Microsatellite marker primers for portunus trituberculatus and application of primers

A technology of portunus trituberculatus and microsatellite marking, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., and can solve the problems of declining breeding area, poor water quality, and damage to the living environment of portunus trituberculatus, etc. problems, to achieve the effect of stable PCR amplification results, good application value, and high polymorphism

Active Publication Date: 2019-11-12
HEBEI UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, industrial pollution in coastal areas has led to poor water quality, which has destroyed the living environment of Portunus trituberculatus, sharply reduced the number of high-quality parents, and greatly reduced the breeding area.

Method used

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  • Microsatellite marker primers for portunus trituberculatus and application of primers
  • Microsatellite marker primers for portunus trituberculatus and application of primers
  • Microsatellite marker primers for portunus trituberculatus and application of primers

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 The acquisition of microsatellite markers and primers

[0041] Portunus trituberculatus was purchased from the original species of Portunus trituberculatus in Huanghua, its muscle tissue was taken under aseptic conditions, total RNA was extracted from the muscle tissue, and sent to a biological company for transcriptome sequencing to obtain transcriptome data; Identify SSRs in unigenes over 1kb. The identification criteria are: the minimum repeats of precise SSR markers containing two, three, four, five, and hexanucleotide types are 9, 6, 5, and 4 times respectively. SSRHunterl. 3 Screening of SSR markers to ensure that the front and rear flanks of the sequence have sufficient length for designing primers. Use Primer Permier 6 to design primers with the screened SSRs; the main parameters of the design are: the optimal length of the primer is 18-25 bp, the length of the PCR product fragment is 90-400 bp, the optimal annealing temperature is 55-60 ° C; GC conten...

Embodiment 2

[0045] Embodiment 2 Utilizes microsatellite primers to the analytical method of Portunus trituberculatus genetic structure

[0046] (1) Genomic DNA extraction: Genomic total DNA was extracted according to the method provided by the marine animal tissue genomic DNA extraction kit. DNA quality inspection: take 2 μL of total genomic DNA and run it on an agarose gel with a mass ratio concentration of 1.5%, and observe whether the DNA is degraded and whether there are protein residues after EB staining and ultraviolet gel imaging system imaging; in addition, the extracted The concentration of the DNA sample was measured with a NanoDrop 2000 ultra-micro spectrophotometer, and the total DNA sample of the genome was uniformly diluted to 50 ng / μL with the measured DNA concentration as a reference.

[0047] (2) Use described microsatellite primers to carry out molecular marker detection: take the uniformly diluted genomic total DNA obtained in step (1) as a template, and use 17 pairs of...

Embodiment 3

[0053] Example 3 Application of Genetic Structure Analysis of Portunus trituberculatus

[0054] The analyzed populations were from Dalian (DL), Huludao (HLD), Qinhuangdao (QHD), Huanghua cultured (HHYZ), wild Huanghua (HHYS), Dongying (DY), Penglai (PL) The portunus trituberculatus of seven groups, each group gets 60 samples, totally 420 samples, according to the (1) step in the embodiment 2, extract the genome total DNA of each sample, take the genome total DNA as amplification template, with The 17 pairs of microsatellite primers that embodiment 1 obtains carry out PCR typing detection, amplification system, amplification condition and concrete amplification steps are the same as the (2) step of embodiment 2, and the mass ratio concentration that obtains amplification product is 10% Polyacrylamide gel separation, through silver staining staining, record amplification result, and detect the fragment size of amplified product.Wherein partial amplified fragment is respectively ...

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Abstract

The invention provides microsatellite marker primers for portunus trituberculatus and application of the primers. The primers comprise 17 pairs of microsatellite primers including NC01F, NC01R; NC02F,NC02R; NC03F, NC03R; NC04F, NC04R; NC05F, NC05R; NC06F, NC06R; NC07F, NC07R; NC08F, NC08R; NC09F, NC09R; ZL01F, ZL01R; ZL02F, ZL02R; ZL03F, ZL03R; ZL04F, ZL04R; ZL05F, ZL05R; ZL06F, ZL06R; ZL07F, ZL07R; and ZL08F, ZL08R respectively. The invention also provides the application of the primers in analysis of population genetic structures of the portunus trituberculatus. The microsatellite markers provided by the invention have characteristics of PRC amplification result stability, high polymorphism and the like, and these molecular markers can be applied to analysis of genetic diversity, analysis of population genetic structures, construction of genetic maps, gene mapping, variety identification, germplasm conservation, analysis of quantitative character genes, study of evolution and genetic relationships and the like, and have very good application values.

Description

technical field [0001] The invention relates to the field of biological genetic breeding of Portunus trituberculatus, in particular to a primer for microsatellite markers of Portunus trituberculatus and application thereof. Background technique [0002] SSR (Simple sequence repeat) is a simple sequence repeat, also known as microsatellite DNA (microsatellite DNA). In eukaryotic genomes, SSR consists of only a few nucleotides (1-6) to form repeating units, such as (( CT)n, (AC)n, (ACT)n (where n is the number of repetitions), etc., the number of repetitions is 10-20, and the same type of microsatellite DNA can be distributed in different positions throughout the genome, due to the difference in the number of repetitions, or The degree of incompleteness forms the polymorphism of each locus. The sequences at both ends of each type of microsatellite DNA are mostly relatively conservative single-copy sequences. A pair of specific primers can be designed according to the sequences...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12N15/11
CPCC12Q1/6888C12Q2600/156
Inventor 康现江段保华穆淑梅管越强李泽健姬新东李书琴李彦芹
Owner HEBEI UNIVERSITY
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