Sex molecular marker primer group, preparation method and application thereof, and cultivation method of all-male largemouth bass

Abstract

The invention discloses a sex molecular marker primer group, a preparation method and application thereof and a breeding method of all-male largemouth bass, and relates to the field of all-male largemouth bass breeding, the sex molecular marker primer group comprises a primer pair I or a primer pair II for screening genetic male largemouth bass, and a primer pair III for screening super-male largemouth bass; in the primer pair I, the sequence of a forward primer F1 is as shown in SEQ ID No: 1, and the sequence of a reverse primer R1 is as shown in SEQ ID No: 2; in the primer pair II, the sequence of a forward primer F2 is as shown in SEQ ID No: 3, and the sequence of a reverse primer R2 is as shown in SEQ ID No: 4; in the third primer pair, the sequence of a forward primer F3 is shown as SEQ ID No: 5, and the sequence of a reverse primer R2 is shown as SEQ ID No: 6. The primer group is designed according to male specific fragments, PCR detection is carried out on the basis of specific primers, super-male micropterus salmoides can be obtained through screening, and then all-male micropterus salmoides groups are obtained through culture.

Description

Technical Field

[0001] This application relates to the field of breeding all-male largemouth bass, and more particularly to a sex molecular marker primer set, its preparation method and application, and a method for breeding all-male largemouth bass. Background Technology

[0002] Largemouth bass belong to the genus *Leptochloa* of the family Spinosauridae in the order Perciformes. They are native to freshwater basins in eastern and central North America. Introduced to China in 1983, largemouth bass are currently farmed in Guangdong, Hunan, Zhejiang, and Jiangsu provinces. They are carnivorous and predatory fish that prefer clear, warm waters with shelter. They are also an important aquaculture species, prized for their delicious, low-bone flesh.

[0003] Largemouth bass exhibit sexual dimorphism, with males growing faster than females. Establishing single-sex populations would help improve reproduction in largemouth bass aquaculture. However, the sex chromosomes of bony fish are often less differentiated than autosomes, lacking significant morphological differences and making them difficult to distinguish. Sex molecular markers, on the other hand, are specific DNA sequences anchored to differences in the male and female genomes and are usually closely linked to genetic sex. Therefore, it is necessary to develop sex molecular markers for efficient identification of the genetic sex of largemouth bass.

[0004] To this end, Chinese invention patent application number CN202511286242.6 discloses a sex-specific molecular marker method for largemouth bass and its application. By using the primers involved in this scheme, male or female largemouth bass can be efficiently identified.

[0005] However, the above method can only systematically identify the xx genotype of females and the xy genotype of males, but cannot accurately distinguish between the xy and yy genotypes of males. For example, if a male with the xy genotype is selected and mated with a female with the xx genotype, the offspring may still produce a female population. Therefore, it is still impossible to establish a single-sex population using the above primers and methods, that is, it is impossible to breed an all-male largemouth bass population. Summary of the Invention

[0006] This invention provides a sex molecular marker primer set, its preparation method, its application, and a method for cultivating all-male largemouth bass. The sex molecular marker primer set is designed based on male-specific fragments. PCR detection using these sex-specific primers can effectively screen for supermale fish, thereby cultivating an all-male largemouth bass population.

[0007] The objective of this invention is achieved through the following technical solution: A sex molecular marker primer set includes primer pair one or primer pair two for screening genetically male largemouth bass, and primer pair three for screening supermale fish. In the primer pair one, the sequence of the forward primer F1 is shown in SEQ ID No: 1, and the sequence of the reverse primer R1 is shown in SEQ ID No: 2.

[0008] SEQ ID No: 1: TGAGCTACAGCCACCCAAAT.

[0009] SEQ ID No: 2: GCCGTGCAGAAGTAACATAACA.

[0010] The template strand DNA sequence corresponding to primer pair one is SEQ ID No: 7.

[0011] In the second primer pair, the sequence of the forward primer F2 is shown in SEQ ID No: 3, and the sequence of the reverse primer R2 is shown in SEQ ID No: 4.

[0012] SEQ ID No: 3: AACACTTCGGGCAATGAGGA.

[0013] SEQ ID No: 4: TCTTGGTCTGGCTGGCTTCT.

[0014] The template strand DNA sequence corresponding to primer pair 2 is SEQ ID No: 8.

