Kit for rapid identification of purity of new sunflower No.19 of oil sunflower

A kit and oil sunflower technology, applied in the field of biological kits for rapid identification of the purity of oil sunflower 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, speeding up the quality inspection process, and the effect of speeding up the identification time

Inactive Publication Date: 2014-11-26
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

[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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  • Kit for rapid identification of purity of new sunflower No.19 of oil sunflower
  • Kit for rapid identification of purity of new sunflower No.19 of oil sunflower

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Kit for quickly identifying the purity of oil sunflower Xinkui No. 19 variety

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

[0041] (1) Oil sunflower DNA rapid extraction kit A configuration: 50mmol / L Tris-HCl pH8, 0.7 mmol / L NaCl, 10 mmol / L EDTA, pH8, 1% CTAB, 20mmol / L 2-mercaptoethanol.

[0042] (2) Configuration of PCR amplification reaction kit B: 5×4ul reaction buffer, 25mmol / L MgCl 2 Solution 2ul, 0.1mmol / L dNTPs solution 3ul, 1umol / L Primer solution 3ul (SSR primer markers 399 and 425), 5u Taq DNA polymerase 0.2ul.

[0043] 1-5ng template DNA; 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, total 30 cycles; extension at 72°C for 8min.

[0044] Primer label 399:

[0045] Left sequence CGTACGGTGTAGTTCTCATGGT;

[0046] Right sequen...

Embodiment 2

[0052] Example 2: Rapid identification of the purity of oil sunflower Xinkui No. 19

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

[0054] (1) Oil sunflower DNA rapid extraction reagent contains 50mmol / L Tris-HCl pH8, 0.7 mmol / L NaCl, 10 mmol / L EDTA, pH8, 1% CTAB, 20mmol / L 2-mercaptoethanol.

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

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

Embodiment 3

[0060] Example 3: Rapid identification of the purity of oil sunflower Xinkui No. 19

[0061] (1) Sunflower DNA extraction: Take the young true leaves of sunflower cultured for 1 week in a mortar, add liquid nitrogen to quickly grind it into a uniform powder, and add 500μl of a 65℃ preheated CTAB extraction kit before the sample is melted. After mixing thoroughly, transfer the sample solution into a 1.5 ml centrifuge tube, place it in a 65℃ water bath for 50 min, invert the centrifuge tube several times during this time, CTAB extraction kit uses 50 mmol / L Tris-HCl pH8, 0.7 mmol / L NaCl, 10 mmol / L EDTA, pH8, 1% CTAB, 20mmol / L 2-mercaptoethanol; take out the sample and cool to room temperature, add equal volumes of phenol / chloroform (1:1) and chloroform / isoamyl alcohol (24:1) respectively Extraction, centrifuge at 12000 r / min at 4°C for 5 min; take the supernatant and add 0.6 times the volume of isopropanol to mix, place at room temperature for 5-10 min, centrifuge at 12000 r / min f...

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Abstract

The invention discloses a kit for rapid identification of purity of new sunflower No.19 of oil sunflower. With a commercial oil sunflower variety new sunflower No.19 and DNAs of parents of the variety as templates, a large number of SSR primer molecular markers are screened and repeated by building an optimal procedure including an oil sunflower SSR technology, optimal combination of various components for PCR amplified reaction, and developing procedures of an amplification kit and an amplification band, so as to obtain primer markers 399 and 425 capable of simultaneously distinguishing the male parent, the female parent and hybrids. The two primer markers can be effectively used for identifying the purity of hybrid seeds of the new sunflower No.19, and distinguishing the authenticity of the seeds, efficient and accurate quality control for the hybrid seeds of the oil sunflower is facilitated, the quality detection process of the seeds of the new sunflower No.19 is accelerated, the kit can be effectively used for detecting the purity of the hybrids seeds of the oil sunflower and distinguishing the authenticity of the seeds, the time from DNA extraction to purity identification of molecular markers is shortened within a workday, and the kit 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 Applications(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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