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Mitochondria PCR-RFLP identification method for Crassostrea nippona and Crassostrea gigas

A PCR-RFLP and identification method technology, applied in the field of identified mitochondrial COI gene primers, can solve the problems of difficulty in developing new species of rock oyster culture, difficult to distinguish juveniles, etc., and achieves rapid detection, low equipment requirements, and cost saving. Effect

Active Publication Date: 2018-03-23
OCEAN UNIV OF CHINA
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Problems solved by technology

In the introduction and breeding, it was found that the adults of rock oyster and long oyster are obviously different, but the spat are not easy to distinguish
Due to the strong plasticity of the shell of oysters and the overlapping of distribution areas, there are strong subjective factors in the identification of oyster species only based on external morphological characteristics, which brings great difficulties to the development of new species of rock oyster.

Method used

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  • Mitochondria PCR-RFLP identification method for Crassostrea nippona and Crassostrea gigas
  • Mitochondria PCR-RFLP identification method for Crassostrea nippona and Crassostrea gigas
  • Mitochondria PCR-RFLP identification method for Crassostrea nippona and Crassostrea gigas

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Embodiment Construction

[0020] The present invention is further explained and illustrated by specific embodiments below.

[0021] The rock oyster and long oyster used in this embodiment were taken from the Shandong Rongcheng breeding base. 18 individuals of each type of oyster, a total of 36 individuals.

[0022] Log in to the National Center for Biotechnology Information (NCBI, http: / / www.ncbi.nlm.nih.gov / ) to download the existing rock oyster and rock oyster mitochondrial COI sequence (Rock oyster Genbank accession number: HM015198, rock oyster Genbank accession number is: AB033687).

[0023] Use Mega6.0 software to compare the downloaded sequences.

[0024] Use the primer design software Primer Premier 5 to design primers in the relatively conservative regions of the sequence. The designed COI primer sequence is:

[0025] COIY-F: 5'-CTATTAGAAATATGCGGTCTG-3',

[0026] COIY-R: 5'-AACAAAGTAAGTATCGTGAAG-3'.

[0027] Use phenol-chloroform method to extract DNA from rock oyster and rock oyster, and dilute it to ...

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Abstract

The invention relates to the technical field of molecular biology and discloses a PCR-RFLP identification method for Crassostrea nippona and Crassostrea gigas and a pair of COI primers used for the identification. The method includes the steps of: a) amplifying 16s rRNAs and COI gene fragments of the Crassostrea nippona and Crassostrea gigas respectively via a 16s rRNA general primer and the COI primers, and sequencing the amplified targeted gene fragments; b) according to the sequencing result, performing restriction endonuclease specific locus analysis to finally select one restriction endonuclease, Alu I, to perform enzyme digestion to the PCR product, and detecting the enzyme digestion result of the PCR product with 2.0% agarose gel. A result proves that after the enzyme digestion withthe Alu I to the PCR products of the Crassostrea nippona and Crassostrea gigas, enzyme digestion fragments in different sizes are generated, and according to enzyme digestion spectrum, the identification between the Crassostrea nippona and Crassostrea gigas is completed. The identification method is simple, reliable and economic and supplies a technical measure for germplasm identification between the Crassostrea nippona and Crassostrea gigas.

Description

Technical field [0001] The invention belongs to the technical field of molecular biology, and specifically relates to a method for identifying mitochondrial PCR-RFLP of rock oyster and rock oyster and a pair of mitochondrial COI gene primers for identification. Background technique [0002] Oyster (Ostreidae) is a worldwide species and an important breeding target for marine aquaculture in all countries in the world. It is also the largest economic shellfish produced in my country and even in the world. In 2016, China's oyster farming output reached 4.83 million tons, ranking first in the world. Crassostrea gigas is an important economic species in my country, and the common species mainly include long oysters ( Crassostrea gigas ), Omi Oyster ( Crassostrea ariakensis ), Hong Kong Giant Oyster ( Crassostrea hongkongensis ), Portuguese oyster ( Crassostrea angulata ), and Kumamoto oysters ( Crassostrea sikamea ). Among them, the Pacific oyster, also known as Pacific oyster, i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/686C12N15/11
CPCC12Q1/686C12Q2521/301
Inventor 李琪刘凯凯
Owner OCEAN UNIV OF CHINA
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