Molecular marker for identifying genetic sex of grass carp and application thereof

Abstract

The application discloses a kind of molecular markers for identifying genetic sex of blue fish and its application, belong to the field of aquatic animal genetics and breeding.The molecular marker includes nucleotide sequence such as the common band of blue fish female and male fish shown in SEQ ID NO:1 and nucleotide sequence such as the unique band of blue fish male shown in SEQ ID NO:2, and the identification method comprises: taking the DNA of the blue fish sample to be identified as template for PCR amplification, after the band of PCR product is determined by agarose electrophoresis, capillary electrophoresis is carried out, if signal appears at 152bp and 168bp simultaneously, then it is male;If only signal appears at 168bp, then it is female.The molecular marker of the application can accurately obtain the genetic sex of different blue fish populations, has the advantages such as simple, fast, efficient, not affected by early undifferentiated gonad of blue fish, etc., is used for blue fish marker assisted breeding, which greatly saves the cost of aquaculture and improves the breeding efficiency.

Description

Technical Field

[0001] This invention belongs to the field of aquatic animal genetics and breeding, specifically relating to a molecular marker for identifying the genetic sex of grass carp and its application. Background Technology

[0002] Grass carp (Mylopharyngodon piceus), also known as black carp, is the only carnivorous fish among my country's four major freshwater fish species and an important freshwater aquaculture fish. It is naturally distributed in large and medium-sized river systems throughout East Asia, ranging from the Heilongjiang River in the north to the Pearl River in the south. According to the China Fisheries Yearbook, my country's grass carp aquaculture production in 2021 was approximately 716,600 tons, an increase of 3.17% compared to 2020. Grass carp have a long sexual maturity cycle; females reach sexual maturity in 5-7 years, and males in 4-5 years. Before sexual maturity, sex cannot be accurately determined by appearance. Therefore, sexing must be determined when the parent fish reach sexual maturity and develop secondary sexual characteristics during the breeding season. During the breeding season, the secondary sexual characteristics of grass carp are: males have tubercles on the inner side of their pectoral fins and gill covers, which feel rough to the touch, while females have smooth pectoral fins without tubercles. In the breeding of black carp broodstock, the inability to control the sex ratio in the early stages often leads to a lack of suitable broodstock for artificial insemination, resulting in reduced reproductive efficiency. Furthermore, during pedigree selection, the inability to determine the sex ratio when choosing high-quality broodstock forces each family to retain more individuals, increasing the cost and difficulty of breeding. Therefore, a method for sex determination at all stages of black carp development is urgently needed.

[0003] Currently, the most common methods for determining the sex of grass carp are: during the breeding season, by touching the pectoral fins to determine if they feel rough; secondly, by squeezing the genital opening and observing the discharge; and thirdly, by examining tissue sections. However, the methods of touching the fins for roughness and examining the discharge from the genital opening are only applicable during the breeding season when secondary sexual characteristics are present. Outside the breeding season, grass carp do not exhibit secondary sexual characteristics, rendering these methods inapplicable. While tissue sections are effective for sexing outside the breeding season, they are ineffective when the gonads of juvenile fish are undifferentiated. Furthermore, tissue sectioning requires euthanizing the fish for the tedious process of preparing the sections, making it costly for large-scale sex determination of grass carp.

[0004] Molecular markers are important tools for fish breeding, and identifying fish-specific molecular markers or DNA fragments is a means to control and identify fish sex. With the rapid development of genome sequencing technology and molecular biology, sequencing costs have significantly decreased, and analytical methods have been continuously upgraded and improved, facilitating the development of sex molecular markers. High-throughput sequencing yields a large number of variant sites, and genome-wide association analysis (GWAS) is used to identify variants associated with the target trait across the entire genome. Specific primers are designed using these identified variant sites to amplify genomic DNA via PCR, yielding polymorphic products specific to these variants. Finally, electrophoresis is used to analyze the polymorphism of the products. This method has advantages such as high density, genetic stability, high representativeness, and ease of automation. Currently, there are no reports on the development and application of sex molecular markers in grass carp. Summary of the Invention

[0005] In view of the above-mentioned shortcomings of the prior art, the main objective of this invention is to provide a molecular marker for identifying the genetic sex of grass carp. Based on resequencing technology, specific markers for the sex of grass carp are screened out, which can be used for the genetic sex identification of grass carp.

