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EST-SSR molecular identification method for E.wushanense and easily mixed species thereof

A technology of molecular identification and Epimedium, which is applied in biochemical equipment and methods, microbiological determination/inspection, DNA/RNA fragments, etc., can solve problems such as differences in the composition and content of medicinal ingredients, and achieve rapid and accurate identification, expansion Stable results and strong specificity

Active Publication Date: 2019-09-06
WUHAN BOTANICAL GARDEN CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there are obvious differences in the morphology of flowers and inflorescences between Epimedium Wushanense and Epimedium Zhenping, Epimedium Jinchengshan, Epimedium pseudo-Wushanii and Epimedium Guizhoubei, the leaves of the treated parts are highly similar in shape. In the use of medicinal materials, they are often mixed, and the composition and content of medicinal components of different species are quite different. Therefore, it is urgent to develop molecular markers that can quickly identify Epimedium wushanense and its easily mixed species.

Method used

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  • EST-SSR molecular identification method for E.wushanense and easily mixed species thereof
  • EST-SSR molecular identification method for E.wushanense and easily mixed species thereof
  • EST-SSR molecular identification method for E.wushanense and easily mixed species thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1: Epimedium SSR primer design

[0028] Use the MicroSatellite (MISA) software to perform SSR searches on the Unigenes in the Epimedium transcriptome above 1kb in the database with the repeat times of 10, 6, and 5 for single nucleotides, dinucleotides, and trinucleotides; The Primer 6 software designed the target primers for the searched SSR sites. The design criteria were: the primer length was 18-26bp, the annealing temperature was 50-65°C, and the length of the final product of PCR amplification was 100-350bp. 5471 pairs of primers were synthesized from the SSR sites compared with the redundant protein database, and each pair of primers consisted of an upstream primer F and a downstream primer R.

Embodiment 2

[0029] Example 2: Extraction and detection of sample genomic DNA

[0030] The extraction and detection of genomic DNA from the leaves of Epimedium wushanense and its four easily mixed species, the specific steps are as follows:

[0031] A. Select the young leaves of the sample to be tested, remove the surface dust with 75% alcohol, preheat CTAB buffer at 65°C, and pre-cool isopropanol at -20°C;

[0032] B. Take 0.1g of dried plant leaves, freeze them in liquid nitrogen, grind them into powder, put them into 2mL centrifuge tubes, add 1mL of CTAB buffer solution preheated to 65°C, mix them upside down, and incubate at 65°C for 30 minutes. Shake well every 10 minutes;

[0033] C. After incubation, centrifuge at 12000r / min for 10 min, take 900μL of supernatant, add an equal volume of chloroform-isoamyl alcohol (24:1 (V / V)), mix well and extract for 5min, centrifuge at 12000r / min 10min, take the supernatant;

[0034] D. Repeat step C, add an equal volume of isopropanol pre-coole...

Embodiment 3

[0040] Embodiment 3: PCR amplification

[0041] Using the synthesized 10 pairs of EST-SSR core primers and universal M13 primers, perform PCR amplification on the genomic DNA of Epimedium wushanense and its four easily mixed species. The reaction system is: 2×Taq PCR Master Mix reaction buffer 5 μL , 0.0125 μL and 0.25 μL of 10 μM upstream and downstream primers, 0.15 μL of M13-ROX / FAM / HEX / TAMRA adapter primer, 3 μL of diluted DNA template, and make up to 10 μL with sterile water; Denaturation for 4min, denaturation at 94°C for 30s, annealing temperature from 65°C to 50°C for a total of 15 cycles, each cycle lowering 1°C, then 22 cycles at annealing temperature of 50°C, extension at 50°C for 30s, final extension at 72°C for 42s , stored at 16°C.

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Abstract

The invention discloses an EST-SSR molecular identification method for E.wushanense and an easily mixed species thereof, and belongs to molecular markers and medicinal material identification in the technical field of molecular biology. An EST-SSR primer group for identification of E.wushanense and the easily mixed species thereof include 10 pairs of EST-SSR core primers and common M13 primers having the sequences of SEQ ID NO:1, 2...20 and 21. The identification method includes the steps: (1) extracting total DNA of a sample by a CTAB method; (2) amplifying by PCR by using total DNA of the sample to be determined as a template; (3) analyzing a product of PCR amplification; and (4) constructing a phylogenetic tree. An application comprises identification of E.wushanense and the easily mixed species thereof. The 10 SSR molecular markers provided by the invention have good polymorphism, high specificity and stable amplification results, can rapidly and accurately identify E.wushanense and the easily mixed species thereof, provide technical support for promotion of the quality standard of epimedium medicinal materials, and are beneficial to development and utilization of the epimediummedicinal materials.

Description

technical field [0001] The invention belongs to molecular markers and identification of medicinal materials in the technical field of molecular biology, and specifically relates to a kind of Epimedium wushanense ( E. wushanense ) and its easily mixed species Epimedium jinchengshan ( E. jinchengshanense ), Zhenping Epimedium ( E. ilicifolium ), Epimedium pseudo-Wusshanense ( E. pseudowishanense ), Qianbei Epimedium ( E. borealiguizhouense ) EST-SSR molecular identification method. Background technique [0002] Epimedium, a traditional Chinese medicinal herb, is the dried leaf of a plant of the genus Epimedium in the family Berberidaceae. There are about 62 known species of Epimedium in the world, and about 52 species in my country (Stearn, WT. The Genus Epimedium and Other Herbaceous Berberidaceae. Portland: Timber Press, 2002.; Ying, TS., Boufford, DE., Brach , AR. Epimedium L. St. Louis:Science Press, MissouriBotanical Garden Press, 2011, 787-799.), each edition o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895C12Q2600/156Y02A50/30
Inventor 张燕君于东悦梁琼王瑛杨路路
Owner WUHAN BOTANICAL GARDEN CHINESE ACAD OF SCI
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