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Primer sequence for identifying resistance of cucumber against alternaria cucumerina and identification method thereof

A primer sequence, black spot technology, applied in the field of DNA sequences, can solve the problems of difficult black spot resistant materials, long breeding cycle, difficult control of the process of identifying resistant materials, etc., and achieves great application value without environmental protection Effects of Conditioning

Inactive Publication Date: 2012-06-27
TIANJIN RES INST OF VEGETABLE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In conventional breeding practice, it is difficult to select materials resistant to black spot disease: on the one hand, the breeding cycle is long; The process of identifying resistant materials is not easily controlled
There have been many reports on molecular marker technology in finding molecular markers linked to target traits, but molecular markers closely linked to genes related to cucumber black spot resistance have not been reported yet.

Method used

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  • Primer sequence for identifying resistance of cucumber against alternaria cucumerina and identification method thereof
  • Primer sequence for identifying resistance of cucumber against alternaria cucumerina and identification method thereof
  • Primer sequence for identifying resistance of cucumber against alternaria cucumerina and identification method thereof

Examples

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Embodiment 1

[0030] A method for identifying resistance to black spot disease of cucumber

[0031] (1) Extracting cucumber genomic DNA;

[0032] ] (2) PCR amplification is carried out, and 20ng of the cucumber genomic DNA, 30ng of the nucleotide sequence described in SEQ ID No1 in the sequence listing, and the nucleus described in SEQ ID No2 in the sequence listing are put into the thin-walled tube for PCR amplification nucleotide sequence 30ng, dNTP 0.2mM, Mg 2+1.5mM, 1 times PCR buffer, Taq DNA polymerase 1.0 unit, add sterile double distilled water to 20μl, put the thin-walled tube for PCR amplification into the PCR machine for amplification, and the amplification conditions are: 94°C Pre-denaturation for 200 seconds, denaturation at 94°C for 60 seconds, annealing at 57°C for 50 seconds, extension at 72°C for 90 seconds, 30 cycles, and extension at 72°C for 350 seconds, the amplification was completed;

[0033] (3) Gel electrophoresis analysis of PCR amplification products: The ampl...

Embodiment 2

[0036] A method for identifying resistance to black spot disease of cucumber

[0037] (1) Extracting cucumber genomic DNA;

[0038] (2) Carry out PCR amplification: put 15ng of the cucumber genomic DNA, 20ng of the nucleotide sequence described in SEQ ID No1 in the sequence listing, and the nucleus described in SEQ ID No2 in the sequence listing in a thin-walled tube dedicated to PCR amplification nucleotide sequence 20ng, dNTP 0.15mM, Mg 2+ 1.2mM, 1 times PCR buffer, Taq DNA polymerase 0.8 units, add sterile double distilled water to 20μl, put the thin-walled tube for PCR amplification into the PCR machine for amplification, and the amplification conditions are: 94°C Pre-denaturation for 180 seconds, denaturation at 94°C for 60 seconds, annealing at 60°C for 40 seconds, extension at 72°C for 60 seconds, 25 cycles, and extension at 72°C for 300 seconds, the amplification was completed;

[0039] (3) Gel electrophoresis analysis of PCR amplification products: The amplification...

Embodiment 3

[0042] A method for identifying resistance to black spot disease of cucumber

[0043] (1) Extracting cucumber genomic DNA;

[0044] (2) Carry out PCR amplification: put 30ng of the cucumber genomic DNA, 40ng of the nucleotide sequence described in SEQ ID No1 in the sequence listing, and the nucleus described in SEQ ID No2 in the sequence listing in a thin-walled tube dedicated to PCR amplification nucleotide sequence 40ng, dNTP 0.25mM, Mg 2+ 2.0mM, 1 times PCR buffer, 1.2 units of Taq DNA polymerase, add sterile double distilled water to 20μl, put the thin-walled tube for PCR amplification into the PCR machine for amplification, and the amplification conditions are: 94°C Pre-denaturation for 300 seconds, denaturation at 94°C for 60 seconds, annealing at 54°C for 60 seconds, extension at 72°C for 120 seconds, 35 cycles, and extension at 72°C for 420 seconds, the amplification was completed;

[0045] (3) Gel electrophoresis analysis of PCR amplification products: The amplifica...

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Abstract

The invention discloses a primer sequence for identifying resistance of cucumber against alternaria cucumerina and an identification method thereof. The primer sequence comprises nucleotide sequences shown as SEQ ID No.1 and SEQ ID No.2 in a sequence table. The method for identifying the resistance of the cucumber against the alternaria cucumerina comprises the following steps of: (1) extracting genomic DNA of the cucumber; (2) performing polymerase chain reaction (PCR) amplification by taking the nucleotide sequences shown as SEQ ID No.1 and SEQ ID No.2 in the sequence table as primers; (3) performing gel electrophoresis analysis on the amplification product; and (4) identifying the resistance of the cucumber against the alternaria cucumerina according to the relative position of each identified sample on a gel strip. By evaluating the resistance of the constructed segregation population of generation F2 and cucumber germplasm resources against the alternaria cucumerina with the primer sequence of the invention, the coincidence rates for the result and artificial inoculation evaluation reach 95.04 percent and 94.74 percent respectively; the linkage genetic distance with the alternaria cucumerina resistance gene is 4.96cm; and the primer sequence has the advantages of high speed, accuracy and freedom from environmental condition and the like, and has great application value for screening the resistance of the resources in the cucumber breeding.

Description

technical field [0001] The invention relates to a DNA sequence in biotechnology and a method for identifying resistance to cucumber black spot disease using the DNA sequence. Background technique [0002] Cucumber alternaria leaf spot, Alternaria cucumerina, commonly known as "yellow spot", is a fungal disease caused by Alternaria cucumerina, and has become one of the important diseases that damage cucumber protected cultivation. The disease mainly damages the leaves, and generally begins to occur on the middle and lower leaves of cucumbers, and then gradually spreads upwards. The disease spots develop along the mesophyll tissue on both sides of the veins, and the main veins are generally not damaged. In the end, only a few green leaves at the top are left, and the diseased plant looks like a fire. Severely diseased plants can be infected except for the heart leaves. The lesions are greenish and nearly round spots at first, and later become round or nearly round lesions wi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 李淑菊王惠哲杨瑞环管炜
Owner TIANJIN RES INST OF VEGETABLE
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