybridization (FISH) technology is an important non-radioactive in situ hybridization technology developed on the basis of the original radioactive in situ hybridization technology in the late 1970s and early 1980s. This technology is based on the principle of nucleic acid base complementary pairing, which combines the labeled exogenous nucleic acid (probe) with the denatured single-stranded DNA of the chromosome to form a specific nucleic acid hybrid molecule. Since the probe itself has a fluorescent substance, or the antibody used in the subsequent detection process has a fluorescent substance, the position and distribution of the target sequence on the cell or chromosome can be directl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6841C12Q1/6895C12Q2563/107
Inventor 高武军李书粉侯翠翠袁金红邓传良
Owner HENAN NORMAL UNIV
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