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Method for developing polymorphic microsatellite molecular markers on basis of multi-sample high-throughput sequencing

A microsatellite marker and molecular marker technology, applied in the field of molecular biology, can solve problems such as low efficiency, and achieve the effect of promoting research and improving efficiency

Inactive Publication Date: 2018-08-24
NANCHANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The present invention aims at the technical defects of the prior art, and provides a method for developing polymorphic microsatellite molecular markers based on multi-sample high-throughput sequencing, so as to solve the problem of low efficiency of conventional screening methods for microsatellite markers in the prior art. low technical issues

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  • Method for developing polymorphic microsatellite molecular markers on basis of multi-sample high-throughput sequencing
  • Method for developing polymorphic microsatellite molecular markers on basis of multi-sample high-throughput sequencing
  • Method for developing polymorphic microsatellite molecular markers on basis of multi-sample high-throughput sequencing

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Embodiment 1

[0027] In this embodiment, taking hexaploid wild camellia oleifera as an example, the specific implementation of the method of the present invention is given.

[0028] Step 1. Sample collection of wild tea leaves. In this example, wild camellia oleifera pieces were collected from Jinggangshan (26.55°N, 114.15°E) and Lushan (29.61°N, 115.98°E), among which, Jinggangshan is the center of the distribution area of ​​wild camellia oleifera, and Mount Lu is the northern edge of the distribution area of ​​wild camellia oleifera . In this example, 4 samples of Camellia oleifera chips at different altitudes were collected in Jinggang Mountain and Mount Lushan, and 3-5 fresh Camellia oleifera chips were collected for each sample. The collected Camellia oleifera pieces are required to have no obvious damage, and they should be stored in liquid nitrogen immediately after collection, and stored in a -80°C refrigerator after arriving in the laboratory.

[0029]Step 2: High-throughput tran...

Embodiment 2

[0038] A method for developing polymorphic microsatellite molecular markers based on multi-sample high-throughput sequencing, the method performs the following steps on the basis of obtaining high-throughput sequencing data of multiple sample genomes of the species to be tested:

[0039] Step 1. Increase the specificity of microsatellite markers: according to the method disclosed in Example 1, the potential amplifiable microsatellite loci are screened, and then the forward and reverse primers are screened for all sequencing reads at the potentially amplifiable microsatellite loci in a single sample In order to increase the specificity of the primers, the microsatellite loci that only appear once in the

[0040] Step 2, exclude complex microsatellite sites: use R script (Simple_SSR) to screen simple microsatellite sites in the sites obtained in step 1.

[0041] Step 3, calculate the number of alleles of each microsatellite locus in each sample, and screen the loci with high PCR...

Embodiment 3

[0045] A method for developing polymorphic microsatellite molecular markers based on multi-sample high-throughput sequencing, comprising the following steps:

[0046] 1) Take several samples of the target species and perform high-throughput sequencing on the genome;

[0047] 2) From the high-throughput sequencing data of each sample, screen potential amplified microsatellite loci;

[0048] 3) From the potentially amplifiable microsatellite loci obtained through the screening in step 2), screening the microsatellite loci whose primer pairs only appear once in all the sequencing reads of the potential amplifiable microsatellite loci of a single sample;

[0049] 4) For the screening results obtained in step 3), use the R script Simple_SSR to perform screening, and the screening results obtained are simple microsatellite sites;

[0050] 5) For the simple microsatellite sites obtained in step 4), use the R script SSR_A to detect the number of alleles in a single sample, and remove...

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Abstract

The invention provides a method for developing polymorphic microsatellite molecular markers on the basis of multi-sample high-throughput sequencing. The method comprises the steps as follows: firstly,high-throughput sequencing data of multiple sample genomes or transcriptomes of to-be-tested species are acquired, potential satellite sits capable of being amplified are screened, microsatellite sites with primer pairs appearing only once in all sequencing reads of the potential satellite sits capable of being amplified in single sample are screened, and markers which appear in multiple samplesand have the allele number larger than 2 in all the samples are screened; finally, those markers appearing in reference genomes or transcriptomes only once are screened by establishing a reference sequence database, and those repeated markers amplifying the same fragments are eliminated. The polymorphic microsatellite markers can be effectively screened by use of high-throughput sequencing data, non-specific and repeated markers can be removed, the length of PCR amplified products of corresponding microsatellite sites can be predicted, and the efficiency of development of microsatellite molecular markers is significantly improved.

Description

technical field [0001] The invention relates to the technical field of molecular biology, and further relates to biomolecular marker development technology, in particular to a method for developing polymorphic microsatellite molecular markers based on multi-sample high-throughput sequencing. Background technique [0002] Molecular markers are fundamental tools for measuring the genetic diversity of organisms. Due to the characteristics of co-dominance and high polymorphism, microsatellite molecular markers have been widely used in the study of population genetic diversity, population genetic structure, and gene flow between populations. It is one of the most commonly used markers in the research of science, animal and plant genetics and breeding. Traditional microsatellite marker development requires creating a library to enrich microsatellite loci, followed by cloning and Sanger sequencing. Traditional methods are expensive, labor-intensive, and often end up with only a f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6811
CPCC12Q1/6811C12Q2535/122C12Q2537/165
Inventor 戎俊杨小强崔相艳黄小毛陈家铭
Owner NANCHANG UNIV
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