PCR amplification primer for chondriosome cytb gene segment of Marsupenaeus japonicus and identification method thereof

A technology for amplifying primers and gene fragments, which is used in biochemical equipment and methods, DNA/RNA fragments, and microbial determination/inspection. , good repeatability

Inactive Publication Date: 2010-04-14
ZHEJIANG OCEAN UNIV
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  • Summary
  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

[0005] The results of a large number of studies by the inventor show that the Fujian population of Penaeus japonicus has a higher genetic diversity index and better breeding potential, and individuals from the Fujian population can be used to carry out fine breed selection, but it is difficult to see from the appearance Therefore, a stable and reliable identification method is needed to accurately screen out the Fujian population of Penaeus japonicus from the breeding parents

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] 1. Design and synthesize a pair of conservative primers with the sequence shown in SEQ No.1 or SEQ No.2:

[0031] L10945 (SEQ No.1): 5'-TGAGGAGGTTTCGCAGTA-3';

[0032] H11563 (SEQ No.2): 5'-AGATGAGGGTGAGTGGGT-3',

[0033] Synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd. according to the inventor's requirements.

[0034] 2. Follow the steps below to identify the Fujian population of Penaeus japonicus from the other 3 populations:

[0035] (1), Select 5 samples of Penaeus japonicus Fujian population, 5 samples of Zhejiang population, 4 samples of Guangdong population and 5 samples of Taiwan population, after using conventional phenol-chloroform extraction method to extract DNA, 4 The mitochondrial DNA-cytb gene fragments of 4-5 individuals in each group were amplified by PCR. The amplification conditions were: pre-denaturation at 94°C for 4 min, denaturation at 94°C for 30 sec, annealing at 50°C for 45 sec, extension at 72°C for 45 sec, and 30 ...

Embodiment 2

[0202] In step 2 of this embodiment, the DNA template is 100 ng, and other operations are the same as in embodiment 1. Nucleotide sequence results showed that in the 552bp mitochondrial cytb gene fragment, the Fujian population of Penaeus japonicus transposed from T to G at position 528, and from A to G at position 534 in Fujian population. The differences of the above two loci can be used as molecular markers to distinguish the Fujian population of Penaeus japonicus from the other three populations. That is, the test samples whose bases at the 528th and 534th positions of the nucleotide sequence are G are from the Fujian population.

[0203] The sequencing results of Example 1-2 show that the amplification results of the four populations of Penaeus japonicus are consistent in size, stable and reproducible.

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Abstract

The invention relates to a PCR amplification primer for a chondriosome cytb gene segment of Marsupenaeus japonicus and an identification method thereof, belonging to the field of shrimps population identification. The invention provides a pair of PCR amplification primers for identifying the geographic group of the Marsupenaeus japonicus, respectively containing the nucleotide sequences of L10945:5'-TGAGGAGGTTTCGCAGTA-3' or H11563: 5'-AGATGAGGGTGAGTGGGT-3'. The identification method comprises the steps: firstly, carrying out PCR amplification on a cytochrome b gene segment in the chondriosome DNA of a sample of four geographic groups to be detected by using the pair of amplification primers; and secondly, purifying the amplification primer and sequencing, wherein a sample to be detected with the 528-locus and the 534-locus bases of the nucleotide sequences to G is from Fujian group. The invention has the characteristics of stable amplification result, favorable repeatability and simple and effective identification method.

Description

technical field [0001] The invention relates to a marker for identification of important shrimp populations, in particular to using mitochondrial DNA molecular markers to identify Penaeus japonicus Fujian population and other three geographical populations. Background technique [0002] Japanese sac shrimp (Marsupenaeus japonicus) belongs to the prawn family and belongs to the genus S. japonicus. It is widely distributed in the coastal areas of Japan, the southeast coast of my country and the coast of Southeast Asia. Among the prawn varieties in my country, Penaeus japonicus can adapt to low dissolved oxygen and has the ability to be suitable for long-distance live transportation, and its economic value ranks first among the prawn varieties. It is one of the important prawn breeding varieties in the southeast coast of my country, mainly distributed in sea areas Including Zhejiang, Fujian, Guangdong and Taiwan provinces. With the increasing depletion of marine resources and t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 徐田军王日昕孟繁星石戈
Owner ZHEJIANG OCEAN UNIV
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