Cucumber variety identification method based on SNP markers

A variety, cucumber technology, applied in the field of agricultural vegetable breeding and application, can solve problems such as affecting the yield and income of growers, insufficient marking density, complicated operation, etc. sexual effect

Pending Publication Date: 2015-08-19
TIANJIN INSTITUE OF QUALITY STANDARD & TESTING OF AGRICULTUAL PRODS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Along with the advantageous development of my country's cucumber seed industry, some decked varieties have appeared on the market, which have been shoddy, which has affected the output and income of growers; even people have illegally stolen breeding materials for breeding, which has seriously affected my country's breeding units. interests and the healthy development of my country's cucumber seed industry, therefore, it is urgent to establish an accurate, high-throughput, high-automation cucumber seed variety identification and purity testing technology system to serve the supervision and testing of seed law enforcement units and the seed quality of breeding units Internal control, among which, the construction of molecular marker fingerprints with strong polymorphism, high density and genetic stability and the supporting high-throughput and highly automated detection mode are the key to the detection technology system
Conventional labeling techniques such as morphological feature labeling, isoenzyme labeling, RAPD labeling, SSR labeling and other technologies have shortcomings such as insufficient labeling density, complicated operation, poor automatic analysis, etc., and cannot meet the requirements of the market

Method used

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  • Cucumber variety identification method based on SNP markers
  • Cucumber variety identification method based on SNP markers
  • Cucumber variety identification method based on SNP markers

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Experimental program
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Effect test

Embodiment 1

[0040] (1) Reagent: GoTaq produced by Promega Master Mix solution; specific amplification primers and sequencing primers synthesized by Shanghai Sangon; Sepharose Bead produced by Biotage.

[0041] (2) Amplification reaction system and amplification program: the total volume of the amplification reaction is 50 μL, and its various components are: 2×GoTaq 25 μL of Master Mix, 1 μL of 10 μmol / L primers, 2 μL of DNA templates (cucumber seed DNA) of the test variety and the control variety, make up to 50 μL with sterilized deionized water; reaction program: 95 °C pre-denaturation for 10 min, 94 °C denaturation for 30 seconds , annealed at 50°C for 30S, extended at 72°C for 45S, cycled 50 times, finally extended at 72°C for 7min, and stored at 4°C.

[0042] (3) Sequencing reaction system: Add 47 μL Binding buffer and 3 μL Sepharose beads to 50 μL PCR product, vortex and mix at 1300 rpm for 15 minutes, separate the single strands by Vacuum prep workstation, and release them into t...

Embodiment 2

[0047] (1) Reagent: GoTaq produced by Promega Master Mix solution; specific amplification primers and sequencing primers synthesized by Shanghai Sangon; Sepharose Bead produced by Biotage.

[0048] (2) Amplification reaction system and amplification program: the total volume of the amplification reaction is 50 μL, and its various components are: 2×GoTaq 25 μL of Master Mix, 1 μL of each 10 μmol / L primer, 2 μL of template DNA (cucumber seed DNA) of the test variety and control variety, filled to 50 μL with sterilized deionized water; Anneal at 50°C for 30S, extend at 72°C for 45S, cycle 50 times, finally extend at 72°C for 7min, and store at 4°C.

[0049] (3) Sequencing reaction system: Add 47 μL Binding buffer and 3 μL Sepharose beads to 50 μL PCR product, vortex and mix at 1300 rpm for 15 minutes, separate the single strands by Vacuum prep workstation, and release them into the PSQ pre-added with 38.8 μL Annealing buffer and 1.2 μL sequencing primers In the 96 sequencing ...

Embodiment 3

[0054] (1) Reagent: GoTaq produced by Promega Master Mix solution; specific amplification primers and sequencing primers synthesized by Shanghai Sangon; Sepharose Bead produced by Biotage.

[0055] (2) Amplification reaction system and amplification program: the total volume of the amplification reaction is 50 μL, and its various components are: 2×GoTaq 25 μL of Master Mix, 1 μL of each 10 μmol / L primer, 2 μL of template DNA (cucumber seed DNA) of the test variety and control variety, filled to 50 μL with sterilized deionized water; Anneal at 50°C for 30S, extend at 72°C for 45S, cycle 50 times, finally extend at 72°C for 7min, and store at 4°C.

[0056] (3) Sequencing reaction system: add 47 μL Binding buffer and 3 μL Sepharose beads to 50 μL PCR product, vortex and mix at 1300 rpm for 15 minutes, separate the single strands by Vacuum prep workstation, and release them into PSQ96 pre-added with 38.8 μL Annealing buffer and 1.2 μL sequencing primer In the sequencing reacti...

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Abstract

The present invention discloses a cucumber variety identification method based on single nucleotide polymorphism markers, wherein 12 single nucleotide polymorphism markers of cucumber are subjected to genotyping analysis by using a pyrosequencing technology so as to achieve authenticity identification on the cucumber variety. With the detection method of the present invention, the cucumber variety authenticity identification can be completed within 5 h, and characteristics of stability, high-throughput, accuracy and the like are provided.

Description

technical field [0001] The invention belongs to the technical field of vegetable breeding and application in agriculture, and specifically relates to a detection method for authenticity identification of cucumber varieties, which is a method for typing and analyzing 12 SNP sites of cucumbers by using pyrosequencing technology. Background technique [0002] Cucumber is one of the top ten vegetable varieties in people's daily life. Because of its unique flavor and taste, it is deeply loved by consumers. my country's cucumber seed industry has always been leading the world, especially the cucumber varieties such as "Jinyou" and "Jinmei" cultivated by Tianjin Kerun Cucumber Research Institute are pillar seed brands in Tianjin, and are exported to 30 provinces, municipalities and autonomous regions. It covers more than 60% of the country's cucumber open-field cultivation area, and its market share is significantly higher than that of international seed companies such as Syngenta. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6827C12Q1/6895C12Q2600/156
Inventor 兰青阔王永赵新李欧静朱珠陈锐
Owner TIANJIN INSTITUE OF QUALITY STANDARD & TESTING OF AGRICULTUAL PRODS
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