[0015] Of the three primer pairs, the sequence of the forward primer F3 is shown in SEQ ID No: 5, and the sequence of the reverse primer R2 is shown in SEQ ID No: 6.

[0016] SEQ ID No: 5: GACTCAGGTCCGACACTTTCAT.

[0017] SEQ ID No: 6: AAGCCTACCCTGGCAAGCAACT.

[0018] The template strand DNA sequence corresponding to primer pair 3 is SEQ ID No: 9.

[0019] This invention also provides a method for preparing a sex molecular marker primer set, the method comprising the following steps: (1) Collect the tail fins of male and female largemouth bass and preserve them in ep tubes containing ethanol; (2) Genomic DNA was extracted from the collected male and female caudal fins; (3) Detect the concentration of genomic DNA. After passing the quality inspection, store the genomic DNA in an environment of -80℃ and perform whole genome sequencing. (4) After obtaining the whole genome mixed sequencing sequence fragments, male-specific fragments are then identified through bioinformatics analysis; (5) Three pairs of male-specific primers were screened out and verified by PCR amplification and nucleic acid electrophoresis in the largemouth bass population, thus obtaining the sex molecular marker primer set.

[0020] As preferred, primer pair one amplified a male-specific band of 431 bp; primer pair two amplified a male-specific band of 524 bp; primer pair three amplified bands that could distinguish between XX, XY, and YY types, with XY type having a 979 bp band and a 388 bp band, YY type male largemouth bass having only a 388 bp band, and XX type female largemouth bass having only a 979 bp band.

[0021] This invention also provides applications of the above-mentioned sex molecular marker primer set, specifically as follows (a1), (a2), (a3), or (a4): (a1) Primer pair one or primer pair two is used to identify genetically male largemouth bass; (a2) Primer pair one or primer pair two are used to prepare a kit for identifying genetically male largemouth bass; (a3) Primer pair 3 was used to identify super-male largemouth bass; (a4) Primer pair 3 was used to prepare a kit for identifying super-male largemouth bass.

[0022] This invention also provides a method for breeding all-male largemouth bass, the method comprising the following steps: S01. Hormone induction was used to induce sex reversal in XY-type males of common largemouth bass juveniles, turning them into pseudo-females. S02. During the breeding season, the hormone-induced population is screened to select largemouth bass with obvious female characteristics. Then, one or two pairs of primers are used to perform genetic sex identification on the selected largemouth bass with obvious female characteristics to screen out XY type pseudo-female fish. S03. Pair pseudo-female fish with normal male fish and use primer pairs for triple screening to select super-male fish in the offspring. S04. When normal female fish mate with super-male fish, male offspring can be produced, thus enabling the cultivation of an all-male largemouth bass population.

[0023] Preferably, in step S01, the hormone is estradiol sex reversal hormone.

[0024] As a preferred option, the hormone induction in step S01 specifically involves adding estradiol sex reversal hormone to the feed during the initial feeding stage of largemouth bass fry, and continuing the administration for 2 months.

[0025] Compared with the prior art, the advantages or beneficial effects of the technical solution of this application include: 1. This invention uses bioinformatics methods to statistically analyze the differences in sequencing depth between male and female individuals, enabling rapid screening of male-specific Y-linked fragments. This method for screening male-specific fragments is simple to operate and widely applicable.

[0026] 2. The sex molecular marker (primer pair one or primer pair two) of the present invention is dominant in male largemouth bass. Male-specific primers designed based on this molecular marker can stably and efficiently identify the genetic sex of largemouth bass.

[0027] 3. By utilizing the sex molecular marker (primer pair 3) binding sex reversal technology provided by this invention, an all-male largemouth bass population can be prepared, which can significantly improve aquaculture efficiency and is of great significance in the aquaculture industry. Attached Figure Description

[0028] Figure 1 Agarose gel image showing the molecular markers for primer pair one; Figure 2 Agarose gel image showing the molecular markers for primer pair 2; Figure 3 Agarose gel image showing the molecular markers for primer pair 3. Detailed Implementation

[0029] The following detailed description of the embodiments of this application, in conjunction with the accompanying drawings, will provide a thorough understanding of how this application uses technical means to solve technical problems and achieve corresponding technical effects, enabling its implementation. The embodiments of this application and the various features within them can be combined with each other without conflict, and all resulting technical solutions are within the protection scope of this application.

