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Method for discriminating two closely-related species of shellfish or identifying their hybrid generation

A species and related technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of cumbersome, high cost, poor accuracy, etc., and achieve the effect of wide application, intuitive results, and simple methods

Active Publication Date: 2014-09-17
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Overcome the shortcomings of cumbersome, time-consuming, high cost and poor accuracy in existing shellfish species identification methods

Method used

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  • Method for discriminating two closely-related species of shellfish or identifying their hybrid generation
  • Method for discriminating two closely-related species of shellfish or identifying their hybrid generation
  • Method for discriminating two closely-related species of shellfish or identifying their hybrid generation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Identification of Portuguese oyster C.angulata and Kumamoto oyster C.sikamea:

[0049] Portuguese oysters are also called mitral oysters in some areas of China. This oyster is only distributed along the coast south of the Yangtze River in China. It is the main economic species in southern China. Some areas, such as Putian, have also established the original breeding grounds and nature reserves of mitral oysters (actually the Portuguese oyster C. angulata). At the same time, the Kumamoto oyster C. sikamea is also the main species distributed in the Yangtze River estuary area and the coastal intertidal zone south of the Yangtze River, and it is often distributed in the same area as the Portuguese oyster. Some people also call this oyster the mitral oyster. Both types of oysters are small and easily confused. At present, the commonly used molecular biology identification method generally uses the mitochondrial COI gene sequence. In this case, six Portuguese oysters and ...

Embodiment 2

[0062] Identification of hybrids of two species:

[0063] Identification of hybrid progenies of Portuguese oyster C.angulata and Kumamoto oyster C.sikamea:

[0064] In many sea areas along the coast of China, oysters of different closely related species often live together in the same sea area. At the same time, due to the introduction of non-native oyster species from different regions, a large number of cultured oysters and local species live together at the same time. , resulting in the possibility of interbreeding. At present, there is no definite data showing the existence of hybrid populations, but in order to prevent the harm of local biological resources due to biological invasion, detection of interspecies hybridization is a top priority. Off the coast of Xiamen, China, Portuguese oysters and Kumamoto oysters often live in the same environment. In this case, artificial hybridization experiments were carried out on two species, and the hybrid offspring of the two spe...

Embodiment 3

[0072] Use this method to identify closely related species: two species of oysters (Portuguese oyster C.angulata or Kumamoto oyster C.sikamea) were bought before the experiment. The oysters were named A oyster and B oyster, respectively, and they were Portuguese oyster C. angulata or Kumamoto oyster C. sikamea from the appearance.

[0073]a. Sequence acquisition: 14 and 12 ITS1 sequences of Portuguese oyster C. angulata and Kumamoto oyster C. sikamea were obtained from the NCBI database (http: / / www.ncbi.nlm.nih.gov / ). Alignment was performed by ClustalX software.

[0074] b. Primer design: Check the aligned sequences, and select InDel sites with stable differences between the two species based on the sequence characteristics of the ITS1 gene of the two species ( Figure 4 ), use Primer Premier5 software to design PCR primers on both sides of this site.

[0075] The primer sequences are:

[0076] ITS1-F1: 5'-AGCGCCTTGGGCCGTCGAA-3'

[0077] ITS1-R1: 5'-TGTACTTAAGGCGACGGAGC-3...

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Abstract

The invention relates to specie discriminating and identification technology and especially relates to a method for discriminating two closely-related species of shellfish or identifying their hybrid generation. Through combination of endonuclear DNA bar code gene PCR amplification technology and high resolution melting (HRM), the method realizes discriminating of closely-related species or identification of their hybrid generation. Through the method, time from sample preparation to result acquisition is a half day and most processes are carried out in a machine without complex experiment operation. Through a dissolving curve showing method, different peak shapes represent different sequence characteristics and thus a visual determination result is obtained. The result obtained by the method is acquired by a genetic material DNA and can be accepted by domestic and international persons in the same industry or experts. A common phenotype identification method for determining specie names and determining if the specie is hybrid generation is accepted by experts difficultly.

Description

technical field [0001] The invention relates to species identification and identification technology, in particular to an identification method for identifying two closely related species of shellfish or their hybrid offspring. technical background [0002] There are many kinds of shellfish in China's coastal areas, and their classification is complex. The research on some economic molluscs has a certain foundation, and some molluscs that are difficult to identify through external morphology have also been studied in depth from the level of molecular biology. However, due to the limitations of technical means, it is difficult to apply these professional knowledge to practical production and research. Many closely related species live together, and it is still difficult to accurately judge and identify them by appearance. In recent years, scientists have invented many new techniques for species identification of shellfish through genetic material. Genetic material includes...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6858C12Q2531/113C12Q2527/107C12Q2563/185
Inventor 许飞李莉张国范
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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