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Oudemansiella radicata SSR molecular marker primer group and application thereof

A technology of molecular markers and long-rooted mushrooms, which is applied in the direction of recombinant DNA technology, microbial measurement/inspection, biochemical equipment and methods, etc., can solve the problems of long time, morphological changes of edible fungus fruiting bodies, etc., and achieve high accuracy and expansion Increase the effect of clear and specific bands

Active Publication Date: 2021-04-30
QINGDAO AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Traditional strain identification methods are mainly based on the phenotype of the strain, such as the morphological structure of fruiting bodies and spores, physiological and biochemical indicators, etc. These identification methods take a long time, and the shape of edible fungus fruiting bodies varies with temperature, humidity and carbon dioxide concentration. Wait for big changes
At present, there is no report on the development of SSR molecular markers based on the whole genome sequence on the genetic diversity of Rhizoma lanceolata germplasm resources

Method used

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  • Oudemansiella radicata SSR molecular marker primer group and application thereof
  • Oudemansiella radicata SSR molecular marker primer group and application thereof
  • Oudemansiella radicata SSR molecular marker primer group and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Example 1: SSR locus analysis, primer screening and cluster analysis of Rhizome lanceolata

[0080] 1. SSR locus analysis of Rhizoma lanceolata

[0081] The whole genome DNA of Rhizome lanceolata samples was extracted by conventional CTAB method, and the whole genome was sequenced by Illumina HiSeq 2000 sequencer, and the obtained genome size was 57.3Mb. The SSRHunter software was used to search for SSR sequences on the Rhizoma lanceolata genome, and the screening conditions were set as follows: the number of repeated bases was 2-6, and the number of repeats was greater than or equal to 5 times, and a total of 1144 SSR sequences were obtained.

[0082] 2. Primer design

[0083] Primers were designed within the range of 200 bp upstream and downstream of the detected SSR site, and the primer design conditions were as follows: the length of the primer was between 18-25 bp, and the annealing temperature was 60±3°C. A total of 20 pairs of primers were designed, and the pri...

Embodiment 2

[0097] Example 2: Application of SSR-labeled primers of Rhizoma lanceolata in strain identification

[0098] 1. The genomic DNA of the strain was extracted by conventional CTAB method. The tested strains were the mycelium of Rhizoma lanceolata, Lentinus edodes, Pleurotus eryngii, Agaricus bisporus, Ganoderma lucidum, Fungus, and Shiji mushroom strains.

[0099] 2. The extracted genomic DNA is used as a template, amplified with SSR-133 primers, and detected by electrophoresis.

[0100] PCR reaction system: Genomic DNA as template 0.5 microliters, SSR-133F upstream primer 2 microliters, SSR-133R downstream primer 2 microliters, 2×pfu mix 12.5 microliters, ddH 2 O to make up to 25 μl.

[0101] PCR reaction program: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 1 min, degradation at 60°C for 1 min, extension at 72°C for 1 min, and repeat the process 35 times; extension at 72°C for 10 min; incubation at 4°C.

[0102] PCR product detection: PCR amplification produc...

Embodiment 3

[0104] Embodiment 3: SSR identification of different parts of the same bacterial strain

[0105] 1. Extract the genomic DNA of the stipe, mushroom root and mushroom part of the fruiting body of the mushroom;

[0106] 2. Using the extracted genomic DNA as a template, use SSR primers to amplify and detect by electrophoresis;

[0107] 3. The electrophoretic bands are consistent with the electrophoretic bands amplified by mycelium, and the layer clustering analysis shows that the similarity coefficient is 100%, and they are completely clustered together, which verifies the effectiveness of the method.

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Abstract

The invention provides an oudemansiella radicata SSR molecular marker primer group and application thereof. According to the invention, 20 pairs of SSR labeled primers with strong specificity, good stability and high polymorphism are obtained by screening based on a whole genome sequence of oudemansiella radicata as a template, and the nucleotide sequences of the primers are shown as SEQ ID NO: 1-SEQ ID NO: 40. The oudemansiella radicata SSR molecular marker primer group can be used for identifying an oudemansiella radicata strain and distinguishing the oudemansiella radicata strain from other non-oudemansiella radicata strains, and can also be used for carrying out traceability detection on the oudemansiella radicata strain. Compared with conventional morphological detection and the like, the method has the advantages of being easy to operate, good in repeatability, stable and accurate in result and the like, and has important reference value and guiding significance for collybia radicata variety identification, genetic diversity analysis, phylogenetic analysis or polymorphism analysis and the like.

Description

technical field [0001] The invention belongs to the technical field of DNA molecular markers of edible fungus strains, and in particular relates to a primer set for SSR molecular markers of Rhizome lanceolata and an application thereof. Background technique [0002] Oudemansiella radicata, also known as Oudemansiella radicata, also known as Oudemansiella radicata, Aureus aureus, and Gallus radicata, etc., belongs to the kingdom of fungi, Basidiomycota, Agaricaceae, Agaricales, and Swellumaceae. The fruiting body of the strain is crisp, smooth and refreshing, with a strong fragrance, rich in nutrients such as protein and polysaccharides, and the unique odermone in the fruiting body has a good inhibitory effect on high blood pressure. It is a food Edible fungus with the same origin as medicine. [0003] With the development of my country's economy and the improvement of living standards, people have higher requirements for the nutrition of food. As an important variety of veg...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/6858C12Q1/04C12N15/11
CPCC12Q1/6895C12Q1/6858C12Q2600/156C12Q2531/113C12Q2565/125
Inventor 于浩胡春辉郭立忠
Owner QINGDAO AGRI UNIV
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