Method for rapidly identifying charybdis feriata sex specific molecular marker and genetic sex

A molecular marker, a technology for the rust-spotted cricket, which is applied in the directions of biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc. problem, to achieve the effect of simple operation, short time-consuming, high identification accuracy

Active Publication Date: 2018-09-28
SHANTOU UNIV
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Problems solved by technology

However, due to the fact that the technical means of screening polymorphic genetic markers through simplified genome sequencing is not widely used, and there is no combination of molecular methods for identifying the sex of rusty spot. Now, in production practice or academic research, only morpholog

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  • Method for rapidly identifying charybdis feriata sex specific molecular marker and genetic sex
  • Method for rapidly identifying charybdis feriata sex specific molecular marker and genetic sex
  • Method for rapidly identifying charybdis feriata sex specific molecular marker and genetic sex

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Embodiment

[0043] A method for quickly identifying sex-specific molecular markers and hereditary sex of rusty spot moth, mainly comprising the following steps:

[0044] 1. Sample collection and extraction of genomic DNA

[0045] A total of 44 samples were collected, including 22 males and 22 females. Take about 50 mg of the muscle tissue of P. rust, put it into a 1.5ml centrifuge tube containing 300 μL of lysate, cut it into pieces until it is homogenized, add 10 μL of RNaseA, mix well, and incubate at room temperature for 2 minutes. Then add 300 μL of lysate and 5 μL of proteinase K to the centrifuge tube, mix thoroughly, and put it in a 55°C water bath for 2 hours for digestion. Then add 300 μL Tris-balanced phenol and 300 μL chloroform for extraction, and centrifuge at 13000r for 10 minutes after 10 minutes. Draw about 500 μL of centrifuged supernatant into a new centrifuge tube, add 600 μL of chloroform to it and extract again. Draw about 350 μL of the supernatant after centrifuga...

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Abstract

The invention relates to a method for rapidly identifying a charybdis feriata sex specific molecular marker and genetic sex. For SNP sites used for charybdis feriata sex identification, the base sequence is SEQ ID NO:1 or SEQ ID NO:2, and the length is 368bp; the SNP sites are the 35 site, the 89 site, the 120 site, the 218 site and the 240 site of SEQ ID NO:1 or SEQ ID NO:2. The identification method comprises the steps that charybdis feriata male and female individuals with known sex is subjected to DNA extraction; genomic library building and high throughput sequencing are simplified; sequence analysis and sex specific SNP site screening are conducted; candidate SNP site enlarged sample validation is conducted; sex specific primers based on the sex specific SNP sitesare designed; by means of the male specific primers, under specific annealing temperature, the genome DNA of charybdis feriata is subjected to PCR amplification, and specific sex determination is conducted according to the agarose gel electrophoresis detection result. A reliable method can be provided for charybdis feriata genetic sex and especially for the charybdis feriata in early developmentstage, and the method has the advantages of being easy to operate, short in consumed time, low in cost, good in repetition, high in accuracy rate and the like.

Description

technical field [0001] The invention belongs to the marine crab sex identification technology in the technical field of aquatic organisms, and in particular relates to a method for quickly identifying sex-specific molecular markers and genetic sex of rusty spot moth. Background technique [0002] SNP (Single Nucleotide Polymorphism) refers to the variation of a single nucleotide on the genome, including transitions, transversions, deletions, and insertions, forming genetic markers with a large number and rich polymorphisms. As the third generation genetic molecular marker, SNP has been widely used in the research of genetic diversity analysis, gene mapping, molecular marker-assisted breeding and functional genomics of many species. [0003] Charybdis feriatus, commonly known as safflower crab, belongs to the class Crustacea, order Decapoda, family Portunidae, genus Charybdis, and is mainly distributed in the tropical, subtropical and temperate regions of the Pacific Ocean an...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12Q1/6879C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q1/6879C12Q1/6888C12Q2600/156C12Q2535/122C12Q2531/113C12Q2537/165
Inventor 马洪雨方少彬管梦云苗贵东石西
Owner SHANTOU UNIV
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