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A kit for rapid identification of the purity of No. 19 oil sunflower variety

A kit and oil sunflower technology, which is applied in the field of biological kits for rapid identification of the purity of new sunflower 19 varieties, can solve the problem that there is no standard procedure for rapid biochemical or DNA molecular detection technology, and achieve the elimination of reagent configuration And specific primer screening process, stable amplification effect, efficient and accurate quality control effect

Inactive Publication Date: 2016-03-16
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

[0006] 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
In the prior art, there is no report about the test kit for identifying the purity of No. 19 oil sunflower variety Xinkui

Method used

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  • A kit for rapid identification of the purity of No. 19 oil sunflower variety
  • A kit for rapid identification of the purity of No. 19 oil sunflower variety

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: A kit for rapid identification of the purity of No. 19 oil sunflower variety

[0040] The present invention provides a kit for identifying the male parent, female parent and hybrid of Xinkui No. 19, which includes:

[0041] (1) Oil sunflower DNA rapid extraction kit A configuration: 50mmol / LTris-HClpH8, 0.7mmol / LNaCl, 10mmol / LEDTA, pH8, 1%CTAB, 20mmol / L2-mercaptoethanol.

[0042] (2) PCR amplification reaction kit B configuration: 5× reaction buffer 4ul, 25mmol / LMgCl 2 Solution 2ul, 0.1mmol / LdNTPs solution 3ul, 1umol / LPrimer solution 3ul (SSR primer markers 399 and 425), 5uTaqDNA polymerase 0.2ul.

[0043] 1-5ng template DNA; the total reaction volume is 20ul, the amplification program is denaturation at 95°C for 2min, denaturation at 94°C for 45s, annealing at 57°C for 45s, extension at 72°C for 60s, a total of 30 cycles; extension at 72°C for 8min.

[0044] Primer Mark 399:

[0045] Left sequence CGTACGGTGTAGTTCTCATGGT;

[0046] right sequence GGATCACGT...

Embodiment 2

[0052] Example 2: Rapid Identification of the Purity of Oil Sunflower No. 19 Variety

[0053] A method for quickly identifying the purity of No. 19 oil sunflower variety, the specific method steps are as follows:

[0054] (1) The oil sunflower DNA rapid extraction reagent contains 50mmol / LTris-HCl pH8, 0.7mmol / LNaCl, 10mmol / LEDTA, pH8, 1% CTAB, 20mmol / L 2-mercaptoethanol.

[0055] (2) PCR amplification reaction reagent contains 4ul of 5× reaction buffer, 25mmol / LMgCl 2 Solution 2ul, 0.1mmol / LdNTPs solution 3ul, 1umol / LPrimer solution 3ul (SSR primer markers 399 and 425), 5uTaq DNA polymerase 0.2ul, 1-5ng template DNA; the total reaction volume is 20ul, and the amplification program is denaturation at 95°C for 2min , Denaturation at 94°C for 45s, annealing at 57°C for 45s, extension at 72°C for 60s, a total of 30 cycles; extension at 72°C for 8min.

[0056] (3) Preparation of the gel plate: the amplified product was subjected to non-denaturing polyacrylamide gel electrophores...

Embodiment 3

[0060] Example 3: Rapid Identification of the Purity of Oil Sunflower No. 19 Variety

[0061] (1) Sunflower DNA extraction: Take the young true leaves of sunflower cultivated for 1 week above in a mortar, add liquid nitrogen and quickly grind them into a uniform powder, add 500 μl of CTAB extraction kit preheated at 65°C before the sample melts , after fully mixing, transfer the sample solution into a 1.5ml centrifuge tube, place it in a 65°C water bath for 50 minutes, and invert the centrifuge tube several times during this period. The CTAB extraction kit uses 50mmol / LTris-HCl pH8, 0.7mmol / LNaCl, 10mmol / LEDTA, pH8, 1% CTAB, 20mmol / L 2-mercaptoethanol; take out the sample and cool it to room temperature, add an equal volume of phenol / chloroform (1:1) and chloroform / isoamyl alcohol (24:1) for extraction, 4°C 12000r / min Centrifuge for 5 minutes; take the supernatant and add 0.6 times the volume of isopropanol to mix, place at room temperature for 5-10 minutes, centrifuge at 1200...

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Abstract

The invention discloses a kit for quickly identifying the purity of No. 19 oil sunflower variety. By establishing the optimal program including the oil sunflower SSR technology, the optimized combination of each component of the PCR amplification reaction and the amplification kit, and the color development program of the amplified band; the commercial oil sunflower variety Xinkui 19 and its parents Using DNA as a template, a large number of SSR primer molecular markers were screened and repeated, and primer markers 399 and 425, which can simultaneously distinguish the male parent, female parent and hybrid species, were obtained. These two primer markers can be effectively used to identify the above-mentioned new sunflower The purity of No. 19 hybrid seeds and the identification of the authenticity of the seeds are beneficial to the efficient and accurate quality control of oil sunflower hybrid seeds and speed up the quality inspection process of Xinkui No. 19 commercial seeds. It can be effectively used to identify the purity of sunflower hybrid seeds, distinguish the authenticity of the seeds, shorten the time from DNA extraction to molecular marker purity verification to within one working day, and has 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 kit for rapidly identifying the purity of the new sunflower variety 19. Background technique [0002] sunflower( Helianthus annuus L. ) is one of the five major economic crops in the world. It has the characteristics of resistance to salt and alkali, drought and barrenness. It is also the main oil crop in northern my country. It is increasingly valued as an oil or protein resource. There are quite a few dry and barren low-yield fields and saline-alkali land in my country, so sunflower plays an important role in agricultural production. All countries in the world have generally carried out three-line research on the utilization of sunflower heterophylls. my country has carried out research on the utilization of sunflower heterozygous species since the 1970s, and began to use hybrids in agricultural production. [0003] Wit...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6858C12Q1/6895C12Q2600/156C12Q2531/113C12Q2565/125
Inventor 段维杨涛王沛政朱文彬刘文杰李晓雷卫泽
Owner 新疆康地种业科技股份有限公司
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