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Molecular markers, primers and methods for estimating the length of the right end of tobacco n-introduced fragments

A technology of molecular markers and fragments, applied in the field of molecular biology, can solve problems such as lack of technical means, limitation of N gene application, unclear length of N-introduced fragments and accompanying redundant genes, etc., to reduce production, reduce burden, and reduce the range Effect

Active Publication Date: 2018-05-15
YUNNAN ACAD OF TOBACCO AGRI SCI
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AI Technical Summary

Problems solved by technology

Although the N gene was cloned 20 years ago, the length of the N-introduced fragment and the accompanying redundant genes have been unclear, limiting the application of the N gene in commercial varieties
Conventional breeding techniques cannot estimate the length of N-introduced fragments, and traits such as yield, yellowing, and roasting are quantitative traits, which are difficult to select in the early stage of breeding
The lack of technical means to estimate the length of tobacco N-introduced fragments has led to a lack of breakthroughs in TMV-resistant tobacco breeding

Method used

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  • Molecular markers, primers and methods for estimating the length of the right end of tobacco n-introduced fragments
  • Molecular markers, primers and methods for estimating the length of the right end of tobacco n-introduced fragments
  • Molecular markers, primers and methods for estimating the length of the right end of tobacco n-introduced fragments

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Embodiment 1

[0058] 1. DNA extraction

[0059] Tobacco genomic DNA was extracted respectively by conventional CTAB method, the method is as follows:

[0060] (1) Weigh about 100 mg of tobacco leaf and place it in a 1.5 mL centrifuge tube, add liquid nitrogen and grind it to powder with a pestle;

[0061] (2) Add 900 μl of 2×CTAB buffer (Tris-HCl pH 7.5100mM, EDTA 20mM, NaCl 1.4M, CTAB mass percentage concentration 2%) preheated to 65°C, take it out of the water bath at 65°C for 20 minutes and cool it ;

[0062] (3) Add 200 μl of chloroform-isoamyl alcohol mixture (the volume ratio of chloroform and isoamyl alcohol is 24:1) and shake well, centrifuge at 4°C for 10 min (7200 rpm) and transfer the supernatant to a 1.5 mL EP tube;

[0063] (4) Add 200 μl of chloroform-isoamyl alcohol mixture again (the volume ratio of chloroform and isoamyl alcohol is 24:1), shake well, and centrifuge at 4°C for 10 min (7200 rpm);

[0064] (5) Take out the supernatant and put it in a new EP tube, add 3M pH5...

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Abstract

The invention relates to a molecular marker, primers and a method for estimating the right-end length of an N introgressed segment of tobacco. The molecular marker is a sequence represented as Seq ID No.1 or Seq ID No2. According to estimating method, PCR (polymerase chain reaction) amplification is performed by adopting a TN5.51 primer pair and a TN5.34 primer pair as the primers and adopting to-be-detected N introgressed segment containing tobacco genome DNA (deoxyribonucleic acid) as a template, then electrophoresis detection is performed, and if a DNA segment with the corresponding size is obtained through amplification, the N introgressed segment of detected tobacco is as long as a disease-resistant control material Coker176 in the molecular marker position; if the DNA segment with the corresponding size is not obtained through amplification, the N introgressed segment of the detected tobacco is shorter than the disease-resistant control material Coker176 in the molecular marker position. The method can be simply, conveniently and rapidly applied to anti-TMV (tobacco mosaic virus) gene mapping of the tobacco and anti-TMV tobacco variety breeding in a high-throughput manner, and reduction of linkage redundancy with the N gene is facilitated.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, in particular to molecular markers, primers and methods for estimating the length of the right end of tobacco N-introduced fragments. The invention also relates to primers for amplifying the molecular markers, and the application of the molecular markers and the primers in the mapping of tobacco TMV disease-resistant genes or in the selection of tobacco TMV-resistant varieties. Background technique [0002] Tobacco mosaic virus (TMV) is an important disease of tobacco in my country, and its annual loss ranks at the top of the list of top ten tobacco infectious diseases. In production, measures such as cultivating non-toxic seedlings, chemical control, and destroying diseased residues in the field are mainly adopted to prevent and control the occurrence and prevalence of TMV. However, TMV outbreaks in local fields still occur from time to time, causing greater economic loss. Therefore,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895C12Q2600/13
Inventor 刘勇陈姝敏李永平黄昌军于海芹肖炳光
Owner YUNNAN ACAD OF TOBACCO AGRI SCI
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