Genetic sex specific molecular marker covering seven InDel sites of spinibarbus hollandi male and application of genetic sex specific molecular marker

Abstract

The invention discloses a genetic sex-specific molecular marker covering seven InDel sites of a spinibarbus hollandi male and application of the genetic sex-specific molecular marker covering the seven InDel sites of the spinibarbus hollandi male. The molecular marker successfully recognizes an inter-sex specific insertion or deletion sequence (InDel) through whole genome re-sequencing in combination with a whole genome association analysis method. Polymerase chain reaction (PCR) amplification is carried out through a conventional primer, and in combination with an agarose gel electrophoresis technology, rapid and stable identification of hereditary sex of spinibarbus hollandi is realized. The molecular marker covers seven InDel loci of the spinibarbus hollandi male, shows the advantages of simplicity and convenience in operation and accurate result in the aspect of genetic sex identification, has important significance in researching the sex determination mechanism of the spinibarbus hollandi, and can provide assistance for a parthenogenesis technology, so that the economic benefit of aquaculture is improved.

Description

Technical Field

[0001] The invention belongs to the fish genetic sex identification technology in the field of aquaculture, and particularly relates to a genetic sex-specific molecular marker covering 7 InDel sites of male black-spined barb and an application thereof. Background Art

[0002] Sex determination is the basis for the reproduction of bisexual organisms. Precise sex differentiation ensures the normal reproduction and continuation of populations. Sex is often related to biological characteristics such as growth rate and metabolic requirements. Fish play an important role in the study of sex determination mechanisms. As a key group in the evolution of vertebrates, they show a variety of sex determination methods. Fish species are abundant and widely distributed, providing an ideal model for revealing the genetic and environmental regulatory mechanisms of sex determination. Many fish have significant differences in sex-related growth rates. The development of precise genetic sex molecular markers can not only assist in single-sex breeding and improve aquaculture efficiency, but also deepen the scientific understanding of sex development and evolutionary adaptation mechanisms. These studies have provided important theoretical support and technical means for biodiversity conservation and the development of aquaculture industry.

[0003] The black-backed spiny barb (Spinibarbushollandi) is a freshwater fish belonging to the order Cypriniformes, family Cyprinidae, genus Spinibarbus. Its meat is delicious and has certain fishery value. In addition, due to its high requirements for water quality, it can also be used as an environmental indicator species to monitor the health of water bodies. In the fields of aquaculture and ecological environmental protection, the black-backed spiny barb shows potential application value. The black-backed spiny barb has sexual dimorphism, and there are differences in body size between female and male individuals. Female individuals are larger than male individuals, which provides a direction for single-sex breeding and aquaculture. By identifying genetic sex and cultivating all-female seedlings, the breeding efficiency of black-backed spiny barb can be significantly improved.

[0004] Sex molecular marker technology is an important tool in biological research and application in recent years, with broad prospects and far-reaching significance. Sex molecular marker technology can greatly improve the efficiency of aquaculture, especially in fish and other aquatic species. By accurately identifying sex, single-sex breeding can be achieved, and individuals of the sex with higher economic value can be selectively bred. Before the gonads mature, male and female individuals of the black-backed spiny barb do not show significant morphological differences. Therefore, the development of a molecular marker that can identify its genetic sex is of great practical significance for the application of single-sex breeding technology. Summary of the invention

[0005] The invention aims to provide a genetic sex-specific molecular marker covering 7 InDel sites of male black-spined barb and application thereof.

[0006] To achieve the above purpose, the present invention adopts the following technologies: The first aspect of the present invention provides a molecular marker for identifying the genetic sex of black-backed spiny barb, wherein the molecular marker is located on chromosome 46 of black-backed spiny barb, covers 7 InDel sites, and has specific position coordinates of 36,036,398 bp to 36,036,651 bp.

[0007] The second aspect of the present invention provides the above-mentioned primers for molecular markers, wherein the nucleotide sequences of the primers are: P11-3-F: 5'-CCCAGTTCAGCTTCAAACCG-3', P11-3-R: 5'-ACAGGTTCAGTAATGTCCTTTAG-3'.

[0008] The third aspect of the present invention provides a kit for identifying the genetic sex of black-backed spinifex, the kit comprising the above-mentioned primers.

[0009] The fourth aspect of the present invention provides the use of the above-mentioned molecular markers, primers and kits in identifying the genetic sex of black-backed spinifex.