[0030] It should be clearly stated that the embodiments described below are merely some embodiments of this application, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application.

[0031] Example 1: This example provides a detailed explanation of the breeding program for all-male largemouth bass: This invention provides three pairs of primers for identifying the genetic sex of males and females. Through PCR amplification and nucleic acid electrophoresis, the results can effectively distinguish between genetic males and females.

[0032] The specific steps for developing sex molecular markers for largemouth bass are as follows: (1) During the breeding season in May, the phenotypic sex of largemouth bass was determined by examining the expelled sperm or eggs. Healthy male and female individuals with normal appearance were selected, and the tail fin rays were cut off and preserved in 1.5ml ep tubes containing 90% ethanol. (2) Genomic DNA was extracted using the TaKaRa MiniBEST Universal Genomic DNA Extraction Kit Ver.5.0 (Code No. 9765) according to the manufacturer’s instructions, and then the genomic DNA was checked for degradation and contamination by 1% agarose gel electrophoresis. (3) The concentration of genomic DNA was detected using a spectrophotometer (NanoDrop ND2000) and a fluorescence meter kit (Qubit® DNA AssayKit in Qubit® 3.0 fluoromete). After passing the quality inspection, the genomic DNA was stored at -80℃ and then subjected to whole genome sequencing. (4) After obtaining the whole-genome mixed sequencing reads, male-specific reads were identified through bioinformatics analysis. Specifically, the male and female reads were aligned with the female reference genome [GCA_014851395.1] at the Scaffold level and the undetermined sex reference genome [GCA_019677235.1] at the chromosome level using BWA mem 0.7.17 software. The aligned sequences were then sorted, grouped, merged, and deduplicated using Picard 2.18.2 software with default settings. The BAM files were filtered using Samtools 1.8 software to remove low-quality reads. Then, the two sex BAM files were used to generate an mpileup file using the samtools mpileup command. The mpileup file was then used by the popoolation mpileup2sync 1.201 software to create a sync file with the minimum quality set to 20. High-density sex-biased SNP regions were identified using PSASS software with a 100 kb sliding window at 10 kb resolution. Finally, regions with sequencing depth differences between males and females were identified by pooled sequencing (Pool-seq) analysis. Sex-specific potential fragments in these regions were extracted using IGV software, and male-specific primers were designed.

[0033] (5) Three pairs of male-specific primers were selected and verified in a largemouth bass population by PCR amplification and nucleic acid electrophoresis. The results showed that... Figure 1-3As shown, primer pair one amplified a male-specific band of 431 bp, while no band of the same size was amplified in females; primer pair two amplified a male-specific band of 524 bp, while no band of the same size was amplified in females; the amplified band of primer pair three could distinguish between XX, XY, and YY types, with XY type having a 979 bp band and a 388 bp band, YY type male largemouth bass having only a 388 bp band, and XX type female largemouth bass having only a 979 bp band.

[0034] Example 2: A method for breeding all-male largemouth bass, the method specifically includes the following steps: This invention provides an application of sex molecular markers in the preparation of an all-male largemouth bass population. The specific steps are as follows: (1) In a population of common largemouth bass juveniles, sex reversal was induced in the XY type males to become pseudo-females through hormone induction.

[0035] (2) During the breeding season, the hormone-induced population is screened, and largemouth bass with obvious female characteristics are selected. Then, the selected largemouth bass with obvious female characteristics are genetically sexed using primer pair one or primer pair two to screen out XY type pseudo-female fish. The selected genetic male largemouth bass are sex-reversed into pseudo-female fish (XY) through hormone induction.

[0036] (3) Pseudo-female fish (XY) were mated with normal male fish (XY), and super-male fish (YY) individuals in the offspring were selected by primer pair triple screening. The nucleic acid electrophoresis results of the super-male fish showed only one 388 bp band.

[0037] (4) Normal female fish (XX) mating with super male fish (YY) can produce offspring with 100% male fish (XY), thus realizing the preparation of an all-male largemouth bass population.

[0038] Conclusion: Controlling the sex ratio of fish in aquaculture is an important technique that can directly improve farming efficiency. Many fish species exhibit significant sexual dimorphism in growth; cultivating monosex populations can effectively address problems such as uneven growth and low feed conversion rates caused by reproduction.

