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SRAP molecule labeled primer for Megalobrama amblycephala family identification, method thereof, and application of primer

A technology of molecular markers and bream, applied in biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of complex operation, long time-consuming, difficult application, etc., and achieve simple operation and cost saving , easy-to-sequence effects

Inactive Publication Date: 2015-01-21
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Bory bream, also known as Wuchang fish, is one of the main freshwater cultured fish in China because of its tender meat and delicious taste. However, the classification and identification of this species’ family has always been a difficult problem in the field of zoology. The operation is complex, time-consuming, and difficult to apply in a laboratory environment

Method used

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  • SRAP molecule labeled primer for Megalobrama amblycephala family identification, method thereof, and application of primer
  • SRAP molecule labeled primer for Megalobrama amblycephala family identification, method thereof, and application of primer
  • SRAP molecule labeled primer for Megalobrama amblycephala family identification, method thereof, and application of primer

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Experimental program
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Effect test

Embodiment 1

[0028] Embodiment 1: Construction of the family sample of bream

[0029] The samples of the 8 bream families were all purchased from the bream breeding base of Huazhong Agricultural University in Ezhou City, Hubei Province. There were 5 male parents and 8 female parents. The parents of the bream families were all from the natural population of Liangzihu. The parent fishes were paired with males and females and artificially propagated, and the hybrid families were established by hybridization. The offspring were numbered L9, L29, L30, L32, L34, L36, L37, and L38. The family information is shown in Table 2. The fins used in the experiment were taken from a small part of the caudal fin of bream and fixed with 95% ethanol.

[0030] Table 2 Information of 8 families of bream used in the experiment

[0031]

Embodiment 2

[0032] Embodiment 2: the screening of primer combination

[0033] The DNA was extracted by conventional methods and diluted to 20 ng / μL, and the most suitable primer pair was selected from the 88 pairs of SRAP primers in Table 1 of the manual. The total volume of the SRAP-PCR reaction system is 15 μL, including TaqDNA polymerase 0.6U (MBI), 10×Buffer 1.5 μL, 2.0 mmol / L Mg2+, 0.25 mmol / LdNTPs (Roche), each primer 0.5 μmol / L, template DNA About 20ng.

[0034] The thermal cycle parameters of PCR were: 94°C pre-denaturation for 5 min; the first 5 cycle parameters were denaturation at 94°C for 1 min, annealing at 35°C for 1 min, extension at 72°C for 1 min; the last 35 cycle parameters were denaturation at 94°C for 1 min, annealing at 50°C for 1 min, and 72 Extend for 1 min at ℃; extend for 10 min at 72°C; cool down to 4°C for storage.

[0035] PCR amplification products were separated by 6% denatured polyacrylamide gel electrophoresis, and the experimental materials used were pr...

Embodiment 3

[0044] Embodiment 3: Genetic diversity and genetic variation analysis of the group head bream population carried out based on the optimal primer combination

[0045] The data processing method is to refer to the 100bp DNA Marker, and count the bands of the obtained SRAP electrophoresis pattern. Each band represents a site, and the band is clearly counted as 1, and no band is counted as 0. A 0, 1 Composed of digital matrix. The POPGENE1.32 program (Yeh et al., 1999) was used to analyze the Nei's gene diversity (h) and Shannon's information index (I) of the 8 families of bream, and calculate the genetic differentiation coefficient among the families (GST), estimated gene flow (Nm) between families based on genetic differentiation coefficients. Molecular analysis of variance (AMOVA) was performed on different families with ARLEQUIN3.11 (Excoffier et al., 2006).

[0046] Using NTSYS-pc2.1 (Rohlf, 2000) to calculate the genetic similarity Dice coefficient (Dice, 1945) of 393 brea...

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Abstract

The invention discloses an SRAP molecule labeled primer for Megalobrama amblycephala family identification, a method thereof, and an application of the primer. The SRAP molecule labeled primer is selected from one of the following primer combinations: Me2 / Em18, Me3 / Em19, Me9 / Em13, Me9 / Em15, Me9 / Em16, Me11 / Em4, Me11 / Em15, Me11 / Em18, Me12 / Em13 and Me12 / Em16, and the sequences of all the primers are respectively shown in a sequence table. The disclosed SRAP molecular marker primer is used to identify the Megalobrama amblycephala family by carrying out SRAP molecule labeling of the Megalobrama amblycephala family, and a band obtained in the invention has the advantages of clearness, good polymorphism, high repeatability, simple operation and broad application prospect.

Description

technical field [0001] The invention relates to an SRAP molecular marker primer used for identification of bream family, and belongs to the technical field of aquatic animal molecular biology. Background technique [0002] Related sequence amplified polymorphism (sequence related amplified polymorphism, SRAP) is a marker system developed based on traditional PCR, which amplifies the reading frame regions (Open Reading Frames, ORFs) of genes through unique primer design. In this method, the 17bp forward primer specifically amplifies the exon, and the 18bp reverse primer specifically amplifies the intron and promoter region. Polymorphisms occur due to differences in the length of the interval between introns and promoters within or between species. Compared with other methods, SRAP molecular markers are characterized by simplicity, rapidity, stability, good reproducibility, medium yield, co-dominant markers, and easy cloning and sequencing of target fragments. They are widely...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6888C12Q2600/156
Inventor 魏开建张桂蓉姬伟冉玮
Owner HUAZHONG AGRI UNIV
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