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A Construction Method of Microsatellite DNA Molecular Marker

A technology of leopard gills and DNA molecules is applied in the field of construction of microsatellite DNA molecular markers for leopard gills, which can solve the problems of limited quantity, time-consuming and labor-intensive, and achieve the effect of short time consumption.

Active Publication Date: 2016-09-21
SUN YAT SEN UNIV
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Problems solved by technology

However, there are only 35 microsatellite molecular markers developed so far for Leopard gillfish (Molecular Ecology Resources 9:1485–1487; Conservation genetics Resources 5:1067–1069; Conservation genetics Resources 2:101–103), the number is very limited , it is urgent to develop more new microsatellite molecular markers
On the other hand, the current microsatellite development of leopard gillfish mainly adopts the enrichment library method, which requires steps such as DNA extraction, genome library construction, probe hybridization enrichment, and clone screening, which is time-consuming and laborious, and usually only dozens of samples can be obtained. mark

Method used

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  • A Construction Method of Microsatellite DNA Molecular Marker
  • A Construction Method of Microsatellite DNA Molecular Marker
  • A Construction Method of Microsatellite DNA Molecular Marker

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Embodiment 1

[0022] A method for constructing a microsatellite DNA molecular marker of leopard gill echinacea, comprising the following steps:

[0023] 1. Transcriptome sequencing of leopard gillfish:

[0024] The total RNA of the sample was extracted by the conventional Trizol method, the DNA in the total RNA was digested by DNase I, and the mRNA was enriched by the mutual adsorption between the magnetic beads with Oligo (dT) and the 3' polyA of the mRNA, and the mRNA was interrupted by heating. After the reaction of the reagent, the product was recovered by ethanol precipitation on ice, double-stranded cDNA was synthesized on the PCR machine, and the end was repaired in the Thermomixer to add base A to the 3' end of the repair product, and then add "A" to the adapter and Product ligation, purification, recovery, amplification and library construction, using Illumina Hi Seq TM 2000 sequenced.

[0025] 2. Acquisition of Unigene and detection of microsatellite loci

[0026] The original ...

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Abstract

The invention discloses a construction method of Plectropomus microsatellite DNA molecular markers. According to the method, Unigene containing Plectropomus microsatellite repeat sequences is firstly screened out by transcriptome sequencing. Then microsatellite sites are detected in the Unigene sequences. Specific primers of microsatellite markers are designed according to the sequences of both ends of the microsatellite sites and are used for detecting polymorphism of the microsatellite sites. The microsatellite markers with abundant polymorphism of Plectropomus are exploited, and seven microsatellite DNA molecular markers of Plectropomus are verified further. The Plectropomus microsatellite DNA molecular markers provided by the invention can be applied to population genetic structure and genetic breeding study of Plectropomus population.

Description

technical field [0001] The invention belongs to the technical field of microsatellite molecular markers, and in particular relates to a method for constructing microsatellite DNA molecular markers of leopard echinacea. Background technique [0002] Plectropomus leopardus belongs to the order Perciformes, the family Anthidae, and the genus Plectropomus, and it is an important farmed fish in the important south of my country. In recent years, the high-intensity fishing pressure has caused the leopard gillfish resource to be overfished. In order to protect and manage the fishery resources of leopard echinacea, it is necessary to study the population genetic structure and genetic variation level. Microsatellite molecular markers have become the most commonly used method for genetic analysis of marine fish populations due to their advantages of high polymorphism and co-dominance. However, there are only 35 microsatellite molecular markers developed so far for Leopard gillfish (...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6888C12Q2600/156
Inventor 蒙子宁谢桢桢于萃平李波刘晓春张勇林浩然
Owner SUN YAT SEN UNIV
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