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Method for quickly identifying purity of cucumber rootstock Heli II by use of molecular marker

A technology of molecular markers and rootstocks, applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems that cannot meet the needs of breeding work and production management, affect the efficiency and accuracy of variety identification, and poor accuracy, etc. Achieve the effects of saving manpower, shortening the cultivation time, and overcoming errors

Active Publication Date: 2017-05-31
华盛农业集团股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The traditional rootstock hybrid seed purity identification work is still mainly based on field planting identification. Through the comparison of the appearance and shape of grafted cucumbers, this method generally takes 2 months for identification, which is long in time, high in cost, difficult and poor in accuracy. It is easily affected by cultivation measures and environmental conditions, which seriously affects the efficiency and accuracy of variety identification, and is increasingly unable to meet the needs of modern breeding work and production management.

Method used

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  • Method for quickly identifying purity of cucumber rootstock Heli II by use of molecular marker
  • Method for quickly identifying purity of cucumber rootstock Heli II by use of molecular marker
  • Method for quickly identifying purity of cucumber rootstock Heli II by use of molecular marker

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Acquisition of Genomic DNA from Young Roots of Cucumber Rootstock Heli Variety 2

[0033] The specific operation method is as follows:

[0034] (1) Put the parents of "cucumber rootstock Heli 2" and F1 seeds into a petri dish covered with damp filter paper, cultivate in the dark at 30°C for 3 days, and then take the young roots;

[0035] Among them, in the process of hybrid seed production of this variety, the female parent needs to be artificially detasselled, and the pollen of the male parent is given to complete the hybridization process, and finally the F1 generation seeds on the female parent are harvested;

[0036] (2) Put a young root in each centrifuge tube, add 500 μL of extraction buffer preheated at 60-65°C, add 4 steel balls, crush the material on a crusher, and bathe in water at 60-65°C for 20-30 minutes;

[0037] The extraction buffer described therein is 2×CTAB extraction solution, and its proportioning is as follows:

[0038]

[0039] Ster...

Embodiment 2

[0045] Embodiment 2 PCR amplification

[0046] Using the RNA enzymatic mixture obtained above as a template, use HELI2 primers for PCR amplification:

[0047] The PCR reaction system is 10 μL, including 1 μL of 10×PCR Buffer, 0.2 mM dNTPs, 5 μM of primers, 0.5U Taq DNA polymerase, 100 ng of template, and make up to 10 μL with sterile ultrapure water;

[0048] According to the present invention, the amplification program is set as follows: pre-denaturation at 95°C for 3 minutes; denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 20 s, 35 cycles; final extension at 72°C for 5 min; PCR products stored at 10°C.

Embodiment 3

[0049] Example 3 Amplification product detection

[0050] 5 μL of amplified product was mixed with 2 μL of loading buffer, and then subjected to non-denaturing polyacrylamide gel electrophoresis with an acrylamide mass volume ratio of 8%, 180v constant voltage electrophoresis for 1.5h, and silver staining for band statistics;

[0051] The loading buffer used is selected from the 10× glycerol gel loading solution;

[0052] The 12% non-denaturing polyacrylamide gel formulation (100mL) described therein is as follows:

[0053]

[0054] Wherein the 30% acrylamide / methylene bisacrylamide configuration process adopted is as follows:

[0055] Acrylamide 29g methylene bisacrylamide 1g distilled water to 100mL and store in the dark at 4°C, with a shelf life of one month;

[0056] The adopted 10╳TBE configuration process is as follows:

[0057] Tris Base 27g, boric acid 13.75g, 0.5M / L EDTA 10mL, distilled water to 250mL, adjust pH to 8.3;

[0058] The 10% ammonium persulfate (APS...

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Abstract

The invention relates to the field of molecular markers and provides a method for quickly identifying purity of cucumber rootstock Heli II by use of a molecular marker. According to the method, a specially designed EST-SSR primer is adopted, and the genome DNA of Heli II serves as a template for amplification, and the purity of the F1-generation seeds of the to-be-detected cucumber rootstock Heli II is detected according to the specific band of the amplification result; by adopting the method, the plant growth of cucumber rootstock Heli II can be identified in any stage, and the hybrid seeds can be distinguished from the female-parent selfing seeds and male-parent selfing seeds; and moreover, the method has high accuracy and broad prospect in application and promotion and provides scientific basis for rootstock seed production and timely selling of improved seeds.

Description

technical field [0001] The invention relates to the field of molecular markers, and specifically provides a method for rapidly identifying the purity of cucumber rootstock Heli Er by using molecular markers. Background technique [0002] Cucumber rootstock Heli No. 2 is the latest high-quality cucumber rootstock variety cultivated by Shandong Huasheng Agriculture Co., Ltd. This variety has good grafting affinity, and after grafting, the cucumber grows vigorously and has enhanced disease resistance. After grafting, the melon strips are black, bright and straight, which improves the commerciality. Suitable for protected spring and autumn and winter planting. During the hybrid seed production process of this variety, the female parent needs to be detasselled manually, and the male parent pollen is given to complete the hybridization process. Incomplete or missing emasculation of the female parent will greatly affect the purity of the seed production. Therefore, it is very imp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6895C12Q2600/156
Inventor 袁晓伟李兴盛汪琳丽
Owner 华盛农业集团股份有限公司
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