Pumpkin SSR labeled primers applied to multiplex PCR reaction and application of pumpkin SSR labeled primers

A technology of labeled primers and reverse primers, applied in the direction of recombinant DNA technology, microbial measurement/inspection, biochemical equipment and methods, etc., can solve problems such as difficulty in reading results, difficulty in multiplex PCR technology, failure of PCR amplification, etc. Achieve the effects of optimizing the reaction system and amplification program, improving analysis efficiency, and high polymorphism

Active Publication Date: 2015-06-10
HUAZHONG AGRI UNIV
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Problems solved by technology

However, due to the existence of unfavorable factors such as the interference of different primer pairs in the same PCR reaction, which leads to the failure of PCR amplification

Method used

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  • Pumpkin SSR labeled primers applied to multiplex PCR reaction and application of pumpkin SSR labeled primers
  • Pumpkin SSR labeled primers applied to multiplex PCR reaction and application of pumpkin SSR labeled primers
  • Pumpkin SSR labeled primers applied to multiplex PCR reaction and application of pumpkin SSR labeled primers

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Embodiment 1

[0025] (1) SSR site selection: The inventor sampled the roots of 2 pumpkin materials and sent them to Huada Company for transcriptome sequencing. The SSR site prediction was included in the sequencing result report, and SSR sites with high sequencing quality were selected from them. The sequence where the point is located is verified for the polymorphism of the SSR site;

[0026] (2) Primer design: Primer3 Plus software was used to design primers on both sides of the SSR site. The primer setting parameters are: the optimal length of the primer is 20bp, the annealing temperature is 58°C, and the size of the amplified product is 100bp-300bp.

[0027] (3) Pumpkin material and DNA extraction: 61 different commercial pumpkin varieties were purchased from the market, and the total DNA of pumpkin leaves was extracted by CTAB method. The concentration and purity of the extracted DNA were detected by Nanodrop2000 (produced by Thermo Company), and high-quality DNA samples with a ratio ...

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Abstract

The invention discloses three groups of pumpkin SSR labeled primers applied to multiplex PCR reaction, belongs to the technical field of plant molecular biology DNA marks, and discloses an application of the primers to identification of pumpkin varieties and a pumpkin variety identification method. A batch of pumpkin SSR marks are developed by using pumpkin transcriptome sequencing results; three marks are selected from the marks and are characterized in that the polymorphism is high, the strip pattern is excellent, the size difference of amplification products is about 50bp, and primers cannot be matched with each other for forming hairpin structure SSR loca; by virtue of optimization of a PCR reaction system and an amplification program, a multiplex PCR analysis technical system with three SSR loca is built; the SSR marks are excellent in multiplex PCR amplification strip pattern, high in polymorphism and easily readable in results; the analysis efficiency of SSR is remarkably improved; the SSR marks can be applied to the fields of identification of pumpkin varieties, construction of fingerprint maps and identification of purity of hybrid seeds.

Description

technical field [0001] The invention belongs to the technical field of molecular biology DNA markers, and in particular relates to a set of pumpkin SSR marker primers suitable for multiple PCR reactions, and also relates to the application of the primers in pumpkin variety identification and a pumpkin variety identification method. Background technique [0002] Simple sequence repeat (simple sequence repeat, SSR) refers to the 1-6bp base tandem repeat sequence existing on the genome, which shows polymorphism among different individuals due to the difference in the number of repeats. Simple repeats have been used extensively to develop SSR markers. Due to the advantages of high polymorphism, good repeatability, co-dominant inheritance and gene coverage, SSR markers are widely used in the fields of genetic map construction, gene positioning and variety identification. [0003] The SSR marker is a sequence-specific marker. To develop an SSR marker, you must know the SSR site a...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6895C12Q2600/156C12Q2600/16
Inventor 别之龙孔秋生刘勇孙士涛刘朋
Owner HUAZHONG AGRI UNIV
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