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.) at the seedling stage or on the market, morphological identification is powerless

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

Embodiment 1

[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 all SSR sites in the Lycium barbarum transcriptome unigenes in the database. The search restriction is: two base repeats with the least repeat 6 times, three bases, four bases, five bases and six bases repeat at least 5 times, if the distance between two SSR sites is less than 100bp, it is considered 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°C, GC content is 40-70%, and the length of PCR product is 100-300bp , select SSR sites that can be compared with the NCBI non-redundant protein database to synthesize 300 pairs of...

Embodiment 2

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

[0086] 1. DNA extraction

[0087] The DNA of the leaves of the above seven Lycium barbarum species was extracted by the improved CTAB method, quantified by a UV spectrophotometer, diluted to 50 ng / μl, and stored at 4°C or -20°C until use;

[0088] Concrete steps are with embodiment 1.

[0089] 2. PCR amplification

[0090] Using the DNA extracted in step 1 as a template, carry out PCR amplification with 10 pairs of EST-SSR core primer pairs developed in Example 1, and the amplification method is the same as in Example 1 (multiple PCR is carried out according to the combination in Table 1, that is, all upstream in each combination Primers and all downstream primers are added to the PCR system, and two or three SSR-labeled amplification products are obtained in the same PCR reaction, specifically:

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

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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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