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Portunus pelagicus polymorphic microsatellite molecular marker, identification method and application

A technology of Portunus pelagics and microsatellite markers, which is applied to the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve problems such as high cost, low efficiency, and unsuitability for non-model animals, and achieve The effects of efficient identification, simple operation, and potential for widespread application

Active Publication Date: 2018-04-20
SHANTOU UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, only 8 microsatellite markers have been developed in Portunus pelagics, which is far from meeting the needs of population genetic diversity and genetic breeding research
The development of traditional microsatellite markers mostly relies on the construction of genome-wide libraries, 5'-anchored PCR, FIASCO, transcriptome sequencing, or NCBI database search, but these development technologies are expensive, inefficient, and mostly Not suitable for non-model animals etc.

Method used

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  • Portunus pelagicus polymorphic microsatellite molecular marker, identification method and application
  • Portunus pelagicus polymorphic microsatellite molecular marker, identification method and application
  • Portunus pelagicus polymorphic microsatellite molecular marker, identification method and application

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Embodiment

[0025] 1. Extraction of Genomic DNA of Portunus pelagics

[0026] Collect 33 adult Portunus pelagics, take about 10g of muscle tissue and preserve it with 95% alcohol. Take about 100mg of muscle tissue, put it into a 1.5mL centrifuge tube filled with 300μL tissue lysate, and homogenate; add 10μL RNase (20mg / mL) to the centrifuge tube in turn, mix well, and incubate at room temperature for 2min; add 5μl proteinase K (20mg / mL), fully mixed, digested in a 55°C water bath until clarified, which can be shaken and mixed several times to accelerate its dissolution; followed by continuous extraction twice with Tris-saturated phenol and chloroform; absorb the supernatant Add about 300-350μL to a new centrifuge tube, add 1ml of pre-cooled absolute ethanol to precipitate DNA, centrifuge at 12000rpm to collect the precipitate, and wash it again with pre-cooled 70% ethanol; finally, dry the DNA (precipitation) at room temperature and dissolve in 40 μL sterile double-distilled water, and s...

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Abstract

The invention relates to a portunus pelagicus polymorphic microsatellite molecular marker, an identification method and application. The identification method comprises the following steps: portunus pelagicus genomic DNA (Deoxyribonucleic Acid) extraction, simplified genomic library construction and high-throughput sequencing, sequence analysis and microsatellite primer designing, and microsatellite marker polymorphism evaluation. According to the identification method disclosed by the invention, 17983 microsatellite loci are identified, and 16 microsatellite markers having molecular polymorphism are screened out. The identification method disclosed by the invention has the advantages that the operation is simple and safe, the accuracy is high, a result is true and reliable, and the like,and is capable of being used for quickly and massively obtaining portunus pelagicus microsatellite markers; the technical method disclosed by the invention can be widely applied to numerous species having no reference genome because of no need to know genomic information in advance, and has a wide popularization and application potential; in addition, a candidate tool can be provided for population genetic variation evaluation, genetic relationship identification and genetic linkage map construction of portunus pelagicus by 16 pairs of polymorphic microsatellite marker primers obtained by themethod.

Description

technical field [0001] The invention belongs to the marine crab microsatellite marker screening and population genetic diversity research technology in the technical field of aquatic organisms; in particular, it relates to a method for identifying polymorphic microsatellite molecular markers of Portunus pelagics. Background technique [0002] Portunus pelagicus, also known as Portunus pelagicus, commonly known as blue crab, sand crab, open sea crab or flower crab, belongs to Arthropoda, Crustacea, Malacostraca, decapod (Decapoda), Reptantia, Portunidae, Portunus. Portunus pelagics are warm-water crabs, mainly distributed in the waters of Hainan, Guangxi, Guangdong, Taiwan, Fujian and Zhejiang provinces in my country, and are also one of the four important economic crabs in the Western Pacific-Indian Ocean. Portunus from the open sea have delicious meat, rich nutrition, large individual size, and have extremely high economic and nutritional value. Since the 1990s, scholars ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/6888C12Q1/6869
CPCC12Q1/6869C12Q1/6888C12Q2600/156C12Q2535/122
Inventor 马洪雨吴清洋苗贵东郑怀平李升康游翠红王树启
Owner SHANTOU UNIV
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