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Method for identifying all chromosomes of poplar by using oligonucleotide probe

An oligonucleotide probe and oligonucleotide technology, which is applied in the field of identifying all the chromosomes of poplar by using oligonucleotide probes, can solve the problems such as the lag of cytogenetic research and the difficulty of accurately distinguishing chromosomes from chromosomes, and achieve the experimental result. Reproducible, high-resolution effects

Active Publication Date: 2019-05-31
NANJING FORESTRY UNIV
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Problems solved by technology

However, what does not match the status of the poplar model tree species is that there is no high-resolution karyotype map for research at home and abroad
This is mainly because the poplar chromosomes are small, and it is difficult to accurately identify each chromosome based on the shape of the mitotic metaphase chromosomes, resulting in a significant lag in cytogenetic research

Method used

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  • Method for identifying all chromosomes of poplar by using oligonucleotide probe

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Embodiment 1

[0036] A kind of method utilizing oligonucleotide probe to identify poplar whole chromosome, comprises the steps:

[0037] (1) Chromosome production

[0038] Collect young poplar root tips of 1 cm, immerse in 2mM 8-hydroxyquinoline solution for pretreatment at room temperature in the dark for 2.5 hours; rinse with water for 10 minutes, transfer to freshly prepared Carnot’s fixative, and store at -20°C until use ;

[0039] The root tip taken out of the fixative was rinsed in water for 10 minutes, the meristem part was cut with a double-sided blade, and the root tip meristem part was enzymatically hydrolyzed in an enzyme mixture of 4% cellulase and 2% pectinase at 37°C 1h; put the root tip meristem into deionized water to terminate the reaction;

[0040] Use a pipette to place the root tip on a clean glass slide, absorb excess water with a filter paper strip; mash the root tip tissue with tweezers and a dissecting needle; quickly add 20 μl of 60% glacial acetic acid dropwise, ...

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Abstract

The invention discloses a method for identifying all chromosomes of poplar by using oligonucleotide probes. The method comprises the following steps: obtaining a specific oligonucleotide sequence of each chromosome through bioinformatics analysis according to genome information of poplar tomentosa; synthesizing an oligonucleotide library corresponding to chromosome 1-19 of poplar; marking the oligonucleotide probes by using biotin or digoxin; identifying the chromosomes of the poplar by using a fluorescence in-situ hybridization technology; and repeatedly carrying out fluorescence in-situ hybridization for 7 times on the same metaphase splitting phase by using the oligonucleotide probes of up to three chromosomes simultaneously in each cycle of hybridization, so that 19 pairs of chromosomes of poplar species can be accurately identified.The method has the advantages of good universality in poplar species, good experimental repeatability and high resolution.The methoddisclosed by the invention provides a novel method for accurately identifying each chromosome of the poplar species, and lays a solid working foundation for molecular cytogenetics research of poplar.

Description

technical field [0001] The invention relates to the field of molecular cytogenetics, in particular to a method for using oligonucleotide probes to identify all the chromosomes of poplar trees. Background technique [0002] Fluorescence in situ hybridization (FISH) is a molecular cytogenetic technique developed in the 1980s. FISH technology can directly locate the DNA sequence on the chromosome, which provides an efficient method for the construction of cytological maps. This technology only needs to use the specific DNA sequence of each chromosome as a probe, and can visually distinguish each chromosome. At present, FISH technology has been widely used in the karyotype analysis of various plants. Chromosome painting is to prepare a probe from the DNA of the whole chromosome, a certain chromosome arm (long arm or short arm) or a certain fragment of the chromosome, and use the FISH method to hybridize the probe to the chromosome. It is a new technology to analyze and study ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6841
Inventor 席梦利辛昊阳赵乙琏尚大鑫宁仪杭
Owner NANJING FORESTRY UNIV
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