Siniperca chuatsi male molecular marker primers and application thereof

A technology of molecular marker and mandarin fish, which can be used in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc. Short time and high efficiency

Active Publication Date: 2020-02-11
广东梁氏水产种业有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is difficult to distinguish the gender of mandarin fish from the external form, and it takes at least three months to identify the gonads through anatomy, and the separate breeding of test cross parents requires a lot of breeding resources, so the method of test cross is time-consuming, laborious and resource-intensive
So far, there are no reports of molecular markers that can successfully identify the genetic sex of Siniperca chinense at home and abroad. Therefore, the development of a molecular marker that can accurately identify the genetic male has great production and application value in the all-female breeding of Siniperca chinense.

Method used

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  • Siniperca chuatsi male molecular marker primers and application thereof
  • Siniperca chuatsi male molecular marker primers and application thereof
  • Siniperca chuatsi male molecular marker primers and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0039] The present embodiment provides the male molecular marker primers for cocked mandarin fish, the primers include primer pair 1 or primer pair 2, the primer pair 1 includes Contig-1 upstream primer and Contig-1 downstream primer, and the primer pair 2 includes Contig-2 Upstream primer and Contig-2 downstream primer, wherein the nucleotide sequence of Contig-1 upstream primer is shown in SEQ ID NO: 1, the nucleotide sequence of Contig-1 downstream primer is shown in SEQ ID NO: 2, Contig- 2 The nucleotide sequence of the upstream primer is shown in SEQ ID NO: 3, and the nucleotide sequence of the Contig-1 downstream primer is shown in SEQ ID NO: 4.

[0040] Specifically, the nucleotide sequences of each primer from 5' to 3' are as follows:

[0041] Contig-1 upstream primer: 5'-GCTGCTTTGAAGTCTGATACATG-3';

[0042] Contig-1 downstream primer: 5'-TGGTTTGTCAGATTGCACCTGTA-3';

[0043] Contig-2 upstream primer: 5'-CATCTCCTCTTAACAGGGACCAT-3';

[0044] Contig-2 downstream primer...

Embodiment 2

[0067] The present embodiment provides the method that utilizes mandarin fish male molecular marker primer to carry out sex identification of cocked mandarin fish, comprising the following steps:

[0068] (1) Design the above-mentioned Siniperca sinensis male molecular marker primer, the specific process is with reference to Example 1;

[0069] (2) DNA sample preparation of mandarin fish with cocked mouth:

[0070] Obtained visually mature mandarin fish from Sanshui Baijin Aquatic Seedling Co., Ltd., Foshan City, Guangdong Province, and obtained 24 male and female mandarin fishes by dissection. Tail fins were cut off separately, and DNA samples were prepared by using the conventional column centrifugation method (Universal Column Genome Extraction Kit, Beijing Kangwei Century Biotechnology Co., Ltd.) and referring to the operation steps in the manual.

[0071] (3) PCR amplification verification:

[0072] Using primers Contig-1F / R and Contig-2F / R, 24 male and female mandarin ...

Embodiment 3

[0076] The present embodiment provides the application of the all-female breeding of mandarin fish with cocked mouth using the above-mentioned cocked mandarin fish male molecular marker primer, including the following steps:

[0077] (1) When the mandarin carp fry are raised to 10 days old, feed the bait fish with the eel feed containing 100 mg / kg of the androgen 17α-methyltestosterone, and then feed the bait fish with the hormone feed to the mandarin fish. Continuously feed for two months, use natural light during the breeding period, cultivate water temperature at 26-28°C, and feed with enough food.

[0078] (2) The mandarin fish fed with androgen was screened by the method in Example 2, and the genotype was obtained as XX male fish, that is, pseudo-male fish. Refer to Example 2 for the specific process.

[0079] (3) Continue raising the XX pseudo-male fishes identified by markers until they reach sexual maturity, and mate with normal XX female fishes during the breeding sea...

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Abstract

The invention provides siniperca chuatsi male molecular marker primers. The primers at least comprise one of a primer pair 1 and a primer pair 2, wherein the primer pair 1 comprises a Contig-1 upstream primer and a Contig-1 downstream primer, and the primer pair 2 comprises a Contig-2 upstream primer and a Contig-2 downstream primer; the nucleotide sequence of the Contig-1 upstream primer is as shown in SEQ ID NO: 1, the nucleotide sequence of the Contig-1 downstream primer is as shown in SEQ ID NO: 2, the nucleotide sequence of the Contig-2 upstream primer is as shown in SEQ ID NO: 3, and thenucleotide sequence of the Contig-2 downstream primer is as shown in SEQ ID NO: 4. The primers can be used for quickly and accurately identifying the sex of siniperca chuatsi. Meanwhile, the invention further provides a method for identifying the sex of the siniperca chuatsi, and the method is short in consumed time and high in efficiency.

Description

technical field [0001] The invention belongs to fish sex identification technology in the technical field of aquatic organisms, and in particular relates to a male molecular marker primer and application thereof. Background technique [0002] In recent years, with the rapid development of molecular biology techniques, sex-specific markers of a variety of fish have been developed, such as Nile tilapia (Oreochromis niloticus), rainbow trout (Oncorhynchus mykiss), yellow catfish (Pelteobagrus fulvidraco), semi-smooth tongue sole (Cynoglossus semilaevis) and redfin puffer (Takifugu rubripes), etc., but these markers are mainly through microsatellite (SSR), restriction site associated DNA (restriction site associated DNA, RAD) and amplified fragment length polymorphism (amplified fragment length polymorphism, AFLP) and other molecular marker development techniques, these molecular marker development methods are time-consuming, heavy workload, and low success rate. The emergence ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12Q1/6879C12N15/11
CPCC12Q1/6879C12Q1/6888
Inventor 张勇韩崇李桂峰李水生韩林强梁建辉林浩然
Owner 广东梁氏水产种业有限公司
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