Molecular genetic identification technique of Eriocheir sinensis and Eriocheir japonica

A technology of Chinese mitten crab and mitten crab, which is applied in the fields of biodiversity conservation and genetics, and can solve the problems of lack of molecular marker technology and methods for identifying mitten crabs

Active Publication Date: 2009-04-29
李思发
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] In summary, there is still a lack of molecular marker techniques and methods to identify different species / geographical groups of mitten crabs

Method used

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  • Molecular genetic identification technique of Eriocheir sinensis and Eriocheir japonica
  • Molecular genetic identification technique of Eriocheir sinensis and Eriocheir japonica
  • Molecular genetic identification technique of Eriocheir sinensis and Eriocheir japonica

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0107] Example 1. Sample Selection

[0108] A total of 125 samples of Chinese mitten crabs from different water systems, 40 samples of mitten crabs from southern Chinese water systems (Nanliu River in Guangxi and Pearl River in Guangdong), and 15 samples of Japanese mitten crabs from Hokkaido, Japan were selected for subsequent experiments . The inventor has selected a sufficient number of mitten crabs, so the obtained results are statistically significant, accurate and reliable.

Embodiment 2

[0109] Example 2.DNA Extraction

[0110] Take 0.2g of the long segment muscle of the third step of each crab, put it into a 1.5ml centrifuge tube, add 400μL STE buffer solution (30mmol / L Tris-HCl, pH8.0, 200mmol / L EDTA, 50mmol / L NaCl) . After mixing, add 90 μL of SDS with a concentration of 10% and 10 μL of proteinase K at 20 mg / mL, digest at 55°C for 8-10 hours; add an equal volume of saturated phenol, rotate slowly on the rotor for 1 hour, centrifuge at 10,000 r / min for 8 minutes, and absorb the supernatant Add an equal volume of mixed solution (phenol: chloroform: isoamyl alcohol = 25:24:1); rotate slowly for 30 min, centrifuge at 10,000 r / min for 8 min, take the supernatant and add an equal volume of chloroform and isoamyl alcohol mixed solution (chloroform : isoamyl alcohol=24:1), rotate slowly for 5min, centrifuge at 10,000r / min for 5min, take the supernatant, add 2 times the volume of absolute ethanol to precipitate DNA, then wash with 70% ethanol, dry, add 300μL Diss...

Embodiment 3

[0111] Example 3.PCR amplification and product cloning, sequencing

[0112] According to the Chinese mitten crab mitochondrial COII gene sequence reported in the 2001 Journal of Nanjing Normal University Natural Science Edition, Volume 24, Phase 4, Page 485-490, a pair of degenerate primers designed to amplify the COII gene are:

[0113] COII-F: 5'-CATCACCTTGTCAAGGTGAAA-3' (SEQ ID NO: 4);

[0114] COII-R: 5'-CATGGTCAGTCTCAGGATTCA-3' (SEQ ID NO: 5).

[0115] The crab genomic DNA prepared in Example 1 was used as a template, and the above-mentioned COII-F and COII-R were used as primers for PCR amplification. The PCR reaction volume is 50 μL, containing 5 μL PCR buffer (10 mmol / L Tris-HCL, pH9.0, 50 mmol / L KCL, 30 mmol / L MgCL2, 0.01% gelatin), 2 μL dNTP mixture (each dNTP 0.1 mmol / L ), 4 μL of primers (0.2 μmol / L), 2 μL of genomic DNA (50 ng / μl), 2 μL of Taq enzyme (2 IU), and 35 μL of distilled water.

[0116] The amplification reaction conditions were: pre-denaturation at 9...

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Abstract

The invention belongs to the field of genetics and biodiversity protection science, and discloses application of a mitochondrion COII gene to identification of Eriocheir sinensis, Japanese Mitten Crabs and / or mitten crabs in a water system of South China. The invention also discloses a method for identifying the Eriocheir sinensis, the Japanese Mitten Crabs and / or the mitten crabs in the water system of South China. The method adopted can effectively and accurately identify the crabs, and requires small sample size, so as to provide a more ideal approach for genetic discrimination and fine breed cultivation of the crabs.

Description

technical field [0001] The invention belongs to the fields of genetics and biodiversity conservation. More specifically, the present invention relates to a molecular genetic identification method for Chinese mitten crabs, Chinese southern water systems (Guangdong Pearl River, Guangxi Nanliujiang) mitten crabs and Japanese mitten crabs . Background technique [0002] Chinese mitten crab (Eriocheir sinensis) and Japanese mitten crab (Eriocheir japonica) both belong to the phylum Arthropoda, the class Crustaceae, the order Decapoda, the family Grapsidae, and the genus Eriocheir ). The genus Eriocheir generally includes four species: Eriocheir sinensis, Eriocheir japonica, Eriocheir mitten and Eriocheir angustifolia. In addition, there are Nanliu river mitten crabs in the southern water system of China. The mitten crabs and mitten crabs are small and have no economic value. Chinese mitten crab and Japanese mitten crab are relatively large individuals, and are economic specie...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11C12N15/12
Inventor 李思发王成辉
Owner 李思发
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