CN-116356006-B - Molecular marker for identifying genetic sex of black carp and application thereof

CN116356006BCN 116356006 BCN116356006 BCN 116356006BCN-116356006-B

Abstract

The invention discloses a molecular marker for identifying the genetic sex of black carp and application thereof, belonging to the field of aquatic animal genetic breeding. The molecular marker comprises a herring male-female fish common band with a nucleotide sequence shown as SEQ ID NO. 1 and a herring male-female fish special band with a nucleotide sequence shown as SEQ ID NO. 2, wherein the identification method comprises the steps of carrying out PCR amplification by taking DNA of a herring sample to be identified as a template, carrying out capillary electrophoresis after the PCR product is determined to have the band through agarose electrophoresis, and if signals appear at 152bp and 168bp simultaneously, the herring male is male, and if signals appear only at 168bp, the herring male is female. The molecular marker can accurately obtain the genetic sexes of different black carp populations, has the advantages of simplicity, convenience, rapidness, high efficiency, no influence of early-stage black carp gonad undifferentiated, and the like, is used for black carp marker assisted breeding, greatly saves the breeding cost, and improves the breeding efficiency.

Inventors

SHEN YUBANG
GUO JIAMIN
LI JIALE
JIANG YUCHEN
WANG YUANHAO
YANG LIN

Assignees

上海海洋大学

Dates

Publication Date: 20260512
Application Date: 20230412

Claims (5)

1. The molecular marker for identifying the genetic sex of the black carp is characterized by comprising a black carp male-female fish sharing band with a nucleotide sequence shown as SEQ ID NO. 1 and a black carp male fish unique band with a nucleotide sequence shown as SEQ ID NO. 2.
2. Use of the primer for amplifying the molecular marker for identifying the genetic sex of the black carp according to claim 1 for identifying the sex of the female and male of the black carp.
3. A primer for amplifying the molecular marker for identifying the genetic sex of black carp according to claim 1, wherein the nucleotide sequence of the primer is shown as SEQ ID NO. 3 and SEQ ID NO. 4.
4. Use of the primer according to claim 3 for identifying sex of black carp.
5. A method for identifying sex of black carp, comprising the following steps: 1) Extracting DNA of a herring sample to be identified; 2) Performing PCR amplification by using the primer of claim 3 by taking DNA of a herring sample to be identified as a template; 3) Detecting PCR products, namely carrying out capillary electrophoresis on the PCR products after the PCR products are determined to have bands through 2% agarose electrophoresis, wherein the black carp to be identified is male if signals appear at 152bp and 168bp simultaneously after electrophoresis, and the black carp to be identified is female if signals appear at 168bp only after electrophoresis.

Description

Molecular marker for identifying genetic sex of black carp and application thereof Technical Field The invention belongs to the field of aquatic animal genetic breeding, and particularly relates to a molecular marker for identifying the genetic sex of black carp and application thereof. Background Black carp (Mylopharyngodonpiceus) is also called black carp, is the only predatory fish in four large domestic fishes in China, is also an important freshwater aquaculture fish in China, and is naturally distributed in a large and medium-sized river system in east Asia, and the distribution area is North Heilongjiang, south to Zhujiang. According to the statistics of the year's authentication of Chinese fishery, the culture yield of the black carp in 2021 is about 71.66 ten thousand tons, and the yield is increased by 3.17 percent compared with the yield in 2020. The sexual maturity period of the black carp is longer, the female fish is sexually mature for 5-7 years, the male fish is sexually mature for 4-5 years, and the sexes of the male and female fish cannot be accurately identified from the aspect before sexual maturity, so that the male and female parents can be identified only when the female parents are sexes of the black carp in the second sexual characteristics in the breeding season. The second characteristic of the black carp in the breeding season is that the inner side of the pectoral fin and the gill cover of the male fish are star-chasing, the female fish feel rough by hand, and the pectoral fin is smooth and star-chasing is avoided. In addition, in the process of breeding the black carp parent fish, the parent fish with proper sex is always lacking in artificial insemination due to the fact that the sex ratio cannot be controlled in the early stage, so that the breeding efficiency is reduced, in the process of breeding the families, more individuals have to be reserved for each family due to the fact that the sex ratio cannot be determined when parents with high-quality breeding values are selected, and the cost and difficulty of breeding are increased intangibly. Therefore, there is a need for a method of sexing a black carp that can be performed at various stages of development. At present, the most common method for identifying the sex of the black carp is to judge the sex by touching whether the pectoral fin has rough feeling in the breeding season of the black carp, then judge the sex according to the effluent by extruding the reproductive hole in the breeding season, and further judge the sex by a tissue slicing method. However, the method of looking for the exudates by touching the fin and squeezing the genital orifice is only used when the second sex is present in the breeding season, and the method is no longer applicable when the black carp is not having the second sex in the non-breeding season. Although the method for tissue slicing can effectively judge the sex in non-propagation seasons, the method for tissue slicing still cannot distinguish the sex when the gonads of young fish are undifferentiated, and the fish is killed in the process of tissue slicing, so that the subsequent complicated slicing can be performed, and the sex identification cost of batch of black carp is high. Molecular markers are important tools for fish breeding, and searching for fish-specific molecular markers or DNA fragments is an implementation way for fish sex control and identification. Along with the rapid development of genome sequencing technology and molecular biology, the sequencing cost is obviously reduced, the analysis method is continuously improved, and the like, thereby providing convenience for the development of sex molecular markers. A large number of mutation sites are obtained through high-throughput sequencing, mutation related to a target property is found out in a whole genome range by adopting whole genome association analysis, a specific primer is designed by utilizing the discovered mutation sites to carry out PCR amplification on genome DNA, a polymorphism product specific based on the mutation is obtained, and finally polymorphism of the product is analyzed through electrophoresis, so that the method has the advantages of high density, stable heredity, high representativeness, easiness in realizing analysis and the like, and no related report about development and application of molecular markers of the sex of the black carp exists at present. Disclosure of Invention Aiming at the defects in the prior art, the main purpose of the invention is to provide a molecular marker for identifying the genetic sex of the black carp, and a specific marker for screening the sex of the female and male black carp based on a resequencing technology can be used for identifying the genetic sex of the black carp. The invention also aims to provide the application of the molecular marker for identifying the genetic sex of the black carp in black carp marker-assisted breeding, which is efficient, simp