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Method for rapidly identifying hot pepper species and golden pepper purity degree by using EST-SSR molecular markers

A molecular marker, golden pepper technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of limited information, difficult hybridization, long cycle, etc., to achieve good repeatability and stability, popularization Promising, easy-to-use effects

Active Publication Date: 2015-09-23
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

Seed morphology is easily affected by environmental conditions, and the accuracy of identification results is poor; field planting identification has a long cycle, high cost, and heavy workload, and the phenotype is easily affected by cultivation measures and environmental conditions, which seriously affects variety identification. The efficiency and accuracy of the method are increasingly unable to meet the needs of breeding work and production management; although the use of isozyme technology to identify the purity of crop varieties is accurate and reliable, but isozyme markers are specific to tissues and organs, and the amount of information is very limited. Polymorphism is not rich enough
As the number of new pepper varieties continues to increase, traditional identification methods are difficult to effectively distinguish hybrids with close genetic relationships

Method used

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  • Method for rapidly identifying hot pepper species and golden pepper purity degree by using EST-SSR molecular markers
  • Method for rapidly identifying hot pepper species and golden pepper purity degree by using EST-SSR molecular markers
  • Method for rapidly identifying hot pepper species and golden pepper purity degree by using EST-SSR molecular markers

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] The acquisition of embodiment 1 golden pepper variety leaf genomic DNA

[0032] The specific operation method is as follows:

[0033] (1) Put the "Golden Pepper" parent and F1 seeds into a petri dish covered with damp filter paper, light intensity 2000lx, light cycle 16h light / 8h dark, light culture at 28°C for 6 days and then take the leaves;

[0034] The process of obtaining the F1 seeds described therein is as follows: after the female parent plant with pollen is pulled out during flowering, the female parent plant and the male parent plant are 4:1, through artificial pollination, the male parent pollen is pollinated to the female parent, and finally harvested F1 generation seeds on the female parent;

[0035](2) Put a young pepper leaf in each centrifuge tube, add 500 μL extraction buffer preheated at 60-65°C, put 4 steel balls, crush the material on a crusher, and bathe in water at 60-65°C for 20-30min ;

[0036] The extraction buffer described therein is 2×CTAB...

Embodiment 2

[0044] Embodiment 2 PCR amplification

[0045] Using the RNA enzymatic digestion mixture obtained above as a template, PCR amplification was performed using ClSH1 primers:

[0046] The PCR reaction system is 20 μL, including 2 μL of 10×PCR Buffer, 0.4 mM dNTPs, 10 μM of primers, 1U Taq DNA polymerase, 100 ng of template, and make up to 20 μL with sterile ultrapure water;

[0047] The amplification program was: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 57°C for 30 s, extension at 72°C for 1 min, 35 cycles; final extension at 72°C for 10 min; PCR products were stored at 10°C.

[0048] Example 3 Amplification product detection

[0049] 5 μL of amplified product was mixed with 2 μL of loading buffer, and then subjected to non-denaturing polyacrylamide gel electrophoresis with acrylamide mass volume ratio of 10%, 160v constant voltage electrophoresis for 1.5h, and silver staining for band pattern statistics;

[0050] The loading buffer used i...

Embodiment 4

[0064] Embodiment 4 identifies the purity of " golden pepper " hybrid

[0065] According to the electrophoresis results of the specific bands, the purity of the "Golden Pepper" hybrid is identified, and the judgment standard is:

[0066] The female parent has specific bands of 120bp, 200bp, 250bp, and 270bp; the male parent has specific bands of 120bp, 200bp, 250bp, and 260bp; the hybrid has specific bands of 120bp, 200bp, 250bp, 270bp, 400bp, and 450bp;

[0067] According to the above-mentioned identification results, the purity of the pepper variety (%)=(1-n / N)×100% can be converted, where N is the number of pepper seeds to be tested, and n is the band characteristic of the male parent alone or the spectrum of the female parent alone The number of plants with band characteristics or different band characteristics from both "Golden Pepper" and parent parent.

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Abstract

The invention relates to the field of molecular markers and provides a method for rapidly identifying hot pepper species and golden pepper purity degrees by using the EST-SSR molecular markers. The method adopts the specially designed EST-SSR primers, augmentation is conducted with the leaf genome DNA of golden pepper, the species purity degree of the golden pepper to be detected is tested through the specific bands of the augmentation result, by the adoption of the method, the identification can be conducted at any period during the growth of golden pepper, and the coeno-species can be separated from female parent selfed seeds and male parent selfed seeds; the requirement on DNA quality is not high, and the needing amount is less; the cost is low, the operation is simple; the repeatability and the stability are good; the fastness and the high efficiency are achieved; the accuracy is high, the application and the popularization prospect is wide, and scientific basis is provided for the development of pepper seed production and good seeds and the in-time selling.

Description

technical field [0001] The invention relates to the field of molecular markers, and specifically provides a method for quickly identifying the purity of hot pepper varieties by using EST-SSR molecular markers. Background technique [0002] Capsicum, belonging to the Solanaceae family, is an annual or perennial herb. Capsicum has become an important worldwide vegetable because of its unique pungent taste, color and nutritional components. A large number of studies have shown that the antioxidant substances contained in pepper fruits can protect biological organisms from oxidative damage and improve the body's immunity. In addition, capsaicinoids in capsicum can accelerate the metabolism of energy and lipids and reduce the incidence of cancer cells. With the public's full understanding of the nutritional and medicinal ingredients of peppers, the cultivation area has increased year by year. [0003] Seed quality directly affects the quality of agricultural products and the y...

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 袁晓伟李兴盛韩永升
Owner 华盛农业集团股份有限公司
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