[0006] Another objective of this invention is to provide the application of the aforementioned molecular markers for identifying the genetic sex of grass carp in marker-assisted breeding of grass carp. This method is highly efficient, simple, and quick, and can effectively identify the genetic sex of grass carp, making it widely applicable in production practice.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] This invention provides a molecular marker for identifying the genetic sex of grass carp, comprising a common band of nucleotide sequences for both male and female grass carp as shown in SEQ ID NO: 1, and a male-specific band of nucleotide sequences as shown in SEQ ID NO: 2, as detailed below:

[0009] TTACGAGCGCATCTGAAAAATAGCGGAGGGCACAGTGAGCTGCTGCGGGGAAAG TGCATAATCCAATTTGTCCTAAACATGAGACATGCTTTATATAAAGGGCTTCAAAATAACT CCTATAGTTCAAAGCGGCTTGCCCTTTCAGATGCTCT (SEQ ID NO: 1);

[0010] TTACGAGCGCATCTGAAAAATAGCGGAGGGCACAGTGAGCTGCTGCGGGGAAAG TGCATAATCCAATTTGTCCTAAACATGAGACATGCTTTATATAAAGGGCTTCAAAATAACT CATAAAATAAATAACGCCTATAGTTCAAAGCGGCTTGCCCTTTCAGATGCTCT (SEQ ID NO: 2).

[0011] This invention also provides primers for amplifying the molecular markers used to identify the genetic sex of grass carp, the nucleotide sequences of which are shown in SEQ ID NO: 3 and SEQ ID NO: 4, as follows:

[0012] Upstream primer Oligo-L: 5'-FAM-TTACGAGCGCATCTGAAAAA-3' (SEQ ID NO: 3);

[0013] Downstream primer Oligo-R: 5'-AGAGCATCTGAAAGGGCAAG-3' (SEQ ID NO: 4).

[0014] The present invention also provides the application of the primers for amplifying the aforementioned molecular markers for identifying the genetic sex of grass carp in marker-assisted breeding of grass carp.

[0015] This invention also provides a method for identifying the sex of grass carp, comprising the following steps:

[0016] 1) Extract DNA from the grass carp sample to be identified;

[0017] 2) Using the DNA of the grass carp sample to be identified as a template, PCR amplification was performed using primers with the aforementioned nucleotide sequences as shown in SEQ ID NO: 3 and SEQ ID NO: 4;

[0018] 3) Detection of PCR products: After confirming the presence of bands by 2% agarose gel electrophoresis, the PCR products are subjected to capillary electrophoresis. If signals appear simultaneously at 152bp and 168bp after electrophoresis, the grass carp to be identified is male; if signals appear only at 168bp after electrophoresis, the grass carp to be identified is female.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] 1. The molecular marker for sex identification of grass carp obtained by the present invention through genome-wide association analysis is accurate and has a short experimental cycle. It can be applied to large-scale sex identification of grass carp, and sex data can be obtained in the early stage of grass carp growth. Through extensive experimental verification, it is expected to be applicable to different grass carp populations. This molecular marker is currently the only molecular marker based on DNA sequence that can effectively identify the genetic sex of grass carp, and can accurately obtain the genetic sex of different grass carp populations, filling the gap in molecular markers for sex identification of grass carp.

[0021] 2. This invention also provides primers for amplifying the aforementioned molecular markers for identifying the sex of grass carp. Based on the specific DNA fragments of male grass carp, these primers have the characteristics of high specificity and high amplification efficiency during PCR amplification. The PCR products can be directly distinguished by electrophoresis. Compared with the method of sex identification by tissue sections, this method has the advantages of being simple, fast, efficient, and unaffected by the undifferentiated gonads of grass carp in the early stage. Marker-assisted breeding of grass carp can greatly save breeding costs and improve breeding efficiency. Attached Figure Description

[0022] Figure 1 This is an electrophoretic identification image of 5 female fish and 5 male fish selected after PCR amplification and capillary electrophoresis of the samples using sex primers in Example 1. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the described embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] The following examples illustrate the screening method for molecular markers to identify the genetic sex of grass carp, as well as the development and validation of primers for grass carp sex identification. The steps are as follows:

[0025] 1) Select male and female grass carp individuals. During the breeding season, determine the sex of the grass carp by observing the discharge from the genital opening when squeezed. Cut off the tail fin of each individual as a sample to be tested.

[0026] 2) DNA samples were extracted using the conventional phenol-chloroform method. The purity of the DNA was detected using a Thermo Nano Drop 2000 spectrophotometer and 2% agarose gel electrophoresis. Qualified male and female grass carp DNA samples were selected. Resequencing libraries were constructed from 30 female and 30 male grass carp DNA samples. Resequencing was performed using the Illumina sequencing platform and the paired-end sequencing method to obtain sequencing data.

[0027] 3) Using the grass carp genome as a reference sequence, all grass carp resequencing data were aligned to the reference genome to obtain all variation data. Then, male-specific fragments were obtained through genome-wide association analysis, and their nucleotide sequences are as follows:

[0028] TTACGAGCGCATCTGAAAAATAGCGGAGGGCACAGTGAGCTGCTGCGGGGAAAG TGCATAATCCAATTTGTCCTAAACATGAGACATGCTTTATATAAAGGGCTTCAAAATAACT CATAAAATAAATAACGCCTATAGTTCAAAGCGGCTTGCCCTTTCAGATGCTCT (SEQ ID NO: 2).

[0029] 4) Primers were designed using Primer5, and the primer with the highest score was selected as the primer for identifying the genetic sex of grass carp.

