Method for developing hypophthalmichthys nobilis sex determination molecular marker by taking Gapdh gene as reference

A molecular marker and sex technology, applied in the field of fish genetics, can solve the problems of long sexual maturity time and inconvenient selection breeding work, and achieve the effect of strong specificity and high accuracy

Active Publication Date: 2021-03-26
INST OF AQUATIC LIFE ACAD SINICA
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because the sexual maturity of bighead carp takes a long time (4-5 years or longer), it brings great inconvenience to the selective breedin

Method used

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  • Method for developing hypophthalmichthys nobilis sex determination molecular marker by taking Gapdh gene as reference
  • Method for developing hypophthalmichthys nobilis sex determination molecular marker by taking Gapdh gene as reference
  • Method for developing hypophthalmichthys nobilis sex determination molecular marker by taking Gapdh gene as reference

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Embodiment 1

[0025] Example 1 Development of Molecular Markers for Sex Identification of Bighead Carp

[0026] Using 5 female and 5 male bighead carp with known genders cultured in our laboratory as samples, the tail fins were cut to extract genomic DNA (gDNA), using BSA XI endonuclease, the sequencing library was constructed according to the 2b-RAD method, and the sequencing library was qualified Then on the machine for sequencing. After filtering the sequencing data, using Stacks v1.31 for sex-specific sequence identification, a specific sequence was obtained that appeared in all 5 male bighead carp but not in 5 female bighead carp.

[0027] (1) enzyme digestion:

[0028] The extracted DNA is used for 2b-RAD library construction with BSA Ⅺ endonuclease, the specific process is as follows: (the following is the amount used for each individual)

[0029] gDNA 5.0 μL

[0030] BSA XI (1U / μL)5.0μL

[0031] CutSmart Buffer 0.5μL

[0032] After mixing, it was incubated at 37.0°C for 1 hour....

Embodiment 2

[0052] Embodiment 2 Gapdh reference sequence primer design and primer concentration optimization

[0053] The reference primers for amplifying the bighead carp housekeeping gene Gapdh were designed to eliminate the influence of factors such as degradation and sample addition errors. The sequences of the designed primers are shown in Table 3.

[0054] Table 3 Information table of primers for Gapdh gene of bighead carp

[0055]

[0056] Using the Gapdh gene as a reference, double PCR amplification was carried out in the same PCR reaction system with the specific primers for sex identification of bighead carp. Due to differences in the amplification efficiency of different primers, it is necessary to optimize the concentration ratio of sex-specific primers and Gapdh primers. In the 25 μL amplification system, when the concentration ratio of HynS-4 and Gapdh-3 primers was 3:1, the amplified bands of the two were clear, and the identification effect was the best. Select 12 tai...

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Abstract

The invention discloses a method for developing a hypophthalmichthys nobilis sex determination molecular marker by taking Gapdh gene as reference, and belongs to the field of fish genetics. Accordingto the method, a specific sequence (SEQ ID NO.2) only existing in a male hypophthalmichthys nobilis genome sequence is found, and primers designed based on the specific sequence can be used for identifying the gender of hypophthalmichthys nobilis. The Gapdh gene is called from a hypophthalmichthys nobilis genome to serve as a reference design primer, the Gapdh gene and the primer for identifying the gender of the hypophthalmichthys nobilis are subjected to double PCR amplification, and the genetic gender of the hypophthalmichthys nobilis can be rapidly and accurately identified through agaroseelectrophoresis. The molecular marker developed by the invention has strong specificity and can be used for accurately identifying the gender of hypophthalmichthys nobilis. The Gapdh gene is used asa reference gene for identification through double PCR, and gender judgment errors caused by DNA degradation, sample adding errors or PCR amplification failures and the like are avoided. The method has the advantages of quickness, simplicity, convenience, high accuracy and the like, and can be used for accurately identifying the genetic sex of hypophthalmichthys nobilis on a large scale.

Description

technical field [0001] The invention belongs to the field of fish genetics, and relates to a method for developing molecular markers for sex identification of fish, in particular to a method for developing molecular markers for sex identification of bighead carp with Gapdh gene as a reference. Background technique [0002] As the most primitive vertebrate, fish is a large group that has rapidly changed to bisexual reproduction in the history of biological evolution, and its sex determination shows primitiveness, complexity and diversity. The primitive nature of sex determination in fish is manifested in the fact that fish do not evolve obvious sex chromosomes and definite sex-determining genes like higher mammals. Most fish have not yet formed obvious sex chromosomes and sex-determining genes are not clear, and have sex chromosomes Only about 10% (176 out of 1700 species) of fish. Fishes determined by genetic factors are mainly divided into two types, namely XX / XY type and ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12Q1/6879C12Q1/6858C12N15/11
CPCC12Q1/6888C12Q1/6879C12Q1/6858C12Q2600/156C12Q2600/16C12Q2600/166C12Q2531/113C12Q2535/101C12Q2537/143
Inventor 周颖童金苟俞小牧廖德杰
Owner INST OF AQUATIC LIFE ACAD SINICA
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