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Microsatellite DNA (Deoxyribonucleic Acid) markers of Chinese three-keeled pond turtle

A technology of DNA markers and microsatellite markers, applied in the field of turtle microsatellite DNA genetic markers, can solve the problem that genetic markers cannot provide enough genetic information

Inactive Publication Date: 2012-12-12
ANHUI NORMAL UNIV
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Problems solved by technology

Some scholars have used different molecular marker techniques (including mitochondrial cytochrome b gene sequence, mitochondrial whole genome sequence, etc.) to study the genetic diversity of tortoises, but the above genetic markers cannot provide sufficient genetic information. Preliminary study of the cytochrome b gene fragment; Nie Liuwang et al., Sequence and structure analysis of the whole genome of tortoise mitochondria

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  • Microsatellite DNA (Deoxyribonucleic Acid) markers of Chinese three-keeled pond turtle
  • Microsatellite DNA (Deoxyribonucleic Acid) markers of Chinese three-keeled pond turtle
  • Microsatellite DNA (Deoxyribonucleic Acid) markers of Chinese three-keeled pond turtle

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Embodiment Construction

[0020] The present invention will be described in further detail below in conjunction with the accompanying drawings.

[0021] 1. Construction of turtle microsatellite DNA enrichment library:

[0022] 1.1 Genome extraction and enzyme digestion: extract the tortoise genome DNA with reference to the method of Sambrook et al. Electrophoresis on % agarose gel, cutting out 400-1000bp DNA fragments under ultraviolet light, and purifying the recovered enzyme-digested products with a gel recovery kit (purchased from Shanghai Sangong).

[0023] 1.2 Add connector:

[0024] Add the two ends of the purified and recovered enzyme-cut product to the adapter:

[0025] SauL-F(5'-GGCCAGAGACCCCCAAGCTTCG-3')

[0026] SauL-R (5'-PO 4 -GATCCGAAGCTTGGGGTCTCTGGCC-3'),

[0027] Under the action of T4 ligase (purchased from Promega), the ligation was performed overnight in a water bath at 16°C.

[0028] 1.3 PCR detection adapter connection:

[0029] Use SauL-F as a primer and the ligation produc...

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Abstract

The invention discloses a microsatellite DNA (Deoxyribonucleic Acid) markers of a Chinese three-keeled pond turtle, which are determined by comprising the steps of constructing a library rich in tortoise microsatellites (CA)n (Carbonic Anhydrase)n and (GATA)n, screening and sequencing positive clones containing microsatellite sequences to obtain 59 clones containing microsatellite repetitive sequences, and determining 10 microsatellite markers with rich polymorphisms, wherein the 10 microstaellite markers are Mclw01, Mclw02, Mclw03, Mclw04, Mclw05, Mclw06, Mclw07, Mclw08, Mclw09 and Mclw10. The invention provides 10 new microsatellite loci of the Chinese three-keeled pond turtle and primer sequences and amplification methods for amplifying the 10 microsatellite loci, can be applied to a study on genetic diversity of different geographical populations of the Chinese three-keeled pond turtles and a study on paternity identification, and the molecular markers are reliable and effective with good repeatability.

Description

technical field [0001] The invention relates to a molecular marker technology, in particular to a turtle microsatellite DNA genetic marker. Background technique [0002] Microsatellite DNA, also known as short tandem repeats (STR), simple repeats (SSR), simple sequence length polymorphism (SSLP), refers to the tandem repeats of 1-6 nucleotides in the genome. Nucleotide sequence; among them, the most common are dinucleotide repeats, such as ((AC / TG)n and (AG / TC)n. Different organisms show polymorphism due to the number of repetitions of the repeat motif Studies have shown that it may be the "strand slippage" phenomenon in the DNA replication process that causes the high polymorphic information content (PIC) of microsatellite DNA. The high mutation and co-dominance of microsatellite DNA Expression and its ubiquity in eukaryotic genomes make it a superior molecular marker for the analysis of population genetic diversity, identification of kinship, genome mapping and genetic br...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11
Inventor 聂刘旺刘罗卜兴江鲁元华汪玲蒋远景万星黄振锋吴立花
Owner ANHUI NORMAL UNIV
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