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Microsatellite primer for macrobrachium nipponensis diversity analysis and application thereof

A diversity analysis and microsatellite technology is applied in the field of microsatellite primers for diversity analysis of Macrobrachium japonicum, and achieves the effects of high polymorphism, good application value and stable PCR amplification results.

Active Publication Date: 2017-02-22
HEBEI UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are few relevant studies on microsatellite markers of Macrobrachium japonicus in the prior art

Method used

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  • Microsatellite primer for macrobrachium nipponensis diversity analysis and application thereof
  • Microsatellite primer for macrobrachium nipponensis diversity analysis and application thereof
  • Microsatellite primer for macrobrachium nipponensis diversity analysis and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] The acquisition of embodiment 1 microsatellite primer

[0035] Macrobrachium japonica was purchased from the Baiyangdian aquatic product market, and its muscle tissue was taken under sterile conditions, total RNA was extracted from the muscle tissue, and sent to a biological company for transcriptome sequencing to obtain transcriptome data; the assembly length of more than 1kb was analyzed using MISA software unigenes for SSR identification, the identification criteria are: the minimum repeats of precise SSR markers containing two, three, four, five and six nucleotide types are 9, 6, 5, and 4 times respectively, and SSR markers are performed using SSRHunter1.3 Screening to ensure that the front and rear flanks of the sequence are of sufficient length for the design of primers. Use Primer Permier 6 to design primers based on 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 10...

Embodiment 2

[0039] Embodiment 2 Utilizes microsatellite primers to the analytical method of Macrobrachium japonicus genetic structure

[0040] (1) Genomic DNA extraction: The total genomic DNA of the sample to be analyzed was extracted by referring to the phenol-chloroform method provided in the "Molecular Cloning Experiment Guide" (Sambrook & Russell, 2001). 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.

[0041] (2) Use the microsatellite primers to detect molecular markers: use the uniformly diluted genomic total DNA obtaine...

Embodiment 3

[0047] Example 3 Application of Macrobrachium japonicus Genetic Structure Analysis

[0048] The analysis population comes from four populations of Macrobrachium japonicus from Hebei Baiyangdian (BYD), Hebei Hengshui Lake (HSH), Shandong Weishan Lake (WSH), and Jiangsu Hongze Lake (HZH). Each population takes 24 samples, a total of 96 Sample, extract the total genomic DNA of each sample according to (1) in Example 2, take the total genomic DNA as the amplification template, and carry out PCR typing detection with 11 pairs of microsatellite primers obtained in Example 1, the amplification system, Amplification conditions and specific amplification steps are the same as step (2) of Example 2, and the amplified products are separated by polyacrylamide gel with a mass ratio concentration of 10%, stained with silver staining, record the amplification results, and detect The fragment size of the amplified product. Some of the amplified fragments are as SEQ ID NO:23 (amplified by MN0...

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Abstract

The invention discloses a microsatellite primer for macrobrachium nipponensis diversity analysis. The microsatellite primer for macrobrachium nipponensis diversity analysis comprises eleven pairs of microsatellite primers, which are respectively MN01, MN02, MN03, MN04, MN05, MN06, MN07, MN08, MN09, MN10 and MN11; meanwhile, the invention also provides application of the microsatellite primer in macrobrachium nipponensis population genetic structure analysis through a following method comprising the steps: (a) extracting genome DNA (Deoxyribose Nucleic Acid) to be analyzed; (b) adopting the genome DNA as a template, and adopting the microsatellite primer for carrying out PCR (Polymerase Chain Reaction) molecular marker detection; (c) carrying out genetic structure analysis. The microsatellite primer provided by the invention has the characteristics of stable PCR amplification results, high polymorphism and the like, can be used to utilize molecular markers for carrying out genetic diversity analysis, population genetic structure analysis, genetic map construction, gene mapping, variety identification, germplasm preservation, quantitative trait gene analysis, evolution and phylogenetic relationship research and the like, and has a better application value.

Description

technical field [0001] The invention relates to the field of biological genetic breeding of Macrobrachium prawn, in particular to a microsatellite primer for diversity analysis of Macrobrachium japonicus and its application. 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 to 6) to form repeating units, such as (GA )n, (AC)n, (GAA)n (where n is the number of repetitions), etc., the number of repetitions is 10-50, and the same type of microsatellite DNA can be distributed in different positions of the whole genome, due to the difference in the number of repetitions or the degree of repetition The incompleteness of each locus forms a polymorphism. Most of the sequences at both ends of each type of microsatellite DNA are relatively conservative single-copy sequences, and a pair of specific primers can be designed accordi...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6888C12Q2600/156
Inventor 康现江穆淑梅武小斌管越强
Owner HEBEI UNIVERSITY
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