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Method for carrying out genetic analysis and paternity test of diversifolious poplar colony by 8-plex PCR (polymerase chain reaction)

A technology of population genetic analysis and paternity testing, which is applied in the field of simultaneously amplifying 8 SSR sites on the whole genome of Populus euphratica, can solve the problems of time-consuming and labor-intensive, reduce the probability of pairing and complementation, and reduce the operation The effect of reducing errors and operating steps

Inactive Publication Date: 2012-09-19
BEIJING FORESTRY UNIVERSITY
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Problems solved by technology

[0007] Aiming at the time-consuming and labor-intensive shortcomings of the traditional PCR method, the present invention provides an efficient and convenient primer and method for amplifying multiple SSR sites of Populus euphratica, realizing the amplification of 8 SSRs with high polymorphism in one reaction loci, providing important technical support for subsequent population genetics analysis and parentage identification in natural populations

Method used

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  • Method for carrying out genetic analysis and paternity test of diversifolious poplar colony by 8-plex PCR (polymerase chain reaction)
  • Method for carrying out genetic analysis and paternity test of diversifolious poplar colony by 8-plex PCR (polymerase chain reaction)
  • Method for carrying out genetic analysis and paternity test of diversifolious poplar colony by 8-plex PCR (polymerase chain reaction)

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Experimental program
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Effect test

Embodiment 1

[0045] The natural population of Populus euphratica was marked by multiplex PCR and the genetic parameters were calculated.

[0046] 1) Select 150 adult Populus euphratica from natural populations in the Korla region of Xinjiang (latitude 41.08°N, longitude 86.11°E).

[0047] 2) The DNA of all Populus euphratica parents was extracted by CTAB method, and the DNA was diluted to 10 ng / μl as a template for multiplex PCR.

[0048] 3) Amplify all DNA samples using the aforementioned reaction system.

[0049] 4) Perform capillary electrophoresis detection on the PCR product, and use GeneMapper (Applied Biosystems) software to read out the fragment length of the PCR product.

[0050] 5) Calculate the corresponding number of alleles, apparent heterozygosity and expected heterozygosity, inbreeding coefficient, etc. according to the molecular marker results of the population. The specific results are as follows:

[0051]

[0052]

Embodiment 2

[0054] The natural population of Populus euphratica was amplified by multiplex PCR method, and the parent-offspring relationship was analyzed according to the results.

[0055] 1) Select 20 adult Populus euphratica female plants in the natural population, collect capsules, and collect 60 individual male plants near the female plants at the same time. The seeds in the female capsule are propagated by tissue culture, and after 3 months, the seedlings are transplanted to the greenhouse for unified cultivation, and young leaves are collected from a single plant.

[0056] 2) The DNA of all Populus euphratica parents was extracted by CTAB method, and 2-9 progeny were selected for each female plant to extract DNA, a total of 126 plants. The DNA was diluted to 10 ng / μl as a template for multiplex PCR.

[0057] 3) Amplify all DNA samples using the aforementioned 8-plex system.

[0058] 4) Perform capillary electrophoresis detection on the PCR product, and use GeneMapper (Applied Bios...

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Abstract

The invention discloses a method for carrying out genetic analysis and paternity test of diversifolious poplar colony by 8-plex PCR (polymerase chain reaction). According to the method provided by the invention, one group of primers are used for multiplex PCR reactions, meanwhile, eight sites are amplified, and the primers comprise primers with sequences of SEQ ID NO: 1-SEQ ID NO: 8. The invention further relates to a primer group formed by the primers with the sequences of SEQ ID NO: 1-SEQ ID NO: 8, and a kit comprising the primer group and application of the primer group. According to the invention, eight pairs of primers with high polymorphism incapable of interacting in pairs are screened, and the multiplex PCR reaction is carried out by optimizing the primer concentration and adjusting amount of DNA (deoxyribonucleic acid) templates. The method provided by the invention is quick and efficient, suitable for extremely trace DNA, easy for automation and wide in application prospects of genetic analysis, paternity test and variety protection of diversifolious poplar.

Description

technical field [0001] The invention belongs to the field of genetics research and discloses a method for simultaneously amplifying eight SSR sites on the whole genome of Populus euphratica (Torr&Gray). Background technique [0002] Polymerase chain reaction (PCR) is the most commonly used amplification technique in biological monitoring and molecular markers today. It can amplify a certain DNA fragment tens to millions of times within a few hours. With the popularization and application of fluorescence-labeled PCR technology, the quantification of target fragments can be accurate to 1bp. However, in general PCR, only one pair of primers is used, the throughput is low, and the cost is high. It is urgent and necessary to establish an efficient and high-throughput method for the genetic research of large populations and multiple loci. [0003] Multiplex PCR (Multiplex PCR) is a PCR technology that improves on the basis of ordinary PCR, adding multiple pairs of specific primer...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 杜芳徐放冯思思
Owner BEIJING FORESTRY UNIVERSITY
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