Method for fast appraising purity of cucumber hybrid seed

A technology for hybrids and seeds, applied in the field of agricultural vegetable breeding and application, can solve the problems of poor stability, increased cucumber breeding speed, and difficult screening, etc., and achieves the effects of high accuracy, simple operation, and simple result judgment.

Inactive Publication Date: 2010-12-29
CENT LAB TIANJIN ACADEMY OF AGRI SCI +1
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  • Abstract
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AI Technical Summary

Problems solved by technology

Conventional seed purity identification technology takes a long time, is seriously affected by factors such as environmental conditions and time conditions, and is difficult to meet the needs of the market
[0003] The current commonly used cucumber seed purity identification technology has morphological identification technology, isoenzyme identification technology and DNA molecular marker (RAPD, AFLP, SSR etc.) identification technology, in combination, mainly has the following deficiencies: (1) polymorphism of marker technology Sex deficiency: With the development of molecular assisted breeding technology, the breeding speed of cucumber has been greatly improved
The search for morphological trait markers requires a wealth of experience and a lot of time, and it is difficult to meet the needs of breeding; the isozyme identification technology relies on the spatiotemporal differences in protein expression during cucumber growth, and the detection target is a specific isoenzyme at a specific time and specific part. Industrial enzymes are difficult to screen and have poor stability; RAPD and SSR are based on PCR technology and have high sensitivity. However, due to the relatively narrow genetic basis of cucumber, the two marker technologies have less polymorphism and cannot meet the increasing breeding speed.
[0004] (2) Unable to overcome the bottleneck of electrophoresis technology: Isozyme identification technology and RAPD, SSR and other molecular marker identification technologies must go through electrophoresis and staining to obtain band information, and the final result can only be obtained through band analysis
(3) Difficulty in high-throughput operation: the current seed purity identification technology detects only a single seed, and the number of each batch of detection is about 100
So far, there has been no report on the successful use of pyrosequencing technology to identify the seed purity of vegetable hybrid varieties

Method used

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  • Method for fast appraising purity of cucumber hybrid seed
  • Method for fast appraising purity of cucumber hybrid seed
  • Method for fast appraising purity of cucumber hybrid seed

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Experimental program
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Embodiment 1

[0047] (1) Reagent: produced by Promega Master Mixes 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: Master Mixes 25 μL, 10 μmol / L primers 1 μL each, template DNA 2 μL (paternal cucumber DNA), make up to 50 μL with sterilized deionized water; reaction program: pre-denaturation at 95°C for 10 minutes, denaturation at 94°C for 30S, annealing at 55°C for 30S , extended at 72°C for 45S, cycled 50 times, and finally extended at 72°C for 7min, and stored at 4°C.

[0049] (3) Sequencing reaction system: The total volume of the sequencing reaction is 100 μL, and the various components are: 50 μL of PCR product, 3 μL of Sepharose Beads, 47 μL of Binding Buffer (10 mmol / LTris-HCl, 2mol / L NaCl, 1 mmol / L EDTA, 0.1% Tween20, pH7.6), 10μmol / L seque...

Embodiment 2

[0052] (1) Reagent: produced by Promega Master Mixes solution; specific amplification primers and sequencing primers synthesized by Shanghai Sangon; Sepharose Bead produced by Biotage.

[0053] (2) Amplification reaction system and amplification program: the total volume of the amplification reaction is 50 μL, and its various components are: 25 μL of Master Mixes Buffer, 1 μL of each 10 μmol / L primer, 2 μL of template DNA (maternal cucumber), make up to 50 μL with sterilized deionized water; reaction program: pre-denaturation at 95°C for 10 min, denaturation at 94°C for 30S, annealing at 55°C for 30S , extended at 72°C for 45S, cycled 50 times, and finally extended at 72°C for 7min, and stored at 4°C.

[0054] (3) Sequencing reaction system: The total volume of the sequencing reaction is 100 μL, and the various components are: 50 μL of PCR product, 3 μL of Sepharose Beads, 47 μL of Binding Buffer (10 mmol / LTris-HCl, 2mol / L NaCl, 1 mmol / L EDTA, 0.1% Tween20, pH7.6), 10μmol / ...

Embodiment 3

[0057] (1) Reagent: produced by Promega Master Mixes solution; specific amplification primers and sequencing primers synthesized by Shanghai Sangon; Sepharose Bead produced by Biotage.

[0058] (2) Amplification reaction system and amplification program: the total volume of the amplification reaction is 50 μL, and its various components are: 25 μL of Master Mixes Buffer, 1 μL of each 10 μmol / L primer, 2 μL of template DNA (hybrid cucumber DNA), make up to 50 μL with sterilized deionized water; reaction program: pre-denaturation at 95°C for 10 min, denaturation at 94°C for 30 seconds, annealing at 55°C 30S, 72°C extension 45S, cycle 50 times, last 72°C extension 7min, 4°C storage.

[0059] (3) Sequencing reaction system: The total volume of the sequencing reaction is 100 μL, and the various components are: 50 μL of PCR product, 3 μL of Sepharose Beads, 47 μL of Binding Buffer (10 mmol / LTris-HCl, 2mol / L NaCl, 1 mmol / L EDTA, 0.1% Tween20, pH7.6), 10μmol / L sequencing primer 1....

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Abstract

The invention discloses a method for fast appraising the purity of cucumber hybrid seed, which comprises the following steps of: taking the genome DNA of a 'Jingyou 38' test seed of a new product of the room temperature cucumber as a template, informing to analyze the differentiation segment of parent DNA of the known 'Jingyou 38', and appraising the purity of the 'Jingyou 38' hybrid seed by applying a pyrophosphoric acid sequencing technology. The method can appraise the purity of the seed within 3h, has the characteristics of being simple and easy to operate, high in sensitivity, fast, low in cost, convenient for popularization and the like, and developing a wide foreground for appraising the purity of the seed.

Description

technical field [0001] The invention belongs to the technical field of vegetable breeding and application in agriculture, and specifically relates to a method for identifying the purity of cucumber hybrid seeds, in particular to a rapid identification method for the purity of "Jinyou 38" hybrid seeds by using pyrosequencing technology. Background technique [0002] Seeds are important means of agricultural production, and their quality directly affects the quality and yield of agricultural products. Seed purity is the key factor to ensure that the potential of high-quality varieties can be increased. In agricultural production, if the seed purity is not high, the heterogeneity of the product will be enhanced, resulting in irregular product shape and reduced harvest, which will directly affect the economic interests of farmers. The development of rapid and accurate seed purity identification technology not only protects the interests of farmers, but also buys time for seed pr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11G01N21/64
Inventor 王永杜胜利兰青阔张桂华赵新韩毅科朱珠魏爱民程奕
Owner CENT LAB TIANJIN ACADEMY OF AGRI SCI
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