[0010] The fifth aspect of the present invention provides the use of the above-mentioned molecular markers, primers and kits in the artificial breeding of black-spined barb.

[0011] The sixth aspect of the present invention provides a method for identifying the genetic sex of black-backed spiny barb, using the above-mentioned primers or kit to perform PCR amplification on the genomic DNA of the black-backed spiny barb to be tested, and detecting the PCR product by agarose gel electrophoresis technology, thereby achieving the identification of the genetic sex of the black-backed spiny barb; Furthermore, the reaction system of the PCR amplification is: 2×Taq Master Mix 10.0 μL, primer P11-3-F 0.8 μL, primer P11-3-R 0.8 μL, DNA template 1.0 μL, and ddH2O is added to make up to 20.0 μL; Furthermore, the reaction procedure of the PCR amplification is: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 25 s; annealing at 56°C for 25 s; extension at 72°C for 30 s, 24 cycles; extension at 72°C for 5 min; cooling and storage at 4°C; Further, when the detection result of the agarose gel electrophoresis shows two bands of 380 bp and 360 bp, it indicates that the black-backed spiny barb to be tested is male; when the detection result of the agarose gel electrophoresis shows only one band of 380 bp, it indicates that the black-backed spiny barb to be tested is female; Furthermore, the concentration of agarose gel electrophoresis is 3%.

[0012] The present invention discloses the following technical effects: The black-backed spiny barb sex molecular marker disclosed in the present invention uses the genomic DNA of the black-backed spiny barb as a template, and realizes the rapid and stable identification of the genetic sex of different black-backed spiny barb individuals through PCR amplification and agarose gel electrophoresis technology. The technology has the advantages of low cost, simple operation, accurate results, etc. Its application is helpful to promote the development of black-backed spiny barb parthenogenesis technology, and then improve the economic benefits of black-backed spiny barb breeding industry. In addition, the molecular marker provided by the present invention provides important theoretical support for basic scientific research such as in-depth study of the black-backed spiny barb sex determination mechanism. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 : GWAS analysis chart of sex of black-backed spiny barb.

[0014] Figure 2 : IGV visualization of sex-specific InDel sites in black-backed spiny barb.

[0015] Figure 3 : Sequence alignment of male and female black-backed barb. The sequence in the box is the primer binding site.

[0016] Figure 4 :The electrophoresis diagram of genetic sex identification of black-spine barb using primers P11-3-F / R. M represents the DNA molecular weight standard DL500, and N is the negative control (ddH2O). DETAILED DESCRIPTION

[0017] Now, a plurality of exemplary implementation steps of the present invention will be described in detail to provide a clearer and more complete explanation of the concept and effect of the present invention. The described embodiments represent only a part of the examples of the present invention, not all examples, and should not be regarded as limiting the present invention.

[0018] Example 1: Acquisition of molecular markers The present invention provides a molecular marker for identifying the genetic sex of black-backed spiny barb. The method for obtaining the molecular marker is as follows: based on the black-backed spiny barb genome of this laboratory, 25 male and female black-backed spiny barb were subjected to whole genome resequencing. Through genome-wide association analysis (GWAS), the genomic regions highly correlated with the sex of black-backed spiny barb were analyzed and identified. After a strict quality control process, a total of 16,774,171 SNP sites were included in the subsequent analysis ( Figure 1 ). Among them, 102 SNP loci were significantly associated with the sex of black-backed spiny barb, and these loci were located on chromosome 46 of black-backed spiny barb. Taking into account the analysis results of GWAS, 7 InDel loci in the range of 36,036,398 bp to 36,036,651 bp were finally selected to develop sex-related molecular markers ( Figure 2 ).

[0019] Example 2: Design and application of specific molecular marker primers Primers were designed based on the positions of the 7 InDel sites in Example 1 on the black-backed barb genome. The expected amplification product contained a fragment including the 7 InDel sites ( Figure 3 ). Finally, a pair of specific primers P11-3-F / R was determined, and the nucleotide sequence was as follows: P11-3-F: 5'-CCCAGTTCAGCTTCAAACCG-3', P11-3-R: 5'-ACAGGTTCAGTAATGTCCTTTAG-3'.

