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A set of primers, detection method and application for detecting the polymorphism of P.

A primer set and polymorphism technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problems of unsuitable scale, poor reproducibility, high cost, etc., and improve accuracy Effect

Inactive Publication Date: 2014-10-15
SICHUAN ACAD OF GRASSLAND SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among these markers, RAPD markers use a random sequence oligonucleotide as a primer, use genomic DNA as a template for PCR amplification reaction, and detect DNA sequence polymorphism by agarose gel electrophoresis. Poor reproducibility; RFLP markers use genomic DNA to produce a considerable number of DNA fragments of different sizes after digestion with specific restriction enzymes, use isotope or biotin-labeled DNA fragments as probes, and hybridize with The fragments related to the labeled DNA are detected to construct a polymorphism map. Simply put, RFLP is the difference in the length of a specific DNA fragment produced by enzyme digestion between individuals. The disadvantage of this marker is that the operation is cumbersome and the detection cycle Long, expensive, and not suitable for large-scale; ISSR markers are mainly based on a DNA sequence that is complementary to microsatellite (SSRs) sequences and contains 2-4 random bases at the 3' or 5' end as primers on genomic DNA. A molecular marker technique for PCR amplification. SRAP markers use primer design to amplify ORFs (open reading frames, open reading frames). Region-specific amplification, because introns, promoters and spacers vary greatly between different species and even different individuals, which makes it possible to amplify SRAP polymorphic markers based on introns and exons, Although ISSR markers and SRAP markers solve the shortcomings of the above two markers to a certain extent, they are both random dominant genetic markers and cannot distinguish dominant homozygous genotypes from heterozygous genotypes

Method used

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  • A set of primers, detection method and application for detecting the polymorphism of P.
  • A set of primers, detection method and application for detecting the polymorphism of P.
  • A set of primers, detection method and application for detecting the polymorphism of P.

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Experimental program
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Effect test

Embodiment 1

[0038] (1) DNA extraction

[0039] The sample material used for the development of SSR primers and the young leaves of the sample material used for the verification of primer polymorphism were used as materials, and the method of Plant Gneomic DNA Kit (Beijing Tiangen Biochemical Technology Co., Ltd.) was used to extract the total DNA of P. It was detected by NanoDrop ND-1000 Spectrophotometer (NanoDrop, USA). Dilute the sample DNA used to develop SSR primers to 100ng / μL, the sample DNA used for primer polymorphism verification was diluted to 25ng / μL, and stored in a -20°C refrigerator for use in the next step.

[0040] (2) DNA digestion and detection

[0041]Use the restriction endonuclease Sau3AI to digest the sample DNA used for the development of SSR primers, and digest in a water bath at 37°C for 4 hours. The enzyme digestion reaction system is as follows:

[0042] Reagent

50μL reaction system

10×buffer (ABI company, item number: 4335613)

5μL ...

Embodiment 2

[0093] The sample materials used for the development of SSR primers and 46 young leaves of wild Pseudomonas spp. were used as materials, and the total DNA of Pseudomonas spp. was extracted by the method of Plant Gneomic DNA Kit (Beijing Tiangen Biochemical Technology Co., Ltd.), and the DNA concentration was determined by NanoDrop ND-1000 Spectrophotometer (NanoDrop, USA) detection. Dilute the sample DNA used to develop SSR primers to 100ng / μL, the sample DNA used for primer polymorphism verification was diluted to 25ng / μL, and stored in a -20°C refrigerator for use in the next step.

[0094] Use the described SSR primers to carry out PCR amplification to the DNA of Pleurotus chinensis, the amplified reaction system and the pcr reaction procedure are the same as the 10th step of Example 1, and the amplified products are separated by denatured polyacrylamide gel electrophoresis, and silver staining is carried out after the electrophoresis is completed. , and then take picture...

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Abstract

The invention provides SSR primers, applications of the SSR primers in detection of polymorphism of saccharum arundinaceum, a design method of the SSR primers, and a method used for detecting the polymorphism of saccharum arundinaceum by using the SSR primers. The detection method is capable of detecting some genomic SSR locus information of saccharum arundinaceum; in addition, according to verification of polymorphism of saccharum arundinaceum by using the designed primers, the designed primers can be used for the research on genetic diversity of saccharum arundinaceum and construction of genetic maps effectively; an advanced gene analyzer is used in the development of the polymorphism, so that accuracy of primer polymorphism detection is increased.

Description

technical field [0001] The present invention relates to a primer set, in particular a set of primers for detecting the polymorphism of P. chinensis and its method and application for detecting the polymorphism of P. chinensis. Background technique [0002] Banmao is a tall grass of the Poaceae Saccharum genus, which has the characteristics of large biomass, strong stress resistance, and rich cellulose content. Because of its strong stress resistance, it can be used for interspecific hybridization with sugarcane, and it is often used as an excellent gene source in sugarcane breeding and is one of the main materials for sugarcane breeding. At the same time, due to its large biomass and high cellulose content, it has been regarded as an excellent biomass energy material in my country, and its energy use potential is huge. There are a large number of wild spotted grass resources distributed in tropical and subtropical regions of my country, and understanding the law of its gene...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11C12N15/10
Inventor 鄢家俊张建波白史且张劲刀志学常丹张昌兵
Owner SICHUAN ACAD OF GRASSLAND SCI
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