[0030] 5) Using the primers designed in step 4), PCR amplification was performed on the individuals whose sexes had been identified in step 1). The PCR amplification system was 25 μL: 2×Taq PCR Master Mix, 12.5 μL; forward and reverse primers, 1 μL each; DNA template, 2 μL; ddH2O, 8.5 μL.

[0031] The PCR amplification program was as follows: 95℃ for 3 min; 95℃ for 30 s, 59.6℃ for 30 s, 72℃ for 1 min, 35 cycles; 72℃ for 10 min. Cool to 4℃.

[0032] 6) After electrophoresis on a 2% agarose gel, observe whether the band has been successfully amplified by checking for the presence or absence of a bright band.

[0033] 7) Perform capillary electrophoresis on the amplified products, and obtain the verification results of the sex identification primers based on the number of signals appearing at the target position and the sex information in step 1).

[0034] Example 1

[0035] This embodiment provides a method for identifying the sex of grass carp, the steps of which are as follows:

[0036] 1) Selection of grass carp: Thirty adult grass carp were selected from the Guangling Yangtze River System Domestic Fish Breeding Farm in Yangzhou City, Jiangsu Province. They were classified by sex according to their external morphology, into 15 males and 15 females.

[0037] 2) Sampling: Take samples of the caudal fin tissue from each grass carp. Using sterilized scissors, cut a small amount of caudal fin along the outer edge of the fin. Use sterilized forceps to place the fin tissue into a petri dish containing anhydrous ethanol for rinsing. Then, place it into a 2mL EP tube containing anhydrous ethanol for dehydration. Mark the tube wall with the number and sex information, and store it in a -80℃ freezer.

[0038] 3) DNA extraction from grass carp: Remove the fin rays from the EP tube, squeeze out the alcohol with filter paper, and extract DNA using the traditional phenol-chloroform method. Use a Thermo Nano Drop 2000 spectrophotometer and 2% agarose gel electrophoresis to detect DNA purity. Take qualified DNA samples for subsequent analysis.

[0039] 4) The PCR amplification system is a 25 μL system: Components include: 12.5 μL of 2×Taq PCR Master Mix; 1 μL each of forward and reverse primers; 2 μL of DNA template; and 8.5 μL of ddH2O. The primer sequences are as follows:

[0040] Upstream primer Oligo-L: 5'-FAM-TTACGAGCGCATCTGAAAAA-3'

[0041] Downstream primer Oligo-R: 5'-AGAGCATCTGAAAGGGCAAG-3'.

[0042] The PCR amplification program was as follows: 95℃ for 3 min; 95℃ for 30 s, 59.6℃ for 30 s, 72℃ for 1 min, 35 cycles; 72℃ for 10 min. Cool to 4℃.

[0043] 5) Capillary electrophoresis was performed on 15 randomly obtained male and 15 female grass carp samples using primer pair Oligo-L / R. The results showed that male grass carp exhibited peaks at both 152bp and 168bp, while female grass carp showed only a peak at 168bp. Figure 1 As shown.

[0044] In summary, this invention, through genome-wide association analysis, screened molecular markers for identifying the sex of grass carp. The results are accurate, the experimental cycle is short, and it can be applied to large-scale sex identification of grass carp. Sex data can be obtained in the early stages of grass carp growth. Extensive experiments have verified that this molecular marker is currently the only one based on DNA sequence that can effectively identify the genetic sex of grass carp. It can be applied to different grass carp populations and can accurately obtain the genetic sex of different grass carp populations, filling the gap in molecular markers and sex identification for grass carp. Compared with methods for sex identification through tissue sections, it has advantages such as simplicity, speed, efficiency, and being unaffected by the undifferentiated gonads in the early stages of grass carp growth. Marker-assisted breeding of grass carp can greatly save breeding costs and improve breeding efficiency.

[0045] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A molecular marker for identifying the genetic sex of grass carp, characterized in that, The molecular markers include a common band of male and female grass carp as shown in SEQ ID NO: 1 and a band specific to male grass carp as shown in SEQ ID NO:

2.

2. The application of the primers for amplifying the molecular markers for identifying the genetic sex of grass carp as described in claim 1 in the identification of the sex of grass carp.

3. A primer for amplifying the molecular marker for identifying the genetic sex of grass carp as described in claim 1, the nucleotide sequence of which is shown in SEQ ID NO: 3 and SEQ ID NO:

4.

4. The application of the primers described in claim 3 in identifying the sex of grass carp.

5. A method for identifying the sex of grass carp, characterized in that, Includes the following steps: 1) Extract DNA from the grass carp sample to be identified; 2) Using the DNA of the grass carp sample to be identified as a template, PCR amplification was performed using the primers described in claim 3; 3) Detection of PCR products: After confirming the presence of bands by 2% agarose gel electrophoresis, the PCR products are subjected to capillary electrophoresis. If signals appear simultaneously at 152bp and 168bp after electrophoresis, the grass carp to be identified is male. If a signal is found only at 168bp after electrophoresis, the grass carp to be identified is female.

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