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Method for indentifying purity of oil sunflower Kangdi No.6 and No.7 varieties

A technology of variety purity and oil sunflower Kangdi, which is applied in the biological field, can solve the problems that there are no rapid biochemical or DNA molecular detection technology standard procedures, and achieve the effects of speeding up the quality detection process, stable amplification effect, and shortened detection time

Inactive Publication Date: 2012-09-26
新疆康地种业科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

To sum up, from the traditional morphological identification, biochemical fingerprint detection to molecular level and genetic level detection of hybrid sunflower variety inspection technology has gone through a process from simple to complex and precise, each method has its own advantages and deficiencies, so there is no rapid biochemical or DNA molecular detection technology standard procedure in the inspection procedures of crop seeds
There is no report in the prior art about the method for simultaneously identifying the purity of No. 6 and No. 7 varieties of Youkui Kangdi

Method used

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  • Method for indentifying purity of oil sunflower Kangdi No.6 and No.7 varieties
  • Method for indentifying purity of oil sunflower Kangdi No.6 and No.7 varieties
  • Method for indentifying purity of oil sunflower Kangdi No.6 and No.7 varieties

Examples

Experimental program
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Effect test

Embodiment 1

[0027] Example 1: Method for identifying the purity of No. 6 and No. 7 oil sunflower Kangdi varieties.

[0028] (1) Extraction of oil sunflower DNA: Take the young and true oil sunflower leaves that have been cultured for 2 weeks in a mortar, add liquid nitrogen to quickly grind them into a uniform powder, and add 500 μl of 65 ℃ preheated CTAB extract before the sample is melted. , After mixing well, transfer the sample solution into a 1.5 ml centrifuge tube, place it in a 65°C water bath for 50 min, invert the centrifuge tube several times during the process; take out the sample and cool to room temperature, add equal volumes of phenol / chloroform and chloroform / isoamyl alcohol. Extraction, in which phenol / chloroform is calculated at a volume ratio of 1:1, chloroform / isoamyl alcohol is calculated at 24:1, centrifuged at 12 000 r / min at 4°C for 5 min; add the supernatant to 0.6 times the volume of isopropyl Mix with alcohol, put it at room temperature for 5-10 min, centrifuge at ...

Embodiment 2

[0034] Example 2: Method for identifying the purity of Youkui Kangdi No. 6 variety.

[0035] (1) Obtain sterile oil sunflower seedlings: take Kangdi No. 6 oil sunflower seeds, soak them in 70% alcohol solution for 10 minutes, rinse twice with sterile water, sterilize with 10% hydrogen peroxide solution for 30 minutes, and then sterilize water Rinse twice, inoculate the embryo end up on 1 / 2MS medium without hormones, the temperature of the culture room is 26℃-28℃, the light time is 16h, the light intensity is 2000LX, and the growth is 14 days.

[0036] (2) Sunflower DNA extraction: Take the two-week-old sunflower young true leaves in a mortar, add liquid nitrogen to quickly grind them into a uniform powder, and add the 65°C preheated CTAB extract (50 mmol / L Tris-HCl pH8, 0.7 mmol / L NaCl, 10 mmol / L EDTA, pH 8, 1% CTAB, 20 mmol / L 2-mercaptoethanol) 500μl, mix well and transfer the sample solution into a 1.5 ml centrifuge tube In the middle, place in a 65℃ water bath for 50 min, duri...

Embodiment 3

[0040] Example 3: Method for identifying the purity of Youkui Kangdi No. 7 variety.

[0041] (1) Obtain sterile oil sunflower seedlings: Take Kangdi No. 7 oil sunflower seeds, soak them in 70% alcohol solution for 10 minutes, rinse twice with sterile water, sterilize with 10% hydrogen peroxide solution for 30 minutes, and then sterile water Rinse twice, inoculate the embryo end up on 1 / 2MS medium without hormones, the temperature of the culture room is 26℃-28℃, the light time is 16h, the light intensity is 2000LX, and the growth is 14 days.

[0042] (2) Sunflower DNA extraction: Take the two-week-old sunflower young true leaves in a mortar, add liquid nitrogen to quickly grind them into a uniform powder, and add the 65°C preheated CTAB extract (50 mmol / L Tris-HCl pH8, 0.7 mmol / L NaCl, 10 mmol / L EDTA, pH 8, 1% CTAB, 20 mmol / L 2-mercaptoethanol) 500μl, mix well and transfer the sample solution into a 1.5 ml centrifuge tube In the middle, place in a 65℃ water bath for 50 min, durin...

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Abstract

The invention discloses a method for indentifying the purity of oil sunflower Kangdi No.6 and No.7 varieties. The method comprises the following steps of: establishing an oil sunflower RAPD (Randomly Amplified Polymorphic DNA) technical program and determining an optimized combination of all components of a PCR (Polymerase Chain Reaction) amplification reaction; and taking oil sunflower Kangdi No.6 and No.7 and DNAs (Deoxyribonucleic Acid) of parents as templates to obtain a primer marker OPB05 which can simultaneously distinguish a male parent, a female parent and a hybrid, wherein the size of a female parent specific marker OPB05 stripe generated by the amplified oil sunflower Kangdi No.7 is 450bp and the size of a male parent specific marker OPB05 stripe is 880bp; a DNA segment of the hybrid contains characteristic bands of male and female parents; the size of a female parent specific marker OPB05 stripe generated by the amplified oil sunflower Kangdi No.6 is 310bp and the size of a male parent specific marker OPB05 stripe is 325bp; and characteristic bands of male and female parents in a DNA segment of the hybrid have the primer marker. Therefore, the method can be effectively used for identifying the purity of the oil sunflower Kangdi No.6 and No.7 varieties, and has an important application value.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular, the invention specifically relates to the technical field of a method for identifying the purity of the oil sunflower variety. Background technique [0002] sunflower( Helianthus annuus L. ) is one of the four major oil crops in the world, and it is also the main oil crop in northern my country. As an oil or protein resource, people pay more and more attention to it. [0003] With the continuous development of sunflower breeding technology and the large-scale promotion of hybrid varieties, variety identification has become an important means to protect new varieties and farmers' interests. Traditional variety identification mostly uses morphological methods, which identify specific genotypes based on the morphological characteristics and agronomic physiological characteristics of crops. Although it is the most primitive method for testing the purity of varieties, it is simple, intuiti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 王沛政段维杨勇刚李新鹏
Owner 新疆康地种业科技股份有限公司
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