Molecular marker for identifying potato virus Y (PVY) resistance of tobacco

A technology of molecular markers and tobacco, which is applied in the field of molecular biology, can solve the problems of low specificity of amplification products, large errors in resistance phenotypes, and long linkage distances, etc., and achieve simple amplification conditions, high accuracy, and linkage close effect

Active Publication Date: 2014-07-02
YUNNAN ACAD OF TOBACCO AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The linkage distance with PVY resistance is relatively long, which will lead to large errors in judging the resistance phenotype using marker data
RAPD markers have disadvantages such as low specificity of amplification products, non-target band interference result judgment, PCR amplification requires as many as 45 cycles, etc.

Method used

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  • Molecular marker for identifying potato virus Y (PVY) resistance of tobacco
  • Molecular marker for identifying potato virus Y (PVY) resistance of tobacco

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: Preparation of Molecular Markers

[0038] 1. Tobacco RNA extraction:

[0039] (1) Take 100 mg of young leaves and put them into a mortar treated with DEPC water;

[0040] (2) Add 1ml Trizol to grind to a homogeneous slurry, transfer to an EP centrifuge tube treated with DEPC water, and let stand at room temperature for 5 minutes;

[0041] (3) Add 0.2ml chloroform (trichloromethane), shake for 15sec, and let stand for 3min;

[0042] (4) 12000rcf, 4°C, centrifuge for 15min;

[0043] (5) Transfer the supernatant (up to 400ul) to a new EP tube;

[0044] (6) Add 0.5ml of isopropanol to the supernatant and let it stand for 10 minutes;

[0045] (7) 12000rcf, 4°C, centrifuge for 10min;

[0046] (8) Discard the isopropanol, use a pipette to suck out the remaining isopropanol and discard it, and add 1ml of 75% ethanol to clean the bottom residue;

[0047] (9) 7500rcf, 4°C, centrifuge for 2min;

[0048] (10) Discard the ethanol, suck out the residual ethanol with...

Embodiment 2

[0065] Example 2: Development of primer pairs for amplifying molecular markers

[0066] According to the molecular marker sequence (Seq ID No.1), in Genbank (www.ncbi.nlm.nih.gov / genbank), the tobacco genome sequence with a sequence identity rate higher than 80% was obtained by using the BLAST method. The primer design software PRIMER5.0 was used to design candidate primer pairs that specifically amplify the sequence (Seq ID No.1) containing all or part of the molecular marker.

[0067] Candidate primer pairs were screened using the genomic DNA of PVY-resistant tobacco variety NC55, and PVY-susceptible tobacco variety Coker176 and Honghua Dajinyuan.

[0068] The PCR reaction system is as follows: the total volume is 20 μL, including 2.5 μL of 50 ng / μL DNA sample, 2.0 μL of 10×PCR buffer, 1.2 μL of dNTPs, 1.5 μL of each primer pair (Seq ID No.2 and Seq ID No.3), Ex- Taq DNase 0.3 μL, ddH2O 12.6 μL. The reagents used were purchased from Bao Biological Company.

[0069] PCR re...

Embodiment 3

[0073] Example 3: Determination of genetic distance between molecular markers and PVY resistance

[0074] 1. Construction of Tobacco F2 Generation Segregation Population

[0075] Male parent: tobacco variety NC55, resistant to PVY. Female parent: tobacco variety Coker176, sensitive to PVY. F2 population construction: male parent and female parent are crossed to obtain F1 generation, and F1 is self-crossed to obtain F2 seeds.

[0076] 2. Phenotypic identification of tobacco F2 generation individuals

[0077] Sow male, female and F2 seeds. A total of 30 single plants of the male parent, 30 single plants of the female parent, and 101 single plants of the F2 generation were obtained in potted plants. When the tobacco seedlings have 4-5 leaves, half a leaf is collected from each plant and stored at -20°C for DNA extraction. After sampling, a high-pressure spray gun was used to inoculate 40 times the diseased leaf juice of PVY virus necrosis strain ZT-5 (the public can obtain i...

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Abstract

The invention belongs to the field of molecular biology, and discloses a molecular marker for identifying potato virus Y (PVY) resistance of tobacco. The molecular marker contains a sequence shown in SeqIDNo.1. The invention also relates to primers for amplifying the molecular marker, and use of the molecular marker and the primers in disease-resistant resource screening and disease-resistant breeding assisted selection of tobacco. A genome DNA sequence and a tobacco PVY resistant gene are associated by the molecular marker disclosed by the invention, which is beneficial to the building of the assisted breeding selection system of the tobacco molecular marker; the molecular marker is tightly interlocked with the tobacco PVY resistant gene and the interlock distance is 0.99cM. The molecular marker and the molecular marker amplification primers disclosed by the invention can be simply and quickly applied to tobacco breeding practice at high flux.

Description

technical field [0001] The invention belongs to the field of molecular biology and relates to a molecular marker, in particular to a molecular marker for identifying tobacco PVY resistance. The invention also relates to primers for amplifying the molecular markers, and the use of the molecular markers and the primers in tobacco PVY resistance gene mapping or tobacco genetic breeding. Background technique [0002] Potato virus Y (PVY) can infect more than 170 kinds of plants belonging to 34 genera, and is very harmful to Solanaceae crops (such as: tobacco, potato, pepper, tomato, etc.). The disease of PVY infecting tobacco, also known as tobacco vein spot disease, is a worldwide epidemic disease. Due to the lack of main resistant varieties and the difficulty in implementing comprehensive control measures, it is seriously harmful in my country's smoking areas. PVY infection can reduce tobacco yield by 7%-18%, and reduce output value by 11%-80%. In 1989, tobacco vein spot dis...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11C12N15/10
CPCC12Q1/6895C12Q2600/13
Inventor 刘勇王丙武宋中邦高玉龙李文正谢贺杨大海陈学军肖炳光
Owner YUNNAN ACAD OF TOBACCO AGRI SCI
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