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High-throughput sequencing-based ilisha elongate SNP (singular nucleotide polymorphism) marker developing method and application thereof

A high-throughput, sequencing technology, applied in the field of molecular biology, can solve the problems of complex sequencing procedures and low purity of target DNA, achieve consistent amplification efficiency, reduce library construction costs, and high screening accuracy.

Inactive Publication Date: 2019-03-12
ZHEJIANG OCEAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the sequencing procedure of this invention is relatively complicated, and the purity of the target DNA is low

Method used

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  • High-throughput sequencing-based ilisha elongate SNP (singular nucleotide polymorphism) marker developing method and application thereof
  • High-throughput sequencing-based ilisha elongate SNP (singular nucleotide polymorphism) marker developing method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] A high-throughput sequencing-based method for the development of SNP markers in squid, comprising the following steps:

[0032] Genomic DNA extraction: Mince the muscle or fin ray tissue samples of the carp and place them in extraction solution I: 70ml of Tris-HCl and disodium hydrogen phosphate with a concentration of 120mM, 0.3g of fatty alcohol ether ammonium sulfate, and 30ml of chlorine with a concentration of 120mM Sodium chloride solution; add 0.5 μg plasmid DNA and 0.1 μg chitin, freeze and thaw 2-3 times, the freezing temperature is -166°C, the melting temperature is 50°C, centrifuge, collect the supernatant and add extract II: 10ml Tris-HCl with a concentration of 100mM and disodium hydrogen phosphate and 30ml of cationic polyacrylamide with a concentration of 0.5ppm were mixed upside down, centrifuged to produce a white precipitate, and the precipitate was washed to obtain a DNA sample for use; circular DNA and chitin The special existence, on the one hand, c...

Embodiment 2

[0039] A high-throughput sequencing-based method for the development of SNP markers in squid, comprising the following steps:

[0040] Genomic DNA extraction: Mince the muscle or fin ray tissue samples of the carp and place them in extraction solution I: 70ml of Tris-HCl and disodium hydrogen phosphate with a concentration of 150mM, 0.5g of fatty alcohol ether ammonium sulfate, and 30ml of chlorine with a concentration of 130mM Sodium chloride solution; add 0.5 μg plasmid DNA and 0.1 μg chitin, freeze and thaw 2-3 times, the freezing temperature is -166°C, the melting temperature is 50°C, centrifuge, collect the supernatant and add extract II: 10ml Tris-HCl with a concentration of 100mM and disodium hydrogen phosphate and 30ml of cationic polyacrylamide with a concentration of 1.6ppm were mixed by inversion, centrifuged to produce a white precipitate, and the precipitate was washed to obtain a DNA sample for use;

[0041] RAD library construction and high-throughput sequencing...

Embodiment 3

[0047] In the process of RAD library construction and high-throughput sequencing, chitin fluid was added during the PCR amplification system, the mass percentage of chitin was 0.16%, and the solvent was L-amino acid ethyl ester; the rest were exactly the same as in Example 2; The special existence of chitin fluid has a strong adsorption force to DNA, which can improve the success rate of PCR. At the same time, chitin fluid is composed of many tiny spheres, which can wrap the reaction system, so that the PCR amplification reaction can be completed in a small independent space. , so as to improve the reaction sensitivity of PCR amplification, make the reaction more sufficient, and improve the PCR amplification effect of trace DNA; in addition, chitin fluid can increase the boiling point of the reaction system so that the system buffer is not easy to evaporate, reducing the amount of time in the PCR amplification process. The amount of reagents used, thereby reducing the cost of t...

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Abstract

The invention discloses a high-throughput sequencing-based ilisha elongate SNP marker developing method and application thereof. The high-throughput sequencing-based ilisha elongate SNP marker developing method comprises genome DNA (deoxyribonucleic acid) extraction, RAD (restriction site associated DNA) library construction and high-throughput sequencing, original sequence data filtering, and SNPinformation site detection and screening; during genome DNA extraction, circular DNA and chitin are added to effectively improve SNP marker screening accuracy and acquiring efficiency. Ilisha elongate high-density SNP sites acquired through the method can be applied to research on ilisha elongate population genetic structures, provide important scientific guidance for illumination adaptive mechanism of groups to local environments as well as reasonable development and management protection of ilisha elongate resources, and be scientifically significant to both declining species resource supplementation and healthy development of ecological systems. The high-throughput sequencing-based ilisha elongate SNP marker developing method has the advantages of being simple, high in efficiency, highin SNP marker screening and acquiring efficiency, good in repeatability, low in cost and the like.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a high-throughput sequencing-based SNP marker development method and application of the carp. technical background [0002] Ilisha elongate, belonging to the order Clupeiformes, Pristigasteridae, and genus Ilisha, is an important economic fish in warm water and pelagics widely distributed along the coast of my country. Both the South China Sea has production, and the sea area with the largest production is located in the East China Sea. Scorpio is mainly plankton, including diatoms, small swimming animals (juvenile fish), copepods, krills, long tails and mysis, etc., and also feeds on benthic organisms and small swimming animals. The prey objects of a variety of high-trophic fish species play an important role in connecting the preceding and the following in the food chain of the marine ecosystem. At the same time, squid has a high meat content, tender meat, delicious ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6869C12Q1/6888
CPCC12Q1/6869C12Q1/6888C12Q2600/156C12Q2531/113C12Q2535/122
Inventor 刘炳舰吕振明柳意樊刘立芹龚理江丽华张伟男朱科桦刘珠段雯
Owner ZHEJIANG OCEAN UNIV
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