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Agaricus bisporus SSR molecular marker specific primer system and application thereof

A molecular marker and specific primer technology, applied in the field of molecular biology, can solve the problem of the same name of Agaricus bisporus, the same name of foreign body and so on

Active Publication Date: 2016-01-20
JILIN AGRICULTURAL UNIV
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  • Abstract
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  • Application Information

AI Technical Summary

Benefits of technology

This patented technology allows us to identify both gram-negative (G) and Gram-positive Bacilluses from samples taken from Gigaspariaceae crops such as Amanita nuxvorum or Laminaria japonica. By comparing these two types with known ones we can make sure that there are no other germs on this type of crop without being detected during testing.

Problems solved by technology

The technical problem addressed in this patented text relates to identifying bacterial genera like Amanita nigricans or Bacillussina diastanutis that contain various valuable compounds such as flavonoids, carbohydrate substances, amino acids, vitamines, mineral salts, phytoalexin gamma steroids, lignocysteine sulfate lyase polypeptides, polyphenols, etc., among others. However, current methods require complicated manipulation techniques, expensive equipment, long periods of incubations before accurate results may appear due to changes caused during natural environments.

Method used

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  • Agaricus bisporus SSR molecular marker specific primer system and application thereof
  • Agaricus bisporus SSR molecular marker specific primer system and application thereof
  • Agaricus bisporus SSR molecular marker specific primer system and application thereof

Examples

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Embodiment 1

[0027] Example 1 Development of Agaricus bisporus EST-SSR marker primers

[0028] 1. Design and synthesis of SSR primers:

[0029] Search and download from the JGI database of Agaricus bisporus H97 genome data (version2.0) (http: / / genome.jgi-psf.org / .), using MISA (http: / / pgrc.ipk-gatersleben.de / tools .php) to search for SSR, the search criteria are: the minimum number of repetitions of two, three, four, five, and hexanucleotides is greater than or equal to 5 times.

[0030] Primers were designed in sequences containing SSR sites using the primer design software Primer3.0. SSR primer setting parameters are: (1) GC content is 45%-60%; (2) annealing temperature is 60°C; (3) expected fragment length is between 150-300bp; (4) primer length is 18-27bp between.

[0031] 2. Screening of SSR primers

[0032] (1) Extract the total DNA of the sample to be identified using Kangwei Century's genome extraction kit;

[0033] (2) Use 10 random Agaricus bisporus strains to conduct prelim...

Embodiment 2

[0045] Example 2 Application of Agaricus bisporus SSR marker primers in species identification

[0046] Select 10 pairs of SSR molecular markers with polymorphism and specificity to construct electronic molecular ID cards for the different strains screened above. Specific steps are as follows:

[0047] 1) The PCR products of 59 strains of each primer were also subjected to 8% polyacrylamide gel electrophoresis in step (3), and then the different electrophoresis band patterns were counted to determine whether the 59 strains were the same strain. The results showed that there were 22 specific strains among the 59 strains, and the remaining 37 strains were divided into 7 categories:

[0048] The first category: the same strain as CCMJA29 is CCMJA9, CCMJA21;

[0049] The second category: the same strain as CCMJA33 is CCMJA6;

[0050] The third category: the same strains as CCMJA22 are CCMJA1, CCMJA2, CCMJA11, CCMJA13, CCMJA14, CCMJA36;

[0051] The fourth category: the same st...

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Abstract

The invention discloses valid combinations of 8 novel SSR molecular markers and 2 known SSR molecular markers. The valid combinations are applied to strain identification. DNAs of 22 specific strains are taken as templates, 10 pairs of SSR primers are respectively utilized for carrying out PCR amplification, each pair of SSR primers is subjected to acrylamide gel electrophoresis after the PCR amplification, banding pattern statistic analysis is carried out according to all electrophoretic bands amplified from 22 samples, and therefore, a standard banding pattern of each pair of primers is determined; then the standard banding patterns of 10 pairs of primers of each sample are combined, and molecular identification cards of the 22 different strains of agaricus bisporus are constructed. According to an agaricus bisporus SSR molecular marker specific primer system and an application thereof, the strain identification effect of agaricus bisporus is improved; the agaricus bisporus SSR molecular marker specific primer system and the application are significant for the resource identification, the protection, the development and the utilization of agaricus bisporus.

Description

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Claims

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Application Information

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Owner JILIN AGRICULTURAL UNIV
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