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Dual-PCR method for detecting tomato TY-1 gene and Mi gene at the same time

A technology of TY-1 and gene, which is applied in the field of simultaneous detection of tomato TY-1 gene and Mi gene, can solve the problems of simultaneous detection of TY-1 gene and Mi gene, unsatisfactory, long identification time, etc.

Inactive Publication Date: 2010-02-03
SHANGHAI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, conventional methods for detecting TY-1 gene and Mi gene, such as Zamir, the method disclosed in the D literature, but there is an obvious defect in this method is that it cannot detect TY-1 gene and Mi gene at the same time, therefore, the identification time Long time, high identification cost, unable to meet the needs of relevant parties

Method used

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  • Dual-PCR method for detecting tomato TY-1 gene and Mi gene at the same time
  • Dual-PCR method for detecting tomato TY-1 gene and Mi gene at the same time
  • Dual-PCR method for detecting tomato TY-1 gene and Mi gene at the same time

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Embodiment 1

[0059] The young leaves of tomato 2300 varieties were taken, and the DNA was extracted respectively by using the method disclosed in the literature of Williamson, V.M, and used as template DNA.

[0060]Tomato 2300 is a high-generation inbred line selected by the tomato group of the Institute of Horticulture, Shanghai Academy of Agricultural Sciences.

[0061] (1) Carry out the PCR reaction with the PCR system according to the following procedures:

[0062] Denaturation at 95°C for 5 minutes, then denaturation at 94°C for 1 minute, annealing at 61°C for 1 minute, extension at 72°C for 2 minutes, 35 cycles, and finally extension at 72°C for 10 minutes to obtain PCR products, and store at 4°C;

[0063] Said PCR system is an aqueous solution, and its components and contents are:

[0064] Template DNA 20ng / L

[0065] Ty-1 primer 0.2umo1 / L

[0066] Mi primer 0.2umol / L

[0067] 10×buffer 2ul / L

[0068] Mg 2+ 3.5mmol / L

[0069] dNTPs 250umol / L

[0070] Taq enzyme 0.2...

Embodiment 2

[0088] Take the young leaves of tomato 2583 or 2697, and use the method disclosed in the literature of Williamson, V.M to extract DNA respectively, which is used as template DNA.

[0089] Tomato 2583 or 2697 is a variety selected by the tomato group of the Institute of Horticulture, Shanghai Academy of Agricultural Sciences.

[0090] (1) Carry out the PCR reaction with the PCR system according to the following procedures:

[0091] Denaturation at 95°C for 5 minutes, then denaturation at 94°C for 1 minute, annealing at 61°C for 1 minute, extension at 72°C for 2 minutes, 35 cycles, and finally extension at 72°C for 10 minutes to obtain PCR products, and store at 4°C;

[0092] Said PCR system is an aqueous solution, and its components and contents are:

[0093] Template DNA 20ng / L

[0094] Ty-1 primer 0.2umol / L

[0095] Mi primer 0.2umol / L

[0096] 10×buffer 2ul / L

[0097] Mg 2+ 3.5mmol / L

[0098] dNTPs 250umol / L

[0099] Taq enzyme 0.2uL / L

[0100] Ty-1 primer ...

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Abstract

The invention provides a dual-PCR method for detecting a tomato TY-1 gene and an Mi gene at the same time, which comprises the following steps: (1) performing PCR reaction on a PCR system comprising the sequences of a Ty-1 primer and an Mi primer to obtain a PCR product; (2) keeping an enzyme cutting system at a temperature of 65 DEG C for 1.5 hours to obtain an enzyme cutting product; and (3) detecting the PRC product and the enzyme cutting product, using EB for dyeing the final result, adopting a Bio-RAD gel imaging system for taking pictures, and analyzing the result. The dual-PCR techniquehas the advantages that: 1, two genes are detected at the same time in the PCR, and the used time is just one half of that for detecting the two genes respectively so that the identification time isgreatly saved; and 2, the medicines used by the PCR are greatly saved, and the experimental cost is reduced.

Description

technical field [0001] The invention relates to a detection method for simultaneously detecting tomato TY-1 gene and Mi gene. Background technique [0002] Tomato Yellow Leaf Curl virus and Root-knotnematode are two worldwide diseases. TY-1 gene and Mi gene are important genes for tomato resistance to yellow leaf curl virus and root-knot nematode respectively. [0003] With the aggravation of tomato yellow leaf curl virus disease and root-knot nematode disease in China in recent years, the breeding of tomato varieties containing TY-1 gene and Mi gene has become more and more urgent. [0004] Currently, literature: Zamir, D., Ekstein-Michelson, I.., Zakay, Y., Navot, N., Zeidan, M., Sarfatti, M., Eshed, Y., Harel, E., Pleben, T. ., Van-Oss, H., Jedar, N., Rabinowitch, H.D. and Czosnek, H.., 1994, Mapping and introgression of a Tomato yellow leaf curl virus tolerance gene, Ty-1. Theoretical and Applied Genetics, 88: 141~ 146 reports: Discovery of the CAPS marker of tomato y...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 于力朱为民万延慧朱龙英
Owner SHANGHAI ACAD OF AGRI SCI
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