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SNP (single-nucleotide polymorphism) marker related to Marsupenaeus japonicus heat resistance and detection method thereof

A detection method and technology of heat resistance, applied in botany equipment and methods, biochemical equipment and methods, microbial determination/testing, etc., can solve few problems such as heat resistance-related SNP screening and detection

Inactive Publication Date: 2016-05-11
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In the research on thermotolerance of Penaeus japonicus, there are few reports on the screening and detection of thermotolerance-related SNPs

Method used

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  • SNP (single-nucleotide polymorphism) marker related to Marsupenaeus japonicus heat resistance and detection method thereof
  • SNP (single-nucleotide polymorphism) marker related to Marsupenaeus japonicus heat resistance and detection method thereof
  • SNP (single-nucleotide polymorphism) marker related to Marsupenaeus japonicus heat resistance and detection method thereof

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Experimental program
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Embodiment 1

[0033] Embodiment 1: cDNA and DNA sequence cloning of Penaeus japonicus HSP60 gene, comprising the following steps:

[0034] 1) Extraction of muscle DNA of Penaeus japonicus;

[0035] 2) Amplification of the intron of the HSP60 gene;

[0036] 3) Obtain the full-length sequence of the MjHSP60 target gene.

[0037] In step 1), the specific method for extracting the muscle DNA of Penaeus japonicus can be: extracting with phenol / chloroform, and then detecting the DNA by agarose TBE gel electrophoresis.

[0038] In step 2), the specific method of the amplification of the intron of the HSP60 gene can be:

[0039] a) Primer design

[0040] According to the MjHSP60mRNA of Penaeus japonicus (GenBank accession number: JQ972715.1) and the complete sequence of Scylla paramamosain HSP60 gene (GenBank accession number: JX262230.1), primers were designed at both ends of the adjacent exons, and finally using crossover Primers verified for unexpanded introns. The primer sequences used for...

Embodiment 2

[0053] Embodiment 2: Screening of SNP markers related to heat tolerance in the HSP60 gene of Penaeus japonicus, including the following steps:

[0054] 1) Experimental prawns

[0055] Genomic DNA extraction (same as above)

[0056] 2) SNP detection of MjHSP60 gene in different populations of Penaeus japonicus

[0057] a) Screening of SNPs by direct sequencing

[0058] A total of 7 pairs of primers were designed according to the obtained full-length sequence of the coding region of the MjHSP60 gene (the sequences of the primers used for SNP detection of the MjHSP60 gene are shown in Table 2), covering the entire length of the gene as much as possible. The experimental prawns included 4 geographical groups: Penghu (PH), Zhaoan (ZA), Beihai (BH), and Sanya (SY), with 30 each. It was found by observing whether the twill stripes on the side of the carapace were fully extended. Penghu belonged to morphological variation type I population (heat-sensitive population), while Beihai ...

Embodiment 3

[0069] Embodiment 3: A detection method for SNP markers related to thermal tolerance, comprising the following steps:

[0070] 1) Preparation of experimental materials;

[0071] 2) Genotyping using PCR-RFLP technology;

[0072] 3) Analysis of individual heat tolerance of different genotypes of Penaeus japonicus.

[0073] In step 1), the specific steps for preparing the experimental materials are:

[0074] a) Heat resistance experiment of Penaeus japonicus

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Abstract

The invention provides an SNP (single-nucleotide polymorphism) marker related to Marsupenaeus japonicus heat resistance and a detection method thereof, relating to detection of shrimp heat-resistant populations in aquiculture. The invention firstly provides an SNP marker related to Marsupenaeus japonicus heat tolerance by using a direct sequencing process. The SNP marker related to Marsupenaeus japonicus heat tolerance is the 3289th site of the HSP60 gene of which the nucleic acid sequence is SEQ ID NO.1, and the base is A or G. The detection method comprises the following steps: carrying out a Marsupenaeus japonicus heat resistance experiment; calculating the heat resistance value; carrying out genotyping by using a PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) technique; and analyzing the heat tolerance of the Marsupenaeus japonicus individuals with different genotypes. The direct sequencing process and the PCR-RFLP process are utilized to give out the polymorphism of the MjHSP60 gene, and the correlation analysis is carried out on the SNP polymorphism and Marsupenaeus japonicus heat tolerance, thereby providing theoretical references for screening high-temperature-resistant SNP molecular markers and developing molecular breeding of Marsupenaeus japonicus heat-resistant lines.

Description

technical field [0001] The invention relates to the detection of heat-resistant populations of shrimps in aquaculture, in particular to a SNP marker related to the heat resistance of Penaeus japonicus and a detection method thereof. Background technique [0002] The English name of Japanese sac prawn (Marsupenaeus japonicus) is Kurumashrimp, commonly known as spotted prawn, bamboo prawn, flower tail prawn, blue tail prawn, and it is an important species of seawater prawn farming in China. Penaeus japonicus is a subtropical species. The optimum temperature range is 25-30°C. It stops feeding at 8-10°C, dies below 5°C, and lives abnormally above 32°C. The culture of Penaeus japonicus is obviously affected by temperature. If the water temperature exceeds a certain critical value, it will cause a large area of ​​death, and the high temperature culture in the southern region is limited in summer. In recent years, in order to improve the growth rate and breeding efficiency of Pena...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/12
CPCC12Q1/6888C12Q2600/124C12Q2600/156
Inventor 王攀攀李丽君钟声平王军苏永全毛勇
Owner XIAMEN UNIV
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