Specific molecular marker for acquiring genders of bighead carps and screening method and application thereof

A technology of molecular markers and screening methods, which is applied in the field of biology, can solve the problems of difficult identification of juvenile bighead carp, etc., and achieve the effects of low cost, simple operation, and overcoming cumbersome operations

Active Publication Date: 2017-07-14
INST OF AQUATIC LIFE ACAD SINICA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Aiming at the defect that it is difficult to identify the sex of juvenile bighead carp in the prior art, the present invention provides a screening method for sex-specific molecular markers of bighead carp, and uses this method to identify a long sequence specific to male and bighead carp, and develops a simple and convenient method based on the long sequence. A fast and reliable method for identifying the sex of bighead carp, laying a technical foundation for batch sex identification of bighead carp

Method used

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  • Specific molecular marker for acquiring genders of bighead carps and screening method and application thereof
  • Specific molecular marker for acquiring genders of bighead carps and screening method and application thereof
  • Specific molecular marker for acquiring genders of bighead carps and screening method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] [Example 1] Identification of specific short sequences for male and bighead carp

[0030] This embodiment mainly includes the following steps, namely restriction enzyme digestion, ligation, amplification, recovery and analysis.

[0031] Digestion:

[0032] 2b-RAD library construction was carried out on 5 female and 5 male bighead carp with known genders cultured in this laboratory. The specific process is as follows (the following is the amount used for each individual):

[0033]

[0034] After mixing, the enzyme digestion reaction was performed at 37.0°C for 4 hours, and then incubated at 65°C for 20 minutes to inactivate the enzyme.

[0035] connect:

[0036] Sequencing adapter ligation. Ligate the above digested products with standard IlluMina sequencing adapters (slx-ad1 and slx-ad2) respectively, the reaction system (27.0μl):

[0037]

[0038] After mixing and centrifuging, place the ligation reaction at 16°C overnight (8h).

[0039] Amplification:

[0...

Embodiment 2

[0049] [Example 2] Whole Genome Sequencing of Male and Bighead Carp and Extension of Specific Short Sequence of Male and Bighead Carp

[0050] A) One of the five male and bighead carp was selected for small fragment library construction and used for IlluMina whole-genome sequencing. 48.2M PE150bp IlluMina original sequence was obtained by sequencing, and then the whole genome was assembled using SOAPdenovo v1.20;

[0051] B) Perform BLAST alignment search on the whole genome of the male and bighead carp-specific short sequence identified in Example 1, and obtain a long sequence with a length of 911bp, and its nucleic acid sequence is SEQ ID NO:2.

Embodiment 3

[0052] [Example 3] Design and verification of sex primers based on male and bighead carp-specific long sequences

[0053] A) Design a pair of gender primers according to SEQ ID NO:2, the sequence of which is:

[0054] ArS-9-1F: GCTCCTTACTCAGCAACT

[0055] ArS-9-1R: TCAGTAAACAGACGAGCA

[0056] B) select 30 tails from the bighead carp population of known sex of this laboratory culture, comprise 15 tails males and 15 are females, carry out PCR verification to its genome DNA sample, PCR reaction procedure is as follows:

[0057] PCR loading information

[0058]

[0059] PCR program settings

[0060]

[0061] The electrophoresis analysis method is as follows: electrophoresis the denatured amplification product with 10% non-denaturing polyacrylamide gel; electrophoresis conditions: voltage 230V, time 2 hours; then pass the electrophoresis product through ethidium bromide (EB) DNA bands were observed, photographed and analyzed by staining. The result is as figure 1 As sho...

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Abstract

The invention discloses a specific molecular marker for acquiring the genders of bighead carps and a screening method and application thereof. The screening method comprises the following steps: firstly, performing reduced-representation sequencing on 5 female bighead carps and 5 male bighead carps by using a 2b-RAD technology to identify a male specific sequence; then, performing genome resequencing and genome assembly on one of the male bighead carps, comparatively searching 2b-RAD specific sequences of the male bighead carps in a male bighead carp genome to obtain a long-fragment male bighead carp specific sequence, and designing a male specific primer. By adopting the screening method, a male specific sequence and a gender specific primer of the bighead carps are screened for the first time, and a PCR (Polymerase Chain Reaction) technology for generic gender determination is built. The method has the advantages of low cost, easiness, rapidness and accuracy in operation, and the like, and a reliable technical measure is provided for the development of a gender marker and gender determination of the bighead carps. The method can be popularized and applied in gender marker development and gender determination research of other fishes.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to fish sex-specific molecular markers, screening methods and applications thereof. Background technique [0002] Among vertebrates, fish have evolved the most diverse mechanisms for sex determination. In some fish, sex is genetically determined, i.e. they hatch with a defined sex; in others, sex is largely influenced by the environment, such as temperature, pH, salinity and other external environmental conditions; the final sex of fish is the result of genetics, environment and their interaction (Devlin et al.Sex determination and sex differentiation in fish:an overview of genetic,physiological,and environmental influences.Aquacμlture,2002,208( 3): 191-364.). Fishes whose sex is determined by genetic factors are mainly divided into two types, namely XX / XY type and ZW / ZZ type. The first type of fish males contain the sex-determining region, or sex chromosome, while the second type of f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/6888C12Q2565/125
Inventor 童金苟刘海洋付北德俞小牧
Owner INST OF AQUATIC LIFE ACAD SINICA
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