[0020] PCR amplification was performed using the genomic DNA of black-backed barb as a template; the PCR amplification reaction system was: 2×TaqMaster Mix 10.0μL, primer P11-3-F 0.8μL, primer P11-3-R 0.8μL, DNA template 1.0μL, and ddH2O was added to make up to 20.0μL; the PCR amplification reaction program was: 95℃ pre-denaturation for 3min; 95℃ denaturation for 25s; 56℃ annealing for 25s; 72℃ extension for 30s, 24 cycles; 72℃ extension for 5min; 4℃ cooling and storage. The PCR product was analyzed by 3% agarose gel electrophoresis to determine the genetic sex of black-backed barb. When the electrophoresis detection results showed two bands of 380bp and 360bp, it indicated that the black-backed barb was male, while when only one band of 380bp appeared, it indicated that the black-backed barb was female.

[0021] Example 3: Actual sample detection The sex of the collected black-backed spiny barb was determined by squeezing out semen or eggs, and 9 male and female black-backed spiny barb of known sex were collected, and genomic DNA was extracted as a template for PCR amplification; wherein, the PCR amplification reaction system was: 2×Taq Master Mix 10.0μL, primer P11-3-F (5'-CCCAGTTCAGCTTCAAACCG-3') 0.8μL, primer P11-3-R (5'-ACAGGTTCAGTAATGTCCTTTAG-3') 0.8μL, DNA template 1.0μL, and ddH2O was added to make up to 20.0μL; the reaction program of PCR amplification was: 95℃ pre-denaturation for 3min; 95℃ denaturation for 25s; 56℃ annealing for 25s; 72℃ extension for 30s, 24 cycles; 72℃ extension for 5min; 4℃ cooling and storage. The PCR product was subjected to 3% agarose gel electrophoresis analysis, and the genetic sex of the black-backed barb was determined according to the determination method of Example 2. Figure 4As shown, the agarose gel electrophoresis results matched the gender of the samples used, and the accuracy of the specific molecular marker primers reached 100%.

[0022] The above embodiments are only preferred embodiments of the present invention, but they cannot be considered as limiting the scope of implementation of the present invention. Several improvements can be made without departing from the principle of the present invention, but they should also be considered as the protection scope of the present invention.

Claims

1. A molecular marker for identifying the genetic sex of black-backed barb, characterized in that: The molecular marker is located on chromosome 46 of black-backed spinifex, covers 7 InDel sites, and the specific position coordinates are 36,036,398 bp to 36,036,651 bp.

2. A molecular marker primer according to claim 1, characterized in that: The nucleotide sequences of the primers are: P11-3-F: 5'-CCCAGTTCAGCTTCAAACCG-3', P11-3-R: 5'-ACAGGTTCAGTAATGTCCTTTAG-3'.

3. A kit for identifying the genetic sex of black-backed barb, characterized in that: The kit comprises the primers according to claim 2.

4. Use of the molecular markers according to claim 1, the primers according to claim 2, and the kit according to claim 3 in identifying the genetic sex of black-backed barb.

5. Use of the molecular marker according to claim 1, the primer according to claim 2, and the kit according to claim 3 in artificial breeding of black-spined barb.

6. A method for identifying the genetic sex of black-backed barb, characterized in that: The primers described in claim 2 or the kit described in claim 3 are used to perform PCR amplification on the genomic DNA of the black-backed spiny barb to be tested, and the PCR product is detected by agarose gel electrophoresis technology, thereby realizing the identification of the genetic sex of the black-backed spiny barb.

7. The method according to claim 6, characterized in that: The reaction system of the PCR amplification is: 2×TaqMaster Mix 10.0 μL, primer P11-3-F 0.8 μL, primer P11-3-R 0.8 μL, DNA template 1.0 μL, and ddH2O is added to make up to 20.0 μL.

8. The method according to claim 6, characterized in that: The reaction procedure of the PCR amplification is: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 25 s; annealing at 56°C for 25 s; extension at 72°C for 30 s, 24 cycles; extension at 72°C for 5 min; and cooling and storage at 4°C.

9. The method according to claim 6, characterized in that: When the detection result of the agarose gel electrophoresis shows two bands of 380 bp and 360 bp, it indicates that the black-backed spiny barb to be tested is male; when the detection result of the agarose gel electrophoresis shows only one band of 380 bp, it indicates that the black-backed spiny barb to be tested is female.

10. The method according to claim 6, characterized in that: The concentration of the agarose gel electrophoresis was 3%.

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