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Crassostrea gigas high temperature response gene HSP70 expression regulation SNP marker and application thereof

A technology of expression regulation and oyster growth, which is applied in the field of genetic engineering and genetic breeding, can solve problems such as economic losses, achieve the effect of improving high temperature resistance and reliable results

Pending Publication Date: 2022-07-29
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But for a long time, the periodic summer mass die-offs that have occurred frequently around the globe have taken a heavy toll on the economies of many countries

Method used

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  • Crassostrea gigas high temperature response gene HSP70 expression regulation SNP marker and application thereof
  • Crassostrea gigas high temperature response gene HSP70 expression regulation SNP marker and application thereof
  • Crassostrea gigas high temperature response gene HSP70 expression regulation SNP marker and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] The SNP marker screening steps associated with high temperature-responsive gene expression are:

[0056] A) Construction of experimental materials: Collect wild individuals of oyster and Fujian oyster, and construct two families ZF2-3 to be backcrossed to F2 generation: the male parent is the offspring of the oyster and Fujian oyster, and the female parent is the collection Wild long oysters in Qingdao.

[0057] B) Genotyping: Genome-wide genotyping of 106 offspring individuals of the family was carried out using the simplified genome (GBS), and a high-density genetic map was constructed. For detailed steps, please refer to the article by Wang et al. (Wang et al. 2016).

[0058] 1. Double restriction endonuclease digestion 2. Design adapters and barcodes 3. Adapter annealing 4. Construct sequencing library 5. Library quality inspection and on-board sequencing 6. Use Stack software to perform SNP typing on the original Reads 7. Use JoinMap 4.0 Build a genetic map.

[...

Embodiment 2

[0062] Use the above obtained site to verify that it is related to HSP70 gene expression:

[0063] A) Pretreatment of experimental samples: 95 individuals of the ZF offspring of the proposed F2 generation family were taken from the oyster and Fujian oyster. Divide into monomers, clean them and place them in seawater at 35°C for stress. After 3 hours, the gill tissues were taken for quick storage in liquid nitrogen, and then stored at -80°C for later use.

[0064] B) DNA extraction: DNA was extracted from 95 oyster individuals according to the instructions of Tiangen marine animal tissue genomic DNA extraction kit. DNA concentration was determined with a Nanodrop 2000 spectrophotometer. Quality identification was carried out by 1.0% gel electrophoresis.

[0065] C) Genotyping: SNaPshot (multiplex single base extension technique) was used to detect the genotype of each individual target site (Marker13973).

[0066] Using the DNA template extracted above, the target fragment i...

Embodiment 3

[0108] The Marker13973 regulatory site obtained above was significantly correlated with the expression of the HSP70 gene to further verify the relationship between this marker genotype and high temperature tolerance:

[0109] The above Marker13973 locus was tested in Fujian oyster (high temperature tolerance) and long oyster (high temperature sensitive) to verify its relationship with high temperature tolerance:

[0110] A) Preparation of experimental samples: 50 wild Fujian oysters and 50 long oysters were collected in Xiamen and Qingdao respectively for subsequent experiments.

[0111] B) DNA extraction: The DNA of these 100 oyster individuals was extracted according to the instructions of the Tiangen marine animal tissue genome DNA extraction kit. DNA concentration was determined with a Nanodrop 2000 spectrophotometer. Quality identification was carried out by 1.0% gel electrophoresis.

[0112] C) Genotyping: SNaPshot (multiplex single base extension technique) was used t...

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Abstract

The invention belongs to the field of genetic engineering and genetic breeding, and particularly relates to a pacific oyster high-temperature response gene HSP70 expression regulation SNP marker and application thereof in identification of a high-temperature tolerant oyster individual. The SNP marker is the 251st basic group in the sequence as shown in SEQ ID NO: 2, and the mutation type is C / G (trans regulation site Marker13973). The invention provides the SNP marker obviously related to HSP70 gene expression, and the SNP marker has the advantages that the genotype of parent shellfish can be identified before offspring seed breeding, and the tolerance of offspring to a high-temperature environment is improved. The SNP marker obtained by the research is high in credibility and stable in result.

Description

technical field [0001] The invention belongs to the field of genetic engineering and genetic breeding, and relates to a long oyster high temperature response gene HSP70 expression regulation SNP marker and its application and a method for identifying high temperature resistant oyster individuals. Background technique [0002] Due to their widespread distribution and ecological importance, bivalve mollusks have long been regarded as good biological indicators for monitoring marine habitat pollution and the impact of global warming. Oysters, as a model species in bivalves, crested chakras and even marine invertebrates, are an excellent choice for studying the impact of climate change on coastal ecosystems. Oysters are the world's most traded aquaculture shellfish, with an annual trade value of more than 1 billion US dollars. Oysters are also the main cultured shellfish in my country, and the annual output accounts for 25% of the marine aquaculture production ( image 3 ), with...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12Q1/6858C12N15/11
CPCC12Q1/6888C12Q1/6858C12Q2600/124C12Q2600/156C12Q2600/172C12Q2531/113C12Q2533/101
Inventor 李莉张渴新黎奥丛日浩张国范
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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