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Microsatellite marker for identifying sepiella maindroni pedigree and application

A technology of squid mansoni and microsatellite markers, which is applied to the determination/testing of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc., and can solve the problem of not paying attention to selective breeding and artificial breeding of squid mansoni germplasm Resource decline, squid premature maturity and other issues

Inactive Publication Date: 2018-01-09
ZHEJIANG OCEAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the long-term breeding process, no attention is paid to selective breeding, and the difference between the breeding environment and the natural environment leads to problems such as precocious puberty of squid, which reduces the germplasm resources of artificially cultured squid and limits the development of its breeding industry.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] The microsatellite marker used for the family identification of Squid Mansoni, the nucleotide sequence of the microsatellite marker is SEQ ID No: 1-19, and its screening method comprises the following steps:

[0037] 1) Genomic DNA extraction: Genomic DNA was extracted from Squid squid, digested with restriction enzyme Mbol at 37° C. for 3 h, and a 400-1000 bp DNA fragment was recovered. Under this condition, the DNA fragment concentration of 400-1000bp in the digestion product is high, which improves the utilization rate of squid mansoni;

[0038] 2) Preparation of the ligation product: Mix linker A (5'-GATCGTCGACGGTACCGAATTCT-3') and linker B (5'-GTCAAGAATTCGGTACCGTCGAC-3') in equal proportions so that the final concentration is 25 μmol / L, incubate at 95°C for 10 minutes, and cool to room temperature, followed by ligation with the recovered DNA fragments at 15°C to obtain ligation products. The adapter is connected to the DNA fragment of Squid Mansoni, which provides...

Embodiment 2

[0044] The nucleotide sequence is the application of the microsatellite marker of SEQ ID No: 1-19 in the identification of the squid mansoni family, comprising the following steps:

[0045] 1) Carry out FAM labeling to the 5' end of the primer designed in step 6) of Example 1;

[0046] 2) Extract the genomic DNA of Squid mansoni, amplify the extracted DNA with the primers in step 1), mix 5 μL 10×buffer, 4 μL dNTP, 2U Taq polymerase, 2 μL upstream primer, 2 μL downstream primer, 2 μL template Mix the DNA, add sterilized water to a final volume of 50 μL, then add 0.1 μL diphenylbromomethane and 0.1 μL 3-methylbenzophenone, and pre-denature the mixture at 95 ° C for 5 min, followed by 35 cycles, each cycle including 94 Denaturation at ℃ for 45s, annealing at 57℃ for 45s, extension at 72℃ for 1min, and extension at 72℃ for 10min after the cycle is completed. Under these conditions, the activity of Taq polymerase is high, the target sequence is completely denatured, and an effecti...

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Abstract

The invention discloses a microsatellite marker for identifying sepiella maindroni pedigree and also discloses an application of the microsatellite marker in identifying sepiella maindroni pedigree. The invention has the beneficial effects that the microsatellite marker for identifying sepiella maindroni pedigree screened according to the invention is in large number of allele, is in high polymorphism and can be used for identifying the population genetic structure of sepiella maindroni, genetic and breeding evaluation, paternity test, and the like. The application of the microsatellite markerwith nucleotide sequence shown as SEQ ID No:1-19 in identifying sepiella maindroni pedigree can make up the limitation of fuzzy genetic background and few molecular markers in the present sepiella maindroni germplasm monitoring. The method is high in efficiency and short in identifying period.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a microsatellite marker used for family identification of the squid mansoni and its application. Background technique [0002] Commonly known as cuttlefish and Japanese needleless squid, it belongs to the phylum Molluscs, Cephalopoda, Dibranchia, Decabranidae, Sepiaidae, Sepiaidae, and Sepia genus. It is widely distributed in the coastal areas of my country. It was once my country's "one of the four major marine products" and has high economic value. With the breakthrough of artificial breeding technology of squid mansoni, the proliferation and release of squid mansoni and the protection and restoration of its spawning grounds are gradually carried out. In the long-term breeding process, no attention is paid to selective breeding, and the difference between the breeding environment and the natural environment leads to problems such as precocious puberty of s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12N15/11
Inventor 郭宝英祁鹏志吴常文
Owner ZHEJIANG OCEAN UNIV
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