EST-SSR core primer group for identifying variety of Chinese wolfberry and identification method and application thereof

A technology of core primers and varieties, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve problems such as powerlessness, achieve stable amplification, good repeatability, and clear amplification results

Active Publication Date: 2016-10-12
SOUTH CHINA BOTANICAL GARDEN CHINESE ACADEMY OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For the identification of wolfberry products (such as dried fruit and drinks, etc.

Method used

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  • EST-SSR core primer group for identifying variety of Chinese wolfberry and identification method and application thereof
  • EST-SSR core primer group for identifying variety of Chinese wolfberry and identification method and application thereof
  • EST-SSR core primer group for identifying variety of Chinese wolfberry and identification method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0053] Example 1: Development of EST-SSR core primer set based on Lycium barbarum transcriptome sequence

[0054] 1. Screening of SSR sites in Lycium barbarum EST sequence

[0055] Use MicroSAtellite (MISA) (http: / / pgra.ipk-gatersleben.de / misa) software to search for all SSR sites in the unigenes of Lycium barbarum transcriptome in the database. The search limit is: the minimum repetition of two bases 6 times, three-base, four-base, five-base, and six-base repeats are repeated at least 5 times. If the distance between two SSR sites is less than 100bp, it is regarded as a composite SSR.

[0056] 2. EST-SSR primer design and synthesis

[0057] Use primer3 software to develop and design target primers for SSR sites in unigenes. The design criteria are: primer length is 18-25bp, annealing temperature is 57-63℃, GC content is 40-70%, PCR product length is 100-300bp , Select SSR sites that can be compared with NCBI non-redundant protein database to synthesize 300 pairs of primers, each pai...

Example Embodiment

[0085] Example 2 Using EST-SSR markers to identify the varieties of Chinese medicine wolfberry

[0086] 1. Extract DNA

[0087] The improved CTAB method was used to extract the DNA of the leaves of the seven species of Lycium barbarum, and the DNA was quantified by an ultraviolet spectrophotometer, diluted to 50ng / μl, and stored at 4°C or -20°C for later use;

[0088] The specific steps are the same as in Example 1.

[0089] 2. PCR amplification

[0090] Using the DNA extracted in step 1 as a template, 10 pairs of EST-SSR core primers developed in Example 1 were used for PCR amplification. The amplification method was the same as that in Example 1 (combined according to Table 1 to perform multiplex PCR, that is, all upstream in each combination The primers and all downstream primers are added to the PCR system, and the same PCR reaction produces two or three SSR-labeled amplification products, specifically:

[0091] Reaction system I: PCR reaction system: 50ng / μl template DNA, 10mM Tris...

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Abstract

The invention discloses an EST-SSR core primer group for identifying the variety of Chinese wolfberry and an identification method and an application thereof. Through screening of EST sequences of lycium ruthenicum and Ningxia wolfberry, a large number of SSR molecular markers are developed, and an SSR marker-meeting fluorescence detection technology system including 10 markers is established and is used for identification of the Chinese wolfberry variety and species. The 10 markers have the advantages of good polymorphism, stable amplification, clear amplification results and good repeatability, can be used for large-scale detection of authenticity of Chinese wolfberry seedlings and the purity of the seedlings, can provide a technical basis for standardization of seedling markets, and are conducive to development and use of the traditional Chinese medicine Chinese wolfberry.

Description

Technical field: [0001] The invention belongs to the field of molecular markers and identification of medicinal materials, and in particular relates to an EST-SSR core primer set for identification of wolfberry varieties, an identification method and application thereof. Background technique: [0002] Lycium barbarum is a deciduous shrub plant of the genus Lycium L. in the family Solanaceae; it is a traditional Chinese medicinal material that nourishes the lungs, clears the liver, nourishes the kidneys, nourishes qi, generates essence, helps yang, nourishes deficiency and labor, and strengthens bones and muscles. , expelling wind, improving eyesight, etc.; the main medicinal ingredient Lycium barbarum polysaccharide (polysaccharide, LBP) has the functions of promoting immunity, anti-aging, anti-tumor, scavenging free radicals, anti-fatigue, anti-ultraviolet radiation, anti-radiation, liver protection, neuroprotection, etc. . Ningxia wolfberry is the most widely distributed ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/6895C12Q2525/151C12Q2537/143
Inventor 王瑛曾少华胡伟明黄珊珊
Owner SOUTH CHINA BOTANICAL GARDEN CHINESE ACADEMY OF SCI
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