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Direct PCR method and application thereof in genetic breeding of cabbage hybrids

A hybrid seed and hybrid seed technology, applied in the biological field, can solve the problems of complex operation, pollution, long extraction time, etc., and achieve the effects of reducing detection time, wide application prospect and space, and simple method

Active Publication Date: 2019-01-18
JIANGSU ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the DNA extraction method is complicated to operate, and the extraction time is relatively long. At the same time, reagents such as phenol, chloroform, and isoamyl alcohol contained in the extraction solution are likely to pollute the environment.

Method used

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  • Direct PCR method and application thereof in genetic breeding of cabbage hybrids
  • Direct PCR method and application thereof in genetic breeding of cabbage hybrids

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] 1. Seed germination

[0026] Put the cabbage "Sugan 20" hybrid in a petri dish and add water to germinate, and after 4-5 days of germination, seedlings will grow for use.

[0027] 2. Direct PCR amplification

[0028] Sections of hypocotyls, radicles and cotyledons with a length of 0.5-1mm, 1-1.5mm and 1.5-2mm were respectively intercepted in a 20 μl PCR reaction system (component content: Tris-HCl 10mM, KCl 50mM, MgCl 2 1.5mM, dNTPs 0.2mM, upstream and downstream primers 0.5μM each, DNA polymerase premixed with anti-Taq DNA polymerase antibody 0.1U / μl). The direct PCR amplification program used was: 98°C for 3min; 95°C for 30s, 54°C for 30s, 72°C for 30s, 35 cycles; 72°C for 7min extension; storage at 10°C. The PCR premix solution containing DNA polymerase premixed with anti-Taq DNA polymerase antibody is called Quick Taq HS DyeMix, and the manufacturer is Toyobo (Shanghai) Biotechnology Co., Ltd.

[0029] 3. Amplified product detection

[0030]The detection of the...

Embodiment 2

[0034] 1. InDel primer design and synthesis

[0035] The InDel primers used in the research were designed based on Brassica oleracea genome sequencing (http: / / ocri-genomics.org / bolbase / ), and were sent to Nanjing Yidao Biotechnology Co., Ltd. for synthesis.

[0036] 2. Seed germination

[0037] Put the seeds of the cabbage "Sugan 20" hybrid and its male parent and female parent in a petri dish and add water to accelerate germination, and after 4-5 days of germination, seedlings will grow for use.

[0038] 3. Direct PCR amplification

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PUM

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Abstract

The invention discloses a direct PCR method and an application thereof in genetic breeding of cabbage hybrids. The direct PCR amplification method provided by the invention is that the cabbage seeds are germinated by 4 Five days later, hypocotyls were taken directly as template and PCR amplification was performed by hot-start method using DNA polymerase of premixed anti-Taq DNA polymerase antibody. The direct PCR amplification method provided by the invention is simple. Compared with the traditional method of extracting DNA from young leaves after germination and PCR detection, this method avoids the complicated steps of extracting DNA and greatly reduces the detection cost and time. The invention has great application prospect in the identification of the genetic purity of the cabbage "Sugan 20" hybrid seed.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a direct PCR method and its application in genetic breeding of cabbage hybrids. Background technique [0002] Heading cabbage (Brassica oleracea var. capitata L.), referred to as cabbage, belongs to the variety of Brassicaceae Brassica species that can form leaf balls. It is one of the important cruciferous vegetables in my country. It is rich in nutrition, has strong adaptability and stress resistance, and is widely planted all over the country. It plays a pivotal role in the vegetable supply. [0003] At present, most of the varieties of cabbage popularized in production are hybrids, and the production of hybrids mainly uses male sterile lines and self-incompatible lines for seed production. However, in the process of seed production, the pseudo-hybrids produced by female parent self-crossing are often mixed with F l In the process, the quality and purity of seeds are...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/686C12Q1/6895
CPCC12Q1/686C12Q1/6895C12Q2521/101C12Q2565/125
Inventor 张伟李建斌王神云余方伟于利唐君王红
Owner JIANGSU ACAD OF AGRI SCI