[0039] Compared to hormone-induced breeding, marker-assisted breeding does not pose drug residue problems, and it is more mature and can be applied on a large scale than gene editing technology. Therefore, using sex molecular markers combined with breeding technology to create an all-male largemouth bass population is the preferred method for large-scale largemouth bass farming in the aquaculture industry, ensuring food safety while significantly improving economic benefits.

[0040] This invention enables rapid screening of male-specific Y-linked fragments using primer pair one and primer pair two. This method for screening male-specific fragments is simple to operate and widely applicable. Sex molecular markers are dominant in male largemouth bass, and male-specific primers designed based on these markers can stably and efficiently identify the genetic sex of largemouth bass.

[0041] Using the sex molecular marker (primer pair 1, primer pair 2, primer pair 3) binding sex reversal technology provided by this invention, an all-male largemouth bass population can be prepared, which can significantly improve aquaculture efficiency and is of great significance in the aquaculture industry.

Claims

1. A sex molecular marker primer set, characterized in that, This includes primer pair one or primer pair two for screening genetically male largemouth bass, and primer pair three for screening supermale fish; In the primer pair one, the sequence of the forward primer F1 is shown in SEQ ID No: 1, and the sequence of the reverse primer R1 is shown in SEQ ID No: 2; In the second primer pair, the sequence of the forward primer F2 is shown in SEQ ID No: 3, and the sequence of the reverse primer R2 is shown in SEQ ID No: 4; Of the three primer pairs, the sequence of the forward primer F3 is shown in SEQ ID No: 5, and the sequence of the reverse primer R2 is shown in SEQ ID No:

6.

2. The method for preparing a sex molecular marker primer set according to claim 1, characterized in that, Includes the following steps: (1) Collect the tail fins of male and female largemouth bass and preserve them in ep tubes containing ethanol; (2) Genomic DNA was extracted from the collected male and female caudal fins; (3) Detect the concentration of genomic DNA. After passing the quality inspection, store the genomic DNA in an environment of -80℃ and perform whole genome sequencing. (4) After obtaining the whole genome mixed sequencing sequence fragments, male-specific fragments are then identified through bioinformatics analysis; (5) Three pairs of male-specific primers were screened out and verified by PCR amplification and nucleic acid electrophoresis in the largemouth bass population, thus obtaining the sex molecular marker primer set.

3. The preparation method according to claim 2, characterized in that, Primer pair 1 amplified a male-specific band of 431 bp; primer pair 2 amplified a male-specific band of 524 bp; primer pair 3 amplified bands that could distinguish between XX, XY, and YY types, with XY type having a 979 bp band and a 388 bp band, YY type male largemouth bass having only a 388 bp band, and XX type female largemouth bass having only a 979 bp band.

4. The application of the sex molecular marker primer set as described in claim 1, characterized in that, The following are options: (a1), (a2), (a3), or (a4): (a1) Primer pair one or primer pair two is used to identify genetically male largemouth bass; (a2) Primer pair one or primer pair two are used to prepare a kit for identifying genetically male largemouth bass; (a3) Primer pair 3 was used to identify super-male largemouth bass; (a4) Primer pair 3 was used to prepare a kit for identifying super-male largemouth bass.

5. A method for breeding all-male largemouth bass, characterized in that, Includes the following steps: S01. Hormone induction was used to induce sex reversal in XY-type males of common largemouth bass juveniles, turning them into pseudo-females. S02. During the breeding season, the hormone-induced population is screened, and largemouth bass with obvious female characteristics are selected. Then, the genetic sex of the selected largemouth bass with obvious female characteristics is determined by using one or two pairs of primers as described in claim 1, and XY type pseudo-female fish are screened out. S03. Mating pseudo-female fish with normal male fish, and using the primer pair described in claim 1 to screen out super-male fish in the offspring. S04. When normal female fish mate with super-male fish, male offspring can be produced, thus enabling the cultivation of an all-male largemouth bass population.

6. The cultivation method according to claim 5, characterized in that, In step S01, the hormone is estradiol sex reversal hormone.

7. The cultivation method according to claim 6, characterized in that, The hormone induction in step S01 specifically involves adding estradiol sex reversal hormone to the feed during the initial feeding stage of largemouth bass fry, and continuing the administration for 2 months.

Images