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

A molecular marker and fragment technology, applied in the field of molecular biology, can solve the problems of lack of breakthrough progress in TMV-resistant tobacco breeding, limited application of N gene, unclear length of N introduction fragment and accompanying burdensome genes, etc.

Active Publication Date: 2018-05-15
YUNNAN ACAD OF TOBACCO AGRI SCI
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  • Abstract
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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 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 N-introduced fragments leads 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 left end of tobacco n-introduced fragments
  • Molecular markers, primers and methods for estimating the length of the left end of tobacco n-introduced fragments
  • Molecular markers, primers and methods for estimating the length of the left end of tobacco n-introduced fragments

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Embodiment 1

[0063] 1. DNA extraction

[0064] Tobacco genomic DNA was extracted respectively by conventional CTAB method, the method is as follows:

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

[0066] (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 ;

[0067] (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 1.0 mL EP tube;

[0068] (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);

[0069] (5) Take out the supernatant and put it in a new EP tube, add 1 / 10 o...

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Abstract

The invention relates to a molecular marker, a primer and a method for estimating the length of the left end of a tobacco N-introduced fragment. The molecule is marked as the sequence shown in Seq ID No.1 or Seq ID No.2. This estimation method uses GL4.06 primer pair or GL3.50 primer pair as primers, and uses the tobacco genomic DNA containing the N-introduced fragment to be detected as a template for PCR amplification, and then electrophoresis detection, such as amplifying the DNA of the corresponding size Fragment, it indicates that the N-introduction fragment of the tested tobacco is equivalent to the disease-resistant control material Samsun NN at the position of the molecular marker; Samsung NN is short. This method can be easily, rapidly and high-throughput applied to the mapping of tobacco TMV resistance genes and the selection of tobacco TMV-resistant varieties, which is beneficial to reduce the burden of linkage with N genes.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular 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 the application of the molecular markers and the primers in mapping tobacco TMV disease resistance genes or breeding tobacco resistant TMV 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, plant...

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12Q1/6895
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