Molecular marker, primers and method for estimating left-end length of N introgressed segment of tobacco

A technology of molecular markers and fragments, which is applied in the field of molecular biology, can solve the problems of restricting the application of N genes, lack of technical means, and lack of breakthrough progress in breeding resistant TMV tobacco, and achieve the effect of reducing yield reduction and reducing burden

Active Publication Date: 2015-12-30
YUNNAN ACAD OF TOBACCO AGRI SCI
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  • Application Information

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 fragm

Method used

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  • Molecular marker, primers and method for estimating left-end length of N introgressed segment of tobacco
  • Molecular marker, primers and method for estimating left-end length of N introgressed segment of tobacco
  • Molecular marker, primers and method for estimating left-end length of N introgressed segment of tobacco

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Embodiment 1

[0063] 1. DNA extraction

[0064] To extract tobacco genomic DNA by conventional CTAB method, the method is as follows:

[0065] (1) Weigh about 100 mg of tobacco leaves and place them in a 1.0 mL centrifuge tube, add liquid nitrogen and grind them with a pestle to powder;

[0066] (2) Add 900μl of 2×CTAB buffer (Tris-HCl pH 7.5100mM, EDTA20mM, NaCl1.4M, CTAB mass percentage 2%) preheated to 65°C, take out and cool in a 65°C water bath for 20 minutes;

[0067] (3) Add 200μl of chloroform-isoamyl alcohol mixture (the volume ratio of chloroform and isoamyl alcohol is 24:1) and shake well, centrifuge at 4°C for 10min (7200rpm) and transfer the supernatant to a 1.0mL EP tube;

[0068] (4) Add 200 μl of chloroform-isoamyl alcohol mixture (the volume ratio of chloroform and isoamyl alcohol is 24:1) again, shake it, and centrifuge at 4°C for 10 min (7200 rpm);

[0069] (5) Take out the supernatant and place it in a new EP tube, add 1 / 10 of the supernatant volume concentration of 3M pH5.2 sodium...

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Abstract

The invention relates to a molecular marker, primers and a method for estimating the left-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 GL4.06 primer pair and a GL3.50 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 Samsun NN 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 Samsun NN 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 TMV-resistant 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, and particularly relates to molecular markers, primers and methods for estimating the length of the left end of tobacco N-introduced fragments. The invention also relates to primers for amplifying the molecular markers, and applications of the molecular markers and primers in tobacco TMV resistance gene positioning or selection of tobacco varieties resistant to TMV. Background technique [0002] Tobacco mosaic virus disease (Tobaccomosaicvirus, TMV) is an important disease on tobacco in my country, and its annual losses are among the top ten tobacco invasive diseases. In production, measures such as cultivating non-toxic seedlings, chemical control, and destroying diseased bodies in the field have been adopted for prevention and control. Certain effects have been achieved in controlling the occurrence and epidemic of TMV. However, the outbreak of TMV in local fields still occurs from time to...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68
Inventor 刘勇李永平方敦煌黄昌军陈姝敏于海芹肖炳光
Owner YUNNAN ACAD OF TOBACCO AGRI SCI
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