Microsatellite molecular marker of mauremys mutica and application thereof

A kind of technology for molecular marker of the yellow-throated terrapin, applied in the field of microsatellite molecular markers of the yellow-throated terrapin

Inactive Publication Date: 2010-11-10
ZHEJIANG NORMAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the development of microsatellite molecular markers for freshwater turtles is still rare, and the micr...

Method used

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  • Microsatellite molecular marker of mauremys mutica and application thereof
  • Microsatellite molecular marker of mauremys mutica and application thereof
  • Microsatellite molecular marker of mauremys mutica and application thereof

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Experimental program
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Effect test

Embodiment 1

[0024] 1. Experimental method

[0025] Genomic DNA was first extracted from the mid-posterior part of the tail of the yellow-throated water turtle by standard proteinase K digestion and phenol / chloroform extraction. Then, the construction of the microsatellite enrichment library, the screening of positive clones, sequence arrangement and primer design were carried out.

[0026] 1. Construction of Microsatellite Enrichment Library

[0027] The construction of the microsatellite enrichment library (CA)n mainly refers to the FIASCO method (Fast Isolation by AFLP of Sequences Containing repeats) of Zane et al. The specific experimental operation is as follows:

[0028] 1.1 Digestion and ligation reaction

[0029] Take the genomic DNA of 2 yellow-throated aquatic turtles, mix them, and carry out enzyme digestion ligation reaction. The system is 25ul, including: 0.8ul mixed DNA, 1ul AFLP adapter acceptor, 0.5ul MseI restriction endonuclease, 2.5ul 1×NEB Buffer, 0.25ul BSA, 2ul AT...

Embodiment 2

[0079] The above pair of microsatellite primers were used to amplify 120 individuals from 8 sampling points to detect their genetic diversity. The specific operation is as follows:

[0080] (1) 120 samples of yellow-throated aquatic turtles for genetic analysis. See Table 2 for sampling points and codes.

[0081] Table 2. Sample information of yellow-throated aquatic turtles

[0082]

[0083] (2) DNA extraction of yellow-throated aquatic turtle

[0084] Genomic DNA was extracted from the tails by standard proteinase K digestion and phenol / chloroform extraction.

[0085] (3) PCR amplification of microsatellite DNA

[0086] The PCR reaction is carried out in a 15ul system, including: about 50-100ng genomic DNA, 7.5ul Ex Taqpremix buffer, 0.4ul each of the upstream and downstream primers of the present invention, 0.4pmol fluorescently labeled M13 primer [either IRD700or IRD800 (LI-COR) ], add ddH 2 O fill up to 15ul. PCR amplification conditions were: 95°C ...

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Abstract

The invention discloses a microsatellite molecular marker of mauremys mutica. The nucleotide sequence of the microsatellite molecular marker is SEQ ID NO:1. The invention also discloses a primer of the microsatellite molecular marker of the mauremys mutica. The microsatellite molecular marker of the mauremys mutica can be used for carrying out genetic structure analysis, genetic map construction, gene location, auxiliary marking seed selection or auxiliary marking seed breeding on the mauremys mutica.

Description

technical field [0001] The invention relates to the three major fields of genomics, bioinformatics and molecular biology, and is a yellow-throated aquatic turtle microsatellite molecular marker and its application developed according to the FIASCO method. Background technique [0002] Since Watson and Crick proposed the DNA double helix structure in 1953 and discovered restriction endonucleases in the late 1960s, molecular biology began to develop. For example, in 1980, Botstein et al. adopted DNA restriction fragment length polymorphism (RFLP) as a new genetic marker, thereby creating a new stage of directly using DNA polymorphism to develop genetic markers. Then some other molecular genetic markers such as microsatellites, DNA fingerprints, RAPD, etc., appeared to directly study DNA polymorphisms. The emergence of these molecular markers opened up a new chapter for the study of genetic diversity. Molecular markers are directly expressed in the form of DNA, and when DNA is...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68
Inventor 郑荣泉章芸俞丹娜李涛
Owner ZHEJIANG NORMAL UNIVERSITY
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